EPA Report Number
September 1985
HEALTH EFFECTS STUDY FOR
THE LUBBOCK LAND TREATMENT PROJECT
Lubbock Infection Surveillance Study (LISS)
by
D. E. Camann1, P. J. Graham2, M. N. Guentzel3, H. J. Harding1,
K. T. Kimball1 B. E. Moore4 R. L. Northrop2,
N. L. Altman , R. B. Harrist5, A. H. Holguin5,
R. L. Mason , C. Becker Popescu , C.A. Sorber
Southwest Research Institute, San Antonio, TX 78284
University of Illinois at Chicago, Chicago, IL 60680
University of Texas at San Antonio, San Antonio, TX 78285
4University of Texas at Austin, Austin, TX 78712
5University of Texas School of Public Health, Houston, TX 77025
CR-807501
Project Officer
Walter Jakubowski
Toxicology and Microbiology Division
Health Effects Research Laboratory
Cincinnati, OH 45268
This study was conducted in cooperation with:
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency, Ada, OK 74820
Lowell Leach, Project Officer
under Grant S806204 to:
LCC Institute of Water Research, Lubbock, TX 79407
Dennis B. George, Project Director
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NC 27711
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NOTICE
The information in this document has been funded in part by the Health
Effects Research Laboratory. United States Environmental Protection Agency
under CR-807501 to Southwest Research Institute and by R. S. Kerr Environmental
Laboratory, United States Environmental Protection Agency under S806204
to LCC Institute of Water Research. It has been subject to the Agency's
review and approved for publication. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
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EPA Report Number
September 1985
HEALTH EFFECTS STUDY FOR
THE LUBBOCK LAND TREATMENT PROJECT
Lubbock Infection Surveillance Study (LISS)
by
D. E. Camann1, P. J. Graham2, M. N. Guentzel3, H. J. Harding1,
K. T. Kimball1 B. E. Moore4 R. L. Northrop2,
N. L. Altman , R. B. Harrist6, A. H. Holguin5,
R. L. Mason1, C. Becker Popescu2, C.A. Sorber4
Southwest Research Institute, San Antonio, TX 78284
University of Illinois at Chicago, Chicago, IL 60680
University of Texas at San Antonio, San Antonio, TX 78285
University of Texas at Austin, Austin, TX 78712
5University of Texas School of Public Health, Houston, TX 77025
CR-807501
Project Officer
Walter Jakubowski
Toxicology and Microbiology Division
Health Effects Research Laboratory
Cincinnati, OH 45268
This study was conducted in cooperation with:
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency, Ada, OK 74820
Lowell Leach, Project Officer
under Grant S806204 to:
LCC Institute of Water Research, Lubbock, TX 79407
Dennis B. George, Project Director
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NC 27711
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FOKEVMD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to enhance
the quality of our environment in order to promote the public health and
welfare and the productive capacity of our Nation's population.
The complexities of environmental problems originate in the deep inter-
dependent relationships between the various physical and biological segments
of man's natural and social world. Solutions to these environmental problems
require an integrated program of research and development using input from
a number of disciplines. The Health Effects Research Laboratory, Research
Triangle Park, North Carolina, and Cincinnati, Ohio, conducts a coordinated
environmental health research program in toxicology, epidemiology, and
clinical studies using human volunteer subjects. Wide ranges of pollutants
known or suspected to cause health problems are studied. The research
focuses on air pollutants, water pollutants, toxic substances, hazardous
wastes, pesticides and nonionizing radiation. The laboratory participates
in the development and revision of air and water quality criteria and health
assessment documents on pollutants for which regulatory actions are being
considered. Direct support to the regulatory function of the Agency is
provided in the form of expert testimony and preparation of affidavits
as well as expert advice to the Administrator to assure the adequacy of
environmental regulatory decisions involving the protection of the health
and welfare of all U.S. inhabitants.
This report describes a 5-year prospective epidemiological study to
investigate potential infectious disease effects from sprinkler application
of wastewater to land. With a better understanding of health effects,
measures can be developed to reduce exposure to harmful materials.
F. Gordon Hueter, Ph.D.
Director
Health Effects Research Laboratory
iii
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PREFACE
The LCC Institute of Water Research (LCCIWR), Lubbock, Texas, conducted
a 5-year (1979-1983) research and demonstration program entitled the Lubbock
Land Treatment Project to expand and study Lubbock's municipal wastewater
land treatment system. A pipeline, storage reservoirs, distribution system,
and spray irrigation equipment were installed at the Hancock farm site,
located about 15 miles southeast of the sewage treatment plant and the
edge of Lubbock. The research programs of the Lnbbock Land Treatment Project
included ground water recovery studies at a farm practicing land application
of wastewater for over 40 years (the Gray site), a health effects study
at the Hancock site, and impact studies on crops, soil and ground water.
As part of the Lnbbock Land Treatment Project, the 5-year study, "Health
Effects Study for the Lubbock Land Treatment Project,'' (Lubbock Infection
Surveillance Study, LISS) was performed to investigate potential infectious
disease effects from sprinkler application of wastewater to land. The
health effects study is the subject of this report.
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ABSTRACT
The Lnbbock Infection Surveillance Study (LISS) was conducted to monitor
infections and acute illness in the primarily rural community surrounding
the Lubbock Land Treatment (Demonstration) System (LLTS) at the Hancock
farm near Wilson, Texas. The LISS objective was to identify possible adverse
effects on human health from slow-rate (sprinkler) land application of
wastewater which contained potentially pathogenic microorganisms.
An epidemiological analytic cohort study of 478 area residents and
Hancock farm workers was maintained during the first 20 months of operation
of the LLTS (February 1982-October 1983) and during the 20-month period
immediately preceding LLTS operation (June 1980-January 1982). Blood samples
collected semiannually were analyzed for antibody titers to 14 enteroviruses,
3 adenoviruses, 2 reoviruses, rotavirus, Norwalk virus, hepatitis A, Legionella.
Entamoeba histolvtica. and influenza A. Routine fecal specimens were collected
regularly to isolate enteric viruses and overt and opportunistic bacterial
pathogens. Electron microscopic examination was performed to detect a
variety of other virus-like particles. Tuberculin skin tests were administered
annually to detect non-tuberculosis mycobacterial infections. Illness
information was provided by study participants on a weekly basis. Concen-
trations of microorganisms also were measured in the wastewater, wastewater
aerosol, and drinking water. Dispersion modeling, participant activity
diaries, and a weekly log of extensive wastewater contact were used to
calculate an aerosol exposure index of relative cumulative exposure of
each participant to the wastewater aerosol within each of the four major
irrigation seasons.
Very high levels of bacteria and enteric viruses were present in the
sprayed wastewater obtained via pipeline directly from the Lubbock sewage
treatment plant. Enteroviruses were consistently found in the wastewater
aerosol in 1982.
Participants in the high and low exposure groups were generally well
balanced with regard to age, gender, previous titer, and time spent in
Lnbbock. However, aerosol exposure was largely confounded with patronage
of a local restaurant and use of evaporative cooler air conditioners.
Disease surveillance did not disclose any obvious connection between
the self-reporting of acute illness and degree of aerosol exposure.
Whenever a sufficient number of infections was observed during an
irrigation season, this infection episode was analyzed by four different
methods: confirmatory statistical analysis, exploratory logistic regression
analysis, confidence intervals of incidence density ratios, and risk ratio
scoring. The association of infection status with wastewater aerosol exposure
and other relevant factors was investigated.
VI
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Comparison of crude seroconversion incidence densities indicated that
some excess risk of viral infection (risk ratio of 1.5 to 1.8) appeared
to be associated with level of aerosol exposure. A symmetric risk ratio
scoring approach provided evidence of a dose-related stable association
(p=0.002) between the infection events in the observed episodes of infection
and aerosol exposure. More than the expected number of statistically signifi-
cant associations of the presence of infection with wastewater aerosol
exposure were found in the confirmatory analysis of independent infection
episodes using Fisher's exact test. Thus, three different statistical
approaches provided similar evidence that the rate of viral infections
was slightly higher among members of the study population who had a high
degree of aerosol exposure.
In the episode of poliovirus 1 seroconversions in spring 1982, some
of the infections were probably caused by wastewater aerosol exposure because
a strong association existed and no alternative explanation could be identi-
fied. Three distinct risk factors (poliovirus immunization in spring 1982,
low polio 1 antibody titer in January 1982, and a high degree of aerosol
exposure) were independently associated with the poliovirus 1 seroconversions
and each appears to have been responsible for some of the poliovirus 1
infections. Weak evidence of association was found between aerosol exposure
and infection by other enteric viruses (specific coxsackie B viruses and
echoviruses) which were simultaneously recovered from the wastewater during
the summer irrigation season of 1982. However, it could not be determined
whether aerosol exposure or identified alternative explanations were the
actual risk factor(s) in these enteric viral infections. The association
of viral infections with aerosol exposure shows a dose effect, since the
study population was exposed to more enteroviruses via the wastewater aerosol
in 1982 than in 1983.
The LISS was conducted by Southwest Research Institute (SwRI), the
University of Illinois (UI), the University of Texas at San Antonio (UTSA)
and the University of Texas at Austin (DTA) . This report was submitted
in fulfillment of CR 807501 and S806204 by SwRI under primary sponsorship
of the U.S. Environmental Protection Agency. This report covers field
activities performed from Hay 1, 1980 to October 31, 1983; work was completed
as of June 30, 1985.
vii
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CONTENTS
Page
Foreword iii
Preface v
Abstract vi
Figures xv
Tables zvi
Abbreviations xxii
Acknowledgement xxiv
1. Int r oduc t ion 1
A. Background 1
Land application and potential infectious disease hazards 1
Recent literature , < . > 2
The Lubbock Land Treatment System (LLTS) expansion 3
The Lubbock Infection Surveillance Study (LISS) 3
B. Study Objective 5
C. Study Design 5
D. Study Organization 10
2. Conclusions. 13
3. Recommendations 17
4. Methods and Materials 20
A. Study Site 20
Description of study area 20
General climatology 20
City of Wilson 21
Rural area 21
Lubbock sewage treatment plants 22
Lubbock land treatment system 23
System design and operation in relation to EPA design
criteria and recommendations 24
Periods of irrigation 27
B. Study Population 28
Sampling 28
Health interview and recruitment 31
Serosurvey 32
Fecal specimens 33
Illness and exposure monitoring 34
Illness specimens 36
Activity diaries 37
Tuberculin skin testing 37
Poliovirus immunization 38
Restaurant patronage survey 39
C. Exposure Estimation 40
Aerosol exposure index (AEI) 40
Additional exposure measures 44
ix
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CONTENTS (CONT'D)
D. Environmental Sampling 46
Wastewater 46
Wastewater aerosol 48
Background runs—1980 baseline year 48
Wastewater aerosol monitoring—1982 irrigation year 51
Microorganism runs 51
Quality assurance runs 54
Enterovirns runs 54
Dye runs 55
Particle size runs 56
Dust storm runs 56
Calculation of microorganism density in air 56
Flies 58
Drinking water 59
Meteorological data 60
Background aerosol runs 60
General climatology 60
Meteorological measurements during aerosol runs 62
E. Laboratory Analysis of Clinical Specimens 62
Serology, , - , , 62
Enteroviruses 62
Adenoviruses 65
Hepatitis A 65
Influenza 65
Legionella bacilli 65
Nor walk virus 66
Entamoeba histolvtica 66
Reoviruses 66
Rotavirus 66
Clinical bacteriology 67
Clinical virology 71
Electron microscopy of fecal specimens 73
F. Laboratory Analysis of Environmental Samples 74
Wastewater samples 74
Microbiological screens 74
Routine wastewater samples 82
Enterovirus identification samples 82
Limited bacterial screen samples 82
Legionella samples 83
Aerosol samples 84
Fly samples 85
Drinking water samples, 85
G. Infection Events and Episodes 87
Bacterial infection event 87
Viral infection event 89
Serological infection event 89
Identification of infection episodes 89
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CONTENTS (CONT'D)
Page
H. Data Management 97
Data processing and verification 97
Data base structure and use 98
I. Quality Assurance 100
Health data and specimens 100
Aerosol measurement precision 100
Laboratory analysis 102
Enterovims serology 102
Hepatitis A serology 104
Clinical bacteriology 104
Clinical virology 105
Electron microscopy 106
Environmental samples 106
Data management 108
Archiving of clinical specimens 108
J. Statistical Methods 108
Preliminary analysis 110
Confirmatory analysis Ill
Testing procedure Ill
Stratification 112
Exploratory analysis 112
Analysis of risk ratio (RR) scores 113
Analysis of incidence density ratios (IDR) using test-
based confidence intervals 116
Other analyses of apparent association of infections
with exposure 119
K. Interpretation of Statistical Results 120
5. Results 125
A. Microorganism Levels in Wastewater 125
24-Hour composite samples—overview 125
24-Hour composite samples—bacterial pathogens 127
24-Hour composite samples—human enteric viruses 131
24-Hour composite samples—geometric mean data 134
30-Minute composite samples 134
B. Microorganism Levels in Air 138
Aerosolization efficiency 138
Size of viable particles in the wastewater aerosol 141
Background microorganism densities in ambient air 143
Microorganism densities in downwind air from
microorganism runs 145
Enterovirus densities in downwind air from virus runs 150
Microorganism exposure via the wastewater aerosol 153
Estimates of aerosol exposure index (AEI) and other
participant exposure measures 156
XI
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CONTENTS (CONT'D)
Page
C. Other Investigated Sources of Microorganisms 162
Microorganism levels on flies 162
Microorganism levels in drinking water 165
Eating food prepared at local restaurants 171
Restaurant A 172
Restaurant B 173
Discussion 173
D. Description of Study Population 174
Onest ionnaire data 174
Population demographics 175
Effect of self-selection on LISS population characteristics 179
Characteristics of subpopulations 179
Characteristics of donor groups 181
Exposure categories based on aerosol exposure indices 182
Samples provided by study population during the health
watch 183
E. Patterns in Self-Reported Illness 183
Baseline 188
Irrigat ion-1982 197
Irrigation-1983 199
Discussion 200
F. Surveillance via Illness and Requested Specimens 201
Illness investigations 201
Group A streptococci 209
Enteric Gram-negative bacteria (EGNB) 210
Viruses 219
G. Clinical Bacteriology of Routine Fecal Specimens 222
Summary data 222
Bacterial infection events 226
Infections by overt pathogens 226
Klebsiella infect ions 227
Infections by non-Klebsiella Category 2 bacteria (other
opportunistic bacteria) . . . 230
Infections by bacteria prominent in wastewater 230
H. Clinical Virology of Routine Fecal Specimens 233
I. Serologic Data and Seroconversion Rates 243
Ant ibody prevalence 243
Incidence densities for serologic agents 244
Identified serologic infection episodes 254
J. Other Infections: Mycobacteria, Parasites, and Coronavirns-
like particles 255
Nontuberculosis mycobacterial (NTM) infections from
tuberculin skin testing 255
Parasite infestation 257
Electron microscopy (EM) of routine fecal specimens 259
xii
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CONTENTS (OONT'D)
K. Observed Episodes of Infection 265
Infection incidence rates of infection episodes 265
Evaluation of association of infections with aerosol
exposure via risk ratio scores 279
L. Statistical Analysis 282
Preliminary analysis 282
Confirmatory analysis 295
Exploratory logistic regression analysis 302
Analysis 1: basic analysis 302
Analysis 2: investigate possible restaurant etiology 317
Analysis 3: exclude AEI to investigate alternative
explanations 317
Analysis 4: investigate route of wastewater exposure 317
Evaluation of the effect of ignoring multiple infection
events on the statistical analysis results 321
M. Evidence of association of specific infection episodes with
wastewater aerosol exposure 325
6. Discussion 339
A. Prior wastewater aerosol health effect studies 339
B. Summary of LISS findings 342
Findings from wastewater and aerosol data 342
Findings from self-reported illness data 342
Findings from nonepisode occurrences of infections 344
Findings from seroconversion incidence density 346
Findings from risk ratio scoring of infection episodes 347
Findings from confirmatory statistical analysis of infection
episodes 348
Findings from exploratory statistical analysis of infection
episodes 349
Evidence of association of specific infection episodes with
wastewater aerosol exposure 349
C. Comparison of Findings to the Literature 351
Self-reported illness 351
Bacterial agent episodes 352
Viral agent episodes 356
D. Significance of Findings 359
References 362
Appendixes
A. Supplemental Figures and Tables for Section 4 (Methods and
Materials)*. 375
B. Initial Personal Interview Questionnaire 425
C. Personal Questionnaire Update in February 1982 441
D. Personal Questionnaire Update in October 1983 449
E. Informed and Parental Consent Forms 461
F. Household Health Diary Booklet (1980) 469
G. Health Diary Forms and Weekly Illness Surveillance Summary (1982
and 1983) 475
xiii
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CONTENTS (CONT'D)
Page
H. Activity Diaries and Haps 483
I. Wilson Eating Establishment Survey Form 493
J. Procedure for Wastewater Sample Collection, Lubbock Southeast
Water Reclamation Plant 497
K. Procedure for Wastewater Sample Collection, Wilson Imhoff Tank
Effluent 503
L. Description of Litton Model H High Volume Aerosol Sampler 507
H. Decontamination Procedure for Model M Samplers 515
N. Collection Efficiency of Litton Model M Large Volume Samplers 519
0. Enterovirus Serology Quality Control: liter Reproducibility (TR)
from Replicate Testing 531
P. Supplemental Tables for Section 5 (Results) 549
Glossary 636
xiv
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FIGURES
Number Page
1 Wastewater irrigation system 4
2 LISS study design: timeframe of monitoring in relation to
major periods of irrigation 6
3 Principal investigators and functional areas 11
4 Hancock farm irrigation system 25
5 Wastewater sprinkler irrigation at Hancock farm during LISS 29
6 Sampling zones comprising the study area 30
7 Relation of activity diary collection weeks to major periods
of irrigation 43
8 Sampler locations for background runs 49
9 Typical sampler configuration for microorganism run 52
10 Typical sampler configuration for quality assurance and
enterovirus runs 52
11 Typical sampler configuration for dye run 53
12 Typical sampler configuration for particle size run 53
13 Drinking water sampling locations 61
14 Isolation and identification of selected organisms from feces.... 68
15 Isolation and identification of organisms from throat swabs 69
16 Viral isolation from clinical specimens 72
17 Isolation of gram-negative enteric bacteria from wastewater 79
18 Analyses of insect vectors 86
19 Incidence density rates by exposure level for total acute
illness by month—1980 191
20 Incidence density rates by exposure level for total acute
illness by month—1981 191
21 Incidence density rates by exposure level for total acute
illness by month—1982 192
22 Incidence density rates by exposure level for total acute
illness by month—1983 192
23 Incidence density rates by exposure level for respiratory
illness by month—1980 193
24 Incidence density rates by exposure level for respiratory
illness by month—1981 193
25 Incidence density rates by exposure level for respiratory
illness by month—1982 194
26 Incidence density rates by exposure level for respiratory
illness by month—1983 194
27 Virus particles observed by EM in illness stool specimens 221
28 Coronavirus-like particles observed by EM in routine stool
spec imens 261
xv
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TABLES
Number Page
1 Suggested prevalence of antibody and seasonal occurrence of
infection for agents potentially present in wastewater 7
2 Frequency of measurement in wastewater of interpretable
infectious agents monitored in the health watch 9
3 Principal participating personnel and areas of activity 12
4 Major irrigation periods at Hancock farm during LISS
surveillance 27
5 Minor irrigation periods at Hancock farm during LISS
surveillance 28
6 Comparison of sentinel population to study population in
October 1982 36
7 Summary of participant poliovirns protection status 39
8 Epidemiologic characteristics of candidate agents for
serologic testing 63
9 Agents and sera selected for use in serologic testing 64
10 Bacterial infection criteria 88
11 Number of cases required for rejection of Pj=P2 i° favor of
Pj
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TAKES (CONI'D)
Number Page
28 Geometric mean of microorganism concentrations in Lubbock
wastewater 135
29 Geometric mean of microorganism concentrations in Hancock
reservoir wastewater . , , , 137
30 Calculated concentrations and corresponding aerosolization
efficiency point estimates for each sampler daring each
dye run 140
31 Summary of aerosolization efficiency of the Hancock farm
irrigation system in 1982 141
32 Standard plate count density of viable particles in air by
distance and particle size 142
33 Geometric mean background densities in ambient air sampled
on background runs 144
34 Estimated densities sampled on microorganism and virus
aerosol runs 146
35 Confirmation of spray irrigation of pipeline wastewater as a
significant source of microorganisms in downwind air:
paired downwind versus upwind densities 147
36 Microorganism densities in air at Hancock farm compared to other
wastewater treatment facilities 149
37 Viruses recovered from aerosol samples during virus runs 150
38 Sampled enterovirus densities on virus runs 151
39 Identification of viral isolates recovered during virus runs.... 152
40 Estimated microorganism densities in air downwind of irrigation
in 1982 relative to ambient background levels near homes and
in fields 154
41 Significant elevation of microorganism density in air downwind
of spray irrigation with pipeline wastewater relative to
ambient background outside participant homes 155
42 Relative aerosol exposure measure to sprayed microorganisms
by irrigation period and downwind distance 157
43 Distribution of participant aerosol exposure index by irrigation
period 158
44 Relative contribution of irrigation seasons to total entero-
viruses sprayed for 1982, 1983 and entire irrigation period... 160
45 Bacterial isolates from flies 164
46 Microorganism densities in drinking water in the study area
by well location and sampling date 166
47 Precipitation by month in the study area 171
48 Frequency distributions of patronage of major food preparation
facilities in Wilson by 117 fecal and illness specimen donors
during irrigation periods 172
49 Variables used in demographic analysis 176
50 Comparison of characteristics: study participants versus
nonparticipants 177
xvii
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TABLES (CONT'D)
Number Page
51 Comparison of characteristics: participants who remained
in the study versus participants who dropped out 178
52 Number of samples collected from health watch activities 184
53 Monthly intervals for self-reported illness data by date and DCP 187
54 Monthly incidence density of self-reported illnesses by type
of illness and exposure level 189
55 Monthly incidence density of self-reported illnesses by type
of illness and exposure group 190
56 Monthly prevalence density of self-reported illnesses by type
of illness and exposure level 195
57 Monthly prevalence density of self-reported illnesses by
type of illness and exposure group 196
58 Bacteriology throat swab series for donors with moderate or
heavy levels of enteric Gram-negative bacteria in an illness
throat swab 204
59 Occurrence of abnormal throat flora in acute and convalescent
illness throat swabs 208
60 Microorganisms found in the oropharynx 210
61 Investigation of various donor exposure variables for associa-
tion with enteric Gram-negative bacteria in illness throat
swabs in summer 1982 212
62 Clinical bacteriology results from requested throat swab surveys
of healthy participants in September 1982 and June 1983 213
63 Investigation of various donor exposure variables for associa-
tion with enteric Gram-negative bacteria in requested throat
swab survey of healthy donors in September 1982 214
64 Occurrence of abnormal levels of flora in acute and
convalescent illness fecal specimens 217
65 Age-specific distribution of abnormal levels of flora in
illness fecal specimens 218
66 Identification and comparison of viral isolates by cell culture
and virus-like particles by EM in illness fecal specimens 220
67 . Organisms isolated from routine fecal specimens during 1980
and 1981 223
68 Organisms isolated from routine fecal specimens during 1982 224
69 Organisms isolated from routine fecal specimens during 1983 225
70 Infections by overt enteric bacterial pathogens 226
71 Prevalence of bacterial infections by collection month 228
72 Exploratory analysis of the association of individual
characteristics with infection prevalance 229
73 Association of level of Klebsiella growth in routine fecal spec-
imens with the incidence of self-reported illness in the prior,
concurrent and subsequent biweekly reporting periods 231
74 Episodes of bacterial infection detected from routine fecal
specimens during irrigation seasons 232
xviii
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TABLES (OONT'D)
Number Page
75 Association of level of growth of prominent wastewater bacteria
in routine fecal specimens with the incidence of self-reported
illness in the prior, concurrent and subsequent biweekly
reporting periods 234
76 Prevalence and identification of viral isolates recovered
from routine fecal specimens by collection month 235
77 Age-specific annual recovery of viral isolates from routine
fecal specimens 236
78 Viral isolates recovered from donors of routine fecal specimens
during baseline monitoring 238
79 Viral isolates recovered from donors of routine fecal specimens
in 1982 239
80 Viral isolates recovered from donors of routine fecal specimens
in 1983 240
81 Association of viral isolates in routine fecal specimens with
the incidence of self-reported illness in the prior,
concurrent and subsequent biweekly reporting periods 241
82 Episodes of infection to viruses detected from routine fecal
specimens during irrigation seasons 242
83 Effect of immunization on participant poliovirus titers by
agent and vacc ine type 244
84 Comparison of baseline and irrigation incidence density rates
by wastewater aerosol exposure level and agent 246
85 Comparison of baseline and irrigation infection incidence
density rates by wastewater aerosol exposure group and agent.. 248
86 Comparison of baseline and irrigation incidence density rates
by wastewater aerosol exposure level and agent grouping 250
87 Comparison of baseline and irrigation incidence density rates
by wastewater aerosol exposure group and agent grouping 251
88 Infection incidence density rates for wastewater aerosol
exposure levels by agent grouping and time interval 252
89 Prevalence of mycobacteria response from initial Hantoux
tuberculin skin test results 255
90 Incidence of mycobacteria infections from tuberculin testing
of study populat ion 256
91 Ova and parasite survey of LISS population 258
92 Aerosol exposure comparison of Giardia-positive and Giardia-
negative fecal donors in ova and parasite survey 259
93 Occurrence of coronavirus-like particles in routine fecal
specimens examined by electron microscopy 262
94 Electron microscopy results for routine fecal specimen series
of donors positive for coronavirus-like particles 263
95 Age-specific prevalence of coronavirus-like particles detected
by electron microscopy in routine fecal specimens 264
96 Average aerosol exposure comparison of coronavirus-like infected
donors versus noninfected donors during irrigation seasons in
1982 264
xix
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TABLES (CONT'D)
Number Page
97 Clinical infection episodes 266
98 Serologic infection episodes to single agents 267
99 Serologic infection episodes to groups of agents 270
100 Infection incidence rates by exposure groups and levels and
risk ratio score of infection episodes classified as exposure
situations 271
101 Infection incidence rates by exposure groups and levels and
risk ratio score of infection episodes classifed as control
situations 276
102 Significance of frequency distributions of risk ratio scores
by group of jointly independent infection episodes 280
103 Comparison of exposure groups with respect to household charac-
teristics by baseline and irrigation season—blood donors 283
104 Comparison of exposure groups with respect to household charac-
teristics by baseline and irrigation year—blood donors 285
105 Comparison of exposure groups with respect to household
characteristics—fecal donors 286
106 Comparison of exposure groups with respect to individual
characteristics—blood donors 288
107 Comparison of exposure groups with respect to individual
characteristics—fecal donors 290
108 Comparison of exposure groups with respect to previous titer
to serologic agents 292
109 Comparison of exposure groups with respect to frequency of
eating food prepared at restaurants A and B—fecal donors 293
110 Comparison of incidence of bacterial infections in low and
high exposure groups. , , 296
111 Comparison of incidence rates of viral infections in low and
high exposure groups 297
112 Comparison of incidence of serologic infections in low and
high exposure groups 298
113 Comparison of incidence of polio infections in low and high
exposure groups stratified by immunization status 299
114 Rate of positive associations detected by the statistical
confirmatory analysis at significance level 0.05 in
independent infection episodes 300
115 Previous titer and response variables for logistic regression
analysis 303
116 Predictor variables for logistic regressions 304
117 Predictor variables used in logistic regression analysis 306
118 Logistic regression results for baseline infection episodes 308
119 Logistic regression results for spring 1982 infection episodes.. 309
120 Logistic regression results for summer 1982 infection episodes.. 310
121 Logistic regression results for spring 1983 infection episodes.. 311
122 Logistic regression results for summer 1983 infection episodes.. 312
123 Logistic regression results for 1982 infection episodes 313
xx
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TABLES (CONT'D)
Number Page
124 Logistic regression results for 1983 infection episodes 314
125 Results of rerun of Analysis 1—investigate infection episodes
with fewer observations deleted 315
126 Results of Analysis 2—investigate possible restaurant etiology. 318
127 Results of Analysis 3—exclude AEI to investigate alternative
explanations 320
128 Results of Analysis 4—investigate route of wastewater exposure. 322
129 Effect of multiple infection events on confirmatory analysis
results 323
130 Effect of multiple infection events on exploratory logistic
regression analysis results 324
131 Summary of findings for control infection episodes: evidence
regarding spurious association of infections with wastewater
aerosol exposure 327
132 Summary of findings for exposure infection episodes: evidence
regarding association of infections with wastewater aerosol
exposure 329
133 Summary of evidence for infection episodes showing strong
association of infections with wastewater aerosol exposure.... 335
134 Summary of findings pertaining to possible association with
wastewater irrigation for occurrences of infections not
classified as infection episodes 345
xxi
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ABBREVIATIONS
AEI
A6I
API
ATCC
BGM
BHI
BOD5
CA
CAL
CDAS
cfu
CI
CMH
CPE
CVLP
CYE
DCP
DE
DFA
DRCH
EGNB
El
ELISA
ELR
EM
EMB
EWS
FA
FHRSEL
FHRSEM
FITC
GI
GMT
GN
BAEI
HAV
HI
HID50
ICU
ID
ID
IDR
IFA
IgG
I HA
IPV
aerosol exposure index
all-glass impinger
Analytab Products, Incorporated
American Type Culture Collection
buffalo green monkey kidney cells
brain-heart infusion
5-day biochemical oxygen demand
confirmatory analysis
cellobiose arginine lysine agar
cassette data acquisition system (Climatronics Corporation)
colony-forming unit
confidence interval
Cochran-Mantel-Haenszel (X^ statistics)
cytopathic effect
coronavirus-like particles
charcoal-yeast extract
data collection period
diatomaceous earth
direct fluorescent antibody
differential reinforced Clostridia medium
enteric Gram-negative bacteria
exposure index
enzyme-linked immunosorbent assay
exploratory logistic regression
electron microscope
eosin methylene blue
electronic weather station (Climatronics Corporation)
fluorescent antibody
level of farm exposure hours
index of farm exposure hours
fluorescein isothiocyanate
gastrointestinal
geometric mean titer
Gram-negative
household aerosol exposure index
hepatitis A virus
hemagglut inat ion-inhib it ion
human infective dose, 50th percentile
intensive care unit
participant identification number
incidence density
incidence density ratio
indirect fluorescent antibody
immunoglobulin G
indirect hemagglutination
inactivated polio vaccine (Salk)
xxn
-------
ABBREVIATIONS (CONT'D)
IR
I SCO
KEC
LIA
LISS
LLTS
LTFP
LVS,
Mac,
MF
MIO
MPN
MRI
NS-PT
NTM
0-P
OPV
PBS
PBS-Man
pfu
PPD-S
PTA
QA
RAEM
RD
RIA
SDA
RR
SeWRP
SIR
SS
TCID50
TKN
TLUBOCK
TOC
TPB
TR
ISA
TSI
TSS
TU
TVSS
URI
WIT
XAEREL
XAEREM
XDIREL
XDIREM
XLD
ZM
LVAS
MAC
incidence rate
Instrumentation Specialties Company
Klebsiella, Enterobacter and Citrobacter
lysine-iron agar
Lubbock Infection Surveillance Study
Lubbock Land Treatment System
Lubbock Trickling Filter Plant 2
large volume air sampler
MacConkey agar
membrane filtration
motility-indole-ornithine
most probable number
Meteorology Research, Incorporated
0.85% sodium chloride with 25 (ig/mL potassium tellurite
non-tuberculosis mycobacteria
ova-parasite
oral polio vaccine (Sabin)
phosphate buffered saline
phosphate buffered saline with 1% mannitol
plaque-forming unit
purified protein derivative-stabilized (tuberculin test)
phosphotungstic acid
quality assurance
relative aerosol exposure measure
rhabdomyosarcoma
radioimmunoassay
Sabouraud dextrose agar
risk ratio
Southeast Water Reclamation Plant
Scientific Information Retrieval
Samonella-Shige1la
tissue culture infective dose, 50th percentile
Total Kjeldahl nitrogen
time spent in Lubbock
total organic carbon
tryptose—phosphate broth
titer reproducibility
trypticase soy agar
triple sugar iron
total suspended solids
tuberculin unit
total volatile suspended solids
upper respiratory illness
Wilson Imhoff tank
level of extensive aerosol exposure
index of extensive aerosol exposure
level of extensive direct wastewater contact
index of extensive direct wastewater contact
xylose-lys ine-deoxycholate
zero-max
xxiii
-------
ACKNOWLEDGEMENT
We would like to acknowledge the patience, understanding and cooperation
of the study participants, especially the 306 participants who stayed with
us until October 1983. Their willingness to provide necessary information
and to comply with our numerous requests for samples is deeply appreciated.
Without their commitment, the study would not have been possible. We are
thankful that they allowed us to intrude into their private lives and are
grateful that we had an opportunity to get to know these very special people.
A special thanks to the City of Wilson officials, especially City
Secretary Naoma (Shorty) Moore, for the help that they provided to the
LISS staff. The city council allowed us to use city facilities to store
project supplies as well as to collect and process blood and fecal specimens.
On occasion, these less than aesthet ically pleasing activities disrupted
city business, and we are grateful for the humor and the patience exhibited
by the city staff during those trying times.
We also acknowledge the vital contribution of the many technicians
and clerical personnel who assisted us in this study. Their competence
and special skills are appreciated. This list includes the technicians
from SwRI who were involved in the wastewater aerosol sampling and fecal
collection, technicians at UTSA and UT-Austin who analyzed clinical and
environmental samples, technicians at UI and UTSA who performed serologic
analyses, phlebotomists from the Lubbock area who drew all of the blood
samples, public health nurses from TDoH who administered both the polio
immunizations and TB skin tests, and the clerical personnel from each organi-
zation who meticulously recorded, transcribed and processed the voluminous
data and who carefully prepared our lengthy reports.
Recognition is due Herbert Pahren, USEPA (Cincinnati) for his foresight
in recognizing the research potential of a health study at the Hancock
site and his guidance in formulating the initial study design. Finally,
we acknowledge the invaluable counsel and support provided by Walter Jakubowski
and Dr. Dennis George. Their guidance and participation in the management
of the LISS greatly exceeded the requirements of their respective responsi-
bilities as project officer and contractor, and has been instrumental in
its successful conduct.
xxiv
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SECTION 1
INTRODUCTION
A. BACKGROUND
Land Application and Potential Infectious Disease Hazards
Land application of wastewater can be an attractive alternative to
traditional waste disposal practices. It avoids contamination of surface
waters, provides additional waste treatment, returns nutrients to the soil,
and reuses the water. The policy of the U.S. Environmental Protection Agency
(EPA) is to ''press vigorously for publicly-owned treatment works to utilize
land treatment processes to reclaim and recycle municipal wastewater''
(Costle, 1977). Applicants for federal construction grants (Section 201)
must show in their requests that they have considered the application of
wastewater to land as an alternative. Financial incentives are provided
to encourage land application (Clean Water Act of 1977). Slow rate application
of wastewater to land by spray irrigation has been and continues to be
one of the most popular application methods. With EPA encouragement, it
is likely that the practice of applying wastewater to land by sprinkler
irrigation according to EPA design criteria (USEPA, 1977 and 1981) will
become more prevalent as a means of final treatment and disposal.
Along with its considerable benefits, land application of wastewater
entails the potential risk of infection from exposure to microorganisms
in the wastewater. A variety of agents of human disease, including many
overt and potentially pathogenic microorganisms, may survive treatment
processes (Guentzel, 1978), and thus could theoretically pose a threat.
There are various environmental pathways by which these agents in the wastewater
and the aerosol produced by its sprinkler application might be introduced
and initiate infection in susceptible exposed individuals. Farmers will
come in direct contact with the wastewater and its sprayed mist in the
course of their work with the irrigation system. Agents in the wastewater
aerosol can be transported by the wind and might be inhaled or ingested
in exposed food while still viable and infective. Other potential environmental
pathways include: 1) ingestion of wastewater-contaminated ground water
used as the domestic water supply, 2) dust storms in which wastewater-irrigated
surface soils are entrained by strong winds, 3) insect vectors (e.g., flies
attracted by the wastewater lagoons), 4) rodents (e.g., feed or food stuffs
contaminated by fecal droppings or urine from field mice, infected by wastewater
spray, which may be spending the winter in farmhouses and barns), and 5)
fomites (e.g., wastewater-contaminated work shoes, clothing, hands, or
doorknobs). Once introduced into the local population, the infectious agents
might be transmitted by contact between infected and susceptible individuals.
-------
Recent Literature
The study of Katzenelson et al. (1976) cautioned that the infectious
disease hazards associated with irrigation of partially treated wastewater
are greater than previously assumed. Existing illness records were analyzed
in a retrospective study of enteric diseases among communal agricultural
settlements (kibbutzim) in Israel. The incidence rates of enteric illness
for kibbutzim utilizing wastewater for spray irrigation were, compared with
other kibbutzim practicing no form of wastewater irrigation. Two- to four-
fold increases in the incidence of shigellosis, salmonellosis, infectious
hepatitis, and typhoid fever were reported for the kibbutzim utilizing
wastewater, whereas the incidence of other diseases not normally associated
with sewage were similar in both groups. A subsequent retrospective study
of Israeli kibbutzim by Shuval et al. (1983) identified serious deficiencies
in the data of the original study, including misclassification of some
kibbutzim regarding wastewater reuse, uncertainties about periods of irrigation,
and the inadequacy of the communicable disease reports used as the basis
for the study. Indeed, the subsequent study failed to find evidence of
excess risk associated with wastewater irrigation except in kibbutzim in
a ''switch'' category (i.e., in kibbutzim practicing two consecutive years
of wastewater irrigation followed by the same period without irrigation
or vice versa). In this category, a significantly increased risk of total
enteric disease was noted only for the 0-4 age group during periods of
wastewater irrigation.
Two prospective epidemiologic studies were conducted among residents
around activated sludge sewage treatment plants near Chicago, Illinois
using the family-based virus watch approach developed by Frost et al. (1941a,b)
and Fox et al. (1957, 1966, 1972, 1974). Both studies included a health watch
of participating households that involved health diaries, serology, and
clinical specimen isolations. Neither Johnson et al. (1980) nor Northrop et al.
(1980, 1981) detected any obvious adverse health effects in residents poten-
tially exposed to wastewater aerosols from aeration basins.
Occupational health effects of wastewater and wastewater aerosols
have also been investigated. A study by Linnemann et al. (1984) of Huskegon
County, Michigan workers exposed to wastewater spray irrigation failed
to show any differences in illness or viral isolation rates between the
workers and a control group. Although antibody titers to coxsackievirus
BS were significantly higher in spray irrigation nozzle cleaners, sereconver-
sions were not documented. Likewise, a prospective seroepidemiologic study
by Clark et al. (1981) of municipal sewer and sewage treatment workers
and controls in three American metropolitan areas failed to support a sig-
nificant risk associated with exposure to the wastewater. However, inexperi-
enced workers reported significantly higher rates of gastrointestinal illness,
and the level of antibody to certain viruses appeared to be related to
level of exposure to wastewater aerosols. In Sweden, Rylander and Lundholm
(1980) found increased incidence of acute febrile illness among workers
exposed to sludge dust (probably due to endotoxins) and also increased
incidence of gastrointestinal symptoms among sewage treatment workers.
-------
None of these studies has investigated the effects on nearby residents'
health of sprinkler irrigation of wastewater over a known broad range of
wastewater quality. The Lubbock Infection Surveillance Study (LISS) was
designed to observe any association of the potential infectious disease
effects with exposure to sprayed wastewater.
The Lubbock Land Treatment System (LLTS) Expansion
A major new land treatment system was constructed as a demonstration
project (George, 1984) to apply wastewater from Lubbock, Texas by sprinkler
irrigation at the Hancock farm near Wilson, Texas (see Figure 1). The
design and operation of this large demonstration project provided for collection
of research data under a wide range of quality of the wastewater that was
used for irrigation. The first four major irrigation periods after the
LLTS expansion commenced operation in February 1982 were monitored. The
quality of the applied wastewater was substantially different in each of
the four periods. The original spray nozzles directed the wastewater upward,
which enhanced the creation and drift of aerosols. Thus, the LISS investigated
the risk of wastewater exposure ranging from conditions representative
of established guidelines [fecal coliforms <1000 MPN/100 mL (DSEPA, 1981)]
to those which explored the relative safety factor of the guidelines.
The LISS was one of several areas of research which were conducted
simultaneously at the land treatment demonstration site. The chemical,
biological and physical conditions of the ground water, soils, and crops
were characterized prior to and during the wastewater irrigation (George
et al., 1985a). The effects of hydraulic, nutrient, and salt mass loading
were assessed on the percolate (Ramsey, 1985) and on the crops and soil
(George et al., 1985b). George has provided a summary of all research
findings (1985c).
The Lubbock Infection Surveillance Study (LISS)
The LISS was conducted to monitor infections in the community surrounding
the new land treatment demonstration system. This prospective observational
study has attempted to determine the association, if any, between the occurrence
of infectious diseases in residents and workers and their exposure to the
wastewater and aerosols produced by wastewater spray irrigation. The initial
two years of operation of the LLTS expansion at the Hancock farm were inves-
tigated. LISS involved a 4-year health watch of nearby residents and micro-
biological monitoring of the wastewater and its aerosol. This site is unique
in that a typical rural community with no prior wastewater exposure was
challenged by the enteric agents active in a much larger urban community
(Lubbock). Persons residing around the Hancock site may have been exposed
to infectious agents indigenous in the Lubbock population but not circulating
in the study area. Thus, many in the study population may have been relatively
susceptible to the pathogens in the wastewater. A health watch of the
rural community was maintained before, during, and after periods of wastewater
spray irrigation. The health watch focused on infections detected serologically
and through isolates recovered from routine fecal specimens. To enhance
-------
HANCOCK IRRIGATION
SITE
KEY:
Pipeline
SeWRP
Hancock Farm
Scale
5
10 km
Figure 1. Wastewater irrigation system
-------
the likelihood of interpreting observed episodes of infection, the likely
routes of introduction and transmission were monitored.
B. STUDY OBJECTIVE
The general objective of the LISS was to identify possible adverse
effects on human health from slow rate (sprinkler) land application of
wastewater which contained potentially pathogenic microorganisms. More
precisely, the objective was to determine the association, if any, between
the occurrence of infectious diseases in residents and workers and their
exposure to the wastewater and aerosols produced by wastewater spray irri-
gation. This objective was accomplished by disease surveillance of the
study population, by description of the distribution of infections, and
principally by evaluation of the incidence of infections for association
with exposure.
C. STUDY DESIGN
The LISS was designed to monitor infections and illnesses occurring
in the study population and concurrent environmental levels of the infectious
agents as illustrated in Figure 2. The diseases, estimated susceptibility,
and seasonal occurrence of the human pathogens potentially present in wastewater
are summarized in Table 1. Disease surveillance was maintained to protect
the population from any obvious untoward effects. However, the study focused
on infections and the infecting agents rather than illness in order to
obtain greater objectivity, sensitivity, specificity, and etiologic evidence.
All participants were asked to provide blood samples semiannually,
usually in June and December. Sera were assayed for antibody titers to
specific enteroviruses and other microorganisms known or suspected to be
present in the sprayed wastewater. A seroconversion, defined as the four-
fold or greater increase in agent-specific antibody titer in simultaneously
tested successive sera from one individual, was considered serologic evidence
that the individual had been infected by the agent during the time interval
between the blood collections. Since mycobacteria were present in the
wastewater, tuberculin skin tests were administered annually to give suggestive
evidence of a non-tuberculosis mycobacterial infection.
An adult from each household and any children under 13 years of age
were designated as fecal donors. Each donor, whether well or ill, was
asked to submit routine stool specimens for microbiological testing during
scheduled weeks which spanned each major irrigation period in 1982 and
1983. A series of three 1-week fecal collection sessions were scheduled
before, during, and near the end of each irrigation period (see Figure
2) to detect infection events occurring in the interim. Clinical bacterio-
logical analyses were performed to isolate overt and opportunistic pathogens.
A semiquantitative measurement of growth (as heavy, moderate, light, or
very light) was obtained by streaking primary plates by a four-quadrant
method. Three categories of bacterial infection events were identified
by comparing results from consecutive monthly specimens from an individual.
Clinical virological analyses were performed to isolate enteric viruses
in the fecal specimens by tissue culture techniques. Electron microscopic
-------
<3\
Z c
— z o
0. O .E
w P «
-------
TABLE 1. SUGGESTED POPULATION SUSCEPTIBILITY AND SEASONAL OCCURRENCE OF
INFECTION FOR AGENTS POTENTIALLY PRESENT IN WASTEWATER
Agent ( human pathogens
potential ly present
In wastewater)
Types
Disease
Percent of
population
susceptible
Time of occurrence
JFMAMJJASOND
Viral
Poliovlrus3
Coxsacklevlrus3
Echovirus3
Reovlrus15
Adenovirus3
Hepatitis A virusc
Rotavlrusd
Norwalk vlrusd
Coronavlrus®
Bactorlal
Salmonella sp.
Shlgel la sp.9
Escherlchla col I,
enteropat hogen I cn
Mycobacterla, non-
tuberculosis'
Klebslella pneumoniae^
YersInI a enteroco1111ca^
Campylobacter sp.J
Leglonella pneumophllak
Staphylococcus aureus*
Streptococcus beta,
hemolytIc^
Pseudomonas sp.'
Proteus sp.f
Fungal
Candida alblcans*
1-3; wild and vaccine Enteritis, meningitis, paralysis
<10$ child '
A 1-24, B1-6
1-33
1-3
1-41
1
1-4
1-3
2
10 groups
4 groups
Serotype 0 and other
4 groups
4 blotypes
4 or more
23 or more
4 of 15 cand Idates
3 or more
A. B groups
Enteritis, meningitis, respiratory, rash >5
Meningitis, conjunctivitis >50$
Unknown >40$
Respiratory >50$
Systemic >70%
Enteritis >90%
Enteritis >50!<
Uncertain, enteritis ?
Enteritis, systemic
Enteritis
Enteritis
Respiratory, adenitis, granuloma
5% respiratory, enteritis
Enteritis, cutaneous
Enteritis, systemic
Respiratory, renal, other
Respiratory, enteric, cutaneous
Respiratory, enteric
Cutaneous, respiratory, other
Cutaneous, respiratory, other
Cutaneous, resolratorv. other
>75$
>75%
>15%
References:
Fox and Hall (1980); b Jackson and Muldoon (1973b); c Szmuness et al (1977); d Cukor and Blacklow (1984); e Gerna et al (1985); f
Lennette et al (1985); g Black et al (1978); h Sack (1975); I Ann et al (1979); j Blaser et al (1983); k Brenner (1984)
-------
examination was performed on about 1/4 of the routine fecal specimens to
detect a variety of virus-like particles, many of which are not recoverable
by tissue culture techniques. Detection of a specific virus by laboratory
cultivation or by electron microscopic examination was considered evidence
of a viral infection. Each non-adenovirns viral infection was regarded
to be new, unless the same agent had been recovered from the individual
in the prior 6 weeks.
Each household was contacted weekly by telephone for a report of any
illnesses during the prior week. When a sufficiently recent respiratory
or gastrointestinal illness was reported, the ill participant was requested
to submit a throat swab or stool specimen to identify the causative agent.
Weekly self-reports of illness and appropriate illness specimens were obtained
over the entire period of irrigation from January 1982 until October 1983
and over baseline periods corresponding to seasons of heavy irrigation.
The types and densities of potentially pathogenic bacteria and viruses
were monitored in the wastewater, wastewater aerosol, and other environmental
routes of introduction and transmission. An effort was made to determine
the fluctuations in levels of every measurable infectious agent utilized
in the health watch, as indicated in Table 2. However, the low densities
of many agents in environmental samples necessitated reliance on indicator
organisms to establish environmental patterns. Wastewater samples of the
effluent from the pipeline and reservoirs to be utilized for spray irrigation,
and of the Wilson effluent, were obtained and analyzed for indicator bacteria
and enteroviruses biweekly to span the major irrigation periods; corresponding
baseline samples had been obtained with the same frequency in 1981 and
at lesser frequency in 1980 to characterize the effluents. Microbiological
screens of indigenous enteric bacteria were conducted on one sample each
from the pipeline and the reservoir per irrigation season. The purpose
of the routine wastewater samples was to document the presence, prevalence,
longitudinal pattern, and passage through the study community of viral
and bacterial pathogens possibly introduced by the wastewater. Extensive
aerosol sampling was conducted to characterize the aerosol density of indicator
microorganisms produced by the spray irrigation of both pipeline and reservoir
wastewater. Virus runs were also conducted to measure the density and
diversity of enteroviruses in aerosols emanating from the sprinkler rigs.
Drinking water, houseflies, and dust storms also were evaluated as other
means of introducing microorganisms into the study population.
An aerosol exposure index (AEI) was devised to measure the degree
of a participant's cumulative exposure to microorganisms in the wastewater
aerosol, relative to all other study participants during a given irrigation
period. When a number of similar infection events were observed either
serologically or microbiologically in the study population within a time
interval corresponding to an irrigation period, this infection episode
was statistically analyzed for association with wastewater aerosol exposure
using AEI. Infection incidence rates were compared among exposure subgroups
and with baseline rates to determine the relative risk of infection.
-------
TABLE 2. FREQUENCY OF MEASUREMENT IN WASTEWATER OF INTERPRETABLE
INFECTIOUS AGENTS MONITORED IN THE HEALTH WATCH
Agents monitored in health watch
Procedure
Infections agents
(serotypes potentially
present in wastewater)
Measurement in wastewater
Sprayed
wastewater
Wilson
wastewater
Data type
Sexology
viruses:
(total enteroviruses:
coxsackie, echo, polio)
Coxsackie A virus (1-24)
Coxsackie B virus (1-6)
Echovirus (1-33)
Adenovirns (1-9. 11, 19, 21)
Reovirus (1-3)
Hepatitis A virus
Rotavirus (1-4)
Norwalk virus (1-2)
R
R
R
R - regular
I - infrequent
Q - quantitative
S - semiquantitative
R
R
R
R
Q
S (by ID)
S (by ID)
S (by ID)
bacteria:
Skin Test
Clinical Bacteriology
bacteria:
fungus :
Clinical Virology
Legionella pneumophila
Mycobacteria (tuberculosis
+ non-tuberculosis)
Salmonella sp.
Shigella sp.
Tersinia enterocolit ica
Campylobacter jejuni
Staphylococcus aureus
Fluorescent Pseudomonas
Klebsiella
Proteus
Serratia and others
Aeromonas hydrophila
Candida albicans
Poliovirnses
Coxsackie A virus (1-24)
Coxsackie B virus (1-6)
Echoviruses (1-33)
Adenovirnses (by group
antigen)
I
R
R
R
R
R
I
R
R (Kl-like)
I
I
I
R
R
R
R
R
R
R
R
R
R
I
R
R
I
I
I
R
R
R
R
R
+/- (will
Q
+/-
+/-
+/- (Q if
+/-
Q
Q
Q
Q
Q
S
Q
S (by ID)
S (by ID)
S (by ID)
S (by ID)
ID)
high)
ID -
present/absent
identification
-------
D. STUDY ORGANIZATION
The LISS involved five major functional activities: project management,
a health watch, environmental sampling, microbiological assay of clinical
specimens and environmental samples, and data analysis. The field activities
(i.e., health watch, environmental sampling, and their management) were
funded by a subcontract to SwRI from LCCIWR (SwRI Project 01-6001). The
other activities (i.e., laboratory analysis, data analysis, and their manage-
ment) were funded by a cooperative agreement between EPA-HERL and SwRI
(SwRI Project 01-6097).
The LISS was conducted by Southwest Research Institute, the University
of Illinois at Chicago, and the University of Texas at San Antonio and
Austin. The following is a listing of participating organizations:
Southwest Research Institute (SwRI)
Department of Environmental Sciences
San Antonio, Texas
University of Illinois at Chicago (UI)
School of Public Health
Chicago, Illinois
University of Texas at San Antonio
(UTSA)
Center for Applied Research and
Technology (CART)
San Antonio, Texas
University of Texas at Austin (UTA)
Austin, Texas
U.S. Environmental Protection Agency
Health Effects Research Laboratory
(EPA-HERL)
Cincinnati, Ohio
Lubbock Christian College
Institute of Water Research (LCCIWR)
Lubbock, Texas
University of Texas
School of Public Health (UTSPH)
Houston, Texas
Naval Biosciences Laboratory (NBL)
Oakland, California
H. E. Cramer Company (EEC)
Salt Lake City, Utah
Texas Department of Health (TDoH)
Public Health Region 2
Lubbock, Texas
Illinois Department of Public
Health (IDPH)
Laboratory Section
Chicago, Illinois
Centers for Disease Control (CDC)
Atlanta, Georgia
University of Massachusetts (UM)
Worcester, Massachusetts
Bletpath Laboratories
Des Plaines, Illinois
The project manager for the LISS was Mr. David E. Camann, SwRI. Each
of the functional activities was directed by a principal investigator who
reported to Mr. Camann as shown in Figure 3. Details regarding principal
participating personnel, participating organizations, and areas of specific
activity are presented in Table 3 for each functional activity area.
10
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PROJECT MANAGEMENT
David E. Camann
SwRI
HEALTH WATCH
Robert L. Northrop, Ph.D.
DI
ENVIRONMENTAL SAMPLING
H. Jac Harding, M.S.
SwRI
UT LABORATORY ANALYSIS
Charles A. Sorber, Ph.D.
CTSA/UTA
DI LABORATORY ANALYSIS
Robert L. Northrop, Ph.D.
UI
DATA ANALYSIS
David E. Camann, M.S.
SwRI
Figure 3. Principal investigators and functional areas
11
-------
TABLE 3. PRINCIPAL PARTICIPATING PERSONNEL AND AREAS OF ACTIVITY
Personnel
PROJECT NMM6EME
D.E. Canann
R.J. Prevost
H.J. Harding
J.K. Morav Its
A. Shelokov
A. Holguin
Organization Specific activity areas
MT (D.E. Camann, Swfil)
SwRI Plannlngi technical and financial statuai meetlngSf re-
ports
SwRI Administration of subcontracts
SwRI Annual reports
SwRI Report preparation
Johns Hopkins Consultant (study design)
UTSPH Consultant (epidemiology)
HEALTH BATCH (R.L. Northrop,
P.J. Graham/C.M. Backer UI
UI)
I. Smlth/S. Stabeno/J. Stelnhauser
C.R. Allen TDoH
EWnDNNEKTAL SANPLIRS (H.J. Harding,
H.J. Harding SwRI
M.A. Chatlgny
S. Schaub
NBL
US Amy
Ft. Detrlck
LCCIHR
D.B. Left«1ch/N. Klein
LABORATORY ANALYSIS (C.A. Sorter, UTSA/UTA, R.L. Northrop, UI)
Environmental Samples
UTSA/UTA
Recrultnent, health surveillance, serum and specimen col-
lection, household health and activity diary collection
On-elte coordinator, Wilson, Texas
Polio vaccination, tuberculin testing
SwRI)
Wasteweter aerosol sample collection, wasteweter and mete-
orological eanpUng
Loan and calibration of LVA samplers
Loan of Andersen samplers
Sample collection
B.E. Moore/C.A. Turk/
M. Ibarra
D.B. Leftwich
R.L. Northrop/
R. Cordell
B.P. Sag1k
Clinical Specimens
LCCIWR
UI
Analysis of wasteweter samples (microbiological screens,
routine wastewater assays! enterovlrus Identification)
Analysis of aerosol and fly samples
Analysis of drinking water
Analysis of Leglonella In wastewater
Drexel Univ. Consultant (virology)
P.J. Graham UI
R. Cordell UI
W. Nunea UI
B.E. Moore/R. DeCresce UTSA/Metpath
N.R. Blacklow UM
G.R. Healy CDC
B.E. Moore/C.A. Turk UTSA/UTA
M.N. Guentzel/ UTSA
C. Herrere
W. Jakuboweki/ EPA-HERL
F. Williams
C. Sweet TDoH
R. Murphy IDPH
M.K. Cooney Univ. Wash.
DATA ANALYSIS (D.E. Camann, SwRI)
Serology
PoUovlrus, coxsacklevlrus, echovlrus, edenovlrue
Reovlrue, rotavlrus, Influenza A
Leglonelle bacillus
Hepatitis A
Nome Ik virus
E. hlstolytlca
Clinical virology
Clinical bacteriology
Electron microscopy of fecal specimens
Ova and parasite one lysis
Consultant (serologic methods)
Consultant (serology)
K.T. Klmball
R.L. Mason/
J. Buckingham
J. Garza/M. Canann
N. Altaian
D.E. Canann
P.J. Graham
A. Anderson
R. Harriet
J. Stobsr
SwRI
SwRI
SwRI
UI
SwRI
UI
HEC
UTSPH
EPA-HERL
Statistical analysis
Logistic regression analysis
Date base management
Data management
Aerosol exposure, bacterial and viral Infection
Seroconverslon Incidence, Illness patterns
Dispersion modeling
Consultant (statistical methods)
Consultant [statistical methods)
patterns
12
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SECTION 2
CONCLUSIONS
1. The LISS employed an epidemiologic analytic prospective cohort study
design which was quite appropriate to measure the strength of association
between exposure to the wastewater used for irrigation and the development
of new infections. The results from the isolation and serology procedures
used to detect infections appear to be adequate. These detection
methods were sufficiently sensitive and specific to observe many episodes
of infection in the study population in which the etiologic agent
was identified. The size of the population was sufficient to analyze
the distribution of observed infections for possible association with
exposure to wastewater irrigation and to control for extraneous variables
via logistic regression analysis. However, the small population size
led to instability of the association. The significance of the study
findings have not been limited to a great extent by such major confounding
factors as age, gender, antibody level, head of household education,
and time spent in Lubbock.
2. The quality of the wastewater to which the study population was exposed
was highly variable during the study. During the initial spring 1982
irrigation period, the quality of the irrigation wastewater approximated
that of a low quality primary effluent, as determined by physical
and chemical analyses. While the quality of the irrigation wastewater
was greatly improved in 1983, its fecal coliform concentration still
exceeded the EPA guideline for controlled agricultural irrigation
as practiced at the study site.
3. Spray irrigation of wastewater obtained via pipeline directly from
the Lubbock SeWRP was a more substantial source of aerosolized microor-
ganisms than spray irrigation of wastewater stored in reservoirs.
Enteroviruses were consistently recovered in the aerosol at 44 to
60 m downwind of irrigation with pipeline wastewater.
4. Microorganism levels in air downwind of spray rigs using pipeline
wastewater were significantly higher than upwind levels: fecal strep-
totocci levels to at least 300 m downwind, and levels of fecal coliforms,
mycobacteria and coliphage to at least 200 m downwind. Levels downwind
were also significantly higher than background levels in ambient air
outside of participants' homes: fecal coliform levels to beyond 400
m downwind, mycobacteria and coliphage levels to at least 300 m and
fecal streptococci levels to at least 200 m.
5. The exposure which most of the study population received to most micro-
organisms via the wastewater aerosol was greater in 1982 than in 1983.
The cumulative enterovirus dose received from aerosol exposure at
13
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a given distance downwind in summer 1982 was estimated to be at least
an order of magnitude greater than in any other irrigation period.
6. Individuals in the high (AEI^.3) and low (AEK3) exposure »groups were
generally well balanced with regard to infection risk factors, including
age, gender and previous antibody titer. The high exposure fecal
donors ate food prepared by a local restaurant very significantly
more often, made greater use of evaporative coolers for air conditioning,
and had more farmers as head of household.
7. The lack of a strong, stable association of clinical illness episodes
with the level of exposure to irrigation wastewater indicates that
wastewater spray, irrigation did not produce obvious disease during
the study period. However, the participants in the high exposure
level (AEI>5) reported a slight excess crude incidence density of
total acute illness shortly after the onset of wastewater irrigation,
both in spring 1982 and in summer 1982, the seasons of initial and
heaviest microbial exposure, respectively. The extent to which this
reflects actual illness versus possible reporting bias by high exposure
participants cannot be ascertained.
8. The occurrence of enteric Gram-negative bacteria (EGNB) at moderate
and heavy levels in the throats of both healthy and ill study participants
was frequent and widespread between July 19 and October 12, 1982.
The household environment was strongly associated with the continuing
EGNB throat infections of one household. Among the ill throat swab
donors, use of an evaporative cooler for home air conditioning was
associated with the EGNB throat infections.
9. Some excess risk of viral infection (risk ratio of 1.5 to 1.8) was
associated with wastewater aerosol exposure, based on comparison of
crude seroconversion incidence densities by aerosol exposure level
and by irrigation vs. baseline period.
10. A symmetric risk ratio score approach provided evidence of a stable
and dose-related association between infection events and wastewater
aerosol exposure in the infection episodes observed by the LISS.
11. Some infection episodes appear to have been related to wastewater
aerosol exposure, because more statistically significant associations
than expected were found in the confirmatory analysis of independent
infection episodes using a one-sided Fisher's exact test. Some imbalances
in the two populations may provide alternate explanations for the
excess associations. On the other hand, the number of detected increases
in incidence rates associated with the wastewater irrigation may be
underestimated, considering the relatively modest power of the tests
to detect small differences.
12. An exploratory logistic regression analysis found significant (p<0.05)
associations between presence of infection and degree of aerosol exposure
while controlling for the effects of extraneous variables in four
infection episodes. More supporting evidence was found for the wastewater
14
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aerosol route of exposure than for direct contact with wastewater
or spending time in the irrigation environment on the Hancock farm.
13. Eight specific infection episodes displayed good or marginally consistent
evidence of association with wastewater aerosol exposure.
a. Two of these episodes were probably unrelated to wastewater exposure
because a more plausible alternative explanation was identified:
o Episode of Klebsiella infections in summer 1983
—alternative: eating at a local restaurant
o Spurious control episode of echovirus 9 seroconversions
in the baseline period
—alternative: within household spread
b. The evidence is inconclusive in five episodes because both aerosol
exposure and the identified alternative explanation(s) are plausible
risk factors:
o Episode of clinical viral isolates excluding adenoviruses
and immunization-associated polioviruses in summer 1982
—alternative: eating at a local restaurant
o Episode of echovirus 11 seroconversions in 1982
—alternatives: o contaminated drinking water
o Caucasian, large household
o Episode of seroconversions to viruses isolated from wastewater
in summer 1982
—alternatives: o contaminated drinking water
o low income, Caucasian
o Episode of seroconversions to viruses isolated from wastewater
in 1982
—alternative: farmer, history of pneumonia
o Episode of seroconversions in summer 1982 to all serum neu-
tralization-tested viruses
—alternative: contaminated drinking water
All five of these infection episodes relate to echo or coxsackie
B viral infections observed primarily in summer 1982 and primarily
to agents recovered from the wastewater at that time.
c. Some of the infections in one episode were probably caused by
wastewater aerosol exposure because a strong association existed
and no alternative explanation could be identified:
o Episode of poliovirus 1 seroconversions in spring 1982
15
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Three distinct risk factors (poliovirus immunization in spring
1982, low polio 1 antibody titer in January 1982, and a high
degree of aerosol exposure) were independently associated with
the poliovirus 1 seroconversions in spring 1982 and each appears
to have been responsible for some of the poliovirus 1 infections.
14. Despite the efforts to obtain a random sample, the study participants
during the irrigation periods were essentially volunteers who were
not representative of the entire population of the study area. Further-
more, the frequency of patronizing local restaurants and the use of
evaporative coolers were factors that were largely confounded with
wastewater aerosol exposure. For these reasons, the LISS findings
cannot easily be generalized to other sites.
15. In summary, a general association existed between exposure to irrigation
wastewater and new infections. A viral dose-response relationship
was observed over the four irrigation seasons, since the aerosol exposure-
associated episodes of viral infection occurred primarily in 1982
during the irrigation seasons of greater enterovirus aerosol exposure.
Some poliovims 1 seroconversions during the spring of 1982 were probably
related to wastewater aerosol exposure. However, even during 1982,
the strength of association remained weak and frequently was not stable.
Wastewater of poor quality comprised much of the irrigation water
in 1982. Of the many infection episodes observed in the study population,
few appear to have been associated with wastewater aerosol exposure,
and none resulted in serious illness.
16
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SECTION 3
RECOMMENDATIONS
1. To minimize exposure, it would be prudent to use wastewater from the
reservoirs at the Hancock farm for irrigation (or to apply equivalent
treatment measures), rather than irrigating directly from the pipeline.
2. Poliovirus serology should be performed on archived sera from June
1982 through October 1983 to identify poliovirus seroconversions in
the study population spanning the summer 1982 and the 1983 irrigation
periods. Any observed poliovirus infection episodes should be fully
analyzed by the inferential methods employed in the LISS. Since summer
1982 and possibly summer 1983 appear to have been seasons of higher
poliovirus aerosol exposure than spring 1982 was, these data would
confirm or dispute the probable relationship of poliovirus 1 sereconver-
sions to wastewater aerosol exposure which was observed in spring
1982.
3. Serological testing of archived sera is recommended for selected entero-
viruses and rotavirus to observe and analyze additional infection
episodes in order to clarify the apparent dose-response relationship
with wastewater aerosol exposure detected in the LISS.
a.' Perform serum neutralization retesting to improve existing infection
episode data. There are 56 echovirus and adenovirns infections
reported for the years 1982 or 1983 that need additional serologic
testing to identify the exact 6-month interval in which the sero-
conversion occurred. Also, there were 28 serologic series in
which infection status was indeterminate due to inconsistent
or contradictory titer results and 33 unconfirmed four-fold or
greater titer rises in unpaired sera; these cases were not used
in the LISS data analysis.
b. Conduct rotavirus and coxsackie B virus serology having a high
probability of yielding additional infection episodes to agents
found in sprayed wastewater. Rotavirus serology should be performed
on the entire serum donor population, since a very high incidence
density of seroconversions to rotavirus was observed throughout
the study period in both the 45 children and the 11 adults tested
in the LISS. Additional serology testing for coxsackieviruses
B2, B3 and B4 is recommended based on their recovery from the
wastewater in 1982 and 1983.
c. Serologic testing of echoviruses 12, 25, 27 and 31 is recommended,
because they were each recovered from wastewater in several of
the irrigation periods.
17
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An exposure assessment should be performed to estimate the range of
cumulative organism exposure dosages that applied to the LISS infection
episodes and other situations in which reasonable evidence of association
with wastewater irrigation was obtained. A dosage to the infections
agent should be estimated for each infected individual and the dosage
range of the high exposure level of participants should be approximated.
Determination of the dosage range in which observed infection effects
were found would provide a crucial missing link in the relationship
between viable aerosol concentration and infection. This would facilitate
transfer the dose-response findings of the LISS to other sites of
wastewater aerosol exposure.
An improved model of microbiological dispersion should be developed
based on the LISS aerosol sampling data. The LISS data provide a
much better basis for model development than the data bases previously
employed. The model would permit the determination of the estimated
range of microorganism exposure dosages at considerable distances
downwind (i.e., 400-800 m) from any spray irrigation source of wastewater
aerosols.
If recommendation 1 is not implemented, a limited program of wastewater
and aerosol sampling should be conducted at the Hancock farm to determine
densities of enteroviruses and indicator bacteria in wastewater and
downwind air and to reevalnate aerosolization efficiency for the current
treatment process and mode of operation. ''Pulsed break-point chlor-
ination'' of pipeline wastewater and installation of proper spray
nozzles to reduce aerosol formation and drift are two major changes
in irrigation practices at the Hancock farm since .1983. The sampling
program would permit determination of where the current irrigation
practices fit into the seasonal dose-effect gradient found in the
LISS.
It is recommended that analyses of existing LISS data be performed
as pilot studies to investigate whether clinically and serologically
detected infections and self-reported illness were associated with
several apparent environmental sources of infection identified in
the LISS:
a. Evaluate bacterial contamination of wells which served as sources
of household drinking water.
b. Evaluate patronage of local restaurants in this rural community
to help to address the extent to which food prepared for public
consumption may be a source of inapparent infections and minor
acute illness.
c. Evaluate the use of evaporative coolers for air conditioning
as a source of bacterial infections and illness, especially when
bacterial contamination of water supplies is quite widespread.
18
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8. Certain additional data analyses are recommended to facilitate proper
interpretation of the LISS results:
a. Calculate incidence density ratios and their confidence intervals
for clinical agents, as was done for serologic agents and self-
reported illness, in order to balance the procedure for selection
of infection episodes with good and marginal evidence of association
with aerosol exposure.
b. Investigate the need to control by logistic regression analysis
for the effects on infection status of three additional factors
which were partially confounded with wastewater aerosol exposure:
evaporative cooler use prior to 1983, rural versus Wilson location,
and children in the household.
c. Conduct a stratified analysis of serologic and illness incidence
densities to control for major potential risk factors, such as
age, gender, previous antibody titer, occupation and education
of head of household, restaurant patronage, and dwelling location.
These analyses would clarify interpretation of apparent associations
with aerosol exposure of seroconversions and self-reported illness
which were based on test-based confidence intervals of crude
incidence density ratios.
d. Determine if there is evidence of association of infections with
residential aerosol exposure when the individuals with occupational
exposure to wastewater irrigation are excluded from the study
population.
19
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SECTION 4
METHODS AND MATERIALS
A. STUDY SITE
Description of Study Area
The Lubbock Land Treatment System is located in Lynn and Lubbock Counties
in northwestern Texas. The source of wastewater for this irrigation project
was the Lnbbock Southeast Water Reclamation Plant (SeWRP), situated in
the southeast portion of the city of Lubbock. The storage and irrigation
facilities were located at the Hancock farm in the north central portion
of Lynn County, 29 km (18 miles) south of Lubbock. Both counties are located
in a plateau area, the South Plains Region of the Llano Estacado of the
High Plains. A regional map of the study area was shown in Figure 1.
Lubbock is the center of the largest cotton producing section of Texas.
Other segments of the agroeconomy of the area included grain sorghum production
and cattle feeding. The Ogallala aquifer, an extensive unconfined aquifer
system stretching from western Nebraska and eastern Colorado south to the
Texas panhandle and eastern New Mexico, has been used for irrigation purposes
as a supplement to natural rainfall to improve crop yields. Withdrawal
of ground water from the Ogallala aquifer has greatly exceeded the natural
recharge. In the Lubbock area, the aquifer is approaching depletion; in
IS years it may no longer be economical to produce irrigation water from
this source.
General Climatology—
The South Plains Region is semiarid, transitional between the desert
conditions to the west and the humid climate to the east and southeast.
The average annual precipitation is 46.8 cm (18.4 inches), most of which
occurs from Hay through September, usually as moderate to heavy afternoon
and evening thunderstorms which may be accompanied by hail. Snow may occur
from late October until April, but is generally light and seldom remains
on the ground for more than 2 or 3 days at any one period.
During the 8-month period from March through October, winds are predom-
inantly from the south. However, during the late winter and springtime,
winds in excess of 11 meters/second (25 MPH) occur for periods of 12 hours
or longer from a westerly direction with the passage of low pressure centers.
These strong winds bring widespread dust, the quantity and amount of which
is influenced by the precipitation patterns of the previous few days and
the agricultural practices of the area (NOAA, 1977).
To anticipate the distribution of wind directions during the major
irrigation periods, wind roses, based on Lubbock Airport data for 1969-1973,
were constructed for those months. The wind roses for March and April
20
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(spring irrigation) and for July and August (summer irrigation) are shown
in Figures A.I and A.2, respectively, in Appendix A.
City of Wilson—
The City of Wilson was the nearest community to the Hancock farm.
It was situated at the southern boundary of the farm. The population of
576 (1980 census) occupied 181 residences ranging from small two bedroom
stucco or frame bungalows to large all-brick homes. Local commerce was
based primarily on agriculture. Support facilities located in Wilson included
three cotton gins, one grain elevator, a welding and machine shop, a pump
service facility, and a combined lumber, hardware and feed store. Other
businesses within Wilson included a bank, two cafes, two service stations,
and a grocery store. During 1982 the grocery store ceased to do business
and one service station was converted into a convenience store. A municipal
building, a school complex for grades 1 through 12, a municipal park, a
post office and six churches were also located within the city limits.
There were no day care centers or medical facilities in the city.
The municipal water supply for city residents was obtained from six
wells which tapped the Ogallala aquifer. A water tower and underground
tank provided storage facilities where the water was intermittently chlorinated
manually prior to distribution. Continuous chlorination of the City of
Wilson water supply system commenced in March 1983.
All but ten of the households within the city limits were serviced
by a municipal wastewater collection and treatment system. The treatment
plant consisted of an Imhoff tank preceded by a bar screen. Plant effluent
was allowed to evaporate from a series of lagoons while the settleable
solids were removed from the tank on a monthly basis and placed in an adjacent
drying bed. Those households not connected to the municipal system had
septic tanks.
Rural Area—
The rural portion of the study area (see Figure 6) lay primarily in
Lynn County (1980 census population, 8,605), with a small portion above
the northern boundary in Lubbock County. Approximately 130 households
were located in this area in 1980 with an estimated population of 450.
Almost every rural household obtained its drinking water from a nearby
private well which tapped the Ogallala aquifer. Treatment of domestic
wastewater was accomplished by septic tank systems in half of the rural
houses while the other half, typically the older homes, utilized cesspools.
In the predominantly agricultural economy of this region, an annual
income of $37.3 million (Lynn County) was derived from a primary crop of
cotton and secondary crops of winter wheat, grain sorghum, sunflowers and
soybeans. Livestock was kept primarily for owner use, though some pasture
land was dedicated to grazing of livestock for market. There was some
production of oil and gas, and some exploration, with attendant drilling
activity, occurring in the area. The value of these mineral resources
and those of a stone quarry amounted to $2 million during 1977 for Lynn
County (Texas Almanac. 1980).
21
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Lubbock Sewage Treatment Plants
The City of Lubbock operated two wastewater treatment plants: the
Southeast Water Reclamation Plant (SeWRP) and the Northwest Water Reclamation
Plant. The SeWRP was in reality three separate systems: two trickling
filter plants (Plants 1 and 2) and an activated sludge plant (Plant 3).
Due to the predominantly agricultural economic base of the Lubbock area,
domestic sewage comprised the bulk (i.e., about 70%) of the wastewater
treated by the SeWRP. The majority of industrial wastes were from cotton
gin operations and industrial plating operations. Industries on a surcharge
contract with the city contributed approximately 22% of the total 5-day
biochemical oxygen demand (6005) mass loading and 15% of the total suspended
solids (TSS) mass loading to the SeWRP. An electroplating plant's discharge
contained high levels of chromium (42 ppm average) and nickel (17.2 ppm
average) and contributed the highest mass loading of heavy metals during
the project period.
Trickling Filter Plant 1 had a hydraulic capacity of approximately
23,000 m^/day (6 mgd). Plant 1 provided most of the water for the Gray
farm, a 1,489 ha farm located east of the City of Lubbock, which comprised
the older part of the Lnbbock Land Treatment System.
Trickling Filter Plant 2 was designed to treat a maximum flow of 76,000
m3/day. Normal flow ranged from 30,000 to 49,000 m3/day (8 to 13 mgd).
During 1980 and 1981, the effluent from Trickling Filter Plant 2 had a
composition equivalent to a typical medium untreated domestic wastewater
as defined by Hetcalf and Eddy (1979). This poor quality effluent was
mainly attributable to the malfunctioning of the anaerobic digestion process
since effective liquid-solid phase separation was not achieved in the second
stage digester. Consequently, the suspension recycled from the anaerobic
process to the head works of the trickling filter plant contained high
levels of ammonia, suspended solids and carbonaceous material. From June
1980 to February 1982, the average effluent total organic carbon (TOO
produced from Trickling Filter Plant 2 was 117.7 mg/L. Total Kjeldahl
nitrogen (TEN) concentration averaged 38.59 mg-N/L of which 67% was ammonia-
nitrogen (25.95 mg-N/L) and 33% was organic nitrogen. Due to high organic
mass loadings and subsequent heterotrophic organism activity, the trickling
filter system was not nitrifying ammonia to nitrate. Approximately 57%
of the total phosphorus (14.43 mg/L) present in the effluent from Plant
2 was orthophosphate phosphorus (PO^. Plant 2 provided the majority of
the water pumped to the Hancock farm.
Treatment Plant 3, an activated sludge system, had a maximum design
hydraulic capacity of 55,000 m3/day (15 mgd). Effluent quality was fairly
good with a 6005 of 25 mg/L and TSS of 18 mg/L. The effluent was dosed
with about 12 mg/L chlorine. Southwestern Public Service (SPS, a power
utility) utilized a major portion of this effluent as cooling and boiler
makeup water. The effluent discharge not utilized by SPS (daily average
of less than 5%) was divided equally between the Gray and Hancock land
application sites.
22
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The Northwest Wastewater Reclamation Plant treated wastewater generated
mainly from the extreme northwest portion of Lubbock and from Texas Tech
University. The 4,000 m3/day (1 mgd) effluent from this plant was used
by Texas Tech University for irrigation studies on the university farm.
Lubbock Land Treatment System
The original component of the Lubbock Land Treatment System (LLTS)
was the Gray farm which has utilized effluent to grow crops since 1938.
As the wastewater discharge increased due to population growth, the Gray
farm was expanded to treat the increased hydraulic and nutrient mass loading.
Eventually, insufficient land was available to adequately assimilate the
hydraulic flow which resulted in a significant rise in ground water level
and subsequent degradation of water quality. Therefore, the Hancock farm
was included in the LLTS to reduce the hydraulic and nutrient overloading
experienced at the Gray farm. In November 1980, construction began on
a pump storage and distribution system to divert 50% of the total flow
pumped to the Hancock farm, a new component of the LLTS.
The total cultivated area of the LLTS land application system was
2,565 ha during the period of study. The Gray farm, located east of the
City of Lubbock, had a total land area of 1,489 ha with about 1,210 ha
in cultivation. The l,478~ha Hancock farm was located 27 km (17 miles)
south of the SeWRP and just north of the community of Wilson. During the
5-year period from 1977 to 1982, the Hancock farm was primarily a dry land
farm with little ground water irrigation.
The completely new system constructed at the Hancock farm consisted
of wastewater conveyance, storage and irrigation facilities. The conveyance
system consisted of a three-pump pumping station located adjacent to the
existing effluent pumping station at the Lubbock SeWRP and 25 km of 0.69
m force main to the Hancock farm. The pumping station and the force main
were designed to accommodate a flow of 28,000 m3/day (7.4 mgd). The average
wastewater flow was 14,000 m3/day in 1982 and 1983.
At the northern boundary of the Hancock farm, the effluent was routed
through three 0.38-m plastic irrigation pipelines to a storage system consisting
of three separate reservoirs. These were constructed on natural playa
lakes with capacities of 1.5 x 10^ m3 (Reservoir 1-east), 6.9 x 105 m3
(Reservoir 2-central), and 7.4 x 10^ m3 (Reservoir 3-west). Irrigation
pump stations were provided at each reservoir. The quality of the stored
wastewater was improved substantially through sedimentation of particulates
and microbial stabilization of organic and nutrient material.
The irrigation system was designed to irrigate 1,153 ha, 991 ha of
which were irrigated by 22 electric-drive center pivot irrigation rigs.
The remaining 162 hectares were irrigated by the furrow flooding technique
to maximize land use in areas not accessible to the center pivot system.
Low pressure Nelson spray nozzles were used to apply the wastewater
along the irrigation rigs. Each nozzle provided a 360° umbrella pattern
with an effective wetted diameter of 8.5 to 9.1 m (28 to 30 ft) to allow
23
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for the greatest application intensity. The energy dissipating deflector
incorporated into the nozzle assembly was a concave plastic plate. Water
discharged through the orifice was deflected upward once it struck the
deflector which enhanced the creation of aerosols during the period of
study and increased drift and evaporation of water. Convex deflectors
were installed on most nozzles after the LISS monitoring period ended (i.e.,
after October 1983) to direct the water downward. This change reduced
aerosol formation and drift. The spray nozzles were situated on drops
3.2 m (10.5 ft) apart on 52.1 to 54.3 m (171 to 178 ft) spans between towers.
Nozzle heights were 1.2 m (4 ft) to 2.1 m (7 ft) above ground, while nozzle
diameters ranged from 2.4 mm (3/32 in.) up to 7.1 mm (9/32 in.) with the
smaller nozzles located near the pivot and the larger ones at the end of
the lateral.
A Rainbird-type end gun on each lateral could be activated to irrigate
all or some of the corners. The height of the end sprinklers was from
3.0 m (10 ft) to 4.6 m (15 ft) depending upon the terrain. When the end
guns were activated, their effective wetted diameter was 18.3 m (60 ft).
The laterals varied in length from 307 m (1007 ft) to 476 m (1562
ft) with six to eight towers per pivot. The speed of traverse of each
lateral was variable, and at maximum speed a pivot could complete a full
cycle in 13 or 14 hours (Sheaffer and Roland, Inc. and Engineering Enterprises,
Inc., 1980).
Each center pivot was designed to irrigate up to 15 cm in 20 days
after allowing for 20% loss due to evaporation. Without the use of the
reservoirs, five to six center pivots could be operated at the same time,
utilizing the flow pumped directly from the SeWRP. Each center pivot had
a centrifugal booster pump which increased the line pressure to an operating
level of 3.1 x 10*> pascals (45 psi). A schematic of the Hancock farm irrigation
system is presented in Figure 4.
The City of Lubbock's wastewater discharge permit required a 46-m
buffer zone along the northern boundary of the farm. In addition, a 400-m
buffer zone was observed immediately north of the city of Wilson. No spray
irrigation was permitted within these buffer zones. Spray irrigation also
was not practiced within 400 m of the homes of non-participants of the
LISS. Plastic tubing measuring 3 m x 1.3 cm (9 ft x 1/2 in.) was attached
to the nozzles on pivots affected by the buffer zone on the northern and
western farm boundaries in order to furrow irrigate these areas, which
consisted of 180 ha.
System Design and Operation in Relation to EPA Design Criteria and Recommen-
dations
The Lubbock Land Treatment System (LLTS) was designed and operated
as a large demonstration project to allow collection of research data under
a wide variety of conditions. The hydraulic conveyance system from Lubbock's
Southeast Water Reclamation Plant (SeWRP) to the Hancock farm was sized
to accommodate a design flow of 28,000 m^/day. The wastewater storage
24
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M3TIIHJTION CAN
MSTIHUTIOM UNI
HANCOCK LAND
DISPOSAL SITE
Figure 4. Hancock farm irrigation system
25
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and distribution system was designed to apply 66 cm (26 in.) of treated
effluent per year to the Hancock farm.
Operational problems associated with wastewater management at SeWRP
and odors emitted during spray irrigation with effluent transported directly
from SeWRP reduced the annual flow to the Hancock farm to only 20% (4,128,000
m3) of the total SeWRP effluent in 1982 and 19% (3,744,000 m3) in 1983.
The City of Lubbock's wastewater discharge permit for SeWRP required
the plant to produce an effluent with a 30-day-average 5-day biochemical
oxygen demand (6005) not greater than 45 mg/L. During the project monitoring
period the effluent BOD5 quality from SeWRP ranged from a monthly high
of 260 mg/L to a monthly low of 27 mg/L:
Average Monthly Effluent 6005
Produced by Lubbock SeWRP
1982 1983
Month mg/L mg/L
January 143 71
February 260 120
March 198 105
April 139 65
May 108 30
June 128 39
July 130 49
August 76 27
September 69 43
October 171 31
November 63 63
December 86 49
The average fecal coliform concentration in the waste stream pumped
to the center pivot irrigation machines exceeded EPA guidelines throughout
the study period. The guidelines issued in November 1978 state:
''Biological treatment by ponds or inplant processes plus control
of fecal coliform count to less than 1000 MPN/100 ml - acceptable
for controlled agricultural irrigation except for human food
crops to be eaten raw." (USEPA, 1981)
The actual flow-weighted average fecal coliform concentrations of the applied
wastewater during the four major irrigation periods were:
Fecal coliform concentration
(colony forming units/100 mL)
Spring 1982 4,300,000
Summer 1982 840,000
Spring 1983 5,200
Summer 1983 120,000
26
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A factor which affected aerosol formation and drift was the energy
dissipating deflector incorporated in the spray nozzle assembly used during
the research study. The deflection pad was a concave plastic plate. Since
water discharged upward, the creation of aerosols was enhanced. The nozzles
were replaced at the conclusion of the research study to direct the wastewater
downward and reduce aerosol formation.
In summary, the LLTS expansion was designed to accommodate specific
research objectives. During system operation, the fecal coliform concentration
of the waste stream from SeWRP and the discharge from the storage reservoirs
greatly exceeded EPA guidelines, especially in 1982. The effluent 6005
concentration produced by SeWRP did not satisfy Texas permit requirements
until Hay 1983. However, the system was operated below hydraulic design
capacity in 1982 and 1983.
Periods of Irrigation
Wastewater spray irrigation commenced at the Hancock farm on February 16,
1982. The infectious disease effects of irrigation occurring through Septem-
ber 20, 1983 were monitored by the LISS. The two major irrigation periods
each year were from mid-February through April, to provide ground moisture
prior to planting, and from July through mid-September, to irrigate the
growing crop. The primary crops were grain sorghum, soybeans, and sunflowers
in 1982 and cotton, grain sorghum, and wheat in 1983. Thus, during the
19-month interval of irrigation whose effect was observed by the LISS,
there were four major periods, or seasons, of sprinkler irrigation with
wastewater at the Hancock farm. Table 4 presents the dates and levels
of sprinkler irrigation with wastewater from the pipeline and from the
reservoirs by the 19 rigs with functional spray nozzles during these major
irrigation periods, based on records maintained by LCCIWR.
TABLE 4. MAJOR IRRIGATION PERIODS AT HANCOCK
Irrigation
period
"Spring 1982"
"Summer 1982"
"Spring 1983"
"Summer 1983"
Start
date
2-16-82
7-21-82
2-15-83
6-29-83
End
date
4-30-83
9-17-82
4-30-83
9-20-83
FARM DURING LISS SURVEILLANCE
Wastewater sprinkler irrigation
(cm applied8)
from pipeline from reservoir
5.83
6.91
0
0.20*
0
3.87
14.87
14.99
Farm average over 19 sprinkler pivots of total centimeters of wastewater
applied during irrigation period (pivots 18, 20, and 21 practicing
furrow irrigation were excluded.
Applied from 7-12-83 to 7-30-83.
Every center pivot rig completed at least one full circular revolution
of wastewater spray irrigation in each of these four irrigation periods.
The irrigation rigs generally completed a circular revolution in 2 to 5
days. Most irrigation rigs made between two and seven circular revolutions
in each of these four irrigation periods. Infection events occurring in
27
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time intervals consistent with these four irrigation periods were analyzed
in the LISS to investigate possible association with aerosol exposure.
In addition to these major irrigation periods, a few irrigation rigs
were operated sporadically at other times, as shown in Table 5. Since
the volume of wastewater applied in these irrigation events was much smaller
than in the major irrigation periods, these additional irrigation events
were generally ignored in the data analysis.
All of the irrigation data from Tables 4 and 5 are plotted versus
time in Figure 5. The ordinate is the wastewater sprinkler irrigation
rate, in centimeters per month, to adjust for varying durations of irrigation.
The area of each rectangle is proportional to the volume of wastewater
applied.
TABLE
5 . MINOR
IRRIGATION PERIODS AT HANCOCK FARM
DURING LISS SURVEILLANCE
Start
date
5-20-82
10-20-82
12-4-82
5-9-83
5-24-83
6-21-83
End
date
5-25-82
11-18-82
12-16-82
5-12-83
5-28-83
6-24-83
Wastewater sprinkler irrigation
(cm applieda)
from pipeline from reservoir
0.10
0.44
0.15
0.81
0.66
0.32
Farm average over 19 sprinkler pivots of total centimeters of
wastewater applied during irrigation period.
The wastewater and aerosol sampling data (see Sections 5A and 5B)
indicate that microorganism levels were substantially higher (by one or
more orders of magnitude) in the pipeline wastewater than in the reservoir
wastewater that was sprayed. Hence, it appears that the LISS population
had greater aerosol exposure to most wastewater microorganisms in 1982
than in 1983. The summer 1982 irrigation appears to have been the highest
period of irrigation-related exposure to many of the microorganisms studied.
B. STUDY POPULATION
Sampling
The rectangular area within 4.8 kilometers (3 miles) to the north,
approximately 4.0 km to the south, and approximately 3.2 km to the east
and west of the perimeter of the spray irrigation rigs on the Hancock farm
was designated as the study area. This area, which includes the small city
of Wilson, Texas and the rural areas north, northwest, and northeast of
Wilson, was divided into six sampling zones (Figure 6). The rectangular
Zone 1 included all rural households located on the Hancock farm and within
approximately 0.8 km (0.5 miles) of its perimeter. Zone 2 contained the
28
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Irrigation from Pipeline
to
O)
S-
S-
s-o
s_
cu
4->
ro
3
OJ
4J
1/5
ra
6--
Irrigation from Reservoir
Figure 5. Wastewater sprinkler irrigation at Hancock farm during LISS
-------
KEY
Rural household participating
during irrigation period(s).
Figure 6. Sampling zones comprising the study area
30
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households located within 0.8 km (0.5 miles) of the Hancock site boundary
within Wilson. Included in Zone 3 were all rural residences located approxi-
mately from 0.8 to 1.6 (E and W) or 2.4 (N or S) km from the Hancock farm.
Zone 4 consisted of the Wilson households which were located 0.8 to 1.6
km from the site. Zone 5 contained the rural households which were approximately
located from 1.6 or 2.4 to 3.2 km (E and W), 4.0 km (S) and 4.8 km (N)
of the Hancock farm boundary. Zone 5 was extended to approximately 4.8
km north of the farm due to the prevailing southerly winds. The households
of the small number of Hancock farm workers who resided outside the study
area were placed in Zone 6. The size of the sampling zones had no impact
on the LISS results, since all data analyses were based on an aerosol exposure
index rather than sampling zone.
Due to the limited number of residences in the rural area (approximately
130), all households within Zones 1, 3, 5, and 6 were invited to participate
in the study. Special emphasis was placed on recruiting all households
located in Zone 1 in order to maximize the amount of information from indi-
viduals who, presumably, would be most highly exposed to wastewater aerosols.
There were approximately 172 households located within Wilson, and
one-half of these were selected for recruitment into the study. Thus, every
other Wilson household was designated a part of the sample. When a refusal
was obtained, the next available house on the block was contacted, according
to a standardized selection procedure.
Households which dropped from the study before June 1982 were replaced
with households in the same sampling zone whenever possible. The study
population is not considered to be transient; however, several households
did relocate within the study area and many individuals temporarily moved
out of the study area. In these cases, the affected households and individuals
were asked to continue their participation in the study as long as they
were residing within the boundaries of the study.
One hundred ninety seven households with 580 members were recruited
into the study. Thirty-four of the households (102 members) which were
recruited in May 1980 never actually participated in the study. One hundred
sixty three households, with 478 members, participated at some level during
the course of the study. One hundred seven (66%) participating households
with 306 (64%) participants remained in the study until its conclusion
in October 1983. Twenty-four percent of the participants dropped out of
the study between June 1980 and January 1982, prior to the onset of irrigation.
Only 12% of the participants dropped out of the study after irrigation
had commenced.
Health Interview and Recruitment
A team of interviewer-recruiters was trained and obtained the medical
history of each family member in the sample households. Each interviewer
received an instruction manual describing methods for conducting the interview
and recording illness history. Interviewers were instructed in methods
of recruiting residents to participate, in maintaining health diaries,
in submitting to tuberculin testing, and in providing stool, illness, and
31
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blood specimens. The purpose, duration, and incentives for participation
in the study were explained to each interviewer to enable him/her to respond
to questions from interviewees during the recruitment period. The incentives
included: 1) continuing information about the health status of each participant,
2) laboratory information regarding infectious agents recovered from specimens
collected during an illness, 3) a brief layman's version of the findings
from the study, 4) a small monetary reward at the end of each study year
for the inconvenience imposed on each participant for cooperating in the
health watch, and 5) small payments for each fecal specimen provided.
A questionnaire was developed to record information on the number
of members in each family, their age, level of education, occupation, income,
chronic health conditions, and relevant medications. This form is presented
in Appendix B. A pretest of the instrument was done to evaluate the inter-
viewee's understanding of and responses to the questions being asked. The
interview required 15 minutes of participant time.
Update questionnaires were administered over the telephone to all
participating households in February 1982 and October 1983. These questionnaires
are presented in Appendices C and D. These questionnaires were designed
to document changes (in chronic health conditions, occupation, use of air
conditioning etc.) during the course of the study. The questionnaire updates
were also used to obtain needed additional information, such as the polio
immunization history of children, the type of air conditioning system,
degree of water consumption, and frequency of contact with large groups.
Serosurvey
Twice each year during the study (usually during June and December),
each participant was contacted (by mail and telephone) and asked to provide
a blood sample at the Wilson Community Center. Blood was collected by veni-
puncture into two sterile 15 mL serum separation vacutainers. Syringes
(10 mL) were used to collect blood from children who were under the age
of two.
Blood specimens were placed on ice and shipped to the serology laboratory
(UTSA in 1980-1981; UI in 1982 and 1983) for serum separation and storage.
Upon arrival at the laboratory, the serum was separated from the clot,
dispensed into four (UTSA) or five (UI) vials, and catalogued. All but
one vial were stored at -70°C. The remaining (UI) vial was heat-inactivated
and stored at -20°C for use in enterovirus serology.
Allowing for variations between participants, approximately 7-8
mL of serum was obtained from each participant. The serum was divided
into five aliquots: two aliquots were allocated for immediate testing (serum
neutralization, Legionella, reovirus, and rotavirus); one aliquot was reserved
for hepatitis A serology (at either UTSA or Metpath); one aliquot was used
for "special testing'' (Norwalk virus, E. histolvtica) at other laboratories;
and, the final 1 mL aliquot was stored and later forwarded to the archive
at EPA HERL. In cases where only small volumes of blood could be collected
from a small child or from a participant with collapsed veins, the archived
specimen was aliquoted first, and the remaining serum was allocated for
32
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as many tests as possible. In cases of severe shortages, children's sera
were reserved for Norwalk virus and rotavirus serology.
Informed and parental consent forms (Appendix E) were signed prior
to collection of the first blood sample from each participant. Consent
forms were updated and administered for a second time in June 1983.
Every effort was made to obtain a blood sample from each person in
every participating household. Participants who could not, or did not,
come to the regularly scheduled blood drawing clinics were contacted by
phone and asked to provide a blood sample in their home or at a follow-up
clinic. These follow-up measures increased the overall number of samples
collected by 10 to 30%.
Fecal Specimens
During 1980 and 1981, regularly scheduled fecal specimens were requested
for children age 12 and under. In cases where the household had only one
child in the age group, the next oldest household member was also recruited
as a donor. Due to the fact that only one of three eligible households
on the Hancock farm and two of five eligible households in Zone 1 regularly
provided specimens in 1981. one randomly selected adult from every study
household was asked to provide a specimen in 1982. If the selected adult
was not willing to provide a specimen, then another family member was asked
to provide a specimen for the household. In households that provided specimens
in 1980 and 1981, the same members were asked to continue providing specimens
in 1982. Only two specimens per household were accepted in 1983 in order
to limit the number of specimens received to 100 per collection period.
Collection of the children's specimens took place over three 2-week
periods in 1980 and six 2-week periods in 1981 (see Figure I.C.I). In 1982,
each of the six collection sessions took place over a 1-week period that
was coordinated with the irrigation schedule. Collections took place over
five 1-week periods in 1983. In order to obtain a maximum amount of information
during periods of irrigation in 1982 and 1983, three consecutive specimens
were solicited. One sample was collected prior to the onset of irrigation
and the remaining two samples were collected during the irrigation period.
A $5 fee was offered for each specimen and a $15 bonus was paid to each
participant who provided the three consecutive specimens.
The Sage stool specimen system was used to collect the fecal specimens.
Each household was provided with a collection kit, a styrofoam ice chest,
and an ice pack. Participants were instructed to keep the specimen cold
until it could be presented at the collection point in the Wilson Community
Center. Participants were also asked to submit specimens as quickly as
possible after collection. In cases where it was not possible or convenient
for participants to bring the specimens to Wilson, a telephone number was
provided to participants so that arrangements could be made for a staff
member to transport the specimen.
The fecal specimens were processed by transferring approximately 10
grams to each of two appropriately labeled sterile containers. Ten mL of
33
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phosphate-glycerol buffer (pH 7) were added to one container to preserve
bacterial viability. The other container was shipped without addition of
any preservative. Processed specimens were stored on wet ice and shipped
in biomailers with icepacks. Host specimens arrived at the UTSA Laboratory
(and the DTA laboratory in 1983) within 24 to 36 hours after actual specimen
collection.
All study participants were asked to provide a specimen for ova and
parasite analysis in conjunction with the regular specimen collections
during the summer of 1983. A subject fee of $5 per specimen was offered
for each of these specimens.
Specimens for ova and parasite analysis were preserved in vials containing
formalin (5% solution) and polyvinyl chloride. All materials for preservation
and shipping were provided by the Texas Department of Health (TDoH). The
preserved specimens were held at room temperature until the end of each
collection week, then shipped to the TDoH laboratory in Austin for analysis.
Illness and Exposure Monitoring
Participating households were asked to record and report information
for any of the following conditions for each participating household member:
o all acute illnesses;
o contacts with wastewater (and aerosols in 1983);
o absences of 2 days or more from the study area.
All diary information was collected by the field representatives and forwarded
to UI on a biweekly basis for review and coding. The coding information
was then forwarded to the Data Management section for data entry. Diary
data were collected from the entire population in the summer of 1980, spring
and summer of 1981, and January through October 1982. Information was collected
from approximately half of the population between November 1982 and October
1983. Methods for collecting the diary information were modified several
times during the course of the study in order to improve the quality of
the data and to minimize the amount of time that was needed to process
the information.
During 1980, each household was provided with a booklet (Appendix
F) to record all illness events. At the end of each 2-week data collection
period (DCP), a field representative collected the booklet and gave the
household a new booklet for the next DCP. This procedure had been used
successfully in a previous study (Carnow et al., 1979); however, the results
were less than satisfactory in the present study. The problems that were
encountered with the 1980 health diaries included:
o Since participants frequently were not at home, field represen-
tatives had to make several trips over a 2- or 3-week period
in order to retrieve a diary from a single household.
o Since participants frequently forgot to complete the diary until
the field representative arrived to collect the diary, the information
34
-------
was often based on recall of events that may have occnred 2 or
more weeks prior to the arrival of the field representative.
o Participants' entries in the diaries often were incomplete and
inconsistent.
o Some hispanic families reported no illnesses because the adults
in the household could not understand the written instructions
or could not write in English.
In 1981, field representatives contacted each household by phone on
a weekly basis. The field representatives were instructed to ask a series
of questions and record the responses on a health diary form. The completed
forms were mailed to UI after each DCP. The procedure modifications improved
the quality and consistency of the illness information. However, contact
with some households continued to be a problem. Field representatives frequently
would try to obtain illness information 3 weeks after the week in question,
resulting in data which was based on recall of events which could have
occurred weeks earlier. It also caused the field representatives to mail
the diary forms to UI a month to 2 months after the end of the DCP in question.
Therefore, the review and coding processes were delayed, and Data Management
received the coded materials several months after the illness events occurred.
The household diary form was modified in 1982 to include questions
about contact with wastewater (see Appendix G). Procedures for collecting
the information were also modified to correct the problems that were experienced
in 1981. The change in procedures allowed illness information to be collected
and analyzed quickly so that illness surveillance could be maintained for
the study population during periods of wastewater irrigation. Field represen-
tatives were instructed to attempt to contact all households by phone within
2 days after the DCP had ended. At the end of this 2-day period, the field
representatives transmitted the following information to UI by phone:
o study participants who reported an illness;
o type of illness;
o dates of onset and conclusion of illness;
o households that could not be contacted;
o study participants who were out of town for 2 or more days during
the week.
The UI staff made an additional attempt to reach the uncontacted households
and then used the information to compile a weekly summary. The weekly summary
(Appendix G) listed the number of participants who were contacted and the
number of new illnesses (by type) that were reported. All illnesses reported
in sampling Zone 1 (Hancock farm families and rural households within one-half
mile of the farm) were also noted in this report. This provided a rapid
method for comparing illness rates of participants who lived in the high
exposure zone to the illness rates for all study participants. The weekly
summary was distributed to all concerned investigators within 4 days after
the week of interest had ended.
35
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Illness information was also reviewed on a weekly basis to determine
if any unusual patterns of illness had developed. Patterns of interest
included geographic distribution of illnesses, age distribution of illnesses,
unexpected increases in respiratory or GI illnesses, and unexpected reoccur-
rences of illness in high exposure households.
Beginning on October 24, 1982, the number of families contacted on
a weekly basis was reduced by approximately half. The distribution of households
which were included as ''sentinel families'' is listed by sampling zone
in Table 6. All households with members who had exposure to wastewater
were included on the sentinel family list. The remainder of the families
were selected on the basis of geographic distribution, family size, and
the family's past record of participation.
TABLE 6. COMPARISON OF SENTINEL POPULATION TO STUDY POPULATION
IN OCTOBER 1982
Study population in Oct. 1982 Sentinel population
Zone Households Adults Children Total Households Adults Children Total
Rural
Wilson
Total
1
3
5
2
4
6
22
9
31
33
33
4
132
37
20
61
57
55
4
234
13
6
30
37
35
3
124
50
26
91
94
90
7
358
22
6
12
11
13
2
66
37
12
23
19
25
4
120
13
3
15
11
16
3
61
50
15
38
30
41
7
181
The weekly diaries were modified again in 1983 to obtain more complete
information about direct contacts with wastewater and to include weekly
information about the sentinel family's exposure to wastewater aerosols.
The modified diary form and exposure questionnaire are included in Appendix
G. Prior to implementation, a draft form of the exposure questionnaire
was submitted to selected study participants and staff members for comments
and suggestions. Comments were used to revise the format and the new question-
naire was implemented in conjuction with the onset of irrigation in February
1983.
Illness Specimens
Field representatives were instructed to request permission to collect
an illness specimen from a study participant whenever the participant reported
the recent onset of an illness. Throat swabs were collected within a 3-day
period after a participant reported the onset of a respiratory illness.
Stool specimens were collected within a 10-day period after a participant
reported the onset of GI or respiratory symptoms. Study participants were
also actively encouraged to contact the field representatives immediately
36
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after the onset of a respiratory or GI illness to request that illness
specimens be collected.
The procedure for collection of throat swabs was taught to the field
representatives by personnel at the Texas Department of Health. The Marion
Culturette II swabs were used for collection and preservation. In most
instances, two swabs were used for each illness specimen.
An illness specimen was labeled ''acute'' if collected while the partici-
pant was displaying symptoms of the illness. A specimen obtained within
1 week after the participant had recovered from symptoms of the illness
was termed a convalescent illness specimen. A follow-up specimen sought
to clarify the etiology of an unusual finding was labeled as ''requested''
specimen. All specimens were kept on wet ice and shipped to UTSA laboratories
as quickly as possible. Abnormal results were promptly reported to the
participants.
Activity Diaries
Each participating household was provided with an activity diary form
and a map during four 1-week periods in 1982 (in March, April, August and
December) and two 1-week periods during 1983 (in April and July). In addition,
Hancock farm residents were asked to provide two additional diaries in
March and August 1983. Participants were asked to use the diaries to record
the amount of time that they spent in each of the designated areas on the
map. They were also asked to record the amount of time that they spent
at home and in Lubbock. This diary information was used to develop a wastewater
aerosol exposure index for each participant during each of the four irrigation
seasons.
The activity diaries which were sent to the households in March and
April were returned to UI in the self-addressed, stamped envelopes which
were mailed with the diaries. Due to the low compliance rate (55% in March,
41% in April) and the high number of incorrectly completed diaries, subsequent
activity diary periods were scheduled to coincide with a fecal collection
or a blood drawing. This scheduling allowed the health watch manager or
the field representatives to be available to help participants correctly
complete the diaries. It also allowed follow-up in cases where participants
did not respond to the request for activity diaries. This modification
resulted in an 80 to 90% response rate which was a marked improvement.
Previous activity diaries were used in cases where the participant indicated
that his activities had not changed since the previous recording period.
The diary form and the maps for the irrigation seasons have been included
in Appendix H.
Tuberculin Skin Testing
Tuberculin skin tests were performed in order to monitor possible
nontuberculosis mycobacterial (NTM) infections. These tests were administered
in June or December 1980, June 1981, December 1982, and October 1983.
The inter dermal Mantoux test (5 TU of PPD-S injected intracutaneously into
the volar surface of the forearm) was performed by Texas Department of
37
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Health nurses. All participants were asked to report back to the Wilson
Community Center within 48 to 72 hours after the test was administered.
The public health nurses or the health watch manager examined all cases
with erythema and measured all indurations. Indurations which were found
to be 10 mm or greater were referred to the Health Department.
Poliovirus Immunization
Based on serological analysis of the first blood sample collected,
a significant proportion of the study population appeared to be susceptible
to at least one of the three poliovirus serotypes. Because poliovirns
was found in the Lubbock effluent, prophylactic immunization of susceptible
residents (particularly those within 400 m of a spray rig) was recommended
and implemented.
All participants who gave a blood sample were notified by mail or
telephone of their poliovirus immune status and as to whether immunization
was recommended. (A susceptible individual was defined as someone who
had a serum titer of less than 8 against one or more of the poliovirns
serotypes by serum neutralization. Individuals with titers greater than
4 for all three serotypes were considered immune.)
Special immunization clinics were conducted at the Wilson City Hall
by the Texas Department of Health, and all susceptible participants were
invited to attend. The first clinic was held in early April 1981 in order
to allow time for immunity to develop before the initiation of irrigation.
Subsequent clinics were conducted in Hay and June and in January 1982.
Study participants could also receive immunization at the Health Department
clinics in Lubbock or Tahoka if they preferred.
In accord with the Texas Department of Health's recommendations, suscep-
tible adults (18 years or over) received four doses of the Salk inactivated
polio vaccine (IPV). Injections were given monthly from April through
June 1981, and a booster shot was administered in January 1982. All susceptible
children received the Sabin oral vaccine (OPV) booster dose in May 1981.
All individuals submitting to the imunization signed the informed
consent form which is used by the Health Department. (Parents signed for
minors.) A copy of this form is presented in Appendix E. All individuals
attending the clinic also received a short polio immunization history ques-
tionnaire. The questionnaire was administered by the field representatives
by telephone to individuals who did not attend the clinic. When an individual
was deemed susceptible by serological analysis but presented proof of immuni-
zation, a booster immunization was recommended.
A summary of the poliovirus protection status of participants is listed
in Table 7.
38
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TABLE 7. SUMMARY OF PARTICIPANT POLIOVIRDS PROTECTION STATUS
(January 1983)
Study populations
Total number tested
Number recommended for immunization
Number receiving complete immunization series
Number receiving incomplete immunization series
Number refusing immunization
Number current study participants who have not
Riven blood
Children
158
71
63 a
0
8
10
Adults
274
123
61
46
16
8
Total
432
194
124
46
24
18
All children who were recommended for immunization had a previous
history of immunization. Therefore, only a booster dose was administered.
Restaurant Patronage Survey
To investigate food preparation as a possible source of the bacterial
infections observed in 1982 and 1983, a restaurant survey was administered
retrospectively by telephone to all available fecal and illness specimen
donors in July 1984. Although the primary intent was to determine how
frequently participants ate food which was prepared at one restaurant during
the summer of 1982, the restaurant survey was designed to include all four
establishments which served food in Wilson during 1982 and 1983. Only
two of the establishments, restaurants A and B, were open for business
during the entire 1982-1983 period of time. The other two establishments
were actually small grocery stores which prepared food (mainly sandwiches)
as a sideline. Restaurant B was the only establishment which served food
that could be eaten on the premises; the other establishments prepared
food on a take-out basis only.
It was anticipated that participants would not remember exactly how
many times they had eaten food prepared by a restaurant 2 years earlier.
Therefore, respondents were asked to estimate how often, if ever, the specimen
donor was likely to have eaten food prepared by each of the restaurants
during the summers (i.e. June-August) of 1982 and 1983. The choices offered
to the respondent were:
o more than once a week;
o once a week to once a month;
o less than once a month;
o never.
The respondent was also asked to compare the frequency of patronage in
stunner to patronage of the restaurant during the rest of the year. The
survey questionnaire is presented in Appendix I. Since this was a small
rural community, most respondents had no difficulty with recall or knowledge
of the donor's patronage frequency.
39
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C. EXPOSUXE ESTIMATION
Aerosol Exposure Index (AEI)
A measure was needed of the degree of a participant's cumulative exposure
to microorganisms in the wastewater aerosol, relative to all other study
participants during a given irrigation period. The aerosol exposure index.
AEI, was used in all LISS data analyses as this measure of relative exposure
to the wastewater aerosol.
AEI was constructed using a microenvironment approach to estimate
the cumulative relative exposure of each participant to the pathogens in
the wastewater aerosols sprayed during each irrigation period. Estimated
aerosol exposures due both to distant transport of aerosols and to extensive
contacts with the aerosol mist and at short distances downwind from an
irrigating rig were accumulated in AEI as a weighted sum:
AEI = El + 0.5 • XAEREM
El, the aerosol transport exposure index, was based on activity diary data
and on dispersion modeling of historical wind data for five microenvironment s,
as discussed below. XAEREM, the index of extensive aerosol exposures,
was based on an exposure log which the sentinel participants provided throughout
the 1983 irrigation period for the downwind aerosol plume microenvironment.
The definition of XAEREM is presented in the next section. Additional Exposure
Measures. El and XAEREM exhibited similar highly skewed distributions,
but XAEREM was much larger than El for the small number of participants
with occupational exposure to the wastewater and its aerosol mist. The
coefficients of 1.0 for El and 0.5 for XAEREM were chosen empirically to
yield an intuitively reasonable ordering of AEI among participants: the
contribution to AEI from documented extensive contacts with the aerosol
mist should dominate the contribution from inferred distant transport of
aerosols. It should be noted that the aerosol densities sampled in the
LISS (see Section 5B) were not used in the calculation of AEI.
For each participant during each of the four periods of irrigation,
a value of El was computed from activity diary data and dispersion modeling
of historical wind data for five microenvironments (h and i = 1, 2, 3,
4) as:
4
El = (PhTh + Z PiTi) (S + l)/2
where h - household location
i=l - blue activity diary map area (Hancock farm)
i=2 - orange activity diary map area (surrounding Hancock farm)
i=3 - white activity diary map area (remainder of study area)
i=4 - outside study area
40
-------
Tjj - weighted average of hours that the participant is at home during
the applicable weeks of the activity diaries
T^ - weighted average of hours that the participant is in microenviron-
ment i (i=l,4) excluding hours at home during the applicable
weeks of activity diaries (Th + £TA = 168)
Ph - predicted relative aerosol concentration at the participant's
home
Pi - average predicted relative aerosol concentration in microenviron-
ment i (i=l,4) calculated as the geometric mean of the Pjj
values for all study households in the microenvironment
S - proportion of days during the irrigation period that the participant
is reported to be in the study area (from the weekly health
report)
As the product of estimated relative microorganism concentration in the
air of a given microenvironment and his time spent in that microenvironment
accumulated over all microenvironments in the study area. El provided a
crude estimate of a given participant's cumulative inhaled dose due to
aerosol transport by the wind, relative to all other participants during
that irrigation period.
The predicted relative aerosol concentration of microorganisms at
a given distance d from the edge of the nearest irrigation rig on the Hancock
farm was estimated according to standard dispersion modeling concepts as
p eXd/u _ n e(-0.005 sec~1)d/(5.0 m/sec) = e-0.001 d
rd ~ "d ~ ud ud
where d - distance in meters from edge of nearest irrigation
rig on Hancock farm
X=-0.005 sec"* - median decay rate of aerosolized wastewater microor-
ganisms determined in Pleasanton, CA study (Camann,
1980)
u - average wind speed = 5.0 m/sec for Lubbock (1965-74)
D(j - normalized aerosol concentration at point d resulting
from diffusion, based on 1965-74 wind patterns
for Lubbock for the months of the irrigation period
The normalized aerosol concentration Dj due to diffusion (i.e., assuming
no microorganism die-off) was estimated for each irrigation period using
a time-averaged dispersion model computer program. Model inputs included
wind speed and wind direction data stratified by stability category and
source emission rates. Source emission rates of the rigs were calculated
assuming uniform areal application so each emission rate was proportional
to the area sprayed by the rig. Rigs with dropped lines were assumed to
produce no aerosol. D
-------
airport were used to calculate D^ isopleths separately for the February-April
and the July-August time periods.
Wind roses from the actual periods of irrigation (see Figures A-3
to A-6 in Appendix A) were later compared to 5 years of the historical
data (see Figures A-l and A-2 in Appendix A) to ascertain the validity
of using the historical data. There were some differences from one year
to another in the distribution of wind directions during the ''spring''
irrigation period (mid-February through April). While the primary wind
direction remained from south-southeast through southwest, the secondary
wind direction for this irrigation period was from the northwest in 1983,
whereas it was from the north through east both in 1982 and in the 1969-73
period. In contrast, the wind direction distributions during the summer
irrigation periods of 1983, 1982 and 1969-73 were very similar. Hence
the El values for the spring 1983 irrigation are probably somewhat less
accurate than for the other three irrigation periods. However, the magnitude
of this effect is relatively small considering the manner in which AEI
is used in the LISS.
An exponential decay factor was multiplied by D,j to estimate the relative
microorganism aerosol concentration P,j. Decay rates X have been observed
to be highly variable both among microorganism groups and for the same
microorganism group under different environmental conditions. A decay
rate of X=-0.005 sec~l was assumed. This value was both the median and
the slowest detectable rate of various microorganism groups obtained for
the sprinkler wastewater aerosol at Pleasanton, CA (Camann, 1980).
The maps used with the activity diary to define the microenvironments
for time reporting (Tj, T2 and T$) are presented in Appendix H. Microen-
vironment i-1 of highest exposure was colored blue on the maps and consisted
of the Hancock farm (excluding the 400-meter buffer area north of Wilson).
The boundary between microenvironment i-2 of less exposure (colored orange)
and microenvironment i=3 of still lower exposure (left white) was chosen
utilizing a Dg diffusion isopleth modified slightly for landmarks recognizable
by participants and for microorganism die-off. The map used with activity
diaries collected during the school year (in March, April and December)
was based on a D
-------
Participants were asked to keep activity diaries during six selected
weeks as a means to estimate their Tfc and T£ values during each irrigation
period. A sample activity diary is presented in Appendix H. The six weeks
in which activity diaries were kept (see Figure 7) were in data collection
periods (DCP) 206 (March 21-27, 1982), 208 (April 20-26, 1982), 216 (August
1-7, 1982), 224 (November 28-December 4, 1982), 308 (April 10-16, 1983).
and 314 (July 10-16, 1983).
WASTEWATER SPRINKLER
IRRIGATION
(cent iseters/month)
8.0
6.0
4.0
2.0
0.0
Activity diary week
Data collection period
1
Spring
1982
2
Sunner
1982
J I
1982
J|F
M|A
AAI
M|J
JlA
A
S|0
206 208 216
N|D
A
224
3
Spring
1983
4
Summer
1983
I i i
1983
M|A
A
308
M|J
J|A
A
314
SO
N|D
LEGEND
Irrigation from pipeline
Irrigation from reservoir
Figure 7. Relation of activity diary collection weeks
to major periods of irrigation
In the last three activity diary periods (i.e., DCPs 224, 308 and
314), participants whose activity pattern was basically unchanged and who
spent little time in the vicinity of the Hancock farm were allowed to certify
that the activity information provided in a prior activity diary was applic-
able. In these cases, the activity information from the applicable prior
activity diary was substituted.
A weighted average of the time reports from the applicable activity
diaries was employed to estimate Tn, TI, T2 and Ij for each irrigation
period. Full weight was given to activities diaries concurrent with the
43
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irrigation period, half-weight to diaries from the same season of the other
year, and quarter weight to other diaries during the same school year.
The resulting weighted averages were:
1. Spring 1982: T = (2T2o6 + 2T2Q8 +
2. Summer 1982: T = (2T21g + T3i4>/3
3. Spring 1983: T « (4T308 + 2T2Q6 + 2T208 + T224>/9
4. Summer 1983: T = (2X314 + T2ig)/3
Hissing activity diaries were excluded in calculating the weighted average.
Cases in which the T^ and Tj time reports from the lesser weighted
activity diaries differed substantially from the concurrent activity diaries
were evaluated to determine whether all the data were applicable to the
weighted average. The reported activity data were checked for errors when
1) the times at home T^ reported on the activity diaries from the same
season differed by more than a factor of 2, or 2) the times spent on the
Hancock farm Tj reported on the activity diaries from the same season differed
by more than a factor of 10. The participant's T value from the most similar
season was substituted if none of the activity diaries in the weighted
average were provided. If a participant provided none of the six activity
diaries, his T values were estimated based on the best available knowledge
of his usual major activities.
When participants were not home during the majority of the activity
diary collection week, they were asked to complete the activity diary for
the week of their return. Hence, a downward adjustment factor (S+D/2
to the T values were included in the El calculation to reduce cumulative
exposure for days during the irrigation period when the participant was
away from the study area.
The relative precision of a participant's AEI estimates was dependent
on his degree of compliance in providing activity diaries and exposure
logs. Accordingly, a quality code was assigned to each AEI estimate based
on the degree of reporting the applicable activity information.
Additional Exposure Measures
Other exposure measures were developed to investigate alternative
routes of exposure to wastewater irrigation besides the wastewater aerosol.
Each sentinel participant was asked to maintain a log of extensive wastewater
contacts from February through September 1983. As part of the weekly illness
report, the most extensive aerosol exposure and direct wastewater contact
of the week and the estimated hours spent on the Hancock farm were also
obtained for each household member. From these data, cumulative measures
of extensive aerosol exposure (XAEREM) and direct wastewater contact (XDIREM)
were calculated using the microenvironment method for each sentinel participant
for both of the irrigation periods in 1983. The hours spent on the Hancock
farm were also averaged as another exposure measure (FHRSEM) .
If a sentinel participant received exposure to the mist or aerosol
from an operating spray rig within 400 yards downwind at least once during
44
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week j, the downwind distance category d(j) and duration category m(j)
of the most extensive aerosol exposure were reported. The index of extensive
aerosol exposures, XAEREM, was calculated as the average of these exposures
for the n weeks comprising an irrigation period:
XAEREM = E cd(j)
The distance category was converted to an aerosol concentration c3
200-400 yards 1.6 cfu/m3
Fecal streptococci were chosen because they are hardy (Camann, 1980) and
may thus serve as a useful model for enteroviruses. The pipeline runs
were chosen because they provided usable data out to 400 yards. The duration
category m(j) was converted to an assumed duration Bm(j) considerably less
than the midrange because of the presumed skewness of the duration data:
Duration. m(.i) u»
<0.5 hr 0.2 hr
0.5-4 hr 1.0 hr
4-12 hr 6 hr
>12 hr 15 hr
Certain individuals who resided near an operating spray rig neglected
to report aerosol exposures received while at home. From available data
on the dates of operation of nearby irrigation rigs, on wind direction
and on time spent at home, these aerosol exposures at home were estimated.
The estimates were included in the XAEREM calculation for weeks in which
aerosol exposure reports were evidently lacking.
If a sentinel participant had direct contact with the wastewater at
least once during week j, the degree category k(j) and the duration category
l(j) of the most extensive direct contact event were reported. The index
of extensive direct contacts with wastewater, XDIREM, was calculated as
the average of these contacts for the n weeks comprising an irrigation
period:
XDIREM . "
wk(j) - t 1(J,
45
-------
The degree of contact was converted to a numerical measure of presumed
severity wfc(j):
Degree of contact. k(j)
on clothing and/or shoes 1
on skin and/or hair 10
in eyes and/or month 100
The duration category was converted to an assumed duration
Duration of contact. 1(1) _jLl(j)_
<5 min 1 min
5-60 min 10 min
>60 min 100 min
The average hours per week spent on the Hancock farm, FHRSEM, was
calculated from the weekly reports for nonresidents of the farm and from
additional activity diaries provided by the farm residents. The weighted
average Tj from activity diaries was used as the FHRSEM value for participants
who did not provide the weekly exposure log data.
Every participant with any anticipated exposure to wastewater piped
from Lubbock was followed as a sentinel participant. Thus, XAEREH and
XDIREM were set to zero for every nonsentinel participant since extensive
wastewater exposures were assumed to be very unlikely for them.
Values of the additional exposure measures and levels for the spring
1982 and summer 1982 irrigation periods were inferred from the corresponding
1983 values except when the participant's activity pattern had changed.
In particular, the XAEREM values for the spring and summer 1982 used in
the AEI calculation for these irrigation periods were the XAEREM values
for corresponding 1983 irrigation season, except for the 14 participants
whose activity patterns had changed. Presumed XAEREM values were substituted
in these cases, based on knowledge of their activities on the Hancock farm.
D. ENVIRONMENTAL SAMPLING
Wastewater
Samples of Lubbock wastewater were collected from three locations:
o the effluent from Trickling Filter Plant 2 (LTFP) at the Lubbock
Southeast Water Reclamation Plant (from June 1980 until February
1982 when the pipeline to the Hancock farm became operational)
o pipeline effluent at the Hancock farm (from February 1982 to
September 1983)
o effluent from the storage reservoirs at the Hancock farm (from
June 1982 to September 1983)
46
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Concurrent samples of Wilson wastewater were also collected from June 1980
to September 1983. The dates of sample collection and the types of microbio-
logical assays performed on each sample are given in Tables A-2, A-3 and
A-4 in Appendix A. The time series of microorganism concentration data
from each sample location characterized each wastewater source. An overview
of the frequency of measurement in the sprayed wastewater and the Wilson
wastewater of each infectious agent monitored in the study population was
presented in Table 2.
Twenty-four-hour composite samples of the LTFP effluent were obtained.
In 1980, six consecutive 4-hour time-weighted samples of effluent were
collected with an ISCO Model 1580 automatic sampler. A flow-weighted composite
sample was prepared based on plant flow data for each 4-hour period. During
collection each 4-hour sample was cooled at 4°C, and after compositing
the final large volume sample was transferred to sterile bottles and shipped
in a 4°C environment to the UTSA-CART laboratories via either airline parcel
or bus express service for analysis within 24 hours. A complete description
of equipment used, sampling procedure, and compositing calculation are
shown in Appendix J. After 1980, three 8-hour samples were collected and
flow-weighted to prepare all 24-hour composite samples.
A pipeline effluent sample at the Hancock farm replaced the sampling
location previously used at the LTFP when the pipeline became operational
in February 1982. Compositing for the 24-hour pipeline sample was accomplished
by a time-weighting method rather than the flow-weighting method previously
used due to the expectation that flows in the pipeline would be more uniform
than the effluent flows experienced at the LTFP.
The pipeline was sampled at Distribution Can 4 at the end of the 19-mile
pipeline and just before distribution onto the Hancock farm at the northern
boundary. The specific sampling point was a faucet attached to the pipe
connecting the pressure gauge in the top of the submerged distribution
can. A 6-foot long, 3/8-inch diameter tygon tube was connected to the faucet
and run outside Can 4's sheltering building to a 4-liter beaker at the
back side. The composite sampler was set inside the building with its sampling
tube running under the building's frame and into the beaker. At time of
sampling, the faucet was turned on, and the wastewater flowed into the
beaker and overflowed onto the adjoining field.
A new sampling location was added at the Hancock storage lagoons beginning
in June 1982 after the reservoirs became operational. Since Reservoir
1 supplied most of the stored wastewater applied from reservoir during
the summer 1982 irrigation season, samples were collected either as a composite
of grabs from various depths and locations in Reservoir 1 or as a time-weighted
composite from Can 1 at Reservoir 1 when irrigation from reservoir was
occurring. During 1983 samples of the reservoir storage system consisted
of volume-weighted composites based on historical and projected use from
individual reservoirs or a 24-hour composite from Reservoir 1 when it was
the only reservoir used for irrigation. An example is: if two reservoirs
were contributing equally to irrigation with no irrigation direct from
line, then the reservoir composite sample would be composed of water, 50%
from each of the two reservoirs. If more than one reservoir was composited,
47
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then grab samples were obtained from the faucets at each reservoir's distri-
bution can. If only one reservoir was being used for irrigation, then a
24-hour composite sampler was set up at the reservoir's distribution can
similar to that for the pipeline sample.
The Wilson wastewater samples were obtained from the Wilson sewage
treatment plant using an automatic sampler in a time-proportional operational
mode. Initially, the effluent from the Imhoff tank (WIT) was sampled prior
to the evaporation lagoons. On November 1, 1982 the Wilson sampling location
was changed to the influent after the bar-screen and grit chamber and prior
to the Imhoff tank inlet. This change was made to enhance recovery of viruses
from this sample source. To collect a WIT sample, an ISCO Model 1580 automatic
sampler was used in a time-weighted mode over a 24-hour collection since
no flow measuring device was available. During collection the sampled wastewater
was cooled to 4°C and at the conclusion of the 24-hour sampling period
was transferred to sterile bottles and shipped with the LTFP effluent samples.
A complete description of equipment used and sampling procedure is given
in Appendix K.
Wastewater Aerosol
Background Runs—1980 Baseline Year—
Four background air sampling runs were performed in August 1980 before
commencement of any spray irrigation at the Hancock farm. The objectives
of these runs were twofold: 1) to estimate the air concentrations of the
microorganisms of concern which residents in the area typically breathed
when outdoors and 2) to identify whether there were any significant aerosol
sources of these microorganisms besides the irrigation system planned for
the Hancock site (e.g., the Wilson effluent pond). The first objective
included determining background air concentration estimates both for Wilson
and for the rural area. The information collected from these runs aided
in the selection of microorganism groups to monitor on the other types
of aerosol runs. Additionally, background exposure information was an important
component of a balanced overall assessment of the significance of participant
exposure to a given microorganism concentration due to wastewater aerosol
sources.
These runs were conducted on four consecutive days during the period
August S through 8, 1980. Aerosol samples were collected by operating nine
Litton Model H large volume samplers (LVS) simultaneously for 30 minutes
before sunrise (0630 to 0700) at nine locations in or near the Hancock
farm. Locations for samplers included three within the city limits of Wilson,
one downwind of the Wilson effluent pond, one at a farm household near
the center of the Hancock farm, and the remaining four at farm households
in quadrants of the study area. Specifically, the sampler locations as
shown in Figure 8 were as follows:
Wilson; Three samplers were placed in fixed predetermined locations
(A, B, C) in the backyards of three Wilson families in the health
watch. These samplers were 400 meters apart, with residences
in all directions from each sampler location.
48
-------
KEY
• Rural household participating
during irrigation period(s)
O Sampler location 0
Figure 8. Sampler locations for background runs
49
-------
Wilson effluent pond: One sampler was located downwind from
the middle of the first effluent pond, 13 meters from the pond
edge (Location D).
Rural area; Five samplers were placed in fixed predetermined
locations approximately 10 meters upwind of the homes of five
rural families participating in the health watch:
E - farm near center of Hancock site
F - farm in northeast quadrant (4 km ME of Hancock site)
G - farm 0.7 km south of Wilson (upwind)
H - farm in southwest quadrant «1 km SW of Hancock site)
I - farm in northwest quadrant (3.5 km NW of Hancock site).
Each sampler location was in an open area at least 10 meters from
any house, farm, or lane. No obvious sources of microorganism aerosols
were located near or upwind of any selected locations near homes. Cotton
was growing on all nearby farmland. There were no cattle or horses at or
within a kilometer upwind of any sampler location. There were hogs near
locations D and H, but they were never upwind during sampling. A few household
and farmyard animals (dogs, cats, chickens, etc.) were observed at nearly
all sampling locations. Sampler operators wore surgical masks and usually
stayed downwind during the air sampling to minimize their effect.
A grab sample of wastewater was taken near the middle of the large,
shallow Wilson effluent pond after each run. During the week of sampling,
the effluent was being diverted to an adjacent pond along a ditch about
12 meters upwind of the air sampler locations. The fecal microorganism
levels were much lower in the pond than in the Imhoff tank effluent.
The wind was from the south-southeast (160° to 168°) on all four background
runs. Winds were fairly strong on Run 2 (5.8 m/sec), but light on the other
runs. Solar radiation was nil «15 W/m2) since sunrise was at 0703. Temperature
ranged from 19°C to 23°C, while the relative humidity varied from 69 to
76%.
Litton Model M large volume samplers were selected for performing
both the background runs and microorganism runs, primarily because the
large volumes of air which can be sampled provide sensitivity to detect
low microorganism levels in the air. These samplers were designed to collect
airborne particles by electrostatic attraction to a rotating disk on which
they are concentrated into a thin, moving film of collection media. A complete
description of the sampler is provided in Appendix L. Collection efficiencies
for electrostatic precipitators depended on the operating high voltage.
Sufficient voltage must be supplied to produce a particle charge; the greater
the voltage, the greater the driving force (particle charge) to effect
particle separation from air. However, very high voltages produced sparking
which in turn disrupted the electrical equipment and electrodes, reducing
the effective voltage.
Field operation of the samplers first required that an effective decontami-
nation be performed followed by suitable storage in this sterile state.
50
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This was accomplished by a cleanup procedure using both absolute ethanol
and a buffered Clorox solution, followed by sealing all sampler openings.
All decontamination procedures, both before commencement of any aerosol
run attempts and at the conclusion of each aerosol run, were performed
in a laboratory at LCCIWR. A copy of the step-by-step cleanup procedure
is found in Appendix M.
Sampler runs were initiated by placing the necessary equipment with
an operator at each sampling site prior to the preagreed start time of
0630. At each site the operator placed the LVS on a table which was leveled
by means of adjustable legs and connected an extension cord to a nearby
power source. At all sampling sites except the Wilson effluent pond where
a gasoline-powered alternator was used, arrangements were made to operate
samplers from a local power outlet. By a predetermined arrangement and
synchronization of watches, all operators started sampler operation at
0630. During these runs sampler operational parameters included an air
flow rate of 1000 liters per minute (1.0 m^/min), a high voltage setting
of 12 to 15 kV (highest voltage obtainable without significant sparking)
and a minimum recirculation rate of brain-heart infusion (BHI) broth, the
collection media, of 10 mL/min. BHI with 0.1% Tween 80 to prevent foaming
was selected as the collection and transfer medium. This medium has previously
been shown to be adequate for sample concentration and for preservation
and assay of the microorganisms (Johnson et al., 1980). At the conclusion
of each sampling run, media containers were tightly capped, appropriately
labeled, cooled to 4°C, and immediately shipped to San Antonio via commercial
airline counter-to-counter parcel service. Sample analyses were initiated
the same day as sample collection.
Wastewater Aerosol Monitoring—1982 Irrigation Year—
In 1982, aerosol monitoring of spray irrigation rigs was conducted
during five monitoring periods covering 6 weeks of irrigation: 2 weeks
during spring irrigation and 4 weeks during summer irrigation. Five types
of aerosol runs comprised aerosol sampling: microorganism runs, quality
assurance runs, virus runs, particle size runs, and dye runs. Diagrams
of typical layouts for each of these runs showing sampler locations relative
to the aerosol source are shown in Figures 9 through 12.
Microorganism runs—A total of 20 microorganism runs were completed
during the preplanting and summer 1982 irrigation periods at the Hancock
farm to characterize the wastewater aerosol. Results from these runs charac-
terized microorganism densities in air under various conditions at the
Hancock site at distances up to 400 meters downwind of the irrigation rig.
To conduct these runs, ten large volume aerosol samplers (Litton Model
M) as used on the background runs were loaned by the Naval Biosciences
Laboratory to SwRI under a subcontract. These were deployed at various
downwind distances up to 400 meters from the rig sampled and upwind of
the primary aerosol source sampled. Initially, samplers were located at
nominal downwind distances of 50 m, 75 m, 150 m (paired), 200 m (paired)
and at an upwind location (paired). Nominal downwind sampler distances
were subsequently adjusted for some microorganism runs to 125 m, 175 m,
300 m (paired) and 400 m (paired) to determine microorganism aerosol levels
51
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Upwind
0
Mean Wind Direction
• 391m-
Plvot
• 50 m (125 m)
• 75 m (175 m)
125m (300 m)
200 m (400 m)
• Location of Model M LVAS
" "" Irrigation Rig
Figure 9. Typical sampler configuration for microorganism run
o
Mean Wind Direction
•391 m
Pivot
50-75 m
30-40 m
Location of Model M LVAS
Irrigation rig
Figure 10. Typical sampler configuration for quality assurance
and enterovirus runs
52
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o
Mean Wind Direction
'391 m-
Pivot
(Point of dye / "" 25 m
injection)
Tower 3 •• 75 m •• Tower 5
Location of AGI samplers
Irrigation rig
Figure 11. Typical sampler configuration for dye run
Upwind
Mean Wind Direction
.391 m,
Pivot
% 25 m
^ l| 50 m
^ 75 m
Location of six-stage Andersen samplers
Irrigation rig
Figure 12. Typical sampler configuration for particle size run
53
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out to the 400-m buffer zone boundary. Actual sampling distances on each
run are given in Table A-5 in Appendix A.
Model H samplers were decontaminated utilizing the same procedure
used for the background runs. BHI plus 0.1% Tween 80 was again used as
the sampling fluid. All runs consisted of a simultaneous 30-minnte sampling
time with sampler operation at 1.0 m^/min air flow and maximum high voltage
obtainable with minimal plate sparking. Field conditions occasionally required
LVS operation below 12 kV to eliminate sparking. It was often difficult
for field operators to maintain an average air intake flow rate for a run
at 1.0 m^/min, since sporadic wind gusts would temporarily alter the air
flow rate.
During the time of aerosol sampling, a simultaneous wastewater composite
sample was collected from the irrigation spray rig being monitored. At
the completion of each run, samples were labeled, cooled to 4°C, and shipped
to the UTSA-CART laboratories for analysis on the following day.
Sampling conditions for the microorganism runs are summarized in Table
A-5 in Appendix A. The operating voltages of the large volume samplers
during these runs are provided in Table A-6 in Appendix A.
Quality assurance runs—Two quality assurance (QA) runs were conducted
to determine assay variability between samplers, between aliquots of BHI
from the same sampler and between replicates split by the receiving laboratory.
These runs consisted of the same cleanup and operational protocols utilized
for the microorganism runs with the exception that all operational samplers
were lined up in a row (2-meter separation) equidistant and parallel to
the orientation of the spray irrigation rig. For QA Run 1, conducted during
spring irrigation at a time of blowing dust, the nozzle line to sampler
line distance was SO meters, whereas for QA Run 2, conducted during the
summer irrigation period, the distance was 75 meters. Sampling conditions
for the quality assurance runs are shown in Table A-7 in Appendix A. After
aerosol collection, but prior to shipment to the CART laboratories, the
100-mL BHI aliquots from each sampler were split into four equal aliquots
to achieve a blind distribution of ''identical'' samples for a predetermined
sequence of microorganism assays.
Enterovirus runs—Since the wastewater contained a high enough level
of enterovirnses for the microbiological dispersion model to predict their
probable detection in aerosols, four special aerosol runs were conducted
to estimate the enterovirus aerosol concentration. To conduct this type
of run, all functional large volume samplers were operated simultaneously
at a downwind distance of about 50 meters from the nozzle line for five
consecutive 30-minute sampling segments. The samplers were aligned parallel
to the nozzle line with a sampler spacing of 1.5 m. The irrigation rig
was operated at a reduced travel setting so it progressed on the dry side
of the field (i.e., toward the samplers) at some minimal rate, typically
5 to 10 m/hr at the tower used for alignment of the sampler line. At the
end of every two 30-minute periods, the sampler position was adjusted to
compensate for the rig movement. The initial and final distances from sampler
line to nozzle line are shown for each segment on the sampling conditions
54
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summary presented in Table A-8 in Appendix A. The BHI collection fluid
was changed after each sampling segment, and all BHI was pooled at the
conclusion of the run. After transport to the UTSA-CART laboratory, the
BHI was concentrated and plaque-assayed for human enteric viruses.
Dye runs—Four dye aerosol runs were conducted to estimate the aerosoli-
zation efficiency (i.e., the fraction of sprayed wastewater that becomes
aerosolized) of the spray irrigation rigs at the Hancock site. The rig
nozzles directed a fairly fine spray laterally and upward in a 360° umbrella
pattern which appeared to enhance aerosol production and drift, due to
improper design. Thus, it was anticipated that the Hancock farm aerosolization
efficiency may differ substantially from the 0.33% (geometric mean) aerosoli-
zation efficiency of the impact sprayers used for wastewater irrigation
at Pleasanton, California (Johnson et al., 1980).
One dye run was conducted in March 1982 while the last three were
completed in July 1982. To perform these runs, a 20% solution of Rhodamine
WT dye mixed with glycerol was injected at a constant rate into the pipeline
supplying the sprayers of the irrigation rig being sampled. The dye was
injected with a Zenith Constant Torque Unit Type ZM coupled with a No.
11 Zenith Metering Gear Pump.
Aerosols were collected using 500-mL graduated all-glass impinger
(AGI) samplers connected to a vacuum pump as indicated in the following
schematic:
Rotameter
0-2 cfm
AGI
Trap
Critical
Orifice
Pump
The rotameter was used only for calibrating the system in the laboratory.
With the critical orifice in line and a pump vacuum of at least IS inches
mercury, the nominal flow rate through AGI sampler was 1.0 CFM (cubic feet
per minute).
To perform a dye run, AGI samplers containing 100-mL deionized water
as collection media were set up in pairs at four locations: two pairs at
25 m and two pairs at 75 m downwind of the monitored rig. One sample set
(i.e., 25-m and 75-m pairs) was aligned with a tower near the center of
the irrigation rig while the other sampler set was aligned at the same
orientation but displaced to the right or left of the center line set depending
upon wind direction by two rig spans. When all equipment was in place,
the Zenith gear pump began injection of dye into the irrigation system,
and when the dye was visible in front of a sampler set, the AGI samplers
commenced operation. Samplers were operated until dye was no longer visible
at the nozzles directly in front of the sampling station which typically
was 6 to 7 minutes. At the conclusion of the sampling period, the water
media was transferred to glass bottles for storage until analysis. As soon
as dye was visible in the wastewater at the nozzle closest to the injection
pump, grab samples were obtained at 1-minute intervals for as long as dye
55
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was visible to determine source strength. Dye concentrations in both the
aerosol samples and wastewater samples were determined using a Turner Spectro-
fluorometer Model 430. Sampling conditions for the dye runs are displayed
in Table A-9 in Appendix A.
Particle size runs—Five particle size runs were performed using Andersen
1 CFM six-stage particle samplers to determine the concentration and particle
size distribution of the wastewater aerosol microorganisms. The samplers
were connected to the orifice system and vacuum pump that was utilized
on the dye runs to maintain a nominal flow rate of 1 CFH through the sampler.
Each run was made with eight samplers deployed in pairs, one upwind of
the sampled source and the remaining three at nominal downwind distances
of 25, 50 and 75 meters. Sampling times ranged from 8 to 10 minutes. A
summary of sampling conditions during each of the particle size runs is
shown in Table A-10 in Appendix A. A composite wastewater sample was collected
simultaneously from the rig being sampled to determine source strength.
Standard plate count agar was used as the collection medium in these
samplers. After sample collection, plates were incubated at the LCCIWR
laboratories for 24 hours at 35 + 0.5°C and counted for colonies.
Dust storm runs—Dust storms that could entrain many sprayed microorganisms
from the spray fields as a particulate aerosol are common in the Lubbock
area, especially in spring. These dust storms may be another wastewater-
associated pathway of infection in addition to the wastewater aerosol.
If dust storms occurred during aerosol monitoring periods, special dust
storm sampling runs were planned. These runs would have been performed
by utilizing AGI samplers with BHI collection medium operated for a brief
period (about 15 minutes). Samplers would have been located both upwind
and downwind of the spray fields on each dust storm run.
No localized dust storms occurred during any of the air sampling weeks
in 1982. However, QA Run 1 took place during a time of blowing dust. On
this run, the aerosol levels of fecal coliforms and fecal streptococci
were higher than expected, based on the results from the microorganism
runs at the same downwind distance involving similar wastewater concentrations.
It is possible that the approximately threefold increase in aerosolized
fecal coliforms and the nearly doubled level of aerosolized fecal streptococci
were due to the blowing dust.
Calculation of Microorganism Density in Air
The microorganism density sampled in air was calculated from the assayed
microorganism concentration in the sampler's collection fluid. For an individual
LVS, the equation is
r = A x V
F x R x D
56
-------
where C - concentration of detectable microorganism units/or of air (e.g..
cfu/m3)
A - concentration of detectable microorganism units assayed in the
collection fluid (cfu/mL)
V - final volume of collection fluid (usually 100 mL)
F - correction factor for LVS operating voltage (reference basis
of 12 kV)
R - air sampling rate (usually 1.0 m^/min)
D - sampling duration (usually 30 min)
LVS were not as efficient as impinger samplers in the collection of
microorganisms in air, and the efficiency varied with the LVS operating
voltage. The collection efficiency of the LVS units employed in the field
sampling was determined relative to AGI samplers in wind tunnel experiments
performed in July 1980 and October 1982. The relative collection efficiencies
(mean + standard error) of the LVS were found in the 1982 tests to be 0.29
+ 0.017 in 18 tests at 12 kV and 0.68 ± 0.022 in 29 tests at 13 to 18 kV.
No attempt was made to adjust the aerosol concentration to the AGI
collection efficiency since there is no standard aerosol sampling method
and since the absolute collection efficiency of AGI samplers was not determined.
Rather, the LVS data were corrected for operating voltage to render these
data as internally consistent as possible.
The applied correction factors F for various operating voltages are
presented in Table A-ll in Appendix A. These correction factors are the
minimum expected correction. Appendix N presents the details on the calibration
studies and evaluation of four candidate operating voltage correction factors.
While other environmental factors such as particle concentration in air
and relative humidity may also influence collection efficiency, no corrections
have been applied to the aerosol data for such factors because the experimental
data were insufficient to develop calibration curves.
The enterovirus density in air was determined during virus runs in
which the collection fluid from many LVS was pooled and all except 100
mL of the fluid was concentrated prior to assay for enteroviruses. The
enterovirns density in air equation is
r = B x U
(V-100 mL) n
- Z Fi x Ri x Di
v i=l
where B - concentration of detectable enterovirus units in concentrated
collection fluid, pfu/mL
U - final volume of concentrated collection fluid, mL
V - final volume of pooled collection fluid, mL
n - number of LVS samplings pooled.
57
-------
For particle size aerosol runs the number of viable aerobic particles
per unit volume of air for each stage in the sampler was calculated using
the formula
C =
R0 x T x 0.0283
where C - concentration in air, cfu/m3
R0 - sampling rate for system from calibrated orifice, (range
of 0.82 to 0.91 ft3/min)
T - sampling time in minutes
0.0283 - conversion factor for ft3 to m3.
Results for each stage were reported as cfn/m3 which represented the mean
number of viable particles detected on standard plate count agar per cubic
meter of air sampled.
The concentration of Rhodamine dye in the aerosol collected in each
downwind impinger during the dye runs was calculated using the formula
C =
R x T x 103
where C - concentration in air (ug/m3)
Cj - Rhodamine concentration in impinger (ug/mL)
V - volume of impinger solution (usually 100 mL)
R - air sampling rate in L3/min
T - sampling time in minutes
103 - conversion factor for L3 to m3.
The geometric mean of all applicable aerosol density values was used
to estimate the middle of the aerosol density distribution in summary tables.
When all values were below the detection limit, the estimate reported in
place of the geometric mean was less than the cumulative detection limit
obtained by pooling the total volume of air sampled. Sometimes the set
of aerosol data included some measured values and some values below the
detection limit. In such cases 1) the geometric mean was calculated with
x/2 substituted for
-------
of the pathway or pathways by which the agent was introduced. Other wastewater-
associated pathways could produce a distance-related pattern very similar
to that of the wastewater aerosol, thus causing the aerosol to be blamed.
Other pathways include: 1) dust storms (discussed above); 2) vectors (e.g.,
flies attracted by the wastewater lagoons); 3) rodents (e.g., feed or food
stuffs contaminated by fecal droppings or urine from field mice, infected
by wastewater spray, which may be spending the winter in farmhouses and
barns); and 4) fomites (e.g., wastewater-contaminated work clothes or door-
knobs). Since the possibility of a fly-insect vector pathway of infection
is frequently cited and the cost was low, a small pilot study was conducted
to investigate this potential route of transmitting infectious agents.
However, lacking an illness/infection distance pattern, the cost of inves-
tigating such other pathways of infection as rodents and fomites could
not be justified.
Houseflies and other flies were trapped at the farmhouses and at effluent
ponds. Using baited traps, flies were collected next to a pig pen near
the Wilson sewage treatment facility and at the several farmhouses in 1980,
collection attempts were made at the reservoirs and at farmhouses in 1982,
and flies were collected in the irrigated fields, at Reservoir 1, and at
the pig pen near the Wilson sewage treatment facility in 1983. An effort
was made to isolate and quantitate the level of enteric bacteria and viruses
in these fly samples. A target number of at least 200 flies per sample
was sought (100 for bacterial analyses and 100 for viral analyses).
To collect flies, a stationary, bait-type trap was located and anchored
in a potentially fly-prone area protected from wind, direct sunlight, children,
animals and other potential disturbances. These traps were baited with
a nonpoisonons bait such as canned cat food and milk. The cat food provided
a perch for the fly to light on and the milk kept it moist longer since
dried-up bait did not attract flies. The traps were checked every 24 hours
at which time the bait was changed since fermented bait (with only milk
added each day) may be harmful to farm pets.
When at least 200 flies were in the trap, it was placed in a large
garbage bag and returned to the laboratory at LCCIWR. Initially (August
1980), flies were killed by using ether, but since this procedure was poten-
tially detrimental to the bacteria of interest, it was discontinued. Thereafter
the entire garbage bag and trap were chilled in a cold room (4°C) for at
least 1 hour. The contents of each trap were emptied on paper, odd species
of flies were discarded, and a maximum of 200 flies was counted out from
each trap. The flies were transferred to a sterile container, appropriately
labeled, and maintained at 4°C until arrival in the DTSA laboratory.
Drinking Water
Samples from drinking water sources on and surrounding the Hancock
farm and the potable water for Wilson were collected and analyzed for total
and fecal coliforms, fecal streptococci, and Salmonella. A total of 13
drinking water wells and one treated drinking water source was sampled
periodically beginning in October 1981. Eight additional drinking water
sample locations, including seven from households in the low exposure group,
59
-------
were added in December 1982 to provide representative data for the entire
rural study area. The original sample locations plus those added are shown
in Figure 13.
For most locations, samples were obtained from the cold water faucet
on the kitchen sink of the residence. When samples could not be obtained
from the kitchen faucet, an outside faucet was used. In either case, the
faucet was cleaned with the tap water by hand using sterile polyethylene
gloves. The outside of the faucet was scrubbed and the inside was cleaned
within finger reach. Then the water was allowed to run for 5 to 10 minutes
to flush loosened debris before collecting 1 liter of water in 1-liter,
autoclave-sterilized, wide-mouth, screw-cap, polyethylene containers.
After sample collection, the sample container was labeled with the sampling
site and placed in an ice chest containing cold packs. Eight to ten samples
were collected per day before the samples were transported to the LCCIWR
lab for immediate sample analysis. Drinking vessels, refrigerated water,
and other beverages were not tested.
Meteorological Data
Background Aerosol Runs—
Various meteorological parameters were observed and recorded during
the four background runs conducted August 4 to 8, 1980 to quantify background
air levels of microorganisms and to identify potential aerosol sources
other than the spray irrigation system. These parameters included wind
direction and wind speed at a 2-meter height utilizing a Meteorology Research,
Incorporated (MRI) Model IH-5810 Mechanical Weather Station, temperature
and relative humidity using a Bendiz Psychron Model 566-2 psychrometer,
and solar radiation using a Belfort Pyrheliograph 5-3850. All of these
parameters were measured at the research plot near the center of the Hancock
farm during the actual run time. Additional parameters obtained from the
National Weather Service at Lubbock included time of sunrise, wind speed,
wind direction, cloud cover, cloud type, and minimum height.
General Climatology—
An electronic weather station (EWS) and cassette data acquisition
system (CDAS) from Climatronics Corporation were installed at the intensive
research plot in March 1981 to measure and record general c limatolog ical
parameters on the Hancock farm. Sensors to measure wind speed and wind
direction were mounted on a 10-meter telescoping tower while sensors for
measuring temperature, dew point, and solar radiation were located on a
2-meter tripod adjacent to the tower. These parameters were recorded contin-
uously on a 5-inch wide chart moving at 1 inch per hour. Instantaneous
values of these parameters were recorded every 5 minutes on a magnetic
cassette tape in the CDAS unit. These tapes allowed cost-effective digitizing
of meteorological data for the irrigation periods. For example, tables
of hourly averages for all parameters plus wind rose plots were obtained.
The meteorological data accumulated in 1982 and 1983 on the CDAS was
processed for the irrigation periods by Envirodata Corporation to produce
hard-copy outputs of hourly averages, daily averages, and daily high and
60
-------
HANCOCK FARM : ft
Rural household participating
during irrigation period(s)
0
L_
KEY
SCALE
1234
L
O Initial sampling location
(1981+) —14 wells
A Added sampling location (Nov 1982+)--8 wells
Figure 13. Drinking water sampling locations
5
i
6 km
61
-------
low values. Wind speed and wind direction data were processed for both
1982 and 1983. Solar radiation, temperature, and dew point data were processed
for 1982 only. Wind rose plots for both the spring and summer irrigation
periods for 1982 and 1983 were generated as shown in Figures A-3 to A-6
in Appendix A. No wind speed data for the 1982 summer period was plotted
due to a malfunctioning anemometer translator board during most of this
period.
Meteorological Measurements During Aerosol Runs—
During aerosol runs, meteorological parameters were measured about
100 meters downwind of the sampled rig to complement measurements made
at the research plot by the Climatronics EWS/CDAS units. Field measurements
included wind speed and wind direction at the 2-meter level, ambient temperature
and relative humidity, and solar radiation by the same instrumentation
utilized on the background runs. Visual observations were made for cloud
type (to determine minimum cloud height) and eighths of the sky with cloud
cover. The Climatronics CDAS unit was programmed to scan and record at
1-minnte intervals during periods of aerosol sample collection.
Summaries of meteorological conditions for the different types of
runs are presented in Tables A-12 through A-16 in Appendix A. Values for
the EWS are averages obtained from the strip chart for the run period.
B. LABOKATOKT ANALYSIS OF SERUM AND CLINICAL SPECIMENS
Serology
Table 8 lists the epidemiologic charactersitics of the agents which
were initially considered for use as serologic antigens in this study.
Table 9 lists the antigens which were selected for testing; also listed,
are the sera which were used for each of the selected antigens. With the
exception of Influenza A and Legionella. all of the listed antigens are
human viruses which infect the gastrointestinal tract, are excreted in
the feces. and are known, or suspected, to be present in wastewater. None
of the viruses selected were considered to be rare or geographically restricted.
Enteroviruse s—
Initial sera from all study participants were tested for neutralizing
antibody to the three poliovirns types. Individuals having low titers (<8)
to any of the three polioviruses were recommended for immunization prior
to the onset of irrigation. The remaining enteroviruses (Coxsackieviruses
A9, B2, B3, B4, B5 and Echoviruses 1, 3, 5, 9, 11, 17, 19, 20, 24) were
selected for use in the study according to the following criteria:
o The enterovirns was isolated from either Lubbock or Wilson wastewater
(except Echoviruses 9 and 17).
o Stock virus for preparation of working virus suspensions was
readily available from either ATCC (American Type Culture Collection)
or CDC.
o The virus produced cytopathic effect (CPE) in Vero cells (except
Coxsackievirus A9 which was grown in RD cells).
62
-------
TABLE 8 EPIDEMIOLOGIC CHARACTERISTICS OF CANDIDATE AGENTS FOR SEROLOGIC TESTING
Virus and type
Hepatitis A
Pol lovlrus 1
2
3
Coxsackle A1
A5
A7
A9
A10
A16
B1
B2
B3
B4
B5
B6
Echo 1
3
5
6
7
9
11
12
13
14
15
17
19
20
21
24
25
27
29
30
31
33
Adenovirus 1
2
3
4
5
6
7
12
14
Reovlrus 1
2
3
Rotavlrus 1-4
Norwalk T
Leqlonel la 1
1 nf 1 uenza A
% Antibody
prevalence3
45
80
85
75
Rare
Rare
Rare
60
Rare
25
25
60
50
70
20
Sporadic
15
25
10
40
50
55
15
35
15
15
15
10
15
15
15
15
15
5
15
15
5
15
40
50
50
20
30
10
10
75 (adults)
20
50
50
50
50
50
10
70
Isolation Occurrence in Seasonal Lubbock-Wl 1 son
Associated disease or symptoms from stool wastewater occurrence wastewater
Inapparent, hepatitis
Inapparent, paralysis
Inapparent, paralysis
Inapparent, paralysis
Inapparent, orphan
Rash, herpangina
Gl
Rash, Gl
Rash, pharyngitis
Rash pneumonia
Pleurodynia
Colds, systemic
Colds, systemic
Colds, rash, systemic
Colds, rash, systemic
Meningitis
Inapparent
Meningitis
Meningitis
Gl, meningitis
Meningitis
Gl, pneumonia
Gl, cold
Gl, rash
Gl
Encephal Itls
Gl
Menlng itis
Gl , pneumonia
Gl , pneumonia
Gl
Gl, meningitis
ARD
Meningitis
Meningitis
Men ingitls
Men Ingitls
Gl
Gl
Gl
Pharynglti s
ARD
Pharyngitis, Gl
Gl
ARD
Inapparent
ARD
Orphan
Orphan
Orphan
Gl
Gl
Respiratory
Resp I ratpry
Yes
Yes
Yes
Common
Yes, sporadic
Seldom
Common
6-year epidemic
Common
Common
Rare
Rare, epidemic
Sporad Ic
Common, epidemic
Common
Most common, no epidemic
Common, no epidemic
Rare, epidemic
Rare, epidemic
5-year cycles
Sporadic
Frequent
Frequent
Frequent
Rare
Frequent
Rare, epidemic
Rare
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Common
Common
Yes
Common
Yes
No
Common
Yes
Yes
Yes
Frequent
Yes
Yes
Yes
Frequent
Yes
Yes
Yes
Yes
Frequent
ND
ND
ND
ND
ND
ND
ND
ND
ND
Yes
Yes
Yes
Yes
Yes
No
Fal 1 /Winter
Al 1 year
Al 1 year
Al 1 year
Fail
Fal
Fal
Fal
Fal
Fal
Fal
Fal
Fal
Fal
Spring/Summer /Fal 1
Fal
Summer
Al 1 year
Fail
Winter
Sprlng/Summer/Fal 1
Fal 1 /Winter
Summer
Summer
Summer
A 1 1 year
Summer
Summer
Winter
A 1 1 year
Winter
Winter
Winter
Winter
Summer
Summer
Wiqter
ND
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
No
ND
ARD - acute respiratory disease
Gl - gastrointestinal illness
ND - not detectable by standard
concentration/assay techniques
a References: Fox and Hall (1980); Szmuness et al (1977); Jackson and Muldoon (1973a,b,c); Blacklow et al (1976, 1979);
Helms et al. (1980).
-------
TABLE 9. AGENTS AND SERA SELECTED FOR USE IN SEROLOGIC TESTING
Serum Collection Period
Agent
Adenovirns 3
Adenovirus 5
Adenovirus 7
Coxsackievirns A9
Cozsackievirns B2
Coxsackievirns B3
Coxsackievirns B4
Coxsackievirns B5
Echovirns 1
Echovirns 3
Echovirns 5
Echovirns 9
Echovirns 11
Echovirns 17
Echovirns 20
Echovirns 24
E. histolytica
Hepatitis A
Influenza A
Legionella 1
Norwalk 1
Poliovirns 1
Poliovirns 2
Poliovirns 3
Reovirns 1
Reovirns 2
Rotavirns
Juna Dec Jnn
1980 1980 1981
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X Xb Xb
X X
X
XC
X
X
X
X
X
XXX
Jan
1982
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Xb
XC
X
X
X
X
X
X
Jnn
1982
X
X
X
X
X
X*
X
X
X
X
X
X
X
X
Dec Jun
1982 1983
X
X
X
X
X
X
X
X
X
X
X
Xb Xb
X
X
X
X
X
X X
Oct
1983
X
X
X
X
X
X
X
X
X
X
X
X
Xb
X
X
X
a In cases where this blood was not available, the first blood obtained
from the participant was nsed.
b These bloods were tested only if previous blood was found to be negative
for antibody.
c These bloods were tested only if June 1983 blood was found to be positive
for antibody.
64
-------
It was determined in advance that testing for antibody to a specific
coxsackie- or echovirus would be continued to the end of the study only
when it was determined that less than half of the population had antibody
to that virus. This was done in order to maximize the number of ''susceptibles''
and therefore to increase the chances of detecting a statistically significant
number of infections in the population. Therefore, only partial results
are available for Cozsackieviruses A9 62, B3, and B4. Additional enterovirases
were added to replace the agents which were dropped.
The serum neutralization test was used to determine antibody titers
for the enteroviruses. This test was selected because it is considered
to be the most sensitive and specific serologic procedure for detecting
antibodies to these particular viruses. In this study, sera were initially
diluted (1:4 for poliovirus titers, 1:10 for cozsackie- and echovirnses),
then serially diluted (1:2) in microtiter plates. A challenge dose (30-300
TCID50) of virus and a suspension of Vero cells were added to each of the
serum dilutions. The antibody titer was determined to be the highest initial
dilution which inhibited the CPE of the virus.
Adenoviruse s—
Since the bentonite adsorption technique which was used in this study
did not allow adenoviruses to be isolated from the wastewater, three adeno-
viruses (Adenoviruses 3, 5, 7) were arbitrarily selected from Table 8 for
use in this study. The serum neutralization procedure which was described
for the enteroviruses was also used to detect antibody to adenoviruses.
Hep-2 cells were used in this procedure.
Hepatitis A—
During the course of this study, no routine method was readily available
to detect hepatitis A in wastewater. However, the presence of hepatitis
A in wastewater was presumed, since it is known to be present in urine
and feces during infection. Screening for hepatitis A antibody was performed
on initial sera from all participants. Only sera from participants who
were found to have no antibody were tested in subsequent blood collection
periods. The analysis of sera for the presence of hepatitis A virus (HAV)
antibody was performed with a commercially available RIA system marketed
by Abbott Laboratories under the name of HAVAB. This test is based on the
principle of competitive binding of anti-HAV in serum with radioact ively
tagged anti-HAV to HAV coated on a solid phase bead.
Influenza—
Influenza virus was included in this study as an epidemiologic control
since it is not ezcreted in the feces and therefore would not be found
in wastewater. Complement fization was the test of choice for measuring
influenza A antibody. Guinea pig complement and sensitized sheep erythrocytes
were used in this test. The antigen for this test was obtained from CDC.
Legionella bacilli—
Legionella organisms occur in the environment and can cause epidemic
and sporadic cases of Legionellosis in man. Of particular interest in this
study was the fact that algae were present in storage reservoirs on the
Hancock farm. Since it is known that Legionella organisms utilize algae
65
-------
as a natural medium (Tison, et al., 1980), it was assumed that the organisms
could be present in aerosols when the stored wastewater was applied to
the land.
The indirect fluorescent antibody (IFA) test was used to determine
the presence of antibody to L. pneumophila serogroup 1. The IFA test is
a ''sandwich'' immunofluorescence technique which uses a two-stage reaction
procedure. In the first stage, the Legionella antigen of interest is overlaid
with dilutions of animal antiserum or human serum; the slides are then
incubated, washed and dried. In the second stage, fluorescent dye-labeled
antibody (to the IgG contained in the animal or human serum which was applied
in the first stage) is placed on the slide. In this manner, Legionella
antigens are rendered fluorescent by positive sera which themselves are
not labeled.
Norwalk virus—
Sera from 25 children (under the age of 10) and 11 high exposure adults
(with a history of self-reported diarrhea during 1982) were tested for
antibody to Norwalk virus. This serology was performed at Dr. Neil Blacklow's
laboratory at the University of Massachusetts. The RIA test developed by
Dr. Blacklow was used to detect the presence of antibody to Norwalk virus.
Entamoeba histolvtica—
Based on a report by Doby et al. (1980) of a higher carriage rate
of E. histolvtica in sewer workers in France, paired sera from 189 participants
were tested for antibody to E. histolyt ica. The testing was performed
under the supervision of Dr. George Healy, at CDC. Indirect hemagglutination
was used to detect the presence of antibody.
Reoviruses—
Since reoviruses are commonly isolated from wastewater, all three
human types were recommended for serologic testing. The hemagglut inat ion-
inhibition (HI) test was used to determine reovirus antibody levels. Antigen
for this procedure was provided by the Biological Products Division of
CDC.
In order to remove nonspecific inhibitors of hemagglutination, sera
were pretreated with kaolin extract. Unfortunately, this treatmment caused
the reovirus 3 agglutination pattern to ''collapse'' prematurely each time
the test was run and titrition endpoints were unreliable. Therefore, only
serology results for reoviruses 1 and 2 were used in this study.
Rotavirus—
Paired sera from 44 study participants under the age of 18 and 10
adults from the high exposure area (with a history of diarrhea in 1982)
were tested for antibody to rotavirus. These reo-like viruses are known
to cause sporadic and epidemic outbreaks of enteritis in children.
Rotavirns antibodies were measured by the enzyme-linked immunosorbent
assay (ELISA). The supply of WA rotavirus stock antigen obtained from Dr. G.
William Gary, CDC, Atlanta, was prepared in HA-104 cells. An ELISA plate
reader was used to measure the spectrophotometric reaction.
66
-------
Clinical Bacteriology
Analyses for selected organisms in fecal specimens and throat swabs
were performed as summarized in Figures 14 and 15. The prevalence of different
microbial types in the specimens was determined in a semiquantitative manner.
All primary plating media were streaked by the same four quadrant method.
and the amount of growth of each microorganism isolated was reported by
determining the highest quadrant in which the organism was isolated as
discrete colonies. The terminology used and respective definitions were:
Very light (VL) - 1 to 10 colonies on the plate
Light (L) - growth in first quadrant
Moderate (M) - growth on first two quadrants
Heavy (H) - growth on three or all quadrants
Fecal specimens which failed to yield any growth, or which yielded
organisms by enrichment only, were excluded from the data set. The lack
of organisms, in these cases, is likely to have been due to problems with
sample processing, shipping or use of antibiotics by participants.
Fecal specimens in the transport medium were used for all isolations,
with the exception of that for Campylobacter jejuni (Figure 14) where the
specimen cup containing the representative portion of the original sample
was used. Contrary to some reports (Lennette et al., 1980), C. .1 e j un i
may survive poorly in buffered glycerol saline (Sack et al., 1980), a widely
used transport medium for most enteric bacterial pathogens. All media
were formulated from the appropriate Difco (Detroit, Michigan) dehydrated
product, with the exceptions of the cellobiose arginine lysine (CAL) agar
of Dudley and Shotts (1979) which was obtained from Scott Laboratories
(Fiskeville, Rhode Island) and plates of Campy-BAP agar which was purchased
from the same source (Aldrich Scientific, San Antonio, Texas) or from BBL
Microbiology Systems (Cockeysville, Maryland).
The procedure for primary isolation and identification of Salmonella.
Shigella. Yersinia enterocolitica. and other enteric bacteria is shown
in Figure 14. CAL agar, a special purpose differential medium for isolation
of Y. enterocolitica. was incubated at room temperature for 48 hours.
The other three media were chosen to represent three levels of selectivity
for the various Enterobacteriaceae and other enteric organisms. These
were a differential medium with little selectivity (MacConkey), a differential,
moderately selective medium (Hektoen enteric), and a highly selective medium
(bismuth sulfite) used primarily in the search for Salmonella (Lennette
et al., 1980). All plates were incubated at 35°C and examined at 24 and
48 hours. Plates were inspected, using a stereomicroscope with oblique
transmitted illumination, and a representative of every colony type observed
was subcultured for a purity check, oxidase testing by Kovac's method (Lennette
et al., 1980), and identification by the API-20E biochemical screen (Analytab
Products). To increase the chance of isolating Shigella (Figure 14), 1
mL of each fecal specimen was transferred to 9 mL of GN broth, incubated
at 35°C for approximately 18 hours, streaked to xylose-lysine-deoxycholate
(XLD) agar, incubated at 35°C for 24 hours, and identified as described
previously. The combination of enrichment in GN with isolation on XLD
67
-------
Homogeneous Suspension Transport Medium
o\
oo
t
STREAK PLATE
,1
t
Selective Media
- Celloblose Arglnlne
Lyslne (CAD Agar
- MacConkey Agar
- Hektoen Enteric Agar
- Bismuth Sulflte Agar
1
t
,j>
*
GN broth
*
Streak to
XLD Agar
Select Representative
Colonies
t
ENRICHMENT
1
J,
*
0.067M Phosphate
Buffered Saline
1
Day 3v Incubate
Day 7>~ at 4eC
t
A 1 ka II Treatment
t
Streak to CAL Agar
1
t
STREAK PLATE
j,
I
Campy -6AP
1
GasPake Jar with
CampyPak M*
r
Select Typical
Colonies
|
Presumptive
Tests
t
STREAK PLATE
(E)
|
Sabouraud Dextrose
Agar
(+ chloramphenl col)
I
Typical Colonies
|
Germ Tube Test
|
Tests for
Chlamydospores,
t
STREAK PLATE
IF)
|
Mannltol Salt
Agar
|
Typical Colonies
*
Gram Stain,
Coagulase Test
Gram Stain and Subculture
Gram Negative Organisms
Oxldase Test
Identification Using
API 20E* Biochemical
Screen
Conf 1 rmatlon
Sucrose
Asslmllatlon
Identification Using
API 20E» Biochemical
Screen
(A) Salmonella, Shlgella, Yerslnla enterocolItlca, other enterics
(B) enrichment for Shlgella
(C) enrichment for Y. enterocolItlca
(D) Campy lobacter jejunl
(E) Candida a Iblcans
(F) Staphy lococcus aureus
Figure 14. Isolation and identification of selected organisms from feces
-------
THROAT SWABS
Place Swab into 1 ml of
Todd-Hewitt Broth for 2 Hours
FLUID
FA Screen for
Group A Streptococci
SWAB
MacConkey Agar
Sheep Blood Agar
I
Select Representative Colonies
Gram Stain and Subculture
I
Gram Positive
Organisms
Catala
Gram Negative
Organisms
se Test
1
Oxidase Test
I
Coagulase
Test
Additional
Tests as
Required, e.g.
Bacitracin
Phadebac$>
Identification Using
API 20E®Biochemical
Screen
Figure 15. Isolation and identification of organisms from throat swabs
-------
has been described as excellent for recovery of Shigella (Taylor and Schelhart,
1975).
Enrichment of Y. enterocolitica (Figure 14) was carried out by inoculating
1 mL of the fecal specimen to 9 mL of phosphate buffered saline followed
by incubation at 4°C for 1 week. At Days 3 and 1, 10 fiL of the enrichment
was mixed in 0.1 mL of 0.5% KOH in 0.5% NaCl, and then streaked to CAL
plates. Representative colonies were picked and identified, as described
previously, after incubation at room temperature for 48 hours.
The procedure for C. ieiuni involved streaking Campy-BAP plates, which
then were incubated at 42°C for 48 hours in a GasPak container with the
CampyPak II (BBL Microbiology Systems, Cockeysville, Maryland) atmosphere
generator. The organism was presumptively identified by the following
criteria: Gram-negative curved rods, characteristic darting motility,
oxidase +, and catalase +. The organisms were confirmed by growth in 1%
glycine, lack of growth at 25°C, and susceptibility to nalidixic acid (30
Hg disk).
The fungal yeast pathogen Candida albicans was isolated (Figure 14)
by streaking plates of Sabouraud dextrose agar supplemented with 50 jig/mL
of chloramphenicol (Calbiochem), followed by incubation at 35°C for 48
hours. Plates were inspected for white, convex, opaque colonies which
were confirmed as C. albicans by germ-tube formation in bovine serum, chlamy-
dospore production on cornmeal Tween 80 agar, and sucrose assimilation
on agar slants of Wickerham's yeast nitrogen base supplemented with the
sugar.
Staphvlococcus aureus was isolated by streaking on plates of mannitol
salt agar. Mannitol-positive colonies were picked for confirmation by
examination of Gram-stained smears for characteristic morphological groups
of Gram-positive cocci and positive coagulase reaction.
Screening for C. albicans in stool specimens was initiated in September
1980 while the C. ieiuni protocol was added in April 1981. The alkali treatment
coupled with plating on CAL agar was substituted for an existing procedure
in April 1981 for the improved detection of Yersinia enterocolit ica . Prior
to that time, fecal samples were analyzed for Y. enterocolitica by enrichment
at 4°C in isotonic saline containing 25 jig/mL of potassium tellurite with
subsequent plating onto Salmonella-Shigella (SS) agar.
Throat swab specimens (Figure 15) were plated onto 5% sheep blood
agar and MacConkey agar. Incubation of the first medium was at 35°C for
24 hours in an atmosphere of 5% C(>2 to facilitate cultivation of Group
A streptococci. The MacConkey agar plates were incubated at 35°C for 24
hours in normal atmosphere. Representative colonies from each medium were
identified using traditional tests as described in Lennette et al. (1980)
in conjunction with commercially available testing systems. Gram-negative
organisms from MacConkey agar plates were identified using the API-20E
(Analytab) system. Beta-hemolytic streptococci were grouped using the
Phadebact (Pharmacia) coagglutination test. Throat swab specimens also
were screened for Group A streptococci using a fluorescent antibody technique.
70
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Clinical bacteriology monitoring, particularly of illness specimens,
provided the most timely mechanism of surveillance for a possible health
effect associated with irrigation operations. Isolation of a pathogen or
any other cause for concern during periods of scheduled sampling was reported
by telephone to health watch investigators at the University of Illinois
within a week of receipt of the sample. The results of all illness specimens
were reported by telephone within a week of receipt of the specimen. In
addition, an illness specimen log, starting with specimens collected during
DCP 212, was updated on the last Friday of each period and sent to the
University of Illinois and the project manager. This mechanism of surveillance
reporting allowed feedback of results to the participants and collection
of follow-up specimens as appropriate.
Clinical Virology
Appropriate enteric and respiratory viral agents were sought via traditi-
onal diagnostic isolation schemes (as illustrated in Figure 16) coupled
with microidentification techniques. Fecal suspensions were prepared by
adding 10 mL of antibiotic diluent (Medium 199 containing penicillin and
streptomycin) to 1 to 2 g of stool sample. Sterile glass beads were added,
and the mixture was vortex-mixed for 1 minute. After centrifugation (8,000 x g)
for 10 minutes in a refrigerated centrifuge, the supernatant fluid was
recovered for inoculation of primate cells in tube culture. Similarly,
an antibiotic diluent was added to the fluid expressed from the throat
swab into the transport medium. If necessary, throat swab eluates were
centrifuged to remove gross particulates prior to inoculation of cultures.
Cell cultures used were primary rhesus monkey kidney (Flow Laboratories,
McLean, Virginia), human rhabdomyosarcoma (RD), African green monkey kidney
(BGM) and HeLa (pretested for adenovirus sensitivity). A 0.1-mL aliquot
of supernatant or eluate was inoculated into two tubes of each cell line.
Tubes were observed microscopically over a 10- to 14-day period for viral
CPE. HeLa cell tube cultures were frozen and thawed prior to a second blind
passage to enhance detection of adenoviruses.
As a result of quality assurance testing conducted during 1981, it
became obvious that the likelihood of recovering viruses from nonillness
(routine) fecal specimens was low. Beginning with Period 201 sampling,
changes in the clinical assay procedures were made to enhance the sensitivity
of viral isolations from routine fecal specimens. The volume of sample
inoculated into each cell line was increased from 0.2 mL to 1.0 mL by inocu-
lating two 60-mm plates when monolayers reached 50 to 75% confluence (0.5
mL/plate). Primary rhesus monkey kidney cells obtained from a commercial
supplier continued to be used as tube cultures.
The identification and typing of viral isolates from clinical specimens
was performed by microneutralization using the Lim Benyesh-Melnick enterovirus
typing pools (NIAID, 1972; NIAID, 1975). Fluorescein conjugated antisera
specific for adenovirus group antigen was purchased from M.A. Bioproducts
(Walkersville, Maryland). Preliminary testing showed that optimal fluorescence
was obtained by using a 1:5 dilution of the conjugate. Prior to use, the
71
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FECES
Prepare Fecal Suspension by
Adding Antibiotic Diluent to
Sample and Vortex Mixing with
Glass Beads .
Pellet Solids by
Centrifugation (8,000 x g)
Recover Supernatant Fluid
1
THROAT SWAB
J
Express Fluid, Add Antibiotics
As Necessary, Clarify Sample
by Centrifugation (8,000 x g)
Recover Supernatant Fluid
Inoculate Tube Cultures
of Appropriate Primate
Cells, e.g.,
- Primary Rhesus Monkey Kidney
- BGM
- RD
- HeLa (Pretested for Adenovirus
Sensitivity)
4
Observe for Cytopathic Effect
over 10-14 day period
4
Freeze Positive Samples, -76°C
4
Identify Isolates by Serological
Procedures
Figure 16. Viral isolation from clinical specimens
72
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conjugate was centrifuged at 2 z 103 RPM for 10 minutes in an IEC tabletop
centrifuge to remove any particulate contaminants.
Those clinical isolates exhibiting CPE characteristic of adenoviruses
and unidentified by enterovirus microneutralization procedures underwent
fluorescent antibody staining. HeLa cells were grown in 125-mm tissue culture
tubes to about 50% confluence and subsequently were inoculated with 0.1
mL of the virus suspension. The tubes were observed daily for evidence
of CPE. When 75% of the mono layer showed viral involvement, the tube was
vortezed to remove infected cells. In the case of negative controls (uninfected
cells), the cells were scraped off of the glass with a rubber policeman.
The tubes were then centrifuged at 6 z 103 RPM in an IEC centrifuge for
10 minutes. The supernatant fluid was decanted and the pelleted cells were
washed three times with 5 mL of phosphate buffered saline (PBS), pH 7.6.
After the last centrifugation the PBS was carefully decanted and the cell
pellet resuspended in a minimal volume of saline (0.1 mL). The cell suspension
was placed on a microscope slide, allowed to air dry, and fized in cold
acetone (-20°C) for 10 minutes. At this point, slides could be stored at
-70°C to await further processing.
After warming to room temperature, fized cells were covered with 0.05
mL of a 1:5 dilution of the adenovirus-specific fluorescein conjugate.
Slides were incubated in a moist chamber for 30 to 45 minutes followed
by two 10-minute rinses in PBS and a final distilled water wash. Cells
were scored for adenovirns antigen production by visually observing fluorescence
using a Zeiss Model 18 microscope equipped with an epifluorescent illumination
and a fluorescein isothiocyanate (FITC) filter set.
Electron Microscopy of Fecal Specimens
Electron microscopic (EM) ezamination of fecal material has been used
to distinguish an increasing number of morphologically distinct viral agents
which have been associated with gastrointestinal illness. The virus particle
types observed by EM in illness stools include: adenovirus, astrovirus,
calicivirns, coronavirus, Norwalk-like or ''small round structured'' virus,
and rotavirus. Routine cell culture techniques cannot currently be used
to isolate many of these agents and specific immunoassays are only capable
of detecting antigenically related viruses. As these agents are frequently
shed by infected individuals in large numbers (1 g of stool may contain
10^0 rotavirus particles), they are detectable by relatively insensitive
EM procedures. Although dependent on virus type, state of aggregation,
adsorption to grids, background material, and other factors, it was considered
that a suspension titer of approzimately 10*> particles/mL would be required
for detection by EM.
Using a negative staining technique, the USEPA HERL-Cincinnati laboratory
has detected a number of these viral agents in illness stool specimens
by EM. This technique was also used to ezamine approzimately 1/4 of the
stool specimens from the LISS. These specimens, labeled with the donor's
name and code number, were shipped by UTSA to the USEPA laboratory in Cincinnati
at various intervals during the intensive health watch. The specimens
were shipped in glass vials on dry ice, in insulated containers. Shipping
73
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time was generally less than 24 hours and samples were cold upon receipt.
All specimens were stored frozen at -70°C until processed as follows:
1) The fecal specimen was thoroughly mixed with a glass rod or pipette.
2) A small amount was removed and enough distilled water added to
give a slightly turbid suspension.
3) A drop of the turbid suspension was placed on a copper EM grid
(carbon substrate) and allowed to stand 1 minute.
4) Excess sample was removed with filter paper and the grid rinsed
with one or two drops of distilled water.
5) The grid was negatively stained with a drop of 2% phosphotnngstic
acid (PTA), pH 7. The excess stain was removed with filter paper.
6) After drying, the grid was examined at 80 Kv on a JEOL 100CX
transmission electron microscope for the presence of virus particles.
The detection of fecal viruses by EM using the negative staining technique
has previously been described by Flewett (1978) and more recently by Field
(1982).
Specimens yielding a Norwalk-like virus identification were sent to
Dr. N. R. Blacklow's laboratory at the University of Massachusetts for
examination of Norwalk-virus antigen by RIA.
F. LABORATORY. ANALYSIS OF ENVIRONMENTAL SAMPLES
Wastewater Samples
Microbiological screens—
Indicator bacteria—Indicator organisms enumerated include total coliforms,
fecal coliforms, and fecal streptotocci. These bacterial groups were detected
using membrane filtration procedures as specified in Standard Methods for
the Examination of Water and Wastewater. 14th Edition (1975) with the following
exceptions. Based on experiences at other field sites, fecal streptococci
were isolated on M-Enterococcus agar instead of KF Streptococcus agar (Sagik
et al., 1980) Fecal coliform plates were incubated for 3-4 hours at 35°C
to allow resuscitation of injured organisms before overnight incubation
at 44.5°C. Additionally, the standard plate count as outlined in Standard
Methods was used to determine the levels of aerobic and facultatively anaerobic,
heterotrophic bacteria in each sample. Results for all indicator bacteria
represent the mean of triplicate platings.
Other bacteria—
a. Salmonella—Prior to March 23, 1981, Salmonella screening was
accomplished by filtering a measured volume of wastewater through a diatomaceous
earth (DE) plug as described in Standard Methods (1975). Portions of the
DE plug as well as aliquots of wastewater (.<25 mL) were placed in separate
bottles of selenite and tetrathionate broths for enrichment at 35°C. Aliquots
from the broths were streaked for isolated colonies onto brilliant green
agar and incubated at 42°C.
74
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In an attempt to improve detection sensitivity, an alternative
procedure described by Kaper and associates (1977) was tested. As described
above, portions of the DE plug (for volumes >25 mL) and aliquots of wastewater
were placed in dulcitol broth and incubated at room temperature for 4 hours
followed by incubation at 3S°C for an additional 18 to 20 hours. An aliquot
from each primary enrichment volume was transferred into selenite cystine
broth and incubated for 24 hours at 42°C. Subsequent plating was as described
above.
Characteristic colonies were counted and tested for ozidase reac-
tivity. Ozidase-negative organisms were transferred to an appropriate
biochemical test screen: triple sugar iron (TSI) agar and lysine-iron
agar (LIA). Based on these results, presumptive Salmonellae were confirmed
with commercially available polyvalent and group-specific antisera.
As shown by results presented in Table A-17 in Appendix A, the
double enrichment procedure yielded better recoveries of Salmonella from
Lnbbock wastewater. On this basis, this procedure was selected to replace
the standard selenite enrichment technique.
b. Shigella—A portion of a DE plug resulting from filtration of
wastewater as described under procedures for Salmonella along with ^.25-mL
portions of the nnconcentrated wastewater were used for detection of Shigella.
Each of these samples was added to a separate bottle of GN broth. After
18 to 24 hours of enrichment at 35°C, aliquots from the bottles were dilution-
plated onto zylose-lysine-deozycholate (XLD) agar and incubated at 35°C.
Ozidase-negative colonies were inoculated to a biochemical screen utilizing
TSI and motility-indole-ornithine (MIO) medium. Shigella isolates were
confirmed using commercially available polyvalent and group-specific antisera.
c. Staphvlococcus aureus—Aliquots of wastewater were spread-plated
onto mannitol salt agar and incubated at 35°C. Typical colonies showing
a yellow zone of mannitol fermentation were counted and identified by micro-
scopic observation of Gram-positive cocci and by testing for coagulase
activity.
d. Mycobacterium—Hycobacteria were assayed quantitatively by a
procedure which almost totally suppresses sewage saprophytes while permitting
recovery of most mycobacteria. The sample was treated for 20 to 30 minutes
with 500 ppm of benzalkonium chloride (Zephiran), diluted and plated onto
the surface of previously prepared plates of Middlebrook 7H11 agar plus
OADC enrichment modified by the addition of 3 iig/mL of amphotericin B. Plates
were incubated at 37°C in a C02 atmosphere and examined over a period of
1 month for the appearance of typical colonies of mycobacteria. Suspect
colonies were identified by ezamination of stained (Ziehl-Neelsen) smears
for acid-fast bacilli. Additionally, all nonchromogens were subcultured
onto Lowenstein-Jensen tubed medium and subsequently tested for niacin
production, a distinguishing characteristic of M. tuberculosis.
If the density of mycobacteria was low, a concentration procedure
was employed to improve detection sensitivity. A 50-mL volume of Zephiran-
treated samples was centrifuged at approzimately 5,000 z g for 20 minutes.
75
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The supernatant fluid was discarded, the pellet resuspended in 1.0 mL of
phosphate-buffered saline, and this volume plated as described above.
e. Klebsiella—Appropriate aliquots of wastewater were dilution-
plated in triplicate to eosin methylene blue (EHB) agar and incubated at
35°C. Hucoid colonies were counted and tested for an ozidase-negative
reaction. Suspect Klebsiella isolates were identified by typical biochemical
reactions in TSI and MIO media.
f. Yersinia enterocolitica—As the detection of this organism was
inconsistent during baseline monitoring using either enrichment or direct
plate procedures, comparative testing of alternative methods was completed
as described below.
Lubbock wastewater (trickling filter composite) was used unseeded
and seeded with approximately 1 x 10^ cfu/mL of Y. enterocolitica ATCC
23715. The different variables tested included the following:
1) Plating media
a) Salmonella-Shigella agar (SS)
b) HacConkey agar (Mac)
c) Cellobiose arginine lysine agar (CAL)
2) Cold enrichment media
a) 0.067H phosphate-buffered saline, pH 7.6 (PBS)
b) PBS with 1% mannitol, pH 7.3 (PBS-Han)
c) 0.85% NaCl with 25 (ig/mL potassium tellurite (NS-PT)
3) Sampling periods
a) Direct
b) 3 days
c) 7 days
d) 14 days
e) 21 days
4) Treatment of inocula
a) Untreated
b) Potassium hydroxide treatment (KOH-NaCl)
Portions (150 mL) of the unseeded and seeded wastewater were
filtered through separate 1-g DE plugs. One third of each plug was placed
into the respective enrichment medium. The enrichment media were incubated
in a refrigerator at 4°C. The seeded and unseeded wastewaters were sampled
prior to filtration and enrichment, immediately after filtration and placement
into the enrichment media (i.e., ''zero time''), and after cold enrichment
for 3, 7, 14 and 21 days. In each case, inocula for the plating media
were untreated or treated by mixing 20 \iL of sample with 0.1 mL of 0.5%
EOH in 0.5% NaCl just prior to plating. The plates were streaked by the
76
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four quadrant plating method and incubated at 25°C for 48 hours. Characteristic
colonies were identified using the API 20E system.
Results of the comparisons of procedures of recovery of Y. entero-
colitica from the seeded and unseeded samples are shown in Tables A-18
and A-19 in Appendix A, respectively. A semiquantitative index of the
numbers of this organism present was obtained by reporting the highest
quadrant in which the organisms were isolated as discrete colonies. It
was apparent from these results that Y. enterocolitica could readily be
isolated from both the seeded and unseeded wastewater samples.
The cold enrichment medium (NS-PT) previously employed (Sonnen-
wirth, 1974) proved to be markedly inhibitory to the organism in both seeded
and unseeded samples; however, both PBS and PBS-Man yielded Y. enterocol it ica
at the different sampling periods, particularly when the inocula were treated
with ZOH-NaCl. Y. enterocol it ica was recovered from each of the plating
media. However, the greatest percentage of isolates picked that proved
to be Y. enterocolitica by the API 20E were from CAL. Colonies of the
organism were very distinctive on CAL in contrast to Mac and SS agars.
Based on these results, Y. enterocolitica was detected by the
following enrichment procedure beginning with samples collected on March
23-24, 1981. A measured amount of wastewater was filtered through a 1-g
DE plug which was subsequently dispersed in PBS (50 mL). A volume was
removed for plating at this time and after 3 days of incubation at 4°C.
Plating volumes were treated with KOH-NaCl and plated onto CAL agar. Typical
colonies were isolated after 48 hours of incubation at room temperature
(22 to 25°C) and identified using API 20E and oxidase tests.
g. Clostridium perfringens—An MPN procedure was used to enumerate
both vegetative and sporulated Clostridia. Prior to analysis, a portion
of the wastewater was heated at 80°C for 30 minutes. Both this heat-shocked
and the untreated sample were diluted appropriately in PBS and inoculated
into three tubes of differential reinforced clostridia medium (DRCM) at
each dilution. Following incubation at 35°C for 72 hours, a loopful of
sample from each DRCM tube was transferred to litmus milk and subsequently
examined for typical stormy fermentation to confirm the presence of C. per-
fringens. Organism densities were computed from the BIPN tables in Standard
Methods (1975).
An alternate membrane filtration (MF) procedure for the enumeration
of C. perfringens as described by Bisson and Cabelli (1979) was evaluated
in parallel with the MPN procedure described above. A volume of wastewater
was filtered through a 0.45-ji membrane filter (Gelman 6C-6) which was placed
onto mCP agar containing cycloserine and polymyxin B sulfate as inhibitory
agents. Plates were incubated anaerobically in the BBL Gas Pak system
at 45°C for 18 to 24 hours. Sucrose positive, cellobiose negative (yellow
colored) colonies were counted and tested for positive reactions for acid
phosphatase and gelatinase. Further confirmation involved subculture to
litmus milk with stormy fermentation followed by testing for lactose, mannose
and sucrose (with gas production) fermentation and nonfermentation of cello-
77
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biose, mannitol and salicin. Additionally, Gram-positive rods were visualized
from litmus milk cultures.
Results of parallel testing are presented in Table A-20 in Appendix
A. The multiple tube technique detected a higher level of vegetative C. per-
frineens (nonheated sample) than the MF method in all of the samples analyzed.
The MF method detected a higher level of sporulative C. perfringens (heated-
treated sample) on two of four samples. This result could be attributed
to the milder heat treatment process used in the MF method. Perhaps more
importantly, the confirmation of C. perfringens by visualization of Gram-
positive, nonmotile rods was nearly equivalent for both procedures.
Due to the nature of the MF technique, this procedure was used
when larger volumes of samples were processed. Specifically, this MF technique
was applied to the recovery of C. perfringens from selected aerosol samples
during 1982. It should be noted, however, that results from the MPN and
MF procedures should not be directly compared.
h. Campylobacter jejuni—Beginning with samples collectd in July
1981, an assay to allow the detection of C. jejuni in wastewater was included
in the microbiological screen. Aliquots of wastewater were spread onto
the surface of Campy-BAP agar plates supplied by San Antonio Biological
Company. This medium consisted of brucella agar base with 5% sheep erythrocytes
and vancomycin (10 mg/L), trimethoprim (5 mg/L), polymizin B (2500 I.U./L),
amphotericin B (2 mg/L), and cephalothin (15 mg/L). Plates were incubated
in a microaerophilic environment (Campy-Pakll) for 48 hours at 37°C. Suspect
colonies were subcultured to 5% sheep blood agar, incubated as before,
and nonhemolytic reactions typical of C. jejuni were noted. Further tests
for this organism included catalase production, ozidase production, growth
in 1% glycine, lack of growth in 3.5% NaCl, sensitivity to nalidizic acid
(30 ug disk) and darting motility as observed microscopically in wet mounts.
i. Candida albicans—Testing for this organism was initiated as
part of wastewater screens in July 1981. Appropriate dilutions of wastewater
were spread onto Sabouraud dextrose agar (SDA) supplemented with 200 ng/mL
chloramphenicol. Plates were incubated at 37°C for 48 hours. Suspect
colonies were subcnltured onto SDA prior to confirmatory testing which
consisted of positive germ tube formation in bovine serum, positive chlamy-
dospore production on cornmeal-Tween 80 agar, and assimilation of sucrose
as the sole carbon source.
j. Fluorescent Pseudomonas sp—Aliquots of wastewater were spread-plated
onto Cetrimide agar (DIFCO) and incubated at 35°C for 24 hours. Plates
were then moved to room temperature for an additional 20-24 hours. Fluorescent
colonies were counted while viewing plates under long-wave ultraviolet
light using a Chromato-Vue cabinet (Ultra-Violet Products, Inc; San Gabriel,
California).
k. Gram-negative enteric bacteria—Both oxidase-negative and oxidase-
positive enteric bacteria including all members of the family Enterobacteriaceae
were sought using the screening procedures diagrammed in Figure 17. Wastewater
samples were diluted appropriately in sterile phosphate-buffered saline
78
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Wastewater
Direct Plating on Selective
Medium, e.g.,
- MacConkey Agar
Counting, Subculture
Ozidase Test
API 20E
Identification by Profile Index
Figure 17. Isolation of Gram-negative enteric
bacteria from wastewater
and spread over three plates per dilution on MacConkey agar. After incubation
at 35°C for 24 hours, all colonies were counted and isolated at a dilution
yielding a total of approximately 100 colonies over three plates. Discrete
colonies were streaked onto quadrants of heart infusion agar plates to
allow growth and confirmation of purity.
Subsequent identification involved oxidase testing and the use
of API 20E identification strips. The API 20E system consists of a preset
battery of 20 microtubes which allows the performance of 22 biochemical
tests for the identification of 49 species/subspecies of Enterobacteriaceae
and 38 group/species of other Gram-negative bacteria.
Bacteriophaees—Coliphages indigenous to wastewater were assayed as
plaque-forming units (pfu) using Escherichia coli K13 as the host organism.
Tests in this laboratory have shown strain K13 to yield the highest coliphage
titers when compared to other E. coli hosts. Appropriate volumes (0.1,
0.5, or 1.0 mL) of the wastewater and 0.5 mL of overnight culture of host
cells were added to 3.5 mL of liquefied tryptose-phosphate soft agar and
poured while warm (45°C) onto 100-mm petri dishes prepared with 10 mL of
solidified tryptose-phosphate agar base layer. When firm, the plates were
79
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inverted and incubated at 35°C for approximately 18 hours prior to counting.
For each sample, a minimum of five plates was used.
Human enteric viruses—During 1980, two concentration techniques were
used in parallel for the recovery of human enteric viruses from wastewater
samples. Both bentonite adsorption and organic flocculation were used to
concentrate indigenous viruses from the five effluent samples. This approach
was deemed necessary due to the nature of the wastewater entering the Lubbock
treatment plant, i.e., both industrial and domestic wastes.
Positive viral recoveries were made consistently from the bentonite
concentrates, while parallel assays of the organic flocculation concentrates
were less successful due to toxicity and contamination. The standard bentonite
concentration procedure performed adequately on both Lnbbock and Wilson
wastewater effluents. Viral concentration efficiencies based on the recovery
of poliovirus 1 (Chat) were relatively consistent with a mean of 67 ± 26%
for Lubbock wastewater (14 samples) and 58 + 16% for Wilson effluent (14
samples) collected during 1980 and 1981. Concentrated volumes were suitable
for both plaque and tube culture assay.
In addition, the bentonite adsorption technique has isolated a wide
spectrum of enteroviruses as shown in Table A-21 in Appendix A. It should
be noted, however, that this concentration technique was not expected to
recover either reoviruses or adenoviruses.
Based on these observations, the bentonite adsorption procedure as
described below was used as the sole viral concentration technique for
wastewater effluents.
For detection of human enteric viruses, a maximum of 4 L of treated
wastewater was concentrated in the laboratory using a standard bentonite
adsorption technique (Moore et al., 1979). Briefly, wastewater was placed
in a vessel of convenient size and 100 mg/L of expanded bentonite was added
along with sufficient CaCl2 to bring the wastewater to approximately 0.01
M. The pH of the sample was adjusted to 6.0 with HC1, and it was mixed
for 30 minutes. After mixing, the virus-solids-bentonite complex was sedimented
by low speed centrifugation. Tryptose-phosphate broth (TPB) was added to
the pellet to facilitate viral elution at a ratio of 10 to 15 mL of TPB
per liter of sample concentrated. Elution was accomplished by sonicating
the TPB-solids-virus suspension for 5 minutes in an ice bath. The suspension
was separated by centrifugation (8,000 x g), and the supernatant fluid
containing the eluted virions was held at -76°C for assay.
Indigenous enteric viruses were enumerated by plaque assay on selected
cell monolayers. Testing conducted as part of the wastewater pathogen screens
during 1980 led to the selection of HeLa and RD cell lines for viral recovery
from environmental samples. Data presented in Tables A-21 and in A-22
in Appendix A substantiate the choice of these cells in a complementary
assay system. In this laboratory HeLa cells recover the greatest variety
of enteric viruses. During baseline monitoring, the RD cell line showed
a preferential recovery of echoviruses, even in the presence of polioviruses
and Coxsackieviruses as evidenced by results from Lnbbock-1 and Lnbbock-2
80
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samples (see Table A-22 in Appendix A). Additional testing has shown that
echoviruses can be isolated as plaques on the RD cell line. To further
enhance the recovery of a broad spectrum of enteroviruses, a portion of
each concentrated volume was neutralized for all three poliovirus serotypes
prior to the assay to avoid overgrowth and interference.
Beginning in January 1981, the assay matrix shown in Table A-23 in
Appendix A was used. To optimize the use of neutralizing antisera, total
enteroviruses were assayed first on He La monolayers. Based on the results
of this analysis, subsequent assays using poliovirus antisera were completed.
At the time of inoculation each series of ten (or five) plates was
assigned a number (1 through 5 or 10, as appropriate). A random ranking
of numbers was created for each assay system by lottery draw. The numbers
were recorded on the assay sheet in the order in which they were pulled.
After the appropriate incubation period, pfu were counted on those plates
yielding countable plaques. Plaques were picked for confirmation and storage
from plates at the dilution which allowed the best separation of pfu and
reflected the viral level to be reported. Selection of pfu from plates
followed the previously recorded order. Thus, if the ranked order of RD
plates (undiluted sample) was 3, 1, 8, 2, etc., all plaques on plate 3
were picked followed by plates 1, 8, etc., until the desired maximum number
of pfu were acquired. If one plate was unacceptable due to overlap of pfu
or contamination, the next listed plate was used. The following guidelines
were followed in picking plaques for confirmation and possible future identi-
fication: 25 pfu from the unaltered HeLa assay and IS pfu from each assay
of polio-neutralized sample on HeLa and RD cells. In those cases when fewer
than the specified number of viral plaques were evident, all pfu were picked.
All pfu were confirmed by passage in the homologous cell line, logged,
and frozen at -76°C if viral identification was indicated.
Poliovirus neutralization was done using commercially available rabbit
antisera (H.A. Bioproducts). During 1981 the commercial supply of specific
poliovirus antisera was discontinued. Subsequently, lyophilized monkey
or equine sera were obtained from the National Institutes of Health for
use in the poliovirus neutralization assays. Each lot of antisera was used
at a level which had previously demonstrated at least a 3.0 log^Q plaque
reduction of homologous laboratory strains of poliovirus. Representative
data showing poliovirus neutralization by monospecific antiserum is presented
in Table A-24 in Appendix A. Sample and diluted antisera against polio
1, 2 and 3 were mixed, incubated in a 37°C water bath for 30 minutes, and
plated.
The generalized procedure for plaque assay consisted of inoculating
confluent cell monolayers grown in 100-mm plates with 1.0 mL of sample.
After a 60-minute infection period, monolayers were overlaid with an agar-based
Eagle's minimal essential medium containing bovine serum and antibiotics.
Infected plates were held at 37°C in a 5% CC>2 humidified incubator. Two
to three days post-infection, a second overlay containing 30 iig/mL of neutral
red was placed on each plate. Plates were read on each succeeding day
and scored for plaques through 5 days.
81
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Possible viral isolates were picked from areas exhibiting character-
istic cytopathic effect (CPE) based on microscopic examination of the stained
monolayer. The removal of plaque-like areas was accomplished by first removing
the second overlay above the area of CPE. Agar overlaying the entire plaque
was aseptically collected using a microspatula. The sample was placed in
0.5 mL of medium 199 containing antibiotics and held at -76°C until confirma-
tion.
Confirmation of potential viral isolates was performed in homologous
tube culture systems. Culture tubes were grown out to 50 to 75% confluence
and inoculated with 0.2 mL of sample. After 48 hours of incubation at
37°C, tubes were observed daily for evidence of CPE. When characteristic
CPE was observed, the sample was removed and frozen at -76°C for viral
identification. After 7 days, all samples not showing CPE were harvested
and blind-passaged. Those isolates that demonstrated CPE after a second
passage also were also reported as viruses (pfu).
Viral isolates were identified using the Lim Benyesh-Helnick pools
for typing enteroviruses (Pools A-H and J-P) in a microneutralization procedure.
Physical-chemical analysis—Total suspended solids (TSS), total volatile
suspended solids (TVSS), and total organic carbon (TOO were analyzed following
procedures outlined in Standard Methods (1975). Values reported are the
mean of triplicate analysis for each parameter.
Routine wastewater samples—
Routine wastewater samples were intended to allow a determination
of potential exposure of the study population when the wastewater was used
in irrigation. Samples were cooled to 4°C in wet ice and shipped to DTSA
at that temperature for analysis.
The routine wastewater samples were analyzed for total and fecal coliforms,
coliphage, fecal streptococci, mycobacteria, enteric viruses, TSS, TVSS,
and TOC. Analytical procedures were those described above under ''Microbio-
logical Screens.''
Enterovirus Identification Samples—
Composite samples were collected from the Lubbock treatment plant
trickling filter effluent or from effluent from the pipeline at the irrigation
site (when available) and from the Wilson Imhoff tank effluent. Samples
were cooled to 4°C and shipped to UTSA/UTA. The enterovirus identification
samples were analyzed for human enteric viruses, fecal coliform, TSS, TVSS,
and TOC following the procedures described above under ''Microbiological
Screens.'' Plaques were picked, confirmed and up to 50 viral isolates
per sample were frozen at -76°C for future identification. Within the
limits of the assay systems employed, the analysis of these samples allowed
the determination of enterovirus types present in the sprayed wastewater
and circulating within the Wilson population.
Limited Bacterial Screen Samples-
Composite samples of Lubbock trickling filter effluent (or when available
pipeline flow) were collected and shipped to DTSA as part of the enterovirus
82
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identification samples described above. In addition to physical-chemical
analyses, the following potential microbiological pathogens were sought
using procedures described under ''Microbiological Screens'': Salmonella.
Shieella. Yersinia. Staphvlococcus aureus. and Klebsiella-like organisms.
On March 23, 1981, both Campylobacter ie iuni and Candida albicans were
added to this list of pathogenic organisms following methods described
above. Beginning June 29, 1982, fluorescent Pseudomonas sp. was substituted
for S. aureus. As part of an effort to characterize Wilson wastewater,
the same limited bacterial evaluation screen covering these seven organisms
was initiated on Imhoff tank effluent beginning in July 1981. The occurrence
of selected organisms with human pahtogenic potential in wastewater destined
for irrigation can thus be documented.
Legionella Samples—
Wastewater from the Lubbock sewage treatment plant was piped to three
reservoirs located on the Hancock site and used for spray irrigation either
directly or from these reservoirs. A total of nine separate wastewater
samples were received by the University of Illinois during 1982. Five
of these samples (one trickling filter effluent sample from March; three
pipeline effluent samples from February, March and June; and one reservoir
sample from June) were processed and inoculated into guinea pigs. Two
samples (pipeline effluent and reservoir samples from July) were examined
by direct fluorescent antibody (DFA) techniques for Legionella antigen.
The two remaining samples (both reservoir samples from August) were not
tested.
Complete testing for Legionella-group agents involved tenfold concentration
of wastewater samples by centrifugation. Aliquots of the samples were
then examined by DFA using available conjugates and diluted (serial tenfold)
for total bacterial counts using standard methods. The purpose of this
latter step was to avoid ''overloading'' guinea pigs with more than 10^
to 10*7 non-Legione 1 la organisms and it was anticipated that samples would
be diluted to this level. However, this concentration was generally found
either in the tenfold concentrated or unconcentrated samples, making further
dilution unnecessary. Guinea pigs were inoculated intraperitoneally with
1.0 mL of samples. Samples seeded with a standard amount of virulent L.
pneumophila 1 were included as controls. Guinea pigs were observed daily
and rectal temperatures recorded. Animals having a fever for two consecutive
days were euthanized. A fever was defined as a 0.5°C increase in rectal
temperature above preinoculation values. Since animals inoculated with
this type of material would be expected to develop fevers unrelated to
Legionella infection after inoculation, fever 3 days postinoculation was
taken as a possible indication of a Legionella infection. All animals
were euthanized on the seventh day postinoculation and were autopsied within
hours of euthanization or dying. Sterile techniques were used to collect
peritoneal exudates and spleens. Samples of these fluids or tissues were
examined by DFA for Legionella and were inoculated onto a variety of non-
selective and semiselective agar media. Potential Legionella colonies
were passed on charcoal-yeast extract (CYE) agar. Second passage material
was inoculated onto trypticase soy agar (TSA) plates. CYE colonies failing
to grow on TSA were considered possible evidence of Legionella.
83
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A number of attempts were made to isolate Legionella directly from
wastewater samples. These included inoculation of samples onto plates
of the semiselective medium BMPAo (Edelstein, 1981) which contained cefamandole,
polymyxin B, anisomycin, an organic buffer, and o-ketoglutarate and pretreatment
of samples with an acid buffer (pH 2.2) as described by Bopp and associates
(1981) followed by inoculation onto BMPAa.
Aerosol Samples
The composite samples of sprayed wastewater taken during the microorganism
aerosol runs were analyzed for the same microorganism groups and water
quality measurements as the routine wastewater samples. The aerosol sampler
fluids from the microorganism aerosol runs and background runs and the
aerosol and wastewater samples from the quality assurance runs were assayed
for fecal coliforms, coliphage, fecal streptococci, and mycobacteria or
Clostridium perfringens. Assays for human enteric viruses were conducted
on the wastewater and aerosol samples from the enterovirus runs. Procedures
for the indicator bacteria, mycobacteria, C. perfringens, coliphages and
human enteric viruses are described in ''Microbiological Screens.''
The aerosol concentration procedures for human enteric viruses described
by Moore et al. (1979) was developed to be performed at a field site.
Due to the relative proximity of the Wilson site and the reduced interval
between sample collection and arrival at the laboratory, organic flocculation
was evaluated as an alternate concentration procedure. It was considered
probable that this procedure might provide higher viral recoveries.
Three enteric viruses were used in the procedure de.velopement and
comparison testing: poliovirus 1, coxsackievirus B3 and echovirus 6.
These viruses were differentiated by using two cell lines and monospecific
antiserum in the following combinations. To determine poliovirus 1 titers
the sample was neutralized for coxsackievirus B3 and assayed on HeLa cells.
Coxsackievirus B3 and echovirus 6 were assayed from samples treated with
poliovirus 1 antisera and titrated on HeLa and RD cells, respectively (echovirus
6 will not plaque on HeLa cells; likewise coxsackievirus B3 will not plaque
on RD cells). This assay scheme allowed all three viruses to be detected
in one sample.
Typically, organic flocculation is performed by adding organics (beef
extract) to a sample. These organics are precipitated out of solution
when the pH is lowered to approximately 3.5. Virions are entrapped in
the organic floe and removed by centrifngation. The amount of organics
present in a solution frequently dictates viral recovery rates; therefore,
experiments were performed to determine the optimal amount of beef extract
that should be added to the sampler fluid (BHI + 0.1% Tween 80).
Poliovirus 1, coxsackievirus B3 and echovirus 6 were added to three
liters of BHI + 0.1% Tween 80 to give a final concentration of approximately
10 to 100 pfu/mL and mixed for 15 minutes. Ten mL of the sample were removed
to establish actual input titers and the remaining sample was divided into
500-mL test volumes. Beef extract was added, resulting in final concentrations
of 0%, 1%, 2% and 3%. The pH of each aliquot was adjusted to 3.5 by the
84
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dropwise addition of IN HC1. The samples then were mixed for 30 minutes
and centrifuged for 10 minutes at 8000 x g. After the supernatant fluid
was decanted, each pellet was resuspended in 10 mL of 0.15M N82HP04 (pH
9.0), and subsequently the pH was adjusted to 7.0. The final volume was
measured and the sample assayed as previously described. For comparative
testing, a 500-mL aliquot of seeded sampler fluid was concentrated by two-phase
separation as described by Hoore et al. (1979).
Results shown in Table A-25 in Appendix A demonstrate that the addition
of 2% beef extract provided optimal recovery when compared to the other
beef extract concentrations evaluated. Organic floccnlation using 2% beef
extract also consistently outperformed two-phase separation, especially
in the recovery of echovirus 6. Therefore, the following protocol was
adopted for the detection of viruses in aerosols.
The total volume of BHI + 0.1% Tween 80 from an aerosol run was measured
and 100 mL of the sample removed for routine organism determinations.
The amount of beef extract added to the sample was calculated on the basis
of total volume minus 100 mL. The beef extract was added to a final concen-
tration of 2% and mixed until the beef extract went into solution. The
pH of the sample was then lowered to 3.5 with IN HC1. After 30 minutes
of mixing the organic floe was recovered by centrifugation at 8000 x g
for 10 minutes. The pellet was resnspended in 140 mL of 0.15M Na2HP04
(pH 9.0). The pH of the final elnate was adjusted to 7 and subsequently
split into two equal portions, one to be assayed on HeLa cells and the
other on RD cells. Prior to being assayed, the sample was treated with
chloroform to reduce bacterial and fungal contamination.
Plaque assay conditions and viral confirmation and identification
utilized the protocols described under ''Microbiological Screens.''
Fly Samples
An effort was made to isolate enteric bacteria and viruses from houseflies
trapped at the farmhouses and at the effluent ponds. The insects were
processed as outlined in Figure 18. The clinical bacteriology and virology
procedures described previously were followed.
Drinking Water Samples
Indicator Bacteria—
Total coliforms, fecal coliforms, and fecal streptococci were enumerated
using membrane filtration techniques described in Microbiological Methods
for Monitoring the Environment (USEPA, 1978). Total coliform bacteria
were assayed on M-Endo agar; fecal coliform, on absorbent pads saturated
with M-FC broth; and fecal streptococci, on KF streptococcus agar.
85
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Horseflies
C02, Packaged; Shipped by Air
- Add 10 mL diluent per 1 g flies
- Homogenize in tissue grinder
I
Bacterial Analysis
Streak plate through clinical
bacterial isolation scheme
(see Figure 14, feces)
1
Viral Analysis
Centrifuge at 8000 x g for
10 min, recover supernatant
fluid
Inoculate through clinical
viral isolation scheme
(see Figure 16)
Figure 18 Analyses of insect vectors
Salmonella—
The presence of Salmonella was determined following procedures described
in Microbiological Methods (1978) and Eaper et al. (1977). Organisms were
recovered by filtering sample aliquots through a membrane filter which
was subsequently incubated in 50 mL of dulcitol broth enrichment medium
for 4 hours at 25°C, followed by 20 hours at 35°C. One mL of this enrichment
medium then was transferred to selective selenite cystine broth and incubated
at 41.5°C for 24 hours. Aliquots from these selenite cultures were streaked
-------
onto brilliant green agar. Salmone1 la colonies, which appeared pink to
white and opaque surrounded by a brilliant red zone were subcnltured to
BHI agar. A dense suspension of bacterial growth was prepared in phenolized
saline on a slide. A drop of polyvalent (A-I) Salmonella antiserum was
added to the cell suspension. Rapid cell agglutination was scored as a
positive response for detection of Salmonella.
6. INFECTION EVENTS AND EPISODES
Bacterial Infection Event
A fecal donor was considered to be having a bacterial infection when
an overt or opportunistic bacterial pathogen was isolated from a fecal
specimen at or exceeding a specified semiquantitative level which might
be associated with enteric disease. The levels equated with bacterial
infection were:
Category 1 any isolate of a major enteric bacterial pathogen (i.e.,
Salmonella or Shigella species. Campylobacter jejuni. or
Yersinia enterocolitica);
Category 2 isolation at the heavy level of a possibly significant oppor-
tunistic pathogen (i.e., API Group I, Candida alb ican s.
Chromobacterium. Citrobacter. Klebsiella. Morganella, Proteus.
Providencia, Serra_tjLa_, and Staphylococcus aureus);
Category 3 isolation at the moderate or heavy level of selected organisms
found to be uncommon in feces but prominent in the sprayed
wastewater (i.e., Ajj£pjnp_na_s hydrpphila and the fluorescent
Pseudomonas group: P. aeruginosa, P. fluorescens. and P. pu-
tida).
The infected donor was considered to have had an infection event since
donation of the prior fecal specimen in the series when the level of the
organism in the prior specimen had been:
1) negative, for major enteric pathogens,
2) negative to light, for possibly significant opportunistic pathogens,
3) negative to light, for organisms prominent in the wastewater.
These criteria for a bacterial infection and for a bacterial infection
event are summarized for all three bacterial pathogen categories in Table
10.
It was of primary interest to determine the bacterial infection status
of a routine fecal specimen donor in relation to a period of irrigation.
Routine specimens were collected from designated donors in scheduled weeks
before, during and near the end of each irrigation period (see Figure 2),
usually at intervals of about 6 and 4 weeks, respectively. Thus, the onsets
of bacterial infection events could be temporally related to wastewater
irrigation periods. When the change in infection status occurred between
87
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TABLE 10. BACTERIAL INFECTION CRITERIA
Infection
Agent Donor infected event
(FROM) > (TO)
Overt Pathogen* + (E,VL,L,M,H) - > +
Salmonella
Shigella
Yersinia enterocolitica
Campylobacter jejuni
Possibly Significant
Opportunistic Pathogens H -.E.VL.L > H
API Group I
Candida albicans
Chromobacterium
Citrobacter
Klebsiella
Morganella
Proteus
Providencia
Serratia
Staphylococcus aureus
Opportunistic Pathogen* UneeMon in
Fee** but Prominent in Wastevater M.H -,E,VL,L > M,H
Aeromonas hydrophila
Fluorescent Pseudomonas
Semiquantitative Levels:
- - negative
E - enrichment
VL - very light (1-10 colonies on plate)
L - light (growth in first quadrant)
H - moderate (growth on first 2 quadrants)
H - heavy (growth on 3 or 4 quadrants)
88
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the two specimens donated during an irrigation period, onset occurred in
the interim (i.e., during the irrigation period). When the change in infection
status occurred in consecutive specimens donated before and during the
irrigation period, it was uncertain whether onset occurred after irrigation
commenced. When a bacterial agent was not recovered at a level equated
with infection in either routine fecal specimen provided during an irrigation
period, the donor was considered to have experienced no infection events
by the agent during the observation period preceding and spanning the collection
dates of the consecutive specimens.
Viral Infection Event
A viral infection event was defined as the detection of a specific
virus by laboratory cultivation or by EH examination in the second and
not the first of paired fecal specimens from the same person. Subsequent
recovery of the same virus in a specimen from the same individual would
be a new event if more than 6 weeks elapsed between sequential recoveries.
Detection of a virus in the first of serial specimens was also considered
a viral infection event. Viral infection status was correlated with an
irrigation period in the same manner as bacterial infection status.
Serological InfectionJEyent (Serological Conversion)
A serological conversion (''seroconversion'') was defined as a fourfold
or greater rise in agent-specific antibody titer in successive sera from
one individual that were tested simultaneously. Since successive sera
from 1982 and 1983 spanned an irrigation period and several additional
months (see Figure 2), it was not possible to determine if the onset of
serologically detected infection events was during the irrigation period.
Identification of Infection Episodes
An infection episode was defined as the observation in the study popu-
lation of a number of similar infection events (either serologically, micro-
biologically, or clinically) within a restricted interval of time. The
minimum number of infections which constituted an infection episode was
set by determining the number of infections that would be needed to reject
the null hypothesis (of no association between infection status and wastewater
exposure), assuming that all of the infections occurred in the high exposure
group and no infections occurred in the low exposure group. Infection
episodes were classified as exposure situations when the observation period
corresponded to one or two major irrigation periods and when the causative
agent was found (or could be presumed) to be present in the wastewater
at that time. Infection episodes were classified as control situations
when the causative agent could not survive in wastewater (i.e., influenza
A) or when the episode preceded the start of irrigation. Each exposure
and control infection episode was statistically analyzed for association
with wastewater aerosol exposure.
To express these ideas more precisely, consider a specified set of
similar agents whose infection events were to be analyzed as a group.
Also consider a specified time interval over which the infection events
89
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were observed (an interval which usually spanned a single irrigation season).
The infection status of each monitored specimen donor (i.e., whether newly
infected or not infected by any agent in the group) was observed over the
specified time interval. Denote by £2 the number of infection events in
the high exposure group of size TO.2 due to a given agent (group) and let
X^ be the number of infection events due to the same agent (group) in the
low exposure group. A ''high'' rate of infections is said to occur when
a sufficient number of infection events (X^ + X2 _> bo) were detected in
the entire monitored study population. The number bo was chosen so if
all these infection events had occurred in the high exposure group and
none in the low exposure group, the appropriate statistical test would
reject the null hypothesis of no association between infection status and
wastewater exposure. The critical number bo of infection events in the
study population sufficient to constitute an infection episode is given
in Table 11 for realistic values of n^ and n2 for the fecal donor sample
(n=100) and for the blood donor sample (n=300). A significance level o=0.05
was chosen if the agent(s) were recovered from the sprayed wastewater during
the specified irrigation season (or could be inferred from the available
wastewater data to have been present, with likelihood exceeding 0.95).
A significance level a=0.01 was chosen if the agent(s) were not recovered
from the wastewater sprayed at that time.
TABLE 11. NUMBER OF CASES (bo) REQUIRED FOR REJECTION OF Pi=P2 IN
FAVOR OF Pi
-------
for agents detected in the blood donor population but not recovered
in the sprayed wastewater.
TABLE 12. INFECTION EPISODE CRITERIA
Subpopulation
Blood donor (n^SOO)
Fecal donor (n«100)
Agent recovered from Required number of
sprayed wastewater? a infection events
Yes
No
Yes
No
0.05
0.01
0.05
0.01
>3
15
>3
>4
The periods of observation of infection episodes were chosen to coincide
as closely as possible with the major irrigation periods:
Period of observation
Irrigation Period of Fecal specimen Paired
season irrigation seri.es sera
1. Spring 1982 2-16 to 4-30-82 1-4 to 4-2-82 1-4 to 6-9-82
2. Summer 1982 7-21 to 9-17-82 6-7 to 9-17-82 6-7 to 12-10-82
3. Spring 1983 2-15 to 4-30-83 1-31 to 4-22-83 12-6-82 to 6-10-83
4. Summer 1983 6-29 to 9-20-83 6-6 to 8-19-83 6-6 to 10-13-83
5. 1982 2-16 to 4-30 and 1-4 to 12-10-82
7-21 to 9-17-82
6. 1983 2-15 to 4-30 and 12-6-82 to 10-13-83
6-29 to 9-20-83
Periods of serological observation which spanned the entire 1982 irrigation
period (i.e., Jan 4-Dec 10, 1982) and the entire 1983 irrigation period
(i.e., Dec 6, 1982-Oct 13, 1983) were employed to utilize serologic infection
events whose time of occurrence could be ascribed to an annual period but
not to a semiannual period. Baseline infection episodes occurring before
irrigation commenced were also defined and analyzed with respect to the
subsequent spring 1982 exposure grouping in order to investigate unmeasured
potential risk factors which might be associated with the wastewater exposure
measure in the study population and hence produce spurious associations
with exposure in the infection episodes after irrigation commenced.
Infection episodes were defined for specific single agents whenever
sufficient infection events to the agent occurred, as indicated in Table
12. Infection episodes were also defined to interpretable groups of specific
agents when the infection events were scattered among the agents in the
group.
The dependent variable defined for each observed participant in every
infection episode was the number of infection events to the agent (or agent
group) detected in the participant during the period of observation. A
participant was seldom observed to experience more than one infection event
91
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to the agent (group) during the observation period of an infection episode,
except in the serologic infection episodes to grouped agents over a 1-year
observation period. To permit use of sensitive statistical methods requiring
that the dependent variable only assume the values 0 or 1, all multiple
infection events were treated as single infection events in most statistical
analyses performed.
The convention used to construct the names of the dependent variables
of all observed infection episodes is presented in Table 13. The dependent
variable name is used throughout Sections 5 and 6 of this report to specify
the infection episode when descriptions, statistical results and findings
regarding the episode are presented.
The clinical (C) bacterial and viral agents and agent groups for which
infection episodes were identified from series of monthly routine fecal
specimens were:
KLB Klebsiella
OOB Other possibly significant Opportunistic Bacteria (all Category
2 opportunistic bacterial pathogens except Klebsiella)
PBW Prominent Bacteria in Wastewater (Category 3 organisms which
were uncommon in feces but prominent in the sprayed wastewater:
Aeromonas hydrophi1a and the fluorescent Pseudomonas group)
VIR all VIRal isolates (excluding adenoviruses and immunization-associated
polioviruses). Adenovirus shedding is sporadic and may represent
a prolonged latent infection. Poliovirus excretion following
immunization is presumably not wastewater associated.
WWI all WasteWater Isolates (all clinical isolates recovered from
the sprayed wastewater during the irrigation period under observation)
For the all wastewater isolate (WWI) infection episodes, each bacterial
and viral pathogen was listed that was isolated from any pipeline or reservoir
wastewater sample taken during the irrigation period. The agents from
the list which were also recovered from clinical specimens during the same
irrigation season are presented in Table 14.
When the pair of fecal specimens from which a clinical infection event
was identified were both obtained between the start and finish of an irrigation
period, the onset of the infection event was clearly during the irrigation
period. However, when the first fecal specimen of the pair preceded the
start of the irrigation period, the infection event onset may have preceded
irrigation (and hence been unrelated to wastewater). Thus, whenever there
were sufficient infection events, a dependent variable was defined and
the statistical analysis was performed both excluding (X variable, see
position 6 in Table 13) and including (W variable) the fecal donors whose
infection event onset may have preceded the irrigation period. In the
statistical analysis, the newly infected donors were contrasted with fecal
donors who were not infected by the agent (group) over the whole period
of observation of the infection episode. A list of the clinical infection
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TABLE 13. INFECTION EPISODE DEPENDENT VARIABLE* NAME KEY
Position
Information
Values and interpretation
2-4
Method of detecting
infections
Agent (group)
C clinical (bacteriologic or virologic)
analysis of routine fecal specimens
S serologic analysis of blood specimens
Clinical agent cronps
KLB Klebsiella
OOB other (non-Klebsiella) opportunistic
bacteria
PBW prominent bacteria in wastewater
WWI all isolates from wastewater
VIR all viruses (excluding adeno and immuni-
zation polio)
Serologic agent groups
ADS adeno 3
ADS adeno 5
AD7 adeno 7
CB2 cozsackie B2
CB4 cozsackie B4
CBS cozsackie B5
E01 echo 1
E03 echo 3
EOS echo S
E09 echo 9
Ell echo 11
E17 echo 17
E19 echo 19
E20 echo 20
E24 echo 24
PL1 polio 1
PL2 polio 2
PL3 polio 3
SNV all serum neutralization-tested viruses,
ezcept polioviruses
FOR sporadic serum neutralization viruses
(too few to be a distinct infection
episode)
all viruses recovered from wastewater
wwv
RE1
RE2
ROT
LEG
INA
reo 1
reo 2
rotavirns
Legionella
influenza a
continued...
93
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TABLE 13 (CONT'D)
Position
Information
Values and interpretation
Period of
observation
6 (clinical
only)
0 baseline
1 Spring 1982
2 Summer 1982
3 Spring 1983
4 Summer 1983
5 1982
6 1983
7-9 nonstandard periods
X onset of all infection events during
irrigation period
W includes infection events whose onset
may have preceded the irrigation period
Value of each dependent variable = number of infection events to agent
(Pos. 2-4) observed in participant by method (Pos. 1) in time interval
(Pos. 5). The values of each dependent variable for each observed par-
ticipant was collapsed to 0 = not infected or 1 = newly infected for
all statistical analyses (i.e., multiple infection events were ignored).
TABLE 14. AGENTS COMPRISING CLINICAL WWI EPISODE BT SEASON:
WASTEWATER ISOLATES RECOVERED* IN ROUTINE FECAL SPECIMENS
DURING SAME IRRIGATION PERIOD
Agent
Klebsiella pneumoniae
Klebsiella ozytoca
Aeromonas hydrophila
Fluorescent Pseudomonas group
Number
bv
CWWI1W
Spring
1982
2
3
of donors infected,
irrigation period
CWWI2W CWWI3W
Summer Spring
1982 1983
12 1
1 1
4 2
CWWI4W
Summer
1983
11
1
2
7
Poliovirns 1
Poliovirus 2
Poliovirns 3
Cozsackievirus B4
Cozsackievirus BS
Echovirus 17
Echovirus 27
1
3
2
1
a Recovered at levels defining an infection event.
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episodes which were observed, defined and submitted to statistical analysis
is presented later in Table 97.
The serological (S) agents and agent groups for which infection episodes
were identified from simultaneously tested paired sera were:
ADS ADenovirus 3.
ADS ADenovirus £
AD7 ADenovirus 7_
CB2 Coxsackievirus B2
CB4 Coxsackievirus B4
CBS Coxsackievirus B5
E01 Echovirus 1
E03 Echovirus 3_
E09 Echovirus £
Ell Echovirus 11.
E19 Echovirus 19.
E20 Echovirus 20
E24 Echovirus 24
PL1 PoLio I
PL2 PoLio 2
PL3 PoLio i
SNV all Serum Neutralization-tested Viruses except polioviruses (serologic
agents listed above plus echoviruses 5 and 17)
POR sPORadic serum neutralization viruses (consists of all SNV agents
for which too few infection events occurred during the period
of observation to constitute a distinct infection episode).
Since wastewater contains many infectious agents, a sporadic
episode to a variety of agents might be the most subtle effect
of wastewater exposure.
WWV all WasteWater Viruses (all SNV agents recovered from the wastewater
sprayed during the period of observation)
RE1 REovirus 1
RE2 REovirus 2
ROT ROTavirus (tested primarily in children)
LEG Legionella
INA INfluenza A (An epidemiologic control agent since influenza A
viruses do not survive in the intestinal tract or in wastewater.)
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Some donors experienced more than one infection event during a serologic
infection episode. This occurred when the period of observation spanned
three or more blood collection periods (allowing detection of multiple
infections to the same agent) or when the infection episode involved a
group of agents (allowing infections to several agents in the group).
The following guidelines were used to determine the value of the dependent
variable for a participant for each of the serologic infection episodes:
o If a person experienced an infection by an agent during a given
interval of time, the number of infection events observed was
coded as the person's infection status. Infection events were
counted and included in analysis of the infection episode even
though that person may not have been observed (i.e., provided
blood samples) during the entire interval of time.
o If no infection was observed in a person but he only provided
a blood specimen for the first portion of the time interval of
interest, then the infection status for that participant during
that interval was coded as ''missing.''
o If no infection was observed, but a person only provided blood
for the last portion of the time interval in question, the coding
for that interval was dependent upon the person's initial titer
for the partial interval. The interval was coded as having ''no
infection'' if the person had either no detectable titer or had
the lowest measurable titer for that agent. If the initial titer
was higher than the lowest measurable titer, then infection status
for the interval was coded as ''missing.''
o For the infection episodes to the agent groups SNV, FOR and WW,
the seroconversion status of a donor may not have been determined
for all agents in the group. If any infection events were observed,
the number of infections experienced by that donor was used as
the value of the dependent variable during the period of observation.
When no infection events were observed, but the seroconversion
status to some of the agents was not determined for that donor,
the donor was excluded from analysis (i.e., infection status
was coded as ''missing'').
A list of the serologic infection episodes which were observed, defined
and submitted to statistical analysis is presented later in Tables 98 and
99.
Many of the infection episodes observed were not independent, primarily
because one episode was a (partial) subset of another, either in time (e.g.,
episodes for seasons 1, 2 and 5) or in agent grouping (e.g., CBS is a subset
of WW which is a subset of SNV). Jointly independent groups of infection
episodes pertinent to comparing exposure and control situations were defined
by classifying episodes by agent category (single and sporadic vs. grouped),
situation (exposure vs. control) and observation period (single season
vs. year). These criteria for the six mutually exclusive and jointly indepen-
dent groups of episodes which were used are presented in Table 15.
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TABLE 15. CLASSIFICATION CRITERIA FOR JOINTLY INDEPENDENT
GROUPS OF INFECTION EPISODES
Jointly
independent
episode
group
A
B
C
D
E
F
Agent
category
single or sporadic
single or sporadic
single or sporadic
grouped agents
grouped agents
grouped agent
Criteria
Situation
exposure
exposure
control
exposure
exposure
control
Observation
period
single season (1,2,3,4)
year (5,6)
all: baseline (0) and
year (for influenza)
single season (1,2,3,4)
year (5,6)
baseline (0)
Notes on
episode
selection
priority
a
a,b
b
For clinical infection episodes with both X and W dependent variables,
the X variable was selected for membership in the jointly independent
episode group since it was more applicable to wastewater irrigation
inferences.
When both WWV and SNV episodes were defined (with WWV infection events
a subset of the SNV infection events), the WWV episode was selected
for membership in the jointly independent episode group since the
WWV episode was more applicable to wastewater irrigation inferences.
H. DATA MANAGEMENT
Data Processing and Verification
The information obtained from the health watch was stored by household
and participant identification numbers on a Control Data Corp. mainframe
computer at SwRI. The Scientific Information Retrieval (SIR) database
management system was chosen for the LISS data base due to its advanced
programming features and its integration with statistical packages such
as Biomedical Computer Programs (BMDP), Statistical Analysis System (SAS)
and Statistical Package for the Social Sciences (SPSS).
Results obtained from UTSA (clinical specimen assays), UI (interviews,
self-reported illness data and serologic assays) and EPA-HERL (electron
microscopy) were keypunched, key-verified, and placed on the SwRI data
base. After logical tests were performed, SIR-generated reports and error
lists were sent to the investigator who had completed the data reporting
forms for further verification and error resolution. The verified data
were also visually inspected for reasonableness by the health watch manager
and the project manager. Another method was implemented later in which
serologic data were coded, keypunched, verified, and corrected at UI; the
entire serologic data file was then placed on the SwRI data base. Computer-
generated labels had been affixed to the container of each sample at each
stage of processing and the label information had been coded along with
the analytical result to further reduce the chance of error. An overview
of the processing of each set of data is given in Table A-26 in Appendix
A.
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Data Base Structure and Use
The LISS data base was arranged into eight record types, which allowed
logical groupings of related variables (see Table 16). Record 1 consisted
of household-based variables. The key variable (sort identifier) of Record
1 was HHID, a three-digit household identification number. The first digit
of HHID represented the zone in which the household was located; households
were numbered consecutively within each zone.
Record 2 contained participant-based independent variables. The key
variables of Record 2 were HHID and ID. a five-digit participant identification
number. The first three digits of ID consisted of the household identification
number (HHID). Adults living in a household were numbered consecutively
from 01, beginning with the head of household; children were numbered
consecutively from 11, beginning with the oldest child. Most of the
variables in Records 1 and 2 were obtained from the recruitment and update
interviews (Appendices B, C and D).
Record 3 contained variables describing fecal and throat culture samples.
Microorganisms isolated and corresponding growth levels were stored in
Record 4. Records 3 and 4 were separate to allow for multiple or no agents
detected in a sample. Key variables in Records 3 and 4 included SAMPRD
(the period of observation), ID, SPECTTP (the type of specimen analyzed)
and in Record 4, ORGNSM (the type of organism isolated).
Record 5 consisted of self-reported illness data from the health watch.
The sort variables in Record 5 were SAMPRD, ID, ILLNESS (the classification
of a reported illness) and ILLNO (an illness repetition code). ILLNO was
used to account for the same illness occurring more than once in the same
sampling period. Inconsistencies in the Record 5 data are discussed in
Section 5E.
Record 6 contained exposure data from the major irrigation periods.
The key variables in Record 6 were ID and SEASON (a number from 1 to 4
corresponding to spring 1982, summer 1982, spring 1983 and summer 1983,
respectively). Methods of exposure estimation and the major exposure variables
were presented in Section 4C. Results from the Wilson restaurant patronage
survey were placed in Record 6 because they had the same sort identifiers.
The variables in Record 7 were the results from serologic analysis
of blood samples. ID and AGENT (the serologic agent tested) were the key
variables in Record 7.
Record 8 contained the dependent variables from each infection episode
used in statistical analyses. The key variable for Record 8 was ID. Con-
struction of the dependent variables in Record 8 was explained in Section
46. Record 8 variables were derived from variables in Records 3, 4 and
7.
A copy of the data base was also placed on the IBM computers at HI
and EPA-HERL. To perform the data analyses, appropriate data files were
abstracted from the data base and transmitted to the cognizant investigator.
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TABLE 16. STRUCTURE OF LISS DATA BASE
Record
type
Description of variables
Sort identifiers
Household independent variables (from
recruitment and update questionnaires)
Participant independent variables
(from recruitment and update ques-
tionnaires) and annual exposure
variables
Clinical specimen description,
virology and electron microscopy
results
Microorganism isolations from
clinical specimens
Self-reported illness data
Exposure and restaurant variables
7 Serology data
8 Dependent variables (number of
infection events) for each
infection episode, previous titer
for serologic infection episodes
HHID (household ID)
a. HHID
b. ID (participant ID)
a. SAHPRD (data collection
period)
b. ID
c. SPECTYP (specimen type)
a. SAHPRD
b. ID
c. SPECTYP
d. ORGNSM (organism)
a. SAHPRD
b. ID
c. ILLNESS (illness category)
d. ILLNO (illness repetition)
a. ID
b. SEASON (irrigation period)
a. ID
b. AGENT (agent tested)
ID
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Retrieval files were created from the SIR data base for use in statistical
analyses via BMDP, SAS and SPSS. Because EPA-HERL did not have the SIR
data base management system, DI generated SAS files of the eight record
types comprising the data base which were transferred by tape to HERL.
I. QUALITY ASSURANCE
Health Data and Specimens
All completed household health diary forms which were forwarded by
field representatives to UI on a biweekly basis were checked for completeness
and coded for data entry. In order to achieve consistency in classification
of illness information, all illnesses were coded according to a standardized
list of illnesses and conditions. Telephone reports and written diaries
were compared for discrepancies, and whenever possible, those discrepancies
were resolved prior to submitting the coded diaries for data entry. Logic
checks were written and forwarded to data management for additional checks
of the health diary information. All discrepancies identified by the logic
checks were resolved, and the receded information was forwarded to data
management for inclusion in the data base.
The health watch manager or one of the field representatives also
supervised the labeling of all specimens received from the study population.
This policy was necessary in order to avoid the problem of technicians
misidentifying similarly named study participants. Computer-generated labels
containing the participant's name and ID number were used to identify samples
as well as to generate packing lists whenever specimens were shipped. A
log was kept for all blood and fecal specimens that were received, so that
an additional source of documentation was available in order to resolve
discrepancies.
All activity and exposure information provided by participants was
checked for completeness and accuracy by the field representatives or the
health watch manager before the information was forwared to SwRI for coding
and data entry. Logic checks were also written and forwarded to data management
for use with the questionnaire information. All discrepancies identified
by this method were resolved and receded. In addition, the health watch
manager reviewed each household and participant record in order to verify
information for responses which could not be addressed by logic checks.
This information, which included important variables such as sex, age,
and occupation, was corrected whenever necessary and any missing information
was obtained by contacting the household in question.
Aerosol Measurement Precision
Inspection of the microorganism aerosol density data showed considerable
variation, even between paired samplers. This measurement variation may
result from differences in many factors, including aerosol density fluctuation,
sampler operating procedures, undetected sampler contamination, shipping
difficulties (e.g., temperature above 4°C), analytical laboratory techniques,
and random error.
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Two quality assurance aerosol runs were conducted to investigate the
amount and source of imprecision of the aerosol density measurements for
each microorganism group. Nine samplers were operated 3 meters apart in
a line at the same distance from a nozzle line, so that all samplers were
theoretically sampling the same aerosol density. The 100 mL of sampler
collection fluid was normally split at the laboratory into four 25-mL portions
for the four microorganism assays. On the quality assurance runs, each
sample was split in the field into 25-mL portions which were labeled for
specific analyses. Three of the four portions were labeled for assay for
the same microorganism group. Hence, portion variation, which reflected
shipping and laboratory-related variation, could be subtracted from measurement
variation to estimate the magnitude of sampling-related variation relative
to shipping/laboratory variation.
The data from the quality assurance runs are presented in Table A-27
in Appendix A. The microorganism density in air determined from portions
from the same sampler often exhibited less variation than the measurements
from different samplers, but there were exceptions.
The precision of a sample of n determinations of the same true concen-
tration can be measured by the coefficient of variation, which is the ratio
of the unbiased sample standard deviation to the sample mean:
CV = ^ s/x
where x - sample mean = Zx/n
s - sample standard deviation = [Z(x-x)2/(n-l)]*/2
OQ - bias correction factor = [ (n-D/2]1/2 T[ (n-1) 12} IT (n/2)
The bias correction factor ctn adjusts for the bias in the sample standard
deviation s as an estimator of the population standard deviation a. The
values of on approach 1.0 as n increases: 02=1.253, 03=1.128, 04=!.085,
and 05=1.064.
To investigate the consistency of measurement variation over the entire
range of aerosol densities sampled in the field, measurement coefficients
of variation were determined for all situations in which several samplers
were theoretically sampling the same true density of microorganisms in
air. These situations were the paired samplers at three locations on each
microorganism run and the samplers assigned the same microorganism assay
on a quality assurance run. Coefficients of variation were calculated when
microorganisms were detected in at least one of the sampler assays, assuming
assays in which no microorganisms were recovered had a value of half the
detection limit. The coefficients of variation for microorganism run pairs
in the same density range were averaged to yield a more stable estimate
of the measurement variation.
The average measurement coefficients of variation throughout the density
range sampled are presented for each microorganism group in Table A-28
in Appendix A. Because the standard deviation calculated from a small sample
is very imprecise, the average coefficients of variation are quite variable
over a microorganism's density range. However, there is no consistent pattern
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in the magnitude of the coefficient of variation with increasing aerosol
density. Hence, average measurement coefficients of variation were determined
over all sample sets, with the values 0.43 obtained for coliphage, 0.46
for fecal streptococci, 0.67 for fecal coliforms, 0.72 for Clostridium
perfringens. and 0.81 for mycobacteria. Therefore, the precision standard
deviation of the aerosol density measurements ranged from 43% of the measured
value for coliphage to 81% of the measured value for mycobacteria.
An investigation of the relative magnitude of the various sources
of the measurement variation was conducted based on the quality assurance
runs. Portion coefficients of variation were determined for each run, where
the values from different samplers were averaged. The assay result reported
by the laboratory is an average, i.e., the total number of colonies or
plaques counted in spreading aliqnots of the sample or its serial dilutions
over m plates (usually m=3 plates for fecal coliforms and fecal streptococci
and m=25 plates for coliphage). To estimate laboratory sources of variation,
an average aliquot coefficient of variation was calculated for all assays
on each quality assurance run using the aliquot standard error s//m~ in
place of the standard deviation to obtain a variability measure comparable
to the measurement and portion coefficients of variation. Since variances
of independent variables are additive, the variation attributable to field
sources was estimated by subtracting the variance for shipping and laboratory
sources from the measurement variance. Similarly the variation attributable
to shipping sources was estimated from the portion and aliquot coefficients
of variation.
Each of these coefficients of variation are presented in Table A-29
in Appendix A. While the aliquot variation estimates are quite stable,
the variation attributed to other sources was highly variable due to the
limited amount of quality assurance data. Although a much broader range
of aerosol densities was sampled in comparison with the Pleasanton study
(Johnson et al., 1980), the average measurement coefficients of variation
determined in the two studies were similar for fecal coliforms, mycobacteria,
and Clostridium perf r ingens. However, the LISS data for fecal streptococci
and coliphage only exhibited about 60% as much measurement variation as
in the Pleasanton study.
Laboratory Analysis
Enterovirus Serology—
Although the serum neutralization test is qualitatively reliable,
the titers that are obtained when this test is used cannot be considered
absolute. Different laboratories using different cell lines, different
virus strains, or other slight variations in procedure, can produce different
titer results for the same positive sera. Results can also be affected
by the virus dose, the age of the tissue culture cells, slight changes
in the pH, etc. Since the titers are known to vary significantly between
tests, an infection was not reported in this study unless a fourfold or
greater increase in titer could be demonstrated in simultaneously tested
sera.
Since all of the sera from the study population could not be tested
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for antibody to a given agent in a single test, results from two or more
tests, which were run at different times in the study, were used to detect
fourfold or greater increases in titer. Whenever a possible fourfold increase
was detected, the sera in question were retested in pairs. Since the initial
screening results were used to determine which sera were selected for retesting,
titer variability was a concern. Therefore, in addition to the usual quality
control concerns in a serology laboratory, such as eliminating potential
sources of contamination or interferences with test results, there were
two additional goals for the enterovirus serology quality control: to limit
the known sources of variability in the serum neutralization test, and
to quantitate the reproducibility of the serum neutralization results.
This was accomplished by increasing the number of controls to include six
replicates of a human sera with a ''high" level of antibody, and six replicates
of a single human sera with a ''low'' or ''intermediate'' level of antibody
to a given agent. The titers obtained from the replicate tests were used
to calculate the geometric mean titer (GMT) and the titer reproduc ibility
(TR) (Wood and Durham, 1980). This information, which can be found in Appendix
0 was used to determine the reliabilty, as well as the variability, of
the results from any given test. With the exception of echovirnses 3 and
24, the between-test variability of the non-polio enterovirus control titers
was within acceptable limits. Low virus dosage in the tests done to confirm
fourfold titer increases in antibody to echoviruses 3 and 24 caused the
control titers to be unusually high. However, the variabilty of the tests
done as a part of the routine screening for antibody to these two agents
were within acceptable limits.
Eighteen sera were forwarded to the University of Iowa Hygienic Laboratory
(UIHL) to determine the antibody titers to poliovirus types 1-3. A listing
of the results is presented in Table A-30 in Appendix A. Comparison of
UI and UIHL results indicates that there was a fourfold or greater difference
in Poliovirus 1 titers in only 12% of the cases. There was a fourfold or
greater difference 23% of the time for Poliovirus types 2 and 3. The UI
titers were generally lower than UIHL titers. If the assumption is made
that the UIHL titers were "correct,'' then the recommendations for immunization
(which were based on the UI titers) may have included participants who
were adequately protected. This situation is preferrable in that there
was less chance of failing to immunize a susceptible participant.
All enterovirus serology was performed by UI personnel under the guidance
and supervision of the Virology Laboratory section of the Illinois Deparment
of Public Health (IDPH). In addition, all enterovirus controls were examined
and verified by IDPH supervisors. The IDPH provided all necessary reagents,
glassware, media preparation rooms, hoods, and environmental chambers for
the enterovirus serology. IDPH routinely tested the distilled water as
well as all new reagent lots for contamination and toxicity. Environmental
chambers were continuously monitored for temperature variation, and the
media preparation and hood rooms were checked for contamination on a routine
basis.
All virus stocks were originally obtained by IDPH from either CDC
or American Type Culture Collection (ATCC), and all subsequent passages
were well documented. All tissue cultures provided by IDPH were similarly
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verifiable. In order to avoid potential labeling or contamination problems,
fresh monospecific antisera from CDC was used to reidentify all of the
stock enteroviruses which were used in this study.
Hepatitis A Serology—
Quality assurance for the determination of antibody to hepatitis A
virus (anti-HAV) was that built into the HAVAB test system. This involved
the use of both positive and negative controls provided by the test system
manufacturer (Abbott Laboratories) and determining that only repeatably
reactive specimens (minimally two tests conducted on separate days) were
considered to be positive for anti-HAV by the HAVAB test. As a further
control measure, each analytical series of 100 tests included two or three
sera from participants whose HAVAB reactivity had been established previously.
Additional quality assurance programs were conducted between Hay 1981
and Hay 1983 at both UTSA and UI. A total of 267 sera (including all positive
sera) were retested in the blind. Only four discrepancies (1.5%) were found
in the retesting. All four discrepancies were found with sera previously
found to be ''borderline'' positives that changed to ''borderline'' negatives
in the retest. Given the variable nature of this test, the reproducib ility
of the HAVAB results was considered excellent.
Clinical Bacteriology—
Quality control in the Clinical Bacteriology Laboratory involved a
program of internal monitoring, seeded unknowns, and replicate, split clinical
specimens. Internal monitoring included testing each new batch of culture
media, testing reagents and stains, and quality control of biochemical
tests. In addition, the plating and enrichment media and biochemical test
media were assigned expiration dates that prevented use beyond the point
where consistent results could be obtained. Periodic seeded, unknown specimens
ensured the proficiency of the laboratory in correctly identifying organisms
and determining the levels of organisms in the specimens.
Selected fecal specimens were split and coded as unknowns for clinical
analysis in April and Hay 1981. A listing of coded split samples (generated
during preanalysis sample handling) was forwarded to the laboratory supervisor
on a weekly basis. Results of this QA testing for clinical bacteriology
are presented in Table A-31 in Appendix A and indicated a very successful
program. Of the 22 split samples, total agreement on both isolate identification
and quantitation was recorded on 14 specimens (64%). In all remaining samples,
the variance between results of known and QA tests involved a difference
of a single quadrant level of microorganism growth. For example, in handling
specimen 55913 (period 108) as a split sample, Escherichia coli was reported
as moderate and heavy, respectively, while Enterobacter cloacae was detected
as no more than 10 colonies on one sample. The results in Table A-31 indicated
excellent repeatability in the clinical bacteriology laboratory.
Results of additional quality assurance unknowns performed in November
1982 are shown in Table A-32 in Appendix A. The unknown samples were given
to the technician as seeded autoclaved fecal specimens in buffered glycerol
saline (from routine fecal specimens that had been preserved by freezing
for use in quality assurance unknowns). Each of the four unknowns was correctly
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identified both with respect to identity of organisms and the level of
seeding.
The concentration of organisms in feces represented by different levels
of growth on HacConkey agar plates streaked by the four quadrant method
is suggested by the results of Table A-33 in Appendix A. Each of the values
represents laboratory reports on ''blind'' (unknown) samples seeded with
known concentrations of three organisms. The unknown samples were given
to the technician as buffered glycerol saline suspensions (E. coli) or
seeded autoclaved fecal specimens in buffered glycerol saline (K. pneumoniae
and P. aeruginosa).
A similar experiment was carried out for throat swabs as shown in
Table A-34 in Appendix A. Four different organisms (Streptococcus pyogenes.
E. co 1 i, Enterobacter c loacae and K. pneumoniae). previously observed in
some illness specimens, were separately diluted in Todd Hewitt broth.
One mL portions of the dilutions were placed into tubes which then received
sterile swabs. The coded samples were then given to the technician who
processed the samples as if they were throat swabs (see Figure 15 and associated
discussion for details of analysis of throat swabs). Results are reported
as levels of growth on replicate plates of blood agar. In general, a light
level (i.e., growth on first quadrant) was observed with suspensions of
greater than approximately 1000 cfu/mL, the moderate level (i.e., growth
on first two quadrants) with suspensions greater than approximately 100,000
cfu/mL, and the heavy level (growth on three or all quadrants) with suspensions
greater than approximately 10,000,000 cfu/mL.
A program of surveillance procedures for selected laboratory equipment
also was used in the clinical laboratory. This included a time schedule
(e.g., each time or use for pH meters, daily for incubators) and set tolerance
limits for incubators, refrigerators, freezers, water baths, and pH meters.
Clinical Virology—
As described above, split samples coded as unknowns were also screened
for viruses in parallel with routine clinical specimens. Unfortunately,
no viruses were recovered from any of the 35 fecal samples received during
April and Hay 1981. Therefore, the split-sample approach did not yield
definitive data concerning laboratory precision for clinical virology.
A similar split-sample program was initiated in August 1981 in an
effort to test the reproducibility of viral isolation in tube cultures
from clinical specimens. Detailed results of this testing are presented
in Table A-35 in Appendix A. Of the 33 participant samples used in this
program, only two specimens yielded virus as part of the routine analysis
while five isolates were recorded in QA testing. Notably, both samples
found to be positive in routine testing were also positive in QA testing,
although in one instance the isolation was made in different cell lines.
These results also highlighted the low likelihood of recovering viruses
from routine specimens when assay volumes were limited by tube culture
inoculation. Subsequently, assay procedures were modified as described
in Section 4E to increase the amount of sample inoculated into susceptible
cell monolayers.
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In addition, a specific quality assurance program was followed for
viral identification. On a quarterly schedule, three ''unknown'' animal
viruses (from laboratory stocks) were handled for identification using
the serological protocols described under ''Laboratory Analysis—Clinical
virology.'' An acceptable performance required the recovery of each unknown
virus in at least one cell line and the correct identification of each
isolate.
Electron Microscopy—
Photographs of each positive specimen were taken for documentation
of visual identification. The electron micrographs were evaluated against
micrographs published in peer reviewed journals with regard to size and
distinctive morphological characteristics. Positive specimen material
is maintained at -70°C for future reference. Poliovirus was used as the
reference standard for size determination. All examinations were performed
on the same JEOL 100CX electron microscope by the same microscopist. The
microscope is maintained under a service contract and undergoes periodic
maintenance and performance checks by qualified personnel.
In order to eliminate possible bias in the EM study, all stool specimens
received from years 1980, 1981 and 1982 (the first year of irrigation)
were coded and examined together. Some duplicates were included so that
equal numbers of pre- and postirrigation specimens were examined. The
identity of individual specimens remained unknown to the microscopist until
all specimens had been examined.
The additional specimens received from the final year (1983) were
examined separately, but included five coronavirus-positive and five negative
specimens from the earlier examination for comparison. This examination
was also performed under code.
Coronavirus-like particles were detected in only two of the five stools
previously found positive for this agent. Subsequent examination of the
three other specimens did reveal particles generally consistent with a
coronavirus-like classification but with poorly defined fringe projections
(perhaps deteriorated or antibody obscured). Such particles are difficult
to detect during routine EM examination as fringed particles of all types
are frequently encountered in stools. Additionally, all the coronavirus-like
particles observed in the specimens to date have not had classical coronavirus
morphology. These particles have an alternate or atypical appearance which
is even more pleomorphic and more variably fringed than the classic propagated
coronavirus. The occurrence of these particles has been widely reported.
although their significance has not been established and it is not clear
that such particles represent actual virus particles.
Environmental Samples—
In addition to the equipment and media performance testing described
above for ''Clinical Bacteriology,'' the internal quality assurance program
for analysis of environmental samples involved two approaches. Wastewater
samples seeded with several laboratory strains of enteric bacteria were
analyzed for the quantitative recovery of the unknowns. Likewise, selected
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known viruses were recovered and identified as described above for ''Clinical
Virology.''
In addition, a series of split analyses for enterovirus concentration
and assay on HeLa cells and for indicator bacteria enumeration by membrane
filtration were incorporated into the enterovirus identification and/or
routine wastewater analyses conducted monthly during April and Hay 1981.
Results are summarized in Table A-36 in Appendix A. Both bacterial and
viral analyses were within an acceptable repeatability range.
QA reprodubility data were generated by compiling data for indicator
bacteria and total organic carbon (TOO in Lubbock wastewater reported
by the LCCIWR laboratory, the UTSA laboratory, and the DT Austin laboratory.
Composite samples were collected by either SwRI or LCCIWR personnel, split
and shipped as part of routine monitoring described previously. Results
for total and fecal coliform bacteria recorded during baseline monitoring
are presented in Table A-37 of Appendix A. Fecal coliform levels reported
by LCCIWR and either UTSA or UTA laboratories during 1982 and 1983 are
shown in Table A-38 in Appendix A. Similarly, split sample values for
fecal streptococci are recorded in Table A-39. In most instances, total
and fecal coliform and fecal streptococci values were well within the vari-
ability expected of a dilution-based bacterial assay. Indeed, when replicate
results were reported by LCCIWR (see Tables A-38 and A-39), the duplicate
value fell closer to that reported as the mean of triplicate platings by
DT laboratories. Perhaps because of the larger number of samples compared,
more interlaboratory discrepancies were observed with fecal coliform results.
To address these differences a series of in-house QA tests were conducted
at DT Austin using samples collected on July 25 and 26, 1983 and August 8
and 9, 1983. Colonies counted as typical fecal coliforms (blue) and/or
nonfecal coliforms (gray to cream-colored) were subcultured onto nonselective
heart infusion agar. An oxidase test was completed on all isolates and
selected bacteria were identified using the API 20E test system. Results
of this QA testing are shown in Table A-40 in Appendix A. With the exception
of three colonieswhose API profile was not definitive, all organisms recorded
as fecal coliforms in samples collected on July 25 and 26, 1983 were identified
as members of the Enterobacteriaceae family. Results for the Wilson wastewater
sample collected on August 8 and 9 were less clear-cut. In this instance,
as Aeromonas hydrophila. Perhaps more importantly, a significant number
of nonfecal coliform colonies were oxidase negative. Of these, at least
half were enteric bacteria. It should be noted that a total of 45 colonies
were subcultured off of a single 47-mm diameter membrane filter and that
some overlap of colonies may have occurred. Nonetheless, based on these
observations, the value of 3.1 x 10? fecal coliform/100 mL reported for
this Wilson sample (see Table P-3 in Appendix P) may be low. Aside from
developmental work, little information identifying ''nonfecal'' coliforms
appears in the published literature. Furthermore, these results are from
a single sample which may or may not be representative of other assays
or wastewater sources.
Overall, the agreement between laboratories for all indicator bacteria
may be considered very good for microbial parameters. Furthermore, these
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comparative QA results show that the procedures used for sample shipment
and analysis within 24-36 hours of collection resulted in valid experimental
data with no remarkable sample deterioration. In addition, chemical analyses
as demonstrated by TOC data shown in Table A-41 of Appendix A were quite
comparable between laboratories.
Data Management
A sample identification system based on a coded label was used to
preserve the integrity of the sample data. A computer-generated label was
affixed to each sample's container (e.g., wastewater, aerosol, blood serum,
fecal specimen, throat swab), each sample aliquot, and each source record
(e.g., medical history, health diary). An alphanumeric code on the label
specified the participant ID number, sample medium (e.g., blood, feces,
wastewater), sampling period, type of sample analysis, etc., so the sample
was uniquely identified. The key elements of the code were also printed
in English on the label to facilitate sample processing. The sample code
was reported to data management along with the analytical result and was
keypunched and placed on the data base with the result. The sample code
also functioned as the index key for the data base.
Data processing errors were minimized by judicious inspection and
editing of participant-furnished data, inspection of field- and laboratory-
reported data, key verification of keypunched data, and reliance on automated
data processing accompanied by checks on the coherence of the data. Key
processing steps were manually double-checked from file listings by the
project manager to ensure they were performed correctly and completely.
The values of key variables on the data base, such as the dependent variable
in infection episodes, the aerosol exposure index and age, were visually
inspected for reasonableness by the health watch manager and the project
manager.
Archiving of Clinical Specimens
A portion of all clinical specimens (blood, feces, and throat swabs)
taken in the health watch were preserved and frozen at -76°C. A cross-referenced
catalog system allowed ready access to specific samples. Master lists of
blood donors and clinical specimen donors were updated each period, reflecting
each individual's cumulative participation in the health watch program.
All illness and virus-positive fecal samples are archived at UIA.
Archived 1 mL aliquots of sera given by all participants during each
blood collection in the entire study were transferred from UI and UTA to
EPA-HERL upon completion of the laboratory phase of the LISS. Prior to
shipment the inventory of archived aliquots was double-checked against
a master listing of all blood samples obtained in the LISS. The archived
sera have been stored between -35°C and -76°C at EPA-HERL.
J. STATISTICAL METHODS
Previous studies of the effect of wastewater and associated aerosols
upon the health of such diverse groups as sewer and sewage treatment workers
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(Clark et al., 1980; Sekla et al., 1980), agricultural workers (Shnval
andFattal. 1980), school children (Camann et al., 1980), and suburban
residents (Johnson et al., 1980; Fannin et al., 1980; and Northrop et al.,
1980) suggested that any health effects seen in the LISS were likely to
be rather subtle. To ensure that the analysis of association of infection
with exposure was sensitive enough to detect such effects, care was taken
to employ statistical tests for which both the level and power could be
calculated. In most instances this led to the use of rather simple tests
of the main hypotheses. More elaborate and sophisticated analyses often
involve tests whose power is unknown or known only approximately. These
were considered to be exploratory techniques and were employed only after
the primary test with controlled error probabilities had been conducted.
Additional comprehensive and ad hoc analyses were performed to address
the association of infection with exposure for data sets which were not
amenable to the standard analysis.
The primary strategy was to divide the study participants into groups
which received high or low exposure to the pathogens through aerosols,
through direct contact with wastewater or by other means, and then to compare
the incidence of infections in these two groups during a period of wastewater
irrigation. Events which indicated an infection of an individual were
either the occurrence of a seroconversion or a significant increase or
detection of a fecal agent according to the definitions of infection events
given in Section 4G. To permit use of sensitive statistical methods requiring
that the dependent variable only assume the values 0 or 1, all multiple
infection events were treated as single infection events in most statistical
analyses performed; the exceptions are noted below. Thus, a value of 0
indicated the donor was not infected during the period of observation while
1 indicated the donor was newly infected. If exposure groups were comparable
in every pertinent respect and the individuals' responses were independent,
the proportion of infections occurring in the groups were compared in a
simple contingency table analysis to determine if there was difference
in incidence rates between the exposure groups. In cases where there was
imbalance between the exposure groups with regard to important variables,
it was necessary to stratify on these variables and compare rates within
strata. Further, such variables were used as predictor variables in a
logistic regression analysis to account for any differential effects they
may have had on the infection rates.
Since individuals were clustered in households, the occurrence of
their infections could have been correlated with those of other household
members. The independence of infection events within households was evaluated
as described below under Confirmatory Analysis.
The standard analysis may be viewed as consisting of these major stages:
1) Preliminary Analysis—comparison of the low exposure group (AEK3)
and the high exposure group (AEI>.3) with respect to individual
and household characteristics in order to determine if the two
exposed groups differed significantly with regard to these factors.
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2) Confirmatory Analysis—comparison of infection rates in the exposure
groups to determine the presence of any association of infection
and wastewater application. This was a major analysis of the
study and resulted in a p-value for the rejection of each null
hypothesis. The principal findings of the study will rest on
the results of these analyses and their consistency with the
other methods of inference employed.
3) Exploratory Analysis — investigation of whether the presence of
infection was associated with a set of potential predictor variables
and in particular with the degree of aerosol exposure.
A careful distinction between these stages was maintained during the
analysis, discussion and conclusion sections of the study report. These
stages of the standard analysis will now be described in more detail.
The analysis of risk ratio scores, incidence density ratios, and various
small data sets are presented later in this section.
Pre1im inary Analys is
Prior to conducting tests for association of infection rates and exposure,
the exposure groups were compared with respect to other characteristics
which could influence the outcome of the tests. The exposure groups therefore
were compared by calculating the proportion in each category of each pertinent
variable for each population to be tested (fecal donors and blood donors)
in each of the six seasons of data and the baseline data set. For the
baseline period comparison, the exposure groups were defined based on subsequent
exposure during the first (spring 1982) irrigation season. A standard
chi-square test for equality of proportions for 2xk contingency tables
was used, where k is the number of categories of the characteristic and
2 is the number of exposure groups (AEK3, AEI>3) . A chi-square test may
be used when fewer than 20% of the cells have an expected frequency of
less than 5 and no cell has an expected frequency of less than 1 (Siegel,
1956). When these requirements were not met, adjacent categories of the
characteristic were combined to increase the expected frequencies. For
2x2 contingency tables, a one-tailed Fisher's exact test was used whenever
the expected frequency for any cell was less than 5. The range of the
p-value, the number of observations in each exposure group, and the proportion
(or percent) in each category of each exposure group was reported for each
chi-square or Fisher's exact test.
From these tests, a judgment was made about the variable(s) to be
used for stratification or included as explanatory variables. The relative
importance, the consistency and magnitude of differences across seasons
and the quality of the data for each variable was considered. To ensure
consistency, a variable was considered for use as a stratifying variable
if and only if 1) the variable was deemed to be epidemiologically important
and 2) the hypothesis of equal proportions was rejected at the 0.01 level
at least once or at the 0.05 level at least twice in the four irrigation
seasons. If a variable met these criteria and if the number of observations
was adequate, the variable was stratified prior to conducting the confirmatory
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analysis (discussed below). If not, the imbalance was reported and the
confirmatory analysis was carried out without correction for that variable.
Confirmatory Analysis
Testing procedure—
Since individuals were clustered in households, the possible dependence
of infections for individuals within households (i.e., intra-household
correlation) was investigated. To determine the proper unit of analysis
(household or individual), we examined whether, say, two individuals in
the same household were more likely to both acquire infections than two
individuals from different households. The approach was to fit a binomial
distribution to the data. The binomial model was chosen because:
1) The data are binary.
2) If individuals are independent (i.e., no correlation within house-
holds) and the probability of infection is constant over individuals,
the binomial is a plausible model.
3) Departures from the binomial can be examined by looking at the
difference in observed and expected numbers of individuals.
For example, in a household with two members donating specimens,
the categories were:
a) both members not infected
b) one member infected
c) both members infected
An excess in Category c indicated significant clustering of infections,
i.e., if one member had the infection, the other was more likely to have
the infection than was predicted by the binomial model. In summary, if
the binomial model fitted (using a chi-square goodness of fit), there was
no reason to suspect correlation.
In cases in which household clustering was not significant, a 2x2
contingency table analysis of infection status observed on individuals
was used in a one-sided test of the hypothesis that the incidence rates
of infection or seroconversion were the same for the high and low exposure
groups. The investigation for each agent and observation period can be
summarized by the following 2x2 contingency table
Exposure
Low High
Yes
Infected
No
nl °2
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wherein the column totals nj and 02 are fixed. The two columns represent
the outcomes of two binomial experiments, with probabilities of becoming
infected being PI and ?2 in the low and high exposure groups, respectively.
The statistic used for testing the null hypothesis P2=Pi against the alternative
?2>Pi was
X ^ = £ (observed-expected)^/expected
or when expected values were small, Fisher's exact test was used according
to the rules stated above. The one-sided alternative was appropriate since
?2Pl was more powerful than the
test against the two-sided alternative, given the same level and sample
size. The range of the p-value, number of infections, and incidence rates
in each exposure group were reported for each chi-square or Fisher's exact
test.
Stratification—
When stratification was indicated in the preliminary analyses, the
study groups were appropriately stratified and a simple contingency table
analysis was conducted within each stratum. The results of the independent
tests within strata were combined by the Hantel-Eaenszel procedure (Kleinbaum
et al., 1982). The range of the p-value, number of infections, and incidence
rates in each stratum of each exposure group were reported for each Mantel-
Haenszel test. Stratification was not performed unless the sample size
criteria suggested by Mantel and Fleiss (1980) were met.
The preliminary and confirmatory analyses discussed above were performed
using the BMDP4F (Dixon et al., 1983), SAS TFREQ (SAS, 1982). and Minitab
(Ryan et al., 1982) statistical packages of computer programs.
Exploratory Analysis
The purpose of the exporatory analysis was to investigate whether
the presence of infection was associated with a set of potential predictor
variables. Primary interest was in determining if an association existed
between the presence of infection and the degree of aerosol exposure.
To achieve this goal, a stepwise logistic regression analysis was performed
for each infection episode in which there was a higher rate of infection
in the high exposure group (AEI2.3) than in the low exposure group (AEK3)
and the high exposure level (AEI>5) than in the low (AEK1) and intermediate
(1
-------
The analyses were performed using the BMDP-LR computer program (Dixon
et al., 1983). LR is a stepwise logistic regression program designed to
investigate the relationship between a binary response variable and a set
of categorical and/or continuous predictor variables (Cox, 1970) . It uses
a maximum likelihood estimation approach for estimating the coefficients
in the prediction equation and testing their significance.
The effects of each predictor variable used in the study were assessed
through the usage of a maximum-likelihood-ratio chi-square test of the
hypothesis that the explanatory power of that variable was zero. At each
step of a given analysis a predictor variable was added to the constructed
regression equation provided that it had the smallest chi-square p-value
among all remaining predictor variables and that the p-value was less than
0.10. Similarly, a term could be removed from the equation at each step
if it had the largest p-value among the predictor variables already entered
into the equation and if the p-value exceeded 0.15. Occasionally, two
or more predictor variables in an equation were so highly correlated that
the regression analysis could not be run. In these cases, one or more
of the collinear variables were deleted based on the magnitude of their
correlation coefficient and the order in which they entered the prediction
equation. This process was repeated for each response variable in every
season.
The goodness of fit of the devised models in describing the relationship
between the probability of infection and the selected predictor variables
was assessed using a test developed by Hosmer and Lemeshow (1980); this
actual test statistic is termed C* in their article. The test is based
on comparing the observed and expected frequencies of subjects having an
infection. These subjects are grouped into ten cells based on their infection
risk. The resultant test statistic has approximately a chi-square distribu-
tion. A small p-value (e.g., p<0.10) indicates that the prediction equation
does not fit the data.
For each constructed model, approximate 90% confidence intervals were
obtained for the odds ratio. This was calculated using an asymptotic normal
approximation. Note that the logarithm of the odds ratio is the estimated
coefficient of the predictor variable of interest. If the constructed
confidence interval contained the value 1, it was concluded that the odds
of having an infection were the same for the various categories of the
predictor variable.
Analysis of Risk Ratio (RR) Scores
Risk ratio scores assigned in a symmetric manner to each independent
infection episode were analyzed to provide a sensitive overview of any
apparent association of infection events with wastewater aerosol exposure.
Since it is based on all infection episodes observed in the LISS, the risk
ratio score analysis provides an overview indication, which is both broad
and sensitive, of any infection effects associated with wastewater spray
irrigation. The risk ratio (RR) for exposure groups in an infection episode
is the ratio of the infection incidence rate in the high exposure group
divided by the infection incidence rate in the low exposure group.
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If wastewater aerosol exposure were a major cause of infections in
the study population, then a certain pattern should be evident in the infection
incidence rates and risk ratios of the exposure groups and levels. The
risk ratio for exposure groups should be large, perhaps RR^S.O for an episode
with 5-8 newly infected donors or RIO2.5 for an episode with 9 or more
newly infected donors. The infection incidence rate should also be larger
in the high exposure level than in the intermediate or low exposure levels,
say by a factor of 2.0 or more both for high-to-intermediate levels and
for high-to-low levels. If these group and level patterns both occurred
in the same infection episode, this would be strong evidence for possible
association of the infection events with wastewater aerosol exposure.
Such an episode was assigned a risk ratio score of ++. The criteria are
formally presented in Table 17. If a somewhat weaker pattern were apparent
both in the risk ratio for exposure groups and in the incidence rates of
the exposure levels, the infection episode was assigned a risk ratio score
of +. The precise criteria for + are also given in Table 17.
Obviously some of the infection episodes assigned risk ratio scores
of ++ or + will be due to chance. To control for this random effect, the
same criteria were applied in a symmetric manner to the infection incidence
rates of the low exposure group and level. Suppose that in an episode
with 9 or more newly infected donors, the infection rate of the low exposure
group exceeded the rate in the high exposure group by a factor of 2.5 or
more and the infection rate in the low exposure level exceeded the rates
in both the intermediate and high levels by more than a factor of 2. This
episode was assigned a risk ratio score of - -, since the pattern was observed
in the low exposure group and level rather than in the high exposure group
and level. In this manner the risk ratio score criteria presented in Table
17 were developed. When no distinct pattern was evident in the group and
level incidence rates (i.e., neither the criteria for a + score nor for
a - score were met), the infection episode was assigned a risk ratio score
of 0. Since smaller proportional incidence rates can be significant
when the overall incidence rate becomes large, an alternate criterion involving
the difference in the group incidence rates expressed as percentage points
was developed for episodes with a large number of infected donors (see
last column of Table 17).
It should be noted that the cutoff values in Table 17 defining a risk
ratio score are arbitrary. If other cutoff values had been chosen, the
scoring of risk ratios would have been different.
In summary, the risk ratio score criteria are symmetric with regard
to the high and low exposure groups and levels (i.e., an infection pattern
that would be scored + if the excess infections occurred in the high exposure
group and level, would be scored - if the equivalent excess infections
occurred in the low group and level). Thus, in the absence of any effect,
random variation should produce an equal number of positive and negative
risk ratio scores.
The risk ratio score criteria presented in Table 17 were applied to
every infection episode. The sign test can be used to determine whether
a preponderance of positive risk ratio scores is statistically significant,
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TABLE 17. RISK RATIO SCORE CRITERIA
Criteria for exposure group and level infection rates
(GlRg and LIRj)a by number infected in episode
(both group and level criteria must be satisified)
Risk ratio
score
3-4
5-8
9 or more
2 groups: GIRLo=0
and
3 levels: LIRHi/LIRInt>3
GIRHi/GIRLo>3
and
LIRHi/LIRInt>2
and
LIRInt/LIRLo>l
GIRHi/GIRLo>2.5
(or GIRei-GIRLo>15% points)
and
LIRHi/LIRInt>2
and
LIRHi/LIRLo>2
2 groups: GIRHi/GIRLo>2.5 GIRHi/GIRLo>2
and
3 levels: LIRHi/LIRInt>2
and
LIRHi/LIRLo>2
and
LIRHi/LIRInt>l
and
LIRHi/LIRLo>l
GIRHi/GIRLo>1.5
(or GIRHi-GIRLo>10* points)
and
LIRHi/LIRInt>l
and
LIRHi/LIRLo>l
No distinct
pattern
No distinct
pattern
No distinct
pattern
2 groups: GIRLo/GIRHi>2.5 GIRLo/GIRHi>2
and
3 levels: LIRLo/LIRInt>2
and
LIRLo/LIRHi>2
2 groups:
and
3 levels: LIRLo/LIRIllt>3
and
LIRLo/LIRInt>l
and
LIRLo/LIRHi>l
GIRLo/GIRHi>3
and
LIRInt/LIRHi>2
and
LIRLo/LIRInt>.l
GIRLo/GIRHi>1.5
(or GIRL0-GIKHi>l°* points)
and
LIRLo/LIRInt>l
and
LIRLo/LIRHi>l
GIRLo/GIRHi>2.5
(or GIRL0-GIRHi>15% points)
and
LIRLo/LIRInt>2
and
LIRLo/LIRHi>2
a GIRg - infection incidence rate of AEI group g, %
LIR1 - infection incidence rate of AEI level 1, %
GIRHi/GIRL0=RR for exposure groups
LIRHi/LIRLo=RR for exposure levels
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provided the observations (i.e., infection episodes) are jointly independent.
Six interpretable groups of jointly independent and mutually exclusive
infection episodes were defined, based on the criteria given in Table 15.
A one-sided sign test of the number of positive scores (++ or +) compared
to the number of negative scores (- - or -) was conducted for each jointly
independent group to determine if there was a significant excess of positive
risk ratio scores for the infection episodes in the group.
Groups C and F of control infection episodes should have symmetric
frequency distributions of RR scores about the score of 0. Under the null
hypothesis of no association between infections and wastewater aerosol
exposure, Groups A, B, D and E of exposure infection episodes should also
exhibit frequency distributions of RR scores which are symmetric about
0, with no excess of positive scores over negative scores. If there were
a significant excess of positive scores (at a=0.05 by the one-sided sign
test) in a group of exposure infection episodes, this would provide an
overall indication of apparent association of infections with wastewater
aerosol exposure.
Analysis of Incidence Density Ratios (IDR) using Test-based Confidence
Intervals
Test-based confidence intervals were constructed to determine if the
ratio of the incidence density (ID) of highly exposed participants to the
incidence density of less exposed participants significantly exceeded one.
This incidence density ratio (IDR) analysis was applied both to new infections
detected serologically and to new self-reported acute illnesses.
The average rates of infection events determined as seroconversions
(i.e., fourfold or greater increases in titer in paired sera) were estimated
as incidence densities for the low (AEK1) , intermediate (15) exposure levels and for the low (AEK3) and high (AEI>3) exposure
groups. The infection ID was expressed as the number of new infections
per hundred person-years of observation:
No. of New Infections in
ID = u ?1f"l5"f"1"^: 7 * (365.25 days/yr) x (100 years)
No. of Person-days Observed ' J J
During Interval
ID was calculated for the seven time intervals defined as serologic periods
of observation in Section 4G and the ''irrigation'' interval from January
1982 to October 1983 spanning all observed periods of irrigation.
In accumulating the numerator and denominator of ID, the participant's
aerosol exposure index for the period of interest was used to categorize
that participant by exposure group or exposure level. For example, if
a person had an infection in the spring of 1982 and was considered to be
in the high exposure level during irrigation in spring 1982 and in the
intermediate exposure level during irrigation for the entire year of 1982,
that person's infection and cumulative person-days of observation would
be included in the high exposure level in the ID calculation for the spring
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of 1982, but in the intermediate exposure level in the ID calculation for
the 1982 time interval.
The exception to this rule was in the ID calculation for the entire
irrigation time interval. In this case, when an individual's exposure
level changed between irrigation periods, his infection events and person-days
of observation were accumulated in the proper exposure level for each irrigation
period. For example, suppose a person had two infection events to a group
of four agents while in the high exposure level in spring 1982, one infection
while in the intermediate exposure level during summer 1982, and no infections
while in the high exposure level in either spring or summer 1983. Suppose
the person had 600 agent-person-days of observation each in spring 1982,
summer 1982, and spring 1983, but 400 agent-person-days of observation
in summer 1983. Then, in the ID calculation for the high exposure level,
the person would contribute 2+0+0=2 infection events and 600+600+400=1600
person-days of observation. He would also increase the numerator and denomi-
nator of ID for the intermediate exposure level by 1 infection event and
600 person-days, based on his summer 1982 experience. As this example
illustrates, a person's experience could be allocated to several exposure
levels or groups in the ID calculation for the irrigation time interval.
In cases where a seroconversion could not be located to a specific irrigation
season, the aerosol index for the appropriate year (1982 or 1983) was used
to categorize the participant's exposure as high, intermediate or low.
The infection and the appropriate high person-days of observation were
then accumulated in that exposure level for the entire year.
Three incidence density ratios (IDRs) were calculated. For exposure
groups, IDR = IDm/IDLo. For exposure levels, two IDRs were calculated:
for the high-to-intermediate exposure levels (IDg^/IDjQ^) and for the high-
to-low exposure levels (IDgi/IDL0). We are interested in testing the null
hypothesis of no association between infections and wastewater irrigation
against the alternative of a positive association. This is equivalent
to determining if IDR is significantly larger than 1.0.
Confidence intervals were constructed for each IDR using Hiettinen's
test-based confidence interval approach as described by Kleinbaum et al. (1982)
on pages 300-302. They point out that the statistical properties of the
test-based confidence interval need additional study. Test-based intervals
tend, on the average, to be a little narrower than Taylor series intervals,
but the discrepancy is usually negligible when 0.25
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to the binomial distribution is used, the expected number of infections
in both exposure groups should be large enough, say np^5 and np2>.5.
Infection IDRs and their 90% and 95% confidence intervals were calculated
for each individual serologic agent and for the six groupings of serologic
agents given in Table 18. Results from the entire baseline and entire
irrigation periods were compared, both by exposure levels and by exposure
groups. Results for individual agents and the six agent groupings were
also calculated for each of the eight time intervals by exposure levels.
Whenever the 90% or 95% test-based confidence interval for IDR did not
include 1.0, this result was reported, provided the expected number of
infections in each of the exposure levels or groups compared was at least
2.0. An IDR was considered to be significant if its 95% confidence interval
did not include 1.0 and if np^>_2 and
TABLE 18. DEFINITIONS FOR AGENT GROUPINGS IN SEROLOGIC DATA ANALYSIS
SNV All nonpolio viruses tested by serum neutralization. This group
includes all coxsackieviruses, echoviruses, and adenoviruses.
WWV All viruses recovered from Lubbock wastewater during the period
of observation (see Tables 25-27 and 39). This group is a large
subset of the SNV grouping, consisting of coxsackieviruses and
echoviruses (see Table 99).
FOR Serum neutralization (SNV) viruses which caused too few infections
during the period of observation to constitute a distinct infection
episode. Since wastewater contains many infections agents, it
was felt that ''sporadic infections'' by a variety of agents
might be the most subtle effect of wastewater exposure.
ADEN Adenoviruses 3,5, and 7. (This grouping was used only for calculating
incidence densities).
COXB Coxsackieviruses B2, B4, and B5. (This grouping was used only
for calculating incidence densities).
ECHO Echoviruses 1, 3, 5, 9, 11, 17, 19, 20, 24. (This grouping was
used only for calculating incidence densities).
The average rates of self-reported acute illness were also estimated
as incidence densities for the three exposure levels and two exposure groups.
The illness ID was expressed as the number of new illnesses per 1000 person-days
of observation. The ID was determined for total acute illnesses and for
the subcategories of respiratory illness, gastrointestinal illness, and
other acute illnesses such as eye and ear infections and skin conditions.
ID was calculated for time intervals of ''months1' which were usually of
4-weeks duration. Otherwise, the illness ID was calculated in the same
manner as the seroconversion ID. Illness IDRs and their test-based confidence
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intervals were also computed for exposure groups and for exposure levels
in the same manner as the seroconversion IDRs.
The assumption in using a test-based confidence interval of a binomial
experiment regarding the allocation of events among the two exposure groups
or levels in the IDR appears reasonably valid, both for serologically-detected
infections and for self-reported acute illnesses. As in most LISS analyses,
the assumption of independence may not be strictly valid because of the
greater likelihood of within-household transmission of the infectious agent.
However, over the 6-month or greater time interval of the seroconversion
ID and the 4-week time interval of the illness ID, this effect is likely
to balance out over the two groups being compared. There are intrinsic
differences among individuals which cause them to respond differently,
regarding the probability of both a seroconversion and of self-reported
illness, to a given challenge by the same agent. The serological overreactors
and underreactors are likely to be evenly distributed throughout the study
population, and hence balanced among exposure levels and groups. Because
self-reporting of acute illness could be biased by the odor of nearby wastewater
irrigation, illness overreactors and underreactors might not be distributed
in a balanced manner by exposure levels and groups.
By using a population-time denominator for ID, the IDR analysis using
a test-based confidence interval takes proper account of periods of
non-observation or nonrisk (i.e., missing reporting periods or days spent
outside the study area). When applied to groups of serologic agents, multiple
sites of acute illness and/or consecutive periods of observation, this
analysis takes proper account of the multiple infection or illness events
which a participant is liable to experience. On the other hand, the IDR
analysis is not valid unless a large number of infection or illness events
occur. Thus, the IDR analysis has most value to the LISS in providing
an overview interpretation of observed gradients in incidence density by
exposure level or exposure group when the infections to groups of serologic
agents are observed over a long time interval (entire baseline or entire
irrigation period) or when total acute illness is observed.
Other Analyses of Apparent Association of Infections with Exposure
For small sets of data, other analyses were performed as appropriate
to investigate the apparent association of the occurrence, prevalence or
incidence of infections with wastewater aerosol exposure and other pertinent
factors. Since incidence data was usually lacking and the data sets were
often small, definitive evidence of association was difficult to establish
through these analyses.
A descriptive analysis was conducted to determine the time period(s)
with the highest rates of occurrence, prevalence or incidence. Unless
there was a higher rate of occurrence during one or more periods of irrigation,
it was decided that there was no apparent association with wastewater exposure.
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When a high rate of occurrence was observed during an irrigation period,
the mean AEI of the infected donors was compared to the mean AEI of the
noninfected donors from the same irrigation period. If the mean AEI of
the infected donors was greater, a one-sided t test of the difference in
the mean aerosol exposure of the populations of infected donors and noninfected
donors was performed. A natural logarithm transformation of AEI was always
necessary to equalize the variances, as determined by the F-test, or to
minimize the variance inequality. The geometric means and the degree of
apparent association indicated by the p-value of the one-sided t test were
reported.
The possible associations of the cluster of occurrences with other
plausible environmental factors were also investigated as alternative explana-
tions. These factors included patronage of local restaurants, use of an
evaporative cooler for home air conditioning, and contamination of the
drinking water wells of rural households. Since each environmental factor
was categorical, the 2x2 contingency table of infection status and environmental
exposure was analyzed for association by a one-sided Fisher's exact test.
The p-value was reported to indicate the degree of apparent association.
K. INTERPRETATION OF STATISTICAL RESULTS
The LISS employed four methods of inference to investigate the possible
association of infections with wastewater aerosol exposure in the specific
episodes of infection which were observed in the study population. These
inferential methods are: 1) risk ratio (RR) scoring, 2) test-based confidence
intervals of the incidence density ratio (IDR) of high-to-intermediate
and high-to-low exposure levels for serologic infection episodes, 3) confir-
matory statistical analysis (CA) , and 4) exploratory logistic regression
(ELR) statistical analysis.
A score was assigned by each of the four methods of inference to every
infection episode. The RR score was assigned by the criteria previously
given in Table 17. The score for the IDR method was based on the signifi-
cance of the IDR confidence .interval (CD, provided the expected number
of infection events in each of the exposure levels compared was at least
2.0:
IDR score = - if IDR<1.0
0 if IDR>1.0, but 90% CI includes 1.0
+ if 90% CI does not include 1.0
++ if 95% CI does not include 1.0
IDR scores were assigned to the IDRs both for the high-to-intermediate
exposure levels and for the high-to-low exposure levels. The score for
the confirmatory analysis method was based on the p-value for the one-tailed
Fisher's exact test:
120
-------
CA score = if pX).95
if 0.95>p>0.15
0 if 0.100.25
0 if 0.10
-------
TABLE 19. CRITERIA FOR STRENGTH AND CONSISTENCY OF APPARENT
ASSOCIATION OF INFECTIONS WITH WASTEWATER AEROSOL EXPOSURE
IN INFECTION EPISODES
Classification
Criteria
Good
Strength of statistically significant association
by at least one of the three methods employed:
a. confirmatory analysis (CA) : p<.0.05 (score
Marginal
1.
b. exploratory logistic regression (ELR): p^.0.05
(score .> ++)
c. Incidence density ratio (IDR) of exposure
levels: 95% CI does not include 1.0, both
for Hi/Int and Hi/Lo (++ and ++)
and 2. Consistency in support for association, either
a. by another method at the degree of strength
in 1 above
or b. by at least three methods at lesser strength:
(1) CA: piO.10 (score >. +) (or p<0.15 if
RR score = ++)
(2) ELR: plO.10 (score 2. +) (or p.<0.15 if
RR score = ++)
(3) IDR either both 90% CIs do not include
1.0 ( + and +) or one 95% CI does not
include 1.0 (++ and 0 or 0 and ++)
(4) risk ratio (RR) score = + or ++
1. Strength of the association approaches statistical
significance by at least one of the three methods
employed:
a. CA: p<0.10 (score >. +)
b. ELR: piO.10 (score >. +)
c. IDR either both 90% CIs do not include 1.0
(+ and +) or one 95% CI does not include 1.0
(++ and 0 or 0 and ++)
and 2. Consistency in support for possible association,
either
a. by another method at the degree of strength
in 1 above
or b. by at least three methods at lesser strength:
(1) CA: plO.15 (score >. 0)
(2) ELR: p<.0.15 (or p.<0.20 if RR score =
(3) IDR: one 90% CI does not include 1.0
(+ and 0 or 0 and +)
(4) RR score - + or ++
122
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TABLE 20. CRITERIA FOR JUDGING QUALITY OF WASTEWATER EVIDENCE FOR EACH MICROORGANISM
Category of quality
1. Excel lent
Quality of evidence of agent In sprayed wastewater
2. Good
3. Fair
4. Presumptive
Source Measurement
(Hancock wastewater)
Frequency
Specificity
Trans*IssIon Measurement
(WfI son wastewater0)
Frequent8
Serotype
Frequent
Species/genus
Occasionalb
Genus/species
None
K>
Frequency
Specificity
Agent Monitored In
clinical specimens
(suitable as a dependent
variable In the statis-
tical analysis)
Frequent
Serotype
Specific coxsacklevlrusc'
Specific echovirus^
Frequent
Species/genus
Salmons Ilae
Shlgella6
YersInI a enteroco1111cae
Campylobacter fetus8
Fluorescent Pseudomonas8
Klebslellae
Mycobacterla (atyplcal)S
Candida alblcans8
None
Leglonella pneumophila*
Staphylococcus aureus9
Proteus/CItrobacter8
Aeromonas/Serratla6
None
Hepatitis A vlrusf
Adenovirus*
Reovirus*
Rotavlrus*
Norwalk virus*
Vlrus-lIke particles"
a Frequent: at least one measurement every four weeks.
b Occasional: about one measurement per irrigation season.
c Feces of rural donors may be substituted when the dependent variable Is serologlc.
d Infections determined from serologlc or fecal Isolate data.
e Infections determined from fecal Isolate/level data.
f Infections determined from serologlc data.
g Infections determined from skin test data.
h Infections determined by electron microscopy of fecal specimens.
-------
of the period of occurrence of the infection episode to the RAEM rank of
the agent's microorganism group in that season can determine whether the
episode occurred in the season of highest exposure to the agent via wastewater
aerosols. Alternative sources of exposure were also investigated. Contaminated
drinking water was evaluated for the subset of under 20 households whose
drinking water wells were being monitored at the time of the infection
episode.
A retrospective survey of routine fecal and requested throat swab
donors was conducted to determine the frequency with which they had eaten
at each of the restaurants in Wilson. A special ELR analysis (Analysis
2) was performed to evaluate the restaurant etiology as an alternative
explanation to wastewater aerosol exposure. Eating at the restaurants
was evaluated both as an alternative and as an additional explanation.
Another ELR analysis (Analysis 3) was performed to investigate alternative
explanations besides the restaurants. AEI was excluded from the eligible
predictor variables for infection episodes in which it had been significant
to determine if another variable would enter the model in its place.
A summary table of the evidence from the additional data sources described
above will be prepared for each of the infection episodes with good or
marginal evidence of wastewater aerosol exposure association. A review
of this evidence regarding an apparently associated episode may discredit
the association by identifying a more plausible alternative explanation.
Any episodes surviving this winnowing process are more likely to be causally
related to wastewater aerosol exposure.
Finally, the separate findings from each observed episode of infection
will be considered together to draw conclusions regarding wastewater aerosol
exposure and the incidence of infection. The relative quality and reliability
of the data upon which each finding was based will be utilized to rank
the findings. Consistency in the pattern of evidence across several infection
episodes would probably be needed to indicate a relationship between infections
and wastewater irrigation.
124
-------
SECTION 5
RESULTS
A. MICROORGANISM LEVELS IN 1ASTE1ATER
24-Hour Composite Samples—Overview
Environmental monitoring spanned a 4-year period beginning in Tune
1980 prior to on-site irrigation and continuing through September 1983.
During the 2 baseline years of the LISS, composite wastewater samples were
collected at both the Lubbock Southeast Trickling Filter Plant and the
Wilson Imhoff tank. Beginning with the delivery of treated Lubbock sewage
to the Hancock farm site in February 1982, pipeline effluent and reservoir
water were analyzed for a variety of microbiological and selected physical-
chemical parameters. Results of these analyses for each sample are presented
in Tables P-l, P-2 and P-3 in Appendix P for Lubbock wastewater (and subse-
quently pipeline effluent), Hancock farm reservoir water and Wilson wastewater,
respectively. To allow a comparative overview of these analytical parameters,
geometric mean values for each of the seasonal irrigation periods also
have been calculated and are summarized in Table 21.
From its sample profile, Lubbock wastewater effluent may be classified
as relatively strong based on both microbial and chemical analyses. A
review of data presented in Table P-l in Appendix P shows that fecal coliform
levels in effluent sampled at the Lubbock treatment plant routinely exceeded
10^ cfu/mL, while total organic carbon (TOO values ranged from 40 mg/L
to over 200 mg/L. During the first 2 years of the study, total enterovirus
levels as measured on HeLa cell monolayers ranged from 0.045 pfu/mL to
over 1.0 pfu/mL in the summer of 1980.
The first pipeline effluent was sampled at the Hancock farm in February
1982 and represented a highly atypical sample microbiologically. Once
a daily wastewater flow to the Hancock site was established, the initial
microbial and physical profile of the wastewater delivered to the irrigation
site was not dissimilar from the wastewater previously characterized at
the treatment plant (see Table P-l). However, the quality of the pipeline
effluent as indicated by TOC and TSS improved considerably after the first
irrigation period (see Table 21). In 24-hour composite samples, maximal
viral levels of about 0.1 pfu/mL were observed during spring monitoring,
while levels approaching 0.5 pfu/mL were detected during the summer 1982
irrigation season. A similar pattern of enteric viruses enumerated on
HeLa cell monolayers was observed during 1983. While viral levels in pipeline
effluent did not reach the highest levels seen in June 1982, the number
of viruses recovered remained relatively constant from late June through
August 1983 at over 0.25 pfu/mL.
125
-------
TABLE 21. QUALITY OF WASTEWATER APPLIED BY SPRINKLER IRRIGATION
Wastewater source
Measurement by
irrigation period
Pipeline
effluent*
Reservoir
effluentb
Total Organic Carbon (mg/L)
Feb-Apr 1982
Jul-Sep 1982
Feb-Apr 1983
Jul-Sep 1983
Total Suspended Solids (mg/L)
Feb-Apr 1982
Jul-Sep 1982
Feb-Apr 1983
Jul-Sep 1983
Fecal Colifons (colony forming units/mL)
Feb-Apr 1982
Jul-Sep 1982
Feb-Apr 1983
Jul-Sep 1983
Bnterovirnses (plaque forming units/mL)
105
61
61
30
149
78
72
26
43,000
13.000
20,000
9,000
22
26
25
27
29
27
130
52
29
Feb-Apr 1982
Jul-Sep 1982
Feb-Apr 1983
Jul-Sep 1983
0.04
0.05
0.07
0.17
—
0.003
0.002
0.001
a Geometric mean of four to eight 24-hour composite samples.
b Geometric mean of four or five grab samples.
126
-------
Similar data for Hancock reservoir water collected beginning in Jane
1982 are shown in Table P-2 in Appendix P and summarized in Table 21.
A comparison of both, indicator bacteria and virus levels shows that, in
general, organism concentrations in reservoir water were two to three orders
of magnitude lower than comparable pipeline effluent. Of the 19 samples
of reservoir water concentrate which were assayed in two cell lines, entero-
viruses were detected in only 12 samples with a maximal level of about
0.06 pfu/mL. In most of the reservoir samples viral levels were at or
below the detection sensitivity of the recovery procedures employed.
Microorganism concentrations in Wilson wastewater are profiled in
Table P-3 in Appendix P. Primarily as a result of the smaller collection
system in the city of Wilson, greater variability in organism levels was
observed in wastewater samples. Fecal coliform densities ranged from 10^
to in excess of 10$ cfu/mL during the monitoring period. Similarly, total
enteric virus levels as assayed on HeLa cell monolayers varied from no
virus detected in five samples to over 3 pfu/mL in two samples.
Of particular interest in the 1982 monitoring period were the unusually
high levels of polioviruses persisting in the Wilson wastewater from March
to May. Viruses did not appear in the Wilson sewage until 3 weeks after
the first Lubbock wastewater (also containing predominantly polioviruses)
was collected at the Hancock farm. Notably, polioviruses 2 and 3 comprised
most of the identified isolates from both sources. Although less dramatic,
a similar pattern of poliovirus prevalence was observed in Wilson wastewater
from March to May during 1983.
Extensive bacterial screens were completed on selected 24-hour composite
samples in an effort to better define the microbial content of wastewater
destined for spray irrigation. Identical analyses were completed on Wilson
sewage to determine if any unique microbial differences existed between
the Lubbock and Wilson wastewaters. Results are presented in Tables 22,
23 and P-4 (Appendix P) for Lubbock wastewater, Hancock reservoir water,
and Wilson wastewater, respectively. The most prevalent Enterobacteriaceae
species encountered in wastewater from either Lubbock or Wilson included
Citrobacter. Enterobacter, Escherichia and Klebsiella . Aeromonas hydrophila
was the most abundant non-Enterobacteriaceae member recovered followed
by Pseudomonas species. In fact, Aeromonas hydrophila was the most prevalent
organism detected in wastewater. No unexpected differences were observed
in microbial profiles. The effectiveness of ponding for the reduction
of microbial numbers was evident both by the lower levels and the reduced
diversity of organisms seen in a single bacterial screen completed on a
sample from the Hancock reservoir (see Table 23).
24-Hour Composite Samples—Bacterial Pathogens
Specific attempts were made during this study to isolate major enteric
bacterial pathogens from wastewater, including Salmonella sp., Shigella
sp., Campylobacter .iejuni. Yersinia enterocolitica. and Legionella pneumophila.
In both Lubbock and Wilson wastewaters. Salmonella sp. were recovered most
frequently with isolations from 62% and 35% of the samples tested, respec-
tively. Campylobacter ieiuni and Yersinia enterocolitica were recovered
127
-------
TABLE 22. BACTERIAL SCREENS8—LUBBOCK, TEXAS
Sampling date
1980
1981
1982
Qrnan 1 ems t103 cfu/mLl
Jun
3-4
Jul
28-89
Nov
3-4
Apr Jul Febb'° Mar Jul
20-21 20-21 15-16 22-23 26-27
Enter
Cltrobacter analonetlcue - -
Cltrobacter diversus - - - -
Cltrobacter fraundll 15 - 10 -
Enterobacter eerogenea 5 10 10 -
Enterobacter agglomerans 16 10 20 10
Enterobecter cloacae 20 30 - 20
Enterobacter aakazakll 5 - -
EacherlcMa coll 20 20 30 20
EacheHchla coli alkalescena - 50
Klebalella oxytoca 7 - 10 20
Klebalelle ozaenae 5 -
Klebalella pneumonias 5 - 10 -
Morganella norganll - -
Provldencla alcallfaclena - - - 10
Provldencla rettgerl - - -
Serratla llquefeclena - -
Sarratia marcescens 5 - -
Sarratle rubldaea 3 10
Vibrio fluvlalla - -
Yeralnla enterocolltlca 10 - -
Yerslnla krlatanaenll - - -
Achronobecter spp.
Achrooiobacter xyloaoxldana
Aclnetobacter calcoacetlcua
var. Lwoffl
AeroBonas hydrophlla
Alcallgenea ap.
API—Group Id
CDC Group II K-2 - -
CDC Group V E-2 - -
Chronobacterlun ap. 5 -
Elkanelle corrodens -
FlavobacteMum odoratum 3
Fluorescent Paeudcmonea gp.
Paeteurelle multoclda 10
Pseudononae cepacla 10 10
Paeudononas fluoreacena 15 10
Peeudononaa maltophUia 5 -
Paeudomonea putlde 30 - 30 20
Pseudomonas putrefaclena 60 20 10 20
Paeudononae etutzerl 10 -
Pseudononae sp., other 25 140
Vibrio alfllnolytlcue - - - -
93
S
20
560
10
10
590 510
10
10
10
10
15
25
5
15
210
10
5
10
10
25
5
0.05
0.05
0.43
0.13
0.4
0.025
0.025
0.025
1.3
0.13
0.025
0.025
2
4
4
2
1
3
5
8
1
1
6.6
3
10
16
10
3
6.6
3
3
56
10
3
150
10
3
10
6.6
20
a Highest levels obeerved on either MacConkey agar or brilliant green agar and Identified
by API 20E blochenlcal testa.
b On February 15, 1982 the sample source was changed from the trickling filter to the
pipeline.
c Chlorlnatlon of waatewater effluent at treatment plant.
d A group of organisms which to date have been described by CDC and have been designated
temporarily by API as API Group I.
128
-------
TABLE 23. BACTERIAL SCREEN8—HANCOCK RESERVOIR
Sampling date
Jul 26-27.
Organisms (103 cfu/mL) 1982
Enterobacteriaceae
Enterobacter cloacae 0.4
Klebsiella oxytoca 0.1
Klebsiella ozaenae 0.1
Non-Bnterobaeteriaceae
Achromobacter xylosoxidans 0.9
Acinetobacter calcoaceticus var. Lwoffi 0.2
Aeromonas hydrophila 4.3
Alcaligenes sp. 0.5
CDC Group V E-2 0.1
Pseudomonas sp. 0.5
Pseudomonas cepacia 0.1
Pseudomonas maltophilia 0.3
a Highest levels observed on either MacConkey agar or
brilliant green agar and identified by API 20E bio-
chemical tests.
in approximately one-third of the Lubbock samples tested for these organisms
while only Yersinia was detected in a single bacterial screen of Wilson
effluent. Shigel la sp. were detected in Lubbock wastewater in 12% of the
samples analyzed. The only major enteric pathogen recovered from reservoir
water was a single isolation of Salmonella.
Table 24 summarizes the results of UI efforts to isolate Legionelja
from wastewater samples. No isolates of these agents were recovered from
any of the seven samples processed, although antigens from a variety of
serogroups and species were repeatedly demonstrated in wastewater samples
and in tissues of guinea pigs inoculated with those samples. Most isolates
of potential Legionella group agents grew readily on TSA or blood agar.
One isolate, not growing on TSA in the UI laboratory, was forwarded to
the Illinois Department of Public Health Bacteriology Laboratory where
it grew on a number of media, including TSA, suggesting the isolate was
not Legionella.
The UI experience in isolation attempts of Legionella from water samples
is not unusual; others have also been unable to recover viable Legionellae
from DFA positive samples. Factors influencing the inability to recover
Legionella include the susceptibility of experimental animals to Leg ionella
infection, viability and virulence of Leg ionella present in wastewater
samples, and the levels of both Leg ionella-group and non-Leg ione 1 la agents
present in those samples.
129
-------
CO
o
TABLE 24. SPECIES OF LEGIONELLA DETECTED8 IN WASTEWATER SAMPLES BY DIRECT FLUORESCENT ANTIBODY
STAINING OF THE ORIGINAL SAMPLES OR TISSUES FROM GUINEA PIGS INOCULATED WITH THOSE SAMPLES
Sample
Febrmary 16. 1982
Pipeline effluent
March 22-23. 1982
Trickling filter
Pipeline effluent
June 29-30. 1982
Pipeline effluent
Reservoir
July 26-27. 1982
Pipeline effluent1*
Reservoir0
L. pneumophila L. L.
12345 6 bozemannii dumoffii
+ - - + NA NA
+ - - + NA NA - +
+ - - - NA NA - +
+ -- + NANA + +
+ - - + NA NA - +
I- + + -- - +
L. L. L. longbeacheae
jtormanii micdadei 1 2
NA NA NA
+ NA NA NA
+ NA NA NA
NA NA NA
NA NA NA
I - I -
NA - conjugates not available.
a All species detected for samples collected on February 16, March 22-23, and June 29-30 came from
guinea pig tissue
b Examination by direct fluorescent antibody staining of wastewater sample only.
-------
The inability to consistently recover L. pneumophila from guinea pigs
inoculated with up to 10^ cfu of yolk-sac passed stock cultures suggests
that there may be differences in susceptibility or that extremely high
doses of Leg ione1 la are needed to infect some animals. The difference
in lethal doses of Leg ionella pneumophila in egg-passed and agar-passed
cultures, reported by HcDade and Shepard (1979), suggests facultative differ-
ences in virulence factors and it is possible that the Leg ione lla observed
in Lubbock wastewater samples were relatively avirulent. It is also possible
that these agents were nonviable, since isolates were not recovered from
samples inoculated onto artificial media. The low levels of Legionella
and high levels of non-Leg ione lla present in the samples undoubtedly influenced
the results. Isolation work using guinea pigs involves a trade-off between
concentrating samples sufficiently to obtain infections doses of Legionella
consistently and diluting samples to nonlethal levels of other agents.
24-Hour Composite SampJ.es--Hum an Enteric Viruses
The basic assay used for the quant it at ion of human viruses beginning
in April 1981 allowed for the estimation of poliovirus levels in any sample
taken as the difference between unaltered and poliovirus neutralized values
enumerated on HeLa cell monolayers (see Tables P-l, P-2 and P-3 in Appendix
P) . In addition, extensive efforts were directed toward the identification
of enteric viruses in selected wastewater samples. In some instances the
presence of given viruses in sprayed wastewater was used in the selection
of viral reagents for serological testing, especially if the study population
showed a low level of immunity to the specific virus.
Specific viral identifications of environmental isolates are provided
in Tables P-5 (in Appendix P), 25 and 26 (Lubbock wastewater), 27 (Hancock
reservoir), and Tables P-6, P-7 and P-8 in Appendix P (Wilson wastewater).
It should be noted that during the later portion of this study problems
were encountered in the use of the Lira Benyesh-Melnick enterovirus typing
pools in the RD cell line. Hence, isolates recovered as plaques on RD
cell monolayers were not identified.
In addition to the expected recovery of all three polioviruses, selected
coxsackie A and the first five coxsackie B viral serotypes were recovered
during this study. Twenty recognized serotypes of echoviruses were also
identified in wastewater samples. Not unexpectedly, seasonal occurrences
of various human viruses were observed. This phenomenon was more pronounced
in Lubbock wastewater, most likely due to the larger contributing population.
The larger wastewater system also resulted in a greater diversity of viral
types being recovered from Lubbock samples.
In general, poliovirus serotypes predominated during spring sampling,
while coxsackie B viruses were more prevalent in the summer and fall.
Poliovirnses also reappeared in selected August-September samples, presumably
reflecting preschool immunizations. Although echoviruses were found year
round, most isolates were recovered during the summer months.
131
-------
TABLE 25. VIRUSES ISOLATED FROM LUBBOCK PIPELINE EFFLUENT DURING 1982
ID
to
Sampl Inq Date
Assay
HeLa (unaltered concentrate)
Concentration (pfu/L)
Virus type
Polio 1
Polio 2
Polio 3
Coxsackle B2
Coxsackle B4
Coxsackle B5
Echo 1 1
Unidentified
TOTAL SAMPLED
HeLa (polio-neutralized)
Concentration (pfu/L)
Virus Type
Polio 3
Coxsackle B5
Echo 1
Echo 31
Unidentified
TOTAL SAMPLED
RO (polio-neutralized)
Concentration (pfu/L)
Virus type
Coxsackle A16
Coxsackle A19
Coxsackle 85
Echo 12
Echo 15
Unidentified
TOTAL SAMPLED
Mar
8-9a
110
3
6
2
1
6
18
22
1
1
1
6
9
<2
Mar
22-23
63
1
4
3
1
1
10
4.0
10
Apr
5-6
17
8
1
9
3.9
1
1
2
44
2
1
1
10
14
Apr
19-20
42
3
6
2
7
2
20
16
5
1
6
10
Jun
29-30
490
23
23
390
11
11
56
1
1
4
7
13
Jul
26-27
60
1
2
5
1
9
30
6
6
6.6
3
3
Sep
13-14
22
1
2
2
1
3
2
11
8.0
4
4
840
a Chior I nation of wastewater effluent at treatment plant.
-------
TABLE 26. VIRUSES ISOLATED FROM LUBBOCK PIPELINE EFFLUENT DURING 1983
Assay
HaLa (unaltered concentrate)
Concentrat 1 on ( pf u/L )
Virus type
Polio 1
Polio 2
Polio 3
Coxsackle A13
Coxsackle 82
Coxsackle B3
Coxsackle B5
Echo 25
Unidentified
TOTAL SAMPLED
HeLa (polio-neutralized)
Concentration (pfu/L)
Virus Type
Coxsackle B2
Coxsackle B3
Coxsackle B4
Coxsackie B5
Unidentified
TOTAL SAMPLED
RD (polio-neutralized)
Concentration (pfu/L)
Virus type
Echo 19
Unidentified
TOTAL SAMPLED
Feb
16-17
44
3
3
4
1
11
20
5
5
6
6
Mar
21-22
31
1
2
1
1
1
1
7
16
2
1
1
4
6
6
Sampl Ing Date
Apr Jul
18-19 11-12
100 280
11
1 1
2
4
15
1
13 22
<4 300
680
7
13
20
Aug
8-9
120
1
10
12
23
130
1
10
1
3
1
16
12
12
Sep
12-13
56
2
1
11
14
180
13
4
17
15
15
TABLE 27. VIRUSES ISOLATED FROM HANCOCK FARM RESERVOIRS DURING 1983
SamplIng Date
Assay
Feb
16-17
Mar
21-22
Apr
18-19
Jul
11-12
Aug
8-9
Sep
12-13
HeLa (unaltered concentrate)
Concentration (pfu/L)
Virus type
Pollo 2
Coxsackle B5
TOTAL SAMPLED
* No Isolates.
-------
During the summer and fall of 1980 and 1983, coxsackie B3 and B5 viruses
were present at high levels in Lubbock wastewater, while only cozsackie
B5 predominated during the same period of 1981 and 1982. In Wilson sewage
coxsackie B3 appeared at substantial levels only during 1980. Coxsackie
BS was prevalent in Wilson during the summer and fall of 1982 and 1983.
The only elevated levels of coxsackie B2 observed during the course of
environmental monitoring occurred in Wilson wastewater during the fall
1983.
Only two of the Hancock reservoir samples designated for viral identifi-
cation analysis yielded viruses (Table 27). Poliovirns 2 and coxsackie
B5 were recovered from this source during 1983.
24-Hour Composite Samples—Geometric Mean Data
To provide a basis of comparison between various irrigation seasons
and to describe the potential microbial aerosol exposure during any given
irrigation period, geometric means were computed for indicator organisms,
viruses and physical-chemical analyses. Calculated values for Lubbock
wastewater and Hancock reservoir water and Wilson wastewater are presented
in Tables 28, 29 and P-9 (in Appendix P), respectively.
Comparing mean organism levels in the spring and summer of 1982 and
1983, one can see a substantially higher viral load in pipeline effluent
during the second year of irrigation (see Table 28). Conversely, geometric
mean data for Hancock reservoir samples collected during the summers of
1982 and 1983 suggest that once established the holding ponds produced
effluent containing lower levels of fecal coliform and enteric viruses
(see Table 29). Therefore, although the levels of microorganisms found
in pipeline effluent increased during 1983, as shown in Figure 5, the actual
aerosol load was reduced during the second year of irrigation since virtually
all irrigated wastewater was drawn from the Hancock reservoirs.
30-Minute Composite Samples
Composite wastewater samples generally of 30 minutes duration were
collected in 1982 during each microorganism, virus and quality assurance
aerosol run and assayed for the microorganisms monitored in the aerosol,
for enteroviruses, and for selected physical-chemical parameters. Results
of these analyses are presented for pipeline wastewater during the irrigation
in spring 1982 (Table P-10 in Appendix P) and summer 1982 (Table P-ll in
Appendix P) and for reservoir wastewater during the summer 1982 irrigation
(Table P-12 in Appendix P).
The 30-minute composite wastewater samples had similar values for
all monitored parameters to those observed in the 24-hour composite samples
for the same wastewater source. Thus, the aerosol sampling data should
be representative of the microorganism levels in air generated by the irrigation
system in 1982. Because aerosol sampling was conducted daily during some
weeks, the 30-minnte composite samples provide an indication of daily vari-
ability. The enterovirns level (5-day assay on HeLa cells) in the pipeline
water was markedly elevated during the 2-day period when virus run V3 was
134
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TABLE 28. GEOMETRIC MEAN OF MICROORGANISM CONCENTRATIONS IN LUBBOCK WASTEWATER
<*>
ut
Lubbock STP effluent
Sampling period
Number of samp lee
Bseterls [cfu/mL)
Standard plate count
Total coll forms
Fecal co 11 forms
Fecal streptococci
May/Jun BO
6-3
1
3,600,000
350,000
67,000
4,700
Summer 80
7-86
1
5,700,000
360,000
72,000
2,000
Fall-Win 60
11-3/1H9
2
3,400,000
92,000
36,000
5,100
Spring 81
2-16/4-20
4
9,600,000
180,000
40,000
6,900
May/Jun 81
5-4/6-15
2
360,000
97 ,000
1,100
Summer 81
6-28/BH7
3
3,000,000
210,000
77,000
4,600
Fall-Win 81
11-17/2-15
2
26,000
11,000
VI
(pfu/mL)
Bacteriophage
En tero viruses
1,400
3,200
1,500
1,600a
2,100°
900a
HeLa, 5-day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Physical Analyses (mg/L]
Total organic carbon
Total suspended solids
Total volatile suspended solids
pH
0.78
83
96
65
6.5
1.2
40
78
52
6.6
0.26
115
199
132
7.1
0.054
0.018
0.008
142
158
123
7.2
0.11
0.020
0.070
70
74
64
7.0
0.063
0.018
0.16
92
53
39
7.0
0.045
0.001
0.065
117
114
91
7.2
continued.
-------
TABLE 28. (CONT'D)
Sampling period
Number of camples
Butarla (cfu/raL)
Standard plate count
Total coll forms
Fecal coli forms
Fecal streptococci
Spring 82
3-1/4-26
8
57,000
43,000
3,400
Hay/Jun 82
6-14/8-29
2
67,000
2,000
Summer 82
7-26/9-^3
4
1,300,000
120,000
13,000
880
Pipeline effluent
Fall-Win 82
11-V12-13
2
190,000
39,000
1,400
Spring 83
2-16/4-18
5
190,000
20,000
4,100
Hay/Jun 83 Summer 83
6-27 7H 1/9^1 2
1 S
59,000 90,000
1,200 160
VI
(pfu/mLj
Bacterlophage
En tero viruses
560
840
180D
4,700°
HeLa, 5-day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Physical AMlyns (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
PH
a Based on a single sample.
b Based on two samples.
0.043
0.009
0.014
105
149
117
7.3
0.11
0.10
0.12
65
92
74
7.2
0.049
0.026
0.011
61
78
60
7.5
0.10
0.041
0.22
54
91
69
7.5
0.071
0.018
0.037
67
72
57
7.6
0.27
0.14
0.34
42
35
25
7.6
0.17
0.20
0.22
30
26
20
7.5
-------
TABLE 89. GEOMETRIC MEAN OF MICROORGANISM CONCENTRATIONS IN HANCOCK RESERVOIR WASTEWATER
May/Jun 82 Summer 82 Fall-Win 82 Spring 83 Hay/Jun 83 Summer 83
Sampling period 6-14/6-29 7-26/9-13 11-1/12-13 2-16/4-18 6-27 7-11/9-12
Number of samples 242515
Bacteria (cfu/mL)
Standard plate count
Total col i forms
Fecal co 11 forms
Fecal streptococci
VI raws (pfu/mL]
Bacteriophage
Enteroviruses
HeLat 5-day (unconnected)
He La, polio-neutralized
RD, polio-neutralized
Physical Analyaaa (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
pH
180
8
16
0.005
0.017
<0.009
26
121
37
7.7
36,000
500
130
3
0.85°
<0.002
0.002
0.004
22
27
23
8.1
3,200
50
2
0.010
0.004
0.006
28
50
42
8.4
1,300
52
54
29°
<0.004
<0.004
<0.004
26
29
20
8.5
300
10
0.004
<0.004
<0.004
17
11
6
8.2
29
1.9
<0.004
<0.004
25
27
20
8.9
a Based on two samples.
-------
conducted (August 3-4, 1982). The polio-neutralized enterovirus assays
indicate that over 90% of the enteroviruses in the pipeline wastewater
on these days were polioviruses . Differential assays also indicate that
the predominant enteroviruses in the sprayed pipeline wastewater were polio-
viruses during the spring 1982 irrigation and nonpolioviruses during the
summer 1982 irrigation (except August 3-5), consistent with the 24-hour
composite results. As expected, sporadic chlorination at the Lubbock treatment
plant reduced bacterial indicator levels in the sprayed pipeline wastewater
but had no apparent effect on enteric virus levels.
B. MICROORGANISM LEVELS IN AIR
Aerosol sampling data from the dye, particle size, background, microor-
ganism, and virus runs follow. The sampling dates and meteorological conditions
(Tables A-12 to A-16 in Appendix A), the sampler layouts (Figures 9 to
12), and the specific sampling conditions (Tables A-5 to A-10 in Appendix
A) were previously presented for each of these runs. Data from the quality
assurance runs were presented in Tables A-27 to A-29. A summary of sampled
microorganism levels in air and inferences regarding downwind transport
of aerosolized microorganisms are presented. Estimated distributions of
AEI and other exposure measures are also presented.
Aerosolization Efficiency
One characteristic of a wastewater spray irrigation system that has
a direct effect on exposure to aerosolized microorganisms is the aerosolization
efficiency of the system. Aerosolization efficiency is defined as the
proportion of the sprayed wastewater that forms droplets small enough to
be carried downwind.
The aerosolization efficiency can be estimated through the use of
a tracer dye. A measured amount of dye is injected into the wastewater
of an operating spray rig. The concentration of dye in the air is measured
at several points downwind of the rig. An atmospheric dispersion model
is then used to estimate the dye concentration at each sampler location
assuming complete aerosolization. The ratio of the measured concentrations
to the calculated concentrations gives an estimate of the aerosolization
efficiency.
Four dye runs were conducted to provide estimates of the aerosolization
efficiency of the center pivot sprinkler system at the Hancock farm as
operated during 1982 and 1983 (i.e., prior to the installation of spray
nozzles which reduced aerosol production and drift). During injection
of the Rhodamine dye, wastewater grab samples were collected at 1-minute
intervals and assayed to determine the source strength of the dye. The
dye concentrations in wastewater are presented in Table P-13 (Appendix
P). Sampling was conducted only for minutes when dye was visible in the
sprayed wastewater. The dye concentrations sampled in air are presented
in Table P-14 (Appendix P). The lowest dye concentrations measured in
air exceeded the method detection limit of 0.2 x 10~*> fig/m^ by a factor
of 2.
138
-------
The dispersion model utilized was the Volume Source Diffusion Models
Program (Cramer et al., 1972) that had been used to calculate the aerosolization
efficiency of the Pleasanton, California, wastewater irrigation system
(Anderson, 1977). Each spray nozzle was considered to be a separate volume
source. The volume source parameters assigned were based on photographs
of the operating spray rigs and on rig design data. The vertical dimension
was estimated to be the nozzle height variation (0.6 m) plus the initial
depth of the spray pattern (0.3 m). It was assumed that the spray rigs
were designed with an overlap of approximately 100%. Therefore, the horizontal
dimension of each volume source was set equal to the distance between the
two nozzles immediately adjacent to the particular nozzle. These dimensions
were divided by 4.3 to get the initial values of the standard deviations
of the crosswind and vertical concentrations for each source. All sources
were assumed to be at 1.8m above the ground. Meteorological input parameters
such as mean wind speed were obtained from field measurements at the run
location and at the electronic weather station (Table A-1S in Appendix
A). The standard deviation of the wind azimuth angle was set equal to
the wind direction range divided by six. The standard deviation of the
wind elevation angle was determined using the solar angle, the cloud cover
and the wind speed. The effect of reflection from the top of the surface
mixing layer was considered to be insignificant and was not included in
the calculations.
An estimate of the concentration of dye that would have been measured
at each receptor had all of the wastewater been aerosolized is presented
in Table 30. The corresponding aerosolization efficiencies calculated
for each of the samplers for each of the dye runs are also given in Table
30.
The aerosolization efficiency data are summarized in Table 31. The
calculated aerosolization efficiency decreased with distance on each run,
as expected, since some of the larger aerosols present at the nearer sampling
distance should have settled out by the farther sampling distance. The
median aerosolization efficiency over the four dye runs was 0.75% for the
nearer samplers (25-40 m), 0.40% for the farther samplers (75-80 m) , and
0.56% overall. This analysis indicates that about 0.40% of the nonvolatile
materials in the wastewater escaped the Hancock farm spray zone as an aerosol
during 1982 and 1983.
As Table 31 shows, the aerosolization efficiency values for the Hancock
farm system in 1982 are about 50% to 100% larger than the corresponding
median aerosolization efficiency values obtained for the Pleasanton, California
irrigation system in 1977. This finding agrees with the visual impression
that Hancock farm rigs appeared to be producing more aerosol. The median
aerosolization efficiency was also higher for the Hancock farm system (0.56%)
than for two other wastewater spray irrigation system which have been similarly
evaluated (Camann, 1980): Fort Huachuca, Arizona (0.29%) and Deer Creek
Lake State Park, Ohio (0.47%). Given the manner in which the Hancock farm
spray nozzles deflected the wastewater upwards, it is not surprising to
find a higher aerosolization efficiency for the Hancock spray system during
the LISS, compared to other spray irrigation sites.
139
-------
TABLE 30. CALCULATED CONCENTRATIONS AND CORRESPONDING
AEROSOLIZATION EFFICIENCY POINT ESTIMATES FOR
EACH SAMPLER DURING EACH DYE RUN
Run
no.
Dl
D2
D3
D4
Sampler
position
3 Near
3 Far
5 Near
5 Far
4 Near
4 Far
6 Near
6 Far
4 Near
4 Far
6 Near
6 Far
3 Near
3 Far
5 Near
5 Far
Concentration
Calculated
244
141
375
191
414
235
630
361
571
326
826
495
471
261
794
436
(uK/m3)
Measured
22
4.5
0.38
1.5
1.1
0.89
1.1
0.96
1.9
7.5
2.3
1.3
80
0.46
0.67
0.87
3.7
0.47
1.9
0.79
2.3
9.7
0.71
0.50
2.5
2.4
1.0
1.8
3.7
6.3
1.3
2.4
Aerosolization
efficiency. %
9.0
1.8
0.27
1.1
0.29
0.24
0.58
0.50
0.46
1.8
0.98
0.55
12.7
0.07
0.19
0.24
0.65
0.08
0.58
0.24
0.28
1.2
0.14
0.10
0.53
0.51
0.38
0.69
0.47
0.79
0.30
0.55
140
-------
TABLE 31. SUMMARY OF AEROSOLIZATION EFFICIENCY OF
THE HANCOCK FARM IRRIGATION SYSTEM IN 1982
Geometric mean
aerosolizat ion efficiency, %
Dve run number
Dl
D2
D3
D4
Hancock farm median
(4 runs, 1982)
Pleasanton, CAa median
(17 runs, 1976-77)
Near pairs
(25-40 m)
1.04
0.94
0.36
0.56
0.75
0.37
Far pairs
(75-80 m)
0.54
0.40
0.21
0.40
0.40
0.26
Total
0.75
0.61
0.28
0.51
0.56
0.33
a See Camann, 1980.
Size of Viable Particles in the Wastewater Aerosol
The distribution of sizes of all the viable particles able to reproduce
on standard plate count agar was determined upwind and at three downwind
distances from the line of spray nozzles during irrigation with pipeline
water using six-stage Andersen samplers. From these data, an estimate
was made of the percentage of viable particles smaller than 5 urn, as this
had been shown to be the range of efficient deposition in the human pulmonary
system (Williamson, 1973). Larger particles (5-7 urn) can also be a factor,
since they can enter the mouth and upper respiratory trace.
The data from the five particle size runs are presented in Table P-15
(Appendix P) and are summarized in Table 32. Fungal spores and aggregate
organisms frequently yielded plates which could not be counted and were
reported as TNTC (too numerous to count). In summarizing the sampling
data, the reported TNTC values in Table P-15 (Appendix P) were inferred
to have been large densities when the corresponding stage from the paired
sampler and of adjoining stages were large, or as probable fungal contamination
when these values were small.
The upwind viable particles had a relatively uniform distribution
of particle diameters, with 52% below 4.7 urn. Spray irrigation of pipeline
wastewater introduced a great number of large viable particles into the
air, but few small viable particles. The density of all viable particles
larger than 2 urn declined rapidly with increasing downwind distance. The
density of smaller viable particles was largely unchanged with downwind
distance. These patterns are consistent with gravitational settling of
heavy low-energy particles and size reduction through drying or desiccation
in the sprinkler aerosol. With these off-setting factors, a relatively
constant percentage (38%-44%) of viable particles were smaller than 4.7
Urn over the limited range of downwind distances investigated. Because
both gravitational settling and size reduction through desiccation continue
to operate in an off-settling manner well beyond 75 m downwind of pipeline
141
-------
TABLE 32. STANDARD PLATE COUNT DENSITY OF VIABLE PARTICLES
IN AIR BY DISTANCE AND PARTICLE SIZE
Andersen
sampler
stage
Range of
particle
sizes, urn
Geometric meana standard plate count
density in air by sampler distance
Downwind
Upwind
20-36 m
45-61 m
70-85 m
1
2
3
4
5
6
All
Percentage
3-5
3-6
X7.0
4.7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
All
1.1-4.7
0.65-4.7
200
56
66
116
70
35
550
46%
52%
1,120
850
760
280
122
22
3,160
37%
38%
350
240
210
116
96
1,740
36%
39%
1,050
40%
44%
a Geometric mean over five particle size runs of the stage arithmetic
means for the paired samplers.
irrigation, it is not possible to estimate the percentage of viable particles
smaller than 5 urn in the downwind air at the much greater distances where
most participants received their aerosol exposure.
The percentage of viable particles between 1.1 and 4.7 um in the ambient
upwind air at the Hancock farm between February and August 1982 (46%) was
very similar to the 48-49% obtained by Bausum et al. (1983) at Deer Creek
Lake State Park, Ohio, in July-August 1976 and the 42% reported by Bausum
et al. (1982) at Fort Huachuca, Airzona, in October 1975. However, there
was a marked difference among the three studies in the proportions of viable
particles in this size range in the air downwind of the spray irrigation
source. Bausum et al. consistently found that, compared to the upwind
air, a much higher proportion (between 66% and 78%) of the viable particles
were between 1 and 5 |im in the air from 21 m to 200 m downwind of the rectan-
gular field source wastewater spray irrigation system at Deer Creek Lake.
In marked contrast, they found that the proportion (43-50%) in this size
range from 46 m to 152 m downwind of a single spray nozzle (a point source
of wastewater aerosol) at Fort Huachuca was very similar to that in the
upwind air. The LISS observed a slightly lower proportion (36% to 40%)
in this size range downwind of the irrigation rig (a line source of pipeline
wastewater aerosol) compared to the upwind air. The configuration of the
wastewater aerosol source, the wastewater quality, the nozzle type, the
operating conditions, and aerosol age may all be factors which affect the
proportion of viable particles downwind of a spray irrigation aerosol source
which are below 5 (im and can be efficiently deposited in the human pulmonary
system.
142
-------
Background Microorganism Densities in Ambient Air
The outdoor air near but in an upwind direction from the homes of
eight participant households was monitored in summer before any irrigation
commenced to measure ambient microorganism levels in the vicinity of homes.
A ninth sampler was located downwind of the Wilson effluent pond to determine
if it was a source of aerosolized microorganisms.
Four background air runs were conducted in nine locations in the study
area before sunrise on the mornings of August 5 through August 8, 1980.
A detailed description of the methodology, sampler locations and sampling
conditions are contained in the Methods Section 4D. All runs were conducted
at the same time of day (6:30-7:00 AM), same season, and with the same
wind direction (from the south-southeast) to minimize sources of variability.
The sampled densities of the standard plate count, fecal coliforms,
fecal streptococci, mycobacteria, and coliphage in the ambient air during
the four background runs are presented in Table P-16 (Appendix P) . The
Wilson effluent pond does not appear to have been an appreciable source
of aerosolized microorganisms. Geometric means calculated over the four
runs are provided in Table 33 to estimate background microorganism levels
in the ambient air just upwind of homes.
Fecal coliforms were only detected in 1 of the 30 air samples near
homes (at location F). Assuming there was a constant background level
near homes throughout the study area, this background level of fecal coliforms
is estimated as 0.01 cfu/m^. As anticipated, no coliphage were detected
in the 30 air samples near homes, yielding a coliphage background level
below 0.005 pfu/m^. Mycobacteria were detected in 9 of the 30 air samples
near homes for an estimated background level of 0.05 cfn/m*. Standard
plate count, monitored as a positive control, indicated that background
bacterial concentrations in the air near homes was about 450 cfu/m*.
Fecal streptococci were prevalent in these background air samples
and were found in 27 of the 30 air samples near homes, at concentrations
ranging from 0.1 cfu/m^ to 11 cfu/m*. Geometric mean air concentrations
of fecal streptococci ranged from about 0.2 cfu/m^ at locations D, E, G
and H to 2 cfu/m^ at location A. The Wilson sites (0.87 cfu/m^ geometric
mean) appear to have differed from the rural sites (0.32 cfu/m* geometric
mean), with locations A, C and F having higher air levels of fecal streptococci
than the other locations.
The sources of the aerosolized fecal streptococci and mycobacteria
are unknown. It is possible that these organisms adhered to dust or particu-
lates, since soil samples were found to contain fecal streptococci. The
prevalence and wide distribution of fecal streptococci densities in air
between about 0.1 cfu/m^ and 1 cfu/m* suggests a normal background of this
order of magnitude throughout the study area. Further, there is no known
feed lot or similar operation south or southeast of the Wilson area which
might produce the observed effect. High air levels of fecal streptococci
were observed consistently at locations A and F and occasionally at C.
Twelve of the fecal streptococci colonies from the first air sample at
143
-------
TABLE 33. GEOMETRIC MEAN MICROORGANISM DENSITIES IN AMBIENT
AIR SAMPLED ON BACKGROUND RUNS8
Background microorganism concentration in air
Sampler location**
(near participant home)
Wilson-A
Wilson-B
Wilson-C
Wilson effluent pond-D
Rural (Hancock) -E
Rural (NE)-F
Rural (SE)-G
Rural (SW)-H
Rural (NW)-I
Wilson (geometric) mean
Rural (geometric) mean
Estimated area background
(A-I, excluding D,
geometric mean)
Standard
plate count
(cfu/m3) _
750
700
430
390
500
510
150
510
390
610
380
450
Fecal
col i forms
(cfu/m3)
<0.03
<0.04
<0.04
<0.04
<0.04
0.09
<0.04
<0.04
<0.04
<0.01
0.02
0.01
Fecal
streptococci
(cfu/m3)
2
0.3
1.1
0.2
0.2
1.5
0.2
0.2
0.3
0.87
0.32
0.47
Mycobacteria
(cfu/m3)
<0.03
0.04
0.07
0.4
0.04
<0.03
0.07
0.04
0.2
0.04
0.06
0.05
Coliphage
(pfu/m3)
<0.04
<0.04
<0.04
<0.04
<0.04
<0.04
<0.04
<0.04
<0.04
<0.012
<0.008
<0.005
NOTE: < indicates none detected in any samples at this location.
a Conducted in August 1980.
b Sampler locations shown in Figure 8.
-------
location C (8 cfu/m^) were characterized: four were classified as S_. durans.
which may be of human origin, and eight were categorized as S. bovis or
S_. eauinus. which are more likely of animal than human origin. A plausible
hypothesis is that the passage of air through Wilson elevates the levels
of aerosolized fecal streptococci of both human and animal origin. The
data at location F suggest there also are comparable isolated local sources
in some rural areas.
A high level of mycobacteria (3.4 cfu/m^) was observed on the fourth
air sample taken downwind of the Wilson effluent pond (location D) ; cows
were grazing approximately 300 to 500 m upwind during this sampling. Repre-
sentative mycobacteria colonies from this sample were speciated. All isolates
tested belonged to the ''M. avium complex,'' consisting of M. avium and
M. intracellulare. of Runyon group III. Traditionally, these species are
the major disease-associated strains of Runyon group III and hence are
classified as pathogens.
The background densities of fecal coliforms and fecal streptococci
in the ambient air were similar to those obtained by Jones and Cookson
(1983) in a Washington, D.C. suburban area over a 24-month monitoring period.
Whereas the LISS obtained ambient geometric mean fecal coliform densities
of <0.01 cfu/m^ for Wilson and 0.02 cfu/m^ for the rural study area, Jones
and Cookson did not detect fecal coliforms in their suburban study area
in 225 nj3 of ambient air «0.004 cfu/m^). The LISS ambient geometric mean
fecal streptococci densities were 0.87 cfu/m^ for Wilson and 0.32 cfu/m^
for the rural area. In the Washington, D.C. suburban area, the 95% confidence
intervals for the mean fecal streptococci density were 0.20 to 0.43 cfu/m^
in 1979 and 0.30 to 0.55 cfo/m3 in 1980, including the winter samples in
which no fecal streptococci were recovered. The Washington, D.C. suburb
had significantly higher densities of airborne bacterial particles in summer
and fall (especially September) than in the winter and spring months.
Microorganism Densities in Downwind Air from Microorganism Runs
The densities of microorganisms in the air upwind and at four distances
downwind from the irrigation nozzle line were determined simultaneously
in each of 20 microorganism runs. The wastewater density and the sampled
densities in air of fecal coliforms, fecal streptococci, mycobacteria,
Clostridium perfringens. and coliphage are presented, respectively, in
Tables P-17 through P-21 of Appendix P. These data are summarized in Table
34 by microorganism group, source of wastewater and irrigation season.
Some caution must be exercised in interpreting Table 34 since the estimated
densities were based on widely varying numbers of air samples and since
environmental conditions were not represented equiva lent ly in the various
distance categories. Nevertheless, Table 34 does provide a good overview
of the extensive air sampling data.
Statistical tests were conducted comparing the downwind and upwind
aerosol data to confirm that the Hancock farm irrigation system was a signi-
ficant source of aerosolized microorganisms. The results shown in Table
35 indicate that irrigation with pipeline wastewater was a significant
145
-------
TABLE 34. ESTIMATED DENSITIES SAMPLED ON MICROORGANISM AND VIRUS AEROSOL RUNS8
Microorganism concentration
Microorganism Wastewater
group Source-season (n
Fecal colifoms (cfu)
Pipeline-spring 1982 109
Pipeline-summer 1982 18
Reservoir-summer 1982
Fecal streptococci (cfu)
Pipeline-spring 1982 5
Pipeline-summer 1982 1
Reservoir-summer 1982
Mycobacteria (cfu)
Pipeline-spring 1982 21
Pipeline-summer 1982 24
Reservoir-summer 1982
Clostridiu» perfringens (cfu)
-Vegetative Pipeline-1982
Reservoir-summer 1982
-Sporulated Pipeline-summer 1982
Reservoir-summer 1982
Coliphage (cfu)
Pipeline-spring 1982 1
Pipeline-summer 1982
Reservoir-summer 1982
Enterovirnses (pfu)
-HeLa cells Pipeline-1982
-RD cells Pipeline-1982
o/mL)
,000
,500
320
,700
.310
11
,000
,000
100
270
3
210
<1
,060
630
2.5
0.22
C >
c Based on one to two air samples.
d Based on three to six air samples.
•
2C and
Dpwind
<0.01
<0.01
<0.03
0.08
0.2
0.04
0.2
0.07
<0.02
0.09
<0.2C
<0.04d
<0.2C
<0.01
0.3
<0.01
detected
* f* *.
, geometric mean"
Aira (no./m3 air)
Downwind of irrigation nozzle line (m)
25-89
180
200d
2
140
200d
0.04
8
0.4d
0.08d
9c
<0.07
<0.07d
11
7d
0.03
0.048d
0.050d
in each
90-149
6
2
0.2
38
5
0.2
2.1
0.6
O.ld
2d
<0.2d
0.5d
<0.2C
4
1
0.06
sample .
150-24J
3
2
0.6
23
5
0.2
0.9
0.08
0.06d
2d
ld
2
0.7
0.07
250-349
4C
0.8
<0.2C
20C
0.7
0.3°
4C
0.2
<0.05C
ld
0.4d
0.9C
0.1
0.06°
350-409
0.5
<0.2C
0.6
0.2C
0.1
<0.07C
0.9d
0.3d
0.07
0.06°
C/2, where C is aerosol concentration.
-------
TABLE 35. CONFIRMATION OF SPRAT IRRIGATION OF PIPELINE WASTEWATER
AS A SIGNIFICANT SOURCE OF MICROORGANISMS IN DOWNWIND AIR:
PAIRED DOWNWIND VS. UPWIND DENSITIES
Significant increases in mean microorganism density
in air at sampled downwind distance?*
Fecal coliforms0
Fecal streptococci
Mycobacteria
Coliphage6
90-149 m
Downwind
Yes
(0.002)
Yes
«0.0005)
Yes
«0.0005)
Yes
(0.002)
150-249 m
Downwind
Yes
(0.002)
Yes
«0.0005)
Yes
(0.05)
Yes
(0.01)
250-349 m
Downwind
Maybe d
(0.06)
Yes
(0.02)
Maybed
(0.08)
Maybed
(0.06)
350-409 m
Downwind
Insufficient
data
Maybed'e
(0.06)
No
O0.25)
Insufficient
data
d
e
Yes if p<0.05
Maybe if 0.050.10
One-sided t-test of difference in population means for paired (downwind-
upwind) observations; In (microorganism air density from average of
sampler pair) transformation of each observation used to reduce variance
inequality.
Signed rank test employed for all distances because of highly skewed
distribution of paired differences.
Lack of significance may be result of insufficient paired observations.
Significant increase using one-sided t-test of difference in two independent
population means.
147
-------
source of the monitored microorganisms to at least the following downwind
distances:
Fecal coliforms at least 200 m
Fecal streptococci at least 300 m
Hycobacteria at least 200 m
Coliphage at least 200 m
Although insufficient data existed for statistical testing, pipeline irrigation
also appeared to be a source of Clostridium perfringens to at least 200
m downwind (see Table P-20 of Appendix P).
These air data provide convincing evidence that spray irrigation of
wastewater directly from the pipeline was a substantial source of each
of the monitored microorganism groups under most conditions of actual operation
of the irrigation system at the Hancock farm. The air densities within
100 m downwind of pipeline irrigation were markedly elevated above upwind
levels, ranging from two orders of magnitude elevation for mycobacteria
to four or more orders of magnitude elevation for fecal coliforms. Under
some conditions of operation, particularly at night or at high wind speeds
(>7 m/sec), sprinkler irrigation of pipeline wastewater appeared to elevate
the ambient (upwind) density in air of fecal coliforms, fecal streptococci,
Clostridium perfringens. and coliphage beyond 400 m downwind and of mycobacteria
to about 300 m downwind.
Irrigation with wastewater which had been stored in a reservoir produced
much lower microorganism levels in air than did irrigation with pipeline
wastewater. Nevertheless, the air sampling data do demonstrate that irrigation
with wastewater stored in Reservoir 1 also was a source of aerosolized
fecal coliforms, fecal streptococci and coliphage. These organisms were
frequently detected at 125 m downwind and may occasionally have been carried
more than 200 m from rigs irrigating with reservoir wastewater.
The aerosolized fecal coliforms exhibited more rapid die-off than
did the other monitored microorganism groups. The aerosol data are consistent
with the hypothesis that a large proportion of the aerosolized colony forming
units of each microorganism were vulnerable and were rapidly inactivated
after aerosolization, while the remaining (hardy or protected) organisms
survived without detectable die-off out to the farthest distances sampled.
Microorganism densities in air downwind of spray irrigation with pipeline
and reservoir wastewater at the Hancock farm are contrasted in Table 36
with densities downwind from other wastewater aerosol sources (both spray
irrigation sites and aeration basins of activated sludge sewage treatment
plants). The geometric mean densities of fecal coliforms, fecal streptococci
and coliphage downwind of Hancock farm irrigation with pipeline wastewater
were at least one or two orders of magnitude higher than at the other sites.
However, downwind mycobacteria densities were comparable or lower. Microor-
ganism densities downwind of reservoir wastewater irrigation at the Hancock
farm were comparable or lower than at the other sites.
148
-------
TABLE 36. MICROORGANISM DENSITIES IN AIR AT HANCOCK FARM COMPARED TO
OTHER WASTEWATER TREATMENT FACILITIES (DSEPA, 1982)
Geometric mean microorganisms/cubic metera
Spray irrigation Aeration basin
Microorganism
Distance downwind
Fecal coliforas
Upwind
10-30 m
31-80 m
81-200 m
Fecal streptococci
Dpwind
10-30 m
31-80 m
81-200 m
Mycobacteria
Upwind
10-30 m
31-80 m
81-200 m
Hancock farm
Wilson^JTX
Pipeline Reservoir
<0.006b
ND ND
180 2
3 0.4
0.07
ND
150
20
0.1
ND
2.1
0.8
ND
0.4
0.3
ND
0.08
0.10
Pleasanton
CA
Schaumburg
IL
Tigard
OR
0.04
2.1
1.0
0.5
0.5
3.0
1.3
0.9
0.4
ND
3.6
1.6
0.2
0.7
0.5
0.3
<2
<2
15
<2
ND
ND
ND
ND
NDC
ND
ND
ND
0.06
5.0
2.7
1.5
<0.02
28
15
5
Coliphage
Upwind
10-30 m
31-80 m
81-200 m
<0.003
ND
10
2
ND
0.03
0.07
0.02
0.7
0.08
0.4
0.02
0.08
0.04
<0.04
<0.04
2.3
1.1
0.06
Enterovirnsea
40-65 m
0.05
ND
0.006
<0.02
<0.002
a Colony forming units (cfu) per m^ for bacteria; plaque forming units
(pfu) per m3 for viruses.
b < = None detected in any samples, yielding the stated cumulative detection
limit.
c ND - no data available—sampling and analysis not performed for this
microorganism or distance.
149
-------
Enterovirus Densities in Downwind Air from Vims Runs
Four special virus runs were conducted to estimate enterovirus levels
in the air downwind from irrigation nozzles spraying pipeline wastewater.
The indigenous enterovirus levels ranged from 0.066 to 2.2 pfu/mL of sprayed
wastewater during these four runs, conducted on March 16, 1982 (Table P-9)
and August 2, 4 and 24, 1982 (Table P-10). As shown in Table 37, enteroviruses
were recovered from the aerosol samples' concentrate on every virus run
and at similar concentrations on the HeLa and RD cell lines.
TABLE 37. VIRUSES6 RECOVERED FROM AEROSOL SAMPLES DURING VIRUS RUNS
Virus runs
VI (3-16-82) V2 (8-2-82) V3 (8-4-82) V4 (8-24-82)
Total Total Total Total
Cell expected expected expected expected
.line pfu/BL E^fi5 pfu/jT^ pfuQ pfu/mL pfu" pfu/mL pfu°
HeLa 0.057 4 0.20 14 310 22,000 0.38 16
(2 pfu) (3 pfu) (5 pfu)
RD 0.029 2 0.32 22 350 25,000 0.31 22
(1 pfu) (9 pfu) (9 pfu)
a Based on confirmed isolates.
b 70 mL of concentrate from each aerosol run (VI: 3416 mL concentrated; V2:
2380 mL; V3: 2690 mL; V4: 2790 mL) . Total number of plaques expected
if all 70 mL of concentrate were plated on a single cell line.
The sampled enterovirus densities in wastewater and air are presented in
Table 38 and compared to those obtained in 1977 in the two virus runs at
the Pleasanton, California, wastewater irrigation system. The range of
enterovirus densities in air observed on three of the LISS virus runs (0.002
to 0.015 pfu/m^) at 46 to 60 m downwind are comparable to those observed
at 63 m downwind of the Pleasanton sprinkler line.
During Virus Run V3 conducted on August 4, the enterovirus density
was elevated in the wastewater sample to 2.2 pfu/mL. However, the enterovirus
density in air at 44 m downwind was exceptionally elevated in the aerosol
sample to a level (17 pfu/L) only one order of magnitude below those generally
observed for the indicator bacteria (see Table 34). The degree of anomaly
is indicated in Table 38 by the ratio of aerosol to wastewater density
of 7.4 for Run V3, compared to ratios ranging from 0.02 to 0.15 for the
other five virus runs. The majority of the aerosolized enteroviruses sampled
on Run V3 appear to have been poliovirus 1, based on neutralization with
monovalent antiserum. Since poliovirus 1 was used in the laboratory to
determine concentration efficiency, a thorough evaluation of laboratory
procedures was conducted. The evaluation indicated that laboratory handling
of aerosol-related samples had not compromised their integrity. Field
contamination of the Run V3 aerosol sample is not a plausible hypothesis
because the aerosol sample contained more plaque forming units than 10
liters of the wastewater and because there was no indication of any irregularity
150
-------
TABLE 38. SAMPLED ENTEROVIRUS DENSITIES ON VIRUS RUNS
Distance
Virus run from spray Cell
Date line (m) line
Enterovims density
in wastewater in air
Lnbbock Infection Surveillance Study
VI
3-16-82
V2
8-2-82
V3
8-4-82
V4a
8-24-82
60
46
44
49
He La
RD
HeLa
RD
HeLa
RD
HeLa
RD
pfu/jnL
0.16
0.10
2.2
0.066
Ratio of aerosol
density to
pfu/m3 wastewater density
Pleasanton Aerosol Monitoring Study**
63 HeLa (5d) 0.036
V2-I
2-26-77
V2-II
4-9-77
63
HeLa (5d)
0.18°
0.0029
0.0015
0.011
0.018
16.2
18.3
0.010
0.013
0.0047
0.0070
0.018
0.11
7.4
0.15
0.13
0.039
a Pipeline wastewater chlorinated at Lubbock SeWRP at rate of 500 Ib/day.
b From Johnson et al., 1980.
c Geometric mean of UTA and UTSA values.
in the field sampling. Hence, there is no laboratory or field evidence
of contamination to cast doubt on the validity of the anomalously high
enterovirus density in air obtained on Run V3.
The identification of viral isolates recovered from the wastewater
and from the aerosol during the virus runs are presented in Table 39.
The specific viruses found in the aerosol sample were nearly always also
recovered from the wastewater being sprayed at the time, despite differences
in procedures used on the wastewater and aerosol samples. Quantitative
interpretation of Table 39 is difficult, because the stability of various
enteroviruses in the aerosol may differ.
The virus runs clearly established that spray irrigation with pipeline
wastewater at the Hancock farm was a substantial source of aerosolized
enteroviruses in both the spring 1982 and summer 1982 irrigation periods.
The geometric mean enterovirus density in air was 0.05 pfu/m^, although
a much higher density (17 pfu/m^) was sampled on one run in August 1982.
It can be inferred from their relative enterovirus concentrations in the
wastewater (see Table 21) that irrigation with reservoir wastewater produced
a much lower enterovirus density in the air downwind of the irrigation
rig than did the sampled irrigation with pipeline wastewater.
151
-------
TABLE 39. IDENTIFICATION OF VIRAL ISOLATES RECOVERED DURING VIRUS RUNS
ro
Source of
isolates
Aerosol
Wastewater
Virus run
Virus
Polio 2
Polio 3
TOTAL
Polio 2
Polio 3
Cox A9
Echo 5
Echo 11
Echo 13
Echo 17
Echo 19
Echo 20
Echo 21
Echo 25
Echo 27
Unidentified
TOTAL
VI
No. of
isolates
2
1
3
4
4
3
1
1
1
1
2
1
2
2
1
_8
31
Virus run V2
Virus
Polio 2
Cox B5
Unidentified
TOTAL
Polio 3
Cox A16
Cox B5
Echo 11
Echo 12
Unidentified
TOTAL
No. of
isolates
1
1
10
12
1
1
24
1
1
_4
32
Virus run V3a
Virus
Polio 1
Polio 2
Polio 3
Cox BS
Echo 11
Unidentified
TOTAL
Polio 1
Polio 2
Polio 3
Cox B5
Echo 11
Echo 12
Echo 24
Echo 25
Unidentified
TOTAL
No. of
isolates
la
18
22
1
1
11
54
1«
3
2
30
1
1
1
1
15
55
Virus run
Virus
Polio 1
Polio 2
Echo 13
Unidentified
TOTAL
Polio 1
Polio 2
Cox B2
Cox B5
Echo 16
Echo 24
Echo 25
Echo 33
Unidentified
TOTAL
V4
No. of
isolates
8
2
1
3
14
3
3
1
18
1
1
1
1
7
36
The majority of the aerosol plaques (94%) were polio 1 based on neutralization with monovalent
antiserum. Only plaques picked from polio 1 neutralized aliquots were selected for identification
using enterovirus pools.
-------
As Table 36 illustrates, the enterovirus density in air downwind of
irrigation with pipeline wastewater at the Hancock farm was an order of
magnitude higher than at the Pleasanton, California, spray irrigation site.
It was also much greater than downwind of the aeration basins at monitored
sewage treatment plants.
Microorganism Exposure Via the Wastewater Aerosol
The increased exposure to aerosolized microorganisms which LISS partici-
pants experienced while within 400 m downwind of a Hancock farm irrigation
rig can be inferred from the air sampling data. In Table 40, the micro-
organism levels in air downwind of an irrigation rig utilizing wastewater
from the pipeline or a reservoir are contrasted with the densities of these
same microorganism groups in the ambient outdoor air in fields and just
upwind of participants' homes. Aerosol densities downwind of the irrigation
nozzle line were determined for both pipeline and reservoir sources of
wastewater from the 20 microorganism runs and four virus runs.
Ambient background densities of the monitored microorganisms in the
air just upwind of eight participant homes were determined in the four
background runs at dawn in early August 1980 prior to irrigation or construction
activities. Ambient background densities in the fields were estimated
from the upwind samplers from 18 of the 20 microorganism runs in 1982 in
which there was no operating irrigation rig or nearby human activity upwind
of the upwind samplers. Ambient background levels of the bacterial indicators,
especially fecal streptococci, were higher near homes than in the fields.
Mycobacteria and vegetative Clostridium perfringen.s were also present in
the ambient air, both with an average level in the fields of about 0.1
cfu/m3. As expected, coliphage was not found in the ambient air near homes
or in fields.
The microorganism densities in air downwind of irrigation with pipeline
wastewater were from two to at least four orders of magnitude higher than
in the ambient background air outside of participants' homes. Statistical
tests established (see Table 41) that the downwind levels were significantly
higher than the background levels in ambient air outside the homes of par-
ticipants: fecal coliform levels to beyond 400 m downwind, mycobacteria
and coliphage levels to at least 300 m downwind, and fecal streptococci
levels to at least 200 m downwind.
The more highly exposed LISS participants received substantial doses
of microorganisms from the wastewater aerosol during four major periods
of wastewater irrigation at the Hancock farm. All of the irrigation wastewater
was obtained via pipeline directly from the Lubbock SeWRP in the spring
1982 irrigation period, since operation of the reservoirs had not been
approved at that time. Pipeline wastewater comprised 64%, 0% and 1%, respec-
tively, of the total applied by spray irrigation in the summer 1982, spring
1983 and summer 1983 irrigation periods. Since microorganism densities
were much higher in the wastewater from the pipeline than from the reservoirs,
the exposure which most of the study population received to most microorganisms
via the wastewater aerosol was greater in 1982 than in 1983.
153
-------
TABLE 40. ESTIMATED MICROORGANISM DENSITIES IN AIR DOWNWIND OF
IRRIGATION IN 1982 RELATIVE TO AMBIENT BACKGROUND
LEVELS NEAR HOMES AND IN FIELDS
Microorganism concentration in air* (no./m3)
Microorganism group/ Ambient background Downwind of irrigation line"
fastewater source Homesc Fields'* 20-89 m 90-249 m 250-409 m
Fecal eolifon* (cfu) 0.01 <0.006
Pipeline 180 3 0.8
Reservoir 2 0.4 <0.08
Fecal streptococci (cfu) 0.5 0.07
Pipeline ISO 20 1
Reservoir 0.4 0.3 -0.3
•rcobacteria (cfu) 0.05 0.1
Pipeline 2.1 0.8 0.3
Reservoir 0.08 0.10 <0.03
Clostridin perfriageas (cfu)
- Vegetative 0.08
Pipeline -921
Reservoir <0.07 <0.2
- Sporulated <0.03
Pipeline 0.8 0.3
Reservoir <0.07 <0.2
Colipkage (cfu) <0.005 <0.003
Pipeline 10 2 0.13
Reservoir 0.03 0.07 ~0.06
Eaterovirases6 (pfu)
Pipeline 0.05
a Geometric mean from aerosol sampling.
b From 20 microorganism runs.
c From background runs.
d From upwind samplers for 18 microorganism runs with no upwind rig in
operation and no nearby human activity.
e From four virus runs.
154
-------
TABLE 41. SIGNIFICANT ELEVATION OF MICROORGANISM DENSITY IN AIR
DOWNWIND OF SPRAY IRRIGATION WITH PIPELINE WASTEWATER RELATIVE
TO AMBIENT BACKGROUND OUTSIDE PARTICIPANT HOMES
Significant increases in mean microorganism density
in air downwind vs. mean background run level
90-149 m 150-249 m
Downwind Downwind
Fecal coliforms
Fecal streptococci
Mycobacteria
Coliphage
Yes
«0.0005)
Yes
«0.0005)
Yes
«0.0005)
Yes
«0.0005)
Yes
«0.0005)
Yes
«0.0005)
Yes
(0.001)
Yes
«0.0005)
250-349 m
_Downwj.nd
Yes
«0.0005)
No
(0.11)
Yes
«0.0005)
Yes
350-409 m
.Downwind
Yes
«0.0005)
No
O0.25)
Maybec
(0.07)
No
(0.25)
Yes if p<0.05
Maybe if 0.050.10
One-sided t-test of difference in means in two independent populations; In
(microorganism air density from average of sampler pair) transformation
of each observation used to reduce variance inequality.
Lack of significance may be result of insufficient observations at 350-409
m downwind.
The relative ranking of the four irrigation periods with regard to
cumulative seasonal dose of microorganisms received by participants from
the air can be inferred at a given distance from the Hancock farm from
the sampling and wastewater application data. A relative aerosol exposure
measure, RAEM, was constructed to provide the basis for ranking. RAEM
is calculated for a given microorganism group, a given irrigation period,
and a given downwind distance (d) by accumulating its component values
for pipeline irrigation and reservoir irrigation, as
RAEM =
\
pipeline
reservoir
where Aas(d)
W.
'as
r
and V
microorganism concentration in air at distance d on aerosol
sampling (as) runs (from Table 34)
microorganism concentration in wastewater on aerosol sampling
runs (from Table 34)
microorganism wastewater concentration in 24-hour composites
(c) during the irrigation period (from Tables 28 and 29)
average wastewater irrigation volume, cm (from Table 4)
155
-------
The RAEM values for the monitored microorganism groups are presented
in Table 42 by irrigation period and downwind distance. The RAEH values
provide a ranking of the four irrigation periods regarding cumulative exposure
via the wastewater aerosol to each monitored microorganism group at a constant
downwind distance. Consider, for example, exposure at 150-249 m downwind,
the farthest distance range at which air sampling was regularly conducted
to determine microorganism densities in air. The irrigation periods in
which the cumulative microorganism dose in air at 150-249 m downwind can
be inferred from RAEH in Table 42 to have been largest and second largest
were:
Irrigation period by rank
1 2
Largest exposure Second largest,exposure
Fecal coliforms Summer 1982 Spring 1982
Fecal streptococci Spring 1982 Summer 1982
Enteroviruses Summer 1982 Summer 1983
(at 44-60 m)
It appears reasonable to extrapolate the relative seasonal exposure to
microorganisms in the wastewater aerosol from the distances in Table 42
to the distance of the residences of the more highly exposed study population
(approximately 1600 m for AEI>5 and <.800 m for AEI>3). For each of the
microorganism groups with adequate aerosol and wastewater monitoring data,
extrapolation from Table 42 indicates that summer 1982 was the irrigation
period when most of the more highly exposed LISS participants received
either their largest or their second largest cumulative dose of the microor-
ganism group from the wastewater aerosol. In particular, the cumulative
enterovirus dose received from the wastewater aerosol was probably at least
an order of magnitude larger during summer 1982 than during any other irrigation
period.
Estimates of A e r o_s o 1 _Exposure Index (AEI) and Other Participant Exposure
Measures
Aerosol Exposure Index—
The aerosol exposure index (AEI) is a measure of the degree of a partici-
pant's cumulative exposure to microorganisms in the wastewater aerosol,
relative to all other study participants, during a given irrigation period.
The procedure for calculating an estimate of AEI for each participant in
each irrigation period was provided in Section 4C.
The distribution of AEI values of all participants is presented in
Table 43 for each of the four major irrigation periods. By design, the
AEI percentile distribution is similar for each irrigation period. Thus,
a participant's AEI value ranks his aerosol exposure relative to all other
participants within that irrigation period. However, one cannot compare
AEI values across irrigation periods because the number of pathogens emitted
in aerosol form varied from one period to another (see Table 42). The
relevant factors, including the volumes of wastewater applied from pipeline
156
-------
TABLE 42. RELATIVE AEROSOL EXPOSURE MEASURE (RAEH) TO SPRAYED
MICROORGANISMS BY IRRIGATION PERIOD AND DOWNWIND DISTANCE
Relative aerosol exposure
RAEMa
Fecal coliforms
Fecal streptococci
Mycobacteria
Coliphage
Downwind
distance, m
25-89
90-149
150-249
250-349
350-409
25-89
90-149
150-249
250-349
350-409
25-89
90-149
150-249
250-349
350-409
25-89
90-149
150-249
250-349
350-409
Spring
1982
410
14
7
~9
490
130
80
-70
40
10
4
-20
34
12
6.2
—3
Summer
1982
970
10
11
~4
-2.5
930
23
23
3.5
3.0
0.7
1.0
0.4
-0.3
-0.2
14
2.1
1.5
0.3
0.2
Spring^
1983
4.8
0.5
1.4
<0.5
<0.5
30
15
15
-20
-15
0.5
1.0
1.2
-1.0
-1.0
measure,
Summer
1983
200
2.2
2.7
-0.9
-0.6
5.9
0.6
0.6
-0.8
-0.5
Enteroviruses
44-60
0.004
1.5
0.0004
or 0.006C
0.04
or 0.02d
RAEM is based on microorganism concentrations in air Aas(d) and wastewater
Was on aerosol sampling (as) runs, in 24-hour composites (c) during
irrigation period (Wc), and average wastewater irrigation volume (V)
sprayed:
RAEM
'/
,
pipeline
reservoir
b Based on Aas and Was values from 1982 aerosol sampling runs for corresponding
season.
c Values based on Aas/Was ratios from spring 1982 and all virus air sampling
runs, respectively.
d Values based on Aas/Was ratios from summer 1982 and all virus air sampling
runs, respectively.
157
-------
TABLE 43. DISTRIBUTION OF PARTICIPANT AEROSOL
EXPOSURE INDEX (AEI) BY IRRIGATION PERIOD
Irrigation period
NO. (%) OP
PARTICIPANTS
AEI Percentile
Distribution
Minimum
10 %ile
25 %ile
50 %ile
75 %ile
90 %ile
Maximum
By Exposure
#Low (AEI
Groups
<3)
#High (AEL>3)
By Exposure
Levels
#Low (AEK1)
#Intermed
. (1-5)
#High (AEI>5)
1
Spring
1982
387
0
0.06
0.31
2.01
3.55
6.55
82.26
260(67)
127(33)
119(31)
222(57)
46(12)
2
Summer
1982
369
0
0.05
0.38
1.68
2.89
7.80
149.22
287(78)
82(22)
124(34)
202(55)
43(11)
3
Spring
1983
335
0
0.08
0.51
2.12
3.79
7.80
82.84
218(65)
117(35)
97(29)
193(58)
45(13)
4
Summer
1983
315
0
0.09
0.64
1.77
2.79
10.15
151.17
248(79)
67(21)
98(31)
175(56)
42(13)
5
1982
365
0
0.07
0.52
1.75
2.96
7.44
139.29
277(76)
88(24)
118(32)
203(56)
44(12)
6
1983
314
0
0.10
0.81
1.93
3.00
8.95
120.53
234(75)
80(25)
91(29)
180(57)
43(14)
7
1982
1983
305
0
0.07
0.69
1.74
2.85
5.95
138.67
241(79)
64(21)
97(32)
172(56)
36(12)
NO. (%) OF
BLOOD DONORS
By Exposure Groups
#Low (AEK3)
321
316
284
265
By Exposure Levels
#Low (AEK1)
llntermed. (1-5)
(AEI>5)
204(64) 244(77) 181(64) 203(77)
117(36) 72(23) 103(36) 62(23)
82(26) 99(31) 70(25) 75(28)
196(61) 178(57) 172(60) 150(57)
43(13) 39(12) 42(15) 40(15)
NO. (%) OF
FECAL DONORS
By Exposure Groups
#Low (AEK3)
#High (AEI13)
By Exposure Levels
#Low (AEK1)
#Intermed. (1-5)
IHigh (AEI>5)
132
133
109
112
82(62) 106(80) 62(57) 84(75)
50(38) 27(20) 47(43) 28(25)
39(30) 37(28) 28(26) 31(28)
72(55) 78(57) 62(57) 60(54)
21(15) 18(15) 19(17) 21(18)
158
-------
and reservoir and the concentrations of the appropriate group of microorganisms
in each wastewater source, varied across irrigation periods and are not
taken into account in AEI. Thus, both RAEH and AEI would be required to
assess the cumulative dose of a given microorganism group received by a
given participant from the wastewater aerosol over a given irrigation period.
Most of the data analyses conducted involved a comparison of infection
rates over an irrigation period among participants stratified by their
degree of aerosol exposure. For these analyses, each participant was placed
in the proper exposure category based on his AEI value during the relevant
irrigation period. To perform each confirmatory statistical analysis,
all participants were placed in either a ''high exposure'' or a ''low exposure''
group for the irrigation period. AEI=3.00 was the cutpoint used as the
boundary between these two exposure groups. Suppose the value AEI=3.0
were obtained from EI=3.0 and XAEREM=0.0, for example. Then this value
AEI=3.0 can be shown (see Section 4C) to be equivalent to staying on the
Hancock farm for 24 hours per week throughout a spring irrigation period
(or 16 hours per week throughout a summer irrigation period) without ever
having extensive aerosol contacts downwind of an irrigating rig. To investigate
a dose-response gradient during an irrigation period, incidence rates and
risk ratios were determined for three aerosol exposure levels: low (AEK1) ,
intermediate (15). The number of participants in
the two exposure groups and the three exposure levels is presented in Table
43 for each irrigation period. It should be noted that many residents
in the central portion of Wilson shifted from the high exposure group in
the spring irrigation periods to the low exposure group in the summer irrigation
periods because of differences in prevailing wind direction between the
spring and summer irrigation periods. Most infections evaluated were determined
from blood or fecal specimens. The breakdown of blood donors and fecal
donors into the exposure groups and levels is also presented in Table 43.
Some analyses involved observation periods of a year or longer, i.e.,
1982, 1983 or the entire irrigation period (1982 and 1983). A participant's
aerosol exposure estimates AEI for each of these observation periods were
calculated as weighted averages of his AEI values for the constituent irrigation
seasons. Since most of the pathogens observed in infection episodes over
these longer observation periods were enteroviruses, the weights for each
irrigation season were calculated to be proportional to WC*V, the total
number of enteroviruses sprayed from irrigating rigs during that irrigation
season. The calculation procedure and resulting weights are presented
in Table 44. For example, Table 44 indicates that the summer 1982 irrigation
contributed 90.65% of the enteroviruses sprayed during 1982. Thus, a partici-
pant 's AEI value for 1982 was calculated as 0.0935 AEIj + 0.9065 AEl2>
where the subscripts 1 and 2 refer to spring 1982 and summer 1982, respec-
tively. Table 43 also presents the distributions of AEI values thus obtained
for 1982, 1983, and the entire irrigation period and the numbers of participants
in the exposure groups and levels based on these values.
A few analyses involved the household as the unit of observation.
A household aerosol exposure index, HAEI, defined as the maximum AEI among
the household members during that irrigation period, was used as the exposure
measure in these analyses. Since these analyses were conducted to take
159
-------
TABLE 44. RELATIVE CONTRIBUTION OF IRRIGATION SEASONS TO TOTAL
ENTEROVIRDSES SPRAYED FOR 1982, 1983 AND ENTIRE IRRIGATION PERIOD
Irrigation season and
irrigation dates
Spring Summer Spring Summer
1982 1982 1983 1983
2-16/4-30 7-21/9-17 2-15/4-30 6-29/9-20
V. Volume of Wastewater
Applied, cm
From pipeline
From reservoir
Wc, Average Enterovirns Cone.,
pfn/mL
Pipeline wastewater
Reservoir wastewater
Wc x V
Pipeline
Reservoir
Both
5.83
—
0.0467
—
0.2723
-
0.2723
6.91
3.87
0,3732
0.0147
2.5788
0.0569
2.6357
0
14.85
0.0594
0.0018
0
0.0267
0.0267
0.20
14.99
0.2692
0.0010
0.0538
0.0150
0.0688
Relative Contribution to
Total Sprayed (weight)8
1982
1983
Entire irrigation period
(1982 + 19&3)
9.35%
9.1%
90.65%
87.8%
28.3%
0.9%
71.7%
2.2%
a From Wc z V for both pipeline and reservoir irrigation.
160
-------
within-household transmission of infection into account, the most highly
exposed household member was considered to best represent the household's
exposure.
AEI cannot be considered an ideal measure of the relative aerosol
exposure of the participants within an irrigation season. Deficiencies
include the lack of knowledge of the precise whereabouts of participants
throughout the irrigation periods, the use of arbitrary weighting factors,
and reliance on historical wind data rather than on actual on-site wind
data from the irrigation periods.
Imprecise information regarding the specific wastewater aerosol exposure
events experienced by each participant during 1982 was the primary limiting
factor in the accuracy of AEI as a relative measure of aerosol exposure.
The activity diary provided valuable information about participant habits
during each irrigation period, especially regarding the amount of time
spent at home, on the Hancock farm, and in Lubbock. However, in deference
to respondent burden and privacy, the activity diary did not request detailed
information about maximal exposure events. The degree to which the week
of activity diary administration was representative of the entire irrigation
period is unknown, although the activity diary weeks were selected to avoid
holidays and school vacation breaks. The log of extensive wastewater contacts
was introduced in 1983 to obtain much better data regarding maximal exposure
events; this information was quantified in the indices of extensive exposures
(XAEREM and XDIREM). XAEREH was incorporated as a component of AEI to
obtain a better ranking of the relative exposure of the more highly exposed
participants.
The sensitivity of the LISS results to alternative assignments of
the arbitrary weighting factors employed in the AEI calculation has not
been investigated, because of the extensive computations involved. However,
other reasonable assignments are unlikely to significantly change the relative
ranking of participants with regard to cumulative aerosol exposure.
Historical wind data was used to calculate the El component of AEI
for reasons of expendiency. This appears to have been justifiable in light
of the greater uncertainty in AEI attributable to imprecise knowledge of
participant exposure events in 1982. Wind roses for the actual irrigation
periods were very similar to the historical wind roses except for the spring
1983 irrigation season. However, spring 1983 was the season of lowest
aerosol exposure (Table 42), the fewest infection events occurred in the
spring 1983 season (Tables 97-99), and there were no apparent associations
with aerosol exposure in spring 1983 (Table 132). Hence, the use of the
historical (rather than actual) wind data in calculating AEI should have
had virtually no effect on the LISS findings.
To investigate the effects of these recognized deficiencies in AEI,
the maximum aerosol exposure value of the household (HAEI) was plotted
at the household location for each irrigation season. The resulting HAEI
exposure isopleths appeared to be intuitively reasonable. In addition,
the AEI values of household members were usually tightly clustered, except
for individuals with occupational exposure to the wastewater. As an additional
161
-------
check, all of the AEI values calculated for every participant were reviewed
for reasonableness by the health watch manager. The review revealed no
significant classification error.
Additional Exposure Measures—
Other exposure measures were obtained to investigate alternative routes
of wastewater irrigation exposure besides the wastewater aerosol. Each
sentinel participant was asked to maintain a log of extensive wastewater
contacts from February through September 1983. As part of the weekly illness
report, the most extensive aerosol exposure and direct wastewater contact
of the week and the estimated hours spent on the Hancock farm were also
obtained for each household member. From these data, cumulative measures
of extensive aerosol exposure (XAEREM) and direct wastewater contact (XDIREM)
were calculated using the microenvironment method for each sentinel participant
for both of the irrigation periods in 1983. The hours spent on the Hancock
farm were also averaged as another exposure measure (FHRSEM). The calculation
procedures were given in Section 4C.
The distributions of values of the additional exposure measures XAEREM,
XDIREM and FHRSEM among all participants in the spring 1983 and summer
1983 irrigation periods are summarized by exposure levels in Table P-22
in Appendix P. Note that the percentage of participants with any extensive
exposure was about 12% for extensive aerosol exposure, 6-8% for direct
wastewater contact, and 19-24% for spending any time on the Hancock farm.
The correlation among the exposure measures is indicated in Table P-23
in Appendix P. Note that the additional exposure measures are quite highly
correlated with AEI (0 .365<.r<.0 .610) and very highly correlated with each
other (0.593
-------
very light to light (see Table 45). Bacterial levels may have been suppressed
by the ether used to inactivate the flies.
A fly population developed following a period of rainfall in early
September. A second fly collection was attempted on September IS and 16
with traps located near the Wilson effluent pond, at two farms on the Hancock
farm, and next to the school's trash can. No flies were collected during
this attempt.
During a third attempt in October with traps at four locations, approxi-
mately 1,200 flies were collected (from October 15 to 17) near the pig
pens adjacent to the Wilson sewage treatment facility, and approximately
65 flies were collected from October 20 to 22 in barns at farmhouses located
near the reservoirs under construction on the Hancock farm. No viruses
were recovered from either sample. Bacterial profiles are compared with
the previous sample in Table 45. Staphylococcus aureus was present in
moderate numbers in both samples collected in October. Additionally, Proteus
vulgar is (in moderate numbers) and Salmonella arizonae were recovered from
the sample collected at the pig pen. A variety of other organisms was
isolated from the flies at low levels.
Fly collection during the irrigation period was attempted concurrently
with the aerosol monitoring in summer 1982. These attempts were performed
utilizing baited fly traps in the same manner as during the baseline year
at locations adjacent to the reservoirs on the Hancock farm and the Wilson
treatment facility. A fly collection attempt in August 1982 yielded insuf-
ficient flies for laboratory analysis. Surveillance for a significant
increase in fly population was maintained until the first freeze, but conditions
never warranted another attempt at fly collection.
Several fly collection efforts were also made during the summer 1983
irrigation. Fly samples were collected from July 19 to 22, 1983 at the
intensive research plot on the Hancock farm, at Reservoir 1 on the Hancock
farm, and next to the pig pen near the Wilson sewage treatment facility.
These fly samples were scavenged by beetles while in the fly traps, then
inadvertently kept cooled at 4°C for 3 weeks and held at room temperature
for 24 hours prior to proper processing and analysis. A second attempt
to collect flies in September 1983 was again unsuccessful because no flies
were present.
The flies collected during July 19-22, 1983 yielded a bacterial profile
that was similar to that observed with the flies collected during the baseline
period. However, the levels of the respective organisms observed were generally
higher in the flies collected during the irrigation period. The increased
levels of organisms observed were undoubtedly affected by the problem in
sample handling. The fly samples collected during the irrigation period
were not analyzed for viruses due to the deteriorated state of the samples.
The fly data from the irrigation period is of questionable significance
due to the problems in sample handling. However, the similarity in bacterial
flora from baseline and irrigation periods suggests that the measurable
flora was not altered by irrigation.
163
-------
TABLE 45. BACTERIAL ISOLATES FROM FLIES
Sample source
No. of
flies
collected
Organism
Quantitation*
of growth
Baseline
Pig pen near Wilson
effluent pond
August 6-7. 1980
Pig pen near Wilson
effluent pond
October 15-17. 1980
Barn near Reservoir 3
Hancock farm
October 20-22, 1980
Irrigation
Hancock farm (Rig 15)
July 19-22, 1983
Pig pen near Wilson
effluent pond
July 19-22, 1983
Hancock farm
(Reservoir 1)
July 19-22, 1983
200b Escherichia coli
Hafnia alvei
Staphylococcus aurens
Klebsiella pneumoniae
Proteus mirabilis
Providencia stuartii
Staphylococcus epidermidis
1200 Proteus vulgaris
Staphylococcus aurens
Escherichia coli I^S"1"
Fluorescent Pseudomonas gp.
Hafnia alvei
Klebsiella ozytoca
Salmonella arizonae
65 Staphylococcus aureus
Escherichia coli
Fluorescent Pseudomonas gp.
Klebsiella ozytoca
Serratia marcescens
17° Klebsiella pneumoniae
Proteus mirabilis
Serratia marcescens
200° Escherichia coli
Providencia stuartii
Serratia rubidaea
Klebsiella pneumoniae
44C Serratia marcescens
Klebsiella pneumoniae
Proteus mirabilis
Serratia odorifera
L
L
L
VL
VL
VL
VL
M
M
L
VL
VL
VL
VL
M
L
L
VL
VL
M
M
M
M
M
H
L
H
M
L
L
a Estimate of prevalence based on growth on primary culture plates
(4 quadrants/plate):
H - heavy—growth in three or all quadrants
H - moderate—growth on first two quadrants
L - light—growth on first quadrant
VL - very light—one to ten colonies on plate
b Flies anesthesized with ether.
c Samples inadvertently held for 3 weeks prior to shipment for analysis.
164
-------
The difficulty in collecting flies, both in the baseline period but
especially after wastewater irrigation commenced, indicates that flies
were not an important route of transmitting infectious agents at the study
site, particularly during summer irrigation periods when the possibility
of flies as an insect vector was most plausible. In marked contrast to
the LISS experience, Echeverria et al. (1983) documented that the flies
in a small rural village in northeastern Thailand frequently carried enteric
pathogens and observed that size of the fly population and the incidence
of diarrhea both increased in the hot dry season.
Microorganism Levels in Drinking Water
To assess contaminated drinking water as a potential source of the
agents of infection episodes, samples of drinking water were obtained from
a cross-section of rural households and from the Wilson water supply (see
Figure 13). The results from analyses of the drinking water samples for
total and fecal coliforms, fecal streptococci, and Salmonella are presented
in Table 46.
Many of the drinking water wells on and adjacent to the Hancock farm
showed evidence of microbial contamination after wastewater irrigation
commenced. Each such well exhibited a high level of bacterial contamination
before wastewater irrigation was initiated. Thus, there is no indication
that wastewater irrigation operations were related to the contamination
of drinking water wells on or near the Hancock farm.
Many of the rural household wells were either periodically or regularly
contaminated, based on the data for the bacterial indicator organisms.
These data indicate that viral and bacterial pathogens may also have been
present quite frequently and sporadically in household drinking water wells
throughout the rural study area. Therefore, microbial contamination of
drinking water was investigated as a possible explanation for observed
episodes of infection and illness, particularly as an alternative explanation
when the pattern of occurrence suggested a possible association with wastewater
irrigation (Section 5M).
The widespread occurrence of bacterially contaminated household drinking
water supplies in the rural study area is consistent with the first national
survey of rural water quality at the point of use conducted recently by
Cornell University. Francis et al. (1984) found that 42% of households
served by individual systems (single connection) had a total coliform density
above 1 cfu/100 mL and that 1.6% of rural households had a fecal coliform
density above 200 cfu/100 mL.
LCCIWR periodically notified each household of the test results on
its drinking water well. Chlorination or other means of disinfection was
recommended when warranted to eliminate bacterial contamination. No investi-
gations were made to determine sources of well contamination or whether
these sources resulted in any other personal exposure. Peak coliform concen-
trations in a well usually did not occur at the same time as peak fecal
streptococci concentrations. This may be an indication that several different
contamination sources were operating.
165
-------
TABLE 46. MICROORGANISM DENSITIES IN DRINKING WATER IN THE
STUDY AREA BY WELL LOCATION AND SAMPLING DATE
Household Dates
Total
conform
[colonies/100 mLl
Fecal
co U form
tcolon1ea/10D mLl
Fecal
streptococcus
[colonies/IPO mil
NOo-N
Salmonella8 [mq/Ll
On Hmcacl
118
120
121
123
(trailer)
125
131
c F»m
10-1 4-81
1-6-62
2-15-82
6-22-82
11-4-82
12-14-82
3-28-83
5-3-83
5-31-83
7-11-83
8-25-83
10-13-83
11-5-81
1-5-82
2-16-82
6-16-82
11-4-82
12-14-82
3-28-83
5-3-83
5-31-83
7-11-83
8-25-83
10-13-83
10-15-81
1-4-82
2-15-82
6-16-82
11-3-82
12-14-82
3-28-83
5-3-83
5-31-83
7-11-83
8-25-83
10-12-83
7-12-83
8-24-83
10-12-83
10-15-81
1-4-82
2-15-82
6-16-82
11-3-82
12-14-82
3-28-83
5-3-83
5-31-83
7-13-83
8-25-83
10-12-83
10-14-81
1-5-82
2-15-82
6-22-82
>2000
200
120
1300
47
0
0
0
0
5
25
3
570
6000
0
60
0
0
0
0
0
0
0
50
>2000
20
1
100
0
0
0
6
0
0
0
37
7
1
60
0
15
1700
1200
0
0
10
0
0
40
0
10
140
100
0
400
14
5
66
25
0
0
0
0
0
2
14
1
20
59
0
0
0
0
0
0
0
0
0
21
400
2
0
50
0
0
0
0
0
0
0
37
1
0
0
0
0
28
NR
0
0
0
0
0
0
0
0
30
0
0
3
Q
OD
0
9
0
0
0
0
4
260
56
5
0
0
0
330
0
0
0
1
0
0
3
122
49
0
0
3
1
1
10
0
0
23
0
1000
4
0
0
0
0
9
1
0
0
0
0
0
0
0
0
3
0
0
1
+
—
-
_
-
_
-
_
_
-
-
-
_
-
-
—
-
-
_
-
-
-
-
-
+
+
-
-
-
-
-
-
-
-
-
-
'_
-
—
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
6.17
0.37
1.99
11.20
2.23
0.74
0.94
4.28
5.71
1.77
8.16
1.90
23.75
25.48
8.32
2.69
0.62
2.35
4.37
4.53
0.45
0.18
1.36
1.00
continued..,
166
-------
TABLE 46. [CONT'O]
Household
131
(Cont'd)
VltklB 401
109
114
116
122
126
Total
conform
Dates (colonies/100 mil
11-3-82
12^14-82
3-28-83
5-3-83
5-31-83
7-13-83
B-25-83
B • of HMCW
10-14-fll
1-5-82
2-16-82
6-16-82
11-3-82
12-14-82
3-28-83
5-3-83
5-31-83
7-11-83
8-24-83
10-12-83
10-14-8
1-6-82
2-16-82
6-22-82
11-3-82
12-14-82
3-fi8-83
5-3-83
5-31-83
7-13-83
8-24-83
10-13-83
10H4-81
1-6-82
2-16-82
6-22-82
11-4-82
12-14-82
3-28-83
5-3-83
5-31-83
7-11-83
8-24-83
10-13-83
12-15-82
3-28-83
5-3-83
5-31-83
7-11-83
8-25-83
10-13-83
10-14-81
1-6-82
2-16-82
6-16-82
11-3-82
12H4-B2
3-28-83
0
0
0
1
0
10
2
* fmm
0
0
0
0
0
0
0
0
0
3
0
0
>2000
800
0
0
1
0
3
0
0
0
0
0
>2000
0
0
300
1
6
0
0
0
28
1
10
9
1
1
90
9
22
10
0
0
0
0
0
0
0
Fecal
collform
(colonies/100 «L)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
0
0
0
0
0
0
0
0
0
0
20
0
0
30
0
6
0
0
0
0
0
0
0
1
0
3
0
0
2
0
0
0
0
0
0
0
Fecal
streptococcus
(colonies/100 ml]
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
22
0
8
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
4
53
27
0
0
0
0
2
300
1
15
73
91
0
3
101
0
0
0
0
0
0
0
NOg-N
Salmonella8 [mo/Ll
_
-
—
_
—
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
—
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.61
0.45
0.10
2.06
0.75
0.47
1.45
0.40
16.36
1.81
1.85
0.95
0.15
2.44
1.07
<0.01
1.45
0.26
3.96
1.70
1.30
continued..
167
-------
TABLE 46. (CONT'D)
Household
126
(Cont'd)
320
Total
collform
Dates [colonies/100 raL)
5-3-83
5-31-83
8-24-83
10-13-83
10-31-81
1-4-82
2-16-82
6-16-82
11-4-82
12-13-82
1-4-83
3-28-83
5-3-83
5-31-83
7-12-83
8-25-83
10-13-83
0
0
0
0
0
5
0
0
0
0
0
2
8
15
2
0
0
Fecal
collform
(colonies/100 raL)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Fecal
streptococcus
[colonies/100 mL)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
N03-N
Salmonella0 [mo/Li
_
-
—
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9.47
1.14
17.28
10.41
4.27
City of Vila**
298 (City
Well 1)
299
(W1 Ison
treated
•ater]
Beyond 400
103
315
10-14-81
1-4-82
2-15-82
6-22-82
11-4-82
12-^3-82
8-24-83
10-13-83
10-31-81
1-4-82
2-16-82
6-22-82
11-3-82
12-13-82
3-28-83
5-3-83
5-31-83
7-11-83
8-24-83
10-12-83
• f FOB Nona
11-6-81
1-5-82
2-15-82
6-22-82
11-3-82
12-13-82
3-28-83
5-3-83
5-31-83
7-^11-83
8-24-83
10-12-83
11-4-82
12-15-82
3-28-83
5-3-83
5-31-83
7-13-83
8-24-83
10-13-83
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ick Far*
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-
-
-
-
-
-
-
-
—
-
—
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5.42
1.55
7.15
9.04
<0.01
7.70
1.21
14.40
7.55
4.53
1.39
10.02
5.30
9.18
3.98
4.27
continued..,
168
-------
TABLE 46. (CONT'D)
Total
conform
Fecal
co 11 form
Fecal
streptococcus
Household Dates (colonies/100 nL] (colonies/100 ml] (colon1e8/1QO oil)
399
504
531
540
545
546
555
a +
10-14-81
1-4-82
2—16—82
6-16-82
11-4-82
12-13-82
3-28-83
5-3-83
5-31-83
7-12-83
8-25-83
10-14-83
12-1 5-82
3-28-83
5-3-83
5-31-83
7-13-83
8-24-83
10-13-83
11-4-92
12-13-82
3-28-83
5-3-83
5-31-83
7-12-83
8-25-83
10-1 2-83
12-15-82
3-28-83
5-3-83
5-31-83
7-13-83
8-24-83
10-12-83
12-15-€2
3-28-83
5-3-83
5-31-83
7-13-83
8-24-83
10-12-83
12-15-82
3-28-83
5-3-83
5-31-83
7-13-83
8-24-83
10-12-83
12-15-82
3-28-83
5-3-83
5-31-83
7-13-83
8-24-83
10-13-83
Salmonella present
500
80
0
19000
21
100
200
0
21
0
0
10
190
0
0
42
92
6
0
0
1
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
2
>8000
= 150
620
135
10
0
48
1100
0
50
10
2000
110
75
(>1 colony/100 nL)
- Salmonella not detected (<1 colony/100
b 0
no colonies detected with a detection
300
0
0
1000
2
3
110
0
2
0
0
1
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
160
0
0
0
0
0
24
3
0
3
5
0
26
30
mL]
Unit of 1
160
0
0
60
3
0
13
0
0
0
0
90
82
0
0
24
0
0
1
0
4
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
27
0
2
0
0
0
1
16
1
2500
1
50
200
42
colony/100 nL
N03-N
Salmonella8 (mg/L)
+ 2.69
0.40
7.46
3.74
1.18
-
-
_
_
-
_
_
_
-
-
-
-
1.90
—
_
-
-
-
-
-
_
-
-
-
-
-
-
-
-
-
^
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
169
-------
The possible association with contaminated drinking water was investigated
for each category of bacterial infection among all fecal donors (Section
56). Contaminated drinking water was also investigated as an alternative
explanation for each infection episode in which there was good or marginal
evidence of a strong association with wastewater aerosol exposure (Table
133). Since the presence of bacterial indicator organisms is so widespread
in rural drinking water supplies nationwide, a conservative definition
of ''contaminated well water1' was employed in classifying each monitored
rural household supply from the data in Table 46 for these analyses. When
four to eight drinking water samples were analyzed during the period of
observation for infections, the well was classified as contaminated if
the average bacterial density per 100 mL of the samples exceeded 20 for
total coliforms, 2 for fecal coliforms, or 5 for fecal streptococci, or
if Salmonella was present in any sample. Since detection of bacterial
contamination is less likely when fewer water samples .are obtained, a less
stringent criterion for contamination was used in this case. When only
one to three drinking water samples were analyzed during the infection
observation period, the well was considered to be contaminated if the average
bacterial density per 100 mL exceeded 5 for total coliforms, 1 for fecal
coliforms, or 2 for fecal streptococci, or if Salmonella was detected.
With these criteria, slightly more than half of the monitored rural wells
in Table 46 were classified as contaminated in most of the observation
periods employed. The small number of participants whose household well
was classified as contaminated but who only drank bottled water (i.e.,
never drank water from the faucet), were excluded from the contaminated
drinking water group in the analyses of association with infection. Since
the drinking water of 20 or fewer households was monitored during each
period of observation, there often were insufficient data to detect an
association of infections with contaminated drinking water, unless the
infection rate was high or the association was very strong.
Monthly precipitation for the study area is presented in Table 47.
There were 4 months of extremely heavy rainfall during the LISS. Rainfall
exceeded the 40-year average by about 12 cm/mo in both May and June 1982
and by about 8 cm/mo in both August and October 1981. The extremely high
densities of indicator bacteria in the rural drinking water (see Table
46) were most commonly observed in the October 1981 and June 1982 surveys
(i.e., during months of excessive rainfall). The proportion of the rural
household wells which were contaminated (by the criterion of the preceding
paragraph) was found to be significantly associated with local rainfall
in the sampling month (r=0.576, p-0.025). Some rural wells were reported
to have been flooded by surface water runoff following heavy rainfall events
in late May and June 1982. At some rural homes, the drinking water well
was located close to the cesspool. Many of these cesspools were constructed
improperly. This combination of circumstances appears to have contributed
to the substantial and widespread contamination of the drinking water supplies
of rural households, which was observed in the study area.
Although never documented through the water sample data, the possibility
cannot be dismissed that the water supplied to households in Wilson was
also contaminated sporadically. Prior to March 1983, the stored water
obtained from six wells was only chlorinated periodically by hand prior
170
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TABLE 47. PRECIPITATION (cm) BY MONTH IN THE STUDY AREA
average 1980 1981 1982 _ _ 1983 _
Lubbock Lubbock Lubbock Hancock Lubbock Hancock Lnbbock
Month _ airport airport airport farm airport farm airport
January
February
March
April
May
Tune
July
August
September
October
November
December
1.2
1.6
2.2
3.2
6.9
6.6
5.5
5.2
6.4
5.2
1.5
1.5
1.4
1.0
0.5
2.9
8.8
4.5
0.5
4.2
9.0
0.5
5.8
1.3
0.8
1.7
3.0
5.2
3.2
2.0
8.5
13.7
4.5
13.6
1.6
0.5
0.8
0.6
3.2
2.2
18.6
19.7
11.3
2.7
4.4
0.8
3.0
3.1
0.1
1.0
1.1
6.4
11.5
12.7
5.3
2.7
3.3
1.2
3.0
5.0
3.2
0.4
0.8
2.6
6.9
3.2
3.1
0
0.6
7.0
0.8
1.4
2.0
3.1
4.5
1.0
0.8
1.0
27.4
1.4
0.9
Annual 47.0 40.3 58.4 70.4 53.3 51.4
to distribution. Those households at the ends of branched 1-inch water
lines in Wilson would have been most subject to the effects of bacterial
contamination, since their drinking water tended to stagnate in the water
lines. Any such effects were not investigated in the LISS.
Eating Food Prepared at Local Restaurants
Responses regarding patronage of the food preparation establishments
in Wilson were obtained retrospectively in July 1984 for 117 routine fecal
and illness specimen donors. Table 48 presents the distribution of responses
by irrigation period for each ''restaurant.''
Since this was a small rural community, the majority of the respondents
had no trouble with recall or knowledge of donor activity. Since all four
establishments were located in the vicinity of the Wilson schools, most
parents knew which ones their children did patronize both during the school
year and in the summer when school was out. Patronage of the restaurants
by the farm families was frequently determined by ''season.'' For example,
some families were more likely to patronize the restaurants during planting
season, some were more likely to patronize the restaurants when weeds were
being sprayed (July-August), while others were more likely to patronize
the restaurants during the harvest. In addition, the unusual weather conditions
during the summer of 1982 made it easier for the respondents to recall
instances when their patterns of restaurant patronage may have deviated.
It should be noted that restaurants A and B were the only ones which
primarily served food, were open for business during both irrigation years,
and were visited at least monthly by more than 10% of the surveyed donors.
Most of the fecal and illness specimen donors reported the same frequency
of eating food prepared at restaurants A and B during all four irrigation
171
-------
TABLE 48. FREQUENCY DISTRIBUTIONS OF PATRONAGE OF MAJOR FOOD
PREPARATION FACILITIES IN WILSON BY 117 FECAL AND ILLNESS
SPECIMEN DONORS DURING IRRIGATION PERIODS
Frequency of patronage
Restaurant A
never
once/week
Restaurant B
never
once/week
Other facilities
never
once/week
Spring
1982
Summer
1982
69
29
7
12
71
28
9
9
63
28
21
5
77
15
20
5
Restaurant C
107
4
0
6
105
3
8
1
Spring
1983
71
28
6
12
71
26
11
9
Summer
1983
65
25
24
3
75
17
20
5
Restaurant D
99
11
0
7
99
8
8
2
periods. However, there was a slightly greater tendency to patronize both
restaurants at least monthly during the summer (i.e., June-August) when
school was out. The demographic characteristics of patrons are compared
to those of nonpatrons below, based on patronage during summer 1982. Very
similar patronage patterns were obtained for summer 1983, but the summer
1982 patterns are reported below because this was the season of initial
interest when the restaurant patronage survey was designed.
Restaurant A—
Twenty-two percent of the 117 illness and fecal specimen donors who
were surveyed reported eating food prepared at restaurant A at least once
a month. Twenty-four percent of the donors reported eating food prepared
by restaurant A less frequently than once a month. Fifty-three percent
of the donors reported that they never ate food prepared by restaurant
A.
The restaurant A patrons differed from the nonpatrons for six of the
seven demographic variables examined. Restaurant A patrons tended to be
younger that nonpatrons (p<0.001), were more likely to be male than female
(p=0.077), were likely to live in households where the head of household's
1979 income was reported to be in the $10,000-19,999 range (p=0.064), and
were more likely to live in Wilson than in a rural area (p=0.030). Hispanic
donors were more likely to patronize the restaurant than Caucasian donors
(p=0.032). There were no differences found between patrons and nonpatrons
for the head of household education variable. The donors in the three
exposure levels differed in their frequency of restaurant A patronage
(p<0.001). Seventy-nine percent of the respondents in the high exposure
172
-------
level reported eating food prepared by restaurant A more frequently than
once a month. Only 6% in the low exposure level reported patronizing restaurant
A more than once a month.
Restaurant B—
Twenty-two percent of the donors reported that they ate food prepared
at restaurant B more frequently than once a month. Thirteen percent of
the donors ate food prepared by restaurant B less frequently than once
a month. Sixty-six percent of the donors reported that they never patronized
the restaurant.
Patrons of restaurant B differed from nonpatrons in four of the seven
demographic characteristics examined. The restaurant patrons were found
to be younger than nonpatrons (p<0.001); patrons lived in Wilson more frequently
than in the rural area (p=0.002); hispanic donors were more likely to patronize
the restaurant than were Caucasian donors (p<0.001); and donors from households
where the head of household's 1979 income was reported to be in the $10,000-
19,999 range were more likely to patronize the restaurant than were donors
from households with higher and lower incomes (p=0.025). Patrons and nonpatrons
did not differ for the variables of sex, head of household education, and
exposure level.
Discussion—
There are common factors which were associated with patronage of restau-
rants A and B. Geographic location of the household in relation to the
restaurant was important in determining restaurant patronage. Those living
in the proximity of Wilson found the restaurants more convenient than did
those donors who lived on the outside edges of the study area. Household
income was also important. Donors from households with low incomes could
not afford to patronize the restaurants, while donors from high income
households were more likely to travel to a larger community for a meal.
Age was important in determining which of the middle income donors (who
lived in or near Wilson) actually patronized the restaurant on a frequent
basis. Children ages 6-17 and adults ages 18-44 were more likely to buy
food from these restaurants. Some children, most of whom were hispanic
residents of Wilson, reported frequenting the establishments on a routine
basis. The adults reported buying food from the restaurants only when
they were ''too tired to cook'' or ''in a hurry.''
Patronage of restaurant A was much greater among surveyed donors with
a high level of wastewater aerosol exposure. Thus, any health effects
of wastewater aerosol exposure may be confounded with any health effects
of eating food prepared by restaurant A in the LISS population. To allow
valid interpretation, it is necessary to investigate eating food prepared
by restaurant A as an alternative explanation to any apparent association
of infections with aerosol exposure. This exploratory analysis was performed
by logistic regression for the surveyed donors and is presented in Section
5L.
173
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D. DESCRIPTION OP STUDY POPULATION
Questionnaire Data
Tables P-24 to P-30 of Appendix P report information derived from
interviews with members of the 163 participating households. The questionnaires
used in these interviews were designed by the University of Illinois School
of Public Health. Interviews were administered in respondents' homes in
1980 and by telephone in 1982 and 1983. Copies of these questionnaires
can be found in Appendices B, C and D. A detailed description of the interview
procedure is presented in Section 4B. Only responses from individuals or
households which actually participated in the study (i.e., provided health
diary information, blood samples or fecal specimens) were tabulated. Every
effort was made to resolve inconsistencies and to correct omissions. However,
four individuals are included in Tables P-25 and P-27 to P-29 in Appendix
P who were considered to be nonparticipants elsewhere in this report, since
they only provided an initial blood sample. The heading NR was used as an
abbreviation for "not recorded" for the few cases where the household withdrew
from the study before the missing information could be obtained. With the
exception of the farm information, the material summarized in the tables
is discussed in greater detail in subsequent portions of this report.
Tables P-24 and P-25 in Appendix P present information concerning
household and individual characteristics of the study population based
on responses to the initial (Hay 1980) and final (October 1983) questionnaires.
Tables P-26 and P-27 in Appendix P present crosstabulat ions of the overall
exposure levels (based on combined 1982-1983 aerosol exposure indices)
with selected household and individual variables of interest in the study.
These crosstabulat ions are used only to provide the reader with an understanding
of the general demographic patterns observed in the study. Since irrigation
patterns varied between the spring and summer seasons as well as between
1982 and 1983, the degree of exposure of individuals in the study population
also varied between time intervals. Therefore, the patterns observed in
Tables P-26 and P-27 only summarize general trends. Table P-28 contains
crosstabulations of selected demographic variables which allow the population
to be characterized by age, sex, race, and household location. Table P-29
summarizes the health history information obtained from participants.
Table P-30 summarizes crop and livestock information provided by participating
farm households. The farm data provide indications that farming activity
in the community declined substantially during the course of the study.
A capsule description of the study population based on participants
remaining with the study until its completion is presented based on Tables
P-24, P-25 and P-28 and other sources. The racial composition of the study
population was 72% Caucasian and 28% hispanic. Hales and females each
comprised about half of the participants in each age group. The size of
households was 22% single member, 37% two member, and 17% with five or
more members.
Farming was the primary occupation and 58% of the heads of household
had completed high school. All participants lived in single family dwellings,
of which 39% had evaporative coolers and 44% had refrigerated air conditioning.
174
-------
Approximately 95% of the study population visited Lubbock at least once
per month, with a median of about 16 hours per month spent there.
The study population included 17 tenant farmers and workers who had
regular direct contact with wastewater and heavy aerosol exposure on the
Hancock farm. An additional 21 participants in 10 households lived within
200 m of the spray irrigation. Eight homes of 19 participants were located
within SO m of a sprinkler irrigation circle and many of them thereby received
substantial aerosol exposure.
Population Demographics
Crosstabulations of specific demographic variables obtained from the
three questionnaires (administered in 1980, 1982, and 1983) were generated
to determine if:
o self-selection altered the characteristics of the LISS population
during the course of the study;
o the major subgroups in the population were similar in terms of
socioeconomic status, age, geographic distribution, family size
and other demographic characteristics;
o the various donor groups differed significantly from the overall
study population with regard to demographic characteristics;
o the two exposure groups and the three exposure levels were balanced
with respect to demographic characteristics.
A description of each variable as well as the value categories for
each of the participant characteristics is contained in Table 49. Variables
of interest included personal information such as age, race, sex, socioeconomic
status, smoking habits, and history of chronic illness. Environmental variables
of interest included household size, presence (or absence) of children
in the household, source of drinking water, air conditioner use, and household
location. Family income and the occupation and education level of the
head of household were used as indicators of socioeconomic status for all
household members. However, since 44% of the study participants lived in
households headed by farmers, and since annual farm income was found to
be unstable during the course of the study, the head of household's education
was considered to be the most reliable of the three socioeconomic indicators.
The appropriate Cochran-Mantel-Haenszel statistics were used to generate
the ''p values'' for all crosstabulations. The ''p values'' are listed
(in Tables 50, 51 and P-31 to P-44) only when equal to 0.10 or less. Each
p-value below 0.05 was interpreted to indicate a significant difference
between the subpopulations being compared. The categories of household
size, income, and education were collapsed to meet the criterion that no
more than 20% of the cells had an expected frequency of 5 or less. In cases
where the same question had been administered in two or more questionnaires
(e.g., household size, smoking, and bottled water consumption), the most
recent response from each participant was used.
175
-------
TABLE 49. VARIABLES USED IN DEMOGRAPHIC ANALYSIS
Household variables
Individual variables
ACOND: Do you have air conditions
in home
1 Yes
0 No
ACSYS: Air conditioning system
0 None
1 Refrigeration
2 Evaporative cooler
3 None
DWATER-B: Drinking water supply (modi-
fied to include bottled water consum-
ers)
0 Bottled water
1 Private well
2 Public supply
GHSIZE: Grouped household size
1 1 person
2 2-4 people
3 >5 people
GINCOME: Grouped income
1 <5.000
2 5000 to 9999
3 10000 to 19999
4 20000 to 29999
5 >30000
HCHILD: Age of youngest child in
household
1 No children
2 Child 6-17
3 Child 15
HOHEDGR: Education category of head
of household
1 0-8
2 9-11
3 12
4 Some college (13-15)
5 College grad (16-18)
(Categories 4 and 5 combined for some
tests.)
HOHOCC: Head of household occupation
group
1 Professional or manager
2 Farmer
3 Other
LOCATE: Dwelling location
1 Rural
2 Wilson
ABDOM: Any abdominal conditions?
0 No
1 Yes
8 Don't know
AGEGRP: Age group (as of June 30.
1982)
1 0-5
2 6-17
3 18-44
4 45-64
5 65+
BOTTLED: Drinks bottled water regu-
larly
0 No
1 Yes
CHRONIC: History of any chronic ill-
ness
0 No
1 Yes
CONTACT: Contacts per week with 10+
people
1 Less than once
2 1 to 5
3 6 to 10
4 11 to 15
5 More than 15
HEART: Any heart conditions
0 No
1 Yes
8 Don't know
OTHERO: Any other chronic conditions
1 Yes
0 No
RESP: Any respiratory illness
0 No
1 Yes
8 Don't know
SEX: Sex
1 Male
2 Female
SMOKES: Smoke cigarettes regularly
in 1983 (or most recent question-
naire)
0 No
1 Yes
continued...
176
-------
TABLE 49. (CONT'D)
Household variables
Individual variables
RACE: Race of respondent
1 Caucasian
4 Hispanic
SENTINL: Sentinel family statns for
1983
1 Yes (sentinel HH)
0 No
ZONE: Household location
1 Rural 0 to 0.5 mile
2 Wilson 0 to 0.5 mile
3 Rural 0.5 to 1 mile
4 Wilson 0.5 to 1 mile
5 Rural 1 to 2 miles
6 Workers >2 miles
TCHEW: Chew tobacco regularly
0 No
1 Tes
WCONSM: Tapwater consumed vs. others
your age
1 Less than average
2 Average
3 More than average
TABLE 50. COMPARISON OF CHARACTERISTICS:
STUDY PARTICIPANTS VS. NONPARTICIPANTS
Variable
ACOND
ACSYS
ABDOM
AGEGRP
BOTTLED3
CHRONIC
DWATER-B
GHSIZE
GINCOME
HCHILD
HEART
HOHEDGR
HOHOCC
LOCATE
OTHERO
RACE
RESP
SEX
SMOKE
ZONE
n
577
577
575
577
575
568
577
578
577
578
574
574
577
578
577
578
577
578
577
578
P value
0.045
0.045
0.014
0.006
0.028
0.038
0.025
0.012
0.042
0.06
0.001
0.017
<0.001
Comment
''none'' associated with nonparticipation
''yes'' associated with participation
''65+'' associated with participation
''yes'' associated with participation
''bottled water'' associated with participation
households with kids ages '6-17' assoc with
nonpart ic ipat ion
''some college'' associated with participation
''prof or manage'' associated with participation
''rural'' associated with nonparticipation
''yes'' associated with participation
''hispanic'' associated with nonparticipation
''yes'' associated with participation
zones 3-5 associated with nonparticipation
177
-------
TABLE 51.
PARTICIPANTS WHO REMAINED
COMPARISON OF CHARACTERISTICS:
IN THE STUDY VS. PARTICIPANTS WHO DROPPED OUT
Variable
ACOND
ACSYS
ABDOM
AGEGRP
BOTTLED3
CHRONIC
DWATER-B
GHSIZE
GINCOME
HCHILD
HEART
HOHEDGR
HOHOCC
LOCATE
OTHERO
RACE
RESP
SEX
ZONE
n
475
339
477
477
478
478
478
468
468
478
477
474
475
478
477
478
477
478
478
p value
0.013
<0.001
0.003
0.001
0.012
0.013
0.003
0.025
0.002
0.034
Comment
higher proportion of
higher proportion of
higher proportion of
pat ion
higher proportion of
pation
higher proportion of
participation
''none'' dropped out
''none'' dropped out
age 45+ continued partici-
' 'yes ' ' continued partici-
''no children'' continued
college education associated with continued
participation
higher proportion of
higher proportion of
pation
higher proportion
out
''other'' dropped out
' 'yes ' ' continued partici-
of ''hispanic'' dropped
higher proportion of zones 2 and 3 dropped
out
178
-------
Effect of Self-selection on LISS Population Characteristics—
Analysis of the questionnaire data indicates that although great efforts
were taken during recruitment to select households which were representative
of the study area, the process of self-selection resulted in some significant
demographic changes during the course of the study. In fact, the characteristics
of the population changed between the time that the initial households
were recruited (May 1980), and the time that the first blood samples and
illness diaries were collected (June 1980). One hundred ninety-six households
with 578 members were initially recruited into the study. Thirty-three
of those households (with 100 members) never actually participated in the
study. Comparison of the nonparticipating households to the 163 participating
households (with 482 members) in Table SO indicates that the two populations
were significantly different for 12 of the 20 variables examined. It can
be seen in Table 50 that residents living more that 1/2 mile from the Hancock
farm (sampling zones 3-5), hispanics, and families with children ages 6-17
were more likely to refuse to participate in the study. People with a history
of chronic illnesses, members of households with high socioeconomic status,
and members of families with children ages 0-5 were more likely to initially
participate in the study.
Sixty percent of the study participants (55% of the households) remained
with the study until its conclusion in October 1983. Twenty-four percent
of the participants dropped from the study prior to the onset of irrigation;
another 12% dropped during the irrigation period. Comparison of the partici-
pants who remained in the study until October 1983 to the participants
who dropped out (Table 51) indicates that the two populations differed
significantly for 10 of the 19 variables examined. Hispanics and participants
under the age of 45 were more likely to drop out of the study before its
conclusion. Participants living in high economic status households, participants
with a history of chronic illness, and participants living in households
with no children were more likely to stay with the study until its conclusion.
As a result of self-selection, the 288 participants who remained in
the study until its conclusion in 1983 probably were not representative
of the community surrounding the Hancock farm. The study participants
were somewhat older, had a higher socioeconomic status, reported more chronic
illnesses, and had less exposure to small children than did the members
of the general community. Since their socioeconomic status was higher
and their exposure to small children in the household was reduced, the
study population's risk of infection (by agents of concern to the LISS)
was probably somehwat lower than the infection risk of the general population.
Due to the increased age and the higher rate of chronic illness in the
study population, it might be expected that symptoms of illness (resulting
from infections by agents which were circulating through the community)
would be more severe in the study population than in the overall population.
Characteristics of Subpopulations—
Tables P-31 to P-33 of Appendix P list the results of the crosstabulations
used to determine if there were demographic differences between subpopulations
stratified on three key characteristics: race (Caucasian vs. hispanic),
sampling zone, and residence location (Wilson vs. rural). These analyses
179
-------
were performed in order to identify the presence of confounding variables
which could affect the interpretation of results of other statistical tests.
Hispanics and Caucasians differed significantly for every variable
tested except sex, head of household occupation, smoking, and use of bottled
water (Table P-31) . Hispanic participants lived in households with more
family members, were generally younger and reported a lower socioeconomic
status than Caucasian participants. Forty percent of hispanic participants
were under the age of 18; only 25% of Caucasian participants were in the
same age group. Only 4% of the hispanics were age 65 or older; 17% of
Caucasians were age 65 or older. One percent of hispanic participants
and 10% of Caucasian participants reported living in single member households.
In contrast, 23% of Caucasian participants and 68% of hispanic participants
reported that they lived in households with five or more members. Sixty-two
percent of Caucasian participants had experienced one or more chronic condi-
tions. Only 28% of hispanic participants reported experiencing any chronic
conditions. The difference in reporting of chronic conditions is not surprising
in view of the fact that almost half of the hispanic participants were
under the age of 18 and had no opportunity to develop many of the chronic
conditions which are associated with aging.
Wilson participants and rural participants were found to be significantly
different for 8 of the 20 variables examined (Table P-32) . The majority
of these differences can be attributed to the fact that 90% of the hispanic
participants lived in Wilson. In addition, 60% of the single member households
were also located in Wilson. The majority of participants living in single
member households were over the age of 65. The clustering of the low income
hispanic population with the elderly population on a fixed income caused
the Wilson participants to have a significantly lower household income
than the rural residents.
Sampling zone residents were found to differ significantly for 10
of the 20 variables examined (Table P-33). Zone 1 reported a higher socio-
economic status, fewer households with children, a higher proportion of
farmers as head of household, and a higher proportion of chronic GI illnesses.
Zone 3 had the highest proportion of participants who drank bottled water,
the highest proportion of chronic illnesses, and the lowest proportion
of smokers. Zone 4 participants drank less bottled water, reported the
fewest chronic illnesses, and had a higher proportion of both single member
and five-or-more-member households.
The presence of significant differences between subpopulations, especially
the differences observed between races, is of some concern in this study. If
hispanic households had been evenly distributed throughout the study area, the
differences between the two races would not have impacted the study. Since
the majority of hispanic households were located in Wilson, the geographic dis-
tribution of the "susceptible'' population was affected. The lower standard of
living, larger household sizes, and more frequent contact with children all
increase the hispanic participants' risk of exposure to infectious agents.
Therefore, the risk of infection (caused by the agents of concern in this
study) was theoretically greater in the Wilson area than in the surrounding
rural area. The presence of a higher standard of living in Zone 1 coupled
180
-------
with the absence of children in over half of Zone 1 households suggests that
the risk of infection was comparatively small for residents and neighbors
of the Hancock Farm. Based on demographic differences (and on the assumption
of no effect of wastewater aerosol), the acute illness rate was expected
to be greater in the Wilson area than in the vicinity of the Hancock farm.
It was also expected that the differences (in illness and infection rates)
between Wilson and the surrounding area would decrease as the process of
self-selection caused the Wilson and Hancock farm residents to become demograph-
ically more similar as the study progressed (Table 51).
Characteristics of Donor Groups—
Four hundred thirty-five (91%) of the 478 participants provided at
least one blood specimen during the course of the study. Thirty-three percent
of the participants provided all eight of the requested bloods, 43% of
the participants provided four to seven of the requested bloods. Twenty-four
percent of the participants provided one to three bloods; this group includes
children who were born during the course of the study and participants
who dropped out of the study prior to the onset of irrigation. Comparison
of the three groups of blood donors (Table P-34) reveals that these groups
differed significantly for 13 of the 20 variables examined. Since the grouping
of blood donors is similar to the grouping used to compare participants
who remained in the study to those who dropped out (Table 51), significant
differences in age, race, chronic illness history, and socioeconomic status
were expected. Blood donor groups differed for two additional characteristics.
drinking water source and household location. Wilson residents and participants
who drank bottled water were more responsive to requests for blood samples.
In terms of transportation and convenience, it was easier for Wilson residents
to provide blood samples. Rural residents who lived on unpaved roads had
more difficulty providing the samples, especially in June and December
1982, when inclement weather frequently caused roads to be impassable.
Table P-35 of Appendix P compares ''sentinel1' participants to the
remainder of the study participants. Sentinel participants were the only
study participants who were asked to continue to provide illness information
between October 1982 and October 1983. All Zone 1 families and all study
participants with wastewater contact were automatically included in the
sentinel group. The remainder of the sentinel families were selected on
the basis of three criteria: their willingness to continue to participate
in the study, a history of chronic illness, and demographic similarity
to the households in Zone 1. Since Zone 1 families differed demographically
from the rest of the study population (Table P-33), all but one of the
significant differences observed between the sentinel family members and
the participant population were expected. The unexpected difference, less
smoking in the sentinel participants than in the overall population, did
not appear to be associated with any of the other demographic variables
except sampling zone (Table P-33). There were more smokers located in Zone
2; however. Zone 2 was adequately represented in the sentinel population.
Tables P-36 to P-38 in Appendix P list the results of analyses which
compared fecal donors from each irrigation season to the remainder of the
participant population. Due to the small number of participants (primarily
children) who provided specimens during 1980-1981, no comparison of donors
181
-------
to nondonors could be made for that period of time. There were no significant
differences between fecal donors and nondonors in the spring of 1982. There
was a higher proportion of fecal donors from low income households in the
summer of 1982. There were also significantly fewer donors from households
with children ages 6-17 during that same period of time. There were signifi-
cantly more fecal donors with chronic conditions and fecal donors living
in single member households during both irrigation periods in 1983. Cigarette
smokers and hispanics were less likely to be donors during 1983.
The gradual increase in demographic differences between fecal donors
and nondonors in 1982 and 1983 can be explained by the fact that the rules
for donating the specimens were changed between 1982 and 1983. The number
of specimens accepted from each household was limited to two in 1983 to
reduce costs; there was no similar restriction in 1982. Therefore, many
children (especially children from hispanic households) who donated specimens
in 1980-1982 were excluded in 1983. Also, the potential fecal donors were
randomly selected as donors in January 1982. Therefore, differences between
donors and nondonors were expected to be minimal at that time. As the
study progressed, it appears that the process of self-selection became
more influential in determining who would donate specimens, and the demographic
differences increased accordingly.
Exposure Categories Based on Aerosol Exposure Indices—
Tables P-39 to P-44 in Appendix P list the demographic differences
observed between the two exposure groups and the three exposure levels
for each of the four irrigation periods, for 1982 and for 1983. Comparison
of the characteristics of the high and low exposure subgroups of blood
donors and fecal donors is provided as the preliminary statistical analysis
in Section 5L.
A quick review of the information in Tables P-39 to P-44 reveals signifi-
cant differences between exposure levels during all periods for the variables
DWATER, LOCATE, and ZONE. These differences can be explained by the fact
that the majority of participants with medium exposure to wastewater aerosols
lived in Wilson. There was also a significant difference between both
exposure groups and exposure levels for type of air conditioning system
in use during all periods of interest. The high exposure group and high
exposure level consistently used more evaporative cooler units for air
conditioning than did the remainder of the study population. There were
no differences between exposure groups or between exposure levels for the
variables age, bottled water consumption, and history of chronic illness.
Overall, there were more significant differences between exposure
levels than between exposure groups for the majority of the variables.
In addition, the variables associated with significant differences between
exposure levels (income, occupation, household size) were the same variables
for which significant differences were found when comparing Wilson residents
to rural residents. However, since portions of the Wilson population were
incorporated into both the high and low exposure groups, fewer significant
differences were observed between exposure groups.
182
-------
The presence of significant differences by exposure level, exposure
group, and subpopulations (i.e., race) necessitated the exploratory statistical
analysis of infection episodes by logistic regression to investigate their
effects and to control the association of infection status with aerosol
exposure for their effects. This analysis is presented in Section 5L.
Samples Provided by Study Population During the Health Watch
Table 52 lists the number of samples obtained from the various health
watch activities by data collection period (DCP) during the course of the
study. This table provides an overview of the scope and extent of the
health watch of the study population which the LISS maintained. Some of
the LISS results are subsequently reported by DCP. The first two columns
of Table 52 give the correspondence between DCP and calendar date for the
interested reader.
B. PATTERNS IN SELF-REPORTED ILLNESS
Study participants were contacted on a regular basis for illness infor-
mation during the study period. All participating households were asked
to keep a written illness diary in 1980; field representatives collected
the illness information by phone in 1981-1983. All households were contacted
for diaries in 1980-October 1982. Only sentinel families were contacted
for illness information after October 1982. The written diaries were collected
at 2-week (data collection period, DCP) intervals in 1980. The households
were contacted by phone on a weekly basis in 1981-1983, and the weekly
information was combined and coded for each DCP. Household members were
asked to report all acute and chronic illness conditions which occurred
during the time interval of interest. Participants were also asked to report
the number of days of illness that they experienced as well as the number
of days that they spent away from the study area.
For purposes of summarization, illnesses have been categorized into
five groups: total acute illness, respiratory illness, gastrointestinal
illness, other acute illness, and chronic conditions. Cases of trauma and
elective surgeries were recorded, but were not used in the data analysis.
An illness with both respiratory and gastrointestinal symptoms was treated
as being two distinct illnesses. Respiratory, gastrointestinal, and other
acute illnesses were included in the category ''total acute illness.''
''Other acute illness'' included all acute illnesses which were neither
respiratory nor gastrointestinal in nature. These illnesses included but
were not limited to eye and ear infections, childhood diseases, headaches
without accompanying symptoms, fevers of unknown origin, genitourinary
infections, and various skin conditions. Newly developed chronic conditions
and flare-ups of existing chronic conditions (such as arthritis) were recorded
whenever reported. However, reporting of chronic conditions in this study
was found to be quite erratic.
183
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TABLE 52. NUMBER OF SAMPLES COLLECTED FROM HEALTH WATCH ACTIVITIES
oo
Data
collection
period
1980
001
002
003
004
005
006
007
008
009
010
011
018
013
014
015
016
017
018
019
020
021
022
023
024
025
026
1981
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
Starting
date
Jan 1
Jan 13
Jan 27
Feb 10
Feb 24
Mar 9
Mar 23
Apr 6
Apr 20
May 4
May 18
Jun 1
Jun 15
Jun 29
Jul 13
Jul 27
Aug 10
Aug 24
Sep 7
Sep 21
Oct 5
Oct 19
Nov 2
Nov 16
Nov 30
Dec 14
Dec 28
Jan 11
Jan 25
Feb 8
Feb 22
Mar 8
Mar 22
Apr 5
Apr 19
May 3
May 17
May 31
Jun 14
Jun 28
Jul 12
Participant
Households Interview Health
Interviewed data diaries
197a 580B
348
366
364
351
342
331
336
402
409
405
386
375
396
401
406
Polio Routine
1«Biin1- Blood Skin fecal
zations BDednens tests specimens
318 265
22
36
47
363 33
49 24
105 11
4 45
76 287 187
1 30
1
Major
Illness Activity irrigation
specimens diaries periods
3
continued.
-------
TABLE 58. (CONT'D)
oo
Data
collection Starting Households
period date Interviewed
116
117
118
119
120
121
122
123
124
125
126
1982
801
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
1888
301
302
303
Jul 26
Aug 9
Aug 23
Sep 6
Sep 20
Oct 4
Oct 18
Nov 1
Nov 15
Nov 29
Dec 13
Jan 3
Jan 17 129
Jan 31
Fab 14
Fab 28
Mar 14
Mar 28
Apr 11
Apr 25
Nay 9
Hay 23
Jun 6
Jun 20
Jul 4
Jul 18
Aug 1
Aug 15
Aug 29
Sep 12
Sep 28
Oct 10
Oct 24
Nov 7
Nov 21
Dec 5
Dec 19
Jan 2
Jen 16
Jan 30
Participant Polio Routine
Interview Heelth Innuni- Blood Skin fecal
data diaries zatlone specimens tests spec linens
407
405
413
350
365 381
387
386
387
388
388
387
389
389
387
370
373
367
367
359
354
352
351
360
357
175
175
175
180
181
181
181
22
11
6 34
3 8
41 330 107
8
3
3
10 127
3
7 127
9
1
6
310 124
3
2
1
119
121
1
10 268 245
1 15 100
Illness
spec leans
2
4
5
1
7
2
5
4
6
7
1
3
16
15
8
4
5
11
6
2
12
5
12
Major
Activity Irrlgetlon
diaries periods
Feb 16-
X
194 X
X
156 X
-Apr 30
Jul 21-
261 X
X
X
-Sep 17
332
continued.
-------
TABLE 52. (CONT'D)
Daia
collection Starting Households
Eeriod date Interviewed
304
305
306
m
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
Feb 13
Feb 27
Mar 13
Apr 10
Apr 24
Hay 8
Hay 22
Jun 5
Jun 19
Jul 3
Jul 17
Jul 31
Aug 14
Aug 28
Sep 11
Sep 25 107
Oct 9
Oct 23
Nov 6
Nov 20
Dec 4
Dec 18
Participant Pullu Homino
interview Health Immuni- Blood Skin fecal
data diaries zatlone specimens tests specimens
181 1
181
181
181 5
182 109
183 5
183
183 5
176 1 273 102
175
175
168 105
168
165 101
156
159
306 161 267 202
Illness
specimens
7
4
2
4
2
17
7
3
2
2
2
Hajor
Activity Irrigation
diaries periods
Feb 15-
X
10 X
X
309 X
-Apr 30
Jun 29-
317 X
X
17 X
X
X
-Sep 20
00
The household head or spouse was Interviewed upon recruitment regarding all household members. One—hundred fifty six
household interviews of 430 members occurred in OCR 011 or 012, but replacement households end new family members were
recruited into the study until DCP 212. Thirty four of the 197 interviewed households (102 of 580 members) which were
recruited In DCP 011 never actually participated in the study.
-------
TABLE 53. MONTHLY INTERVALS FOR Two measures of illness were
SELF-REPORTED ILLNESS DATA BY employed to characterize the self-reported
DATE AND DCP illness. Incidence density, defined
as the number of new illnesses per
1000 person-days of observation,
was used to measure the occurrence
of new illness in the population.
Prevalence density, defined as the
number of person-days of illness
per 1000 person-days of observation,
is a period prevalence measurement
which was used to characterize the
burden or duration of the illnesses
which were observed during a given
period of time. These rates were
calculated for both the two exposure
groups and the three exposure levels
(based on AEI calculations) for ''mon-
thly'' intervals of time. The AEI
values from the spring 1982 irrigation
period were used to determine exposure
groups and levels for the illness
data from July 1980 through Hay 1982.
The summer 1982 AEI values determined
exposure groupings for the June through
December 1982 illness data. For
1983, the correspondence used was:
spring 1983 AEI for January-May 1983
and summer 1983 AEI for June-September
1983. Since all data were collected
on a 2-week basis, the DCPs did not
always correspond with the exact
beginning and ending of each of the
months. Table 53 lists the DCPs
which correspond with each of the
months used to present the self-reported
illness information.
It should be noted that all
of the self-reported illness information,
especially the baseline information, should be interpreted with extreme
caution. In addition to the normal problems and biases that are encountered
with self-reported data, the methodology for collecting this information
was revised several times during the course of the study in order to improve
the consistency, reliability, and completeness of the information. Thus,
these data should be regarded as varying in consistency, reliability, and
completeness. The illness information may be too unreliable to permit
secular comparisons (i.e., comparison of rates in the same month of different
years) due to the revisions in methodology. Illness information was only
collected for a three month period (July-September) in 1980. Information
which was collected during this period of time was at best incomplete.
since many households did not provide any illness data due to collection
Month
Jul 1980
Aug 1980
Sep 1980
Apr 1981
May 1981
Jun 1981
Jul 1981
Aug 1981
Sep 1981
Jan 1982
Feb 1982
Mar 1982
Apr 1982
May 1982
Jun 1982
Jul 1982
Aug 1982
Sep 1982
Oct 1982
Nov 1982
Dec 1982
Jan 1983
Feb 1983
Mar 1983
Apr 1983
May 1983
Jun 1983
Jul 1983
Aug 1983
Sep 1983
DCPs
014-015
016-017
018-020
108-109
110-111
112-113
114-115
117-118
119
201-202
203-204
205-206
207-209
210-211
212-213
214-215
216-217
218-219
220-222
223-224
225
301-302
303-304
305-306
307-309
310-311
312-313
314-315
316-317
318-320
Dates
Jun 2 9- Jul 26
Jnl 27-Aug 23
Aug 24-Oct 4
Apr 5 -May 2
May 3-May 30
May 31- Jun 27
Jun 2 8- Jnl 25
Aug 9-Sep 5
Sep 6-Sep 19
Jan 3-Jan 30
Jan 31-Feb 27
Feb 28-Mar 27
Mar 28-May 8
May 9- Jun 5
Jun 6- Jul 3
Jul 4- Jul 31
Aug 1-Aug 28
Aug 29-Sep 25
Sep 26-Nov 6
Nov 7-Dec 4
Dec 5-Dec 18
Jan 2-Jan 29
Jan 30-Feb 26
Feb 27-Mar 26
Mar 27-May 7
May 8- Jun 4
Jun 5-Jul 2
Jul 3-Jul 30
Jul 31-Aug 27
Aug 28-Oct 8
187
-------
problems. Information which was obtained between April and September 1981
was more complete but should still be regarded with caution. Also of note
is the fact that there is no baseline information available for the October
to March interval of time. Therefore, interpretation of illness rates from
October 1982 to March 1983 is limited since there is no basis for comparison.
Finally, for purposes of consistency, AEI values from the spring of 1982
were used to classify participants into the three exposure levels and two
exposure groups during the baseline period. The subpopulations in the
''spring'' exposure levels differ slightly from the subpopulat ions in the
"summer" exposure levels (The high exposure level is comprised of essentially
the same participants, but the low and intermediate populations shift drama-
tically between ''spring1' and ''summer.''). Thus, two slightly different
populations are being compared when the irrigation year illness rates (based
on ''summer'' exposure levels) are compared to baseline rates (based on
''spring'' exposure levels) for the same monthly intervals.
Illness incidence density ratios and their associated test-based 90%
and 95% confidence intervals were calculated as described in Section 4T.
These ratios and associated confidence intervals were used to identify
the consistent patterns in the data and to identify stable ratios (i.e.,
those for which the confidence intervals were tight). It should be empha-
sized that the various problems with the illness data limit the extent
to which these results can be extrapolated or directly compared to data
from other studies.
Table 54 summarizes the monthly incidence densities by type of acute
illness and by exposure level. Table 55 summarizes the same information
by exposure group. Cases where the 90% or 95% confidence interval for the
incidence density ratio did not include the value 1 have been indicated,
provided the expected illness incidence in each exposure category was 2.0
or more. Figures 19-26 present the total acute illness and respiratory
illness rates from Table 54 in a bar graph format. Tables 56 and 57 summarize
the prevalence density rates. Since the prevalence density rates followed
a trend similar to the incidence density rates, this information is not
presented in a graphic format.
Baseline
Illness information was collected between July and September in 1980.
The high exposure level experienced the highest rate of illness during
the month of July: the illness rate in the high exposure level was twice
the rate of both the low and intermediate exposure levels. Both ratios
of incidence densities were found to be stable and possibly significant
using 90% confidence intervals. The low exposure level experienced the
highest rate of illness during August and September.
Illness information was collected from April-September in 1981. The
high exposure level had the highest rate of illness during May and July.
Illnesses reported in May and June had symptoms which were primarily gastro-
intestinal in nature. Although the rate of new GI illnesses appeared to
be higher in both the high and intermediate (primarily Wilson) exposure
levels, the prevalence density information in Table 56 indicates that the
188
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TABLE 54. MONTHLY INCIDENCE DENSITY OF SELF-REPORTED ILLNESSES BY TYPE OF ILLNESS AND EXPOSURE LEVEL
(Number of New Illnesses Per 1000 Person-days)
[Number of New Illnesses Indicated In Brackets]
oo
ve
Total acute Respiratory
Low
Exp
Level
Mad
Exp
Level
61
Other acute
High Low Med High Low Hed High Low Med
Exp Exp Exp Exp Exp Exp Exp Exp Exp
Level Level Level Level Laval Level Level Level Level
High
Exp
Level
1980
Jul 4.9[131 5.2F23] 9.9[10]a'b 2.7(7} 1.6[7] 5.0[5]c 1.5[4] 2.0[9] 2.0[2] 0.8[2] 1.6[7] 3.0[3]
Aug 7.0M8] 3.2tl5l 3.2(3] 3.5[9] 1.9[9] 2.1[2] 2.3[6] 0.6[3] 0.0[0] 1.2[3] 0.8[3] 1.1[1]
Sap 7.8[31] 2.0[13] 5.0[6]e 3.5(14] 1.4[9] 1.7[2] 3.3[13] 0.6[4] 1.7[2] 1.0[4] 0.0[0] 1.7[2]
1881
Apr 9.4[29] 5.2[27] 6.3[7] 6.5(20] 4.0(21] 1.8(2] 2.3
Hey 4.4(13] 5.2(27] 7.7(8] 1.4(4] 2.7(14] 1.0(1] 2.0(
Jun 1.7(5]
2.6(13] 1.0(1] 0.0(0] 0.8(4] 0.0(0] 1.4
Jul 5.0(15] 0.8(4]
Aug 5.2(12] 0.7(3]
Sep 8.0(8]
1888
0.9(2]
6.1(6] 0.7(2] 0.4(2] 3.1(3] 4.0
1.1(1] 2.6(6] 0.7(3] 0.0(0] 1.7
7] 0.8(4] 1.8(2] 0.6(2] 0.4(2] 2.7(3]
6] 1.7(9] 4.8[5]a 1.0(3] 0.8(4] 1.9(2]
4] 1.6(8] 1.0
12] 0.2(1] 1.0
4] 0.0(0] 0.0
6.6(3] 1.8(2] 0.9(2] 4.4(2] 6.3(7] 0.0(0] 0.0
[1] 0.3(1] 0.2(1] 0.0(0]
1] 0.3(1] 0.2(1]
2.0(2]
0] 0.9(2] 0.0(0] 1.1(1]
0] 0.0(0] 0.0(0] 2.2(1]
Jan 8.2(24] 8.4(45] 9.5(10] 7.2(21] 6.5(35] 9.5(10] 1.0(3] 0.7(4] 0.0(0] 0.0(0] 1.1(6] 0.0(0]
Fab 11.4(34]
Mar 8.2(25
Apr 11.1(48
May 4.4(13
Jun 1.9(6]
6.7(35]
5.9(29
8.7(70
4.9(25
13.0[13]c 7.7(23] 3.4(18] 10.0(10]° 3.4(10] 2.3(12] 2.0(2] 0.3(1] 1.0(5] 1.0(1]
10.3(11] 6.3(19] 3.2(16] 8.4[9]c 1.3(4] 1.4(7] 1.9(2] 0.7(2] 1.2(6] 0.0(0]
5.4(9] 7.6(33] 5.7(46] 4.8(8] 2.5(11] 2.5(20] 0.0(0] 0.9(4] 0.5(4] 0.6(1]
7.3(8] 1.3(4] 2.1(11] 5.5[6]8'd 2.7(8] 2.3(12] 0.0(0] 0.3(1] 0.4(2] 1.8(2]
2.5(12] 4.0(4] 0.3(1] 1.3(6] 2.0(2] 0.3(1] 0.6(3] 2.0(2] 1.3(4] 0.6(3] 0.0(0]
Jul 4.4(13] 9.2(46] 5.6(6] 2.4(7] 3.6(18] 2.8(3] 1.4(4] 3.8(19] 0.9(1] 0.7(2] 1.8(9] 1.9(2]
Aug 4.4(12] 6.9(34] S.3(8]b 3.3(9] 3.2(16] 3.5(3] 1.1(3] 3.0(15] 3.5(3] 0.0(0] 0.6(3] 2.3(2]
Sep 11.4(34] 10.8(55] 12.5(12] 6.7(20] 6.5(33] 5.2(5] 3.7(11] 3.0(15] 6.3(6] 1.0(3] 1.4(7] 1.0(1]
Oct 12.9(49] 11.4(75] 4.8(7] 8.2(31] 4.9(32] 4.8(7] 4.0(15] 5.0(33] 0.0(0] 0.8(3] 1.5(10] 0.0(0]
Nov 17.3(25] 10.8(27] 15.6(15] 13.8(20] 6.4(16] 10.4(101 2.8(4] 3.2(8] 1.0(1] 0.7(1] 1.2(3] 4.2(4]
Dec 3.8(3]
1883
6.3(8]
11.2(6] 0.0(0] 4.0(5] 9.3[5]° 1.3(1] 2.4(3] 1.9(1] 2.6(2] 0.0(0] 0.0(0] .
Jan 14.6(22] 11.9(26] 9.2(11] 12.6(19] 10.1(22] 7.6(9] 2.0(3] 1.4(3] 0.0(0] 0.0(0] 0.5(1]
Feb 11.0(17] 15.1(34] 2.5(3] 7.1(11] 9.3(21] 1.6(2] 3.2(5] 4.4(10] 0.8(1] 0.6(1] 1.3(3
Mar 6.9(10] 10.0(22] 5.1(6] 6.2(9] 8.6(19] 2.5(3] 0.0(0] 0.0(0] 0.8(1] 0.7(1] 0.9(2
Apr 7.7(17] 3.2(10] 7.5[13]c 5.9(13] 2.3(7] 5.2(9]a 1.4
May 6.6(10] 5.7(12] 5.8(7] 4.7(7] 1.9(4] 5.8[7]B 1.3
Jun 7.1(8]
Jul 6.6(7]
Aug 2.8(3]
3.0(6]
4.3(9]
3.8(8]
6.9(7] 2.7(3] 3.0(6] 4.9(5] 1.8
7.1(7] 3.8(4] 1.9(4] 6.1(6]c 1.9
2.9(3] 0.0(0] 2.4(5] 1.9(2] 1.9
Sep 7.6(13] 8.5(26] 7.5(11] 4.7(8] 3.6(11] 2.1(3] 1.8
3] 0.6(2] 0.6(1] 0.5(1] 0.3[i:
2] 3.3(7] 0.0(0] 0.7(1] 0.5(1!
1.7(2]
0.0(0]
1.7(2]
1.7(3]
0.0(0]
2] 0.0(0] 0.0(0] 2.7(3] 0.0(0] 2.0(2]
2] 1.4(3] 1.0(1] 0.9(1] 1.0(2] 0.0(0]
2] 1.4(3] 1.0(1] 0.9(1] 0.0(0] 0.0(0]
3] 4.6(14] 4.1(6] 1.2(2] 0.3(1] 1.4(2]
a The SOX confidence Interval of the incidence density ratio of hlgh-to-lntemediate exposure levels does not include the value 1.
b The 90% confidence Interval of the incidence density ratio of high-to-low exposure levels does not Include the value 1.
c The 95% confidence Interval of the Incidence density ratio of h1gh-to-1ntemediate exposure levels does not Include the velue 1.
d The 95% confidence interval of the incidence density ratio of high-to-low exposure levels does not Include the value 1.
-------
TABLE 55. MONTHLY INCIDENCE DENSITY OF SELF-REPORTED ILLNESSES BY TYPE OF ILLNESS AND EXPOSURE GROUP
(Number of New Illnesses Per 1000 Person-days)
[Number of New Illnesses Indicated In Brackets]
Total Acute Respiratory
61
Low High Low High Low High
exp exp exp exp exp exp
group group group group group group
1880
Other acute Chronic
Low
exp
group
High Low High
exp exp exp
group group group
Jul 4.9[27] 7.5[19] 1.8[10] 3.5[9] 1.5[B] 2.7(7] 1.6[9] 1.2(3] 0.2[1] 0.4[1]
Aug 4.8[27] 3.6[9] 2.9[16] 1.B[4] 1.3(7] 0.8[2] 0.7[4] 1.2[3] 0.0[0] 0.0[0]
Sep 5.0[42] 2.5[8] 2.5[21] 1.2[4] 2.0[17] 0.6(2] 0.5(4] 0.6(2] 0.1(1] 0.3(1]
1881
Apr 7.B
Nay 4.8
Jun 2.7
Jul 2.9
Aug 3.1
50] 4.3(13] 6.0(38] 1.6(5] 1.6(10] 1.0(3] ,_ 0.3(2] 1.6[5]a 0.0[l
30] 6.2(18] 2.4(15] 1.4(4] 1.6(10] 3.5(10]° 0.8(5] 1.4(4] 0.2(1
17] 0.8(2] 0.6 4] 0.0(0] 1.8(11] 0.8(2] 0.3(2] 0.0(0] 0.0[l
18] 2.7(7] 0.6(4] 1.1(3] 1.9(12] 0.8(2] 0.3(2] 0.8(2] 0.0[(
15] 0.4(1] 1.8 9] 0.0(0] 0.8(4] 0.0(0] 0.4(2] 0.4(1] 0.0[(
)] 0.0(0]
] 0.3(1]
I] 0.0(0]
)] 0.0(0]
)] 0.0(0]
Sep 3.8(10] 3.1(4] 1.2(3] 2.3(3] 2.7(7] 0.0(0] 0.0(0] 0.8(1] 0.0(0] 0.0(0]
1982
Jan 8.0
Fob 8.3
Mar 7.1
Apr 9.2
Hay 4.5
51] 9.428] 6.6(42] 8.0(24] 0.9(6] 0.3(1] 0.5(3] 1.0(3] 0.0(0] 0.0(0]
53] 10.3 29] 5.3(34] 6.0(17] 2.2(14] 3.6(10] 0.8(5] 0.7(2] 0.0(0] 0.0(0]
45] 7.3 20] 4.9(31] 4.8(13] 1.3(8] 1.8(5] 0.9(6] 0.7 2] 0.0(0] 0.0(0]
88] 8.7(39] 6.0(57] 6.7(30] 2.6(25] 1.3(6] 0.6(6
28] 6.1 18] 1.9(12] 3.0(9] 2.4(15] 1.7(5] 0.2(1
Jun 2.2(16] 3.7(6] 0.7(5] 2.5(4] 0.6(4] 1.2(2] 1.0(7
Jul 7.6(55] 5.7(10] 3.3(24] 2.3(4] 3.2 2
Aug 6.3(44] 6.5(10] 3.6(25] 1.9(3] 2.3 1
Sep 11.3(63] 10.7(18] 7.1(52] 3.6(6] 3.1 2
Oct 11.8(112] 8.2(19] 6.4(61] 3.9(9] 4.3(4
Nov 13.1(49] 15.3(18] 9.4(35] 9.3(11] 2.7(1
Dec 4.7(9] 11.9[8]B 2.1(4] 8.9[6]a 1.6(2
1888
3] 0.6(1] 1.1(8
6] 3.2(5] 0.4(3
3] 5.3(9] 1.1(8
0.7(3] 0.0(0] 0.2 1]
1.3 4] 0.0(0] 0.0 0]
0.0(0] 0.0(0] 0.0 0]
2.8 5]b 0.0(0] 0.0 0]
1.3 2] 0.0(0] 0.0 0]
1.8(3] 0.0(0] 0.0 0]
H] 3.0(7] 1.1(10] 1.3(3] 0.0(0] 0.0 0]
0] 2.5(3] 1.1(4] 3.4(4] 0.0(0] 0.0(0]
] 3.0(2] 1.1(2] 0.0(0] 0.0(0] 0.0 0]
Jan 13.4(41] 9.9(18] 11.4(35] 8.3(15] 2.0(6] 0.0(0] 0.0(0] 1.7(3] 0.0(0] 0.0(0]
Fob 13.1(41] 6.9(13] 8.3(26] 4.3(8] 3.8(12] 2.1(4
Mar 9.7(29] 5.0(9] 8.7(26] 2.8(5] 0.0(0] 0.6(1
Apr 5.2(23] 6.4(17] 3.8(17] 4.5(12] 1.1[S
Hey 7.0
Jun 4.6
Jul 5.0
Aug 3.3
21] 4.4(8] 3.7(11] 3.8(7] 3.0[S
14] 6.1(7] 3.0(9] 4.45] 0.7[£
15] 7.0(8] 2.7(8] 5.3 6] 1.3(4
10] 3.3(4] 1.7(5] 1.7 2] 1 .3 4
] 0.4(1]
1 0.0(0
1.0(3
1.0(3
0.2 1
0.3(1
] 0.0(0] 1.0(3
\] 1.8(2] 1.0(3
0.5(1] 0.0(0] 1.1(2]
1.1(2] 0.0(0] 0.0 0]
1.5(4] 0.0(0] 0.0 0]
0.5(1] 0.0[(
1.7(2] 0.0[l
] 0.0(0] 0.0 (
U 1.7(2] 0.3(1] 0.0(0] 0.0((
Sep 8.2(37] 7.6(13] 4.2(19] 1.83] 3.3(15] 4.7(8] 0.7(3] 1.2(2] 0.0 I
)] 0.0(0]
1] 0.0(0]
)] 0.0(0]
)] 0.0(0]
)] 0.0(0]
a The 95% confidence Interval of the Incidence density ratio of high-to-low exposure groups does not Include
the value 1.
b The BOX confidence interval of the Incidence density ratio of high-to-low exposure groups does not Include
the value 1.
-------
zg -
17 -
16 -
15 -
14 -
13 -
12 -
11 -
10 -
9 -
8 -
7 -
S -
5 -
4 .
3 -
2 -
1_
™
0 -
n
r
.
s
N
S
s
s
s
s
s
s
s
JMN FEB k
]
_
s1
s
s
s
s
s _
s
s
s
s
s
s
»
i
•
rfl
IB
MR APR MAY JUN JUL AUG SEP
[RRIG
IRRIG
_
^
s
s
s.
s
s
s
s
s
s
s
s
s
OCT NCV
•
DEC
I LOW EXP
| INTERNED. EXP
I HIGH EXP
Figure 21. Incidence density rates by exposure level
for total acute illness by month—1982
19 -
18 -
17 -
IS -
14 -
13 -
12 -
11 -
10 -
9 -
8 -
7_
"
6 -
5 -
4 -
3 -
2 -
1 -
0 -
i
i
V
s
S
V
s
s
s
V
s
s
s
s
s
s
s
s
*
VI
J
•a
HI
J
^fl_ .
_
JAN FEB MAR APR
IRRIG
MAY JUN
I
1 1 1 I
JW. AUC SEP OCT NOV DEC
IRRIG
LOW EXP
INTERMED. EXP
HIGH EXP
Figure 22. Incidence density rates by exposure level
for total acute illness by month—1983
192
-------
I
13
20
19 -
18 -
17 -
16 -
15 -
14 -
13 -
12 -
11 -
10 -
9 -
8 -
7 -
6 -
3 -
4 -
3 -
2 -
1 -
0
1
JAN FEB MAR APR MAY JUN JUL AUC SEP OCT NCV DEC
LOW EXP CS INTERNED . EXP •§ HIGH EXP
Figure 19. Incidence density rates by exposure level
for total acute illness by month — 1980
20
19 -
18 -
17 -
16 -
15 -
14 -
13 -
12 -
11 -
10 -
9 -
8 -
7 -
6 -
3 -
4 -
3 -
2 -
1 -
0
JAN FEB MAR APR MAY JUN JUL AUC SEP OCT NCV DEC
l~~l LOW EXP
INTERNED. EXP
HIGH EXP
Figure 20. Incidence density rates by exposure level
for total acute illness by month—1981
191
-------
10 -
14 -
13 -
s 11 "
w 10 -
UJ
»• 9 -
i * -
§ ? ^
8 6-
§ 5 -
> 4 -
z 3 -
2 -
1 -
0 -
111
JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC
l~"l LOW EXP C33 INTERNED. EXP •• HIGH EXP
Figure 23. Incidence density rates by exposure level
for respiratory illness by month — 1980
14 -
13 -
I «-
' 11 -
g 10 -
0. 9 -
1 • -
^ 7 -
8 6-
3 5 -
i ;:
%
2 -
1 -
il
^ In 1
1 1 nl
I"! n rt! i III , , , ,
JAN FEBMARAPRMAYJUNJULAUCSEPOCTNOV DEC
f— 1 LOW EXP
INTERNED. EXP
HIGH EXP
Figure 24. Incidence density rates by exposure level
for respiratory illness by month — 1981
193
-------
14 -
13 -
12 -
11 -
10 -
9 -
e -
7 -
6 -
5 -
4 -
3 -
2 -
1 -
-
«
s
v
s
s
s
s,
s
s
s
\
\
s
s
s
JAN FE
•
s
*
s
V
s
s
^BlG***
c
^N
rv
Is
•
1
£•
rfl
MAY JON
m
>«
>3
l_
SB
SB
J
^|
s|
1
•
_
_•
^B
sB
sB
J
<>•
J
1
S
s
s
s
\
s
s
s
s
•>
s
s
s
ML AUC SEP
L IRRIG
-1
.^
^H
sH
^^1
^^1
^^H
^1
^H
vfl
9
s
V
s
s
s
s
V
s
OCT NOV DEC
f~l LOW EXP
C3INTERMED. EXP
HIGH EXP
Figure 25. Incidence density rates by exposure level
for respiratory illness by month—1982
13
14
13
12
11
10
0
8
7
6
5
4
2
1
0
JAM FEB MAR APR
IRRIG
MAY JUM
JUL AUC SEP OCT MOV DEC
IRRIG
II LOW EXP
INTERNED. EXP
HIGH EXP
Figure 26. Incidence density rates by exposure level
for respiratory illness by month—1983
194
-------
TABLE 56. MONTHLY PREVALENCE DENSITY OF SELF-REPORTED ILLNESSES BY TYPE OF ILLNESS AND EXPOSURE LEVEL
(Number of New Illnesses Per 1000 Person-days)
[Number of New Illnesses Indicated In Brackets]
*o
1980
Jul
Aug
Sap
1881
Apr
May
Jun
Jul
Aug
Sep
1882
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
1888
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Low
Exp
Level
59.5M57]
24.2(62]
52. 9 [211]
51 .4(159]
62.0(183]
11.0(32]
B7. 9(204]
21.8(50]
35.7(40]
50.7(148]
65.1 [194]
64.9 [197]
63.6(276]
40.1(119]
13.3(41]
36.1(106]
23.7(65]
76.0(226]
78.6(298]
144.5(209]
85.9(67]
127.7(192]
169.7(262]
113.8(164]
52.3(116]
67.2(101]
52.5(59]
48.8(52]
13.2(14]
55.8[95L
Total acute
Mad
Exp
Level
24. Of 106]
16.5(76]
14.1(92]
32.8(171]
26.1(135]
11.6(58]
4.9(24]
2.9(12]
7.3(17]
46.2(248]
46.6(244]
33.3(165]
59.6(480]
29.7(153]
24.8(118]
75.9(379]
61 .9(305]
80.7(410]
80.0(527]
77.0(193]
81.7(103]
79.2(173]
115.6(260]
90.6(199]
31.5(98]
37.0(78]
14.3(29]
21.9(46]
15.5(33]
31.7(97]
Respiratory
High
Exp
Level
Low
Exp
Level
52.6(53] 8.7(23]
27.7(26] 14.1(36]
23.5(28] 32.1(128]
20.5(23] 38.5(119]
36.7(38] 8.8(26]
4.1(4]
0.0(0]
45.8(45] 5.0(15]
7.8(7]
8.3(19]
54.7(25] 8.9(10]
77.1(81] 48.3(141]
95.9(96] 50.3(150]
75.6(81] 60.3(183]
33.8(56
47.6(52
71.6(71
47.0(50
54.5(47
53.0(230]
23.6(70]
7.4(23]
9.9(29]
9.5(26]
50.1(48] 48.7(145]
51.3(75] 59.6(226]
99.8(96] 128.6(186]
215.0(115] 66.7(52]
52.9(63] 115.0(173]
36.3(44] 129.5(200]
49.9(59] 104.1(150]
65.9(114] 45.1(100]
44.3(53] 59.8(90]
70.0(71] 24.0(27]
34.4(34] 36.6(39]
30.8(32] 1.9(2]
30.1 [44] 38.2(65]
Med
Exp
Level
8.2(36]
11.9(55]
8.6(56]
26.3(137]
17.2(89]
2.0(10
2.3(11
2.9(12
7.3(17
39.5(212]
35.7(187]
18.4(91]
45.0(363]
14.6(75]
12.6(60]
22.4(112]
27.2(134]
48.4(246]
37.2(245]
51.8(130]
65.9(83]
71.5(156]
84.4(190]
86.9(191]
23.8(74]
19.5(41]
14.3(29]
13.3(28]
10.4(22]
15.7(46]
High
Exp
Level
35.7(36]
6.4(6]
6.7(8]
8.3(7]
7.7(8]
0.0(0]
20.4(20]
0.0(0]
24.1(11]
77.1(81]
85.9(86]
71.9(77]
30.8(51]
32.9(36]
42.3(42]
16.9(18]
17.4(15]
30.3(29]
51.3(75]
83.2(80]
129.0(69]
47.1(56]
12.4(15]
33.0(39]
40.4(70]
44.3(53]
54.2(55]
33.4(33]
27.9(29]
6.2[9]
Low
Exp
Level
6.1(16]
7.0(18]
16.3(65]
10.0(31]
42.0[124]
9.6(28]
60.6(182]
5.2(12]
26.8(30]
2.4(7]
8.7(26]
1.6(5]
6.0(26]
10.8(32]
0.3(1]
3.1(9]
4.0(11]
9.7(29]
11.6(44]
10.4(15]
9.0(7]
5.3(8]
22.7(35]
4.9(7]
5.0(11]
6.6(10]
2.7(3]
1.9(2]
7.5(8]
8.2(14]
61
Med
Exp
Level
8.4(37]
2.4(11]
2.9(19]
4.0(21]
4.4(23]
8.0(40]
0.6(3]
0.0(0]
0.0(0]
2.2(12]
6.7(35]
5.7(28]
7.4(60]
10.5(54]
1.5(7]
18.4(92]
13.0(64]
10.6(54]
20.5(135]
13.6(34]
15.9(20]
5.0(11]
26.7(60]
0.0(0]
1.3(4]
12.8(27]
0.0(0]
3.8(8]
5.2(11]
14.1(43]
High
Exp
Level
Low
Exp
Level
5.0(5] 44.7(118]
0.0(0] 3.1(8]
3.4(4] 4.5(18]
1.8(2] 2.9(9]
11.6(12] 7.5(22]
4.1(4] 1.4(4]
3.1(3] 2.3(7]
0.0(0] 8.3(19]
0.0(0] 0.0(0]
0.0(0] 0.0(0]
6.0(6
3.7(4
0.0(0
0.0(0
6.0(18]
3.0(8]
4.6(20]
5.7(17]
5.0(5] 5.5(17]
7.5(8] 23.2(68]
15.1(13] 10.2(28]
17.7(17] 17.5(52]
0.0(0] 7.4(28]
2.1(2] 5.5(8]
5.6(3] 10.3(8]
0.0(0] 7.3(11]
0.8(1] 17.5(27]
0.8(1] 4.9(7]
1.2(2] 2.3(5]
0.0(0] 0.7(1]
0.0(0] 25.8(29]
1.0(1] 10.3(11]
2.9(3] 3.8(4]
8.2(12] 9.4(16]
Other acute
Med
Exp
Level
7.5(33]
2.2(10]
2.6(17]
2.5(13]
4.4(23]
1.6(8]
2.1(10]
0.0(0]
0.0(0]
4.5(24]
4.2(22]
9.3(46]
7.1(57]
4.7(24]
10.7(51]
35.0(175]
21.7(107]
21.7(110]
22.3(147]
11.6(29]
0.0(0]
2.7(6]
4.4(10]
2.3(5]
6.4(20]
4.7(10]
0.0(0]
4.8(10]
0.0(0]
2.0(6]
High
Exp
Level
11.9(12]
21.3(20]
13.4(16]
12.5(14]
17.4(18]
0.0(0]
22.4(22]
7.8(7]
30.6(14]
0.0(0]
4.0(4]
0.0(0]
3.0(5]
14.6(16]
24.2(24]
22.6(24]
22.0(19]
2.1(2]
0.0(0]
14.6(14]
80.4(43]
5.9(7]
23.1(28]
16.1(19]
24.3(42]
0.0(0]
15.8(16]
0.0(0]
0.0(0]
15.7(23]
-------
TABLE 57. MONTHLY PREVALENCE DENSITY OF SELF-REPORTED ILLNESSES BY TYPE OF ILLNESS AND EXPOSURE GROUP
(Number of New Illnesses Per 1000 Person-days)
[Nuaber of New Illnesses Indicated In Brackets]
vo
o\
1880
Jul
Aug
Sep
1881
Apr
May
Jun
Jul
Aug
Sep
1882
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
1888
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Total
Low
exp
arouo
Acute
High
exp
aroup
40.3(222] 36.9(94]
19.6(110]
21.4(54]
33.6(283] 14.7(48]
45.0(287] 21.6(66]
42.4(266] 31.1(90]
12.7(79]
5.7(15]
36.2(225] 18.3(48]
12.7(62]
17.7(46]
48.0(305
56.8(364
48.4(306
56.9(544
31.8(198
17.8(129
2.9(7]
27.8(36]
57.6(172
60.4(170
50.1(137
59.7(288
42.4(126
Respiratory
Low
exp
group
19.2(35]
36.4(77]
51.0(164]
44.2(227]
34.9(86]
12.4(10]
41.5(26]
82.7(31]
60.4(16]
21.9(280]
19.9(284]
8.4(245]
13.8(415]
19.5(113]
63.0(101] 35.6(56]
58.3(422] 64.3(113] 13.7(124]
46.9(328] 57.5(89]
80.1(587] 57.5(97]
1.6(142]
4.2(379]
81.8(778] 52.4(122] 6.4(461]
104.1(389] 92.5(109] 6.1(309]
73.7(140] 214.8(145] 44.1(114]
105.3(323] 58.0(105] 44.3(293]
146.7(459] 57.0(107] 38.7(344]
112.9(339] 45.7(83]
43.4(320]
39.6(175] 57.9(153] 18.1(155]
56.7(169] 34.5(63]
29.1(88]
31.8(96]
13.2(40]
61.8(71]
31.6(36]
32.5(39]
41.2(1861 29.3(501
7.7(131]
17.1(56]
20.3(67]
51.7(24]
48.5(113]
High
exp
aroup
61
Low
exp
aroup
23.5(60] 6.5(36]
7.9(20] 3.8(21]
8.6(28] 10.0(84]
11.8(36] 8.0(51]
12.8(37] 20.9(131]
0.0(0]
9.9(62]
7.6(20] 29.3(182]
0.0(0]
2.5(12]
17.0(22] 11.5(30]
51.6(154] 2.0(13]
49.4(139] 6.9(44]
38.8(106] 2.5(16]
51.0(229] 7.3(70]
22.9(68] 10.6(66]
43.0(69] 1.1(8]
19.9(35!
21.3(33!
24.3(41!
36.5(85!
73.9(87!
133.3(90
14.0(101]
9.3(65]
10.2(75]
17.2(164]
11.5(43]
9.5(18]
50.8(92] 6.2(19]
32.5(61] 25.6(80]
33.0(60] 2.3(7]
33.7(89] 3.4(15]
29.0(53] 12.4(37]
47.9(55] 1.0(3]
29.0(33] 2.7(8]
24.2(29] 4.0(12]
5.3(9]
11.3(51]
Other acute
High
exp
group
8.6(22]
3.2(8]
1.2(4]
1.0(3]
9.7(28]
3.8(10]
2.3(6]
0.0(0]
0.0(0]
2.0(6]
8.2(23]
7.7(21]
3.6(16]
6.7(20]
3.1(5]
4.6(8]
14.8(23]
14.8(25]
6.4(15]
6.8(8]
17.8(12]
0.0(0]
8.5(16]
0.6(1]
0.8(2]
0.0(0]
0.0(0]
2.6(3]
8.3(10]
10.5(181
Low
exp
group
27.4(151]
2.1(12]
4.2(35]
1.4(9]
6.1(38]
1.1(7]
2.7(17]
3.9(19]
0.0(0]
1.9(12]
5.6(36]
7.1(45]
6.2(59]
3.0(19]
9.0 65]
27.2 197]
17.3 121]
18.2 133]
18.1(153]
9.9(37]
4.2(8]
3.6(11]
11.2(35]
4.0(12]
1.1(5]
0.3(1]
9.6(29]
7.0(21]
1.3(4]
4.9(221
High
exp
grouo
4.7(12]
10.3(26]
4.9(16]
8.8(27]
8.6(25]
1.9(5]
8.4(22]
2.9(7]
10.8(14]
4.0(12]
2.8(8]
3.7(10]
5.1(23]
12.8(38]
16.8(27]
39.8(70]
21.3(33]
18.4(31]
9.4(22]
11.9(14]
63.7(43]
7.2(13]
16.0(30]
10.5(19]
23.5(62]
5.5(10]
13.9(16]
0.0(0]
0.0(0]
13.5(23]
Chronic
Low
exp
group
High
exp
group
0.7(4] 0.0(0]
0.0(0] 0.0(0]
3.9(33] 0.0(0]
0.0(0] 0.0(0]
0.0(0] 4.8(14]
0.0(0] 0.0(0]
0.0(0] 0.0(0]
0.0(0] 0.0(0]
0.0(0] 0.0(0]
0.0(0] 0.0(0]
0.0(0] 0.0(0]
0.0(0] 0.0(0]
0.0(0] 2.7(12]
0.0(0] 0.0(0]
0.0(0
0.0(0:
0.0(0
0.0(0
0.0(0]
0.0(0]
0.0(0]
0.0(0]
0.0(0] 0.0(0]
0.0(0] 0.0(0]
0.0(0] 0.0(0]
0.0(0] 0.0(0]
0.0(0] 13.3(25]
0.0(0] 0.0(0]
0.0(0
0.0(0
0.0(0
0.0(0
0.0(0
0.0(0]
0.0(0]
0.0(0]
0.0(0]
0.0(0]
0.0(0]
0.0(01
-------
low exposure level was reporting GI illnesses which continued over longer
durations during May. Yersinia enterocolitica was isolated from the routine
stool specimens of two Wilson children in June and from the routine stool
specimen of another Wilson child in July (see Table 70). None of these
participants resided in the same household or were related to each other.
Illnesses reported during July-September were primarily respiratory and
occurred mostly in rural areas.
In summarizing the self-reported illnesses which were reported during
the primarily ''summer'' months of the baseline period, the high exposure
level was found to have the highest rate of self-reported illness during
3 of the 9 months investigated. The low exposure level had the highest
rate of illness during 5 of the 9 months. The intermediate level, primarily
the city of Wilson, had the highest rate of illness during only 1 of the
9 baseline months investigated (i.e., June 1981); it may have been associated
with the GI illness which affected the entire study area. It appears, therefore,
that the participants living in rural areas tended to report a higher rate
of illness during the baseline period. Furthermore, the rural residents
in the low exposure area tended to report a higher rate of illness than
did the rural residents in the high exposure area. Given the fact that
the majority of the ''susceptibles'' (the lower socioeconomic status families
and the families with children) resided in the city of Wilson, this result
would not have been predicted.
Irrigation-1982
With the exception of the last 2 weeks of the year, illness information
was collected during all of 1982. Only the sentinel families were contacted
for information after October 23. The high exposure level participants
reported the highest rate of total acute illness in 8 of the 12 months.
The low exposure level reported the highest rate of total acute illness
in 3 of the 12 months in 1982. The intermediate exposure level reported
the highest rate of total acute illness during 1 month in 1982.
A high rate of respiratory illness was reported for all three exposure
levels prior to the initial irrigation, with the highest rates of illness
being reported in the low exposure level. During the 2-week interval after
the onset of irrigation (February 14-27, 1982), the rates of illness (primarily
respiratory) reported by the high exposure level participants increased
to a level twice as high as the illness rate reported by the low exposure
level participants and three times the rate reported by the intermediate
exposure level participants. The incidence density ratio between the high
and intermediate exposure levels was found to be significant when the 95%
confidence interval was calculated. The high-to-low exposure level ratio
was not found to be stable. This illness pattern continued through March.
In April, the rate of illness in the high exposure level decreased
as the illness rate in both the low and intermediate exposure levels increased.
While it is possible that the respiratory illnesses which were experienced
by the high exposure level after the onset of irrigation were transmitted
to the other exposure levels, comparison of April 1982 incidence density
rates to April 1981 incidence density rates suggests that April 1982 incidence
197
-------
density rates were not unusual. The prevalence densities for the same periods
of time do suggest that the respiratory illnesses reported in April 1982
lasted for a longer period of time.
The incidence density of self-reported illnesses increased in the
high exposure level during Hay, after major irrigation had ceased. This
illness pattern is similar to the pattern observed in Hay 1981, except
that the high exposure level participants reported respiratory symptoms
while the low and intermediate exposure participants reported respiratory
and GI symptoms. A Norwalk viral particle was identified in an illness
fecal specimen collected from an 18-month-old participant from Wilson during
this period of time (see Table 66). The rates of illness decreased in
June; however, the high exposure level continued to report the highest
illness rate. Of note is the fact that the prevalence of self-reported
acute illness in June 1982 was quite high when compared to June 1981, especially
in the high and intermediate exposure groups. Only 58 person-days of total
acute illness were reported for the intermediate exposure level in June
1981; 118 person-days of illness were reported for the same period in June
1982. Four person-days of illness were reported for the high exposure level
in June 1981; 71 person-days of illness were reported in June 1982. The
prevalence of illness in June 1982 may have been associated with the heavy
rainfall which occurred from the last week of Hay through June 1982 (see
Table 47) and the resultant flooding which appeared to have contaminated
many rural drinking water wells (Table 46). The intermediate exposure
level experienced a sharp increase in incidence and prevalence density
rates during the month of July. The illness observed in the intermediate
exposure level (primarily the northern section of Wilson) during July appears
to be unusual. However, since irrigation did not commence until July 21,
the unexpected increases cannot be attributed to wastewater aerosol exposure.
It should be noted that enteric Gram-negative bacteria (EGNB) were first
isolated at unusually high levels from the throats of a family living in
the northern sector of Wilson during July and prior to the summer irrigation.
This unexpected EGNB phenomenon, which was also observed by September in
both ill and healthy participants throughout the study area and lasted
into October, is discussed in Section 5.F.
The illness rate increased in the high exposure level during August
after the start of summer irrigation. Using the 90% confidence interval,
the incidence density ratio of the high-to-low exposure level was found
to be stable and possibly significant. Three weeks after irrigation commenced
(during August 15-28, 1982), the incidence density rate of total acute
illness in the high exposure level was twice the rate found in the low
and intermediate exposure levels. Using the 90% confidence interval, the
incidence density ratio of the high-to-low exposure levels was found to
be possibly significant for total acute illnesses in this 2-week period.
When prevalence density rates for total acute illness in August 1982 are
compared to rates for the same month in 1980 and 1981, it can be seen that
the low exposure level reported approximately the same rate of person-days
of illness during August for all 3 years. The prevalence rate for the
intermediate exposure group in 1982 was three times higher than the rate
reported in August 1980, and twenty times higher than the rate reported
during August 1981. The high exposure level reported a rate twice as high
198
-------
as the rate in 1980, and seven times greater than the rate reported in
1981.
Total acute illness incidence density rates increased for all exposure
levels during the month of September. The high exposure level continued
to report the highest rate of illness, especially GI illness, during this
period of time. The rate of illness in the high exposure level decreased
in October after irrigation was completed and then increased in November.
Illness rates during this period of time appeared to be quite high, especially
in the rural areas. However, an increase in respiratory illnesses was expected
during this time of year. The illness rates for all three exposure levels
decreased during December. The low exposure level reported the largest
decrease in illness; the high exposure level experienced a smaller decrease
and reported the highest rate of respiratory illness during this period
of time. Symptoms reported by the high exposure level, in combination with
the high prevalence density rates, suggest the onset of the ''flu season.''
The respiratory illness incidence density ratio of the high-to-low exposure
levels in December was found to be significant.
In summary, it appears that the high exposure level reported the highest
monthly rate of total acute illness more frequently during 1982 than in
the months observed during 1980 or 1981. The high exposure level reported
the highest rate of illness during four distinct periods of time in 1982:
after the onset of irrigation in both the spring and the summer, in late
spring, and in December. There is no basis for comparing illness rates
after the onset of the spring irrigation. Comparison of the rates of illness
after the onset of summer irrigation suggests that rates for the high and
intermediate exposure groups in August 1982 were much higher than the rates
observed during the same period of time in 1980 and 1981. The high rate
of illness in May and June occurred after spring irrigation had concluded
and followed extremely heavy rainfall. The Hay 1982 pattern was similar,
though not identical, to the May 1981 pattern, but the June prevalence
patterns in the high level were very different. Therefore, there is no
real evidence that the illnesses which were observed in the late spring
were associated with exposure to wastewater aerosols. Finally, the illness
episode during December 1982 appeared to be associated with the onset of
the ''flu season.''
Irrigation-1983
Illness information was collected from sentinel families between January
and September in 1983. The high exposure level reported the highest rate
of illness during one of the nine months that were observed (i.e., July
1983). The low and intermediate exposure levels each reported the highest
rate of illness for 4 of the 9 months.
A high rate of respiratory illness was observed in January through
March. As in December 1982, the prevalence density rates and the reported
symptoms suggested that influenza was circulating through the community.
The low exposure level participants reported the highest rate of illness
in January; the intermediate exposure participants (mainly Wilson residents)
reported the highest rate of respiratory illness in February and March.
199
-------
There was a slight increase in the total acute illness incidence density
rate for the high exposure level in March after the onset of irrigation.
However, the rate was lower than the rates for low and intermediate exposure
levels and lower than the incidence density rates observed in March 1982.
The high exposure level illness rate increased again in April, and remained
at a consistent level until August. The low and high exposure levels reported
approximately the same rate of illness between April and September, with
both exposure levels reporting a drop in illness rates in August. The
prevalence density rate for the high exposure level did not decrease in
parallel with the incidence density rate in August. The intermediate exposure
level participants reported a lower rate of illness than the high and low
exposure level participants between April and July.
In summary, it does not appear that there was an increase in the illness
rates of the high exposure level at the onset of irrigation in either February
or July 1983. After the apparent outbreak of influenza had subsided, there
appeared to be a higher rate of illness in the rural areas than in Wilson
in April, June, and July. The illness rates were similar for all three
exposure levels during May, August, and September. The pattern of illness
which was observed in 1983 bore little resemblance to the overall illness
patterns which were observed in either 1982 or the baseline years.
Discussion
Disease surveillance did not disclose any obvious connection between
illness and degree of wastewater exposure. The self-reported illness data
varied in consistency, reliability, and completeness over the July 1980-
September 1983 period of surveillance, with the better quality data obtained
during the years of wastewater irrigation. In addition, self-reports of
illness are always subject to respondent bias.
Nevertheless, it is of interest and may be significant that the partici-
pants in the high exposure level reported the highest density of illness
shortly after the onset of wastewater irrigation, both in spring 1982 and
in summer 1982. The excess total acute illness among high exposure level
participants during the spring 1982 occurred primarily during February 14-27,
1982, in the initial 2 weeks of wastewater irrigation at the Hancock farm.
The extent to which this reflects actual illness vs. reporting bias by
high exposure participants has not been ascertained. The high exposure
level participants also reported a significant excess of total acute illness
in August 1982, primarily during August 15-28 (after more than 3 weeks
of wastewater irrigation had elapsed). The high exposure level participants
did not report a comparable excess of acute illnesses during either irrigation
period in 1983. This pattern of excess illness during both irrigation
periods is consistent with the hypothesis of an association of illness
with exposure to wastewater irrigation in that the pattern appeared both
upon initial wastewater exposure and in the summer 1982 irrigation period
which produced highest exposure to microorganisms in the wastewater aerosol
(see Table 42). However, the patterns did not persist throughout either
irrigation period in 1982. The total acute illness incidence density ratios
of the high exposure level to the intermediate and low exposure levels
were less than 1.5, both for the entire spring 1982 and summer 1982 irrigation
200
-------
periods. Thus, if not a reporting artifact, the excess rate of illnesses
which might be associated with the initial and heaviest periods of microorganism
emission from wastewater irrigation was small.
Since the agents which the LISS monitored clinically and serologically
show a very high proportion of asymptomatic infection, it is difficult
to correlate the self-reported illness data with the infection episodes
which were observed. However, it is of interest and probably of health
significance that the incidence density of self-reported total acute illness
increased among high exposure level participants during the initial and
heaviest periods of microorganisms exposure via wastewater irrigation.
F. SURVEILLANCE VIA ILLNESS AND REQDESTED SPECIMENS
To determine the causative agent in self-reported respiratory and
gastrointestinal illnesses, the ill participant was asked to submit a throat
swab or stool specimen for clinical bacteriologic, virologic and electron
microscopic analyses, as appropriate. Acute illness specimens were collected
while the participant displayed symptoms. If the specimen was obtained
within 1 week after recovery from the symptoms of the illness, it was termed
a convalescent illness specimen. Follow-up specimens were also sought
to clarify the etiology of unusual bacterial findings; these were termed
requested specimens. Unusual illness within a household was investigated
using requested specimens as a primary source of information. Three substantive
illness investigations were performed in 1982.
Illness Investigations
Salmonella Investigation: Household 540, June-August 1982—
Invest igation report—Heavy growth of Salmonella sp. Group Cj was
detected in the routine fecal specimen collected from the father (54001)
on June 8, 1982. His prior routine fecal specimen collected on March 31
had contained normal fecal flora. The household was contacted on June 18
to request additional fecal specimens from all five family members and
to obtain information concerning the source of the Salmonella infection.
The father reported that he was currently being treated for a bladder
infection. He reported no other symptoms which would indicate that he
was experiencing a Salmonella infection. Exposure information was similarly
negative. He reported no exposure to wastewater and could not recall any
unusual activities in the weeks prior to collection of the fecal specimens.
He did indicate, however, that heavy rainfall and subsequent runoff had
infiltrated the well which was the source of the family's drinking water.
After consultation with the Texas Department of Health, it was also
decided that treatment of the father for a Salmonella infection was unnecessary
since he was not experiencing any symptoms. LCCIWR was asked to obtain
a sample of water from the family's well. No bacterial contamination was
found in the well water samples collected. Results of the requested fecal
specimens collected from the family on June 22 and 23 indicated normal
fecal flora in all family members except the father, whose specimen contained
a medium growth of Salmonella sp.
201
-------
The father reported a flare-up of the bladder infection on June 28.
A urine specimen was collected and sent to UTSA on July 1. Insignificant
levels of E. coli and Citrobacter sp. were recovered from this sample only
by enrichment.
Follow-up fecal specimens were obtained from all family members on
July 13 and forwarded to UTSA. Salmonella sp. was isolated at the very
light level from the specimen provided by a son, age 17 (54011). No unusual
bacteria were found in the specimens provided by the other family members.
Follow-up stool specimens were again collected from the entire family
on August 2. All specimens were found to contain normal fecal flora.
A final set of four follow-up fecal specimens was collected on Septem-
ber 15 and 16 from all family members except the father. A possibly significant
API Group I infection of the son was indicated by isolation at the heavy
level. The specimens provided by the three other family members contained
normal fecal flora.
Convalescent-phase blood was obtained from the father on August 11.
This serum was paired with acute phase serum which was obtained during
the regular blood collection clinic on June 8. UTSA obtained serological
confirmation that his infection was to Salmonella Group Cj_.
Discussion—The Salmonella infections experienced by the father in
June 1982 and by his son in July 1982 were the only infections by overt
enteric bacterial pathogens detected in the study population after wastewater
irrigation commenced. The father was being treated for a concurrent bladder
infection. However, the Salmonella infections experienced by the father
and son appear to have been asymptomatic.
Household 540 was located more than 2 km from the Hancock farm. The
aerosol exposure index values of both infected participants were low for
the summer 1982 irrigation period: AEI=0.48 for the father and AEI=1.61
for his son. The Salmonella Group Cj infection of the father preceded
the start of the summer 1982 irrigation and he reported having no exposure
to wastewater. The onset of the Salmonella infection in the son was presumably
between June 22 and July 13, prior to commencement of wastewater irrigation
operations on July 21. Since heavy rainfall runoff had recently infiltrated
the family's drinking water well, contaminated drinking water remains a
possible source of the infections, despite lack of evidence of bacterial
contamination of the water. Alternatively, the consumption of contaminated
food could be a plausible explanation for the Salmonella infection (Benenson,
1975). The genus Salmonella has an exceptionally wide host range which
would suggest a variety of possible sources. Wastewater aerosol exposure
is considered an extremely unlikely source of these Salmonella infections.
Enteric Gram-negative Bacteria (EGNB) Investigation: Household 210, June-
November 1982—
Investigation report—The mother (21002) reported on June 26, 1982
that her 3-year old son (21012) had a cold which began on June 23. A throat
swab was obtained on June 29. The son was placed on antibiotic therapy
202
-------
by his physician on Tune 30. Laboratory analysis of the throat swab yielded
normal flora on blood agar, including E. cloacae at the very light level,
but a very light level of Group A streptococci was detected by fluorescent
antibody (see Table 58).
The mother was contacted on July 8 and given the results of the son's
throat swab. She reported that he had recovered from his cold on July 2.
She also reported that she had a cold which commenced on July 7. She recovered
from the cold on July 12.
On July 13, the 3-year old and his 7-year old brother (21011) went swimming
in the Tahoka public swimming pool. The younger son developed a fever and a
sore throat later the same evening. The older son developed a fever on July 17
and complained of a headache and a stomachache. Throat swabs were collected
from both boys on July 19. It was reported that both boys recovered from their
illnesses on July 24, 1982. Moderate to heavy levels of E. coli and Entero-
bacter cloacae were found in the throat swabs from both boys, and Klebsiella
oxytoca was isolated from the younger son's throat swab (see Table 58).
Due to the unusual nature of the July 19 throat swab results, the
entire family was asked to submit additional throat swabs on July 29.
High (i.e., heavy or moderate) levels of E. coli and E. cloacae were found
in the throat cultures of all family members.
It was reported that the father (21001) slept in the living room in
front of the evaporative cooler every night during ''hot spells,'' and
that the children frequently played in front of the evaporative cooler
during the day. The evaporative cooler water and the family's drinking
water were supplied by the Wilson water system. Samples of the family's
drinking water and reservoir water from the evaporative cooler were collected
and sent to UTSA for bacterial screening on August 9. No fecal bacteria
were isolated from either sample. Investigation of other possible bacterial
sources were essentially negative. However, it was observed that the family
frequently shared drinking glasses and eating utensils. Otherwise, no
unusual sanitation problems could be identified.
Requested throat swabs were again collected from the family on August 11
and 13. E. coli, E. cloacae and K. oxytoca were found at moderate levels
in the throats of all family members except the older son, who had been
at his grandmother's house for the week prior to collection of the throat
swabs. Based on this finding, it was recommended that the family make
an effort to avoid the practice of sharing eating utensils in order to
reduce spreading of these fecal bacteria among family members.
The father reported a sore throat and cold which began on August 21
and ended on September 1. He reported that he was taking antibiotics for
the condition; however, he had not consulted a physician. A throat swab
was obtained on August 30 and forwarded to UTSA for analysis. A moderate
growth of E. cloacae was recovered from this swab, but not the Group A
streptococcus.
203
-------
TABLE 58. BACTERIOLOGY THROAT SWAB SERIES FOR DONORS WITH MODERATE OR HEAVY
LEVELS OF ENTERIC GRAM-NEGATIVE BACTERIA IN AN ILLNESS THROAT SWAB
to
o
Age
Donor on
ID 6-30-82
Household 210
21001 " 32
21002 28
21011 7
21012 3
Household 403
40301 43
Throat
swab
category8
R
R
A
R
R
R
A
R
A
R
R
R
R
A
A
R
R
R
R
A
A
Specimen
collection
date
7-29-82
8-11-82
8-30-82
9-15-82
7-29-82
8-11-82
9-14-82
11-23-82
7-19-82
7-29-82
8-13-82
9-14-82
11-82
6-29-82
7-19-82
7-29-82
8-12-82
9-15-82
11-82
2-8-83
6-10-83
Abnormal
flora?
Tes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
No
No
No
Yes
Yes
Yes
Yes
No
No
No
Yes
Clinical bacterioloKv results
Gram-negative bacteria
(level of Krowthb) Other abnormal flora
E. coli (H)
E. cloacae (M)
E. cloacae (M)
E. coli (M)
K. ozytoca (M)
E. cloacae (M)
E. cloacae (H)
E. cloacae (M)
E. coli (M)
K. ozytoca (M)
E. cloacae (H)
E. coli (H)
E. cloacae (M)
Pseudomonas sp. (L)
E. cloacae (VL) Group A strep (VL)
E. cloacae (M)
E. coli (M)
K. oxytoca (M)
E. cloacae (M)
E. coli (M)
K. ozytoca (M)
Pseudomonas sp. (M)
K. ozytoca (VL)
E. aRRlomerans (M)
continued.
-------
TABLE 58. (CONT'D)
K>
O
Age
Donor on
ID 6-30-82
40312
Household
44702
Household
50902
Household
53312
Household
54502
Household
55701
55713
55714
55715
6
447
25
509
47
533
8
545
54
557
27
10
5
2
Throat
swab
category8
A
A
C
C
R
A
R
A
A
C
A
A
A
A
A
A
A
A
Specimen
collection
date
8-17-82
9-13-82
9-18-82
2-6-83
9-19-82
10-7-82
6-8-83
9-29-82
10-12-82
1-83
7-19-83
9-20-82
9-20-82
9-20-82
11-82
12-82
9-20-82
12-82
Abnormal
flora?
Yes
No
No
No
Tes
Tes
No
Tes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Clinical bacteriology
Gram-negative bacteria
(level of «rowthb)
Achromobacter
zylosoxidans (H)
E. agglomerans (H)
E. cloacae (H)
S. liqnefaciens (H)
E. cloacae (H)
A. hydrophila (M)
E. cloacae (H)
K. pneumoniae (H)
K. ozytoca (H)
Psendomonas sp. (H)
E. agglomerans (L)
Pseudomonas sp. (L)
CDC Or. V E-2 (H)
results
Other abnormal flora
Group A strep (H)
Group A strep (L)
Group A strep (H)
Grono A strep (L)
Throat swab categories:
A - acute illness specimen collected while donor was displaying symptoms of a respiratory illness
C - convalescent illness specimen collected within 1 week after recovery from symptoms of the
respiratory illness
R - requested throat swab for follow-up or special study
Quantitation of growth on primary culture plates
H: Heavy - growth on three or all quadrants L: Light - growth on first quadrant
M: Moderate - growth on first two quadrants VL: Very Light - one to ten colonies on plate
-------
The mother and two sons spent the week of September 5-12 in Houston.
The mother reported a sore throat which began on September 12 and ended
on September 25; she received antibiotic therapy. Throat swabs were collected
from the whole family on September 14 and 15. Heavy levels of E. cloacae
were isolated from the throat of the father, but the other family members
including the mother were found to have normal throat flora.
Follow-up throat swabs were collected from the mother and sons on
November 23. These throat swabs were found to contain normal flora. The
father was unavailable at the time that throat swabs were collected and
thereafter refused to allow any more swabs to be collected.
Discussion—Enteric Gram-negative bacteria (E6NB), namely E. coli.
E. cloacae and K. oxytoca, were repeatedly recovered at moderate or heavy
levels throughout the summer from all four members of household 210. EGNB
were recovered most regularly and at highest levels from the father (see
Table 58). The levels of EGNB recovered from the throat swabs were comparable
to those routinely observed with fecal specimens. Isolation of EGNB at
these levels in throat swabs may possibly be significant, since these organisms
are uncommon in the normal human oropharynz (Youmans et al., 1980). In
two separate instances (August 11-13 and September 14-15), all family members
who spent the week prior to throat swab collection away from home had normal
throat flora, whereas all family members who stayed at home had EGNB throat
infections. Clearly, the home environment was associated with the EGNB
throat infections. The observed practice of sharing eating utensils may
have spread EGNB from one family member to another.
The initial means by which EGNB were introduced into the throats of
family members was not clearly established. E. cloacae was recovered at
very light levels along with Group A streptococci in the initial June 23
acute illness throat swab from the younger son. The public swimming pool
was a possible source, since he developed a respiratory illness attributable
to EGNB the same day that he swam there. The evaporative cooler was another
possible source, despite failure to recover fecal bacteria from the evaporative
cooler reservoir water on August 9. The evaporative cooler hypothesis
would explain both the high EGNB recovery rate and levels in the father
(due to his habit of sleeping in front of it) and the persistence of EGNB
in the throats of all household members while at home during the hottest
summer months.
Household 210 was located in the northeastern part of Wilson, approximately
750 m south of the nearest wastewater irrigation rig. All family members
received moderate aerosol exposure while at home during the summer 1982
irrigation. Their AEI values were 2.64 for the father, 2.91 for the mother,
2.90 for the older boy and 2.87 for the younger boy. However, the initial
recovery of E. cloacae (very light) from the younger boy during a cold
which began on Tune 23 preceded the brief irrigation for aerosol sampling
which commenced on July 7. The initial recovery of possibly significant
levels of EGNB from the throats of the boys was during illnesses whose
onsets on July 13 and July 17 preceded the start on July 21 of the large-scale
summer irrigation. Thus, the wastewater aerosol is a very unlikely source
206
-------
of introduction of the EGNB agents compared to the more plausible hypotheses
discussed above.
Investigation of Respiratory Illnesses Following Aerosol Exposure: Households
109 and 403, August 1982—
Investigation report—The members of household 403 visited household
109 (located across the road from the eastern edge of the Hancock farm)
on the evening of August 8, 1982. It was reported that the visit lasted
approximately 2 hours and the children, an 8-year old girl from household
109 and a 6-year old boy from 403, played outside during the visit. It
was also reported that irrigation rig 7 which was closest to household
109 was in operation that evening.
On August 9, the girl (10913) reported a sore throat. A culture was
taken that day and coxsackievirus B4 was subsequently isolated from her
throat swab (see Table 59, footnote e).
A routine stool specimen was collected from the boy (40312) on August 10,
1982 during the regularly scheduled fecal collection. Coxsackievirus B4
was subsequently isolated from that specimen also (see Table 79).
On the evening of August 13 the members of household 403 again visited
household 109. The visit lasted approximately 3 hours and the children
played outside for the entire visit. (The children rode their bikes along
the nearby roads in their outdoor play during one or both visits.) On August 17,
the boy reported a sore throat. A throat swab was collected and a heavy
level of Achromobacter xylosoxidans was isolated from his throat swab (see
Table 58).
Assessment of aerosol exji_p_sjttre._to_ jj..ajLS_at iy_e_. organisms—The a e ro s ol
exposure index values during the summer 1982 irrigation were high for the
girl (AEI=11.2) and intermediate for the boy (AEI=2.25), based on the standard
exposure estimation methodology and data sources. However, the aerosol
exposure of the boy relative to other study participants may have been
considerably higher in summer 1982 than AEI=2.25 would indicate. The exposure
estimation methodology as applied in 1982 gave virtually no weight to irregular
visits to households which were downwind of an operating irrigation rig
on the Hancock farm, unless such events also occurred during 1983 when
better exposure records were kept (see section 4C). However, better information
exists concerning the aerosol exposure of the children in the vicinity
of household 109 for the days preceding their illness onsets.
Household 109 was located across the road from an irrigation rig which
passed within 120 m of the homestead as it traversed its irrigation circle.
This rig sprayed wastewater supplied via pipeline directly from the Lubbock
sewage treatment plant on many of the days preceding onset of the illness
events. Estimated daily irrigation and aerosol drift patterns from the
two nearest rigs were determined for the period from August 1 to August 16.
It appears that the girl received substantial exposure to pipeline wastewater
aerosol while at home on August 6 and occasional exposure on several other
days. However, the daily aerosol drift patterns were approximations, because
of limitations in the available data sources: rig operation records did
207
-------
TABLE 59. OCCURRENCE OF ABNORMAL THROAT FLORA IN ACUTE8 AND
CONVALESCENT1* ILLNESS THROAT SWABS
to
o
00
Number (Percent)
Collection
period
ACUTE ILLNESS THROAT
1980
Jul-Sep
1982
Jan-Mar
Apr-Jnn
Jul-Sep
Oct-Dec
1983
Jan-Mar
Apr-Jun
Jnl-Sep
ALL ACUTE
CONVALESCENT ILLNESS
1982
Jan-Mar
Apr-Jun
Jnl-Sep
Oct-Dec
1983
Jan-Mar
Apr-Jun
Jul-Sep
ALL CONVALESCENT
ALL ILLNESS TS
Number of
illness Group A
throat strep-
swabs tococci
SYABS
3 0 (0)
10 0 (0)
6 1 (17)
34 8 (24)
34 5 (15)
22 1 (5)
16 5 (31)
4 0 (0)
129 20 (15.5)
THROAT SWABS8
8 0
2 0
6 0
3 1
6 0
8 4
1 0
348 5 (14.7)
163 25 (15.3)
a Swab obtained while donor was displaying
b Swab obtained within 1 week after donor
c Enteric Gram-negative bacteria isolated
d Enteric Gram-negative bacteria isolated
e Cozsackievirus B4
f NA - not analyzed
Clinical bacteriology
Possibly
significant
bacteria0
0 (0)
0 (0)
0 (0)
10 (29)
2 (6)
0 (0)
1 (6)
1 (25)
14 (10.9)
0
0
0
0
0
0
0
0 (0)
14 (8.6)
symptoms of a
recovered from
at the moderate
at the light or
isolated from donor 10913 (age 8) in
Clinical virology of
g Includes four illness throat swabs whose
Probably
insignificant
bacteriad
3 (100)
0 (0)
1 (16)
2 (6)
1 (3)
1 (5)
2 (13)
0 (0)
10 (7.8)
1
0
0
0
0
0
0
1 (3)
11 (6.7)
Clinical virology
isolates
0
0
0
1« (Cox B4)
0
NA*
NA
NA
1/64 (1.6)
0
0
0
0
NA
NA
NA
0/15 (0)
1/79 (1.3)
respiratory illness.
symptoms of the respiratory illness.
or heavy levels.
very light levels
acute throat swab
and Neisseria spp.
obtained on 8-9-82.
throat swabs discontinued on 10-23-82.
illness phase
was not reported.
-------
not correlate rig location with hour of the day, yet hourly variation in
wind direction frequently was substantial.
Enterovirus levels in the pipeline wastewater were relatively high
from August 2 to 10. ranging from 0.06 to 2.2 pfu/mL (see Tables P-3 and
P-ll in Appendix P). Virus runs V2 and V3 were performed to monitor pipeline
wastewater aerosols on August 2 and 4 respectively during the week preceding
the viral isolations of cozsackie B4 from the children. The enterovirus
density of the wastewater aerosol sampled on August 4 was extremely high:
16.2 pfu/m3 on HeLa cells and 18.3 pfu/m^ on RD cells (primarily poliovirus
1) at 44m downwind from the irrigation rig (see Table 38). While coxsackie-
virus B4 was not isolated from the aerosol or wastewater samples in early
August 1982 (see Table 39), it was isolated from pipeline wastewater sampled
in September 1982 (see Table 25). Due to the high levels of poliovirus
in wastewater sampled on August 3 and 4, the detection of a lower level
of coxsackievirus B4 could have been masked. Furthermore, although it
was not as prevalent as coxsackieviruses B3 and B5, coxsackie B4 was isolated
during summer monitoring of Lubbock wastewater in 1980, 1981 and 1983 as
well (see Tables P-5 in Appendix P and 26).
Achromobacter xylosoxidans was a prevalent bacterium in both the pipeline
and reservoir wastewater during the summer 1982 irrigation. This agent
was one of the more frequently isolated bacteria in screens of pipeline
and reservoir wastewater samples obtained July 26-27, 1982 (see Table 22).
Discuss ion—This illness surveillance report documents respiratory
illnesses attributable via clinical isolates to coxsackievirus B4 and Achromo-
bacter xylosoxidans. both of which were presumably present in irrigated
wastewater. The temporal pattern of wastewater irrigation and illness
or agent isolation is consistent with aerosol exposure in this investigation.
Assuming an initially low dose of coxsackievirus B4, a minimal incubation
period of 24-48 hours would be required to allow multiple cycles of viral
replication prior to the onset of clinical symptoms. Exposure of participant
10913 on August 6 and 40312 on August 8 fall within this anticipated time
frame. Likewise, colonization of the throat by Achromobacter xylosoxidans
to a heavy level would require several days. Thus, the evidence of this
illness episode is consistent with the hypothesis that wastewater microorganisms
transmitted by wastewater aerosol from spray irrigation infected and produced
respiratory illness in the subject children. However, since plausible
alternative modes of transmission such as person-to-person spread and contam-
inated drinking water were not investigated, the evidence for the aerosol
exposure hypothesis is inconclusive.
Group A Streptococci
All illness and requested throat swabs were examined for Group A strep-
tococci by the fluorescent antibody technique and also by isolation and
identification of p-hemolytic colonies on sheep blood agar. Group A strepto-
cocci were isolated from 15.3% (25) of 163 respiratory illness throat swabs
as shown in Table 59. The isolation rate of Group A streptococci was about
15% in throat swabs from both the acute and convalescent phases of the
209
-------
illness. Table 58 indicates that Group A streptococci occurrence in respiratory
illness throats displayed a seasonal pattern: lowest (1/46=2%) in January-
March, highest (10/32=31%) in April-June, and intermediate for the duration
of the calendar year (8/48=17% in July-September and 6/37=16% in October-
December) .
The rate of isolation of Group A streptococci in illness throat swabs
was highest (9/24=38%) during April-June 1983. Seven of these specimens
were collected on or after Hay 23 and were presumably unrelated to the
spring 1983 irrigation which terminated on April 30, 1983.
The second highest isolation rate of Group A streptococci was 8/40=20%
in July-September 1982. Illness throat swabs were collected between July 27
and September 20, 1982 from 26 ill donors whose illness onset may have
been between July 21 and September 17, 1982 during the summer 1982 irrigation
period. The mean aerosol exposure of the five donors with Group A streptococcal
infections (AEI=1.29) was less than the mean AEI of the 21 ill donors who
were negative for Group A streptococci (AEI=2.04). Thus, the Group A strep-
tococcal infections which produced respiratory illness during the summer
1982 irrigation appear to have been unrelated to wastewater aerosol exposure.
Enteric Gram-Negative Bacteria (EGNB)
EGNB in Throats—
All illness and requested throat swabs were also plated onto MacConkey
agar to detect unusual levels of enteric organisms. Enteric Gram-negative
bacteria (EGNB) isolated at the moderate or heavy level in throat swabs
were considered to possibly be significant (and were interpreted as an
EGNB throat infection), since these organisms are uncommon in the normal
human oropharynx, as shown in Table 60 (Youmans et al., 1980).
TABLE 60. MICROORGANISMS FOUND IN THE OROPHARYNX
Range of prevalence
Microorganisms
Staphylococcus aureus 35-40
Staphylococcus epidermidis 30-70
Aerobic corynebacteria (diphtheroids) 50-90
Streptococcus pyogenes (Group A) 0-9
Streptococcus pneumoniae 0-50
Alpha- and nonhemolytic streptococci 25-99
Branhamella catarrhalis 10-97
Neisseria meningitidis 0-15
Haemophilus influenzae 5-20
Haemophilus para influenzae 20-30
Gram-negative bacteria, e.g.,
Uncommon
Youmans et al., 1980
210
-------
Various members of the Enterobacteriaceae or Pseudomonas occasionally are
found in small numbers from oropharyngeal swabs of healthy humans. However,
heavy colonization of the upper respiratory tract by these organisms, as
seen in Table 58, at levels similar to those occasionally observed in routine
fecal specimens, is a situation that occurs under unusual circumstances.
Data and investigation—EGNB were isolated at the moderate or heavy
levels considered possibly significant in 14 (10.9%) of the acute illness
throat swabs, but were not found at these levels in any of 34 convalescent
illness throat swabs (see Table 59). There was a marked seasonality to
the occurrence of these possibly significant isolates in acute illness
throat swabs, with 12 occurring between July 19 and October 12, 1982.
The other two occurred in June and July 1983. To investigate this phenomenon,
bacteriology results were assembled in Table 58 for all throat swabs provided
by the 14 donors with EGNB throat infections during the acute phase of
a respiratory illness.
The source of all EGNB throat infections in acute illnesses occurring
in the study population during the summer of 1982 was pursued. The degree
of exposure of throat swab donors with acute illness who had moderate or
heavy levels of these bacteria was compared with those who did not (see
Table 61). No apparent association was observed with degree of wastewater
aerosol exposure or with frequency of eating food prepared at restaurants
A or B in Wilson. However, all six of the ill donors with EGNB throat
infections lived in homes which used evaporative coolers for air conditioning.
The association of EGNB throat infections with evaporative cooler use at
home was significant (p=0.02) among the illness throat swab donors. However,
since many of the EGNB infected donors were in household 210, the association
with evaporative cooler use is not significant (p=0.23) using the household
as the unit of observation.
In an additional attempt to characterize this phenomenon, 23 throat
swabs were obtained from three groups of healthy adult and teenage participants
in mid-September: Hancock farm residents and workers, Wilson residents
living at least 800 m from the Hancock farm spray irrigation (Zone 4),
and distant rural residents (Zone 5). Surprisingly, EGNB throat infections
were about as prevalent in the healthy participants (6/23=26%) in September
as they had been in the participants with acute respiratory illness from
July to September (8/34=24% from Table 59). Table 62 shows that while
the Hancock farm sample had a higher recovery rate (3/7=43%) in the September
survey, EGNB were also recovered from the throats of healthy participants
in Wilson (1/8) and Zone 5 (2/8). Hence, the phenomenon of moderate and
heavy levels of EGNB in the upper respiratory tract appears to have been
prevalent throughout the study area, in both ill and healthy participants.
The degree of exposure to potential environmental sources of enteric
bacteria of the six healthy throat swab donors surveyed in September 1982
who had EGNB throat infections was compared to the exposure of the 17 who
had normal throat flora (see Table 63). Healthy donors with inapparent
EGNB throat infections had a higher average aerosol exposure index for
summer 1982 than did the healthy donors without EGNB infected throats,
but the difference was not statistically significant (p=0.18). The healthy
211
-------
TABLE 61. INVESTIGATION OF VARIOUS DONOR EXPOSURE VARIABLES FOR
ASSOCIATION WITH ENTERIC GRAM-NEGATIVE BACTERIA IN
ILLNESS THROAT SWABS IN SUMMER 1982
Number of illness
throat donors by
EGNB infection status
M or H Negative8
(infected) (not infected)
Period of
observation
Apparent
association
Wastewater Aerosol Exposure
7-19 to 9-20-82
Low AEI «1) 4
Intermediate (1-5) 4
High AEI (>5) 0
Mean AEI 1.5
Frequency of Eating at Restaurant A
7-19 to 10-12-82
Never 3
1 to 2 times 3
At least once 0
per month
Frequency of Eating at Restaurant B
7-19 to 12-7-82
Never 6
1 or 2 times 0
At least once 0
per month
Use of Evaporative Cooler for
Air Conditioning
7-19 to 10-12-82
No A/C system 0
Refrigeration A/C 1
Evaporative cooler 5
A/C
6
10
1
2.0
9
7
2
7
7
4
5
16
9
No
No
No
Yes
(p=0.02)b
Includes six donors with very light or light EGNB in illness throat
swabs.
One-sided Fisher's exact test, with no A/C and refrigeration A/C rows
combined. There is no significant association (p=0.23) using the household
as the unit of observation.
212
-------
TABLE 62. CLINICAL BACTERIOLOGY8
SURVEYS OF HEALTHY PARTICIPANTS
RESULTS FROM REQUESTED THROAT SWAB
IN SEPTEMBER 1982 AND JUNE 1983
Throat Normal
Group of healthy participants swabs flora
FIRST SURVEY; Sep 19-22. 1982
Hancock farm residents
and workers
Wilson residents (Zone 4)
Distant rural residents
(Zone 5)
TOTALS
SECOND SURVEY: Jun 6-8. 1983
Hancock farm residents
and workers
Wilson residents (Zone 4)
Distant rural residents
(Zone 5)
TOTALS
23
6
7
19
17
6
5
17
Positive for enteric Gram-
negative bacteria
3 (43%)
diversus-levinea (H),
E. aerogenes (H)
- E. coli (M)
- E. cloacae (H),
E. agglomerans (M)
1 (13%)
- E. agglomerans (M)
2 (25%)
- E. cloacae (H)
- Acinetobacter calcoacet-
icns var. anitratus (H),
K. oxytoca (H)
6 (26%)
0 (0%)
0 (0%)
2 (29%)
- E. aerogenes (VL)
- E. cloacae (VL)
2 (11%)
a Bacteriology only; fluorescent antibody screen not done.
213
-------
TABLE 63. INVESTIGATION OF VARIOUS DONOR EXPOSURE VARIABLES FOR
ASSOCIATION WITH ENTERIC GRAM-NEGATIVE BACTERIA IN REQUESTED
THROAT SWAB SURVEY OF HEALTHY DONORS IN SEPTEMBER 1982
Number of healthy throat swab
donors by EGNB infection status
M or H Negative
(infected) (not infected)
Apparent
association
(p-value)
Wastewater Aerosol Exposure (in summer 1982)
Low AEI «D
Intermediate (1-5)
High AEI OS)
Mean AEI
Geometric mean AEI
2
1
3
35,8
3.64
7
6
4
6.9
1.17
Frequency of Eating at Restaurant A (in summer 1982)
Seldom or never
At least once per month
2
4
8
3
Frequency of Eating at Restaurant B (in summer 1982)
Never 4
At least once per month 2
Use of Evaporative Cooler for
Air Conditioning (A/C)
Refrigeration or no A/C 2
Evaporative cooler A/C 4
Contaminated Private Drinking Water Well
(in June 1982 and/or Nov/Dec 1982)
9
2
9
6
Acceptable
Contaminated0
2
2
2
1
No
Insufficient
data (?)
No (p=0.18)a
No (p=0.14)b
Insufficient
data (?)
No
No (p=0.22)b
Insufficient
data
a One-sided t-test of difference in means in two independent populations;
In(AEI) transformation used to reduce variance inequality.
b One-sided Fisher's exact test.
c Total coliforms, fecal coliforms, or fecal streptococci >1 cfn/100 mL.
214
-------
donors with EGNB throat infections also tended to eat at restaurant A more
often, bat this difference also was not significant (p=0.14). The donors
with inapparent EGNB throat infections were more likely to reside in a
household using an evaporative cooler for air conditioning, but again there
was not a significant association (p=0.22). Because of the small sample
sizes, none of these three exposure variables nor contaminated private
drinking water wells can be ruled out as possible risk factors. The frequency
of eating at restaurant B was not a risk factor.
A second throat swab survey of 19 healthy donors was performed in
June 1983. None of them had throat infections with moderate or heavy levels
of EGNB (see Table 62), although two of the distant rural participants
had very light (probably insignificant) levels of these bacteria in their
throats. Thus, the prevalence of EGNB throat infections in the acute upper
respiratory illness population reflected the prevalence in the healthy
population during each survey. Respiratory ill and well participants both
had an EGNB throat infection prevalence above 25% in September 1982 and
both had a lower prevalence of these bacterial infections (approximately
10% in the illness population and below 5% in the healthy population) in
June 1983.
Discussion—The remarkable aspect of the results of illness specimen
throat swabs of some LISS participants during July to October 1982 (Table
58) is not the mere presence of Gram-negative enterics, but the unusually
high levels of the organisms. EGNB had been observed occasionally before
and after these dates at the VL or L level, but seldom at the M or H levels.
The oropharynx of healthy humans is not commonly assumed to be an environment
favoring growth or persistence of EGNB. For example, one study (Johanson
et al., 1969) examined the oropharyngeal flora (presence/absence only)
of five groups of adult subjects. Only 2% of normal subjects, whether
hospital or nonhospital associated, and 0-2% of patients on the psychiatry
service yielded throat cultures positive for EGNB. However, the levels
of positive cultures in a single culture survey of moderately ill and moribund
patients was 16% and 57%, respectively. Other evidence suggests that increased
oropharyngeal colonization by EGNB may be associated with upper respiratory
illness (DRI). In a study carried out in a Puerto Rican hospital (Ramirez-
Rhonda et al., 1980), presence of EGNB was found in the oropharynx of 14%
of normal adult outpatients. Colonization of the oropharynx of hospital
staff with EGNB ranged from 12 to 18% in the absence of illness, but increased
to 38 to 60% in individuals with URI, presumably of viral origin. K. pneumoniae
was the most frequent isolate, followed by E. coli and Enterobacter spp.
Although high levels of EGNB were observed in acute illness throat
swabs of LISS participants, they were largely confined to specimens obtained
in the summer months, which would tend to argue against an association
with URI of other etiology, particularly viral. Also, high levels of EGNB
were observed in requested throat cultures of a similar proportion of healthy
LISS participants during the same period.
Oropharyngeal EGNB levels appear to have been much higher in the infected
LISS participants than in infected subjects in the Puerto Rican study.
Ramirez-Rhonda et al. (1980) determined the total numbers of EGNB/mL of
215
-------
oropharyngeal fluid of hospital staff with URI (151 subjects). The levels
of EGNB/mL in positive individuals were <10 cfu (9%), 10 to 100 cfu (54%),
100 to 300 cfu (38%), and >300 cfu (1%). For LISS participants with high
levels of oropharyngeal EGNB, it would appear from quality assurance studies
(see Table A-34 in Appendix A) that isolation at the M or H level would
require >10^ to 10^ cfu/mL of the organisms. Such numbers would be incon-
sistent with all but the 1% of subjects with URI in the study of Ramirez-
Rhonda et al. (1980) who may have had comparable levels of organisms (i.e.,
the >300 cfu/mL group).
Use of antibiotics could conceivably reduce susceptible components
of the normal flora that would normally prevent colonization of the oropharynz
by EGNB through bacterial interference. For example, pharyngeal colonization
with a-hemolytic streptococci, the most prevalent group of organisms observed
on throat cultures, appears to protect neonates in a hospital environment
from pharyngeal colonization with EGNB (Goldmann, 1981). However, the
seasonal incidence of high levels of EGNB in LISS participants and their
isolation from healthy subjects would argue against this interpretation.
Also, the role of antibiotics as a predisposing factor for colonization
of the oropharynx by EGNB is a subject of some controversey, since there
have been studies in which use of antibiotics was (Haverkorn and Michel,
1979) and was not (Johanson et al., 1969, 1972) correlated.
The factors (perhaps use of evaporative coolers) responsible for the
high levels of EGNB in LISS participants remain unresolved. The studies
of Philpot and HacDonald (1980) suggested that pharyngeal carriage rates
of EGNB may differ substantially between different groups of normal individuals
and challenged the common assumption that a high rate of carriage of the
organisms exclusively is associated with hospitalization or debility.
The prevalence of all EGNB (presence/absence) recovered from throat swabs
of healthy Australian adults (31 subjects), Malaysian adults (25 subjects),
and Malaysian children (25 subjects) were 9%, 36% and 4%, respectively.
The prevalence of the organisms in Malaysian adults (28% of 25 subjects)
and children (12% of 25 subjects) with sore throats was not markedly different
from that observed for the healthy counterparts. It is interesting and
perhaps relevant to the LISS EGNB throat data that the investigators noted
that ''in each case the numbers of these bacteria detected were not great.1'
They suggested that the higher carriage rate in Malaysian as opposed to
Australian adults might be due to ''food preferences or other social habits.''
Abnormal fecal levels (AFL) of selected EGNB—
Clinical bacteriologic analysis (see Figure 14) was performed on 34
gastrointestinal and respiratory illness stool specimens. The results
are presented in Table 64. Normal fecal flora were absent or present at
abnormally low levels in 8 (24%) of these illness stools, especially in
convalescent specimens. This probably indicates antibiotic therapy, but
may reflect problems with sample processing or shipping. AFL of selected
EGNB were observed at the moderate or heavy level in 5 (15%) of these illness
fecal specimens. Occurrence of AFL of EGNB was higher in the illness fecal
specimens of adults age 18-44 (50%) than in children or older adults (see
Table 65). All five isolates were Klebsiella pneumoniae or K. oxytoca.
The M or H Klebsiella levels were from illness fecal specimens collected
216
-------
TABLE 64. OCCURRENCE OF ABNORMAL LEVELS OR FLORA IN ACUTE* AND
CONVALESCENT11 ILLNESS FECAL SPECIMENS
Number (Percent)
Clinical bacteriology
Number of Absence or
illness decrease Possibly
fecal of normal significant
specimens fecal flora bacteriac
Viruses
Clinical
virology
isolates
Electron
microscopy
detections
1982
Jan-Mar 10 0
Apr-Jun 4<* 2 (50) 1 (25)
Jnl-Sep 3 1 (33) 0
Oct-Dec 20 0
1983
Jan-Mar 12 3 (25) 0
Apr-Jun 11 1 (9) 4 (36)
Jul-Sep 1 1 (100) 0
All acute 10 1 (10) 0
All convalescent 8 4 (50) 1 (13)
Illness phase 16 3 (19) 4 (25)
not reported
TOTAL 34 8 (24) 5C (IS)
0
0
2 (67)
NAd
1 (8)
2 (18)
1 (100)
2d (25)
1 (13)
3 (19)
0
1^ (33)
0
2 (100)
1 (8)
0
0
4 (40)
0
0
6/32<* (19) 4/33<* (12)
a Specimen obtained while donor was displaying symptoms of a gastrointestinal
or respiratory illness.
b Specimen obtained within 1 week after donor recovered from symptoms
of the gastrointestinal or respiratory illness.
c Enteric Gram-negative bacteria isolated at the moderate or heavy level.
All isolates were Klebsiella (pneumoniae or oxytoca).
d Illness fecal specimens not analyzed (NA): 2 by tissue culture virology
and 1 by EM.
217
-------
TABLE 65. AGE-SPECIFIC DISTRIBUTION OF ABNORMAL LEVELS OR
FLORA IN ILLNESS FECAL SPECIMENS
No. positive/No.specimens analyzed
(percent)
Donor age Klebsiella
on 6-30-82, at M or H Isolated by Detections
years iev®i £e 11_cuj.ture by EM
0-5
6-17
18-44
45-64
65+
1/9 (11)
0/9 (0)
4/8 (50)
0/5 (0)
0/3 (0)
3/8 (38)
2/8 (25)
0/8 (0)
0/5 (0)
1/3 (33)
3/9 (33)
1/9 (11)
0/7 (0)
0/5 (0)
0/3 (0)
5/34 (15) 6/32 (19) 4/33 (12)
on June 24, 1982, May 17, 1983, and three on June 1, 1983. Each of these
illness onsets followed termination of the spring irrigation period and
preceded the start of the summer irrigation. The prevalence of moderate
or heavy Klebsiella was 33% in 15 illness fecal specimens from May and
Jane.
Surprisingly, the occurrence of moderate or heavy levels of EGNB was
not much higher (15%) in fecal specimens collected during gastrointestinal
and respiratory illness than in throat swabs collected during the acute
phase of respiratory illness (11%). In addition, the seasonal pattern
of occurrence was somewhat different: moderate or heavy levels of these
EGNB were only found in illness stools during May and Tune, whereas they
were most prevalent in acute illness throat swabs from July to early October.
Some of the preceding discussion concerning factors influencing coloni-
zation of the oropharynx by EGNB also is applicable to AFL of selected
EGNB. However, an important difference is that organisms such as Klebsiella.
Enterobacter. and Citrobacter along with the almost ubiquitous E. coli
are common and significant components of the facultatively anaerobic normal
flora of the gut (Lennette et al., 1980), in contrast to the rarer occurrence
of the organisms in smaller numbers in the oropharynx. Various members
of the family Enterobacteriaceae. aside from the overt pathogens Salmonella,
Shigella and Yersinia. are commonly encountered as pathogens only in special
circumstances (e.g., as the major causes of nosocomial infections). However,
toxin-producing E. colj^ are a common cause of diarrhea in normal subjects
and other toxin-producing coliforms may at times be associated with acute
diarrhea. In addition, increased prevalence and levels of intestinal coloni-
zation by organisms such as Klebsiella in a hospital environment have been
associated with illness, duration of hospitalization, and use of antibiotics
(Haverkorn and Michel, 1979; Goldmann et al., 1978; Selden, et al., 1971).
218
-------
Viruses
Viruses in Illness Throat Swabs—
Illness throat swabs were examined for viruses by tissue culture techniques
as diagrammed in Figure 16. As Table 59 illustrates, viruses were rarely
isolated from illness throat swabs; a single viral isolate was recovered
from 79 specimens (1.3%). Coxsackievirus B4 was isolated in a throat swab
collected on August 9, 1982 from 10913 while she had a sore throat. The
circumstances are thoroughly discussed in the respiratory illness investigation
above, since it may have been associated with wastewater aerosol exposure.
Because of the low viral recovery rate, clinical virologic analysis of
illness throat swabs was discontinued in October 1982.
Viruses in Illness Fecal Specimens—
Fecal specimens collected during gastrointestinal and respiratory
illnesses were examined for viruses both by tissue culture techniques and
by electron microscopy (EM). Viral prevalence is summarized in Table 64.
Viral isolates were recovered from 6 (19%) of 32 illness fecal specimens
analyzed by tissue culture. The recovery rates from acute and convalescent
phase specimens were similar. Viral recovery showed a seasonal pattern:
markedly higher for July-September (3/4=75%) than in earlier calendar quarters
(8% for January-March and 13% for April-June). Three of the viral isolates
were identified by fluorescent staining as adenoviruses, but the other
three could not be identified by enterovirus typing pools or fluorescent
staining (see Table 66). Viral recovery appears to show an age-related
pattern (see Table 65), with higher recovery rates from children and the
elderly.
Illness onset associated with three of the viral isolates (two from
illness fecal specimens and one from an illness throat swab) occurred during
the summer 1982 irrigation period. Both of the ill children with fecal
isolates received an intermediate level of aerosol exposure (see Table
66). Only one donor provided a negative illness fecal specimen during
the summer irrigation. These data are insufficient to address the question
of possible association of the illness viral isolates with wastewater aerosol
exposure.
Virus-like particles were detected in 4 (12%) of the 33 illness fecal
specimens examined by EM (see Table 64). All of the detected virus-like
particles were in acute illness specimens (40% detection rate). The detection
of virus-like particles was strongly associated with illness specimens
from young children (see Table 65), with a positive rate of 33% in ill
donors of age 0-5. The types of virus-like particles detected by EM are
presented in Table 66 and Figure 27.
Norwalk-like particles were detected in an acute illness specimen
from one boy (21112) in May 1982. This specimen, a simultaneous specimen
from his older sister (21111), and four pairs of sera were sent to Dr. N. R.
Blacklow's laboratory at the University of Massachusetts for examination
by EIA. Both stools were negative for Norwalk antigen and no sereconversions
to Norwalk virus were detected.
219
-------
TABLE 66. IDENTIFICATION AND COMPARISON OF VIRAL ISOLATES BY CELL CULTURE AND
VIRUS-LIKE PARTICLES BY EM IN ILLNESS FECAL SPECIMENS
Donor AEI.
Collection
date
5-18-82
8-4-82
9-24-82
11-16-82
12-6-82
2-14-83
5-17-83
6-1-83
9-8-83
a Illness
Donor
ID
21112
60111
40312
20211
21112
51013
53101
60111
21111
phase :
Age
on Illness
6-30-82 t>hasea
1
0
6
13
1
0
74
0
8
A -
A
A
C
A
A
A
?
?
?
Decrease
in normal
(if onset
Clinical virology
fecal flora?b agent isolated
Yes
No
Yes
No
No
No
No
No
Yes
acute, C - convalescent.
b A decrease in normal fecal flora
sample
processing or
c AEI value may
to
N>
o
shipping.
probably
underestimate aerosol exposure
(none)
Adenovirus
Unidentified virus
(not analyzed)
(not analyzed)
Adenovirus
Unidentified virus
Adenovirus
Unidentified virus
? - not reported.
Virus-like particles
detected bv EM
Norwalk virus-like
(none)
(none)
Astrovirns-like
Calicivirus-like
Adenovirus- like
(none)
(none)
(none)
indicates antibiotic therapy, but may reflect
during
irrigation)
1.70
2.25C
2.85
problems
with
(see Illness Investigation involving household 403).
-------
Figure 27. Virus particles observed by EM in illness stool specimens.
(a) Norwalk-like particles in the first illness stool (5-82) of 21112.
(b) Calicivirus-like particles in the second illness stool (12-82) of 21112.
(c) Astrovirus-like particles in the stool of 20211 (11-82).
(d) Adenovirns-like particles in the stool of 51013 (2-83).
Bar = 100 nm for a-d.
221
-------
Calicivirus-like particles were detected in a second illness specimen
from the same boy in December 1982. Astrovirus-like particles were detected
in a November 1982 illness specimen from another girl (20211). Requested
stools received in January 1983 from these children were negative for virus-like
particles.
As shown in Table 66, adenovirus-like particles were detected by EH
in one of the three illness fecal specimens from which an adenovirus was
isolated by tissue culture. This 33% adenovirus detection rate by EM in
adenovirus-positive specimens is similar to the 40% detection rate of corona-
virus-like particles by EM in routine specimens previously found to be
positive (see EM Quality Assurance).
The onset of each of the four illnesses for which EM analysis detected
virus-like particles was during times when there was no sustained wastewater
irrigation. Thus, these EM-detected viral infections presumably were unrelated
to wastewater irrigation operations.
Of the enteric viruses frequently associated with gastroenteritis,
only human rotaviruses have been reprodncibly cultivated outside the human
host. Therefore, their involvement in diarrheal illness is far from certain.
To date, only rotaviruses and Norwalk virsns are recognized as medically
important agents of human gastroenteritis. Recently, enteric adenoviruses
have been recognized for their possible role in diarrheal illness (Cukor
and Blacklow, 1984).
While astroviruses are found in stool specimens obtained from cases
of intestinal illness, experimental ingestion of astrovirus-containing
fecal filtrates by nine volunteers resulted in viral shedding by only two
individuals, neither of whom developed diarrhea or vomiting (Kurtz et al.,
1979). In a prospective study involving 447 children hospitalized with
infectious gastroenteritis, Ellis and associates (1984) found no significant
association of astrovirus with this disease when compared to childred treated
for respiratory infections. Conversely, rotavirns (p<0.0001), adenovirus
(p<0.01) and calicivirus (p<0.01) were associated with diarrheal illness
in young children.
6. CLINICAL BACTERIOLOGY OP ROUTINE FECAL SPECIMENS
Summary Data
Routine fecal specimens provided by donors in scheduled collection
weeks were analyzed for bacteria using procedures summarized in Figure
14. In all cases, the organisms isolated were reported as a function of
the level of growth (very light to heavy) observed on primary plating media.
Results from 268 specimens collected during 1980 and 1981 are presented
in Table 67. Approximately 90% of these baseline specimens were obtained
from children age 12 or less. Beginning in January 1982 one randomly selected
adult from each household was also asked to donote specimens. The results
from 725 specimens collected in 1982 and from 517 specimens collected in
1983 are shown in Tables 68 and 69, respectively.
222
-------
TABLE 67. ORGANISMS ISOLATED FROM ROUTINE FECAL SPECIMENS DURING 1980 AND 1981
(268 Specimens)8
to
K>
o*
Quant itat ion of growth1* [percent (
Oreanism
Aeromonas hydrophila
Candida albicansd
Citrobacter diversus
Citrobacter freundii
Citrobacter spp.
Enterobacter aerogenes
Enterobacter cloacae
Enterobacter sakazakii
Escherichia coli
Hafnia alvei
Klebsiella ozytoca
Klebsiella pneumoniae
Klebsiella spp.
Morganella morganii
Proteus mirabilis
Providencia alcalifaciens
Fluorescent Psendomonas gr.
Pseudomonas spp.
Serratia liquefaciens
Serratis odorifera
Staphylococcns aureus
Staphylococcns epidermidis
Yersinia enterocolitica
Heavy
_
—
-
0.7 (2)
-
-
0.4 (1)
-
40.7 (109)
-
-
1.1 (3)
-
-
-
-
-
-
-
-
0.4 (1)
-
-
a From Data Collection Periods 015, 017. 019
b Quant itat ion of growth, on
Heavy - growth on three
Moderate - growth on first
primary culture
Moderate
_
0.9 (2)
-
2.6 (7)
-
0.4 (1)
2.6 (7)
0.4 (1)
44.8 (120)
-
0.7 (2)
2.6 (7)
-
-
-
0.4 (1)
-
-
-
-
2.2 (6)
-
0.4 (1)
Lieht
0.4 (1)
7.2 (15)
-
3.4 (9)
-
0.7 (2)
4.1 (11)
0.7 (2)
11.9 (32)
0.7 (2)
4.9 (13)
9.3 (25)
0.7 (2)
0.7 (2)
0.4 (1)
0.4 (1)
1.5 (4)
0.4 (1)
0.4 (1)
0.7 (2)
23.9 (64)
0.4 (1)
-
, 108, 110, 112. 114, 117 and
plates
or all gnadrants
two quadrants
number) positive]
Very light
0.4 (1)
12.9 (27)
0.4 (1)
4.5 (12)
0.4 (1)
0.7 (2)
4.5 (12)
1.1 (3)
1.9 (5)
0.7 (2)
2.6 (7)
9.3 (25)
-
0.7 (2)
-
0.7 (2)
2.6 (7)
—
—
-
8.2 (22)
1.1 (3)
-
118.
Light - growth on first
Very Light - one
Totalc
0.7 (2)
21.5 (45)
0.4 (1)
11.2 (30)
0.4 (1)
1.9 (5)
11.6 (31)
2.6 (7)
99.6 (267)
1.5 (4)
8.2 (22)
22.4 (60)
0.7 (2)
1.5 (4)
0.4 (1)
1.5 (4)
4.1 (11)
0.4 (1)
0.4 (1)
0.7 (2)
34.7 (93)
1.5 (4)
1.1 (3)
quadrant
to ten colonies on plate
c Includes positives by enrichment only
d Based on 209 specimens (procedures for
019)
isolation of C. albicans began
in Data Collection Period
-------
TABLE 68. ORGANISMS ISOLATED FROM ROUTINE FECAL SPECIMENS DURING 1982
(725 Specimens)8
N>
to
Quant it at ion of growth" [percent (number) positive]
Organism
API Group I
Aeromonas hydrophila
Candida albicans
Chromobacterium
Citrobacter amalonaticus
Citrobacter diversus-levinea
Citrobacter freundii
Citrobacter spp.
Enterobacter aerogenes
Enterobacter agglomerans
Enterobacter cloacae
Enterobacter sakazakii
Enterobacter spp.
Escherichia coli
Hafnia alvei
Klebsiella oxytoca
Klebsiella pneumoniae
Morganella morganii
Proteus mirabilis
Proteus rettgeri
Proteus vnlgaris
Providencia alcalifaciens
Fluorescent Pseudomonas gr.
Pseudomonas aeruginosa
Pseudomonas spp.
Salmonella spp.
Serratia fonticola
Serratia marcescens
Serratia odorifera
Staphylococcus aureus
Heavy
.
—
0.1 (1)
-
-
-
-
-
0.7 (5)
0.3 (2)
1.8 (13)
-
-
36.7 (266)
0.1 (1)
0.1 (1)
4.6 (33)
-
-
-
-
-
0.1 (1)
-
-
0.1 (1)
0.1 (1)
-
-
-
a From Data Collection Periods 201, 205, 207,
b Quant itat ion of growth on
Heavy - growth on three
Moderate - growth on first
Moderate
L
—
1.0 (7)
0.1 (1)
-
0.7 (5)
1.1 (8)
-
0.8 (6)
-
3.2 (23)
0.1 (1)
-
44.1 (320)
0.1 (1)
1.8 (13)
7.3 (53)
-
0.6 (4)
0.1 (1)
-
0.1 (1)
1.2 (9)
-
-
-
-
-
-
2.1 (15)
LiKht
L_
0.1 (1)
3.0 (22)
0.4 (3)
0.1 (1)
0.1 (1)
1.2 (9)
0.1 (1)
1.7 (12)
0.3 (2)
4.0 (29)
0.7 (5)
-
13.9 (101)
0.1 (1)
3.4 (25)
8.1 (59)
0.3 (2)
0.3 (2)
-
0.3 (2)
-
1.7 (12)
0.6 (4)
0.1 (1)
-
-
0.3 (2)
0.1 (1)
6.3 (46)
Verv littht
0.1 (1)
0.1 (1)
10.2 (74)
-
-
-
1.1 (8)
0.1 (1)
0.4 (3)
0.1 (1)
3.0 (22)
-
0.1 (1)
3.0 (22)
0.1 (1)
1.2 (9)
3.3 (24)
0.4 (3)
0.3 (2)
0.1 (1)
0.1 (1)
0.1 (1)
1.7 (12)
-
—
-
-
-
-
6.5 (47)
Total0
0.3 (2)
0.3 (2)
14.3 (104)
0.7 (5)
0.1 (1)
0.8 (6)
3.4 (25)
0.3 (2)
3.7 (27)
0.7 (5)
12.4 (90)
0.8 (6)
0.1 (1)
98.6 (715)
0.6 (4)
7.3 (53)
25.5 (185)
0.7 (5)
2.2 (16)
0.4 (3)
0.4 (3)
0.3 (2)
5.2 (38)
0.7 (5)
0.1 (1)
0.1 (1)
0.1 (1)
0.3 (2)
0.1 (1)
14.9 (108)
212. 216 and 219
primary culture plates
or all guadrants
two quadrant s
Light - growth on first
Very Light - one
quadrant
to ten colonies on plate
c Includes positives by enrichment only
-------
TABLE 69. ORGANISMS ISOLATED FROM ROUTINE FECAL SPECIMENS DURING 1983
(517 Specimens)6
K>
Quant itat ion
Oreanism
API Group I
Aeromonas hydrophila
Candida albicans
Chromobacterium
Citrobacter amalonaticns
Citrobacter diversus-levinea
Citrobacter freundii
Enterobacter aerogenes
Enterobacter agglomerans
Enterobacter cloacae
Enterobacter sakazakii
Escherichia coli
Hafnia alvei
Klebsiella ozytoca
Elebsiella ozaenae
Klebsiella pneumoniae
Morazella spp.
Morganella morganii
Plesiomonas sbigelloides
Proteus mirabilis
Proteus rettgeri
Psendomonas aeruginosa
Pseudomonas spp.
Serratia liquefaciens
Serratia odorifera
Staphylococcus aureus
Heavy
0
0
0
0
0
0
1
0
50
0
4
0
.2 (1)
.2 (1)
-
-
-
.4 (2)
.2 (1)
.6 (3)
.2 (1)
.5 (8)
.4 (2)
.1 (259)
-
.8 (4)
-
.1 (21)
-
.2 (1)
of nrowthb
[percent (number)
Moderate Light
0
0
0
0
0
0
1
0
4
0
35
1
9
0
.2
.4
.6
.2
.2
-
.4
.7
.2
.6
.4
.8
-
.7
-
.3
.4
-
(1)
(2)
(3)
(1)
(1)
(2)
(9)
(1)
(24)
(2)
(185)
(9)
(48)
(2)
0.2
0.2
6.4
-
0.2
-
0.8
1.5
0.2
4.6
1.2
8.3
0.4
0.8
-
8.1
0.2
—
(1)
(1)
(33)
(1)
(4)
(8)
(1)
(24)
(6)
(43)
(2)
(4)
(42)
(1)
2 by enrichment
0
0
0
a From Data Collection Periods 303,
b Quant itat ion of growth on
Heavy - growth on three
Moderate - growth on first
primary
or all
.2 (1)
-
-
-
.2 (1)
-
.6 (3)
308, 312,
0
0
0
0
2
315
.4
.4
.2
.6
-
-
.1
and
(2)
(2)
(1)
(3)
(11)
317
0.4
-
0.2
0.4
0.2
-
8.1
(2)
(1)
(2)
(1)
(42)
Verv
0.4
-
9.1
0.2
-
-
1.0
-
-
1.5
0.4
1.7
0.2
0.8
0.2
2.7
0.2
—
only
-
-
0.2
-
-
0.2
4.8
positive]
liKht
(2)
(47)
(1)
(5)
(8)
(2)
(9)
(1)
(4)
(1)
(14)
(1)
(1)
(1)
(25)
Total6
1.0
0.8
16.1
0.4
0.4
0.4
2.5
3.9
0.8
12.8
2.3
96.7
0.6
4.3
0.2
24.4
1.0
0.2
0.4
1.2
0.4
0.8
1.4
0.4
0.2
15.7
(5)
(4)
(83)
(2)
(2)
(2)
(13)
(20)
(4)
(66)
(12)
(500)
(3)
(22)
(1)
(126)
(5)
(1)
(2)
(6)
(2)
(4)
(7)
(2)
(1)
(81)
culture plates
guadrants
two quadrants
Light
Very
—
Light -
growth on
one to ten
first
quadrant
colonies on plate
c Includes positives by enrichment only
-------
Bacterial Infection Events
Infection events for bacterial agents have been defined in Section
4G. An infection event is not equated with disease, the latter being indicated
by detectable alterations in normal tissue functions (i.e., clinical manifes-
tations of illness). Infection is used in the broader sense of the entrance
and multiplication of a microbe in the body.
Specimens which failed to yield any growth, or which yielded organisms
by enrichment only, were excluded from the data set in defining bacterial
infections and infection events. The lack of organisms, in these cases,
is likely to have been due to problems with sample processing, shipping
or use of antibiotics by participants.
The densities of the overt and opportunistic pathogens in bacterial
infection event Categories 1-3 and of indicator bacteria in the sprayed
pipeline and reservoir wastewater were monitored regularly. The environmental
data previously presented indicate that the overt and opportunistic pathogens,
except Shigella. were present periodically. Aeromonas hydrophila. the
fluorescent Pseudomonas group, and Klebsiella consistently were prominent
organisms in the wastewater. Pipeline wastewater always had much higher
microorganism levels than reservoir wastewater.
Infections by Overt Pathogens
The results for Category 1 organisms (overt enteric bacterial pathogens)
are presented in Table 70.
TABLE 70. INFECTIONS BY OVERT ENTERIC BACTERIAL PATHOGENS
(CATEGORY 1)
Baseline Irrigation
Period11 Periodb
Fecal specimens 369 1,091
Infections by major enteric 3C (1%) ld (0.1%)
bacteria
a Fecal collection periods from June 1980 through January
1982.
b Fecal collection periods from March 1982 through August
1983.
c Three Y. enterocolitica, two by enrichment only:
June-July 1981.
d Salmonella Group Cj_, heavy level, June 1982.
No major bacterial enteric pathogens were isolated from the direct platings
of the 369 routine fecal specimens collected during the baseline preirrigation
periods. However, Y. enterocolitica was isolated after enrichment from
three different individuals in June and July 1981. Likewise, the analysis
of 1,091 routine fecal specimens collected from participants after commencement
226
-------
of spray irrigation failed to reveal major bacterial enteric pathogens,
except for the isolation of a serologically confirmed SalmoneHa group
Cj. The organism was isolated at the heavy level from an adult male in
June 1982. Subsequent requested fecal specimens also yielded this organism
from the same individual and his son (see Illness Investigations in Section
5F). Because so few infections by overt enteric bacterial pathogens were
observed from routine fecal samples during the preirrigation and irrigation
periods, the data were not subjected to futher analysis.
The overt enteric pathogens are of major clinical significance because
they often are associated with disease and even inapparent or subclinical
infections may provide a source for infection and disease in others. In
spite of a rigorous search for overt enteric bacterial pathogens, the number
of isolations from the routine fecal specimens was small in baseline monitoring
(three) and periods after commencing of irrigation (one). Overt pathogens
often were detected in the wastewater sampling with the exception of Shigella.
which may have been below the level of detection by the direct plating
and enrichment procedures used. The size of inoculum required to produce
disease in humans varies widely for enteric pathogens (Gangarosa, 1978),
ranging, for example, from as few as 10 organisms for Shigella to 10& for
most seretypes of Salmonella. Thus, while most of the major enteric bacterial
pathogens were present in the sprayed wastewater, the reduced rate of infections
by these pathogens after irrigation commenced indicates that no increased
risk of these infections was associated with exposure to wastewater.
Klebsiella Infections
A single genus, Klebsiella. produced most of the observed infections
by the possibly significant opportunistic bacterial pathogens (Category
2). Since more definitive risk factors and etiology might be identified
for a more specific group of organisms, the Klebsie Ha infections were
analyzed separately from the infections by the other opportunistic pathogens.
Klebsiella pneumoniae was the agent recovered in 91% of the Klebsiella
infections. The remaining infections were due to K. oxvtoca.
The prevalence of Klebsiella infections is presented in Table 71.
Although they were infrequent during the baseline period, Klebsiella infections
occurred throughout 1982 and 1983 and were especially prevalent during
both of these summers.
An exploratory analysis was conducted to identify possible risk factors
for Klebsiella infections. During the time interval from January 1982
through August 1983 when most of the Klebsiella infections were observed,
donors having Klebsiella infections were compared to the donors who were
not infected with regard to demographic, socioeconomic, lifestyle, drinking
water and health history characteristics. The association of Klebsie Ha
infection status (infected at least once vs. never infected) with each
characteristic was evaluated by a chi-square test using Cochran's cell
size rule and Yates' continuity correction for 2x2 tables. When a difference
was observed at p<0.05, the characteristic was considered a possible risk
factor.
227
-------
TABLE 71.
Spec imen
collection
month
1980
Jul
Aug
Sep
1981
Apr /May
Jun
Jul
Aug /Sep
1982
Jan
Mar
Mar /Apr
Jun
Aug
Sep
1983
Feb
Apr
Jun
Jnl
Ang
PREVALENC
Rout ine
fecal
donors
22
36
47
27
44
29
35
105
125
118
124
107
110
97
107
100
103
101
PREVALENCE OF BACTERIAL INFECTIONS BY COLLECTION MONTH
rate (Infections per 100 donors)
Opportunistic pathogens Bacteria prominent
Klebsiella Others in wastewater
0
5.6
0
0
2.3
0
0
1.0
1.6
0.8
8.1
10.3
8.2
0
0
0
3.7
0
0
5.7
0
0
0
0
1.9
0
4.1
1.9
3.0
4.9
9.9
1.0
4.7
0
1.9
2.0
0
0
0
0
0
0
0
1.0
0.8
1.7
0.8
1.9
2.7
5.2
3.7
0
8.7
3.0
In contrasting the 37 fecal donors having Klebsiella infections in
1982 and 1983 with the 71 donors not experiencing Klebsiella infections
during the same period of observation, gender was the only factor which
appeared to be significantly associated with the infected donors (see Table
72). Whereas 34% of all donors had Klebsiella infections, 44% of the female
donors experienced KJebsiella infections, which is a nominally signifi-
cant association at the p=0.02 level. This excess of Klebsiella infections
among female donors relative to male donors occurred at all age levels.
An equally high proportion (i.e., 50%) of males aged 65 and above had Klebsjella
infections, but this association was only of borderline significance (p=0.07).
Repeated Klebs iella infections were observed over intervals ranging up
to 20 months in 12 donors, 10 of whom were females. Hence being female
appears to be a risk factor for infection by Klebsiella in the population
studied.
The clinical significance of Klebsiella infection was also investigated.
The incidence densities of self-reported respiratory, gastrointestinal,
and skin illnesses in the 2-week periods prior, concurrent and subsequent
to the fecal collection were compared for all routine fecal specimens with
heavy Klebs iella growth (i.e., ''infected''), with moderate Klebsiella
228
-------
TABLE 72. EXPLORATORY ANALYSIS OF THE ASSOCIATION OF INDIVIDUAL
CHARACTERISTICS WITH BACTERIAL INFECTION PREVALENCE
Klebsiella
Other
Opportunistic
pathogens
Bacteria prominent
in wastewater
Period of observation
Donors infected
Donors not infected8
Jan-Sep 1982
Feb-Aug 1983
37 (34%)
71
Characteristics associated
with infected donors:
Associated subgroup Female
% infected (p-value) 44% (0.02)
Elderly male
50% (0.07)
Apr-Sep 1981
Aug-Sep 1982
Feb-Aug 1983
14 (16%)
72
Ate at
restaurant B
31% (0.007)
Jan-Sep 1982
Feb-Aug 1983
19 (18%)
85
Elderly
38% (0.02)
Drinks much water
47% (0.004)
Lives alone*
50% (0.001)
At home
during day*
39% (0.04)
Seldom in
large groups*
32% (0.007)
Gastrointestinal
condition history**
43% (0.003)
Heart condition
history*
36% (0.01)
a No infection detected during period of observation; fecal specimens
were observed in at least half (i.e., six) of the specimen collection
periods.
b Confounding among age, household size, occupation, group contact, heart
conditions, and gastrointestinal conditions; only one of these factors
may actually be related to donors infected with bacteria prominent
in wastewater.
229
-------
growth, and with negative to light growth of all bacteria recovered except
E. coli (i.e., ''normal''). These data are presented in Table 73. Heavy
Klebsiel la levels in feces may be associated with an increased risk of
gastrointestinal illness during the 2-week period of fecal donation and
in the subsequent 4 weeks. However, since the illness rates for the heavy
Klebsiel la level are variable due to the small number of person-days observed,
this observation of a risk ratio of about 3 for subsequent gastrointestinal
illness in persons with a Klebsiel la infection should be cautiously interpreted.
Episodes of Klebsiella infection coincided with two of the major wastewater
irrigation periods: summer 1982 and summer 1983. Table 74 characterizes
these infection episodes and presents the infection rates by aerosol exposure
level. The statistical analysis of these infection episodes, denoted CKLB2X
and CKLB2W for summer 1982 and CKLB4X and CKLB4W for summer 1983, for associa-
tion with wastewater exposure is presented later.
Infections by NQn-Klebsiella Category 2 Bacteria (Other Opportunistic Bacteria)
Infections by a variety of other possible opportunistic microbial
pathogens also were detected: Staphylococcus aureus (4), Citrobacter freundi i
(3), Citrobacter diversus (2), and one each by API Group I, Candida albicans.
Morganella morganii. Proteus mirab 11 is . Serrat ia fouticola. and Serrat ia
liquef ac iens . These infections occurred sporadically throughout the study
(see Table 71). Donors who ate at restaurant B experienced significantly
more of these infections (see Table 72). While not significantly associated
(p-0.11) perhaps because of the small sample size, two (33%) rural donors
drinking contaminated well water (see Section 5C for contamination criteria)
had these opportunistic bacterial and fungal infections, while none of
11 rural donors drinking well water of better quality were infected.
An episode of infections by these opportunistic microorganisms occurred
in the early spring of 1983 (see Table 74). While unrelated to any measure
of wastewater exposure, it did appear to be associated with eating at least
once per month at restaurant B (p=0.009).
In feet ions...b_Y.-Bacteria.. Prominent,. in_Wastewater
The donor population experienced 27 infections by Aeromonas hydropMJa
and the fluorescent Pseudomonas species, some of the most prevalent enteric
bacteria in the sprayed wastewater. Most (89%) of these infections were
by the fluorescent Pseudomonas group (P. aeruginosa, P. flucrescens, and
P. putida) . As Table 71 shows, these infections occurred throughout 1982
but were more prevalent in 1983 when all of the A. hydrophila infections
occurred.
The characteristics associated with the donors experiencing fluorescent
Pseudomonas and A. hydrophila infections are presented in Table 72. The
infected donors exhibited a pattern of characteristics associated with
the elderly: age 65 and above, living alone, retirees and homemakers who
spent the day at home, infrequent contact with large groups of people,
previous gastrointestinal conditions, and previous heart conditions. Because
many of these were characteristics of the same infected donors, the data
230
-------
TABLE 73. ASSOCIATION OF LEVEL OF KLEBSIELLA GROWTH IN ROUTINE FECAL
SPECIMENS8 WITH THE INCIDENCE OF SELF-REPORTED ILLNESS IN THE PRIOR,
CONCURRENT AND SUBSEQUENT BIWEEKLY REPORTING PERIODS
Level of
Klebsiella
growth
Heavy
Moderate
Neg to Lightb
Heavy
Moderate
Neg to Light
Heavy
Moderate
Neg to Light
Heavy
Moderate
NCR to Light
Period of
illness
observation
DCP-1 c
DCP-1
DCP-1
DCpd
DCP
DCP
DCP+1 e
DCP+1
DCP+1
DCP+2 *
DCP+2
DCP+2
Person
days
observed
674
1254
8460
679
1318
9997
674
1335
9429
672
1335
9530
Incidence of self-reported illness
(New illnesses/1000 person days)
Rate (No. of new illnesses)
Respiratorv Gastrointestinal Skin
7.
4.
5.
4.
7.
6.
1.
7.
4.
4.
3.
6.
4
0
2
4
6
5
5
5
9
5
0
3
(5)
(5)
(44)
(3)
(10)
(65)
(1)
(10)
(46)
(3)
(4)
(60)
3
1
2
4
2
1
5
1
2
7
2
2
.0
.6
.2
.4
.3
.5
.9
.5
.2
.4
.2
.4
(2)
(2)
(19)
(3)
(3)
(15)
(4)
(2)
(21)
(5)
(3)
(23)
1.
0
0.
0
0
0.
1.
0
1.
1.
0.
0.
5
6
2
5
2
5
7
2
(1)
(0)
(5)
(0)
(0)
(2)
(1)
(0)
(11)
(1)
(1)
U)
a Includes routine fecal specimens donated from January 1982 (DCP 201) to
Angust 1983 (DCP 317).
b Negative, very light or light for all bacteria except E. coll.
c Two-week illness observation period prior to donation of rontine fecal
specimen.
d Two-week illness observation period in which fecal specimen was donated.
e Two-week illness observation period after period of specimen donation.
f Two-week illness observation period after DCP+1.
231
-------
TABLE 74. EPISODES OF BACTERIAL INFECTION DETECTED FROM ROUTINE
FECAL SPECIMENS DURING IRRIGATION SEASONS
to
u>
N>
Episode
dependent Total Number
Period of Irrigation variable donors not
observation oeriod name observed infected*
Infection rates, %, by
Number (%) aerosol exposure level
newly Inter-
infected^ Low mediate High
ELBBSIELLA INFECTION EPISODES
1982
Jun 7-Sep 17 Jnl
(Ang 9-Sep 17)
1983
Jun 6-Aug 18 Jun
(Jul 18-Aug 18)
OTHER OPPOiaiiNlSTIC
1983
Jan 31 -Apr 22 Feb
21-Sep 17 CKLB2W
CKLB2X
29-Sep 20 CKLB4W
CKLB4X
BACTERIA INFECTION
15-Apr 30 COOB3
88
80
93
89
EPISODE
107
75
75
81
81
102
13 (14.8) 13.6
5C (6.3) 5.0
12 (12.9) 7,7
8« (9.0) 4.0
5 (4.7) 3.8
20.4 0
9.3 0
10,4 26.3
6.5 22.2
4.8 5.3
INFECTION EPISODES BY PROMINENT BACTERIA IN WASTEWATER
1982
Jan 4-Apr 2 Feb
Jun 7-Sep 17 Jul
(Aug 9-Sep 17)
1983
Jun 6-Aufl 18 Jnn
16-Apr 30 CPBW1W
21-Sep 17 CPBW2W
CPBW2X
29-Seo 20 CPBW4W
113
89
88
94
110
85
85
85
3 (2.7) 5.7
4 (4.5) 0
3C (3.4) 0
9 (9.6) 7.7
0 5.6
4.1 11.1
4.1 5.9
10.2 10.5
a Neither specimen from the individual during irrigation period contained the pathogen at a level
classified as infected.
b Individuals infected during irrigation period, but not infected in previous month. Onset of
the infection event was during the period of observation.
c Individuals whose infection event onset was definitely during irrigation period.
-------
do not permit inference as to which one(s) may be actual susceptibility
or exposure risk factors. Repeated or prolonged infections were observed
in seven donors, six of whom were older than 60.
Drinking more water than others their age also appeared to be significantly
associated with the infected donors. However, the quality of the drinking
water of rural households with private wells was not associated with these
infections in the subset of donors whose well water was monitored. Whereas
two (20%) of the donors whose private wells were contaminated with the
bacterial indicators experienced infections by these prominent wastewater
bacteria, three (38%) of the donors who drank well water of better quality
also had these infections.
The association the fluorescent Pseudomonas and A. hydrophila infections
with self-reported illness is presented in Table 75. No patterns of associa-
tion are evident, but only a small number of person-days of observation
were available for donors with infections to these bacteria prominent in
the wastewater. Footnotes h and i indicate that most (i.e., 6) of the
illnesses in infected donors were reported by a single individual before
and after one fluorescent Pseudomonas infection.
Episodes of infection by bacteria prominent in the wastewater occurred
during three of the four wastewater irrigation periods monitored (see Table
74). The statistical analysis of these infection episodes is reported
later.
H. CLINICAL VHOLOGY OF ROUTINE FECAL SPECIMENS
Viral isolates were recovered from routine fecal specimens by traditional
tissue culture methods (see Figure 16). Enteroviruses were identified
and typed by microneutralization procedures, while adenoviruses were identified
by a group antigen-specific, fluorescent staining procedure. The prevalence
and identification of viral isolates is presented in Table 76 by specimen
collection period. The annual viral isolation rates are not directly compar-
able, both because of the addition of numerous adult donors in 1982 and
1983 to the predominantly child donor population of 1980 and 1981 and because
of the different seasonal distribution of the specimens. The age-specific
rates of viral recovery are presented in Table 77. Donors who were 0-5
years of age had substantially higher viral isolation rates than other
age groups in each collection year. Older children (ages 6-17) also had
higher virus recovery rates than adults. The viral isolation rate in the
0-5 age group was constant at 16-17% in 1981, 1982 and 1983. The higher
isolation rates for children in 1980 (32% for ages 0-5 and 18% for ages
6-17) may be partially due to the restriction of the specimen collection
to the summer months during 1980. Viral isolates were much less prevalent
during 1983 than they had been in 1982 in all adult age groups and in school-age
children.
The distribution of identified viral types differed by year, as Table
76 illustrates. Adenoviruses were the most prevalent type in 1982 and
1983, with the highest number of isolates recovered in January 1982. Coxsackie
B and polioviruses were the most prevalent types in 1980, while polio-
233
-------
TABLE 75. ASSOCIATION OF LEVEL OF GROWTH OF PROMINENT WASTEWATER
BACTERIA8 IN ROUTINE FECAL SPECIMENS1* WITH THE INCIDENCE OF SELF-REPORTED
ILLNESS IN THE PRIOR, CONCURRENT AND SUBSEQUENT BIWEEKLY REPORTING PERIODS
Level of growth
of fl. Pseudo- Period of
monas or A. illness
hvdroohila observation
Heavy /Mode rate DCP-1*
Neg to Light0 DCP-1
Person
days
observed
216
8460
Incidence of self-reported illness
(New illnesses/1000 person days)
Rate (No. of new illnesses)
Respiratory Gastrointestinal Skin
4.6 (l)h 4.6 (I)* 0 (0)
5.2 (44) 2.2 (19) 0.6 (5)
Heavy/Moderate DCP® 225 0 (0) 4.4 (1) 0 (0)
Neg to Light DCP 9997 6.5 (65) 1.5 (15) 0.2 (2)
Heavy/Moderate DCP+1* 228 8,8 (2)1 13.2 (3)* 0 (0)
Neg to Light DCP+1 9429 4.9 (46) 2.2 (21) 1.2 (11)
Heavy/Moderate
Neg to Light
DCP+28
DCP+2
214
9530
0
6.
3
(0)
(60)
0
2.
4
(0)
(23)
0
0.
2
(0)
(2)
a Fluorescent Psendomonas and Aeromonas hydrophila.
b Includes routine fecal specimens donated from January 1982 (DCP 201) to
August 1983 (DCP 317).
c Negative, very light or light for all bacteria except E. coli.
d Two-week illness observation period prior to donation of routine fecal
specimen.
e Two-week illness observation period in which fecal specimen was donated.
f Two-week illness observation period after period of specimen donation.
g Two-week illness observation period after DCP+1.
h Both illnesses reported by ID 45201 in DCP 218.
i Both respiratory illnesses and two of the three gastrointestinal illnes
ses were reported by ID 45201 in DCP 220.
234
-------
TABLE 76. PREVALENCE AND IDENTIFICATION OF VIRAL ISOLATES RECOVERED
FROM ROUTINE FECAL SPECIMENS BY COLLECTION MONTH
Specimen
collection
period
1980
Jul
Aug
Sep
1980 Total
1981
Apr/May^
Jnn
Jul
Aug/Sepa
1981 Total
1982
Jan 4-8
Mar 1-5
Mar 29-Apr 2
Tun 7-11
Aug 9-13
Sep 13-17
1982 Total
1983
Jan 31-Feb 4
Apr 18-22
Jun 6-10
Jul 18-22
Aug 15-19
1983 Total
a Some donors
Rout ine
fecal
donors
22
36
47
105
27
45
30
35
137
107
127
127
124
118
121
724
100
109
102
105
99
515
Viral isolation
Number
prevalence rate
Number
7
9
7
23
0
5
6
6
17
11
9
14
5
3
12
54
0
3
2
4
2
11
Percent
32
25
15
21.9
0
11
20
17
12.4
10.3
7.1
11.0
4.0
2.5
9.9
7.5
0
2.8
2.0
3.8
2.0
2.1
Adeno
0
0
0
0
0
2
2
0
4
8
2
3
4
0
1
18
0
2
1
1
0
4
of samples yielding
designated
Cox
3
3
2
8
0
0
0
0
0
0
0
0
0
1
3
4
0
0
0
2
0
2
provided more than one fecal specinx
B Echo
0
2
1
3
0
0
1
1
2
3
2
2
0
0
5
12
0
0
0
1
1
2
;n over
viral
Polio
3
3
2
8
0
3
1
1
5
0
3
5
0
0
1
9
0
0
0
0
0
0
this
type
Unidentified
1
1
2
4
0
0
2
4
6
0
2
4
1
2
2
11
0
1
1
0
1
3
extended col-
lection period. Tabulation based on first specimen donated.
235
-------
TABLE 77. AGE-SPECIFIC ANNUAL RECOVERY OF VIRAL ISOLATES FROM ROUTINE FECAL SPECIMENS
Donor
age.
1980
years Specimens
0-5
6-17
18-44
45-64
65+
All
ages
34
65
6
105
1981
Isolates (%) Specimens
11
12
0
23
(32)
(18)
(0)
(21.9)
54
97
9
160
1982
Isolates (%) Specimens
9
9
0
18
(17)
(9)
(0)
(11.3)
98
190
141
161
134
724
Isolates (%) Specimens
17
19
4
9
5
54
(17)
(10)
(3)
(6)
(4)
(7.5)
62
111
86
150
106
515
1983
Isolates (%)
10
1
0
0
0
11
(16)
(1)
(0)
(0)
(0)
(2.1)
K>
-------
and adenoviruses were most frequently recovered in 1981. Eight of the
22 poliovirus isolations were considered to be immunization-associated,
in that the donor had received Sabin oral polio vaccine during the preceding
month.
These patterns of viral recovery from healthy populations are consistent
with other published studies conducted in the United States. In an early
study reported by Honig and associates (1956), 92% of the enteric viruses
isolated from healthy preschool children in Charleston, West Virginia were
isolated over the period of June to October. In the lower socioeconomic
group, 8.3% of specimens yielded viruses while only 3.1% of the samples
from an upper middle class district were positive. Among the viruses isolated
over a 29 month period, 44% were echoviruses; 37%, coxsackieviruses; and
19%, polioviruses.
Similarly, data collected by Gelfand and co-workers (1963), showed
a seasonal pattern of enterovirus isolations among healthy children in
six major U.S. cities over a two year period. In southern cities (Atlanta
and Miami) enteroviruses were recovered year-round, albeit at lower frequencies
in the winter season. In northern cities (Minneapolis, Buffalo and Seattle),
virtually no viral isolations were made during late winter and early spring
months. Positive viral isolation rates, excluding vaccine-derived polioviruses,
ranged from 1% to as high as 22% among lower socioeconomic status children.
Rates of viral isolation from males (12.6%) statistically exceeded that
of females (9.5%) over the two year study. Excluding immunization-associated
polioviruses, echoviruses accounted for 46% of the viral isolates; coxsackie-
viruses, 33%; polioviruses, 9%; and nntypable isolates, 12%. Notably,
the procedures used in this study to cultivate viral agents were not optimal
for adenovirus recovery.
The occurrence of viruses within family units was described as part
of the extensive Seattle Virus Watch program. Isolation rates of coxsackie-,
echo- and adeno-viruses from fecal specimens provided by children 0-5 years
of age averaged 5.3% as compared to 1.4% for children 6-9 years of age
and 1% for mothers (Cooney et al., 1972), During this monitoring program
a preponderance of isolates were vaccine-derived polioviruses. Of the
nonpoliovirnses recovered, adenoviruses accounted for appoximately 64%
of the total fecal isolates while coxsackieviruses and echovirnses accounted
for 20% and 16%, respectively.
The viral isolation results of all routine fecal specimens donated
during each year of the LISS are presented in Tables 78 through 80 by par-
ticipant for all individuals from whom a viral isolate was recovered. In
some instances, the same viral type was shed and recovered in consecutive
specimens collected approximately 4 weeks apart.
The association of viral infection, as determined by viral recovery
from a routine fecal specimen, with self-reported illness was also investi-
gated. The incidence densities of self-reported respiratory, gastrointestinal,
and skin illnesses in the 2-week periods prior, concurrent, and subsequent
to the fecal collection were compared for all routine fecal specimens with
a viral isolate and with no viral isolate. This analysis was accumulated
237
-------
TABLE 78. VIRAL ISOLATES RECOVERED FROM DONORS8 OF ROUTINE FECAL SPECIMENS DURING BASELINE MONITORING
(July 1980 to September 1981]
ID
number8
21111
43414
22712
42711
30612
40812
53913
53911
32412
56211
20211
21916
21915
45314
45313
10414
55715
55714
40411
32112
32111
21012
21011
53313
43511
40216
45112
12211
43614
Period 015
mi
unidentified
-
polio 3D
Coxseckie B-2
Coxeeckie B-3
Coxeackie B-3
polio 3
polio 3
Period 017
Coxeackle B-3
-
polio 1
-
unidentified*
echo 11*
polio 1b
-
Coxeeckie B-5
_
Coxeackle B-3
_
-
—
-
echo 24
—
-
polio 1
Period 019
• —
polio 1
unidentified
—
polio 1
Coxeeckie B-5
Coxeackle B-5
-
-
_
-
unidentified
-
echo 24
_
-
—
Viral Isolates from fecal
Period 108 Period 110 Period
— —
- -
-
— —
- -
-
- adeno
-
—
—
_
polio
_ _
- -
- -
— —
_ _
- polio
_ _ _
adeno
polio
- - -
—
specimens
112 Period 114 Period 117
unidentified
unidentified polio 1
— —
adeno
3b
adeno
- -
echo 5
polio 3
—
_
3b
—
1b
—
—
Period 118 Period 119
-
echo 11
unidentified
unidentified
-
-
-
-
unidentified
-
unidentified
unidentified
-
—
- No viral isolate recovered from fecal specimen
* Illnees convaleecent specimen
(Blank) No specimen obtained
8 Only donore with viral Isolates are listed.
b Recipient of oral vaccine during preceding month.
-------
TABLE 79. VIRAL ISOLATES RECOVERED FROM DONORSa OF ROUTINE
FECAL SPECIMENS IN 1982
Fecal collection period in 1982
ID
numbe ra
10201
10414
10901
11402
11902
12211
12501
12602
13211
13212
20502
20713
21012
21112
21301
21611
21915
21916
22712
23112
23614
23615
32202
32411
32412
40312
41302
41601
42801
45113
45312
45313
45314
50501
53901
53911
53912
54502
60111
(Blank)
201
(Jan 4-
adeno
adeno
-
-
adeno
-
adeno
echo 5
—
adeno
adeno
-
echo 11
-
adeno
adeno
-
-
-
-
-
echo 5
-
-
-
-
-
-
-
-
-
205
8) (Mar 1-5)
_
—
-
polio 3
-
-
-
-
—
polio 3b
-
adeno
-
-
adeno
-
echo 24
+
-
-
-
polio 1
-
-
-
-
-
-
+
-
echo 17
-
-
-
-
-
207 212 216
(Mar 29-Apr 2) (Jnn 7-11) (An* 9-13)
polio 1 - -
+ +
_ _ _
_
polio 1 - -
_ _ _
_ _ _
_ _ _
adeno -
polio 3
-
adeno -
echo 27
_ _ _
_
_ _ _
- adeno -
_ _ _
_ _
- adeno -
adeno - -
echo 17 - -
adeno
adeno - -
CB 4
-
- - -
polio 1 - -
+
+
_ _ _
- +
_
+
+
_ _ _
_ _ _
polio 3b
219
(Sep 13-17)
_
—
echo 27
-
echo 31
echo 30
-
-
adeno
-
-
CB 5
-
-
-
-
-
+
-
-
-
CB 5
polio 2
-
-
+
—
CB 5
-
'-
-
echo 30
echo 31
-
No fecal specimen obtained
- No viral isolate recovered
+ Unidentified
a Only
b Recij
donors
)ient of
viral isolate
from fecal specimen
recovered from fecal specimen
with viral isolates are listed
oral vaccine
during preceding month
239
-------
TABLE 80. VIRAL ISOLATES RECOVERED FROM DONORSa
OF ROUTINE FECAL SPECIMENS IN 1983
Fecal, co lie cation period in 1983
303 308 312~ 315 317
ID (Jan. 31rFeb 4_).... iApr_18r221 . . (Jun 6-10) (Jul 18-22) . (Aug 1S-19J
20713 - adeno -
20714 - adeno -
21112 - - - - echo 27
32413 - +
40216 - - - CB 5
45411 - - echo 15
45412 + - CB 1
60111 - _ adeno adeno
(Blank) No fecal specimen obtained
- No viral isolate recovered from fecal specimen
+ Unidentified viral isolate recovered from fecal specimen
a Only donors with viral isolates are listed.
over all routine fecal specimens provided in 1982 and 1983, when the donors
represented all age groups and the illness data were more reliable. The
results presented in Table 81 show that viral recovery from feces may be
associated with an increased risk of respiratory illness during the 2-week
period of fecal donation and during the subsequent 2-week period. Although
the illness rates for positive viral isolates are variable due to the small
number of person-days observed, this observation of a risk ratio of about
2 for concurrent and subsequent respiratory illness in persons with a viral
isolate is consistent with the literature (Fox et al., 1977).
A viral infection event was defined as the isolation of a specific
virus by laboratory cultivation in the second and not the first of consecutive
routine fecal specimens from the same person. Subsequent recovery of the
same virus in a specimen from the same individual was considered to be
a new event if more than 6 weeks elapsed between sequential recoveries.
Detection of a virus in the first of serial specimens was also considered
a viral infection event.
Adenoviruses are often shed sporadically over an extended period of
time. Thus, the time of onset of an adenovirus infection cannot be determined
reliably from an adenovirus recovery in a specimen series. A poliovirus
isolate recovered from a donor who received Sabin oral polio vaccine during
the prior month was presumed to result from the immunization. Thus, the
infection events to viruses other than adenoviruses or immunization-associated
poliovirnses whose onset was during periods of wastewater irrigation were
identified to investigate their possible association with the donor's wastewater
exposure.
Five episodes of infection by viruses other than adenoviruses and
immunization-associated polioviruses that occurred during seasons of irrigation
240
-------
TABLE 81. ASSOCIATION OF VIRAL ISOLATES IN UOl/ilNE FECAL SPECIMENS*
WITH THE INCIDENCE OF SELF-REPORTED ILLNESS IN THE PRIOR.
CONCURRENT AND SUBSEQUENT BIWEEKLY REPORTING PERIODS
Viral
isolation
Positive
Negative
Positive
Negative
Positive
Negative
Positive
Negative
Period of Person
illness days
observation observe*
DCP-lb
DCP-1
DCPC
DCP
DCP+ld
DCP+1
DCP+2e
DCP+2
409
8429
600
9558
588
9342
581
9405
Incidence of self-reported illness
(New illnesses/1000 person days)
Rate (No. of new illnesses)
i Respiratory Gastrointestinal Skin
4
5
11
6
10
4
5
6
.9
.1
.7
.2
.2
.6
.2
•?_
(2)
(43)
(7)
(59)
(6)
(43)
(3)
(58)
2
2
3
1
3
2
0
2
.4
.1
.3
.4
.4
.1
.4
(1)
(18)
(2)
(13)
(2)
(20)
(0)
Ml-.
0
0
0
0
0
1
1
0
.6
.2
.2
.7
.1
(0)
(5)
(0)
(2)
(0)
(11)
(1)
(1)
c
d
e
Includes routine fecal specimens donated from January 1982 (DCP 201} tc
August 1983 (DCP 317).
Two-week illness observation period prior to donation of routine fecal
specimen.
Two-week illness observation period in which fecal specimen was donated.
Two-week illness observation period after period of specimen donation.
Two-week illness observation period after DCP+1.
were detected from the routine fecal specimen virology. These viral infection
episodes are described in Table 82. Three viral infection episodes occurred
during periods of irrigation. Fifteen of the 120 donors monitored throughout
the spring 1982 irrigation had at least one new viral infection. The onset
of the viral infection definitely occurred after irrigation commenced for
at least 9 of these 15 infected individuals (i.e., those nine in Table
79 in which the period 205 specimen was negative but a virus other than
adeno or an immunization-associated polio was recovered from the period
207 specimen). The dependent variables for this episode were named CVIR1W
for the observation period in which 15 individuals were infected and CVIR1X
for the shorter observation period during irrigation in which 9 individuals
were infected. Twelve of the 106 donors monitored during the summer 1982
irrigation period had at least one viral infection event (episode CVIR2) .
A viral infection episode (CVIR4W) also occurred in summer 1983. Viral
infection episodes CVIR8 and CVIR9 occurring during summer 1980 and summer
1981 were also evaluated as nonirrigation control situations.
Infection rates are also presented by level of aerosol exposure in
Table 82. Observed donors with a high level of aerosol exposure (AEI>5)
during the summer 1982 irrigation exhibited a higher rate of viral infections
(23.5%) than did donors with less aerosol exposure. The viral infection
episodes occurring at other times did not show this pattern. The statistical
analysis of these infection episodes for possible association of viral
infections with wastewater irrigation is presented later.
241
-------
TABLE 82. EPISODES OF INFECTION TO VIRDSES (EXCLUDING ADENOVIEUSES AND IMMUNIZATION-ASSOCIATED
POLIOVIRUSESa) DETECTED FROM ROUTINE FECAL SPECIMENS DURING IRRIGATION SEASONS
Episode
dependent
Period of Irrigation variable
observation period name
1980
Jol 20-Sep 17 (None) CVIR8
1981
Jon 1-Sep 2 (None) CVIR9
Infection rates, %, by
Total Number Number (%) aerosol exposure level
donors not newly Inter-
observed infected** infected0 Low mediate High
28 16 12 (42.9) 43 54 (0)
29 20 9 (31.0) 60 18 (0)
1982
Jan 4 -Apr 2
(Mar 1-Apr 2)
Jon 7-Sep 17
(Aog 9-Sepl7)
1983
Jon 6 -Aug 18
Feb 16-Apr 30
Jol 21-Sep 17
Jon 29-Sep 20
CVIR1W
CVIR1X
CVIR2W
CVIR2X
CVIR4W
120
114
106
105
97
105
105
94
94
92
15 (12.5)
9d (7.9)
12 (11.3)
lld (10.5)
5 (5.2^
10.5
8.1
7.7
7.7
0
13.8
8.2
9.5
8.1
7.8
11.8
6.3
23.5
23.5
5.3
a Recipient of Sabin oral polio vaccine doring prior month.
b Both of the individual's specimens daring irrigation period were negative for viruses (other
than adenoviroses).
c Individual had at least one viral infection event to a viros other than an adenoviros or immuniza-
tion-associated polioviros; onset was doring the period of observation.
d Individuals whose infection event onset was definitely daring irrigation period.
-------
I. SEROLOGIC DATA AND SEROCONVERSION RATES
Antibody Prevalence
The frequency distribution of titers to all serologic agents which
were used in this study is summarized in Table P-45 in Appendix P. It
should be noted that the serologic testing protocol required assay of all
of the bloods from only certain collection periods. Table 9 should be consulted
to determine which bloods were included in the testing for each agent.
With the exception of Nor walk and rotavirus, frequency distributions with
small sample sizes (N<50) usually contain titers from individuals who provided
blood samples on an irregular basis. Periods with small sample sizes also
include titers which were obtained during retesting. Retesting was performed
to confirm fourfold increases in titer and, whenever possible, to determine
the exact interval of time when seroconversions occurred. It should be
noted that 15% of the participants who provided paired bloods during the
baseline period dropped out before the end of the study. Therefore, changes
in the distribution of antibody titers between blood collection periods
(for each of the agents) reflect the slight changes in the population as
well as changes in antibody titer that resulted from infections in indivi-
duals that remained in the study.
Based on the first blood obtained from each study participant, approxi-
mately 85% of the entire study population had influenza A antibody. More
than half of the study population had antibody to coxsackie B2, coxsackie
B4, Legionella. and reovirus 2. Eighty-nine percent of the selected subpopu-
lation (consisting of children under the age of 10 and high exposure adults
with diarrhea in 1982) had antibody to rotavirus. Forty-two percent of
the population had antibody to hepatitis A. The majority of the participants
with hepatitis A antibody were over the age of 45 or resided in a lower
socioeconomic status household. Seven of the 24 children (29%) and all
12 of the adults who were tested were found to have antibody to Norwalk
virus. Less than 20% of the population had antibody to echoviruses 1,
17, 19, 20 and 24. Only about 1% of the population had antibody to E. his-
tolytica. As would be expected, only a small portion of the population
had no antibody to polioviruses 1 and 2. Forty percent of the participants
had no detectable antibody to poliovirus 3.
Table P-45 in Appendix P should not be used to determine the efficiency
of the poliovirus immunizations which occurred during the course of this
study. The low poliovirus titers in blood samples that were collected
in January and June 1982 were observed in infants, in adults who refused
to be immunized, and in adults who had not provided a blood sample for
testing earlier in the study. Table 83 illustrates the effect of immunization
on the poliovirus titers of participants who were immunized (and provided
paired bloods) during the baseline interval. The table also illustrates
that the Salk vaccine series (without the booster) was more effective than
the Sabin booster in increasing the level of antibody titer during the
baseline period. Using Cochran-Mantel-Haenszel (nonzero correlation) statis-
tics, the difference in vaccine effectiveness was found to be significant
for all three poliovirus types (p=0.003 for polio 1, p=0.001 for polio
2 and p<0.001 for polio 3) during the baseline period. Since irrigation
243
-------
TABLE 83. EFFECT OF IMMUNIZATION ON PARTICIPANT POLIOVIRUS
TITERS BY AGENT AND VACCINE TYPE
(FOR PARTICIPANTS WHO PROVIDED BLOOD IN BOTH THE
BASELINE PERIOD AND IN JANUARY 1982)
Poliovirus 1
Poliovirus 2
Poliovirus 3
Salk Vaccine*
#
#
#
immunized
twofold increases in titer
fourfold or greater
increases in titer
68
11
50
(16%)
(74%)
68
11
52
(16%)
(76%)
68
9
57
(13%)
(84%)
Sab in Vaccineb
#
#
#
immunized
twofold increases in titer
fourfold or greater
increases in titer
39
12
17
(31%)
(44%)
38
7
19
(18%)
(50%)
37
11
15
(30%)
(41%)
Adults who were recommended for immunization received the complete Salk
series. The majority of the adults (47/68) had received the first three
Salk injections before the January 1982 blood was collected. The third
injection was administered in June 1981. The booster was administered
immediately after the blood sample was collected in January 1982.
Children who were recommended for immunization received only the Sabin
booster dose in May 1981. because all had previously received their
basic immunization series.
began soon after the Salk booster was administered, the titer increases
observed in the participants who received the boosters may have been caused
either by the Salk booster or by exposure to wastewater aerosols. An analysis
of their relative importance is presented later in the statistical results.
The frequency distribution of antibody titer by age group is listed
in Table P-46 in Appendix P. Inspection of this table reveals that antibody
presence remains constant among age groups for adenovirus 5; coxsackievirus
B2; echoviruses 9, 11 and 20; Legionella; the polioviruses and reovirus
1. Antibody presence definitely increases with age for echoviruses 1 and
19, hepatitis A, influenza and reovirus 2. Antibody occurrence appears
to increase from young children to older age groups for rotavirus, but
the small sample sizes of the adult age categories render this impression
uncertain.
Incidence Densities for Serologic Agents
The incidence density of infections (defined as a fourfold or greater
increase in titer in paired sera), the incidence density ratio (IDR) and
its 95% and 90% test-based confidence invervals were calculated as discussed
in Section 4J. An infection incidence density ratio was considered to
be significant if its 95% confidence interval did not include 1.0, provided
the expected number of infections in both exposure groups compared was
244
-------
2.0 or larger. The IDR was considered possibly significant if its 90%
confidence interval did not include 1.0 and at least 2 infection events
were expected in both groups compared.
No incidence density calculations or any statistical analyses were
performed on results for coxsackieviruses A9 and B3, Norwalk agent, E.
histolytica or hepatitis A. Due to a high prevalence of antibody to coxsackie-
viruses A9 and B3, serology testing was discontinued and no analysis was
performed on the partial serologic results. There were three fourfold increases
in titer to Norwalk agent. Two increases were observed during the irrigation
period: one fourfold increase occurred in a high exposure level participant;
the other increase occurred in a low exposure level participant. Unfortunately,
the small sample size prevented interpretation of this information, and
no further analyses were performed for the Norwalk data. There were two
fourfold increases in E. histolytica titer: one during the baseline period
and one during the January 1982-June 1983 time interval. The only hepatitis
A infection identified during the course of the study occurred in the baseline
period between June and December 1980. Thus, neither E. histolyt ica nor
hepatitis A was included in further analyses.
Results were modified somewhat before incidence densities could be
calculated for the polioviruses, the reoviruses, and Legionella. Only those
participants who were not immunized were included in incidence density
calculations for the three polioviruses. Thirty-four fourfold titer increases
to reovirus 1 and 17 fourfold increases in titer to reovirus 2 were detected
in the summer of 1982. Unfortunately, none of these particular fourfold
increases were tested in pairs. Consequently, the titers associated with
the unconfirmed infections were coded as missing and not included in either
incidence density calculations or in any other statistical analyses. Therefore,
although it appears that there were no reovirus infections in the summer
1982 irrigation season, in fact all of the (possible) positive results
have been excluded. To conserve January 1982 blood for virus testing, bloods
which were selected for use in the Legionella serologic testing (see Table
9) created interpretation problems because the exact 6-month interval in
which the infection occurred was not identified. Whether the Legionella
infections that occurred between June 1981 and June 1982 were incurred
before or after irrigation commenced has not been determined. However,
there were not enough Legionella infections to detect a significant difference
(between exposure groups or between exposure levels) even if the exact
6-month interval of each seroconversion were known.
Table 84 compares the infection incidence densities to individual
agents which were observed in the three aerosol exposure levels during
the baseline and irrigation periods. The high exposure level was found
to have the highest incidence density of infection for adenovirus 7 and
echovirus 5 during the baseline period. The high exposure level was found
to have the highest incidence density of infection for eight (coxsackieviruses
B2 and B4, echoviruses 3, 11, 19, 20 and 24, and rotavirus) out of the
nineteen agents during the irrigation period. As indicated in the table,
the incidence density ratio of the high to the intermediate exposure levels
was found to be possibly significant for coxsackievirus B4, as indicated
by the 90% confidence interval. However, the incidence density ratios for
245
-------
TABLE 84. COMPARISON OF BASELINE AND IRRIGATION INCIDENCE DENSITY
RATES8 BY WASTEWATER AEROSOL EXPOSURE LEVEL AND AGENT
(NUMBER OP INFECTION EVENT INDICATED IN PARENTHESES)
Baseline^
Low exp
level
(AEK1)
Adeno 3
Adeno 5
Adeno 7
Cox B2
Cox B4
Cox B5
Echo 1
Echo 3
Echo 5
Echo 9
Echo 11
Echo 17
Echo 19
Echo 20
Echo 24
Reo 1
Reo 2
Influenza A
Rotavirus
2.07
3,16
0,84
7.14
5.07
0,82
0.85
8.29
0,96
1.64
5.85
1.05
0,00
1.05
2.15
14.22
7.53
3.24
0.00
(2)
(3)
(1)
(7)
(5)
(1)
(1)
(7)
(1)
(2)
(7)
(1)
(0)
(1)
(2)
(17)
(9)
(3)
(0)
Med exp
level
(11AEJX5)
11.40
5.27
2.51
5,10
11.15
6.57
5,11
4,15
0.96
4.11
6.69
1.05
3.21
4.19
6.46
12.55
17.57
12.96
151.24
(11)
(5)
(3)
(5)
(11)
(8)
(6)
(4)
(1)
(5)
(8)
(1)
(3)
(4)
(5)
(15)
(21)
(12)
(7)
Hi exp
level
(AEI>5)
0.00
0,00
3.38
3.32
0.00
3.44
0,00
1.80
1.82
3.44
3.47
0.00
0.00
0.00
1.80
5,07
11.80
7.34
23.50
(0)
(0)
(2)
(2)
(0)
(2)
(0)
(1)
(1)
(2)
(2)
(0)
(0)
(0)
(1)
(3)
(7)
(4)
(4)
Low exp
level
(AEK1)
0,57
1.15
0.00
0.00
7.93
1.67
0.57
3.98
0.00
1.19
4.48
0.00
0.00
1.18
2.98
2.99
2.93
10.33
8.75
(1)
(2)
(0)
(0)
(6)
(3)
(1)
(7)
(0)
(2)
(8)
(0)
(0)
(2)
(5)
(3)
(3)
(10)
(1)
Irrigation6
Med exp
level
(1CAEIX5)
1.91
2.58
0.00
4.51
5.63
3.62
0.00
4.19
0.28
0.00
3.79
0.83
0.83
1,97
2.77
5.75
5.19
13.29
10.89
(7)
(9)
(0)
(7)
(9)
(13)
(0)
(15)
(1)
(0)
(14)
(3)
(3)
(7)
(10)
(13)
(12)
(27)
(7)
Hi exp
level
(AEI>5)
0.00 (0)
1.17 (1)
0.00 (0)
5.80 (2)
13.91 (5)d
2.28 (2)
0.00 (0)
5.75 (5)
0.00 (0)
0.00 (0)
7.91 (7)
0.00 (0)
1.18 (1)
2.31 (2)
4.66 (4)
1.94 (1)
0.00 (0)
8.04 (4)
23.91 (8)
Infection incidence density is expressed as the number of new infections per hundred person-years
of observation:
Infection ID
No. Fourfold Increases in Time Interval
No. Person-days Observed During Interval
x 36525
Spring 1982 aerosol exposure values were used for the baseline period (June 1980 to January
1982).
Since an individual could have different exposures during the irrigation period (January 1982
to October 1983), the infection rate was calculated by summing results from each of the four
irrigation seasons. Aerosol exposure values for 1982 or 1983 were used when it was not possible
to determine the exact irrigation season in which the infection had occurred.
The 90% confidence interval for the high to intermediate incidence density ratio does not include
the value 1.
-------
rotavirus and the other six enterovirnses was not found to be significant.
In contrast, the incidence of influenza A infection (our epidemiologic
control) was lowest in the high exposure level during the irrigation period. The
majority of the ''susceptible'' study participants (i.e., children, adults
over the age of 60, lower socioeconomic status families) was located in
the intermediate and low exposure levels. Thus, this finding of elevated
incidence of infections during the irrigation period to viruses recovered
from the wastewater was not expected.
Table 85 compares the individual agent incidence densities for the
two aerosol exposure groups during the baseline and irrigation periods.
Since the ''susceptible'' population was more evenly divided between the
two exposure groups, it was expected that there would be an even distribution
of infections between the two groups. Nine agents were found to have a
higher infection density in the high exposure group during the baseline
period. The risk of echovirus 9 infection was six times greater for the
high exposure group than the low exposure group; this ratio was found to
be significant. The elevated risk of adenovirus 7 infection in the high
exposure group was possibly significant during the baseline period. Eight
agents were found to have a higher rate of infection in the high exposure
group during the irrigation period. Infection rates were noticeably higher
during the irrigation period for coxsackievirus 62, echoviruses 11 and
19, and rotavirus. The risk of infection for the high exposure group was
found to be five times as great for coxsackievirus B2, twice as great for
echovirus 11 and rotavirus, and seven times as great for echovirus 19,
during the irrigation period. The elevated risks of infection by coxsackievirus
B2 and echovirus 11 in the high exposure group were significant.
The agent groupings which were used in the serologic data analysis were
defined in Table 18. Agents were grouped in order to increase the number
of infections observed, thereby increasing the chances of detecting an
association between infection and wastewater exposure that was operative
for all agents in the group. For purposes of calculating incidence densities,
incidence density ratios, and the associated 90% and 95% confidence intervals,
it was assumed that the infections caused by the members of an agent grouping
were independent events. Therefore each person was at risk of infection
by each agent in the agent grouping during each period of observation.
Thus, the person-days for each agent (agent-person-days) were considered
to be additive. For example, if a person was observed for 100 days and
there were three agents in the agent grouping, then that person was considered
to be at risk to infection by the members of the agent grouping for 300
agent-person-days.
The assumption of independence of the infection events to the agents
in each group is probably valid. Consideration was given to the possible
confounding effects of virus-host interaction. While mixed infections
with more than one enterovirus have been frequently observed in warm climates
and under poor hygienic conditions (Parks et al., 1967), such multiple
infections were found infrequently among normal families in the United
States (Cooney et al., 1972). On the other hand, as demonstrated during
live poliovirus vaccine trials, multiplication of one virus can effectively
interfere with the growth of a second enterovirus (Sabin et al., 1960).
247
-------
TABLE 85. COMPARISON OF BASELINE AND IRRIGATION ENTEROVIRUS INFECTION
INCIDENCE DENSITY RATES8 BY WASTEWATER AEROSOL EXPOSURE GROUP AND AGENT
(NUMBER OF INFECTION EVENTS INDICATED IN PARENTHESES)
Baseline0
Irrigation6
Low ezp group
(AEK3)
High ezp group
(AEL>3)
Low ezp group
(AEK3)
High ezp group
(AEI13)
Adeno 3
Adeno 5
Adeno 7
Coz B2
Coz B4
Coz B5
Echo 1
Echo 3
Echo 5
Echo 9
Echo 11
Echo 17
Echo 19
Echo 20
Echo 24
Reo 1
Reo 2
Influenza A
Rotavirus
3
2
0
3
3
1
1
3
0
0
3
0
0
1
2
10
9
5
18
.96
.04
.69
.90
.87
.76
.42
.96
.39
,70
,48
.80
.80
.60
,43
.07
.01
.96
.24
(10)
(5)
(2)
(10)
(10)
(5)
(4)
(9)
(1)
(2)
(10)
(2)
(2)
(4)
(5)
(29)
(26)
(14)
(4)
2
2
2
2
4
4
2
2
1
4
4
0
0
0
2
4
7
4
20
.10
.17
.65
.76
.07
.13
.15
,16
.44
.82
.78
.00
.72
.72
.10
.03
.41
.75
.85
(3)
(3)
(4)e
(4)
(6)
(6)
(3)
(3)
(2)
(7)d
(7)
(0)
(1)
(1)
(3)
(6)
(11)
(5)
(7)
1
2
0
1
6
3
0
4
0
0
3
0
0
2
2
4
4
11
10
,53
.48
.00
.58
.63
.08
.22
.41
.22
.44
.45
.66
,22
.03
.70
.18
.10
.42
.11
(7)
(11)
(0)
(3)
(13)
(14)
(1)
(20)
(1)
(2)
(16)
(3)
(1)
(9)
(12)
(11)
(11)
(29)
(6)
0.56
0.59
0.00
7.72
8.79
2.23
0.00
4.03
0.00
0.00
7.21
0.00
1.72
1.14
3.98
5.00
3.27
11.86
20.07
(1)
(1)
(0)
(6)d
(7)
(4)
(0)
(7)
(0)
(0)
(13)d
(0)
(3)
(2)
(7)
(6)
(4)
(12)
(10)
Infection incidence density is ezpressed as the number of new infections
per hundred person-years of observation:
Infection ID =
No. Fourfold Increases in Time Interval
No. Person-days Observed During Interval
z 36525
Spring 1982 aerosol ezposure values were used for the baseline period
(June 1980 to January 1982).
Since an individual could have different ezposures during the irrigation
period (January 1982 to October 1983), the infection rate was calculated
by summing results from each of the four irrigation seasons. Aerosol
ezposnre values for 1982 or 1983 were used when it was not possible
to determine the exact irrigation season in which the infection had
occurred.
The 95% confidence interval for the high to low group incidence density
ratio does not include the value 1.
The 90% confidence interval for the high to low group incidence density
ratio does not include the value 1.
248
-------
It was considered unlikely however that simultaneous, multiple infections
would occur within the confines of a normal study population exposed to
a presumably low viral infectious dose via environmental (aerosol) pathways.
Table 86 compares incidence densities of the three exposure levels
for infections caused by agent groupings during the baseline and irrigation
periods. It can be seen in Table 86 that the high exposure level had the
lowest infection density for all agent groupings during the baseline period.
Of more interest is the fact that the high exposure level had the highest
infection density during the irrigation period for two of the three independent
agent groupings: coxsackie B viruses and the echoviruses. The wastewater
viruses, which consisted of coxsackie B and echoviruses (see Table 99),
also caused the highest infection incidence density in the high exposure
level during the irrigation period. The high exposure level's density of
infection by wastewater viruses was found to be twice as great as the density
of the intermediate exposure level; this result was significant because
the 95% confidence interval for the high to intermediate WWV incidence
density ratio exceeded 1.0. Since the wastewater viruses are a large subset
of the serum neutralization viruses, it was not surprising to find that
the high exposure level also had the highest rate of infection for the
SNV grouping. The rate of infection to the SNV group in the high exposure
level was found to be greater than the rate of infection in the low exposure
level. Using the 90% confidence interval, the ratio of the incidence densities
of the high exposure level to the low exposure level is possibly significant
for the serum neutralization viruses. Given the demographics of the the
study population and the distribution of infections during the baseline
period, the higher rates of infection during the irrigation period were
expected in the low or intermediate exposure levels. The high incidence
density of infection observed in the high exposure level participants by
the viruses which were recovered from the irrigation wastewater indicates
an apparent association between exposure to irrigation wastewater aerosols
and infection.
Table 87 compares incidence densities for the two exposure groups
for infections caused by the same agent groupings. The high exposure group
was found to have a slightly higher density of infection by all agent groupings
during the baseline period. Comparison of Table 87 to Table 86 discloses
that the higher baseline density of infections occurred among participants
in the upper portion of the intermediate exposure level (3
-------
to
en
o
TABLE 86. COMPARISON OF BASELINE AND IRRIGATION INCIDENCE DENSITY RATES8
BY WASTEWATER AEROSOL EXPOSURE LEVEL AND AGENT GROUPING
(Number of infection events indicated in parentheses)
[Number of infected individuals indicated in brackets]
Baselineb
Low exp
level
(AEK1)
SNV
WWV
POR
ADEN
COXB
ECHO
2.62
2.99
1.61
4.08
2.46
(41H31]
(25) [23]
(5)[5]
(13) [12]
(23) [19]
Med ezp
level
2.28
2.65
2.63
3.31
1.82
(82H57]
(51H37]
(19H17]
(24) [22]
^39) [30]
Hi ezp
level
(AEI>5)
1.53 (13)[10]
1.32 (6)[5]
1.19 (2)[2]
2.24 (4)[3]
1.39 (7)[51
Irrigation"
Low ezp
level
(AEK1)
1.55
5.46
0.45
0.57
2.76
1.62
(37)[34]
(24) [22]
(4) [4]
(3)[3]
(9) [9]
(25) [24]
Med ezp
level
1.94
4.68
0.79
1.39
4.31
1.63
(97) [84]
(44) [42]
(15) [15]
(15) [14]
(29) [28]
(53) [45]
Hi ezp
level
(AEI>5)
2.42
8.34
0.74
0.38
5.73
2.44
(29)[23]d
(17)[15]e
(3)[3]
<1)[1]
(9) [8]
(19H161
Infection incidence density is expressed as the number of new infections per hundred person-years
of observation:
Infection ID =
No. Fourfold Increases in Time Interval
No. Agent-Person-days Observed During Interval
z 36525
Spring 1982 aerosol ezposure values were used for the baseline period (June 1980 to January
1982).
Since an individual could have different ezposures during the irrigation period (January 1982
to October 1983), the infection rate was calculated by summing results from each of the four
irrigation seasons. Aerosol ezposure values from 1982 or 1983 were used when it was not possible
to determine the exact irrigation season in which the infection had occurred.
The 90% confidence interval for the high to low level incidence density ratio does not include
the value 1.
The 95% confidence interval for the high to intermediate incidence density ratio does not include
the value 1.
-------
TABLE 87. COMPARISON OF BASELINE AND IRRIGATION INCIDENCE DENSITY
RATESa BY WASTEWATER AEROSOL EXPOSURE GROUP AND AGENT GROUPING
(Number of infection events indicated in parentheses)
[Number of infected individuals indicated in brackets]
Baseline"
Irrigation6
Low ezp group High ezp group Low ezp group High ezp group
(AEK3) (AEI>3) (AEK3) (AEI>3)
SNV
WWV
POR
ADEN
COXB
ECHO
2.08 (82)[62] 2.52 (54)[36]
2.42 (51)[44] 2.72 (31)[21]
2.04 (16)[15] 2.32 (10)[9]
3.13 (25H23] 3.66 U6H14]
1.74 (41H341 2.20 (28H2Q]
1.79 (112)[99] 2.09 (51)[42]
4.88 (55)[51] 6.23 (30)[28]
0.73 (17)[17] 0.54 (5)[5]
1.25 (17H16] 0.38 (2)[2]
3.59 (30H29] 5.07 U7)[16]
1.60 (65)[59] 2.03 (32)[26]
a Infection incidence density is ezpressed as the number of new infections
per hundred person-years of observation:
Infection ID
No. Fourfold Increases in Time Interval
No. Agent-Person-days Observed During Interval
z 36525
b Spring 1982 aerosol exposure values were used for the baseline period
(June 1980 to January 1982).
c Since an individual could have different exposures during the irrigation
period (January 1982 to October 1983), the infection rate was calculated
by summing results from each of the four irrigation seasons. Aerosol
exposure values for 1982 or 1983 were used when it was not possible
to determine the ezact irrigation season in which the infection had
occurred.
251
-------
TABLE 88. INCIDENCE DENSITY RATES OF INFECTION FOR WASTEWATER AEROSOL
EXPOSURE LEVELS BY AGENT GROUPING AND TIME INTERVAL
(Number of infections indicated in parentheses)
[When different than number of infections, number of infected
individuals indicated in brackets]
Ageat group
Interval
SNV
0-Baseline
1-Spring 1982
2-Snmmer 1982
3 -Spring 1983
4 -Summer 1983
5-1982
6-1983
7-Irrigation
1W?
0-Baseline
1 -Spring 1982
2-Suraner 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
FOR
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4 -Summer 1983
5-1982
6-1983
7-Irrigation
ADEN
0-Baseline
1-Spring 1982
2-Snmmer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
Low ezp level
(AEK1)
2.62 (41) [31]
1.40 (6)[51
0.61 (4)
1.04 (4)
0.55 (5)
1.98 (25) [22]
1.97 (13H11]
1.55 (37H34]
2.99 (25H231
1.97 (4)
1.36 (3)
2.14 U8)[17]
1.92 (2)
5.46 (24) [22]
0.81 (3)
0.19 (1)
0.20 (1)
0.48 (2)
0.45 (4)
1.61 (5)
1.14 (1)
0.75 (1)
0.00 (0)
0.00 (0)
1.18 (3)
0.00 (0)
0.57 (3)
Med exp level
(KAEK5)
2.28 (82) [57]
1.15 (13)
1.20 (14H13]
0.72 (7)
1.64 (28) [20]
2.13 (51)[[45]
2.81 (43)[27]
1.94 (97) [84]
2.65 (51) [37]
1.53 (8)
2.05 (8)[7]
2.19(35)[32]
3.36 (8)
4.68 (44) [42]
0.82 (8)
0.75 (7)
0.42 (4)
0.74 (7)
0.79 (15)
2.63 (19H17]
0.88 (2)
0.87 (2)
1.34 (3)
0.25 (1)
2.52 (12H11]
1.17 (4)
1.39 (15)[14]
High exp level
(AEI>5)
1.53 (13) [10]
0.86 (2)
2.75 (7)[5]a
0.41 (1)
1.54 (7)[4]t>
3.87 (19)[14]c
3.34 (13)[9]t>
2.42 (29)[23]b
1.32 (6)[5]
0.00 (0)
7.02 (6)[5]c
5.47 (18)[13]°
1.76 (1)
8.34 (17)[15]d
0.98 (2)
0.49 (1)
0.00 (0)
0.41 (1)
0.74 (3)
1.19 (2)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
1.06 (1)
0.38 (1)
continued. . .
252
-------
TABLE 88. (CONT'D)
Ageat growp Low exp level Hed ezp level High exp level
Interval (AEK1) (KAEK5) (AEI>5)
COXB
0-Baseline 4.08 (13)[12] 3.31 (24)[22] 2.24 (4)[3]
1-Spring 1982 1.18 (1) 2.68 (6) 2.08 (1)
2-Summer 1982 0.75 (1) 2.15 (5)[4] 7.75 (4)[3]c
3-Spring 1983 0.00 (0) 1.34 (1) 0.00 (0)
4-Summer 1983 2.76 (2) 4.59 (6) 0.00 (0)
5-1982 3.11 (8) 3.92 (19)[18] 9.79 (10)[8]"
6-1983 3.77 (2) 5.92 (7) 3.73 (1)
7-Irrigation 2.76 (9) 4.31 (29)128] 5.73 (9)[8]
ECHO
O-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
2
1
0
1
0
1
2
1
.46
.56
.51
.51
.48
.85
.42
.62
(23)
(4)
(2)
(4)
(3)
(14)
(11)
(25)
[19]
[13]
[9]
[24]
1.
0.
1.
0.
1.
1.
2.
1.
82
74
00
44
77
40
99
63
(39)
(5)
(7)
(3)
(21)
(20)
(32)
(53)
[30]
[14]
[19]
[19]
[45]
1
0
1
0
2
3
4
2
.39
.72
.97
.59
.23
.08
.10
.44
(7)[5]
(1)
(3)
(1)
(7)14]
(9)[8]
(ll)[8]b
(19) [16]
The 95% confidence interval for the high to low level incidence density
ratio does not include the value 1.
The 90% confidence interval for the high to low level incidence density
ratio does not include the value 1.
The 95% confidence intervals for both the high to low level and high
to intermediate incidence density ratios do not include the value 1.
The 95% confidence interval for the high to.intermediate incidence density
ratio does not include the value 1.
253
-------
year of 1982 (see Table 88). The majority of the infections which were
observed during this period of time were caused by echovirns 11, and coxsackie-
viruses B4 and BS. These same agents were isolated from the irrigation
wastewater at that time. Inspection of Table P-47 in Appendix P reveals
that the high exposure level participants' incidence densities of infection
by echovirus 11 and coxsackievirus B4 were significantly higher for 1982.
The unimmunized high exposure level participants had a noticeably
higher rate of infection to poliovirus 1 during spring 1982 as shown in
Table P-47. Poliovirus 1 was also isolated from the irrigation wastewater
during spring 1982 (see Table 25). Poliovirus infections can occur as
a result of exposure to a young child who has been recently immunized with
oral polio vaccine. There were two cases, one during the baseline and
the other in the high exposure level during spring 1982, where the infected
adult lived in the household with a recently immunized child. However,
since 64 of the 69 oral polio immunizations (administered to the study
participants) occurred between Hay 1981 and July 1981, it would be expected
that the poliovirus infection rate in non-immunized participants would
have been higher during the baseline period than during the spring 1982
interval. This was not the situation which was observed: the infection
incidence densities in unimmunized adults were higher in spring 1982 (see
Table P-47).
During 1983 the high exposure level participants experienced the highest
incidence density of infection by the serum neutralization viruses and
echoviruses. Using a 90% confidence interval, the risk of infection by
the serum neutralization viruses was found to be slightly greater and possibly
significant for high exposure level participants (compared to the low exposure
level) during 1983. The majority of the 1983 infections were caused by
echoviruses 3, 11, 20, and 24. The risk of infection by echoviruses 20
and 24 was found to be seven times greater for high exposure level participants
than for low exposure participants during the summer of 1983. None of those
viruses were isolated from the wastewater in 1983, but less effort was
placed on wastewater viral isolation in 1983 than in prior years.
Identified Serologic Infection Episodes
A serologic infection episode was defined as the observation of a
sufficient number of fourfold (or greater) increases in antibody titer
to an agent (or group of agents) within a given interval of time. The minimum
number of infection events required to constitute a serologic infection
episode was determined to be:
3 for agents recovered from the sprayed wastewater,
5 for agents not recovered from the sprayed wastewater.
A list of the serologic infection episodes which were observed, defined,
and submitted to statistical analysis is presented later in Tables 98 and
99. Some donors experienced more than one infection during an infection
episode. This occurred when the period of observation spanned three or
more blood collection periods (allowing detection of multiple infections
to the same agent) or when the infection episode involved a group of agents
254
-------
(allowing infections to several agents in the group). The guidelines used
to determine the value of the dependent variable for a participant for
each of the infection episodes were presented in Section 4.G.
J. OTHER INFECTIONS: MYCOBACTERIA, PARASITES AN) CORNONAVIRDS-LIIE PARTICLES
Non-tuberculosis Mycobacterial (NTM) Infections from Tuberculin Skin Testing
Mycobacteria infections were inferred from serial Mantonx tuberculin
testing of the study population. The distribution of initial induration
diameters of all tested participants is presented in Table 89. An increase
in induration diameter from less than 5 mm to 5 mm or more was considered
evidence of a new mycobacteria infection occurring in the interim. An
increase in induration diameter from less than 5 mm to between 5 and 9
mm inclusive was treated as presumptive evidence of a new non-tuberculosis
mycobacteria (NTM) infection. Indurations smaller than 5 mm in diameter
are usually of non-mycobacterial origin, often due to trauma (A. Holguin,
personal communication).
TABLE 89. PREVALENCE OF MYCOBACTERIA RESPONSE FROM
INITIAL MANTOUX TUBERCULIN SKIN TEST RESULT
Number (percent]
Size of JLnduratjon of responses
0 mm 367 (92.0)
1-4 mm 1 (0.3)
5-9 mm 8 (2.0)
110 mm 19 (4.8)
Self-reported previous reactor 4 (1.0)
TOTAL SURVEYED 399
TLe incidence of mycobacteria infections is summarized in Table 90.
The tuberculin testing detected nine new mycobacteria infections in the
study population during the study period, five of which were presumably
due to NTM. Seven of the nine new mycobacteria infections observed occurred
in the first year of the study, including four of the five presumed NTM
infections. The incidence of mycobacteria infections was higher in the
baseline period than in the irrigation period, both for the NTM and for
all mycobacteria infections. There were insufficient mycobacteria infections
after irrigation commenced to warrant statistical analysis. Only one of
the detected mycobacteria infections clearly occurred after irrigation
commenced. In a second case it is uncertain whether the onset of infection
followed irrigation; in a third case (see footnote a of Table 90) it is
uncertain whether there was a new infection. All three of these cases
were Wilson residents with intermediate aerosol exposure and no direct
wastewater contact. In summary, no evidence of association between mycobacteria
infections and wastewater sprinkler irrigation was found.
255
-------
TABLE 90. INCIDENCE OF MYCOBACTERIA INFECTIONS FROM
TUBERCULIN TESTING OF STUDY POPULATION
Presumed non-
tuberculosis
mycobacteria (NTM)
infections
All mycobacteria
infections
Infection criterion
Change in induration diameter
Nnber of new infections by
tmberomlin testing interval
<5 mm —> >5 mm
<5 mm —> 5-9 mm
DCP
012-113
113-225
225-320
012-225°
TOTAL
Months
6-80/6-81
6-81/12-82
12-82/10-83
6-80/12-82
7
0
1»
!b
9a
4
0
1
0
lafeotion rate (= no. new
infections/100 person-years
at risk)
Baseline (012-113)
Irrigation (225-320)
3.6
0.7a
2.1
0.7
a Excludes ID 40201 with an induration series 8 mm, 0 mm, 11 mm, where
the rise from 0 mm to 11 mm occurred from December 1982 to October 1983.
b New infection occurred between June 1980 and December 1982 (no tuberculin
test obtained in June 1981).
c Including ID 40201.
256
-------
Parasite Infestation
Previous studies of parasitic infections in occupational groups exposed
to wastewater have produced variable results (Clark et al., 1984; Knob loch
et al., 1983). However, one study in France found higher carriage rates
of Entarooeba histolytica and Giardia intestinalis in sewer workers as compared
to controls (Doby et al., 1980).
Stool specimens were collected from 206 participants during June,
July or August 1983 to detect acute parasitic infestation. One of two
portions of each specimen was mixed with polyvinyl alcohol and the other
with 5% formalin to preserve trophozoites and cysts, respectively, for
microscopic evaluation. The reagents were prepared and procedures for
the ova-parasite (0-P) analyses were performed by Dr. Charles Sweet, Texas
Department of Health.
Concurrently, 567 sera samples from 189 participants (3 sera from
each participant obtained during June 1980, January 1982 and June 1983)
were sent to Dr. George Healy, CDC, Atlanta, Georgia, for analysis of E. histo-
lytica antibody. An indirect hemagglutination test (IHA) was used to detect
invasive amebic disease.
The primary purpose of the 0-P analysis and serosurvey was to determine
if there was an association between contact with irrigation wastewater
and having acute infestation or invasive infection by E. histolyt ica.
The prevalence of other pathogenic protozoa and helminths was also of interest.
The results of the 0-P survey are presented in Table 91. Protozoa
were found in the fecal specimens from 21 (10.2%) of the routine specimen
donors, which was relatively high for a population survey in Texas (C. Sweet,
personal communication). Giardia Iambiia were isolated from 5 (2.4%) of
the specimens, but Entamoeba histolytica was not found.
Some clustering of protozoa within families was observed. G. lamblia
was recovered from three of four members of household 122. Entamoeba coli
was isolated from all five tested members of household 219. Two of the
positive June donors from household 122 were retested in August with identical
results.
The Giardia-positive donors had a significantly higher average aerosol
exposure (p=0.03) than the Giardia-negative donors (see Table 92). However,
all three donors with AEI>1 from whom G. lamblia was recovered were members
of the same household (i.e., 122). The drinking water well of household
122 was contaminated with indicator bacteria during the survey months (see
Table 46). While the two Giardia-positive children in this household were
reported to drink bottled water only, ingestion via water used for food
preparation or other household activities is still plausible. Since fecal
contamination of the water supply and hand-to-mouth transfer of cysts from
the feces of an infected individual are the major known modes of transmission
of giardiasis (Benenson, 1975), they appear more likely routes than wastewater
exposure. Also, in these circumstances, the members of household 122 cannot
be considered independent observations, as assumed in the t-test. Thus,
257
-------
TABLE 91. OVA AND PARASITE SURVEY OF LISS POPULATION
Number of donors of
tested fecal specimens
Positive results:
Chilomastix mesnili
Endolimaz nana
Entamoeba coli
Ent amoeba hartmanni
Giardia lamblia
lodamoeba bntschlii
Parasite infestation
prevalence
donors (%) positive
Data
312
Jun 6-10
101
12202
53201
11812
12202
21915
21916
23602
42901
45411
12211
9 (8.9%)
collection
315
Jnl 18-22
87
40211
40211
23614
45101
45312
40211
12201
12212
40214
55501
52002
9 (10.3%)
period
317
AUK 15-19
18*
12202b
12202^
21902
21913
21914
1221lt>
3a (17%)
Total
206«
1 (0.5%)
3a (1.5%)
13» (6.3%)
1 (0.5%)
5a (2.4%)
1 (0.5%)
21" (10.2%)
a Excludes positive specimens from persons with previous positive specimen.
b Retest result.
258
-------
TABLE 92. AEROSOL EXPOSURE COMPARISON OF GIARDIA-POSITIVE
AND GIARDIA-NEGATIVE FECAL DONORS IN OVA AND PARASITE SURVEY
Routine fecal donors in ova and
parasite survey
Giardia lamblia Negative for Apparent Association
recovered G.lamblia (p-value)
Number of donors
Mean AEI
Geometric mean AEI
5a
20.6
3.02
201
6.6
1.53
Yesa (0.03)b
E Three of the fecal donors were from high AEI household 122 whose drinking
water well was contaminated during the survey months.
" One-sided t test of difference in means in two independent populations; In
(AEI) transformation used to equalize variances.
the 0-P results for G. lamblia are less likely to be associated with wastewater
irrigation than with contaminated household drinking water and/or hand-to-mouth
transfer of cysts.
The prevalence of antibody to E. histolvtica in the IHA serosurvey
was only about 1% (see Table P-45 in Appendix P). Only two seroconvers ions
in adult males were determined in 189 participants tested (1.1%), which
was a rather low rate. One conversion (ID 45101) occurred between June
1980 and January 1982 before irrigation began and the other (ID 21901)
occurred between January 1982 and June 1983 after irrigation had started.
Participant 21901 did not report any direct contact with wastewater and
had an intermediate level of aerosol exposure in all three irrigation periods
between January 1982 and June 1983. Neither acute nor invasive E. histplytica
infestations were of an unusual magnitude. Thus, there was no evidence
that wastewater contact was a source of E. histolytica infection to the
participant population tested.
Electron Microscopy (EM) of Routine Fecal Specimens
HERL-Cincinnati received 370 routine fecal specimens for electron
microscopic (EM) examination. Fecal viruses were visualized by EM using
a negative staining technique.
The routine fecal specimens examined by EM were selected in a nonrandom
proportional manner at UTSA from among those provided during each fecal
collection period. Hence, they cannot be considered a representative sample
of all routine fecal specimens donated.
In marked contrast to the variety of virus-like particles detected
in illness specimens (see Table 66), coronavirus-like particles (CVLP)
were the only virus-like particles detected in routine fecal specimens.
Coronaviruses are pleomorphic, enveloped, RNA viruses which possess a fringe
of distinctive projections resembling a solar corona. In humans, coronaviruses
259
-------
have chiefly been associated with respiratory illness, although as in several
animal species they may have a role in gastroenteritis. The CVLP detected
by EM in the Lubbock stools were of a highly pleomorphic type (see Figure
28) and possessed thin, knobbed-type projections rather than the more classical
bulbous or petal-shaped projections. CVLP of the type detected here have
been observed by other investigators; however, their significance as agents
of human illness has not been firmly established (Macnaughton and Davies,
1981; Sitbon, 1985).
The occurrence of the CVLP positives observed in the routine fecal
specimens examined is presented in Table 93. The detection rate was 7%
to 8% in 1980 and 1981, 12% to 18% in 1982, and 0% to 2% in 1983. The
specimen selection problem complicates interpretation of these prevalence
rates, because the CVLP-pos it ive donors tended to be closely followed in
1982, whereas few of their specimens from 1983 were selected for EM examination
(see Table 94). Nevertheless, the data on positive donors still suggest
that the prevalence of CVLP-like infections may have increased somewhat
in 1982 and decreased somewhat in 1983.
All EM results for donors with CVLP-like detections are presented
in Table 94. The persistence of positive results in most individuals over
extended time periods is noteworthy (see IDs 21915, 21916, 40214, 40215,
45302 and 45314 for example). The clustering of infected donors within
certain households (i.e., 207, 219, 402 and 453) is also apparent.
The age-specific prevalence of the CVLP infections is presented in
Table 95. The prevalence of CVLP infections was inversely related to the
age of the specimen donor. The occurrence in all routine specimens examined
ranged from 18% in donors aged 0-5, to 8% in ages 6-17 and to 3% in adults.
Because certain donors provided a substantial number of the positive detections
(Table 94), age-specific prevalence among donors is also presented in Table
95 and the same age-related pattern was observed. The percentage of examined
donors with CVLP detected was 21% in 0-5 year olds, 11% for ages 6-17 and
3% for adults. These rates are similar to the age-specific EM-positive
prevalence rates for illness specimens (see Table 65), despite differences
in the types of particles detected.
Comparison by inspection of the donors infected with CVLP to the donors
whose routine fecal specimens were negative by EM suggests other characteristics
may be associated with the infected donors. The more strongly associated
characteristics of CVLP infected donors were a low socioeconomic status
lifestyle and residence in Wilson. Most infected donors were also hispanics.
The occurrence of CVLP infections was high throughout 1982 and highest
in the summer of 1982 (see Table 93), which were the year and season in
which the study population had the highest exposure to wastewater irriga-
tion. Table 96 compares the average aerosol exposure index (AEI) of donors
detected to be shedding CVLP in routine fecal specimens during an irrigation
period to the average AEI of donors of EM-negative routine fecal specimens
during the same period. The donors with CVLP infections had less aerosol
exposure than the EM-negative donors during the spring 1982 irrigation.
While CVLP infected donors had a somewhat higher mean AEI than the EM-negative
260
-------
a
Figure 28. Coronavirus-1ike particles observed by EM in routine stool
specimens. (a) Two particles (arrows) in the stool of 45314 (5-81).
(b) A particle from the same individual collected over a year later (8-82).
(c) A particle from 2191$ showing the highly pleomorphic nature of the
coronavirus-like particles detected in this study. Bar = 100 nm for a-c.
261
-------
TABLE 93. OCCURRENCE OF CORONAVIROS-LIKE PARTICLES IN
ROUTINE FECAL SPECIMENS EXAMINED BY ELECTRON MICROSCOPY (EM)
Specimen
collection
Quarter
1980
Jul-Sep
1981
Apr-Jun
Jul-Sep
1982
Jan-Mar
Apr-Jun
Jul-Sep
1983
Jan-Mar
Apr-Jun
Jul-Sep
TOTAL
Routine fecal
specimens examined
by EM
39
25
27
60
35
50
27
45
62
370
a Other characteristic virus-like i
Infection
Coronavirus-like
Number
3
2
2
7
5
9
0
1
0
29
prevalence
particles
Percent
8
8
7
12
14
18
0
2
0
7.8
^articles which were observed
rate
Other*
fart iclfts
0
0
0
0
0
0
0
0
0
0
by electron
microscopy of illness stools include adeno-like, astro-like, calici-like,
corona-like, NorwaIk-like, and rota-like particles.
262
-------
TABLE 94. ELECTRON MICROSCOPY RESULTS FOR ROUTINE FECAL SPECIMEN SERIES
OF DONORS POSITIVE FOR CORONAVIRDS-LIKE PARTICLES
ID
number*
20713
20714
21514
21611
21915
21916
30102
40214
40215
43414
45302
45312
45314
Fecal collection period
015 017 019 108 110
+
-
+
0
+ 0 0
0 0
0 +
000
00 +
112
0
-
0
0
0
0
114 117 118 119 201 205
0
- -
0 0
+ 0
+ 0
0 +
0 0
0
+ 00 00
0 + 0 +0
207
0
+
-
+
0
+
+
0
0
0
212
0
0
+
+
+
0
0
+
0
0
216 219
0
0
+ 0
+ 0
+ +
0
+
+
+ +
0 0
+ 0
303
0
-
0
0
0
308
0
0
-
0
+
0
312
T
0
0
0
0
0
315
0
0
0
0
0
0
317
_
0
0
0
-
(Blank) No fecal specimen obtained
0 Fecal specimen obtained, but not analyzed by EM
- Negative by EM
+ Coronavirus-like particles detected by EM
a Only donors with virus-like particles detected by EM are listed.
-------
TABLE 95. AGE-SPECIFIC PREVALENCE OF CORONAVIRUS-LIKE PARTICLES
DETECTED BY ELECTRON MICROSCOPY IN ROUTINE FECAL SPECIMENS
Donor age
on 6-30-82,
years
Occurrence in
routine fecal specimens
Examined Corona-like particles
by EM No. positive Percent
Age-specific prevalence
Corona-like
Donors infected donors
Examined Number Percent
0-5
6-17
18-44
45-64
65+
All ages
71
134
41
65
59
370
13
11
3
2
0
29
18
8
7
3
0
7.7
24
53
23
30
21
151
5
6
1
1
0
13
21
11
4
3
0
8.6
TABLE 96. AVERAGE AEROSOL EXPOSURE COMPARISON OF CORONAVIRUS-LIKE
INFECTED DONORS VERSUS NONINFECTED DONORS DURING IRRIGATION SEASONS IN 1982
Irrigation
season
Spring 1982
Summer 1982
Routine fecal
collection periods
205, 207
216, 219
Mean AEI (No.
Coronavirus-like
infected donors8
2.28 (4)
3.47 (7)
of donors examined)
Donors
negative Apparent
by EMb association
5.33 (32) No
2.25 (32) No (p=.12)c
a Particles detected in one or both routine fecal specimens from observation
period.
b All EM-examined routine specimens from donor in the period were negative.
c One-sided t test of difference in means in two independent populations;
In(AEI) transformation used to equalize variances.
264
-------
donors during the summer 1982 irrigation, the difference was not statistically
significant (see Table 96). Thus, the CVLP detections by EM provided no
evidence of association with wastewater aerosol exposure.
K. OBSERVED EPISODES OF INFECTION
Infection Incidence Rates (IR) of Infection Episodes
The infection episodes detected by the LISS are presented in Tables
97-99. Procedures for defining infection events, infection status, and
infection episodes were presented in Section 4.G. Each infection episode
was uniquely specified by the method of detecting infections, the etiologic
agent or agent group, and the period of observation relative to periods
of irrigation. Acronyms of the specified components comprised the name
of an infection episode's dependent variable (see Table 13). The value
of the infection status dependent variable for each observed participant
was the number of infection events detected in that individual during the
observation period of the infection episode. A participant was seldom
observed to experience more than one infection event to the agent (group)
during the observation period of an infection episode, except in the serologic
infection episodes to grouped agents over observation periods of 1 year
or more (see the numbers of infection events and infected donors in Tables
97-99). To permit use of sensitive statistical methods requiring that
the dependent variable only assume the values 0 or 1, all multiple infection
events were treated as single infection events in most statistical analyses
performed. Thus, a value of 0 indicated the donor was not infected during
the period of observation while 1 indicated the donor was newly infected.
The numbers of observed donors who were not infected and who were newly
infected are provided in Tables 97-99 for each infection episode. These
tables also present the infection incidence rates (IR) as percent infected
for each infection episode. IR values varied widely during LISS observation
periods, ranging from 1.0% for SE19S (echovirus 19 seroconversion rate
for 1982) up to 42.9% for CVIR8 (clinical viral isolation rate for summer
1980). Most infection incidence rates were below 10%.
Infection episodes were classified as exposure situations when the
observation period corresponded to one or two major irrigation periods
and when the causative agent was found (or could be presumed) to be present
in the wastewater at that time. The exposure infection episodes are listed
in Table 100. Infection episodes were classified as control situations
when the causative agent could not survive in wastewater (i.e., influenza
A) or when the episode preceded the start of irrigation. The control infection
episodes are given in Table 101. Each exposure and control infection episode
listed in Tables 100 and 101 was statistically analyzed for association
with wastewater aerosol exposure (see Section 5.L).
The infection incidence rates of both the low (AEK3) and high (AEI>3)
exposure groups and of all three exposure levels [low (AEK1), intermediate
(liAEIiS) and high (AEI>5)]are also presented in Tables 100 and 101 for
each infection episode. The risk ratio (RR) for exposure groups is the
ratio of the infection rate in the high exposure group divided by the rate
in the low exposure group. RR=IRg£/IR^o values are presented in Tables
265
-------
TABLE 97. CLINICAL INFECTION EPISODES
to
ON
O\
Clinical (C) aiemt croup Recovered
Onset of
Dependent Number of
Irrigation Period of from sprayed infection variable infection
period
11
ebaii
2
4
code observation wastewater?
.iir, rtn
Summer 19828
Summer 1983 i
Other Qpportuaiatic Bacteria.
3
Spring 1983h
Yes
Yesc
OOB
No
events
Xa
Wb
X
W
X
name
CKLB2X
CKLB2W
CKLB4X
CKLB4W
COOB3
events
5
13
8
12
5
Fecal
donors
infected
5
13
8
12
5
Donors Infection
not incidence
infected
75
75
81
81
102
rate.
6.
14.
9.
12.
4.
%
3
8
0
9
7
Preaiae&t Bacteria in Vastcvater. PBY
1
2
4
Spring 1982f
Summer 1982
Summer 1983
Yes
Yes
Yes°
All Yimaea (excluding adeno a«d imma
1
2
4
Sum 80 BLd
Sum 81 BLe
Spring 1982
Summer 1982
Summer 1983
-
-
Some
Some
Some
W
W
X
W
niiation po
-
-
X
W
X
W
W
CPBW1W
CPBW2X
CPBW2W
CPBW4W
lio). VHt
CVIR8
CVIR9
CVIR1X
CVIR1W
CVIR2X
CVIR2W
CVIR4W
3
3
4
9
12
11
9
15
11
14
5
3
3
4
9
12
9
9
15
11
12
5
110
85
85
85
16
20
105
105
94
94
92
2.
3.
4.
9.
42.
31.
7.
12.
10.
11.
5.
7
4
5
6
9
0
9
5
5
3
2
All Waatewater Isolate*. Ill
a
b
c
d
1
2
3
4
X -
W -
by
Sum
Spring 1982
Summer 1982
Spring 1983
Summer 1983
Yes
Yes
Yes
Yesc
onset of all infection events
X
W
X
W
X
W
W
CWWI1X
CWWI1W
CWWI2X
CWWI2W
CWWI3
CWWI4X
CWWI4W
during irrigation period
includes infection events whose onset may
the irrigation period
have preceded
inference from available wastewater data
80 BL-Baseline: 7-21/9-17-80
7
13
12
22
4
8
22
e Sum 81
f Spring
g Summer
h Spring
i Summer
7
12
12
20
4
8
22
98
98
66
66
100
73
73
6.
10.
15.
23.
3.
9.
23.
7
9
4
3
8
9
2
BL-Baseline: 6-1/9-2-81
1982:
1982:
1983:
1983:
1-4/4-2-82
6-7/9-17-82
1-31/4-22-83
6-6/8-19-83
-------
TABLE 98. SEROLOGIC INFECTION EPISODES TO SINGLE AGENTS
to
Serolocie (S)
Irrigation
period code
Adeao 3. ADS
0
5
Ademo 5, AD5
0
5
Ademo 7. AD7
0
Cox««ckie B2.
0
5
Co»»ackie B4.
0
2
5
Coztackie B5.
0
1
2
5
4
6
Echo 1. E01
0
Echo 3. B03
0
5
4
6
Echo 9. E09
0
Echo 11. Ell
0
1
2
5
4
6
••eat
Period of
observation
Baseline8
1982*
Baseline
1982
Baseline
CB2
Baseline
1982
CB4
Baseline
Summer 1982C
1982
CBS
Baseline
Spring 1982b
Summer 1982
1982
Summer 1983 f
19838
Baseline
Baseline
1982
Summer 1983
1983
Baseline
Baseline
Spring 1982
Summer 1982
1982
Summer 1983
1983
Recovered
from sprayed
wastewater?
—
-
-
-
-
-
Tes
-
Yes
Yes
-
Yes
Yes
Yes
Yes
Yes
-
-
No
No
No
-
-
Yes
Yes
Yes
No
No
Dependent
variable
name
SAD30
SAD35
SAD50
SADS5
SAD70
SCB20
SCB25
SCB40
SCB42
SCB45
SCB50
SCB51
SCB52
SCB55
SCB54
SCB56
SE010
SE030
SE035
SE034
SE036
SE090
SE110
SE111
SE112
SE115
SE114
SE116
Number of
infection
events
13
7
7
8
6
14
9
16
5
20
11
4
4
8
8
9
7
13
9
11
18
9
17
4
7
19
6
10
Blood
donors
infected
13
7
7
8
6
14
9
16
5
19
11
4
4
8
8
9
7
12
9
11
18
8
17
4
7
19
6
10
Donors
not
infected
242
297
239
285
297
230
284
227
284
281
276
305
304
288
248
247
285
247
288
241
239
268
271
298
296
283
249
249
Infection
incidence
rate. %
5.1
2.3
2.8
2.7
2.0
5.7
3.1
6.6
1.7
6.3
3.8
1.3
1.3
2.7
3.1
3.5
2.4
4.6
3.0
4.4
7.0
2.9
5.9
1.3
2.3
6.3
2.4
3.9
continued..
-------
TABLE 98. (CONT'D)
Sero Ionic (S) agent
Irrigation Period of
period code observation
Echo 19.
5
Echo 20.
0
4
6
Echo 24.
0
5
4
6
Polio 1.
0
10
S i
Polio 2.
0
1
B19
1982
E20
Baseline
Summer 1983
1983
E24
Baseline
1982
Summer 1983
1983
PL1
Baseline
Spring 1982
PL2
Baseline
Spring 1982
Recovered
from sprayed
wastewater?
Yes
-
No
No
-
Yes
No
No
-
Adults Salt
Adults not
Children Sab in
Children not
Yes
Polio
Not
-
Adults Salk
Adults not
Children Sab in
Children not
Yes
Polio
Not
Dependent Number of Blood Donors Infection
variable infection donors not incidence
name events infected infected rate, %
SE195
SE200
SE204
SE206
SE240
SE245
SE244
SE246
SPL10
immunized:
immunized:
immunized:
immunized:
SPL11
immunized:
immunized:
SPL20
immunized:
immunized:
immunized:
immunized:
SPL21
immunized:
immunized:
3
5
6
9
9
7
7
12
70h
50
2
17
1
13*
8
5
73h
52
0
19
2
9h
7
2
3
5
6
9
8
7
7
10
70
50
2i
17
I1
13
8
5
73
52
0*
19
21
9
7
2A
291
265
241
241
261
287
244
242
175
18
97
22
38
234
53
181
169
16
98
19
36
235
54
181
1.0
1.9
2.4
3.6
3.0
2.4
2.8
4.0
28.6
73.5
2.0
43.6
2.6
5.3
13.1
2.7
30.2
76.4
0
50.0
5.3
3.7
11.5
1.1
continued. . .
-------
TABLE 98. (CONT'D)
to
9\
VO
Serolocio
(S) atemt
Irrigation Period of
period code observation
Polio 3.
0
1
Rcovirvs
0
1
ReoviruB
0
1
PL3
Baseline
Spring 1982
1, RBI
Baseline
Spring 1982
2. RB2
Baseline
Spring 1982
Recovered Dependent
from sprayed variable
wastewater? name
SPL30
Adults Salk immunized
Adults not immunized
Children Sabin immunized
Children not immunized
Yes SPL31
Polio immunized
Not immunized
SRE10
SRE11
SRE20
SRE21
Number of Blood Donors Infection
infection donors
events
72h
: 57
: 0
: 15
: 0
7h
: 7
: 0
35
16
37
13
not incidence
infected infected
72
57
Oi
15
Oi
7
7
Oi
35
16
37
13
169
11
98
22
38
236
54
182
246
297
241
297
rate, %
29,9
83.8
0
40.5
0
2.9
11.5
0
12.5
5.1
13.3
4.2
Rot*viru». ROT
0
1
2
5
3
4
6
Lecionell
Influenza
0
1
3
Baseline
Spring 1982
Summer 1982
1982
Spring 1983e
Summer 1983
1983
«. LEG
6-81/6-83
A. INA
6-80/6-81
6-81/6-82
6-82/6-83
a Baseline: 6-80/1-82
b Spring
c Summer
d 1982:
1982: Jan-Jun
1982: Jun-Dec
Jan-Dec 1982
SROTO
SROT1
SROT2
SROT5
SROT3
SROT4
SROT6
No SLEG7
SINAO
SINAI
SINA3
e Spring
1982 f Summer
1982 g 1983:
13
3
4
7
3
6
9
6
19
6
35
1983: Dec
11
3
4
7
3
6
9
6
19
6
35
1982-Oct 1983
19
45
50
45
45
39
35
207
167
229
219
36.7
6.3
7.4
13.5
6.3
13.3
20.5
2.8
10.2
2.6
13.8
1983: Jnn-Oct 1983
Dec 1982-Oct 1983
h Includes polio immunization seroconversion
i Not an
infection
episode (too few
infected
donors)
-------
TABLE 99. SEROLOGIC INFECTION EPISODES TO GROUPS OF AGENTS
10
Seroloitio (S) agent Oromi
Irrigation
period Period of
code observation
Sporadic Sera Neml
0
1
2
5
6
All
1
2
5
6
All
tralizi
»
Specific aeents included
Adeno
ttion T«
Cox B
Echo
>ated Viruses.
Baseline
Spring
Summer
1982
1983
1982
1982
3.5.7
3.5.7
7
3.5,7
2.4
2
FOE
5,17.19
1.3.5.
19.20,
1.3.5.
19.20.
1.5.9.
1.5.9.
9.17.
24
9.17.
24
17,20
17,19
Dependent
variable
name
SPORO
SPOR1
SPOR2
SPOR5
SPOR6
Number of
infection
events
8
13
9
5
10
Blood
donors
infected
8
13
9
5
10
Donors Infection
not incidence
infected8 rate. %
207
175
199
232
218
3.7
6.9
4.3
2.1
4.4
Viruses in Sprayed Wastewater. WWV
Spring
Summer
1982
1983
1982
1982
Serai Neutralization
0 Baseline
1
2
5
3
4
6
Spring
Summer
1982
Spring
Summer
1983
1982
1982
1983
1983
Teated
3.5.7
3.5.7
3.5,7
3.5.7
3.5.7
3.5.7
3.5.7
5
2,4.5
2.4.5
5
1,5.11
19,20
11.24
1.5.11
19.20.
19
.17.
.17.
24
SWWV1
SWWV2
SWWV5
SWWV6
12
16
70
11
12
15
62
11
210
235
173
235
5.4
6.0
26.4
4.5
Viruses. SNV
2.4,5
2.4,5
2.4.5
2.4,5
5
5
5
1,3,5.
17.19.
1.3.5.
17.19.
1.3,5,
17.19.
1.3.5.
17.19.
1.3.5.
17.19,
1.3.5,
17,19.
1.3.5.
17.19.
9.11,
20.24
9.11.
20,24
9,11,
20.24
9.11.
20,24
9,11,
20,24
9,11.
20.24
9,11.
20.24
SSNVO
SSNV1
SSNV2
SSNV5
SSNV3
SSNV4
SSNV6
136
21
24
94
12
40
69
98
20
22
81
12
29
47
110
163
168
144
200
180
174
47.1
10.9
11.6
36.0
5.7
13.9
21.3
a Donors without seroconversions excluded unless their seroconversion status to all specific agents
listed was observed.
-------
TABLE 100. INFECTION INCIDENCE RATES8 BY EXPOSURE GROUPS AND LEVELS AND RISK RATIO SCORE
OF INFECTION EPISODES CLASSIFIED AS EXPOSURE SITUATIONS
to
-j
Infection incidence rates (IR) and
and risk ratios (RR=IRm/IRi „) , %
J
"Exposure'' infection episode ind
Period of Dependent e
Aeent observation variable R
ointly
ependen
pisode
TOUPD
bv two AEI groups
t Infection
llIP !**>•»*»
NO?
"-*--
Low
«3)
IR
High
(>l)
IR
AEI
Group
RR
bv
Low
«D
IR
three AEI levels
Inter-
mediate
IR
High AEI
(>5) Level
IR RR
Risk
ratio
scorec
Clinical (C)
KLB
OOB
PBW
VIR
WI
(Klebsiella)
2 (Sum 82) CKLJm
l&um oz; CKLB2W
A i c,,— aa \ CKLB4X
4 (Sum 83) CKLB4W
(Other opportunistic bacteria)
3 (Spr 83) COOB3
A
A
A
5
13
8
12
5
6.3
14.8
9.0
12.9
4.7
5.1
13.8
4.6
7.5
3.3
9.5
17.4
20.8
26.9
6.4
1.9
1.3
4.5
3.6
1.9
5.0
13.6
4.0
7.7
3.8
9.3
20.4
6.5
10.4
4.8
0 0
0 0
22.2 5.6
26.3 3.4
5.3 1.4
0
0
++
++
0
(Prominent bacteria in wastewater)
1 (Spr 82) CPBW1W
J(S»82) »J«|
4 (Sum 83) CPBW4W
(Viruses, excluding adeno and
1
-------
TABLE 100. (CONT'D)
Infection incidence rates (IR) and
and risk ratios (RR=IRm/IRi rt) , %
' 'Exposure ' ' infect
Period of
Agent observation
WWI (Cont'd)
3 (Spr 83)
4 (Sum 83)
Serolofic (S)
AD3 (Adeno 3)
5 (1982)
ADS (Adeno 5)
5 (1982)
CB2 (Coxsackie B2)
N> 5 (1982)
** CB4 (Coxsackie B4)
2 (Sum 82)
5 (1982)
CBS (Coxsackie B5)
1 (Spr 82)
2 (Sum 82)
5 (1982)
4 (Sum 83)
6 (1983)
E03 (Echo 3)
5 (1982)
4 (Sum 83)
6 (1983)
ion episode
Dependent
variable
CWWI3
CWWI4X
CWWI4W
SAD35
SAD55
SCB25
SCB42
SCB45
SCB51
SCB52
SCB55
SCB54
SCB56
SE035
SE034
SE036
Jointly
independent Infection
episode incidence8
gronp^ No. %
D
D
B
B
B
A
B
A
A
B
A
B
B
A
B
4
8
22
7
8
9
5
18
4
4
8
8
9
9
11
18
3.8
9.9
23.2
2.3
2.7
3.1
1.7
6.1
1.3
1.3
2.7
3.1
3.5
3.0
4.4
7.0
bv two AEI groups
Low
IR
3.4
5.0
17.4
2.7
3.6
1.8
1.3
5.4
1.0
0.8
2.3
4.1
4.3
3.6
4.1
5.9
High
IR
4.4
23.8
38.5
1.4
0
6.9
3.0
8.1
1.8
2.8
4.2
0
1.4
1.4
5.2
9.9
AEI
Group
RR
1.3
4.8
2.2
0.5
0
3.7
2.3
1.5
1.8
3.3
1.8
0
0.3
0.4
1.3
1.7
bv
Low
IR
3.8
4.3
15.3
1.1
2.2
0
1.1
8.0
1.3
0
1.1
2.6
2.9
4.4
1.4
4.3
three AEI levels
Inter-
mediate
IR
3.3
7.1
22.0
3.5
3.7
4.2
1.2
4.1
1.6
1.2
2.5
4.2
4.0
2.4
5.7
7.4
High
IR
5.6
25.0
36.8
0
0
5.6
5.9
11.1
0
5.1
8.1
0
2.6
2.8
5.4
10.3
AEI
Level
RR
1.4
5.8
2.4
0
0
Large
5.2
1.4
0
Large
7.5
0
0.9
0.6
4.0
2,4
Risk
ratio
score0
0
++
0
0
+
+
0
0
+
0
0
0
-
0
continued.
-------
TABLE 100. (CONT'D)
10
-1
to
Infection incidence rates (IR) and
and risk ratios (RR=IRnj/IRi „) , %
Jo
''Exposure'' infection episode inde
Period of Dependent ep
Agent observation variable nr
Ell
El 9
E20
£24
PL1
(Echo 11)
1 (Spr 82)
2 (Sum 82)
5 (1982)
4 (Sum 83)
6 (1983)
(Echo 19)
5 (1982)
(Echo 20)
4 (Sum 83)
6 (1983)
(Echo 24)
5 (1982)
4 (Sum 83)
6 (1983)
(Polio 1)
1 (Spr 82)
61
SE111
SE112
SE115
SE114
SE116
SE195
SE204
SE206
SE245
SE244
SE246
SPL11
polio immunized:
186 not immunized:
PL2
PL3
(Polio 2)
1 (Spr 82)
61
(Polio 3)
1 (Spr 82)
61
SPL21
polio immunized:
SPL31
polio immunized:
intly
pendent Infection
isode incidence*
oupb No. %
A
A
B
A
B
B
A
B
B
A
B
A
A
A
A
4
7
19
6
10
3
6
9
7
7
10
13
8
5
9
7
7
7
1.3
2.3
6.3
2.4
3.9
1.0
2.4
3.6
2.4
2.8
4.0
5.3
13.1
2.7
3.7
11.5
2.9
11.5
bv two AEI groups
Low
IR
1.0
2.1
4.8
1.5
2.7
0.5
2.1
3.9
2.7
1.6
2.2
2.0
3.6
1.6
1.3
3.6
2.0
10.7
High
IR
1.9
2.9
10.8
5.1
6.9
2.7
3.6
2.8
1.4
6.9
8.6
10.4
21.2
4.8
7.4
18.2
4.2
12.1
AEI
Group
RR
1.9
1.4
2.2
3.3
2.6
6.0
1.7
0.7
0.5
4.4
3.9
5.2
5.9
2.9
5.7
5.1
1.4
1.1
bv
Low
IR
2.5
2.1
5.3
1.4
4.3
0
0
3.0
4.7
0
1.5
0
0
0
3.8
10
1.9
10
three AEI levels
Inter-
mediate
IR
1.1
1.7
4.7
2.8
3.3
1.2
2.9
3.5
1.2
3.5
4.0
4.5
14
1.7
2.0
8
3.3
14
High
IR
0
5.9
16.2
2.6
5.0
2.9
5.3
5.1
2.8
5.4
7.9
15.8
20
13.0
10.5
20
2.6
7
AEI
Level
RR
0
2.9
3.0
1.9
1.2
Large
1.8
1.7
0.6
Large
5.1
Large
Large
Large
2.8
2.0
1.4
0.7
Risk
ratio
score0
0
0
+
0
+
+
0
0
-
+
+
++
+
+
++
+
0
0
continued.
-------
TABLE 100. (CONT'D)
to
-j
Infection incidence rates (IR) and
and risk ratios (RR=IRn;/IRi „) , %
Jointly
''Exposure'' infection episode independent
Period of
Agent observation
RE1 (Reo 1)
1 (Spr 82)
RE2 (Reo 2)
1 (Spr 82)
ROT (Rotavirus)
1 (Spr 82)
2 (Sum 82)
5 (1982)
3 (Spr 83)
4 (Sum 83)
6 (1983)
Dependent episode
variable group"
SRE11
SRE21
SROT1
SROT2
SROT5
SROT3
SROT4
SROT6
LEG (Legionella pneumophila
1981-83
FOR (Sporadic serum
1 (Spr 82)
2 (Sum 82)
5 (1982)
6 (1983)
SLEG7
A
A
A
A
B
A
A
B
1)
B
bv two AEI groups
Infection
incidencea
No. %
16
13
3
4
7
3
6
9
6
5.1
4.2
6.3
7.4
13.5
6.3
13.3
20.5
2.8
Low
«3)
IR
5.4
5.0
4.2
2.8
9.4
4.8
12.5
20.0
2.7
High
(13)
IR
4.5
2.7
8.3
16.7
20.0
7.4
14.3
20.8
3.1
AEI
Group
RR
0.8
0.5
2.0
6.0
2.1
1.6
1.1
1.0
1.1
bv
Low
«1)
IR
3.7
3.8
0
10
10
0
0
25
0
three AEI levels
Inter-
mediate
IR
6.3
5.3
7
0
11
6
12
17
4.1
High
OS)
IR
2.5
0
8
21
21
7
19
25
3.0
AEI
Level
RR
0.7
0
Large
2.1
2.1
Large
Large
1.0
0.7
Risk
ratio
score0
0
0
0
+
+
0
0
0
0
neutralization viruses)
SPOR1
SPOR2
SPOR5
SPOR6
WWV (Viruses isolated from
1 (Spr 82)
2 (Sum 82)
5 (1982)
6 (1983)
SWWV1
SWWV2
SWWV5
SWV6
A
A
B
B
wastewater)
D
D
E
E
13
9
5
10
12
15
61
11
6.9
4.3
2.1
4.4
5.4
6.0
26.1
4.5
7.3
4.9
2.7
4.9
5.5
5.2
22.3
5.1
6.3
2.3
0
3.0
5.3
8.8
38.2
2,9
0.9
0.5
0
0.6
1.0
1.7
1.7
0.6
6.5
1.5
1.4
3.3
6.8
3.8
23.6
3.1
6.9
6.0
3.0
5.3
6.0
4.9
23.5
5.5
7.7
3.8
0
2.8
0
17.9
43.3
2.8
1.2
2.5
0
0.8
0
4.8
1.8
0.9
0
0
0
0
0
+
+
o
continued.
-------
TABLE 100. (CONT'D)
Infection incidence rates (IR) and
and risk ratios (RR=IRn;/IRi ,J . %
Jointly
''Exposure'' infection episode independei
Period of Dependent episode
Agent observation variable aroup"
SNV
a
b
(All serum neutralization viruses)
1 (Spr 82) SSNV1
2 (Sum 82) SSNV2
5 (1982) SSNV5
3 (Spr 83) SSNV3 D
4 (Sum 83) SSNV4 D
6 (1983} SSNV6
Based on all observed individuals for
Classification criteria for the joint:
by two AEI croups
it Infection Low
incidence8 «3)
No. % IR
20
22
81
12
29
47
whom an
Ly indept
10.9
11.6
36.0
5.7
13.9
21 f 3
AEI
inden
9.8
11.3
33.9
7.3
14.4
21 T0
High
(13)
IR
13.1
12.5
42.6
2.7
12.2
21.9
AEI
Group
RR
1.3
1.1
1.3
0.4
0.9
1.0
exposure estimate was
t groups of exposure
bv
Low
«D
IR
10.9
6.3
31.0
7.3
8.3
18.0
three AEI levels
Inter-
mediate
IR
11.4
12.5
36.3
5.5
16.8
21.4
High
IR
8.7
21.7
46.7
3.3
13.3
26.5
available.
infection episodes
AEI
Level
RR
0.8
3.4
1.5
0.5
1.6
1.5
were giv<
Risk
ratio
score0
0
0
0
0
0
an in
Table 15.
c Risk ratio score criteria were given in Table 17.
-------
TABLE 101. INFECTION INCIDENCE RATES8 BY EXPOSURE GROUPS AND LEVELS AND RISK RATIO SCORE
OF INFECTION EPISODES CLASSIFIED AS CONTROL SITUATIONS
Infection incidence rates (IR) and
and risk ratios (RR=IRn;/IRi „) , %
"Control'' infection episode
Period of
Agent observation
Clinical (C)
Dependent
variable
VIR (Viruses, excluding adeno
8 (Sum 80)
9 (Sum 81)
Strolofic (S)
ADS (Adeno 3)
Baseline
ADS (Adeno 5)
Baseline
K> AD7 (Adeno 7)
3\ Baseline
CB2 (Cozsackie E2)
Baseline
CB4 (Coxsackie B4)
Baseline
CBS (Coxsackie B5)
Baseline
E01 (Echo 1)
Baseline
E03 (Echo 3)
Baseline
E09 (Echo 9)
Baseline
Ell (Echo 11)
Baseline
CVIR8
CVIR9
SAD30
SAD50
SAD70
SCB20
SCB40
SCB50
SE010
SE030
SE090
SE110
Jointly
independent Infection
episode incidence8
group^ No. %
by two AEI group s
Low
«3)
IR
High
(>3)
IR
AEI
Group
RR
bv
Low
«1)
IR
three AEI levels
Inter-
mediate
IR
High AEI
(>5) Level
IR RR
Risk
ratio
score*5
and immunization polio)
C
C
C
C
C
C
C
C
C
C
C
C
12
9
13
7
6
14
16
11
7
12
8
17
42.9
31.0
5.1
2.8
2.0
5.7
6.6
3.9
2.4
4.6
3.0
6.0
35.7
34.8
6.1
2.5
1.0
6.4
6.4
2.7
2.1
5.5
1.1
5.3
62.5
16.7
3.3
3.4
3.8
4.5
6.9
6.4
3.1
3.2
6.4
7.4
1.8
0.5
0.5
1.4
3.8
0.7
1.1
2.4
1.5
0.6
5.6
1.4
43
60
3.3
3.2
1.2
11.9
8.5
1.3
1.3
11.9
2.8
9.1
54
18
6.8
3.4
1.6
3.3
7.4
4.8
3.5
2.4
2.5
4.6
(0)
(0)
0 0
0 0
5.1 4.2
5.7 0.5
0 0
5.3 4.2
0 0
2.8 0.2
5.4 1.9
5.7 0.6
0
—
0
0
++
0
0
+
0
—
+
0
continued.
-------
TABLE 101. (CONT'D)
Infection incidence rates (IR) and
and risk ratios (RR=IRm/IRi „) , %
J
''Control" infection episode ind
Period of Dependent e
Agent observation variable ft
£20
£24
PL1
PL2
PL3
RE1
RE2
ROT
INA
(Echo 20)
Baseline SE200
(Echo 24)
Baseline SE240
(Polio 1)
Baseline SPL10
67 Salk immunized adults:
34 Sabin immunized children:
(Polio 2)
Baseline SPL20
67 Salk immunized adults:
33 Sabin immunized children:
(Polio 3)
Baseline SPL30
67 Salk immunized adults:
32 Sabin immunized children:
(Reo 1)
Baseline SRE10
(Reo 2)
Baseline SRE20
(Rotavirus)
Baseline SROTO
(Influenza A)
0 (1980-81) SINAO
1 (1981-82) SINAI
3 (1982-83) SINA3
ointly
ependent Infection
pisode incidence*
roupb No. %
C
C
C
C
C
C
C
C
C
C
C
C
C
C
5
8
69
49
17
72
51
19
71
56
15
35
37
11
19
6
35
1.9
3^0
28.4
73.1
43.6
30,0
76,1
50.0
29.7
83.6
40.5
12.5
13.4
36.7
10.2
2.6
13.8
bv two AEI groups
Low
IR
2.3
2.9
28.0
72
46
28.4
78
48
26.5
81
38
15.6
14.4
30.8
10.6
2.5
15.2
High
IR
1.0
3.1
29.0
74
36
32.6
74
55
34.8
87
45
6.3
11.5
41.2
9.3
2.8
11.1
AEI
Group
RR
0.4
1.0
1.0
1,0
0.8
1,1
1,0
1.1
1.3
1.1
1.2
0.4
0.8
1.3
0.9
1.1
0.7
bv
Low
IR
1.5
3.1
22.6
62
33
26.9
62
67
25.5
77
38
22.7
12.7
—
5.8
1.6
14.3
three AEI levels
Inter-
mediate
IR
2.4
3.0
28.1
79
44
29.8
87
42
25.8
82
33
8.9
12.4
33
11.1
3.7
14.4
High
IR
0
2.8
37.8
69
60
35.1
63
60
51.4
94
80
8.1
19.4
44
15.4
0
10.5
AEI
Level
RR
0
0.9
1.7
1,1
1.8
1,3
1.0
0.9
2.0
1.2
2.1
0.4
1.5
Large
2.7
0
0.7
Risk
ratio
score6
0
0
0
0
0
0
0
0
0
0
0
—
0
+
0
0
0
continued.
-------
TABLE 101 (CONT'D)
Jointly
''Control'' infection episode independent Infection Low
Period of Dependent episode
Infection incidence rates (IR) and
and risk ratios (RR=IRHi/IRLo). %
bv two AEI groups by three AEI levels
AEI
High AEI Low Inter- High AEI Risk
incidence* «3) (>3) Gronp «1) mediate (>5) Level ratio
Agent observation variable
group''
No.
IR
IR
RR
IR
IR
IR
RR
SNV (All serum neutralization viruses)
Baseline SSNVO F
score*
FOR (Sporadic serum neutralization viruses)
Baseline SPORO C 8 3.7 3.8 3.7 1.0 4.4 3.6
3.1 0.7 0
98 47.1 44.9 51.4 1.1 57.4 44.9 37.0 0.6 0
a Based on all observed individuals for whom an AEI exposure estimate was available.
b Classification criteria for the jointly independent groups of control infection episodes were given in
Table 15.
c Risk ratio score criteria were given in Table 17.
to
-4
oo
-------
100 and 101 both for exposure groups and for exposure levels. The risk
ratios vary widely, as expected for the low incidence of infections. About
half of the group and level risk ratios for the control infection episodes
in Table 101 exceed 1.0, as expected. However, a large majority (about
2/3) of both the group and level risk ratios for the exposure infection
episodes in Table 100 exceed 1.0. Since this suggests a potential correlation
of infections with wastewater aerosol exposure in exposure infection episodes,
this phenomenon is investigated more carefully below.
Evaluation of Association of Infections with Aerosol Exposure via Risk
Ratio Scores
A risk ratio score was assigned to each infection episode observed
in the LISS as described in Section 4J. The risk ratio score criteria
were symmetric with regard to the high and low exposure groups and levels
(i.e., an infection pattern that would be scored + if the excess infections
occurred in the high exposure group and level, would be scored - if the
equivalent excess infections occurred in the low group and level). Thus,
in the absence of any effect, random variation should produce an equal
number of positive and negative risk ratio scores.
The assigned risk ratio scores are presented in Tables 100 and 101.
A preponderance of positive (+ or ++) scores over negative (- and - -)
scores is seen for the exposure situations (see Table 100), but not for
the control situations (see Table 101).
The distribution of risk ratio scores obtained for each group of inde-
pendent infection episodes was analyzed to provide a sensitive overview
of any apparent association of infection events with wastewater aerosol
exposure. The criteria for six mutually exclusive and jointly independent
groups of episodes were presented in Table 15. The infection episodes
placed in each of these groups are shown in Tables 100 and 101. The frequency
with which each risk ratio score occurred was determined for all six groups
of independent episodes. The frequency distributions are presented in
Table 102. If aerosol exposure had no effect on infections, one would
expect random variations to produce a symmetric distribution of risk ratio
scores about 0, with approximately equal numbers of positive and negative
scores and of ++ and - - scores. Symmetry would be expected because of
the symmetric treatment of ''high'' and ''low'' exposure groups and levels
in the risk ratio criteria. A one-sided sign test of the number of positive
scores (++ or +) compared to the number of negative scores (- - or -) was
conducted for each jointly independent group (see lower portion of Table
102), to determine if there was a significant excess of positive risk ratio
scores for the infection episodes in the group.
Let us first consider the findings in Table 102 from the risk ratio
scores for infection episodes to single or sporadic agents (Groups A, B
and C) . The frequency distribution of risk ratio scores for the control
infection episodes (Group C) were symmetric about 0, in accord with our
expectation for this group. However, among the exposure infection episodes
occurring in single seasons (Group A), there were nine episodes with positive
risk ratio scores, but none with negative scores. This excess of positive
279
-------
TABLE 102. SIGNIFICANCE OF FREQUENCY DISTRIBUTIONS OF RISK RATIO
SCORES BY GROUP* OF JOINTLY INDEPENDENT INFECTION EPISODES
FREQUENCY DISTRIBUTIONS OF RISK RATIO SCORES
TOTAL
Single and sporadic agent
infection episodes
Grouped agent
infection episodes
Exposure
situations
Risk ratio
score
—
-
0
+
++
Group A
(single
seasons)
0
0
22
7
2
Group B
(years)
0
2
10
7
0
Control
situations
Group C
(baseline*
inf luenza)
1
2
20
3
1
Exposure
situations
Group D
(single
seasons)
0
1
5
1
1
Group E
(years)
0
0
1
1
0
Control
situations
Group F
(baseline)
0
0
1
0
0
31
19
27
8
SIGNIFICANT EXCESS OF + (OR ++) RISK RATIO SCORES IN FREQUENCY DISTRIBUTION11
Group A Group B Group C Group D Group E Group F
Total negative 02 3100
scores
(- or —)
Total postive 97 4210
scores
(+ or ++)
Significant
excess of posi-
tive scores?
(p-value)p
Yes
(0.002)
Maybe
(0.09)
No
No
No
No
See Tables 100 and 101 for episode assignment to jointly independent
groups. See Table 15 for group classification criteria.
One-sided sign test of total positive scores vs. total negative scores.
280
-------
scores was highly significant (p=0.002). Among exposure episodes of 1-year
duration (Group B), there were seven positive RR scores versus two negative
scores. The excess of positive scores in Group B approaches significance
(p=0.09), considering the smaller number of infection episodes in Group
B. The RR score results for single and sporadic agent episodes of infection
suggest that an excess risk of infection was associated with wastewater
aerosol exposure.
The observation periods in which the Group A exposure infection episodes
with positive RR scores occurred was (see Table 100):
Spring 1982 - 3 episodes (PL1 immunized, PL1 not immunized, PL2 immunized)
Summer 1982 - 4 episodes (VIR, CB4, CBS, ROT)
Spring 1983 - 0 episodes
Summer 1983 - 2 episodes (KLB, E24)
This seasonable distribution is consistent with the hypothesis of association
of viral infections with wastewater aerosol exposure. The relative aerosol
exposure measure to enteroviruses and indicator organisms from the wastewater
spray irrigation was greater in the 1982 irrigation periods, especially
summer 1982, and lowest in the spring 1983 period (compare RAEM for entero-
viruses by irrigation period in Table 42). (Since poliovirus seroconversions
were investigated only for the spring 1982 irrigation period, it was not
possible to observe additional polio infection episodes in later seasons.)
Thus, the seasonal distribution of Group A episodes with positive RR scores
is correlated with seasonal microorganism (especially enteroviruses) aerosol
exposure from wastewater spray irrigation, suggesting a dose-response rela-
tionship.
The excess Group B exposure infection episodes with positive RR scores
occurred both in 1983 (three excess positive episodes) and in 1982 (two
excess positive episodes). The relative aerosol exposure measure data
in Table 42 suggests greater aerosol exposure to enteroviruses and indicator
organisms from spray irrigation in 1982 rather than in 1983. The excess
Group B episodes with positive RR scores lack both statistical evidence
of excess positive episodes and the dose-response pattern anticipated for
wastewater irrigation effects.
There were fewer independent infection episodes to groups of agents.
Consequently, there were insufficient negative and positive RR scores by
which to detect a significant excess of positive scores using the.sign
test. The only control infection episode (Group F) had no distinct exposure
pattern of infection incidence rates (RR score=0) . The independent single
season exposure episodes to grouped agents (Group D) had a fairly symmetric
distribution of RR scores about 0, with one excess positive score. The
positive score episodes in Group D occurred in summer 1982 and summer 1983,
while the negative score episode occurred in spring 1983. One of the two
Group E exposure infection episodes had a positive risk ratio score; SWWV5
occurred in 1982. The results from the RR scores of independent grouped-agent
infection episodes (Groups D-F) are consistent with the findings for the
single agents (Groups A-C). Accumulation of the single agent episodes
with RR scores of 0 in the grouped agent episodes and the smaller number
281
-------
of grouped agent episodes may have reduced the sensitivity of the distribution
of the RR score method to detect wastewater irrigation effects in the grouped-
agent episodes.
L. STATISTICAL ANALYSIS
The standard statistical analyses of infection episodes were performed
in three major stages:
1) Preliminary Analysis—comparison of the low exposure group (AEK3)
and the high exposure group (AEI>.3) with respect to individual
and household characteristics in order to determine if the two
exposure groups differed significantly with regard to these factors
2) Confirmatory Analysis—comparison of infection rates in exposure
groups to determine the presence of any association of infection
and wastewater aerosol exposure
3) Exploratory Analysis — investigation of whether the presence of
infection was associated with a set of potential predictor variables,
and in particular with the degree of aerosol exposure.
Preliminary Analysis
Prior to conducting tests for association of infection rates and exposure,
the exposure groups were compared with respect to other characteristics
which could influence the outcome of these tests. In the high and low
exposure groups, the proportion in each category of a characteristic was
calculated. A standard chi-square test for equality of proportions (or
Fisher's exact test) was done for each characteristic in each population
(fecal donors and blood donors) in the six seasons of data plus a baseline
data set. The fecal donor and blood donor populations were defined for
each season as those individuals or households donating the necessary series
of specimens to determine the infection status (see Table 43). Characteristics
which were known to be constant over a household were tested using the
household as the unit of observation. Household exposure to wastewater
aerosols was defined as the maximum participant exposure level observed
in the household. The results of these comparisons of exposure groups
are given in Tables 103 through 107 for most individual and household charac-
teristics, in Table 108 for previous titer, and in Table 109 for eating
at local restaurants. Both the percentage in each category of the variable
and the range of the probability value for each test are shown in the tables.
The exposure groups tended to differ in certain characteristics on a seasonal
basis (i.e., in both spring seasons or in both summer seasons) because
many residents in the middle of Wilson shifted exposure groups by season
due to seasonal differences in the prevailing wind direction.
For these tables, a judgment was made about the variable(s) to be
used for stratification prior to comparison of infection rates in exposure
groups. The relative importance, consistency and magnitude of differences
across seasons and quality of the data for each variable were considered.
To ensure consistency, a variable was considered for use as a stratifying
282
-------
to
oo
u>
TABLE 103. COMPARISON OF EXPOSURE GROUPS WITH RESPECT TO HOUSEHOLD CHARACTERISTICS
BY BASELINE AND IRRIGATION SEASON—BLOOD DONORS
(Entries are percent of households with each characteristic in each exposure group)
Baseline*
exposure
Characteristic
Number of Households
Rao*
% Caucasian
% hispanic
Household Size
% 1-2 members
% 3-4 members
% 5+ members
Head of Household Education
% 0-11 years
% 12 years
% 13+ years
Most Educated Family Member
% 0-11 years
% 12 years
% 13+ years
Head of Household Occupation
% professional or manager
% farmer
% other
Income, in 1979
% less than $9,999
% *10. 000-119,999
% i20, 000-129, 999
% |30,000+
Air Conditioning System
% none
% refrigeration
% evaporative cooler
pa Low
72
83
17
53
28
19
48
31
21
18
28
54
18
32
50
38
22
13
27
13
53
34
HiKh
56
82
18
63
23
14
43
32
25
11
33
56
12
43
45
44
28
15
13
17
36
47
Spring0
1982
exposure
D Low
69
83
17
57
23
20
50
29
21
18
30
52
16
33
51
38
24
12
26
11
55
34
Hich p
59
83
17
61
24
15
42
32
26
11
34
55
*
14
42
44
44
26
18
12
16
37
47
Summer0
1982
exposure
Low
86
83
17
55
25
20
48
27
26
19
30
51
19
30
51
41
25
13
21
12
52
36
Hitth
41
81
19
66
19
15
40
43
17
6
36
58
10
53
37
35
27
20
18
16
31
53
Spring®
1983
exposure
p Low
60
80
20
52
27
21
50
27
23
17
24
59
18
35
47
36
24
13
27
+
12
57
31
High
53
87
13
64
21
15
34
40
26
14
39
47
15
42
43
39
29
19
13
11
37
52
Summer1
1983
exposure
p Low
78
81
19
56
23
21
*
47
26
27
18
27
55
+
18
32
50
36
26
16
22
**
12
56
32
High
31
90
10
64
23
13
29
55
16
10
42
48
13
55
32
40
23
17
20
10
26
64
continued..
-------
TABLE 103. (CONT'D)
Characteristic
Baselineb
exposure
Spring0
1982
exposure
Summer"
1982
exposure
pa Low High p Low High p Low High
Spring6
1983
exposure
Loi
Summer'
1983
exposure
High p Low High
to
oo
•u
Use of Air Conditioning
% all or most of time
% some each day
% only when very hot
% never
Drinking Water Supply
% private well
% public supply
32
17
39
12
**
63
37
40
13
31
16
36
64
32
19
38
11
**
61
39
38
16
31
15
36
64
31
20
36
13
49
51
43
15
27
15
46
54
35
17
37
11
*
60
40
42
13
32
13
40
60
na 38
17
33
12
50
50
42
6
39
13
52
48
c
d
e
f
Blank if p>0.10, + if 0.05
-------
TABLE 104. COMPARISON OF EXPOSURE GROUPS WITH RESPECT TO HOUSEHOLD
CHARACTERISTICS BY BASELINE AND IRRIGATION YEAR—BLOOD DONORS
(Entries are percent of households with each characteristic
in each exposure group)
Baseline"
1982°
exposure exposure
Characteristic
Nnbor of Household*
Race
% Caucasian
% hispanic
Household Size
% 1-2 members
% 3-4 members
% 5+ members
Head of Household Bduemtioa
% 0-11 years
% 12 years
% 13+ years
Host Educated Family Heater
% 0-11 years
% 12 years
% 13+ years
Head of Household Occupation
% professional or manager
% farmer
% other
Income, in 1979
% less than $9,999
% JlO, 000-119, 999
% $20.000-*29,999
% 130,000+
Air Conditioning System
% none
% refrigeration
% evaporative cooler
Use of Air Conditioning
% all or most of time
% some each day
% only when very hot
% never
Drinking fater Supply
% private well
% public supply
a Blank if p>0.10, + if 0.05
-------
TABLE 105. COMPARISON OF EXPOSURE GROUPS WITH RESPECT TO HOUSEHOLD CHARACTERISTICS—FECAL DONORS
(Entries are percent of households with each characteristic in each exposure group)
to
oo
0\
Basel ineb
exposure
Characteristic
Number of Households
Race
% Caucasian
% hispanic
Homsehold Size
% 1-2 members
% 3-4 members
% 5+ members
Head of Household Education
% 0-11 years
% 12 years
% 13+ years
Most Educated Family Member
% 0-11 years
% 12 years
% 13+ years
Head of Homsehold Occupation
% professional or manager
% farmer
% other
Income, in 1979
% less than $9,999
% ilO,000-il9,999
% i20,000-j29,999
% $30,000
Air Conditioning System
% none
% refrigeration
% evaporative cooler
pa Low
21
62
38
na
0
48
52
na
48
28
24
na
21
36
43
na
29
38
33
na
29
19
19
33
na
29
43
28
High
10
60
40
10
60
30
30
10
60
0
25
75
30
60
10
33
45
11
11
22
45
33
Spring**
1982
exposure
t> Low
52
85
15
54
23
23
44
33
23
16
30
54
+
23
25
52
39
20
16
25
16
55
29
Hieh
41
88
12
61
24
15
44
27
29
11
30
59
14
49
37
45
22
23
10
16
37
47
Summer"
1982
exposure
t> Low
60
87
13
57
21
22
45
27
28
18
28
54
***
23
22
55
na
47
19
14
20
*
16
53
31
Hiah
24
83
17
54
29
17
37
38
25
4
31
65
8
67
25
33
29
25
13
9
30
61
Spring®
1983
exposure
o Low
40
83
17
48
25
27
40
33
27
16
24
60
22
30
48
+
33
21
13
33
**
16
60
24
High
42
88
12
64
22
14
44
27
29
17
38
45
10
46
44
49
24
17
10
15
29
56
Summer'
1983
exposure
p Low
55
84
16
58
22
20
44
25
31
19
26
55
*
18
27
55
na
41
22
17
20
***
8
56
26
Hiah
24
88
12
54
29
17
37
42
21
12
38
50
8
63
29
48
22
17
13
8
21
71
continued.
-------
TABLE 105. (CONT'D)
N>
00
Spring"
Baselineb 1982
exposure exposure
Characteristic pa Low High p Low High
Use of Air Conditioning na
% all or most of the time 29 50 31
% some each day 24 0 17
% only when very hot 28 30 39
% never 19 20 13
Drinking Water Swpply * *
% private well 48 10 64
% public supply 52 90 36
a Blank if p>0.10, + if 0. 05
-------
TABLE 106. COMPARISON OF EXPOSURE GROUPS WITH RESPECT TO INDIVIDUAL CHARACTERISTICS—BLOOD DONORS
(Entries are percent of individuals with each characteristic in each exposure group)
to
00
oo
Characteristic
Number of individuals
Age group
% 0-5 years
% 6-17 years
% 18-44 years
% 45-64 years
% 65+ years
Gender
% male
% female
Tap water consumed — vs. others
your age
% less than average
% average
% more than average
Time spent in Lnbbock
% 0-1 hours/week
% 2-11 hours/week
% 12+ hours/week
Contacts per week with MO people
% 0-5 contacts
% 6-10 contacts
% 11+ contacts
Smokes cigarettes regularly
% no
% yes
Soring
1982
Exposure
pa Low Hich
203
2
27
32
25
14
47
53
12
71
17
36
45
19
41
34
25
90
10
118
6
24
30
24
16
48
52
19
71
10
36
47
17
42
29
29
85
15
Summer 1982
Exposure
p Low High
247
5
28
30
24
13
47
53
14
73
13
33
46
21
40
33
27
88
12
69
4
23
30
28
15
51
49
21
60
19
37
48
15
54
27
19
88
12
Sprinit
1983
Exposure
p Low Hiah
181
3
28
29
27
13
46
54
11
74
15
35
46
19
40
35
25
90
10
103
9
26
27
23
15
49
51
20
67
13
35
51
14
47
26
27
86
14
Summer 1983
Exposure
p Low High
207
5
27
27
28
14
46
54
14
73
13
35
42
23
42
34
25
87
13
58
7
24
33
26
10
48
52
19
64
17
33
48
19
54
25
21
90
10
continued..
-------
TABLE 106. (CONT'D)
N>
oo
Spring 1982
Characterist ic
Exposure
Pa Low High
Summer 1982
Exposure
p Low High
Spring 1983
Exposure
p Low High
Summer 1983
Exposure
p Low High
Smokes cigarettes regularly 1983
% no
* yes
Chews tobacco regularly
% no
% yes
Any respiratory illness
% no
% yes
Ever had pneumonia
% no
% yes
Any heart condition
% no
% yes
Any abdominal condition
% no
% yes
Any other condition
% no
% yes
Polio immunization
% no
% yes
88
12
94
6
73
27
91
9
79
21
84
16
69
31
**
84
16
87
13
88
12
72
28
94
6
77
23
81
19
67
33
70
30
88
12
**
95
5
74
26
92
8
81
19
85
15
70
30
87
13
83
17
71
29
93
7
72
28
80
20
64
36
88
12
*
95
5
75
25
92
8
80
20
85
15
69
31
88
12
88
12
70
30
92
8
77
23
82
18
68
32
88
12
*
94
6
86
24
91
9
80
20
82
18
68
32
89
11
86
14
67
33
95
5
78
22
84
16
71
29
a Blank if p>0.10, * if 0.01
-------
to
VO
O
TABLE 107. COMPARISON OF EXPOSURE GROUPS WITH RESPECT TO INDIVIDUAL
CHARACTERISTICS—FECAL DONORS
(Entries are percent of individuals with each characteristic in each exposure group)
Spring 1982
Characteristic i
Number of individuals
Age group
% 0-5 years
% 6-17 years
% 18-44 years
% 45-64 years
% 65+ years
Gender
% male
% female
Tap water consumed — vs. others
your age
% less than average
% average
% more than average
Time spent in Lubbock
% 0-1 hours/week
% 2-11 hours/week
% 12+ hours/week
Contacts per week with 2.10 people
% 0-5 contacts
% 6-10 contacts
% 11+ contacts
Smokes cigarettes regularly
% no
% ves
Exposure
j* Low High
82
12
32
17
21
18
45
55
11
71
18
26
57
17
f
36
41
23
f
98
2
50
16
20
20
24
20
42
58
20
67
13
40
50
10
50
20
30
90
10
Summer 1982
Exposure
p Low High
106
14
29
21
18
18
42
58
b
17
69
14
24
55
21
+
39
36
25
93
7
27
15
18
26
30
11
44
56
19
62
19
26
63
11
62
15
23
93
7
Spring 1983
Exposure
p Low High
62
11
26
15
32
16
42
58
+
9
69
22
23
56
21
+
38
38
24
92
8
47
15
15
21
26
23
47
53
24
58
18
30
59
11
59
23
18
94
6
Summe r 1
.983
Exposure
p Low High
84
14
23
12
31
20
45
55
14
68
19
23
57
20
45
34
21
95
5
28
14
18
25
29
14
50
50
18
64
18
29
50
21
52
26
22
93
7
continued..
-------
TABLE 107. (CONT'D)
Spring 1982
Characteristic
Smokes Cigarettes Regularly 1983
% no
% yes
Chews Tobacco
% no
% yes
Any respiratory illness
% no
% yes
Ever had pneumonia
% no
% yes
\c Any heart conditions
% no
% yes
Any abdominal conditions
% no
% yes
Any other conditions
% no
% yes
Exposure
Pa Low Hi«h
+
95
5
92
8
80
20
95
5
80
20
87
13
71
29
87
13
89
11
76
24
92
8
68
32
80
20
62
38
Summer 1982
Exposure
P Low Hiah
90
10
+
96
4
73
27
93
7
81
19
86
14
69
31
88
12
85
15
70
30
89
11
70
30
78
22
70
30
Spring 1983
Exposure
P Low Hiah
93
7
93
7
77
23
94
6
79
21
84
16
71
29
93
7
87
13
70
30
91
9
66
34
79
21
60
40
Summer 1983
Exposure
P Low HiKh
96
4
94
6
75
25
92
8
74
26
82
w
62
38
89
11
86
14
71
29
96
4
71
29
86
14
75
25
a Blank if p>0.10, + if 0.05
-------
Table 108. COMPARISON OF EXPOSURE GROUPS WITH RESPECT TO PREVIOUS TITER TO SEROLOGIC AGENTS
[Entries ere number of Individuals observed followed In parentheses by percent of individuals with
previous titer below indicated titer level)
Baseline8
Titer exposure
Aaent level o" Low HI ah
$1 N RIB) mm
AD7 10 155(76] 84(81)
CB2 10 37(24) 21(24]
CB4 10 48(31) 25(29]
CBS 10 128(68) 63(67)
E01 10 + 178(92) 83(86)
£03 10 132(81) 72(78)
E09 10 111(63] 60(64)
E11 10 125(66) 56(60)
E19 10
E20 10 148(86) 79(81)
E24 10 149(87) 88(90)
RE1 8 130(70) 65(68)
RE2 8 87(48) 44(46]
ROT 4 2(15) 2(12)
LEG* 64
INAJ 4 23(17) 5(9)
PL1 4 22(15) 17(18)
PL2 4 20(14) 15(16)
»j» PL3 4 + 60(41) 50(54)
to
e Baseline titer: Jun 1980.
b Spring 1982 titer: Jan 1982.
c Summer 1982 titer: Jun 1982.
d Spring 1983 titer: Dec 1982.
e Summer 1983 titer: Jun 1983.
f 1982 titer: Jan 1982.
g 1983 titer: Dae 1982.
h Blank If p>0.10, + If 0.05
-------
TABLE 109. COMPARISON OF EXPOSURE GROUPS WITH RESPECT TO FREQUENCY
OF EATING FOOD PREPARED AT RESTAURANTS A AND B--FECAL DONORS
(Entries are percent of individuals with each
frequency in each exposure group)
Characteristics
Number of Individuals
Restaurant A
% >pnce /month
% pnce /month
% 0.10, ** if O.OOKpOO.Ol, *** if plO.OOl in chi-square test.
variable if and only if 1) the variable was deemed to be epidemiologically
important and 2) the hypothesis of equal proportions was rejected at the
0.01 level at least once or at the 0.05 level at least twice in the four
irrigation seasons. If the variable met these criteria, stratification
was used if the number of observations was large enough to permit statistical
analysis in the stratified groups. While all of the variables listed in
Tables 103 through 109 could have some individual or collective influence
on infection rates, six variables were considered to be epidemiologically
important enough to warrant stratification should they be imbalanced over
exposure groups (Criterion 1). These were household size, head of household
occupation, age, gender, previous titer for serological variables, and
immunization status for polioviruses.
In the household-based analyses of the blood donor population (Tables
103 and 104), none of the variables met both criteria for stratification,
i.e., neither household size nor head of household occupation met Criterion
2 for statistical significance. Note that in the summer 1982 and summer
1983 seasons, head of household occupation of the blood donors was near
the criterion for statistical significance. This near-significant imbalance
reflects the fact that the proportion of farmers in the high exposure group
(53% in summer of 1982 and 55% in the summer of 1983) was greater than
the proportion of farmers in the low exposure group (39% in the summer
of 1982 and 32% in the summer of 1983). The hypothesis of equal proportions
was rejected at the 0.01 level in the summer 1983 season for type of air
conditioning system, because a majority (56%) of households in the low
exposure group had refrigerated air conditioning while most (64%) of the
households in the high exposure group had evaporative coolers. Also, drinking
water supply was sufficiently different across exposure groups to be statis-
tically significant at the 0.01 level in the baseline and spring 1982 seasons
and at the 0.05 level in spring 1983. Although not significant, these
293
-------
proportions were sometimes reversed in the summer 1982 and summer 1983
seasons.
In the household-based analysis of the fecal donor population (Table
105), the high exposure group also contained significantly more farmers
in summer 1982 and summer 1983 (67% and 63%, respectively, as shown by
the head of household occupation variable) than the low exposure group
(25% and 29%). Although this variable meets both criteria for stratification,
the number of households in the fecal donor population was not large enough
to permit statistical analysis in stratified groups as discussed in Section
4J, Statistical Methods. The exposure groups were also imbalanced with
respect to type of air conditioning system, with more households in the
high exposure group having evaporative coolers.
Comparison of exposure groups with respect to individual characteristics
in the blood donor and fecal donor populations are shown in Tables 106
and 107. Two of the Criterion 1 variables (age and gender) were not statis-
tically significant in any season. The exposure groups were significantly
imbalanced with respect to a third Criterion 1 variable, polio immunization
status (p=0.005) in spring 1982, with a larger proportion immunized in
the high exposure group. A larger proportion of individuals regularly
chewed tobacco in the high than in the low exposure group (Table 106).
This difference was significant at p=0.01, 0.05 and 0.05 in summer 1982,
spring 1983 and summer 1983, respectively. Tobacco chewing represents
a possible hand-to-month exposure factor.
An imbalance in previous titer levels of individuals in the exposure
groups could bias the tests for association between infection rates and
wastewater exposure if one exposure group was significantly less susceptible
to the agent than the other exposure group. Table 108 shows the comparison
of exposure groups with respect to previous titer to the serologic agents
for which titer levels were measured. Two agents, influenza A in June
1981 and echovirus 3 in January 1982, showed imbalance at the 0.05 level
in one season, and these did not meet the criteria outlined above as justifi-
cation for stratification prior to the confirmatory analysis. The exposure
groups were significantly imbalanced for previous titer to poliovirus 3
for both the baseline and the spring 1982 periods of observation.
The exposure groups were very significantly imbalanced with respect
to frequency of eating food prepared at restaurant A (Table 109) among
the fecal donors surveyed. Those individuals in the fecal donor population
who were in the high exposure group ate significantly more often at restaurant
A than individuals in the low exposure group. This gives an alternative
explanation for infections (especially bacterial) which could have been
transmitted in food handling. For this reason, eating food prepared at
restaurant A was considered a possible alternative explanation whenever
a positive statistical association between infection rates and wastewater
exposure is found, because this could negate the implication of the apparent
association with wastewater exposure. The number of observations is too
small for stratification into groups with respect to frequency of eating
food prepared at the restaurant. Therefore, patronage of restaurant A
was explored by logistic regression as an alternative explanation whenever
294
-------
an apparent association between infections and wastewater exposure was
found (especially when they were bacterial infections).
In conclusion, when comparing infection rates in exposure groups,
stratification on household or individual characteristics was done only
for polioviruses on polio immunization status. However, all of these individual
and household variables were considered in the exploratory logistic regression
analyses of infections on degree of aerosol exposure and other potential
predictor variables.
Confirmatory Analysis
Fisher's exact test was used to test the hypothesis that the incidence
rates within the low and high exposure groups were equal for each agent
in each irrigation season, with the one-sided alternative being that the
high exposure group had a larger incidence rate than the low exposure group.
One of the major requirements for the validity of this test is that the
infections occurred independently in individuals. An individual could
become infected either from the wastewater (primary exposure) or from another
household member (secondary exposure). If secondary infections occurred
frequently among members in large households, the validity of the statistical
analysis could be questionable. Since there usually was more than one
blood donor per household (and often more than one fecal donor), the indepen-
dence of the responses was investigated. The data in Tables P-48 and P-49
in Appendix P showing the number of households by size with 0, 1 or 2 infections
are not inconsistent with the hypothesis that the infections occurred inde-
pendently. This can be seen from the fact that in only a few instances
were there more than one seroconversion per household. Thus, it was concluded
that the binomial was a suitable model for the occurrence of infections
and that Fisher's exact test or a chi-square test for equality of the binomial
proportions in the low and high exposure groups could be used.
In Tables 110 to 112, the incidence rates for bacterial, viral and
serologic infections in the low (AEK3) and high (AEI>3) wastewater aerosol
exposure groups, were compared for the baseline period and for each of
the four or six seasons of data. The study design specified that each
individual be measured for serum titer and serologic infection status at
the beginning of each season and at the end of each season. New infection
events were defined in terms of seroconversions or changes in infection
status. The serologic data did not permit inference as to whether the
time of onset of observed serologic infection events was before, during
or after the irrigation period for which association with aerosol exposure
was being investigated. From the clinical data based on routine fecal
specimens, it could be determined that the onset of many bacterial and
viral infection events was during a period of irrigation (i.e., when the
change in infection status occurred between two specimens donated during
the irrigation period). Clinical infection status variables were constructed
(denoted by ''-X'' in Tables 110 and 111) in which only the infection events
with onset during an irrigation period were retained. For bacterial and
viral infection events occurring between the fecal specimens collected
prior to and shortly after an irrigation period commenced, it could not
be determined whether the onset of the infection event preceded or followed
295
-------
TABLE 110. COMPARISON OF INCIDENCE OF BACTERIAL INFECTIONS IN LOW AND HIGH EXPOSURE GROUPS
(Entries are number of infections observed followed by incidence rates expressed as
percents (in parentheses), risk ratios (RR = high/low), and probability levels6)
K>
Spring" 1982 Summer6 1982
Agent
KLB-X*
KLB-WS
OOB-X
OOB-W
PBW-X
PBW-W
a
b
c
d
e
f
g
Blank
Spring
Summer
Spring
Summer
Low
0
2(3)
0
0
1(1)
2(3)
if p>0
1982
1982
1983
1983
X: Onset of
W: Includes
exposure
HiKh
0
0
0
0
1(2)
1(2)
.10, * if
period of
period of
period of
period of
exposure
RR Low HiKh RR
3(5) 2(10) 1.9
0 9(14) 4(17) 1.3
0 0
1(2) 1(4) 2.7
1.7 2(3) 1(4) 1.4
0.8 2(3) 2(8) 2.7
0.01
-------
TABLE 111. COMPARISON OF INCIDENCE RATES OF VIRAL INFECTIONS IN LOW AND HIGH EXPOSURE GROUPS
(Entries are number of Infections observed followed by Incidence rates expressed as percente (In parentheses),
risk ratios (RR = high/low), and probability levels8)
Summer 1980°
baseline
expoeu re
Summer 1981C
baseline
exposure
Spr1ngd 1982
exposure
Summer6 1982
exposure
Spr1ngf 1983
exoosu re
Hloh RR Low High RR Low H1oh RR Low High RR Low High RR Low
Summerfl 1983
exposure
High
RR
VIR-Xh
VIR-W*
WWI-X
WWI-W
5(36)
5(36)
5(63)
5(63)
1.8
1.8
8(35) 1(17)
8(35] 1(17)
0.5
0.5
6(8]
11(14)
4(6]
8(12)
3(7)
4(9]
3(8)
4(10)
0.9
0.7
1.2
0.8
6(8]
7(9]
8(14)
13(20)
5(19) 2.5+
5(19) 2.2
4(21) 1.B
7(32] 1.6
0
0
2(3)
2(3)
1(2]
1(2)
2(4)
2(4)
__
—
1.3
1.3
1(1)
4(6]
3(5]
12(17)
1(4]
1(4)
5(24)
10(38)
2.8
0.7
4.8*
2.2*
e Blank 1f p>0.10, + If 0.05
-------
TABLE 118. COMPARISON OF INCIDENCE OF SEROLOGIC INFECTIONS IN LOW AND HIGH EXPOSURE GROUPS
[Entries are number of Infections observed followed by Incidence rates expressed as percents
(In parentheses), risk ratios (RR = high/low), and probability levels8]
to
oo
Baseline0 Spr1ngc 1982 Summerd 1982
exoosure exposure exposure
Agent Low High RR Low H1qh RR Low High RR
ADS 10(6] 3(3} 0.5 0 0 — 1(0] 0 0
ADS 4(3] 3(3} 1.4 3(2] 0 0 2(1] 0 0
A07 2(1) 4(4) 3.8 0 0 — 0 0 —
CB2 10(6) 4(5) 0.7 0 1(1] — 1(0} 0 0
CB4 10(6] 6(7) 1.1 1(1] 2(2) 3.5 3(1) 2(3) 2.3
CBS 5(3] 6(6} 2.4 2(1) 2(2) 1.8 2(1) 2(3] 3.3
E01 4(2) 3(3) 1.5 1(1) 0000 —
£03 9(5] 3(3) 0.6 0 0 — 3(1] 0 0
EOS 1(1} 2(2) 3.7 1(0] 0000 —
£09 2(1] 6(6) 5.6* 00—00 —
E11 10(5) 7(7) 1.4 2(1) 2(2) 1.9 5(2] 2(3) 1.4
El 7 2(1] 0 0 1(1} 0000 —
E19 2(1) 1(1] 0.9 0 0 — 0 1(1} —
E20 4(2) 1(1) 0.4 1(1] 0 0 1(0] 0 0
E24 5(3] 3(3) 1.0 1(1) 1(1] 1 .7 0 0 —
RE1 29(16] 6(6] 0.4 11(5] 5(5] 0.8
RE2 26(14) 11(11) 0.8 10(5) 3(3) 0.5
ROT 4(31) 7(41) 1.3 1(4) 2(8] 2.0 1(3} 3(17) 6.0
INA1 14(11] 5(9) 0.9
INAJ 4(2) 2(3) 1.1
INAk 25(15) 10(11) 0.7
LEG1 4(3) 2(3] 1.1
POR 5(4) 3(4} 1.0 9(7) 4(6] 0.9 8(5] 1(2) 0.5
WMV 8(5) 4(5) 1.0
SNV 62(45) 36(51) 1.1 12(10) 8(13) 1.3 17(11) 5(13) 1.1
Spring8 1983
exposure
Low
1(1)
2(1)
0
1(1]
0
3(2]
0
1(1)
0
0
0
0
2(1]
0
1(1)
1(5]
10(5]
10(7]
a Blank If pX).10, + If 0.05
-------
the start of irrigation. The time of onset of these infection events was
termed "questionable," and the analysis was conducted with these observations
included and again excluding these observations. Variables excluding and
including these observations were denoted ''-X'' and f'-W' in Tables 110
and 111. If the two analyses agreed, the result was accepted without change.
If the two analyses disagreed, the result of the analysis excluding the
questionable observations was accepted. Usually only a few infected individuals
were in the questionable category as can be determined by comparing the
entries in Tables 110 and 111; in all cases, the results using the X and
W variables were similar.
Table 113 shows the incidence rates of poliovirus infections in the
baseline and spring 1982 periods. Mantel-Haenszel tests were used to test
for association between infection and wastewater exposure with the individuals
stratified on immunization status.
TABLE 113. COMPARISON OF INCIDENCE OF POLIO INFECTIONS IN
LOW AND HIGH EXPOSURE GROUPS STRATIFIED BY IMMUNIZATION STATUS
[Entries are number of infections observed followed by incidence
rates expressed as percents (in parentheses), risk ratios
(high/low), and probability levels8 of the stratified
Mantel-Haenszel (MH) test and Fisher's exact test]
Agent
PL1
PL2
PL3
Immunization
status
MH test
Yes
No
MH test
Yes
No
MH test
Yes
No
Basel
Low
39(61)
3(3)
41(65)
0
39(63)
0
inc° exposure
High _
27(64)
0
29(69)
1(2)
32(76)
0
RR p
1.1
1.1
1.2
Spring
Low
1(4)
2(2)
1(4)
1(1)
3(11)
0
J=282°_
High
7(21)
3(5)
6(18)
1(2)
4(12)
0
exposure
RR "p
*
5.9 •
2.9
5.1 +
2.0
1.1
b
c
Blank if p>0.10, + if 0.05
-------
o all wastewater isolates in summer 1983 at p=0.02 and 0.03 (WWI-X
and -W),
o coxsackie B2 in 1982 at p=0.05 (CB2),
o echovirus 9 in baseline at p=0.02 (E09),
o echovirus 24 in summer 1983 and 1983 at p=0.05 and 0.03 (£24),
o all viruses in sprayed wastewater in 1982 at p=0.02 (WWV),
o poliovirus 1 in spring 1982 at p=0.02 (PL1).
Each of these agents was significant in only one season, since in the echo
24 case, summer 1983 infections are a subset of 1983 infections.
The possibility of false positive associations should be considered
when interpreting these results. False positive associations are possible
only when the infection incidence rate in the population is large enough
to detect a difference between exposure groups. Thus, as recommended by
Gart et al. (1979), the rate of false positives should be based only on
independent infection episodes in which by definition the infection incidence
rate of the population was large enough to possibly reject the null hypothesis.
Gart et al. also point out that the expected rate of false positives in
independent infection episodes is the average actual a-level. This will
be considerably less than 5% when Fisher's exact test is used at a=0.05,
since the cumulative distribution function of a discrete random variable
is a step function which does not increase monotonieslly.
The actual rates of positive association in the six groups of independent
infection episodes defined in Table IS and identified in Tables 100 and
101 are presented in Table 114.
TABLE 114. RATE OF POSITIVE ASSOCIATIONS DETECTED BY THE STATISTICAL
CONFIRMATORY ANALYSIS AT SIGNIFICANCE LEVEL 0.05 IN INDEPENDENT
INFECTION EPISODES11
No. of significant
Il
ei
M
A
B
C
D
E
F
a
b
[dependent Number of c
tisode independent
•oupb episodes
31
19
(Control) 27
8
2
(Control) 1
From Tables 100 and 111.
See Table 15.
onfirmatory analysis
results (p<0.05)
2
2
1
1
1
0
%
6%
11%
4%
13%
(50%)
(0%)
Significant episodes
(CKLB4X, SPL11)
(SCB25, SE246)
(SE090)
(CWWI4X)
(SWWV5)
300
-------
In the 27 independent control infection episodes involving single and sporadic
agents (Group C) which were tested, one spurious positive association for
echovirus 9 was found (4% positive rate). Two of the 31 independent exposure
episodes to single or sporadic agents spanning a single irrigation period
were associated with wastewater aerosol exposure, a 6% positive rate for
Group A. Of the 19 Group B exposure episodes to single or sporadic agents
which spanned several irrigation periods, 2 (11%) were significantly associated
with exposure. For independent infection episodes involving grouped agents,
the rates of positive associations were 0/1 for the control episode, but
1/8=13% for single season exposure episodes (Group D) and 1/2=50% for year-long
exposure episodes (Group E) . The actual rate of positive association in
control episodes was approximately equal to the expected false positive
rate. In contrast, the actual rate of significant associations exceeded
the false positive rate in each of the four independent groups of exposure
episodes. The actual rate of positive associations in the exposure episodes
appears to be at least twice as large as the false positive rate.
The possibility must also be recognized that important differences
in incidence rates may exist, but were not detected by the statistical
test. The probability of such a false negative result is determined by
the true (and unknown) incidence rates in each of the two exposure groups
and the number of individuals observed in each exposure group. In accord
with intuition, the power of the test, that is, the probability of detecting
a given difference in the two incidence rates p^ (low exposure) and p2
(high exposure), increases as the number of individuals increases. Further,
the power to detect differences in pj and P2 tends to increase as PJ becomes
very small. Table P-50 in Appendix P displays the actual sample sizes
(HI in the low exposure group, 02 in the high exposure group) and pj_, the
observed incidence rates in the low exposure group. It is then assumed
that PI=PI and P2=P1 + A (where A = 0.05, 0.07, 0.10, 0.15, 0.20 or 0.25).
With n^, &2> Pi a°d P2 thus specified, the power of the test for which
a=0.05 is calculated and displayed in the body of Table P-50. This shows
that in most cases only relatively large differences in p^ and P2 can be
detected from these data and these statistical procedures with a power
of 0.90 or greater. This means that the lack of a significant test result
in a given instance could result either from the absence of important differ-
ences in PI and p2 or the lack of power to detect a difference which is
in fact present.
In conclusion, an excess of statistically significant associations
of the presence of infection with wastewater aerosol exposure was found
in the confirmatory analysis. The interpretation of the epidemiological
importance of these significant associations must be moderated by recognition
of the possibility that some of the tests may be significant only by chance
and that some imbalances in the two populations may provide alternate explana-
tions for the observed differences. On the other hand, the number of detected
increases in incidence rates associated with the wastewater irrigation
may be underestimated, considering the relatively modest power of the tests
to detect small differences. The certainty of the results is also lessened
when the observational nature of the study and the difficulty inherent
in determining appropriate assignment of individuals to the exposure groups
are considered.
301
-------
Exploratory Logistic Regression Analysis
The exploratory logistic regression analysis investigated whether
the presence of infection was associated with a set of potential predictor
variables, and in particular with AEI, the degree of aerosol exposure.
An analysis was performed for each infection episode in which there was
a higher rate of infection in the high exposure group than in the low exposure
group and in the high exposure level than in the low and intermediate exposure
levels.
The effects of each predictor variable added in a stepwise manner
to the logistic model were assessed by means of a maximum-likelihood-ratio
chi-square test of the hypothesis that the explanatory power of that variable
was zero. The goodness-of-fit of the devised models in describing the
relationship between the probability of infection and the selected predictor
variables was assessed using a test developed by Hosmer and Lemeshow (1980).
A small p-value (e.g., p<0.10) indicates that the prediction equation does
not fit the data.
For each constructed model, approximate 90% confidence intervals were
obtained for the odds ratio. If the constructed confidence interval contained
the value 1 it was concluded that the odds of having an infection were
the same for the various categories of the predictor variable.
Four different analyses were performed in order to analyze the relationship
between rate of infection and the chosen predictor variables. These four
analyses are described below.
Analysis 1: Basic Analysis—
A stepwise logistic regression was performed to investigate whether
the presence of infection was associated with a selected set of predictor
variables. This analysis was repeated for each of the six seasons of data
plus a baseline data set. The response variables used in each season are
listed in Table 115 preceded by the previous titer predictor variable corres-
ponding to each serologic single-agent response variable. The response
and previous titer variables were described in more detail in Tables 97-99
and P-45 in Appendix P, respectively. Descriptions of the predictor variables
under consideration are presented in Table 116. Table 117 lists the candidate
set of predictor variables (besides previous titer) chosen from Table 116
for usage in the various stepwise regressions.
The restaurant variables and the alternative exposure variables were
not included in the basic analysis. Since the restaurant variables were
observed only for a small subset of the individuals, the investigation
of a possible restaurant etiology was analyzed separately (see Analysis
2). Since the alternative exposure variables (FHRSEM, XDIREL and XDIREM)
were highly correlated with AEI (see Table P-23 of Appendix P), a separate
analysis of the route of wastewater exposure was performed (see Analysis
4) when AEI was found to be a significant variable. The polio immunization
variables (IH1, SABINO, SALKO) were regressed only against the respective
polio infection response variables (SPL11, SPL21 and SPL30).
302
-------
TABLE 115. PREVIOUS TITER AND RESPONSE
REGRESSION ANALYSES
VARIABLES FOR LOGISTIC
u>
Baseline Spring
1980-81 1982
CVIR8 CWWI1X
PAD703 PROT13
SAD70 SROT1
PCB503 PPL113
SCB50 SPL11
PE0903 PPL213
SE090 SPL21
PINAO3
SINAO
PRE203
SRE20
PROTOa
SROTO
PPL303
SPL30
Summer
1982
CPBW2X
CPBW2W
CVIR2X
CVIR2W
CWWI2X
PCB423
SCB42
PCB523
SCB52
PE1123
SE112
PROT23
SROT2
SWWV2
SSNV2
Spring Summer
1983 1983
COOB3 CKLB4X
CWWI3 CKLB4W
PROT33 CWWI4X
SROT3
CWWI4W
PE0343
SE034
PE1143
SE114
PE2043
SE204
PE2443
SE244
PROT43
SROT4
Year
1982
PCB253
SCB25
PCB453
SCB45
PCB553
SCB55
PE1153
SE115
PE1953
SE195
PROT53
SROT5
SWWV5
SSNV5
Year
1983
PE0363
SE036
PE1163
SE116
PE2463
SE246
SSNV6
3 The log (base e) of
analyses .
Prefix
these previous
titer variables were
used in the
regression
P Previous titer
C Clinical dependent variable
S Serologic dependent variable
-------
TABLE 116. PREDICTOR VARIABLES FOR LOGISTIC REGRESSIONS'1
Predictor variable
1. AEI, Aerosol Exposure Index
2. AGE82, age on June 30, 1982
3. SEX, sex
4. RESP, history of respiratory
conditions
5. PNEU, history of pneumonia
6. HEART, history of heart conditions
7. ABDOM, history of gastrointestinal
conditions
8. OTHERO, history of other chronic
conditions
9. SMOKE/SMOKE3, current cigarette
smoker
10. TCHEW, tobacco chewer in 1983
11. RACE, race
12. HHSIZGR, household size group
13. HOHEDGR, education group of
household head
14. HOHOCC, occupation of household
head
15. INCOME, income group (1979
family income)
16. ACSYS, type of air conditioning
system
17. ACUSE, frequency of air conditioning
use (in summer)
See ''Aerosol Exposure Index''
in Section 4C
r •
Age, in years
1
2
0
1
Hale
Female
No
Yes
0 No
1 Yes
0 No
1 Yes
0 No
1 Yes
0 No
1 Yes
0 No
1 Yes
0 No
1 Yes
1 Caucasian
4 Hispanic
1 1-2 members
2 3-4 members
3 5 or more members
1 Grades 0-8
2 Grades 9-11
3 Grade 12
4 Some college
5 College graduate
1 Professional or manager
2 Farmer
3 Other
1 J30,000
1 None
2 Refrigeration
3 Evaporative cooler
1 All or most of time
2 Some time each day
3 Only when very hot
4 Never or no air conditioning
continued...
304
-------
TABLE 116. (CONT'D)
Predictor variable
Code
18. DWATER, drinking water source
19. WCONSM, tap water consumed vs.
others your age
20. CONTACT, contacts per week with
ten or more people
21. TLUBOCK. time in Lubbock
22. LNP_, natural logarithm of
previous serologic titer to
response variable agent
23. RESTA, frequency ate food prepared
at restaurant A
24. RESTS, frequency ate at restaurant B
25. FHRSEM, time on Hancock farm
26. XDIREM, index of extensive direct
wastewater contacts
27. XDIREL, level of direct wastewater
contact
28. SALKO, Salk inactivated polio
immunization in 1980-81
29. SABINO, Sabin oral polio
immunization in 1980-81
30. IM1, polio immunization in
spring 1982
1 Private well (rural)
2 Pub lid- supply (Wilson)
1 Less than average
2 Average
3 More than average
1 Less than once
2 1 to 5 times
3 6 to 10 times
4 11 to IS times
5 More than IS times
Average hours per week spent
in Lubbock
In (previous titer)
1 >0nce/week
2 Once/week to once/month
3 <0nce/month
4 Never
1 >0nce/week
2 Once/week to once/month
3 <0nce/month
4 Never
Average hours per week spent
on Hancock farm (see ''Additional
Exposure Measures'' in Section
4C)
See ''Additional Exposure Measures"
in Section 4C
1 None (XDIREM=0)
2 Low (O.liXDIREMaO)
3 High (XDIREM>10)
0 No
1 Yes
0 No
1 Tes
0 No
1 Yes (Salk or Sabin)
a All predictor variables with more than two codes were treated as interval
variables, except HOHOCC and ACSYS which were treated as categorical
variables.
305
-------
TABLE 117. PREDICTOR VARIABLES USED IN LOGISTIC REGRESSION ANALYSIS
to
o
o\
Baseline
AEI
AGES 2
SEX
RESP
PNEU
HEART
ABDOM
OTHERO
SMOKE
RACE
HHSIZGR
HOHEDGR
HOHOCC
INCOME
ACUSE
DWATER
TLUBOCK
SALKO
SABINO
RESTA
RESTB
FHRSEM
XDIREL
Spring
1982
AEI
AGE82
SEX
RESP
PNEU
HEART
ABDOM
OTHERO
SMOKE 3
RACE
HHSIZGR
HOHEDGR
HOHOCC
INCOME
ACUSE
DWATER
TLUBOCK
IM1
RESTA
RESTB
FHRSEM
XDIREL
Summer
1982
AEI
AGE82
SEX
RESP
PNEU
HEART
ABDOM
OTHERO
SMOKE 3
RACE
HHSIZGR
HOHEDGR
HOHOCC
INCOME
ACUSE
DWATER
TLUBOCK
RESTA
RESTB
FHRSEM
XDIREL
Spring
1983
AEI
AGE82
SEX
RESP
PNEU
HEART
ABDOM
OTHERO
SMOKE3
TCHEW
RACE
HHSIZGR
HOHEDGR
HOHOCC
INCOME
ACSYS
ACUSE
DWATER
WCONSM
CONTACT
TLUBOCK
RESTA
RESTB
FHRSEM
XDIREM
Summer
1983
AEI
AGE82
SEX
RESP
PNEU
HEART
ABDOM
OTHERO
SMOKE3
TCHEW
RACE
HHSIZGR
HOHEDCR
HOHOCC
INCOME
ACSYS
ACUSE
DWATER
WCONSM
CONTACT
TLUBOCK
RESTA
RESTB
FHRSEM
XDIREM
1982
AEI
AGE82
SEX
RESP
PNEU
HEART
ABDOM
OTHERO
SMOKE 3
RACE
HHSIZCR
HOHEDGR
HOHOCC
INCOME
ACUSE
DWATER
TLUBOCK
RESTA
RESTB
FHRSEM
XDIREL
1983
AEI
AGE82
SEX
RESP
PNEU
HEART
ABDOM
OTHERO
SMOKE 3
TCHEW
RACE
HHSIZGR
HOHEDGR
HOHOCC
INCOME
ACSYS
ACUSE
DWATER
WCONSM
CONTACT
TLUBOCK
RESTA
,., RESTB
FHRSEM
XDIREL
-------
The results of the initial stepwise logistic regression runs are summarized
in Tables 118 through 124 for each season of data. Included is a list
of the tested agent, the significant predictor variables from the stepwise
runs, approximate 90% confidence intervals on the odds ratios for the signifi-
cant variables, and the p-value for the Hosmer goodness-of-fit test. Also
given are the p-values from the chi-square test f6r the significance of
the AEI variable both at the initial step (when AEI is the only variable
in the equation) and at the final step (regardless of whether or not AEI
entered the equation). Finally, indicated with each significant predictor
variable is the category that had the higher infection rate.
A few observations, ranging up to 10% of the individuals observed
per response variable, were deleted from each initial analysis because
the values of certain predictor variables were missing. For those response
variables providing good or marginal evidence of aerosol exposure association
(see Tables 131 and 132), the basic analysis was rerun, deleting predictor
variables with missing values when these variables were not significant
in the initial analysis and estimating missing values of important variables
where possible. These rerun analyses are presented in Table 125.
While controlling for the effects of significant monitored covariates,
the logistic regression analysis identified four infection episodes in
which the infections were associated with AEI at a final step p-value below
0.05:
o SE090—echovirus 9 in baseline (p=0.01)
o SPL11—poliovirus 1 in spring 1982 (p=0.01)
o SWWV2—seroconversions to wastewater isolates in summer 1982
(p=0.02)
o SSNV2—all seroconversions to serum neutralization-tested viruses
in summer 1982 (p=0.04)
The significant covariates are presented in Table 125. The goodness-of-
fit of each of these models was excellent.
The effect of excluding some of the observations in the initial runs
can be seen by comparing the AEI significance p-values for the episodes
in Table 125 with the same values for the initial run of the episode in
Tables 118-124. When the excluded observations are influential, the effect
can be major. This is illustrated by SCB42 with a p-value of 0.16 in Table
125 using all 289 observations, but a p-value of 0.01 in Table 120 for
the run with one infected donor and 14 noninfected donors excluded. However,
the effect on AEI significance of excluding some of the observations usually
was minor (see SE090 and SE115, for example) or trivial (e.g., SPL11, CVIR2X
and CKLB4X).
The poliovirus 1 infections in spring 1982 (SPL11) are shown in Table
125 to be significantly associated with three predictor variables: IM1—polio
immunization in spring 1982, low LNPPL11—polio 1 antibody titer in January
1982, and high AEI—aerosol exposure in spring 1982. This infection episode
was subsequently found to be the only episode consistently associated with
307
-------
TABLE 118. LOGISTIC REGRESSION RESULTS FOR BASELINE
INFECTION EPISODES
o
00
AEI Significance
Agent
CVIR8
SAD 70
SCB50
SE090
SINAO
SRE20
SROTO
SPL30
Initial3
p>0.25
p=0.12
p>0.25
p=0.02
p>0.25
p=0.12
p>0.25
p>0.25
Finalb
p>0.25
p=0.23
p>0.25
p=0.02
p>0.25
p>0.25
p>0.25
p>0.25
Significant0
predictor variable
AGES 2 (young)
HOHEDGR (college educ. HOH)
LNPCB50 (high antibody level)
AEI (high aerosol exposure)
LNPINAO (low antibody level)
RACE (hispanics)
HOHOCC (farmer)
HEART (heart history)
LNPROTO (low antibody level)
INCOME (high)
LNPPL30 (low antibody level)
SALKO (salk vaccination in
Baseline)
AGE82 (young)
HOHEDGR (little educ. HOH)
90% Confidence
interval for
the odds ratio
(0.68,
(1.07,
(1.07,
(1.02,
(0.25,
(1.02,
(1.38,
(1.04,
(0.23,
( 1 . 04 ,
(0.20,
(72.54
(0.92,
(0.50,
0.99)
3.13)
3.41)
1.11)
0.84)
1.97)
3.56)
4.30)
0.75)
8.19)
0.69)
, 1304.85)
0.98)
0.93)
Goodness
of fitd
0.79
0.21
0.48
0.38
0.11
0.96
0.63
0.45
This is the p-value of AEI at the initial step; i.e., when AEI would be the only
variable in the prediction equation.
If p^.10, then p-value indicates X2 to remove AEI at last step in model selection;
otherwise p-value indicates X2 to enter AEI at last step in model selection.
Predictor variables in regression model at last step of model selection;
the subgroup in parentheses had the higher infection rate.
p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
o
vo
SROT1
SPL11
SPL21
TABLE 119. LOGISTIC REGRESSION RESULTS FOR SPRING 1982
INFECTION EPISODES
AEI significance
Agent
CWWI1X
Initial3
p>0.25
Final^
p>0.25
Significant0
predictor variable
INCOME (low)
90% Confidence
interval for
the odds ratio
(0.01, 0.53)
Goodness
of fitd
0.81
RESP (respiratory history)
HHSIZGR (small HH)
(2.83, 89.68)
(0.08, 1.06)
p>0.25 p>0.25
p=0.01 p=0.01
OTHERO (history of other
chronic conditions) (2.86, 764.98)
TLUBOCK (little time in Lubbock) (0.55, 1.10)
p=0.11 p>0.25
IMl (polio immunization in
Spring 82)
LNPPL11 (low antibody level)
AEI (high aerosol exposure)
IMl (polio immunization in
Spring 82)
LNPPL21 (low antibody level)
SEX (males)
(7.18, 101.98)
(0.14, 0.48)
(1.02, 1.10)
(6.56, 144.53)
(0.10, 0.47)
(0.04, 0.93)
0.65
0.92
0.31
a This is the p-value of AEI at the initial step; i.e., when AEI would be the only
variable in the prediction equation.
b If p_<_. 10, then p-value indicates X2 to remove AEI at last step in model selection;
otherwise p-value indicates X2 to enter AEI at last step in model selection.
c Predictor variables in regression model at last step of model selection;
the subgroup in parentheses had the higher infection rate.
d p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
TABLE 120. LOGISTIC REGRESSION RESULTS FOR. SUMMER 1982
INFECTION EPISODES
Agent
CPBW2X
CPBW2W
AEI
significance
Initial3
p>0.
p>0.
25
25
Final"
p>0.
p>0.
25
25
Significant0
predictor variables
AGE82 (elderly)
ABDOM (gastrointestinal
history
90% Confidence
interval for
for odds ratio
(0.
(1.
99,
98,
1.
98
15)
.98)
Goodness
of fitd
0.05
CVIR2X
CVIR2W
CWWI2X
SCB42
p=0.16 p=0.16
p=0.19 p=0.09
p>0.25
p>0.25
p>0.25
p=0.01
AGE82 (young)
AEI (high exposure)
OTHERO (history of other
chronic conditions)
DWATER (public water supply)
ACUSE (regular A/C users)
AGES2 (young)
HOHEDGR (college educ. HOH)
SMOKE3 (current smoker)
AEI (high exposure)
RESP (respiratory history)
(0.95, 0.99)
(1.00, 1.04)
(3.69, 78.35)
(2.82, 88.21)
(0.19, 0.80)
(0.54, 0.92)
(1.15, 10.78)
(8.31, 1.25E7)
(1.01, 1.21)
(0.86, 461.39)
0.55
0.73
0.35
SCB52
SE112
SROT2
SWVJV2
SSNV2
p>0.25 p>0.25 none
p=0.11 p=0.11 INCOME (low)
p>0.25 p>0.25 TLUBOCK (much time in
Lubbock)
p=0.04 p=0.05 AGE82 (young)
AEI (high exposure)
INCOME (low)
RACE (Caucasians)
p=0.19 p=0.05 AGE82 (young)
INCOME (low)
SMOKE3 (smoker)
AEI (high exposure)
DWATER (public water supply)
RACE (Caucasians)
(0
(1
(0
(1
(0
(0
(0
(0
(1
(1
(1
(0
.14,
.01,
• 93,
.00,
.19,
.40,
.93,
.19,
.21,
.01,
.16,
.49,
1.
1.
0.
1.
0.
0.
0.
0.
13
1.
8.
0.
06)
13)
98)
04)
79)
93)
98)
71)
.74)
04)
31)
99)
0.25
0.44
0.58
0.27
This is the p-value of AEI at the initial step; i.e., when AEI would be the only
variable in the prediction equation.
If p£. 10, then p value indicates X^ to remove AEI at last step in model selection;
otherwise p-value indicates X^ to enter AEI at last step in model selection.
Predictor variables in regression model at last step of model selection;
the subgroup in parentheses had the higher infection rate.
p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
SROT3
TABLE 121. LOGISTIC REGRESSION RESULTS FOR SPRING 1983
INFECTION EPISODES
AEI significance
Agent
COOB3
CWWI3
Initial3
p>0.25
p>0.25
Finalb
p>0.25
p>0.25
Significant0
predictor variables
none
ABDOM (gastrointestinal
907» Confidence
interval for Goodness
the odds ratio of fitd
p>0.25
history)
INCOME (low)
TCHEW (tobacco chewers)
p=0.21 HOHEDGR (college educ. HOH)
(2.89, 1.90E3)
(0.02, 0.83)
(1.39, 1.26E3)
(0.91, 6.05)
0.36
0.79
This is the p-value of AEI at the inital step; i.e., when AEI would be the only
variable in the prediction equation.
If p£. 10, then p-value indicates X^ to remove AEI at last step in model selection;
otherwise p-value indicates X^ to enter AEI at last step in model selection.
Predictor variables in regression model at last step of model selection;
the subgroup in parentheses had the higher infection rate.
p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
TABLE 122. LOGISTIC REGRESSION RESULTS FOR SUMMER 1983
INFECTION EPISODES
Agent
CKLB4X
CKLB4W
CWWI4X
CWWI4W
SE034
SE114
SE204
SE244
SROT4
AEI
significance
Initial3
p=0.
p=0.
p=0.
p>0.
p>0.
p>0.
p>0.
P>0.
p>0.
10
18
11
25
25
25
25
25
25
Final13
p=0.
p=0.
p=0.
p>0.
p>0.
p>0.
p>0.
p>0.
p>0.
13
18
16
25
25
25
25
25
25
Significant0
predictor variables
WCONSM (drinks a lot of water)
none
WCONSM (drinks a lot of water)
CONTACT (infrequent group
contact)
OTHERO (no history of other
chronic condition)
SEX (females)
HHSIZGR (large HH)
DWATER (private wells)
HOHEDGR (college educ. HOH)
CONTACT (frequent group
contacts)
INCOME (high)
AGE82 (young)
LNPROT4 (low antibody level)
AGES 2 (young)
90% Confidence
interval for
the odds ratio
(1.
(1.
(0.
(0.
(1.
(1.
(0.
(1.
(1.
(0.
(0.
(0.
(0.
08,
11,
26,
03,
10,
63,
02,
23,
08,
90,
86,
07,
84,
10
11
0.
1.
15
42
0.
6.
4.
10
0.
0.
1.
.02)
.09)
75)
01)
.10)
.94)
82)
98)
19)
.81)
97)
50)
03)
Goodness
of fitd
0.16
0.12
0.29
0.89
0.63
0.07
0.53
0.50
a This is the p-value of AEI at the initial step; i.e., when AEI would be the only
variable in the prediction equation
b If p£. 10, then p-value indicates X^ to remove AEI at last step in model selection;
otherwise p-value indicates X^ to enter AEI at last step in model selection.
c Predictor variables in regression model at last step of model selection;
the subgroup in parentheses had the higher infection rate.
d p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
SE195
SROT5
SWWV5X
SSNV5X
TABLE 123. LOGISTIC REGRESSION RESULTS FOR 1982
INFECTION EPISODES
AEI significance
Agent
SCB25
SCB45
SCB55
SE115
Initial3
p>0.25
p>0.25
p>0.25
p=0.04
Final13
p>0.25
p>0.25
p>0.25
p=0.06
Significant0
predictor variable
SEX (males)
AGE82 (elderly)
AGE82 (young)
AGE82 (young)
RESP (no respiratory history)
HOHOCC (farmer)
DWATER (public water supply)
AEI (high aerosol exposure)
90% Confidence
interval for
the odds ratio
(0
(1
(0
(0
(0
(1
(1
(1
.02,
.00,
.96,
• 92,
.03,
.35,
.35,
.00,
0
1
0
0
1
5
9
1
.69)
.06)
.99)
.99)
.06)
.18)
.35)
.03)
Goodness
of fitd
0.74
0.32
0.39
0.33
p>0.25 p>0.25
p>0.25 p>0.25
p=0.17
p>0.25
p>0.25 p>0.25
none
ACUSE (regular A/C users)
HOHOCC (other occupation)
HHSIZGR (small HH)
HOHOCC (farmer)
PNEU (pneumonia history)
PNEU (pneumonia history)
ACUSE (infrequent A/C user)
(0.01, 0.77)
(1.31, 281.8)
(0.03, 0.94)
(1.23, 2.52)
(1.04, 5.55)
(1.81, 10.40)
(1.06, 1.64)
0.95
0.83
0.84
This is the p-value of AEI at the initial step; i.e., when AEI would be the only
variable in the prediction equation.
If pjC.10, then p-value indicates X^ to remove AEI at last step in model selection;
otherwise p-value indicates X^ to enter AEI at last step in model selection.
Predictor variables in regression model at last step of model selection;
the subgroup in parentheses had the higher infection rate.
p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
SE116
SE246
SSNV6
TABLE 124. LOGISTIC REGRESSION RESULTS FOR 1983
INFECTION EPISODES
AEI significance
Agent
SE036
Initial3
p>0.25
Final"
p>0.25
Significant0
predictor variables
LNPE036 (high antibody)
90% Confidence
interval for
odds ratio
(1.21, 2.35)
Goodness
of fitd
0.56
p>0.25 p>0.25
p>0.25 p>0.25
HEART (young)
HHSIZGR (large household)
DWATER (private wells)
(0.04, 1.14)
(1.25, 5.96)
(0.05, 0.65)
AGE82 (young) (0.91, 0.99)
WCONSM (drinks little water) (0.01, 0.31)
RACE (hispanics) (1.69, 7.72)
HOHEDGR (college education, HOH) (1.43, 4.93)
p>0.25 p>0.25
ABDOM (No GI history)
SMOKE3 (nonsmoker)
AGE82 (young)
(0.02, 0.60)
(0.03, 0.80)
(0.97, 0.99)
0.17
0.17
0.34
This is the p-value of AEI at the initial step; i.e., when AEI would be the only
variable in the prediction equation.
If p£.10, then p-value indicates X^ to remove AFI at last step in model selection;
otherwise p-value indicates X^ to enter AFI at last step in model selection.
Predictor variables in regression model at last step of model selection;
the subgroup in parentheses had the higher infection rate.
p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
TABLE 125. RESULTS OF RERUN OF ANALYSIS 1 - INVESTIGATE
INFECTION EPISODES WITH FEWER OBSERVATIONS DELETED
AEI significance
Season/Agent Initial3 Final0
BASELINE
SE090 p=0.03 p=0.01
SPRING 1982
SPL11 p=0.01 p=0.01
SUMMER 1982
CVIR2X p=0.16 p=0.16
CVIR2W p=0.18 p=0.07
SCB42 p>0.25 p=0.16
SCB52 p=0.12 p=0.12
SWWV2 p=0.04 p=0.02
SSNV2 p=0.17 p=0.04
Significance
predictor variables
Race (hispanics)
AEI (high aerosol
exposure
RESP (respiratory
history)
IM1 (polio
immunization in
Spring 1982)
LNPPL11 (low anti
body level)
AEI (high aerosol
exposure)
none
HHSIZGR (large
household)
AEI (high aerosol
exposure)
AGE82 (young)
HHSIZGR (small
household)
OTHERO (history
of other chronic
conditions)
none
AGE82 (young)
AEI (high aerosol
exposure)
AGE82 (young)
DWATER (public water
supply)
AEI (high aerosol
exposure)
90% Confidence
interval for
the odds ratio
(1.52,
(1.02,
(1.37,
(6.98,
(0.14,
(1.02,
(1.14,
(1.00,
(0.75,
(0.01,
(1.27,
(0.94,
(1.01,
(0.95,
(1.43,
(1.01,
4.16)
1.13)
21.36)
98.18)
0.48)
1.10)
4.72)
1.04)
0.92)
0.33)
62.90)
0.99)
1.04)
0.99)
8.87)
1.04)
Goodness
of fitd
0.71
0.70
0.53
0.17
0.65
0.86
continued. .
315
-------
TABLE 125. (CONT'D)
AEI significance Significant0
Season/Agent Initial3 Final" predictor variables
SUMMER 1983
CKLB4X
CKLB4W
CWWI4W
SE244
1982
SCB25
SCB55
SE115
SWWV5
1983
SE246
p=0.09 p=0.13 WCONSM (drinks alot
of water)
p=0.17 p=0.21 HHSIZGR (small
household)
p>0.25 p>0.25 CONTACT (infrequent
group contact)
HOHEDGR (little
educ. HOH)
p>0.25 p>0.25 AGE82 (young)
SEX (females)
p>0.25 p>0.25 SEX (males)
AGE82 (elderly)
p=0.20 p=0.10 AGE82 (young)
p=0.02 p=0.11 RESP (no respiratory
history)
HOHOCC (farmer)
p=0.13 p>0.25 HOHOCC (farmer)
PNEU (pneumonia
history)
p>0.25 p>0.25 AGE82 (young)
WCONSM (drinks
little water)
907. Confidence
interval for
the odds ratio
(1.
(0.
(0.
(1.
(0.
(0.
(0.
(1.
(0.
(0.
(1.
(1.
(1.
(0.
(0.
16,
23,
22,
00,
86,
85,
02,
00,
93,
03,
21,
13,
03,
90,
05,
11
1.
0.
1.
0.
31
0.
1.
0.
0.
4.
2.
5.
0.
0.
.0)
03)
68)
82)
97)
.82)
30)
05)
99)
84)
03)
25)
34)
98)
47)
Goodness
of fitd
0.13
0.75
0.15
0.41
0.23
0.27
0.29
0.78
0.75
a This is the p-value of AEI at the initial step; i.e., when AEI would be the only
variable in the prediction equation.
b If p£.10, then p-value indicates X^ to remove AEI at last step in model selection,
otherwise p-value indicates X2 to enter AEI at last step in model selection.
c Predictor variables in regression model at last step in model selection;
the subgroup in parentheses had the higher infection rate.
d p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
high AEI for which no other explanation could be found to explain the aerosol
exposure effect (see Section 5M). For the SPL11 episode, the cross-product
terms of the significant variables (i.e., AEI x IH1, AEI x LNPPL11, IM1
x LNPPL11 and AEI x IM1 x LNPPL11) were also constructed as predictor variables
to investigate interaction effects. None of the cross-product terms were
significant predictor variables. Thus, concurrent polio immunization,
low polio 1 antibody titer, and high aerosol exposure were independently
associated with the polio 1 seroconversions in spring 1982 as three distinct
risk factors. Each risk factor appears to have been responsible for some
of the 13 poliovirus 1 infections observed between January and Tune 1982.
Analysis 2: Investigate Possible Restaurant Etiology—
The possible association of the infection episode with the frequency
of eating food prepared at the two restaurants in Wilson was investigated
in Analysis 2. This was done only for those response variables providing
good or marginal evidence of aerosol exposure association. The predictor
variables RESTA and RESTB (see Table 48) were added to the set of variables
used in Analysis 1 and the same methodology used there was again employed.
Variables RESTA and RESTB were obtained primarily from fecal donors (see
Section SO. Thus, for the serologic response variables, Analysis 2 was
based on less than half of the observations used in Analysis 1.
The results of Analysis 2 are presented in Table 126. RESTA was a
significant predictor variable for CVIR2W and especially for CKLB4X. RESTB
was a significant predictor variable for CVIR2X. This analysis suggests
frequent patronage of restaurant A as the probable explanation for the
Klebsiella infection episode during the summer 1983 irrigation period.
Analysis 3: Exclude AEI to Investigate Alternative Explanations—
Analysis 1 was repeated, excluding AEI as a predictor varible, for
those response variables in which AEI was a significant predictor variable
in Analysis 1. The purpose of this analysis was to determine if other
predictor variables would play the same explanatory role in the logistic
regression as did AEI. Such variables could be considered alternative
explanations to AEI as the possible cause of the infection episode. The
results of Analysis 3 are given in Table 127.
Comparison of the results in Table 127 with the prior run for the
response variable shows that no replacement variable for AEI was found
in the SE090, SPL11, CVIR2W and SSNV2 episodes. For SWWV2, low income
and Caucasian replaced high AEI. For SE115, Caucasian and large households
replaced high AEI. The replacement variables can be considered alternative
explanations to high AEI for SWWV2 and SE115.
Analysis 4: Investigate Route of Wastewater Exposure—
Exposure to the wastewater aerosol, direct contact with the wastewater,
and spending time in the irrigation environment on the Hancock farm are
three alternative routes by which infectious agents in the wastewater could
be transmitted to initiate an infection episode. The relevant measures
of these exposures, AEI, XDIREM (or XDIREL), and FHRSEM, were highly correlated
in the study population in each exposure season (see Table P-23 of Appendix
P). Thus, AEI, which was considered to be the best single measure of wastewater
317
-------
TABLE 126. RESULTS OF ANALYSIS 2 - INVESTIGATE POSSIBLE RESTAURANT ETIOLOGY
AEI significance Significant0
Season/Agent Initial3 Final" predictor variables
BASELINE
SE090 p=0.001 p=0.001 AEI (high aerosol exposure)
SAD70 p=0.21 p>0.25 HOHEDGR (college educ. HOH)
SPRING 1982
SPL11 p=0.01 p=0.002 AEI (high aerosol exposure)
LNPPL11 (low antibody
level)
IM1 (polio immunization
in Spring 82)
SUMMER 1982
CVIR2X p=0.18 p=0.13 RESTS (ate frequently at
restaurant B)
CVIR2W p=0.22 p>0.25 HHSIZGR (large household)
RESTA (ate frequently at
restaurant A)
SCB42 p>0.25 p>0.25 none
SCB52 p=0.11 p=0.11 none
SWWV2 p=0.02 p=0.002 AEI (high aerosol exposure)
AGE82 (young)
SSNV2 p=0.08 p=0.001 AGE82 (young)
AEI (high aerosol exposure)
PNEU (pneumonia history)
INCOME (low)
SUMMER 1983
CKLB4X p=0.11 p>0.25 RESTA (ate frequently at
restaurant A)
WCONSM (drinks a lot of
water)
SEX (females)
TLUBOCK (little time in
Lubbock)
SE244 p>0.25 p>0.25 AGE82 (young)
DWATER (private wells)
90% Confidence
interval for
the odds ratio
(1
(1
(1
(0
(1
(0
(1
(0
(1
(0
(0
(1
(4
(0
(0
(1
(1
(0
(0
(0
.17,
.18,
.04,
.03,
.92,
.20,
.21,
.25,
.00,
.73,
.75,
.02,
.96,
.02,
.05,
.97,
.90,
.74,
.86,
.02,
11
5.
1.
0.
.28)
91)
17)
68)
Goodness
of fitd
0.50
0.49
217.29)
0.
5.
0.
1.
1.
0.
1.
3.
0.
0.
56
80)
89)
93)
14)
04)
96)
14)
21E6)
85)
44)
.24)
0.14
0.74
0.60
0.88
0.01
118.60)
1.
0.
0.
01)
97)
78)
0.78
continued. . .
318
-------
1983
SE246
TABLE 126 (CONT'D)
AEI significance
Season/Agent
1982
SCB25
SE115
SWWV5
Initial3
p>0.25
p=0.02
p>0.25
Final0
p>0.25
p>0.12
p>0.25
907. Confidence
Significant0 interval for Goodness
predictor variables the odds ratio of fitd
AGE82 (young) (0.23, 1.19)
SEX (males) (0.01, 0.25)
none
p>0.25 p>0.25 AGE82 (young)
(0.86, 0.98)
0.58
This is the p-value of AEI at the initial step; i.e., when AEI would be the only
variable in the prediction equation.
If p£.10, then p-value indicates X2 to remove AEI at last step in model selection;
otherwise p-value indicates X2 to enter AEI at last step in model selection.
Predictor variables in regression model at last step of model selection;
the subgroup in parentheses had the higher infection rate.
p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
TABLE 127. RESULTS OF ANALYSIS 3 - EXCLUDE AEI TO INVESTIGATE
ALTERNATIVE EXPLANATIONS
Season/Agent
Significant3
predictor variables
90% Confidence
interval for
the odds ratio
Goodness
of fitb
BASELINE
SE090
none
SPRING 1982
SPL11
IM1 (polio immunization in
Spring 1982)
LNPPL11 (low antibody level)
(7.45, 97.23)
(0.14, 0.48)
0.36
to
K>
O
SUMMER 1982
SWWV2
CVIR2W
1982
SE115
AGES2 (young)
INCOME (low)
RACE (Caucasians)
HHSIZGR (large household)
RESP (no respiratory history)
HOHOCC (farmer)
DWATER (public water supply)
RACE (Caucasians)
HHSIZGR (large household)
(0.93, 0.98)
(0.38, 0.87)
(0.18, 0.74)
(1.01, 3.59)
(0.03, 0.98)
(1.76, 9.68)
(2.17, 31.96)
(0.31, 0.85)
(1.02, 3.39)
0.50
0.72
0.82
Predictor variables in regression model at last step of model selection;
the subgroup in parentheses had the higher infection rate.
p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
irrigation exposure, was the only exposure measure employed in Analysis
1. For those response variables whose regression equation in Analysis
1 contained the predictor variable AEI, Analysis 4 also was performed.
In Analysis 4, predictor variables FHRSEM and XDIREL (or XDIREM when
available) were included with the previous predictor variables used in
Analysis 1. The methodology of Analysis 1 again was utilized in performing
the logistic regression analysis.
The results of Analysis 4 are presented in Table 128. Of the six
response variables investigated during periods of irrigation, the irrigation
exposure measure selected was AEI for four episodes (SPL11, SCB42, SSNV2
and SE115), XDIREL for episode CVIR2W and FHRSEM for episode SWWV2. Wastewater
irrigation cannot be implicated as the source of exposure using only the
logistic regression evidence. However, if wastewater irrigation was found
to be a causative factor of the infection episodes investigated, the results
of Analysis 4 provide evidence supporting all three exposure routes, with
the aerosol exposure route having the most supporting evidence.
Evaluation of the Effect of Ignoring Multiple Infection Events^on the Statis,-
tical Analysis Results
To conduct the confirmatory analysis using Fisher's exact test and
the exploratory analysis using logistic regression, it was necessary to
ignore multiple infection events. These analyses made the assumption that
persons experiencing more than one infection event in the period of observation
of an infection episode provided the same information regarding the distribution
of infections as did persons experiencing a single infection event in the
observation period.
The effect on each confirmatory analysis result of ignoring the multiple
infection events is presented in Table 129 for each episode in which multiple
infection events occurred. By noting which exposure group would have had
more infection events or a higher rate of increased infection events in
each such episode, the direction of the effect on the reported p-valne
was determined. No confirmatory analysis results would have been changed
substantially. Two associations reported to be significant at p=0.02 (i.e.,
for echovirus 9 in the baseline period and for all serologically detected
infections in 1982 to viruses recovered from the wastewater) were probably
somewhat more significant (p<0.02).
The effect on each exploratory logistic regression result of ignoring
multiple infection events is shown in Table 130. The AEI means of all
participants with 2, 3 and 4 infection events were compared to the mean
AEI of all participants with a single infection event to determine the
direction of the effect of ignoring the multiple infection events. There
were four infection episodes with multiple infection events in which the
p-valne of AEI on the final step of model construction was less than 0.10.
The p-value accounting for multiple events would probably have been more
significant for one of the four: SSNV2 (all serum neutralization-tested
viruses in summer 1982) with p<0.05. Taking multiple events into account
would likely have made these associations less significant: SE090 (p>0.01),
321
-------
TABLE 128. RESULTS OF ANALYSIS 4 - INVESTIGATE ROUTE OF
WASTEWATER EXPOSURE
Season/Agent
AEI significance
Initial3Final"
Significant0
predictor variables
907. Confidence
interval for Goodness
the odds ratio of fitd
BASELINE
SE090
SPRING 1982
SPL11
SUMMER 1982
CVIR2W
SCB42
SWWV2
SSNV2
1982
p=0.02 p=0.02 AEI (high aerosol exposure)
p=0.01 p=0.01
p=0.19 p>0.25
p>0.25 p=0.01
p=0.04 p>0.25
p=0.19 p=0.05
IM1 (polio immunization in
Spring 1982)
LNPPL11 (Low antibody
level)
AEI (high aerosol exposure)
AGE82 (young)
XDIREL (extensive direct
wastewater contact)
AGE82 (young)
HOHEDGR (college educ. HOH)
SMOKE3 (smoker)
AEI (high aerosol exposure)
RESP (respiratory history)
FHRSEM (frequent Hancock farm)
AGE82 (young)
AGE82 (young)
INCOME (low)
SMOKE3 (smoker)
AEI (high aerosol exposure)
DWATER (public water supply)
RACE (Caucasians)
(1.02, 1.11)
(7.18, 101.98) 0.93
(0.95, 0.99)
(1.20, 5.84)
(0.54, 0.92)
(1.15, 10.78)
(8.31, 1.25E7)
(1.02, 1.21)
(0.86, 461.39)
(1.01, 1.03)
(0.94, 0.99)
(0.93, 0.98)
(0.25, 0.85)
(1.21, 13.74)
(1.01, 1.04)
(0.98, 6.61)
(0.49, 0.99)
This is the p-value of AEI at the initial step; i.e., when AEI would be the only
variable in the prediction equation.
If p<.10, then p-value indicates X2 to remove AEI at last step in model selection;
otherwise p-value indicates X2 to enter AEI at last step in model selection.
Predictor variables in regression model at last step of model selection;
the subgroup in parentheses had the higher infection rate.
p-value for Hosmer's chi-square goodness-of-fit; a large value (i.e., .10
-------
TABLE 129. EFFECT OF MULTIPLE INFECTION EVENTS ON CONFIRMATORY ANALYSIS RESULTS
Dependent
variable
Agent
VIR-X
VIR-W
WWI-W
CB4
£03
E09
£24
ROT
wwv
SNV
Season
Sun 1981
Sum 1982
Spr 1982
Sam 1982
1982
Baseline
Baseline
Baseline
1983
Baseline
Sam 1982
1982
Baseline
Spr 1982
Sam 1982
1982
Sam 1983
1983
Number of observations
bv infection status
Exposure
group
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
0«
15
5
73
21
61
37
51
15
213
68
156
91
176
87
167
94
178
64
9
10
183
52
139
34
77
33
111
52
133
35
113
31
137
43
124
50
1 or
more8
8
1
7
5
8
4
13
7
13
6
9
3
2
6
5
3
4
6
4
7
10
5
41
21
62
36
12
8
17
5
58
23
23
6
33
14
1
6
1
5
5
7
4
11
7
13
5
8
3
2
5
4
3
3
5
3
6
9
5
37
18
50
23
11
8
16
4
51
20
19
3
23
9
234
2
2
1
2
1
1
1
1
1
1
1
1
1
4
2 1
723
931
1
1
1
6 1
2 1
3 1
111
8 2
212
Confirmatory Direction of effect on reported
analysis p-valne if multiple infection
p-valne events had been taken into account
>0.25
0.13
>0.25
0.21
>0.25
>0.25
0.02
>0.25
0.02
>0.25
>0.25
0.02
>0.25
>0.25
>0.25
0.22
>0.25
>0.25
Less significant
Less significant (p>0.13)
Less significant
Less significant
More significant
Less significant
More significant (p<0.02)
Less significant
Little effect
Little effect
Less significant
,->
Slightly more significant (p<0.02)
Slightly more significant
Less significant
Slightly more significant
Slightly more significant
More significant
More significant
a Values used in analysis.
-------
TABLE 130. EFFECT OF MULTIPLE INFECTION EVENTS ON EXPLORATORY
LOGISTIC REGRESSION ANALYSIS RESULTS
Dependent Mean AEI
( numbe r]
variable Recoded observations
by season 0 1
BamllM
SED90
SuBMr 11
CVIR2W
SMWV2
SSNV2
1882
SCB45
SMWV5
SSNVS
1983
SE246
SSNV6
3.84(863]
182
6.44(94)
4.53(835)
5.17(168)
5.53(881)
5.05(173)
5.48(144)
6.82(242)
6.01(174)
13.09(8)
17
16
11
7
9
7
3
7
,
.10(12)
.93(15)
.98(22)
.01(19)
.16(62)
.80(81)
.75(10]
.64(47]
of observations by Infection status
Actual observations
1
14.32(7)
20.17(10)
17.98(14)
11.75(20]
3.25(18)
7.98(55)
7.04(71)
3.92(8)
9.97(32]
4
1
2
14
74
17
12
3
2
2 34
.53(1]
.75(2]
.25(1)
.20(2]
.71(1)
.04(6) 26.51(1)
.95(8] 26.51(1) 1.79(1)
.09(2)
.35(10) 2.04(3) 5.08(2)
Exploratory
logistic
regression
final step
p-velue
0
0
0
0
X)
X)
X)
X)
X)
.01
.07
.02
.05
.25
.25
.25
.25
.25
Direction of effect
on reported AEI p-value
If multiple Infection events
had been taken Into account
Less
Less
Less
More
Much
More
More
significant
significant
significant
significant
(p>0.01)
(pX).07)
(pX).02)
(p<0.05)
more significant (p<0.25?)
significant
significant
(p<0.25?)
(p<0.25?)
Little effect
Less
significant
(pX).2S)
to
-------
CVIR2W (p>0.07) and SWWV2 (p>0.02). Because there was a small proportion
of multiple infection events in each of these episodes, the magnitude of
the change in p-value is unlikely to have been large. For only three of
the episodes (SWWV5, SSNVS and SSNV6) did enough multiple infection events
occur to have allowed a valid exploratory analysis of their effect using
a weighted least squares approach.
M. EVIDENCE OF ASSOCIATION OF SPECIFIC INFECTION EPISODES WITH 1ASTEWATBR
AEROSOL EXPOSURE
The LISS has employed four methods of inference to investigate the
possible association of infections with wastewater aerosol exposure in
the episodes of infection which were observed in the study population.
These inferential methods were: 1) risk ratio (RR) scoring (see Section
5K) , 2) the incidence density ratio (IDR) of high-to-intermediate and high-to-
low exposure levels for serologic infection episodes (see Section SI),
3) confirmatory statistical analysis (CA) (see Section 5L) , and 4) exploratory
logistic regression (ELR) statistical analysis (see Section 5L) . Five
scores were assigned to every infection episode based on the results obtained
by each of the four methods.
The RR score is a classification of an infection episode by comparison
of the infection incidence rates in the low (AEK3) and high (AEI>.3) exposure
groups and in the low (AEK1), intermediate (liAEI<5) and high (AEI>5)
exposure levels. The high and low exposure groups and levels are treated
in a symmetric manner in assigning the RR score.
Two incidence density (ID) ratios for the exposure levels (i.e.,
and IDgi/IDL<>) were calculated for each serologic infection episode. The
90% and 95% confidence intervals (CI) were constructed for each IDR for
which two or more infection events were expected in both of the compared
levels to determine if the intervals included the value 1.00. Two IDR
scores which are assigned on this basis also evaluate the possible association
of infections with aerosol exposure.
The confirmatory statistical analysis used Fisher's exact test to
test the hypothesis that the infection rates within the low and high exposure
groups were equal for each infection episode, against the one-sided alternative
that the high exposure group had a larger infection rate. The confirmatory
analysis score is assigned based on the p-value of this test.
The exploratory statistical analysis used the stepwise logistic regression
method to investigate whether the presence of infection was associated
with the degree of exposure measured by the aerosol exposure index (AEI) ,
controlling for the effect of significant monitored covariates. An analysis
was performed for each infection episode for which a higher infection rate
was observed in the high exposure group than in the low exposure group
and in the high exposure level than in the intermediate and low exposure
levels. A multiple linear logistic regression model was formed in a stepwise
fashion, with one predictor variable with a chi-square p-valne below 0.10
entering the model or one predictor variable with chi-square p-value above
0.15 removed from the model at each step. The exploratory analysis score
325
-------
is based on the p-value of chi-square to enter or remove the AEI predictor
variable at the last step of the model selection process.
A summary containing the scores from each of these inferential methods
is presented for each control infection episode in Table 131 and for each
exposure infection episode in Table 132. The actual p-value of the CA
result is given in parentheses after the score when p^.0.15. The actual
p-values of the AEI predictor variable are given in parentheses for the
ELR results both initially and at the final step, whenever the respective
p<0.25. The initial step p-value suggests the apparent degree of association
of infections with AEI, uncontrolled for other factors. In contrast, the
final step p-value indicates the degree of association of infections with
AEI, controlling for the other significant predictor variables (which are
also in the model at the last step).
Tables 131 and 132 indicate that, as expected, a number of the statis-
tically significant associations found by the methods employed in certain
infection episodes were not supported by the results from the other inferential
methods. It is important to identify the infection episodes for which
there is strong and consistent evidence of association among the inferential
methods, since these infection episodes warrant additional scrutiny.
The four inferential methods complement each other to provide a balanced
assessment of the association of infection events with wastewater aerosol
exposure in a specific infection episode. Since each method also has its
deficiencies, all four methods are needed to achieve a proper interpretation
about the strength of the association.
The RR score, CA and ELR all ignore multiple infection events in the
episode, in that they place each participant with one or more infection
events in the same group, the ''infected donors.'' In contrast, the IDR
takes multiple infection events properly into account. However, the IDR
confidence intervals will be inaccurate, and thus are not used, when the
number of observed infection events is small.
The confirmatory analysis is conducted with known power to permit
assessment of the frequency of positive associations found. . However, CA
lacks the ability to investigate association with degree of exposure.
Thus, participants with very high (e.g., AEI>50) and intermediate (3
-------
TABLE 131. SUMMARY OF FINDINGS FOR CONTROL INFECTION EPISODES:
EVIDENCE REGARDING SPURIOUS ASSOCIATION OF INFECTIONS WITH WASTEWATER AEROSOL EXPOSURE
to
"Control" Jointly
Infection episode Indep.
Agent Depend, episode
Ob s period var. group8
Clinical (C)
VIR (Viruses, excluding
8 (Sun 80) CVIR8
9 (Sun 81] CVIR9
SaroUgle (8]
ADS (Adeno 3)
Baseline SAD30
ADS (Adano 5)
Baseline SAD50
AD7 (Adeno 7)
Baseline SAD70
CB2 (Coxsackle B2)
Baseline SCBSO
CB4 (Coxsackle B4)
Baseline SCB40
CBS (Coxsackie BS)
Baseline SCBSO
E01 (Echo 1)
Baseline SE010
EOS (Echo 3)
Baseline SE030
EOB (Echo 9) V
Baseline SEOBO
E11 (Echo 11)
Baseline SE110
E20 (Echo 20)
Baseline SE200
E24 (Echo 24)
Baseline SE240
PL1 (Polio 1)
Bass line SPL10
67 Salk Imnun adults:
34 Sab In Innun children:
PL2 (Polio 2]
Baseline SPL20
67 Salk Inunun adults:
33 Sabln Imnun children:
adeno
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
Infection
risk ratios0
1nf!b
Exp
group
RR
Exp
level
RR
Risk
ratio
score"
and Immunization polio)
12
9
13
7
6
14
16
11
7
12
8
17
5
8
68
49
17
72
51
19
1.8
0.5
0.5
1.4
3.8
0.7
1.1
2.4
1.5
0.6
5.6
1.4
0.4
1.0
1.0
1.0
0.8
1.1
1.0
1.1
—
-
0
0
4.2
0.5
0
4.2
0
0.2
1.9
0.6
0
0.9
1.7
1.1
1.8
1.3
1.0
0.9
0
—
0
0
*+
0
0
•f
0
—
+
0
0
0
0
0
0
0
0
0
Scores of Statistical Strength end
serologlc analysis results consistency
Incidence Confirm. Exploratory: of apparent
density ratio analysis AEI significances association
of exp levels6 score' score (p— value) of Infections
Hl/Int HI/Lo [p-value) Initial Final with exposure"
j
nd1 nd -
nd nd - (-)
- (-)
- (-)
0 00 (0.11) (0.12) 0 (0.23)
- (-)
- (-)
00 (0.12)
- M
_ _ (_j
^
0 ++ (0.02) (0.03) -H- (0.01) Good
- - M
- (-)
- (-)
0 0 (-)
0 0 -
0 0 -
0 0 (-)
0 0 -
- -
continued...
-------
TABLE 131. (CONT'D)
o*
K>
oe
"Control" Jointly
Infection episode Indep.
Agent Depend, episode
Ob s period ver. aroup8
PL3 (Polio 3)
Baseline SPL30
67 Se Ik Irnmin adults: C
32 Sab in immun children: C
RE1 [Reo 1]
Baseline SRE10 C
RE2 (Reo 2)
Baseline SRE20 C
ROT (Rotavirus) f
Baseline SROTO C
INA (Influenza A)
0 (60-81) SINAO C
1 [81-82] SINA1 C
3 (82-83) SINA3 C
Infection
risk ratios0
Exp Exp Ri
No. group Level ra
inf.0 RR RR sc
71 1.3
56 1.1
15 1.2
35 0.4
37 0.8
11 1.3
19 0.9
6 1.1
35 0.7
FOR (Sporadic serum neutralization viruses)
Baseline SPORO C 8 1.0
SNV (All serum neutralization
Baseline SSNVO F
vi ruses)
98 1.1
2.0
1.2
2.1
0.4
1.5
Large
2.7
0
0.7
0.7
0.6
Sc
at
ir
sk dens
t1o of t
tores of Statistical Strength and
iro logic analysis results consistency
icldence Confirm. Exploratory: of apparent
iity ratio analysis AEI significance0- association
ixp levels6 score" score (p-value] of Infections
ored Hi/Int Hi/Lo (p-value) Initial Final with exposure"
0 0
0 0
0 0
_ _
0
+ -
0
0
0
0 nd
0
+ -
0
0
- — (-)
0 - (0.12)
0 -
0 -
- - (-)
- - (-)
nd - (-)
(-]
a Classification criteria for the jointly independent groups of control Infection episodes are given in Table 15.
b Based on all observed individuals for whom an AEI exposure estimate was available.
c RR^Rui/IRLg.
d From Table 101.
e From Tables 88 and P-47 in Appendix P. The score criterion for the incidence density ratio (IDR) is based on its
confidence interval (CI):
- IDR<1.0 + 90% CI does not include 1.0
0 IDR>1.0, but 90% CI includes 1.0 ++ 95% CI does not include 1.0
From Tables 110 to 113. The confirmatory analysis score criterion is based on the p-value for the one-tailed Fisher's
exact test:
— p^0.95 + 0.05p>0.15 -H- 0.010.25 +++ p<0.01
0 0.10
-------
TABLE 132. SUMMARY OF FINDINGS FOR EXPOSURE INFECTION EPISODES:
EVIDENCE REGARDING ASSOCIATION OF INFECTIONS WITH WASTEWATER AEROSOL EXPOSURE
u>
to
vo
Infection
''Exposure11 Jointly risk ratios0
infection episode indep. Exp Exp Risk
Agent Depend, episode No. group level ratio
Ob s period ver. group8 inf. RR RR score"
Scores of Statistical Strength and
eero logic analysis results consistency
incidence Confirm. Exploratory: of apparent
density ratio analysis AEI significances association
of exp levels8 score' score (p-value] of infections
Hi/Int
Hi/Lo [p-value] Initial Final Kith exposure"
Clinical (C)
KLB
2
4
OOB
3
PBW
1
2
4
VI R
1
2
4
WWI
1
2
3
4
(Klebsiella)
(Sum 82)
(Sum 83)
[Other
(Spr 83)
CKLB2X
CKLB2W
CKLB4X
CKLB4W
opportunistic
COOB3
(Prominent bacteria
(Spr 82)
(Sum 82)
(Sum 83)
CPBW1W
CPBW2X
CPBW2W
CPBW4W
(Viruses, excluding
(Spr 82)
(Sum 82)
(Sum 83]
(Agents
[Spr 82]
(Sum 82)
(Spr 83]
(Sum 83)
CVIR1X
CVIR1W
CVIR2X
CVIR2W
CVIR4W
A
A
5
13
8
12
1.
1.
4.
3.
9
3
5
6
0
0
5.
3.
6
4
0
0
£
nd
nd
nd
nd - (-)
nd - [-)
nd -H- (0.03) (0.09) 0 [0.13] Good
nd ++ (0.02) [0.17] 0 (0.21)
bacteria)
A
in
A
A
A
5
wastewater)
3
3
4
9
1.
0.
1.
2.
1.
9
8
4
7
3
1.
1.
4
0
Large
Large
1.
adeno and immunization
A
A
A
9
15
11
12
5
0.
0.
2.
2.
0.
9
7
5
2
7
0.
1.
3.
3.
4
polio]
8
1
1
1
Large
0
0
0
0
0
0
++
0
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd -
nd - (-)
nd -
nd -
nd - (-]
nd - (-)
nd - (-)
nd + (0.10) [0.16] 0 (0.16)
nd 0 (0.13) (0.18) + (0.07) Marginal
nd - (-)
isolated from wastewater)
CWWI1X
CWWI1W
CWWI2X
CWWI2W
CWWI3
CWWI4XJ
CWWI4W
D
D
D
D
7
12
12
20
4
8
22
1.
0.
1.
1.
1.
4.
2.
2
8
6
6
3
8
2
2.
1.
2.
1.
1.
5.
2.
0
2
7
4
4
8
4
0
0
0
0
0
-H-
nd
nd
nd
nd
nd
nd
nd
nd -
nd - (-)
nd -
nd - (-)
nd -
nd -H- (0.02) (0.11) 0 [0.16] Marginal
nd -H- (0.03)
Sera logic (S)
ADS
5
ADS
5
CB2
5
(Adeno
(1982)
(Adeno
[1982]
3)
SAD35
5)
SAD 55
B
B
7
8
0.
0
5
0
0
0
0
-
—
(-)
(-)
(Coxseckie B2)
(1982)
SCB25
B
9
3,
7
Larqe +
0
0 ++ [0.05]
continued,
-------
TABLE 132. (CONT'DJ
"Exposure" Jointly
infection episode indep.
Agent Depend, episode
Ob 8 period var. nroup8
CB4 [Coxsackle B4)
2 (Sum 82] SCB42
5 (1982) SCB45
CBS (Coxsackie 85)
1 (Spr 82) SCB51
2 (Sum 82] SCB52
5 (1982) SCB55
4 (Sum 83) SCB54
6 (1983) SCB56
E03 (Echo 3)
5 (1982) SE035
4 (Sum 83] SE034
6 (1983) SE036
E11 (Echo 11)
u> 1 (Spr 82] SE111
to
0 2 (Sum 82) SE112
5 (1982) SE115
4 (Sum 83] SE114
6 (1983) SE116
E19 (Echo 19)
5 (1982] SE195
A
B
A
A
B
A
B
B
A
B
A
A
B
A
B
B
No
1nf.D
5
18
4
4
8
8
9
9
11
18
4
7
19
6
10
3
Infection
risk ratios0
Exp Exp
group level
RR RR
2.3
1.5
1.8
3.3
1.8
0
0.3
0.4
1.3
1.7
1.9
1.4
2.2
3.3
2.6
6.0
5.2
1.4
0
Large
7.5
0
0.9
0.6
4.0
2.4
0
2.9
3.0
1.9
1.2
Large
Risk
ratio
score"
+
0
0
+
0
0
0
-
0
+
0
0
+
0.
+
+
Scon
serol
inclc
denalt)
of exp
H1/Int
0
•H-
-
0
0
-
-
0
-
0
-
0
++
-
0
0
58 of Statistical Strength and
Loalc analysis results consistency
lance Confirm. Exploratory: of apparent
/ ratio analysis AEI significances association
levels6 score" score (p-value) of Infections
Hi/Lo (p-value] Initial Final with exposure11
0 - 0 (0.16)
0 -
(-]
0 - (0.12) 0 (0.12)
0 - (0.20] 0 (0.10)
(-)
(-)
(-)
0 -
0 -
_ (_)
0 - [0.11] 0 (0.11)
++ + (0.07) (0.02) 0 (0.11) Good
0 0 (0.14)
0 0 (0.11)
0 - ,:
E20 (Echo 20)
4 (Sum 83) SE204 A
6 (1983) SE206 B
E24 (Echo 24)
5 (1982) SE245 B
4 (Sum 83) SE244 A
6 (1983) SE24B B
PL1 (Polio 1]
1 (Spr 82) SPL11
61 polio Immunized: A
186 not Immunized; A
6
9
1.7
0.7
1.8
1.7
7 0.5 0.6
7 4.4 Large
10 3.9 5.1
13 5.2 Large
8 5.9 Large
5 2.9 Large
0
0
0
0
0
0
0
0
0
0
0
0
(0.05)
- (0.03}
- (0.02)
- (0.04)
(0.01)
(0.01)
Good
continued..,
-------
TABLE 132. (CONT'D)
UJ
Infection
"Exposure" Jointly risk ratios0
Infection episode Indep. Exp Exp
Agent Depend, episode No. group level
Ob s period var. aroup8 1nf.D RR RR
PL2 [Polio 2)
1 (Spr 82] SPL21
61 polio Immunized: A
PL3 [Polio 3)
1 (Spr 82] SPL31
61 polio Immunized: A
RE1 (Reo 1)
1 (Spr 82] SRE11 A
RE2 (Reo 2)
1 (Spr 82) SRE21 A
ROT (Rotavlrus)
1 (Spr 82] SROT1 A
2 (Sum 82] SROT2 A
5 (1982] SROT5 B
3 [Spr 83} SROT3 A
4 (Sum 83) SROT4 A
6 (1983] SROT6 B
LEG (Leglonella pneumophl la
1981-83 SLEG7 B
9
7
7
7
16
13
3
4
7
3
6
9
1)
6
5.7
5.1
1.4
1.1
0.8
0.5
2.0
6.0
2.1
1.6
1.1
1.0
1.1
2.8
2.0
1.4
0.7
0.7
0
Large
2.1
2.1
Large
Large
1.0
0.7
Risk
ratio
score11
•H-
+
0
0
0
0
0
+
+
0
0
0
0
Scor
esro
Incl
denait
of exp
H1/Int
0
0
-
—
—
-
0
0
0
0
0
0
0
68 of Statistical Strength and
logic analysis results consistency
dence Confirm. Exploratory: of apparent
y ratio analysis AEI significances association
levels6 score' score [p-value] of Infections
Hi/Lo [p-value] Initial Final with exposure"
0 + (0.07) [0.11]
0 + (0.08)
0
- -
(-)
(-)
0 -
0 0 (0.10)
0 -
0 0 [0.21]
0 -
o - (-)
0 - (-)
POR (Sporadic serum neutralization viruses)
1 (Spr 82) SPOR1 A
2 [Sum 82) SPOR2 A
5 (1982) SPOR5 B
6 (1983) SPOR6 B
WWV (Viruses Isolated from
1 (Spr 82) SWWV1 D
2 (Sum 82] SWWV2 D
5 (1982] SWWV5 E
B (1983) SWWV6 E
13
9
5
10
wastewater]
12
15
61
11
0.9
0.5
0
O.B
1.0
1.7
1.7
0.6
1.2
2.5
0
0.8
0
4.8
1.8
0.9
0
0
0
0
0
+
+
0
0
-
-
-
-
-H-
++
-
0 - [-]
o - (-)
- (-]
(-)
(-)
-n- - (0.04) -H- [0.02] Good
+•»• ++ (0.02) (0.13) - Good
3
[_]
continued...
-------
TABLE 132. (CONT'D)
1'Exposure''
infection episode
Agent Depend.
Oba period var.
Jointly
Indep.
episode
group8
No.
1nf.b
Infection
risk ratios0
Exp Exp
group level
RR RR
Scores of
sero logic
Incidence
Risk density ratio
ratio of exp levels8
score" Hl/Int HI/Lo
Statistical
enalysis results
Confirm.
analysis
score'
[p-valuel
Exploratory:
AEI significance0-
score [p-valuel
Initial Final
Strength and
consistency
of epparent
association
of Infections
with exposure"
to
w
to
SNV (All serum neutralization viruses)
1
2
5
3
4
6
(Spr 82]
(Sum 82]
(1982)
(Spr 83]
(Sum 83)
(1983)
SSNV1
SSNV2
SSNV5
SSNV3
SSNV4
SSNV6
20
22
81
D 12
D 29
47
1
1
1
0
0
1
.3
.1
.3
.4
.9
.0
0
3
1
0
1
1
.8
.4
.5
.5
.6
.5
0
0
0
-
0
0
-
0
•H-
-
-
0
(-)
•H- - (0.17) ++ (0.04) Marginal
•H- 0 (0.15)
(-)
+ - (-)
+ -
a Classification criteria for the jointly Independent groups of exposure Infection episodes are given In Table 15.
b Based on all observed Individuals for whom an AEI exposure estimate was available.
c RR=IRu.j/IRLQ.
d From Table 100.
e From Tables 88 and P-47 In Appendix P. The score criterion for the Incidence density ratio (IDR] Is based on Its
confidence Interval (CI):
- IDFK1.0 + 90% CI does not Include 1.0
0 IDR>1.0, but 90% CI includes 1.0 ++ 95% CI does not Include 1.0
f From Tables 110 to 113. The confirmatory analysis score criterion Is based on the p-valua for the one-tailed Fisher's
exact test:
— pX).95 + 0.05p>0.15 -H- 0.010.25 +++ p<0.01
0 0.10
-------
when it is selected in the final model, since spurious variables can enter
stepwise regression models.
The risk ratio score provides a good overview because it examines
the infection incidence rates of both the exposure groups and the exposure
levels simultaneously. However, the RR score cannot assess the statistical
significance of the apparent associations which it identifies.
The strength of the association of infections and exposure in an infection
episode was determined based on the most statistically significant result
from the CA, ELR and IDR methods. Consistency in support of the association
among the other inferential methods (CA, ELR, IDR and RR score) was also
required. The precise criteria which were employed to classify the strength
and consistency of the evidence of association in a specific infection
episode as ''good'' or ''marginal'' based on the four inferential methods
were given in Table 19.
The infection episodes classified as having good or marginal evidence
of a strong and consistent association are identified in the last column
of Tables 131 and 132. The six infection episodes for which good evidence
of a strong and consistent association was found are:
''Good'' evidence;
o SE090 (echovirus 9 seroconversions in baseline)
o CKLB4X (Klebsiella infections in summer 1983)
o SE115 (echovirus 11 seroconversions in 1982)
o SPL11 (poliovirus 1 seroconversions in spring 1982)
o SWWV2 (seroconversions to viruses isolated from wastewater in
summer 1982)
o SWWVS (seroconversions to viruses isolated from wastewater in
1982)
The infection episodes for which marginal evidence of a weaker or less
consistent association was present are:
''Marginal'* evidence:
o CVIR2W (clinical viral infections excluding adeno and immunization
polio in summer 1982)
o CWWI4X (clinical infections to agents isolated from wastewater
in summer 1983) (Note: all eight CWWI4X infections were Klebsiella
infections)
o SSNV2 (all seroconversions to serum neutralization-tested viruses
in summer 1982)
It should be noted that SE090 is a control infection episode. This
obviously spurious asociation with aerosol exposure demonstrates the necessity
of investigating whether the apparent associations identified for the episodes
listed above may also have alternative explanations.
The infection events of some listed infection episodes are subsets
of the infection events of other listed episodes. For example, SE115 overlaps
SWW2; both are partial subsets of SWWV2, which is itself a subset of SSNV2.
333
-------
The eight CWWI4X infection events are the eight Klebsiella infection events
which comprise CKLB4X. Since Klebsiella was the agent of the CWWI4X infection
episode and since CKLB4X provided better evidence of association, CWWI4X
will be dropped from further scrutiny in deference to CKLB4X.
The LISS obtained additional pertinent information which was not employed
in the inferential methods used to compile the list of eight infection
episodes with good or marginal evidence of association with aerosol exposure.
Enteroviruses recovered from regular wastewater samples were identified
(see Tables P-5 in Appendix P, 25-27 and 39). Thus, whether the specific
agent(s) of the infection episode were recovered from the wastewater during
the irrigation period can be ascertained. A relative aerosol exposure
measure (RAEM) was calculated for each microorganism group monitored in
the aerosol sampling (see Table 42). Comparison of the period of occurrence
of the infection episode to the RAEM rank of the agent's microorganism
group in that season can determine whether the episode occurred in the
season of highest exposure to the agent via wastewater aerosols. Alternative
sources of exposure were also investigated. Contaminated drinking water
was evaluated for the subset of under 20 households whose drinking water
wells were being monitored at the time of the infection episode (see Table
46). The definition of a contaminated well and the procedures used to
determine association with infected donors were given in Section 5C.
A retrospective survey of routine fecal and requested throat swab
donors was conducted to determine the frequency with which they had eaten
food prepared at each of the restaurants in Wilson. Eating frequently
at restaurant A was found to be highly associated with aerosol exposure
among fecal donors (see Table 109). A special ELR analysis (Analysis 2)
was performed to evaluate the restaurant etiology as an alternative explanation
to wastewater aerosol exposure (see Table 126). Eating at the restaurants
was evaluated both as an alternative and as an additional explanation.
Another ELR analysis (Analysis 3) was performed to investigate alternative
explanations besides the restaurants. AEI was excluded from the eligible
predictor variables for infection episodes in which it had been significant
to determine if another variable would enter the model in its place.
A summary of the evidence from all of the additional data sources
described above is presented in Table 133 for each of the eight infection
episodes with good or marginal evidence of wastewater aerosol exposure
association. A review of this evidence regarding an apparently associated
episode may discredit the association by identifying a more plausible alter-
native explanation. Any episodes surviving this winnowing process are
more likely to be causally related to wastewater aerosol exposure.
For several of the episodes in Table 133, a more plausible alternative
explanation was identified. For CKLB4X, frequently eating food prepared
by restaurant A was identified by ELR Analysis 2 as the most significant
predictor variable of the Klebsiella infections. In addition, the episode
occurred in summer 1983, which was only the third highest season of aerosol
exposure to fecal coliforms. Thus, eating food prepared by restaurant
A is considered the more likely explanation for this Klebsiella infection
episode. For the spuriously associated episode SE090, there was evidence
334
-------
TABLE 133. SUMMARY OF EVIDENCE FOR
OF INFECTIONS WITH
INFECTION EPISODES SHOWING STRONG ASSOCIATION
WASTEWATER AEROSOL EXPOSURE
Inf. episodes with
strong end conslsten
evidence of aerosol
exposure assn
Agent Depend.
Ob s period var.
Evidence of aerosol
t exp. association
IDR
No. RR scores
1nf. score H/I H/L
Stet anal
CA ELR
final
p o
Recovery
of agent
Evidence 1n Irrlg.
of AEI weste-
Bssn8 water"
Rank of
Irrlg.
period
by RAEM
aerosol
dosec
Assoc.
with
contain.
drink.
water?"
p
Alternative explanations
GOOD EVIDENCE OF ASSOCIATION
Control Situation
E09 (Echo 9]
Baseline SE090
Exposure Situations
KLB (Klebslelle)
4 (Sum 83) CKLB4X
E11 (Echo 11)
5 (1982) SE115
PL1 (Polio 1)
1 (Spr 82) SPL11
£J 61 polio Immunized:
wi 186 not Immunized:
8 + - 0
8 ++ n1h n1
19 + ++ -H-
13 -H- 0 -H-
8 + 00
5 + 00
0.02 0.01
0.03 0.13
0.07 0.11
0.02 0.01
0.04
0.21
Good No
Good Presumed
Good Yes:
3-8-82
3-16-82
3-22-82
8-2-82
8-4-82
Good Yes:
3-8-82
3-22-82
4-19-82
nae
3
Higher
year
3
na
No
Maybe
0.21
No
.,-.,»
Within family spree dS
Eating frequently at
restaurant Af
Caucasians and large
households'
Nonef
WWV (Viruses Isolated from wasteweter)
2 (Sum 82) SWWV2
5 (1982) SWWV5
15 + ++ ++
61 + ++ -H-
0.24 0.02
0.02 XI .25
Good By def.
Good By def.
1
Higher
year
Maybe
0.23
[HH125]
No
Low Income end
Caucasians'
Fanners and pneumonia,-,
history'
continued...
-------
TABLE 133. (CONT'DJ
u>
u>
9\
Inf. episodes irlth Evidence of aerosol
strong and consistent exp. association
evidence of aerosol Stat anal
exposure asen IDR CA ELR Evidence
Agent Depend. No. RR scores final of AEI
Obs oerlod var. Inf. score H/I H/L o o aeon8
WflBDML EVDBBE OF ASSOCIATION
Exposure Situations
VIR (Viruses, excluding adeno and immunization polio)
2 (Sun 82) CVIR2W 12 + nl nl 0.13 0.07 Marginal
SNV (All serum neutralization viruses)
2 (Sum 82) SSNV2 22 0 0 ++ >0.25 0.04 Marginal
a From Tables 131 and 132.
b From Tables 22, 23, P-5 In Appendix P, 25-27
and 39.
c From Table 42. For single Irrigation periods, rank
of 1 is if period of observation covers irrigation
season of highest aerosol dose, rank of 4 if observa-
tion period covers Irrigation season of lowest aerosol dose.
Rank of Assoc.
Recovery Irrlg. with
of agent period contam.
In irrlg. by RAEM drink.
waste- aerosol water?"
water" dosec p Alternative explanations
Yes. 1 No Eating frequently
Agents of at restaurant A*
5-10 Inf.
recovered
Some 1 Maybe None''
recovered 0.22
[HH125]
d From Table 46.
e na - not applicable.
f From exploratory logistic regression
126 or 127 vs. Table 125)
g Three infected donors in same household.
h n1 - not investigated.
1 From Table 125.
at
(Tables
-------
of within household spread of the echo 9 infections in ''high exposure''
household 451: the youngest child had two seroconversions and his two
next older siblings had one seroconversion each to echo 9 among the six
family members observed during the baseline period.
Episode CVIR2W had only marginal evidence of aerosol exposure association.
CVIR2W occurred in summer 1982, the season of maximum aerosol exposure
to enteroviruses. For five of the ten infections to coxsackieviruses and
echoviruses in CVIR2W, the specific agent was also recovered and identified
from the wastewater sprayed during the summer 1982 irrigation. However,
eating frequently at restaurant A was identified by ELR as an alternative
explanation to AEI. The statistical evidence does not permit an inference
whether eating at restaurant A or aerosol exposure is a more probable expla-
nation for the CVIR2W episode.
Episodes SE115, SWWV2 and SWWV5 displayed good evidence of aerosol
exposure association, but alternative explanations were identified by ELR
for each of these episodes. Echovirus 11 was recovered from the wastewater
on five occasions during the 1982 irrigation periods. All SWWV2 and SWWV5
agents were also recovered, because this was the definition of the WV
episodes. All three episodes also occurred in the year or season of highest
enterovirus aerosol exposure. However, two SWWV2 infected donors in a
very high exposure household on the Hancock farm obtained their drinking
water from a well which was heavily contaminated in June 1982. The SE115
infections also might be associated with contaminated drinking water.
The infection rate among donors who drank contaminated water was much higher,
both for SE115 and SWWV2, but the p-values for the association were only
0.21 and 0.23, respectively, possibly due to the small sample sizes. Explora-
tory logistic regression identified Caucasians and large households as
a better fitting alternative explanation to high aerosol exposure for SE115.
Low income households and Caucasians were selected by ELR as a poorer fitting
alternative explanation to high AEI for SWWV2. ELR found that SWWV5 infections
were not related to degree of aerosol exposure. Instead, ELR selected
farmers and a history of pneumonia as predictor variables for SWW5. The
evidence of episodes SE115, SWW2 and SWWV5 is inconclusive regarding whether
aerosol exposure or the identified alternative explanation(s) were the
actual risk factors in these episodes.
There is only marginal evidence from the four inferential methods
that episode SSNV2 was associated with wastewater aerosol exposure. The
episode occurred in summer 1982, the season of highest aerosol exposure
to enteroviruses. Based on very fragmentary contaminated drinking water
data (including the two donors from the very high exposure household on
the Hancock farm in the SWWV2 episode above), there is an indication, albeit
nonsignificant at p=0.22, that episode SSNV2 might be associated with contami-
nated drinking water. However, no alternative explanations to AEI were
identified by ELR for SSNV2. The available evidence indicates the association
of SSNY2 with aerosol exposure is better than marginal, but still inconclusive
because the alternative explanation of contaminated drinking water is quite
plausible.
337
-------
There is strong evidence that the poliovirus 1 seroconversions in
spring 1982 were associated with wastewater aerosol exposure. Furthermore,
SPL11 is the only infection episode in which all four inferential methods
provided evidence of a significant association. The Cochran-Mantel-Eaenszel
confirmatory analysis showed a significant association (p=0.02) of polio
1 seroconvers ions between January and June 1982 with the high aerosol exposure
group in the spring 1982 irrigation, when controlling for the effects of
polio immunizations during this time period. The groups were balanced
regarding previous polio 1 titers. ELR selected polio immunization in
spring 1982, low prior antibody level, and a high degree of aerosol exposure
as strong predictor variables for SPL11 seroconvers ions in a well-fitting
logistic model. Each variable may be considered a distinct risk factor
for polio 1 seroconvers ions since each made a strong contribution to the
ELR model. No alternative explanations to high AEI were identified by
ELR. Poliovirus 1 was recovered three times from the pipeline wastewater
sprayed in spring 1982. Therefore, the poliovirus 1 seroconversions in
spring 1982 provide substantial evidence of a causal association with wastewater
aerosol exposure.
It is noteworthy that spring 1982 is estimated to be one of the irrigation
periods in which the more highly exposed LISS participants received a relatively
low cumulative dose of enteroviruses from wastewater aerosol exposure (see
Table 42). Because poliovirus serology was not performed after June 1982,
any poliovirus seroconversions occurring thereafter were not observed by
the LISS. Summer 1982 appears to have been the season of highest poliovirus
aerosol exposure (see Tables 39 and 42), with summer 1983 a distant second.
Therefore, in order to fully assess the relationship between infections
and wastewater aerosol exposure, it would be necessary to perform the poliovirus
serology through October 1983 and to analyze any observed poliovirus infection
episodes.
338
-------
SECTION 6
DISCUSSION
A. PRIOR WASTEWATER AEROSOL HEALTH EFFECT STUDIES
Measuring the effect of wastewater aerosol exposure on an individual's
health is complicated by the variety of potential infectious agents as
well as the range of host responses. Unless disease symptoms are manifested,
the interaction between microbial agent and host would pass unnoticed.
Only by clinical observation can microbial infection be demonstrated, and
then only if the correct analyses are being done. These qualifications
must be considered in evaluating existing literature on the association
of wastewater aerosols and disease.
Previous efforts to link wastewater exposure with human health effects
have utilized a variety of observational approaches including retrospective
and prospective studies at sewage treatment plants and wastewater irrigation
sites. Using data collected between 1965 and 1971 as part of an intensive
community health study, Fannin and associates (1980) evaluated the occurrence
of acute gastrointestinal and respiratory diseases in families residing
within 2400 m of an activated sludge treatment plant (1 HGD) in Tecumseh,
Michigan. While persons living within 600 m of the plant had reported
excess illnesses during summer months when compared to more distant households,
the researchers concluded that this elevated illness rate was more likely
to have been related to the high density of low socioeconomic families
in that area rather than to the treatment plant.
Two prospective studies which utilized both clinical and environmental
monitoring in areas around wastewater treatment plants have been reported.
Johnson et al. (1980a) collected both baseline and operational year (9
months) data from families residing 350 m to 5 km from a new 30 MOD activated
sludge treatment plant in Schaumburg, Illinois. Air sampling at the plant
site showed that while indicator organisms were elevated, their numbers
dropped to background levels at residential distances. Furthermore, enteric
viruses were not detected in the air sampling. Self-reported illnesses
as well as clinical microbial isolation and viral serology against 31 agents
were used as tools to investigate the effect of wastewater aerosols on
the study population. Although nearby residents reported a higher incidence
of skin disease and gastrointestinal disorders during the operational year,
virtually no serologic or clinical evidence was associated with proximity
to the treatment plant. However, the pattern of echovirus 29 antibody
response showed a slight association with aerosol exposure.
Working in a 1.6-km area surrounding an established sewage treatment
plant (200 MOD), Carnow et al. (1979) conducted a similar study following
a more intensive clinical sampling regime over an 8-month period. While
aerosol sampling showed elevated fecal coliform counts within the plant,
339
-------
downwind distances of 0.8 km and 1.6 km showed background levels of this
indicator. No correlations were fonnd between calculated exposure indices
and the rate of self-reported illnesses or the microbial infection rates
determined by agent isolation or antibody response.
Finally, an environmental monitoring program was "coupled with an evaluation
of retrospective school attendance records to investigate the potential
health hazard posed by the operation of a new advanced wastewater treatment
plant (approximately 10 MGD) located adjacent to an elementary school in
Tigard, Oregon (Camann et al., 1980). The plant's aeration basin (approximately
400 m from classrooms and 250 m from the school playground) was noted as
a source of indicator bacteria and coliphage, but no enteric viruses were
detected. No overall effect of plant startup and operation was seen on
school attendance relative to baseline school years and to five control
schools. It was noted, however, that several periods of increased absenteeism
occurred among the youngest students (first and second grade) after the
treatment plant began operation.
Taken together, no definitive evidence can be found linking wastewater
aerosol exposure to either illness or infection in the general population
residing in areas around wastewater treatment plants in the United States.
A similar conclusion was reached by Clark et al. (1981) after observing
a population with high occupational exposure, namely sewer and sewage treatment
workers and their families. In a 3-year prospective seroepidemiologic
study involving workers in three metropolitan areas (Cincinnati, Chicago
and Memphis), there was no consistent evidence for increased parasitic,
bacterial or viral infections based either on agent cultivation or on antibody
surveys. In a few instances, level of antibody to certain viruses in wastewater
workers appeared to be related to level of exposure to wastewater aerosols.
An increased level of minor gastrointestinal illness was noted during the
spring season among inexperienced, sewage-exposed workers.
A study by Linnemann and coworkers (1984) of Muskegon County, Michigan,
workers exposed to wastewater spray irrigation failed to show any differences
in illness or viral isolation rates between the workers and a control group.
Although antibody titers to coxsackievirus BS were significantly higher
in spray irrigation nozzle cleaners, seroconversions were not documented.
Aerosol exposure as a result of irrigation with wastewater provides
yet another setting in which health effects on the surrounding community
can be evaluated. An initial retrospective study in Israel implicated
wastewater use in kibbutzim with an increased incidence of illness (Katzenelson
et al., 1976). However, a more complete retrospective study of the incidence
of enteric disease associated with wastewater utilization by kibbutzim
in Israel (Shuval et al., 1983) raised serious questions about the results
of the original study of Katzenelson et al. (1976). An excess risk of
enteric disease was not associated with wastewater irrigation except in
the 0-4 age group of kibbutzim of a ''switch'' category during periods
of wastewater irrigation compared to periods during which wastewater was
not used. This excess risk of total enteric disease ranged from 32 to
112% in this single group, a finding far different from the two- to fourfold
increase of cases of salmonellosis, shigellosis, typhoid fever, and hepatitis
340
-------
reported from the kibbutzim practicing wastewater irrigation in the first
study. Subsequently, Fattal et al. (1984) reported a prospective epidemic-
logical study in 30 kibbutzim having varying degrees of wastewater utilization
for irrigation. Paired sera were drawn approximately 1 year apart (1980-81)
and tested for antibody to eight enteroviruses and varicella-zoster virus
(as a negative epidemiologic control). Emphasis was placed on obtaining
samples from young children (6 months to 5 years old) who would be more
susceptible to viral infection. Serological results indicated that antibody
to echovirus 4 was statistically more prevalent in kibbutzim practicing
spray irrigation of wastewater within 600 m of the residential area (Category
A) when compared to similar settlements in which wastewater irrigation
was at a distance of 2.1000 "> (Category B) or in which noneffluent water
was used for irrigation (Category C) . Notably, this increased antibody
prevalence was observed in those Category A kibbutzim using wastewater
from neighboring communities (as opposed to wastewater generated within
the kibbutz itself).
Jakubowski (1983) has critically reviewed and evaluated previous wastewater
health effects studies and has noted that the preponderance of data was
negative. However, he observes that interpretation of the significance
of the data, whether negative or positive, of all the studies is limited
by the low numbers of highly exposed persons and the inability to adequately
and quantitatively determine that exposure. None of the previous studies
has investigated the health effects on residential populations exposed
to sprinkler systems that apply wastewater to land according to EPA design
criteria. The LISS was designed for this purpose and to answer many of
the criticisms of previous studies, such as the reliance on self-reported
illness, long-recall surveys or retrospective analysis of health data.
The LISS involved a variety of health watch activities including serology
for viruses present in the wastewater, routine fecal specimens for bacterio-
logical and virological analyses, analyses of illness specimens, tuberculin
skin testing, household self-reports of illness and activity diaries.
The health watch activities were supplemented by environmental monitoring
of aerosols, wastewater, and drinking water. This study differs from previous
U.S. studies in that, while both illness and infection were monitored,
primary emphasis was placed on intensive infection surveillance.
Placed alongside these studies which used various epidemiological
approaches to evaluate the effects of wastewater exposure on human health,
the LISS has several unique attributes. The spray irrigation system at
the Hancock farm was new, thus allowing baseline monitoring of the surrounding
population. Once irrigation commenced, temporal exposure and infection
data were collected over the course of multiple exposure/irrigation events.
Perhaps more importantly, considering the positive findings of the Israeli
study reported in 1984 and the lack of an association in the treatment
plant studies, the wastewater used for irrigation on the Hancock farm was
imported from a large metropolitan area. Thus, the LISS population was
exposed to microorganisms circulating within another community, thereby
increasing the likelihood of detecting an episode of infection introduced
by wastewater irrigation. Another similarity between the Israeli studies
and the first year of irrigation on the Hancock farm was the relative microbial
341
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strength of the wastewater sprayed directly from the pipeline during 1982.
Finally, unlike the other health effects studies completed to date in the
United States, aerosol sampling at the Hancock farm repeatedly demonstrated
the presence of human viruses downwind of the spray source. Thus, it would
appear that of the studies completed to date, the LISS was most likely
to demonstrate a health response to wastewater aerosol exposure.
B. SUMMARY OP LISS FINDINGS
Wastewater spray irrigation at the Hancock farm commenced on February 16,
1982. The LISS monitored infection events and acute illness in the study
population from July 1980 through September 1983 for possible association
with irrigation.
Findings from Wastewater and Aerosol Data
The LISS monitored four major periods of wastewater irrigation at
the Hancock farm. These periods were termed spring 1982 (February 16-April 30,
1982), summer 1982 (July 21-September 17, 1982), spring 1983 (February 15-
April 30, 1983), and summer 1983 (June 29-September 20, 1983). The quality
of the wastewater used for irrigation varied substantially by irrigation
period. All of the irrigation wastewater was obtained via pipeline directly
from the Lubbock SeWRP in the spring 1982 irrigation period, since operation
of the reservoirs had not been approved at that time. The quality of this
pipeline effluent was similar to that of a low quality primary effluent,
as determined by physical and chemical analyses (see summary Table 21 and
source Table P-l in Appendix P). Pipeline wastewater comprised 64%, 0%
and 1%, respectively, of the total applied by spray irrigation in the three
following irrigation periods. There was some improvement in pipeline wastewater
quality during summer 1982 and spring 1983, but it did not reach the quality
expected of secondary effluent until summer 1982. Reservoir wastewater
was more consistently of secondary effluent quality in all three of these
periods. This observation is important, since the majority of irrigation
wastewater used during 1982 came via pipeline directly from the SeWRP,
while essentially all the wastewater applied during 1983 was from the irrigation
reservoirs.
The wastewater utilized at the Hancock farm contained a broad spectrum
of enteric bacteria and viruses. Spray irrigation of wastewater received
via pipeline directly from the Lubbock SeWRP was found to be a substantial
aerosol source of each group of microorganisms monitored in the aerosol
sampling (i.e., fecal coliforms, fecal streptococci, mycobacteria, Clostridium
perfringens. coliphage, and enteroviruses) . Microorganism levels in air
downwind of spray rigs using pipeline wastewater were found to be significantly
higher than upwind levels: fecal streptococci levels to at least 300 m
downwind, and levels of fecal coliforms, mycobacteria and coliphage levels
to at least 200 m downwind. The downwind levels were also significantly
higher than the background levels in ambient air outside the homes of par-
ticipants: fecal coliform levels to beyond 400 m downwind, mycobacteria
and coliphage levels to at least 300 m downwind, and fecal streptotocci
levels to at least 200 m downwind. Operation at night and at high wind
speeds appeared to elevate microorganism levels to greater downwind distances.
342
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Enterovirnses were recovered in the aerosol at 44 to 60 m downwind of irrigation
with pipeline wastewater on each of four virus runs. The geometric mean
enterovirus density in air was 0.05 pfn/m*, although a much higher density
(17 pfu/m^) was sampled on one run in August 1982. Spray irrigation of
reservoir wastewater was also found to be source of aerosolized fecal coliforms,
fecal streptococci and coliphage, sometimes to d-ownwind distances of at
least 125 m.
Since microorganism densities were much higher in the wastewater from
the pipeline than from the reservoirs, the exposure which most of the study
population received to most microorganisms via the wastewater aerosol was
greater in 1982 than in 1983. The irrigation period in which aerosol exposure
at a given distance downwind was estimated to be highest was: summer 1982
for enteroviruses, summer 1982 for fecal coliforms, and spring 1982 for
fecal streptococci (see Table 42, using estimates for 150 to 249 m downwind
when available). For each of the microorganism groups with adequate aerosol
and wastewater monitoring data, summer 1982 was the irrigation period when
most of the more highly exposed study population received either their
largest or their second largest cumulative dose from the wastewater aerosol.
Findings from Self-reported Illness Data
Disease surveillance did not disclose any obvious connection between
illness and degree of wastewater exposure. The self-reported illness data
varied in consistency, reliability and completeness over the July 1980-
September 1983 period of surveillance, with the better quality data obtained
during the years of wastewater irrigation. In addition, self-reports of
illness are always subject to respondent bias.
Nevertheless, it is of interest and may be significant that the partici-
pants in the high exposure level (AEI>5) reported the highest rate of illness
shortly after the onset of wastewater irrigation, both in spring 1982 and
in summer 1982. The excess total acute illness among high exposure level
participants over the spring 1982 irrigation period occurred primarily
during February 14-27, 1982, in the initial 2 weeks of wastewater irrigation
at the Hancock farm. The extent to which this reflects actual illness
as opposed to reporting bias by high exposure participants has not been
ascertained. The high exposure level participants also reported a significant
excess of total acute illness in August 1982, primarily during August 15-28
(after more than 3 weeks of wastewater irrigation had elapsed). The high
exposure level participants did not report a comparable excess of acute
illnesses during either irrigation period in 1983. This pattern of excess
illness during both irrigation periods in 1982 is consistent with the hypothesis
of an association of illness with exposure to wastewater irrigation: the
pattern appeared both upon initial wastewater exposure and in the summer
1982 irrigation period which produced highest exposure to microorganisms
in the wastewater aerosol. However, the patterns did not persist throughout
either irrigation period in 1982. In addition, the effects of known risk
factors such as age and socioeconomic status have not been taken into account.
For total acute illness, the crude incidence density ratios of the high
exposure level to the intermediate (1
-------
periods. Thus, if not a reporting artifact, a small excess rate of illnesses
might have been associated with the initial and heaviest periods of microor-
ganism emission from wastewater irrigation. Since the agents which the
LISS monitored clinically and serologically show a very high proportion
of asymptomatic infection, it is difficult to correlate the findings for
self-reported illness with those for the clinically and serologically detected
infections.
Findings from Nonepisode Occurrences of Infections
The LISS detected the occurrence of a variety of infections which
could not be analyzed as infection episodes. Many of these infections
were detected in a nonsystematic manner (e.g., from illness or requested
specimens) which precluded a determination of incidence for the study popu-
lation. Other infections occurred too infrequently to constitute an infection
episode. The results obtained from such occurrences of infection are summarized
in Table 134 by infectious agent.
The occurrence of enteric Gram-negative bacteria (EGNB) at moderate
and heavy levels in the throats of both healthy and ill study participants
was both frequent and widespread between July 19 and October 12, 1982.
This phenomenon was first identified in an extended illness investigation
of a household in Wilson. The illness investigation established that the
household environment was strongly associated with the continuing EGNB
throat infections and identified the evaporative cooler as a potential
source of infections. Among illness throat swab donors during the July 19-
October 12 time period, use of an evaporative cooler for home air conditioning
was associated (p=0.02) with the EGNB throat infections. A throat swab
survey of healthy donors in September 1982 established an EGNB throat infection
prevalence of 2ffh in healthy adults and teenagers at that time. The prevalence
of these inapparent EGNB throat infections was higher in donors who frequently
ate food prepared at restaurant A, who had high wastewater aerosol exposure,
and whose homes used evaporative coolers for air conditioning. However,
none of these potential risk factors were significantly associated with
the inapparent EGNB throat infections.
Host of the infection occurrences presented in Table 134 appear to
have been unrelated to wastewater irrigation. The highest or only period
of occurrence of some infections was in the LISS baseline before irrigation
commenced. In this category were Yersinia enterocolit ica infections, non-
tuberculosis mycobacteria infections, and hepatitis A infections. Entamoeba
histolvtica infections occurred too infrequently to identify a period of
higher incidence. The highest period of occurrence of other infections
was between irrigation periods. The infections to Group A streptococci,
Salmonella. EGNB in illness stools, and virus-like particles detected by
EH in illness stools belong in this category. Other infections occurred
primarily during an irrigation period, but in donors with lower average
wastewater aerosol exposure (i.e., mean AEI) than the noninfected donors.
In this category were the throat infections to Group A streptococci and
EGNB among ill donors in summer 1982.
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TABLE 134. SUMMARY OF FINDINGS PERTAINING TO POSSIBLE ASSOCIATION WITH NASTEWATER
IRRIGATION FOR OCCURRENCES OF INFECTIONS NOT CLASSIFIED AS INFECTION EPISODES
Aaent
BACTERIA
Group A
streptococci
Salmonella
Other major enteric
bacterial pathogens
Enteric Gram-negative
bacteria (EGNB)
(M or H level]
Non-tuberculosis
mycobacteria (NTM]
VIRUSES
Viruses-isolates
Vi ruses-EM detections
Hepatitis A
Coronavi rus-like
particles (CVLP)
uintHS
Parasites
Repiratory illness
following aerosol
exposure
Methods of
observation
Illness TS
Illness investiga-
tion
RF
RF
RF
Illness fecal
Illness investiga-
tion
Illness TS
Healthy donor TS
survey
Illness fecal
Tuberculin skin
tests
Illness fecal
Illness fecal
Serosurvey
EM of RF
Serosurvey
OBJ3 survey-RF
Illness investiga-
tion and RF
Period of greatest
occurrence (0),
prevalence (P)
or incidence [I]:
period [ratet %]
0: Apr- Jun 1983 (31X)
0: Jul-Sep 1982 (24X]
0: Jun-Jul 1982
P: Jun 1982 (1XJ
Y. entertocolitica,
0: Jun-Jul 1982 (4%)
C. jejuni and Shigella
not found
None found
0: Jul-Sep 1982
0: Jul 19-Oct 12,
1982 (24%)
P: Sep 19-22, 1982
(26*)
0: May-Jun 1983 (36*)
I: Jun 1880-Jun 1981
(2%)
0: Jul-Sep 1982 (67%)
(None)
I: Jun-Dec 1980 (0.3%)
0: Jul-Sep 1982 [18%]
E. histolytica (I):
G. lamblia (P): Jun-
Aug 1983 (2%)
Aug 6-17, 1982
Apparent
association with
wastewater aerosol
exposure? [p-value]
No-between irrig.
periods
No- lower mean AEI
No [extremely
unlikely]
(see Illness Inves-
tigation]
No-baseline
(No)
(No)
No (unlikely)
No-lower mean AEI
Unlikely [0.18]
No-between irrig.
periods
No-baseline
Unknown-i nsuf f 1 ci ent
data
No-between irrig.
periods
No-baseline
Unlikely (0.12)-onset
unknown
No
Unlikely, despite
(0.03)
Possible (evidence
consistent with
aerosol hypothesis]
Alternative explanation(s)
[p-valua]
Contain, drinking water
(?), food (?)
Evaporative cooler,
Public swimming pool (?)
Evaporative coolers (0.02)
Eating at rest. A? (0.14)
Evaporative coolers? [0.22]
f-^
Household cluster
Contain, drinking water
Person-person spread (?j
Contaminated drinking
water (?)
References
Table 59
Section 5.F
Table 70
Tables 70
and 71
Table 70
Table 64
Table 68
Section 5.F
Tables 58
and 61
Tables 62
and 63
Table 64
Table SO
Tables 64
and 66
Tables 64
and 66
Section 5.1
Tables 93
and 86
Section 5.1
Tables 81
and 92
Section 5.F
TS - throat swab
RF - routine fecal specimens
EM - electron microscopy
-------
There are insufficient data to determine whether other infection occur-
rences presented in Table 134 were associated with wastewater irrigation.
Insufficient illness fecal specimens were obtained during the summer 1982
irrigation to determine if the mean AEI of donors with viruses recovered
was higher than for the virus-negative donors.
<• -
Although not significantly associated (p=0.18), the inapparent EGNB
throat infections detected in the September 1982 survey of healthy donors
might be related to aerosol exposure. However, since the concurrent EGNB
throat infections in ill donors were associated with evaporative cooler
use at home, the evaporative cooler hypothesis may also be a more likely
explanation for the inapparent infections (despite the lack of significant
association with evaporative coolers: p=0.22).
The occurrence of coronavirus-like particles (CVLP) in routine fecal
specimens in summer 1982 is unlikely to have been related to wastewater
irrigation. The CVLP-infected donors had a higher average aerosol exposure
than the EM-negative donors, but the difference was not significant (p=0.12).
In addition, since many CVLP-infected donors were persistently positive,
the onset of these CVLP infections may have preceded the summer irrigation.
The prevalence of Giardia lamblia in routine fecal specimens in summer
1982 is also unlikely to have been related to wastewater irrigation. All
three of the five Giardia-posit ive donors who had high aerosol exposure
were members of the same household. Since their Giardia infections cannot
be considered independent, the apparently significant association (p=0.03)
with wastewater exposure is invalid. The household's contaminated drinking
water well or hand-to-month transfer of cysts are considered more probable
routes of exposure.
The investigation of respiratory illnesses in children following aerosol
exposure (see Section 5F) suggests a more likely association with wastewater
irrigation than any of the other infection occurrences summarized in Table
134. Respiratory illnesses attributable via clinical isolates to coxsackie-
virus B4 and Achromobacter xylosesidans were documented. Both of these
agents were presumably present in the wastewater to which the ill children
appear to have been exposed by spray irrigation of pipeline wastewater.
The evidence of this illness incidence is consistent with the hypothesis
that wastewater microorganisms transmitted by wastewater aerosol from spray
irrigation infected and produced respiratory illness in the subject children.
However, since plausible alternative modes of transmission such as person-
to-person spread and contaminated drinking water were not investigated,
the evidence for the aerosol exposure hypothesis is inconclusive.
Findings from Seroconversion Incidence Densities
An overview of the association of serologically detected infections
with exposure to wastewater aerosols was obtained by comparison of the
seroconversion incidence densities for serum donors in the three levels
(or two groups) of aerosol exposure, for both the entire baseline (June
1980-January 1982) and the entire irrigation (January 1982-October 1983)
periods of observations. The high exposure level participants had a higher
346
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incidence density of coxsackievirus B4 infections versus intermediate level
participants during the entire irrigation period (see Table 84). In contrast,
the high exposure level had no elevated infection incidence density to
specific agents in the baseline period. Based on test-based 95% confidence
intervals for the crude incidence density ratios, the high exposure group
(AEIX3) had a significantly greater incidence of infections to coxsackievirus
B2 and echovirus 11 over the irrigation period, but a significantly greater
infection incidence only to one agent, echovirus 9, during the baseline
period (see Table 85). While extraneous variables were not investigated
as alternative explanations, these results do appear to suggest an association
between enterovirus infections and wastewater irrigation exposure.
A more sensitive analysis can be performed on groups of agents, provided
the agent-person-time observations are independent. In the baseline period,
the high exposure level had the lowest infection incidence densities of
the three exposure levels to all of the adenoviruses tested, to all coxsackie
B viruses tested, and to all echoviruses tested. In the irrigation period,
the high exposure level had the highest incidence densities of infection
by all coxsackie B viruses tested and by all echoviruses tested. Moreover,
in the irrigation period the high exposure level also had the highest incidence
density of infections to all of the tested viruses which had been recovered
from the irrigation wastewater; the incidence density ratio of the high
to the intermediate exposure level was significantly greater than 1.0 (see
Table 86). Again, extraneous variables are not taken into account in this
simplistic analysis. Nevertheless, these crude incidence densities suggest
a probable association between seroconversions (especially to viruses recovered
from the wastewater) and wastewater aerosol exposure. The crude incidence
density ratios of the high exposure level to the intermediate and low exposure
levels during the irrigation period were 1.8 and 1.5, respectively, for
the viruses recovered from the wastewater, indicating some excess risk
of viral infection from wastewater aerosol exposure.
Findings from Risk Ratio Scoring of Infection Episodes
A risk ratio score was assigned to each infection episode based on
the infection incidence rates in the exposure levels and in the exposure
groups (see Tables 100 and 101). The risk ratio score was symmetric with
respect to the high and low exposure categories, with a positive score
assigned if a pattern of excess infections occurred in the high exposure
subjects and a negative score assigned if the same pattern of excess infections
occurred in the low exposure subjects. Frequency distributions of risk
ratio scores were formed for six jointly independent and mutually exclusive
groups of infection episodes (see Table 102). For single and sporadic
agents, the risk ratio scores of the control episodes (Group C) were symmetric
about 0, as expected. However, there was a highly significant (p=0.002)
excess of positive scores among exposure episodes whose duration spanned
single irrigation periods (Group A) and a borderline significant (p=0.09)
excess of positive scores among exposure episodes of 1-year duration (Group
B) . These results suggest that an excess risk of infection was associated
with wastewater aerosol exposure. The seasonal distribution of positive
scores in Group A was correlated with seasonal microorganism dose via aerosol
exposure. The results from the risk ratio score distributions for grouped
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agents were similar. The risk ratio score approach provided evidence of
a stable and dose-related association between infection events and wastewater
aerosol exposure in the infection episodes observed by the LISS.
Findings from Confirmatory Statistical Analysis of Infection Episodes
r^ •
The preliminary analysis found that the high (AEI>3) and low (AEK3)
exposure groups were generally well balanced with regard to infection risk
factors, including age, gender and previous titer. The high exposure group
of serum donors had a significantly higher rate of polio immunizations
during spring 1982. The high exposure group of fecal donors did contain
significantly more farmers in the summer irrigation seasons. The high
exposure fecal donors also ate food prepared at restaurant A very significantly
more often in all four irrigation seasons. The exposure groups were stratified
on polio infection status in comparing poliovirns seroconversion rates.
No other stratification was done, because the number of observations was
too small. After looking at the distribution of infected donors within
households to investigate within household transmission, it was decided
that the distribution in any single episode was not inconsistent with the
hypothesis that the infections occurred independently in that episode.
A one-sided Fisher's exact test was employed in the confirmatory analysis
(CA) to determine if the high exposure population had a larger infection
incidence rate than the low exposure population. The test was applied
to each agent in all exposure seasons for every agent which produced an
infection episode in any of the seasons. The tests for association of
infection incidence and wastewater exposure were significant at the a=0.05
level for seven infection episodes:
o CKLB4X—Klebsiella in summer 1983 (p=0.03)
. o CWWI4X—clinical isolates of wastewater agents in summer 1983
(p=0.02)
o SCB25—coxsackievirns B2 in 1982 (p=0.05)
o SE090—echovirus 9 in baseline (p=0.02)
o SE246—echovirus 24 in 1983 (p=0.03)
o SPL11—poliovirus 1 in spring 1982 (p=0.02)
o SWWV5—seroconversions to wastewater isolates in 1982 (p=0.02)
The actual rate of positive associations in the exposure episodes
appears to have been at least twice as large as the false positive rate.
Among infection episodes involving single and sporadic agents, the positive
rates were 4% in 27 independent control episodes (Group C), 6% in 31 independent
single season exposure episodes (Group A), and 11% in 19 independent year-
duration exposure episodes (Group B). For infection episodes involving
grouped agents, the positive rates were 0/1 for the control episode, 1/8=13%
for independent single season exposure episodes (Group D) and 1/2=50% for
independent year-long exposure episodes. The actual rate of positive associ-
ations in control episodes was approximately equal to the expected false
positive rate. In contrast, the actual rate of significant associations
exceeded the false positive rate in each of the four independent groups
of exposure episodes.
348
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In conclusion, an excess of statistically significant associations
of the presence of infection with wastewater aerosol exposure was found
in the confirmatory analysis. The interpretation of the epidemiological
importance of these significant associations must be moderated by recognition
of the possibility that some of the tests may be significant only by chance
and that some imbalances in the two poulations may prdvide alternate explana-
tions for the observed differences. On the other hand, the number of detected
increases in incidence rates associated with the wastewater irrigation
may be underestimated, considering the relatively modest power of the tests
to detect small differences. The certainty of the results is also lessened
when the observational nature of the study and the difficulty inherent
in determining appropriate assignment of individuals to the exposure groups
are considered.
Findings from Exploratory.Statistical, Analysis of Infection Episodes
The exploratory logistic regression (ELR) analysis was conducted to
investigate the association, if any, between presence of infection and
degree of aerosol exposure (i.e., AEI), while controlling for the effects
of other variables. Significant associations with AEI at a final step
p-value below 0.05 were identified in four infection episodes:
o SE090—echovirns 9 in baseline (p=0.01)
o SPL11—poliovirus 1 in spring 1982 (p=0.01)
o SWWV2—seroconversions to wastewater isolates in summer 1982
(p=0.02)
o SSNV2—all seroconversions to serum neutralization-tested viruses
in summer 1982 (p=0.04)
The significant covariates are presented in Table 125. The goodness-of-
fit of each of these models was excellent.
The ELR analysis investigated alternative explanations to AEI (including
eating food prepared at the restaurants in Wilson) for the infection episodes
showing good or marginal evidence of aerosol exposure association by the
four inferential methods employed. The alternative explanations identified
are summarized in Table 133. Investigation of the route of wastewater
exposure in the infection episodes where AEI was a significant predictor
variable provided some evidence supporting all three routes (i.e., wastewater
aerosol, direct contact with wastewater, and spending time in the irrigation
environment on the Hancock farm). However, the aerosol exposure route
received the most supporting evidence.
Evidence of Association of Specific Infection Episodes with Wastewater
Aerosol Exposure
Specific infection episodes which displayed good or marginal evidence
of association with wastewater aerosol exposure were identified by comparison
of results from four methods of investigation (i.e., confirmatory statistical
analysis, exploratory logistic regression analysis, confidence intervals
of incidence density ratios, and risk ratio scoring). Additional evidence
was considred regarding recovery of the infectious agent from the irrigation
349
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wastewater, seasonal correspondence of the infection response to aerosol
dose, association with contaminated drinking water, alternative risk factors
identified by ELR, and within-household transmission of infections.
A summary of this evidence was presented in Table 133 for each of
the eight infection episodes with good or marginal evidence of wastewater
aerosol exposure association. Any episodes in which a more plausible alter-
native explanation was not identified are more likely to have been causally
related to wastewater aerosol exposure.
The eight infection episodes were placed in three categories based
on the likelihood of causal association of the infection events with wastewater
aerosol exposure:
1) More plausible alternative explanation identified:
o Episode CKLB4X (Klebsiella infections in summer 1983)
—alternative: eating food prepared at local restaurant A
o Spurious control episode SE090 (echovirus 9 seroconversions
in the baseline period)
—alternative: within-household spread
2) Both aerosol exposure and identified alternative explanation(s)
are plausible risk factors (evidence inconclusive):
o Episode CVIR2W (clinical viral isolates excluding adenoviruses
and immunization-associated polioviruses in summer 1982)
—alternative: eating food prepared at local restaurant A
o Episode SE115 (echovirus 11 seroconversions in 1982)
—alternatives: o contaminated drinking water
o Caucasian, large household
o Episode SWWV2 (seroconversions to viruses isolated from
wastewater in summer 1982)
—alternatives: o contaminated drinking water
o low income, Caucasian
o Episode SWWV5 (seroconversions to viruses isolated from
wastewater in 1982)
—alternative: farmer, history of pneumonia
o Episode SSNV2 (seroconversions in summer 1982 to all serum
neutralization-tested viruses)
—alternative: contaminated drinking water
3) Strong evidence of aerosol exposure association and no alternative
explanation identified:
o Episode SPL11 (poliovirus 1 seroconversions in spring 1982)
It should be noted that all five of the infection episodes in Category
2 relate to echo or coxsackie B viral infections observed primarily in
summer 1982 and primarily to agents recovered from the wastewater at that
350
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time. Hence, it is reasonable to consider these to be five manifestations
of a single nonpolio enterovirus episode centered on the summer 1982 irrigation
season. With the heavy rainfall, rural drinking water contamination and
other unusual circumstances which occurred during this summer, it is not
surprising that fragmentary evidence of various alternative explanations
surfaced for this nonpolio enterovirus episode. '-'
There is strong evidence that the poliovirus 1 seroconversions in
spring 1982 were associated with wastewater aerosol exposure. Furthermore,
SPL11 is the only infection episode in which all four inferential methods
provided evidence of a significant association. The Cochran-Hantel-Haenszel
confirmatory analysis showed a significant association (p=0.02) of polio
1 seroconversions between January and June 1982 with the high aerosol exposure
group in the spring 1982 irrigation, when controlling for the effects of
polio immunizations during this time period. The groups were balanced
regarding previous polio 1 titers. ELR selected polio immunization in
spring 1982, low prior antibody level, and a high degree of aerosol exposure
as strong predictor variables for SPL11 seroconversions in a well-fitting
logistic model. Each variable may be considered a distinct risk factor
for polio 1 seroconversions since each made a strong contribution to the
ELR model. No alternative explanations to high AEI were identified by
ELR. Poliovirus 1 was recovered three times from the pipeline wastewater
sprayed in spring 1982. Therefore, the poliovirus 1 seroconversions in
spring 1982 provide substantial evidence of a causal association with wastewater
aerosol exposure.
C. COMPARISON OF FINDINGS TO THE LITERATURE
Self-reported Illness
Due to the paucity of prior data linking wastewater exposure to either
microbial disease or infection, there is virtually no basis for evaluating
the findings of the LISS relative to those previously described. The finding
of excess self-reported illnesses among high exposure LISS participants
after irrigation commenced is similar to findings observed in other studies
(see Discussion in Section 6A). Although this in itself raises the suspicion
of association with wastewater irrigation, it was difficult to evaluate
epidemiologically and was not thoroughly analyzed biometrically. One problem
is that the definition of illness varies from person to person. Last (1983)
stated that, ''The words disease, illness and sickness are loosely inter-
changeable—but not wholly synonymous.'' H. Susser (1973) suggested, the
following definitions:
Disease is a physiological/psychological dysfunction.
Illness is a subjective state of the person who feels aware of
not being well.
Sickness is a state of social dysfunction, i.e., a role that
the individual assumes when ill.
351
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Therefore the self-reported information collected could be biased by the
participants' attitude towards the project and perception of odor, as well
as by personal situations that arose over the study period.
The monthly incidence density of total acute illnesses reported by
the LISS participants varied from 2 to 13 per 1000r-person-days observed.
The National Health Interview Survey (National Center for Health Statistics,
1984) which also collected information on self-reported acute conditions
through household interviews obtained an annual density of 6.3 acute conditions
per 1000 person-days of inquiry in 1980-81. The density varied inversely
with age in a nearly linear manner, from 8.8 for persons under 17 years
old to 3.3 for persons over 65 years of age. Besides age, these rates
varied inversely with family income. An additional consistency found in
the LISS self-reported illness data that has been found repeatedly in a
number of surveys (Fox et al., 1972; Elveback, et al., 1966; Monto and
Koopman, 1980; Northrop et al., 1980) was the higher incidence of reporting
respiratory illness than gastrointestinal conditions. Thus, it appears
that the incidence of self-reported illness obtained from this study population
was generally consistent with epidemic logic expectations of acute (including
infectious) disease occurrence.
Given the inherent weaknesses associated with the collection of such
data and the uncertainty surrounding biased reporting, it is not possible
to draw firm inferences about wastewater irrigation health effects from
the LISS data on self-reported acute illness. The resolution of wastewater-
related health effects must rely on independent objective infection responses
as measured by either isolation of infectious agents or serologic response.
Bacterial Agent Episodes
It was assumed that apparent disease might constitute only a small
part of the total number of infections that might occur during wastewater
irrigation. Thus, methods were designed to rigorously search not only
for overt enteric bacterial pathogens such as Salmonella. Shigella. Yersinia
enterocolitica. and Camuvlobacter ieiuni. but also for heavy colonization
by important opportunistic pathogens and for unusual occurrences of organisms
which were prominent in wastewater but rare in fecal specimens from initial
baseline monitoring.
Two major points must be emphasized that concern the approach and
results of the bacteriological monitoring of health watch participants
in the LISS. Firstly, we did not equate the term ''infection'' with ''dis-
ease,'' the latter being indicated by detectable alterations in normal
tissue functions (i.e., clinical manifestations of illness). Infection
was used in the broader sense of the entrance and multiplication of a microbe
in the body. Secondly, the health significance of the organisms sought
covered a wide spectrum, ranging from highly significant to little or no
health significance. Organisms of three categories were chosen in order
to provide a more sensitive indicator of possible wastewater risk, rather
than disease, resulting from wastewater exposure.
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The organisms of our first category, overt enteric pathogens, are
of major clinical significance because they often are associated with disease
and even inapparent or subclinical infections may provide a source for
infection and disease in others. In spite of a rigorous search for overt
enteric bacterial pathogens, the number of isolations from the routine
fecal specimens was small in baseline monitoring (three) and periods after
commencing of irrigation (one).
Thus, given the constraints of the size of the fecal donor population
at risk, the results of this study do not appear to support an increased
risk of acquisition of overt enteric bacterial pathogens associated with
wastewater exposure. Relevant to this conclusion was the fact that overt
pathogens often were detected in the wastewater sampling, with the exception
of Shigella. which may have been below the level of detection by the direct
plating and enrichment procedures used. Lack of infection by these organisms
may have been due to failure to achieve an infections dose through aerosol
or direct contact. The size of inoculum required to produce disease in
humans varies widely for enteric pathogens (Gangarosa, 1978), ranging,
for example, from as few as 10 organisms for Shigella to 10** for most seretypes
of Salmonella.
The clinical significance of fecal isolates of the organisms at levels
defining the other two categories is questionable. However, opportunistic
pathogens were infrequently isolated at levels defining Category 2 during
baseline fecal sampling and only 0.3% of the baseline samples yielded isolates
meeting the definition of Category 3. These observations coupled with
the prominence of some of the organisms (particularly Aeromonas hydrophila.
the fluorescent Pseudomonas group, and Klebsiella pneumoniae) in wastewater
led us to believe that the two categories might provide a sensitive indicator
of a possible health risk associated with exposure to wastewater. In addition,
the organisms may be associated with enteric disease if isolated in large
numbers from stools. For example, enterotoxin-producing Klebsiella. Entero-
bacter. Proteus. Citrobacter. Serrat ia. and Aeromonas have been isolated
from the stools of children and infants with acute gastrointestinal symptoms
(Wadstrom et al., 1976). Some K. pneumoniae and Enterobacter cloacae produce
heat stable (ST) and heat labile (LT) enterotoxins, the latter of both
organisms being immune logically related to cholera toxin and Escher ichia
coli LT (Klipstein and Engert, 1977). K. pneumoniae ST recently has been
purified to homogeneity and found to have the same potency as E. coli ST
in the suckling mouse assay and immunological cross-reactivity with the
E. coli toxin (Klipstein et al., 1983). Likewise, A. hydrophila produces
an enterotoxin, and the organism has been associated with diarrhea in American
travelers, but not in Thais (Pitarangsi et al., 1982). A large percentage
(41%) of A. hydrophila isolates from diarrheal stools were negative for
enterotoxin in a recent study (Turnbull et al., 1984) and enterotoxic ity
was approximately equally divided (i.e., 58% and 53%) among fecal and environ-
mental isolates. An interesting observation in the present study was that
heavy levels of Klebsiella in feces and moderate or heavy levels of the
prominent bacteria in wasteawater (primarily fluorescent Pseudomonas species)
appeared to be associated with increased incidence and period prevalence
of self-reported 61 illness. Heavy levels of other opportunistic bacteria
were not. It is apparent, however, that the quantitative importance of
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the organisms of Categories 2 and 3 in enteric disease is probably small
and the etiological role of many of the organisms as enteric pathogens
is not well established. Many of the organisms of Categories 2 and 3 do
have unquestioned roles as major nosocomial pathogens (Guentzel, 1982).
A number of observations relating to nosocomial infections (NIs) by
organisms of Categories 2 and 3 are perhaps relevant to the present study.
The association of Klebsiella infections with elderly males, albeit borderline
significant, and the significant association of prominent wastewater bacterial
infections with the elderly (see Table 72) may be related to the observation
that the elderly are at increased risk for acquiring NIs. Gross et al. (1983)
noted that of all NIs, 64% occurred after 60 years of age even though the
elderly group represented only 23% of hospitalized patients. Increased
prevalence and levels of intestinal colonization by organisms such as Klebsiella
in a hospital environment have been associated with severity of illness,
duration of hosp italizat ion, and use of antibiotics (Haverkorn and Michel,
1979; Goldmann et al., 1978; Selden et al., 1971).
At least six possible causes of the elevated levels of Klebsiella
and other opportunistic pathogens and the unusual isolations of organisms
in Category 3 are suggested by the observations from NIs, other reports,
and the present study. These include:
1) antibiotic selection of resistant organisms or promotion of growth
as a result of reduction of competing flora by prior use of anti-
biotics,
2) ingestion of organisms on garden vegetables,
3) exposure to Gram-negative bacteria associated with heavily contami-
nated cotton,
4) fecal contamination of drinking water or food,
5) aerosols created by contaminated evaporative coolers,
6) wastewater irrigation operations.
Antibiotic selection or promotion of growth is an unlikely cause of
the isolations in Categories 2 and 3 since the isolations were observed
with routine rather than illness specimens. Ingestion of contaminated
garden vegetables also is an unlikely cause, even though Wright et al. (1976)
reported that salads may be heavily contaminated with Gram-negative bacilli.
Wright et al. (1976) studied the flora of foods served to patients in a
hospital and recovered enteric bacteria and Pseudomonas aeruginosa from
vegetable salads. The organism most frequently isolated was Enterobacter
agglomerans (85% of samples. 10^-10^ CFU/g). Other organisms isolated
frequently and mostly at high counts were E. c loacae (48%) and Klebsie 1 la
(46%). The studies of Casewell and Phillips (1978) and Cooke et al. (1980)
challenge some of the interpretations of Wright et al. (1976). Casewell
and Phillips (1978) observed that food prepared for intensive care patients
was frequently contaminated with Klebs ie lla but noted that the hospital
was the main source of contamination. Likewise, Cooke et al. (1980) examined
hospital food for the presence of Klebs ie 1 la . Salads and cold meat were
the most frequently contaminated foods. However, Klebsiella also was widely
354
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distributed in the hospital kitchen environment which was considered, at
least in part, to be the source of the organisms found in the food. It
should be noted that E. agglomerans. the most frequent isolate from salads
in the study of Wright et al. (1976), was isolated at any level of growth
from less than 1% of the fecal specimens of LISS participants.
c -
Exposure to Gram-negative bacteria associated with heavily contaminated
cotton also is an unlikely cause of the unusual isolations. Horey et al. (1983)
recently reported that seed cotton and cotton plants collected from Lubbock,
Texas, were heavily contaminated with Gram-negative bacteria. The organisms
were not identified; however, the investigators noted that E. agglomerans
was the predominant species in other similar studies. E. agglomerans is
a relatively recent designation for a group of organisms which include
the former Herbicola-Lathyri bacteria which were included in the plant
associated genus Erwinia. The nature of the flora of the contaminated
cotton and the lack of relationship to the isolations from specimens of
LISS participants make contaminated cotton an unlikely source. It should
also be noted that while K. pneumoniae is widely distributed in the environment,
strains isolated from humans and animals may be routinely different in
properties. Bagley and Seidler (1977) noted that 85% (49/58) of K. pneumoniae
of human and bovine origin were fecal coliform (FC) positive whereas 16%
(19/120) of environmental strains were FC positive. Strains of K. pneumoniae
that are FC positive have been shown to have other unique properties (Edmondson
et al., 1980).
The fact that the unusual isolations of organisms of Categories 2
and 3 occurred over a defined period also tends to argue against possibilities
1 through 3, but not 4 through 6. Fecal contamination of drinking water
as a consequence of contaminated individual wells and city of Wilson water
is a possibility since most of the isolations occurred following a period
of unusually heavy (>10 in.) rainfall in the study area in May and June
1982.
Klebsiella has been reported to be the most prevalent, potentially
pathogenic Gram-negative bacterium in the air surrounding sewage treatment
plants (Kenline and Scarpino, 1972; Randall and Ledbetter, 1966) and in
air samples of wastewater used for spray irrigation (Linnemann et al.,
1984) . The organism also is found at very high levels in textile finishing
plant effluents (Dufour and Cabelli, 1976) and in pulp and paper mill effluent
discharge (Kanarek and Caplenas, 1981), and thus may be expected in the
aerosols of those sources as well. Examinations of microorganism levels
in air in the present study revealed unusually high levels of certain indicator
organisms (fecal coliforms and fecal streptococci) that were carried long
distances downwind from irrigation nozzle lines. These levels were greatest
when irrigation was directly from the pipeline in the spring and summer
of 1982. Presumably the aerosols also contained high levels of Klebsiella .
However, Klebsiella infections were not associated with degree of aerosol
exposure during this period of presumably greatest exposure in 1982.
Much of the interest in aerosols associated with sewage treatment
and land application of wastewater has centered around small particle aerosols
(i.e., 5 urn or less) which may be carried to deep areas of the lungs.
355
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However, it has been proposed that most human bacterial pneumonia is due
to microorganisms that have colonized the oropharynz, and that aspiration
of such organisms may be the principal mechanism underlying nosocomial
pneumonia (Sanford and Pierce, 1979). An interesting observation in experi-
mental animals (mice and monkeys) was that K. pneumoniae administered by
aerosol was significantly less virulent than when given by intranasal or
intratracheal instillation (Berendt, 1978). These observations suggest
that large particle aerosols containing the organism may lead to colonization
of the nasopharynx associated with seeding of the gut by the organisms.
The use of evaporative coolers at home was identified as a potential
source of infection by enteric Gram-negative bacilli (E6NB) at high levels
in the throats of some health watch participants between July 19 and October 12,
1982. The authors are not familiar with studies describing transmission
of EGNB, presumably via aerosolized particles, by this route. It is very
unlikely that EGNB such as E. coli would be free living in the water or
evaporative coolers. However, if fecal contamination of the well water
used for this purpose had occurred, then this could be a potential source
of infection. Given that the well water was contaminated, ingestion would
remain quantitatively the most significant route of infection by enteric
organisms.
An apparent association of Klebsiella infections with wastewater aerosol
exposure occurred in summer 1983. However, frequently eating food prepared
at restaurant A was more strongly associated with this infection episode
and in the same individuals. The restaurant etiology may be more compelling
for two reasons. Firstly, a part-time food handler at restaurant A was
infected by Klebsiella during the same period, and secondly, the Klebsiella
infections in summer 1982 were not associated with aerosol exposure, even
though wastewater aerosol levels of Klebsiella were higher in summer 1982.
However, the summer 1982 association could have been obscured by heavy
rainfall—associated contamination of drinking water which occurred in that
period.
In summary, the results of bacteriological analysis reported in this
study dealing with the incidence of infection inferred by isolation of
either overt or opportunistic pathogens from fecal specimens do not appear
to suggest an increased risk associated with exposure to wastewater.
Viral Agent Episodes
Human viruses cannot replicate outside a susceptible host and hence
their concentration in wastewater decreases due to dilution and eventually
inactivation. However, the relative environmental stability of numerous
enteric viruses shed into wastewater by infected individuals enhances their
potential transmission to susceptible populations by wastewater aerosols.
Dispersion modeling developed by Camann (1980) and based on limited data
collected at a wastewater irrigation site in Pleasanton, California, predicted
that median impact factors reflecting enhanced organism survival were approxi-
mately 20 times greater for viruses when compared to even the hardiest
indicator bacteria (fecal streptococci). Indeed, aerosol monitoring during
1982 LISS irrigation periods repeatedly detected human enterovirnses in
356
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downwind air samples. Enterovirus survival in Hancock farm aerosols from
pipeline wastewater irrigation was at least as great as that observed at
Pleasanton.
In addition to their relative stability, the minimal infectious dose
of various human enteric viruses is low when compared to most pathogenic
bacteria found in treated wastewater (Akin, 1983). A comprehensive review
by Ward and Akin (1984) evaluated numerous studies directed at determining
the infectious doses of both respiratory and enteric viruses. The 50%
human infectious dose (111059) for respiratory agents such as coxsackievirus
A21 and adenovirus type 4 in aerosols was reported as 34 and 0.5 TCID5Q,
respectively. Notably, the dose of coxsackievirus A21 required to cause
illness was apparently less when the infections agent was delivered to
the upper respiratory tract than when the virus was delivered to the lower
portion of the gastrointestinal tract.
Infectious dose studies with enteric viruses known to replicate in
human intestinal cells have been limited to polioviruses and echovirus
12. Without exception, poliovirus studies have measured infections in
infants and young children, representing perhaps the most highly susceptible
population. In one such study 2-month-old infants were fed doses of 7
to 280 TCID5o of attenuated poliovirus 1 (Sabin) (Minor et al., 1981).
Based on viral shedding the HID59 was determined to be 72 TCIDso- Earlier
studies with polioviruses 1 and 3 which introduced the virus either directly
into the stomach or employed gelatin capsules to transport viruses to the
intestinal tract had demonstrated HID5Q of less than 10 TCID5Q or pfu,
respectively (Katz and Plotkin, 1967; Koprowski et al., 1956).
Healthy male subjects (18-45 years of age) initially lacking detectable
antibody to echovirus 12 were challenged with various doses of this virus
suspended in drinking water (Schiff et al., 1984). The HID50 of echovirus
12 was determined to be 919 pfu while the HIDQi (dose required to infect
1% of the volunteers) was predicted as 17 pfu. In this study most viral
shedding occurred during the first week after inoculation, regardless of
the viral dose. The duration of viral shedding (up to 28 days) was also
independent of dose. In a second experimental challenge in individuals
seropositive for echovirus 12, 72% became reinfected (as determined by
detection of virus in stool specimens) when 1500 pfu (HIDgg) were ingested.
Thus, the presence of serum antibody caused no significant change in the
number of volunteers infected with echovirus 12.
Considered as a whole, response to infection by viral agents as measured
either by fecal shedding or seroconversion probably provides the most sensitive
measure of wastewater aerosol exposure currently available. Thus, the
serological identification of discrete infection episodes occurring mostly
in 1982 is feasible. Furthermore, these LISS findings are consistent with
conclusions reached by Fattal and coworkers (1984) who suggested a wastewater
exposure route for infection by echovirus 4.
However, further analyses of viral infection episodes as well as other
infections possibly associated with wastewater exposure have identified
alternative explanations in selected cases (see Tables 133 and 134) which
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should be weighed in the light of epidemiological consistency. Intrafamilial
transmission of enterovirnses and adenoviruses has been well documented
(Fox and Hall, 1980). In the New York virus watch program, the spread
of coxsackieviruses to susceptible household members was high (76%) while
echovirus transmission was somewhat lower (46%). Notably, while larger
families of lower socioeconomic status yield enterovirnses more frequently,
intrafamilial spread appears to be independent of family size. A more
important correlation of infections among family members has been shown
to be the duration of fecal shedding by infected individuals. Reinfection
by both coxsackieviruses and echoviruses, even in the presence of specific
antibody, also occurs (Fox and Hall, 1980; Schiff et al., 1984). A similar
pattern of transmission between family members has been observed with adeno-
viruses in both the New York and Seattle virus watch programs (Fox and
Hall, 1980). However, because of the relatively prolonged and intermittent
excretion of adenoviruses, long continuing intrafamilial spread is not
uncommon. For these reasons, the alternative explanation of within-family
spread as applied to infection episode SE090 attributed to echovirus 9
(three of eight infections) should reasonably supercede the association
of these events with wastewater exposure.
Ingest ion of contaminated drinking water from private and public wells
has been documented in several outbreaks of viral disease in the United
States including hepatitis A, Norwalk virus and rotavims (Bergeisen et
al., 1985; Olivieri, 1984; Hopkins et al., 1984). Presumably, subclinical
infections with other enteroviruses having similar environmental stability
can occur, particularly if drinking water wells were contaminated with
wastewater from septic tanks.
The involvement of selected enteric viruses in common-source foodborne
disease outbreaks has been well documented. Cases of hepatitis A traced
to the consumption of shellfish harvested from contaminated coastal waters
is well known. Additionally, ingestion of uncooked or cold foods such
as salads (Latham and Schable, 1982), meats and cheeses (Gustafson et al.,
1983) have been linked to hepatitis A outbreaks. However, of the 1,097
confirmed foodborne outbreaks reported to CDC between 1972 and 1978, only
3% were attributed to viruses, while 66% were due to bacteria (Sours and
Smith, 1980) . Twenty-nine of these viral outbreaks accounting for 1,346
cases were attributed to hepatitis A, while a single outbreak caused by
echovirus 4 involved 80 cases. Thus, while poor personal hygiene of a
food handler can cause the viral infections of restaurant patrons, relatively
few foodborne outbreaks of viral etiology have been documented.
The remaining alternative explanations identified in Tables 133 and
134 for viral infections were race (Caucasians) and previous medical history
(pneumonia). In studying the response within households to poliovirus
infection. Fox and Hall (1980) noted that socioeconomic group showed a
greater influence on the percentage of individuals with specific antibody
than did race. Specifically fewer of the whites in an upper economic group
had neutralizing antibody than blacks and whites in the lower economic
group who developed parallel seroimmunity to poliovirus with increasing
age. Previous disease occurrences, especially if tissue damage resulted,
can predispose an individual to subsequent infection by viral agents.
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D. SIGNIFICANCE OF FINDINGS
Assessment of the significance of the findings from the LISS requires
that particular attention be given to the possible limitations of the study.
The design employed, an epidemiologic analytic cohort study, was quite
appropriate to measure the strength of associatidn between exposure to
the wastewater used for irrigation and the development of new infections.
As a guide to the following discussion, the more frequent, important limitations
that may occur with the prospective cohort study design are presented first,
followed by the major advantages of this design. Then, the specific limitations
of the LISS are presented and discussed.
A major limitation in interpreting the strength of association, i.e.,
relative risk, from this type of study design can arise from bias introduced
by uncontrolled confounding factors. Another limitation may be imposed
by instability of the association when the sample size is small. By using
consenting study participants, the findings maybe inferred to the study
population only with caution, since volunteer populations are known to
differ from nonparticipants in risk factors related to viral infections
(Francis et al., 1955). Unless the study population were representative
of the general situation involving exposure to wastewater for irrigation
purposes, it would be unwise to generalize from the LISS findings. Finally,
bias may be introduced during ascertainment of the study variables due
to missing values or transcription errors or the methods employed for measuring
may produce misclassifications.
If these limitations are either prevented or controlled, the prospective
cohort design may have several important strengths in assessing causality
of associations. Since this is a study of incidence, exposure is known
to precede infection. The hypothesis of causal inference may be strengthened
by: a strong association that is stable, the demonstration of a dose-effect,
an association that is consistent at different times, an association in
agreement with biologic and epidemic logic theory, and an association which
is specific.
A major limitation in interpreting the significance of the findings
from the LISS involves the selection of participants. Of necessity all
participants were volunteers. The study sample was not representative
of the study population. Further, we can only assume that self-reported
illness was accurately reported during the study. The source of irrigation
wastewater varied during the study, making interpretation of findings difficult,
since the dose of exposure varied within the exposure levels by irrigation
period. Because of these factors, the results cannot easily be generalized
to other sites.
The preliminary analysis compared the low exposure group and the high
exposure group with respect to several individual and household characteristics
that could confound the interpretation of the significance of the findings.
Of the six variables considered important enough epidemiologically to warrant
stratification for an imbalance, only polio immunization and fecal donor
head of household occupation met the criteria for stratification. For
the serum donor sample, type of air conditioning and drinking water supply
359
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were found to be different. Preexisting antibody titers to only three
agents (influenza A in June 1981, echo 3 in January 1982 and polio 3 in
January 1982) were not balanced. The exposure groups were significantly
imbalanced with respect to frequency of eating food prepared at restaurant
A. In general, the two exposure groups were quite similar in risk factors
that could confound interpretation of the relative risk. Poliovirus sero-
conversion rates were stratified on polio immunization status. Sample
size was too small to permit stratification for air conditioning, drinking
water supply and patronage of restaurant A. Therefore, each relative risk
analysis with a value greater than 1 had to be reviewed with these character-
istics as an alternative explanation. Exploratory analysis using a stepwise
logistic regression model served this purpose (except for air conditioning
system in 1982). The significance of the study findings have not been
limited to a great extent by the major confounding factors.
The size of the study sample has limited the ability to interpret
the stability of the strength of association in most instances. Therefore,
it was necessary to rely more on the consistency of the findings. The
three outcome variables selected for the study varied in sensitivity to
detect infection, ranging from low sensitivity for clinical disease, inter-
mediate sensitivity for infectious agent isolation, to high sensitivity
for serologic determination of infection. The self-reported illness data
of the disease surveillance varied in consistency, reliability and completeness,
which makes interpretation difficult. High but unstable incidence density
ratios of acute illness for the high exposure level followed wastewater
irrigation in the spring and the summer of 1982. According to the aerosol
results, microorganism dosage was greater in 1982 than 1983, with the summer
of 1982 being greatest for enterovirus exposure. Disease surveillance
did not disclose any obvious consistent association between acute illness
reports and the degree of wastewater exposure.
The results from isolation and serologic determination are more reliable
and accurate. During the baseline period the high exposure group had the
lowest conversion rates to all the adenoviruses, coxsackie B viruses, and
echoviruses tested; however, in the irrigation period the high exposure
group had the highest seroconversion rates to all coxsackie B viruses tested,
to all echoviruses tested, and to all the tested viruses recovered from
irrigation wastewater. The risk ratios were greater than 1 but less than
2. When the risk ratio scores of each infection episode were displayed
graphically, the baseline distribution was symmetrically centered about
zero; however, an excess of positive scores occurred in the episodes whose
duration spanned single irrigation periods.
Using the one-sided Fisher's exact test in the confirmatory statistical
analysis revealed seven infection episodes with stable risk ratios. When
the results were compared using the four statistical approaches, eight
specific infection episodes were identified which displayed marginal to
good evidence of association with wastewater aerosol exposure. The two
episodes which had a more plausible alternative explanation occurred in
the baseline period and during the summer of 1983. Of the remaining six
episodes, all occurred during 1982 and one episode had strong evidence
of aerosol exposure association.
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Except for illness ascertainment, the results from isolation and serology
appear to be adequate. There is no evidence that the results were biased
by additional efforts in detection. The laboratory methods would underestimate
infections in general, but not by exposure group. Classification of participants
into exposure groups was done employing a reasonable model which estimated
exposure level by distance from the irrigation sprayers, wind direction,
and risk of direct contact. Review of participants revealed no significant
classification error.
In summary, the results indicate that a general association between
exposure to irrigation wastewater and new infections existed, especially
for 1982. However, even during 1982, the strength of association remained
weak and frequently was not stable. Wastewater, directly from the pipeline,
comprised much of the irrigation water in 1982. The isolation of enteroviruses
from pipeline wastewater was greater than that observed when the wastewater
had been retained in reservoir. The methods employed resulted in the observa-
tion of a large number of infection episodes, none of which resulted in
serious illness. The voluntary nature of participation and the unrepresentative
circumstances of the study area make generalization of the results unwise.
A larger sample size with greater comparability of the exposure groups
on the basis of drinking water source and frequency of visiting the same
eating establishments would have reduced their confounding effects.
From the public health standpoint, the lack of a strong, stable association
of clinical illness episodes with the level of exposure to irrigation wastewater
indicates that wastewater spray irrigation produced no obvious disease
during the study period. However, when more sensitive indicators of infection
were used, a general association was found to exist, especially for 1982.
A particular concern is the evidence that the poliovirus 1 seroconversions
were probably related to wastewater aerosol exposure during the spring
of 1982, even when the effects of polio immunizations were controlled.
Because of the low prevalence of poliovirus antibody observed during the
baseline period, the study population had been immunized, and thus was
probably better protected against polio than other rural populations.
Very high concentrations of both bacteria and enteric viruses were observed
in the 1982 wastewater applied as received via pipeline directly from the
Lnbbock sewage treatment plant. Much lower concentrations were observed
in wastewater obtained from the reservoir. Although the LISS found no
obvious evidence that disease was associated with using treated wastewater
for irrigation during the study period, as a public health measure it would
be prudent to allow the wastewater to settle in a reservoir before use
if other conditions remain the same.
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Land Treatment of Municipal Wastewater. EPA-625/1-81-013, Cincinnati,
Ohio.
U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory. 1978. Microbiological Methods for Monitoring the Environ-
ment. EPA-600/8-78-017, Cincinnati, Ohio.
U.S. Environmental Protection Agency, Office of Research and Development.
1982. Estimating Microorganism Densities in Aerosols from Spray Irrigation
of Wastewater. EPA-600/9-82-003, Cincinnati, Ohio.
U.S. Environmental Protection Agency, U.S. Army Corps of Engineers, U.S. Depart-
ment of Agriculture. 1977. Process Design Manual for Land Treatment
of Municipal Wastewater. EPA-625/1-77-008, Cincinnati, Ohio.
Wadstrom, T., A. Anst-Kettis, D. Habte, J. Holmgren et al. 1976. Enterotoxin-
producing Bacteria and Parasites in Stools of Ethiopian Children with
Diarrheal Disease. Arch. Dis. Childhood. 51:865-870.
Ward, R. L. and E. W. Akin. 1984. Minimum Infective Dose of Animal Viruses.
CRC Critical Reviews in Environmental Control. 14:297-310.
Williamson, S. J. 1973. Fundamentals of Air Pollution. Addison-Wesley,
Reading, Massachusetts, pp. 39-41.
Wood, R. J., and T. M. Durham. 1980. Reproducibility of Serological Titers.
J. Clin. Microbiol. 11:541-545.
Wright, C., S. D. Kominos and R. B. Tee. 1976. Enterobacteriaceae and
Pseudomonas aeruginosa Recovered from Vegetable Salads. Appl. Environ.
Microbiol. 31:453-454.
Yonmans, G. P., P. I. Paterson, and H. M. Sommers. 1980. The Biologic
and Clinical Basis of Infectious Diseases, 2nd Edition. W. B. Saunders,
Philadelphia, Pennsylvania, p. 88.
373
-------
APPENDIX A
SUPPLEMENTAL FIGURES AN) TABLES FOR SECTION 4
(METHODS AND MATERIALS)
375
-------
NNW
337.5°
0/360C
NNE
22.5°
NW
315
WNW
292.
SW
225
SE
135C
SSW
202.5°
ESE
112.5
NOTE: Three-hour observations are from the 5-year period, 1969-1973,
Radiating-bar lengths indicate the percent of the period that
winds blow from the indicated directions.
Figure A-l. Wind frequencies for the 2-month period of
March-April, Lubbock, Texas
377
-------
WNW
292.5°
WS1
247.5°
SW
225°
NNW
337.5°
N
0°/360C
NNE
22.5°
SSW
202.5°
SSE
157.5°
ESE
112.5°
NOTE: Three-hour observations are from the 5-year period, 1969-1973,
Radiating-bar lengths indicate the percent of the period that
winds blow from the indicated directions.
Figure A-2. Wind frequencies for the 2-month period of
July-Angust, Lubbock, Texas
378
-------
WIND ROSE OBSERVED WIND FREQUENCY FOR Z/16/82 TO 5/04/8Z
Lubbock Infection Surveillance
SENERRL CLIHflTOLOGY NUN
HANCOCK FHRM
NH
•INM
HSH
MISSING
VSRIRBLE
POSSIBLE HOURS
NUMBER 3F HOURS 1627
DflTR CRPTURE 88.30X
NNE
NE
ENE
ESE
PLOT LEGEND
RVERflSE MINO SPEED
PERCENT MIND
CRL.1
1.61
13B-.903 I
2.831 !
1-1.98
4.491
Z-Z.9I
8.48Z
3-5.98
46.71
6-9.98 [
38.3Z
18-15.9
5.98Z
16- 25 ! 25.1- 99
.7991 I 8t
Figure A-3. Wind frequencies for the 1982 spring irrigation
period: Hancock farm meteorological station
379
-------
HIND ROSE OBSERVED WIND FREQUENCY FOR 7/26/82 TO 9/17/82
N
Lubbock Infection Surveillance
GENERAL CLIMATOLOGY NNH
HANCOCK FARM
NH
MNH
HSM
HISSING
VARIABLE
POSSIBLE HOURS
NUMBER OF HOURS 11*6
3ATR CAPTURE 88.431
NNE
NE
ENE
ESE
sse
m
SE
PLOT LEGEND
PERCENT MIND
Figure A-4. Wind frequencies for the 1982 summer irrigation
period: Hancock farm meteorological station
380
-------
HIND ROSE OBSERVED WIND FREQUENCY FOR 2/15/83 TO 4/30/83
u
Lubbock Infection Surveillance
GENERAL CLIMATOLOGY NNH
HRNCOCK FARM
NM
NE
MNH
HSM
HISSING
VARIABLE
POSSIBLE HOURS
NUMBER OF HOURS 1799
ORTR CRPTURE 1881
ENE
ESE
SSE
m
PLOT LEGEND
AVERAGE MIND SPEED
PERCENT NINO
CflLM
.It
.138-.90B
1.881
i-1.90 I
4.391
2-2.98
18.81
3-5.98
AS. IX
6-9.98
38.21
18-15.9
7.391
16- 25
.5561
25.1- 99
Figure A-5. Wind frequencies for the 1983 spring irrigation
period: Hancock farm meteorological station
381
-------
HIND ROSE OBSERVED WIND FREQUENCY FOR 6/29/33 TO 9/20/83
N
Lubbock Infection Surveillance
GENERAL CLIMRTOLOGY NNH
HPNCOCK FRRM
HNH
MSM
SH
MISSING 9>
VRRIR8LE 31
POSSIBLE HOURS 2016
NUMBER OF HOURS
ORTR CRPTURE .
SSH
I)NE
NE
ENE
ESE
PLOT LEGEND
SVERRGE HIND SPEED i
PERCENT MIND
I
CHLM
67.at
3.481
1-K9B
11.41
2-Z.9B
23.41
3-5.98
47. SI
6-9.9B
1 a-1 s.9
1.33X
16- 25
az
25.1- 99 ;
31 i
Figure A-6. Wind frequencies for the 1983 summer irrigation
period: Hancock farm meteorological station
382
-------
TABLE A-l. VALUES OF PREDICTED RELATIVE AEROSOL CONCENTRATION, Pd
Irrigation period and
dates of irrigation
2-16/4-30-82
7-21/9-17-82
2-15/4-30-83
6-29/9-20-83
Activity diary map
(1965-1974 wind data)
Range of household values. Pha
Blue map area (Hancock farm)
Orange map area (surrounding
Hancock farm)
White map area (remainder of
study area)
Outside map area
Feb-Apr
0.00004-0.19
Pj = 0.1207
i>2 = 0.0244
£3 = 0.0011
§4=0
Jul-Aog
0.00003-0.30
PI = 0.1806
P£ = 0.0221
§3 = 0.0017
Feb-Apr
0.00004-0.19
PI = 0.1207
§2 = 0.0243
§3 = 0.0012
§4=0
Jul-Aug
0.00004-0.30
PI = 0.1806
?2 = 0.0219
^3 = 0.0017
w NOTE: PI, ?2 and P3 are geometric means of
w respective colored map areas.
values of all study participant households in the
= predicted rlative aerosol concentration at participant's home
-------
TABLE A-2. WASTEWATER SAMPLING AND ASSAY SCHEDULE: 1980-81
CO
00
Lubbock trickling filter effluent Wilson Imhoff
Full
microbiological
Sampling dates screen
1910
6-3/6-4 x
7-28/7-29 x
11-3/11-4 x
1981
1-19/1-20
2-16/2-17
3-9/3-10
3-23/3-24
4-20/4-21 x
5-4/5-5
5-18/5-19
6-1/6-2
6-15/6-16
6-29/6-30
7-20/7-21 x
8-17/8-18
9-14/9-15
11-17/11-18
x - performed on composite wastewater
0 - viral identification performed on
EV - enterovirns assay
FC - fecal coliform assay
Limited
bacterial
screen
x
x
X
X
X
X
X
X
X
X
X
sample from
this sample
EV Full
and microbiological
FC screen
xO x
xO x
xO
x
X
X
X
xO
X
xO
X
xO
xO
X
designated source
tank effluent
Limited EV
bacterial and
screen FC
xO
xO
x
x
X
X
X
X
X
X
xO
X
X X
x xO
X •"' X
X X
-------
TABLE A-3. WASTEWATER SAMPLING AND ASSAY SCHEDULE: 1982
oo
in
Collection
date
2-16/2-16
3-1/3-2
3-8/3-8
3-15/3-16
3-22/3-23
3-28/3-30
4-6/4-6
4-18/4-20
4-28/4-27
5-2/5-3
6-17/6-18
6-14/6-15
6-28/6-30
7-18/7-20
7-26/7-27
8-8/8-10
8-30/8-31 °
8-13/8-14
8-27/8-28
10-11/10-12
11-1/11-2
12-13/12-14
Pipeline effluent
Full LIB 1 ted
•Icroblologlcal bacterial
screen screen
x«-
X
X
x>-
X
X
x»-
x"-
X
X
X
Reservoir effluent Wilson effluent
Routine
assay8
•
X
X
X
•
X
X
X
X
X
X
•
X
X
X
X
EV
end
FC
•
•
•0
•
•0
•
•0
•0
•
•
•0
•0
X
•
•0
Seiple
tvDeb
6
8
C
C
C
C
6
6
Full Limited EV Limited
Microbiological bacterial Routine end bacterial
screen screen assava FC screen
X
X
X
X
X •
x*- x • x
X
XL ...
X XX
X X • X
X X • X
X
X
X
X
EV
and
FC
x
X
xO
X
xO
X
X
X
X
xO
X
xO
X
xO
X
X
X
X
x - mstemter sample collected for Indicated assay
• - assay performed as subset of another assay
0 - viral Identification performed on this sanple
XL - LegloneIla assay performed In addition to regular assay
EV - enterovirus assay
FC - fecal collfora assay
Sane organlens nonltored on aerosol runs (fecal colifom* fecal streptococci, collphege, total enteroviruses, and C.
perf rlngene/siycobaoterla).
C - composite sanplai G - grab saopla.
Chlorlnation of pipeline effluent of Lubbock nastewter treatBent plant.
-------
TABLE A-4. WASTEWATER SAMPLING AND ASSAY SCHEDULE: 1983
oo
Pipeline effluent
Collection Routine
dates assav* Coliohaee
1983
2-16/2-17 xO
3-7/3-8 x
3-21/3-22 xO x
4-4/4-5 x
4-18/4-19 xO x
5-16/5-17
6-27/6-28 x
7-11/7-12 xO x
7-25/7-26 x
8-8/8-9 xO x
8-22/8-23 x
9-12/9-13 xO
9-26/9-27
x - composite water sample collected
Limited Sample
screen tvueb
C
C
x G
C
x G
C
C
G
G
G
C
for indicated assay
Reservoir effluent
Rout ine
assava Coliohane
xO
x
xO x
X
xO x
X
xO x
X
xO x
X
xO
Wilson
Imhoff
influent
Limited Routine
screen assay*
xO
x
x xO
X
x xO
xO
X
xO
X
xO
X
xO
xO
0 - viral identification performed on this sample
a Total coliforms, fecal coliforms,
fecal streptococci.
total enterovirnses.
b C - composite sample; G - grab sample.
-------
TABLE A-5. SUMMARY OF SAMPLING CONDITIONS—AEROSOL RUNS—OPERATIONAL YEAR 1982
to
oo
Aeroso 1
Run
no.
Ml
M2
M3
M4
M5
M6
M7
M8
M9
M10
Mil
M12
M13
M14
M15
M16
M17
M18
M19
M20
No.
9
2
15
12
15
3
11
15
15
4
4
8
8
7
10
12
14
14
9
10
Sampled
Orien-
tation
315°
130°
290°
315°
230°
50°
325°
70°
70°
330°
280°
80°
80°
55°
125°
500°
30°
20°
90°
130°
rig
End gun
status
On
Off
Off
On
Off
Off
Off
Off
Off
Off
Off
Off
Off
Off
Off
On
On
Off
Off
Off
Position/
tower
Outer /6
Center/3
Center/4
Outer/5
Outer/5
Inner/3
Outer /6
Inner/3
Inner/3
Outer /6
Center/4
Center/4
Center/4
Center/3
Center/4
Center/4
Center/4
Center/4
Outer/5
Outer/5
Line
Angle
esia
35°
80°
60°
30°
80°
45°
50°
75°
75°
70°
85°
90°
90°
75°
65°
65°
90"
90°
65°
85°
samp 1 er
location
Distance to
Single
39
35
49
55
64
50
61
50
55
50
125
125
125
125
125
50
125
125
23
80
Single
64
60
80
80
115
75
87
75
80
75
175
175
175
175
175
75
175
175
23
130
rlq,
Pair
139
135
140
148
174
125
155
125
130
125
300
300
300
300
300
125
290
275
48
255
(m)
Pair
214
210
203
225
288
200
236
200
205
200
400
375
375
365
400
200
400
400
98
323
Rig
movement Mean wind
during direction
run (m) 8wb
+2
-2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
90
25°
100°
75°
23°
113°
60°
60°
50s
90°
80°
130°
105°
90°
80"
60°
65°
90"
85°
50°d
110"
Other rigs In
operation
Possibly Not
upwind upwind
3
None
None
2,7
2,7
12,19
None
None
None
None
None
None
None
4
6,7,8
22
7
None
21C
6.7
15
3,5
3,5,8,11
6
None
None
None
None
None
None
None
6,11,12,13
17,19
11,13,17,
19.20C
7,17,19,
20C
2,4,8,9,
12,18,c
20,c2ic
3,4,7,8,
9,11,12,
18,c20,c 2ic
2,8,11,15,
18,19
15.18.19
Wastewater
Source
Pi pel Ine
Plpel Ine
Pi pel ine
Pipe! Ine
Plpel Ine
Pipel Ine
Reservoir
PI pel Ine
Reservoir
Reservoir
Plpel Ine
Pipel Ine
Reservoir
Plpel I ne
Plpel Ine
Reservoir
Pipel Ine
Pipel ine
Reservoir
'?
Plpel ine
Temp
.
-
-
-
-
-
-
-
-
-
27
-
-
27
-
-
26
24
-
28
a 6S| - angle of sampler I Ine with rig (0° _<_ »s1 <_ 90°)
b 6y, '- mean angle of wind with the rig during the run, measured In same direction from rig as 9sI
c Rig with drops
d From ClImatron Ics Weather Station at the tech plot
-------
TABLE A-6. SAMPLER OPERATING VOLTAGE ON THE MICROORGANISM AEROSOL RUNS
Operating high voltage of large volume samplers (kV)
Aerosol Upwind of
run Irrigation
number rig 20-39 m
PREPLANTING IRRIGATION
Ml ? ? 14
M2 13 11.5 11
M3 12 9
M4 14 14
M5 13 13
M6 12 14
SUMMER CROP PIPELINE IRRIGATION
M7 12.7 10.7
M8 14 12
Mil 15 12.5
M12 12 12
M14 13 13.5
M15 13 13
00 M17
08 M18
M20
SUMMER
M9
M10
M13
M16
M19
12
12
12
12
12
12
Downwind of
40-59 m 60-89 m
14
11
10 6
11 10
12
10 11
12 12
12 12
Irrigation nozzle line
90-149 m
12.
12.
12.
12
12
12
12.
2
8
5
9
8
12
12
10
12
13
14.
14
13.8
12.8
12.5
12
13
12
12.4
5
150-249 m 250-349 m 350-409 m
14
14 15
10 12.5
12.5 12.5
12 12 12.5 13
12 12.5
11 12
12 12
12 12 11 14
12 12.8 12.5 13
12 13.2 12.8 11.5
12 12.5 13 12.8
12
16
14
.5 12.8 12.8 13
.5 12.8 14
16.4 11 14
13
12
12
11
13
13
14
CROP RESERVOIR IRRIGATION
15
13
12
12.5
12
12
13
11.5
13.5
12 14 14.5
12 12
12 12
12 12
12 14.5
11.
12.
12.
14
5
8
12
6
12.4
12.5
13.2
14
12.7
12.5
11
12.5
12.7
12
12.5 12.8 12
12.8
13
? - Voltage not recorded.
-------
«*»
oo
vo
TABLE A-7. SUMMARY OF SAMPLING CONDITIONS—QUALITY ASSURANCE RUNS—OPERATIONAL YEAR 1982
Sampled rl^
Run
no.
01
02
No.
11
15
Orien-
tation
340°
65°
End gun
status
Off
On
Ae r oso 1 samp 1 er 1 ocat 1 on
Position/
tower
Rlght/4-5
Center/3
Distance to
rig (m)
75
50
Rig
movement
during
run (m)
0
0
Mean wind
direction
Qva
110°
75°
Other rigs In
operation
Possibly
upwind
None
None
Not
upwind
None
None
Wastewater
Source
PI pel Ine
PI pel Ine
a 9W - mean angle of wind with the rig during the run, measured In same direction from rig as
-------
TABLE A-8. SUMMARY OF SAMPLING CONDITIONS—VIRUS RUNS—OPERATIONAL YEAR 1982
vo
o
Aerosol sampler location
Run
No.
VI
V2
V3
V4
Segment
No.
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
No.
4
it
ti
it
it
17
tl
11
II
tt
14
it
ii
ii
n
14
ti
n
it
n
Sampled
Or 1 en-
tat Ion
320°
320"
325°
325°
325°
60"
60°
58°
58°
56°
70°
70°
68°
68°
66°
35°
32°
30°
27°
25°
rlq
End gun
status
Off
n
n
it
n
Off
ii
n
it
n
Off
ii
n
n
"
On
n
M
n
"
Position/
tower
Rlght/4-5
n
it
n
n
Center/5
it
n
n
n
Center/4
it
n
n
n
Center/5
n
n
ii
n
Distance
to rig (m)
Start
60
60
55
55
55
50
45
50
45
52
47
44
50
47
50
60
51
55
46
50
Finish
60
60
55
55
55
47
42
47
42
49
44
41
47
44
47
54
45
49
40
44
Rig movement
during
segment (m)
0
0
0
0
0
3
3
3
3
3
3
3
3
3
3
6
6
6
6
6
Mean wind
direction
e«
so-
so0
105°
110"
110°
105°
110°
105°
110°
115°
80°
85°
55°
80°
55°
-
45°
75°
60°
35"
Other rigs In
operation
Possibly
upwind
None
ii
n
n
n
None
M
n
ii
n
4,7
n
n
»
n
7
ii
n
it
n
Not
upwind
None
it
it
n
n
2,4,6,7,
11,12,13
17,19
ti
n
n
n
6,11,13,
17,20
ii
n
n
it
2,4,8,9,
12,18,20,
21
it
n
n
n
Wastewater
Source
PI pel Ine
n
n
M
ti
Pipe! Ine
n
"
n
n
Pipe) Ine
n
n
n
n
FM pel Ine
(27CC)
ii
M
n
n
-------
TABLE A-9. SUMMARY OF SAMPLING CONDITIONS—DYE RUNS—OPERATIONAL YEAR 1982
Sampled rig
Tower
Run
No.
D1
02
D3
04
No.
15
4
4
15
Orien-
tation
230°
330°
330°
65°
End gun
status
Off
Off
Off
On
Left
pos 1 t 1 on
3
6
6
3
Right
position
5
4
4
5
Aerosol sampler
Line
angle
$sla
65°
70°
70°
90°
location
Distance to rig (m)
Left
position
25 75
25 75
25 75
40 80
Right
position
25 75
25 75
25 75
40 80
Mean wind
direction
ewt>
80°
90°
80°
90°
Wastewater
Source Temp
Pipeline
Pipeline
Pipeline
Pipeline 25.
(°C)
5
a 8sl - angle of sampler line with rig (0° _<_ &s1 _<_ 90°)
b 0W - mean angle of wind with the rig during the run, measured In same direction from rig as
-------
TABLE A-10. SUMMARY OF SAMPLING CONDITIONS--PARTICLE SIZE RUNS—OPERATIONAL YEAR 1982
Aerosol sampler location
Run
no.
PI
P2
P3
P4
P5
a 6S 1
b 6W
Sampled
Or 1 en-
No, tat Ion
2 130°
1 1 330"
15 70°
4 280°
14 30°
- angle of
rig
End gun
status
Off
Off
Off
Off
Off
samp 1 er
Line
Position/ Angle
tower 9C i a
Center/3 80°
Rlght/6 85°
Inner/3 75"
Center/4 85°
Center/5 60°
line with rig (0°
Distance to
Pair
36
33
20
35
35
vo
-------
TABLE A-ll. CORRECTION FACTOR FOR LVS OPERATING VOLTAGE
(Referenced Basis of 12 kV)
Operating voltage (kV) Correction factor (F)
6 0.33
8 0.36
9 0.38
10 0.42
11 0.47
11.5 0.80
12 1.00
12.5 1.15
13 1.25
13.5 1.32
14 1.33
14.5 1.32
15 1.29
16 1.24
17 1.22
18 1.21
393
-------
TABLE A-12. SUMMARY OF METEOROLOGICAL CONDITIONS—AEROSOL RUNS—OPERATIONAL YEAR 1982
Mean wind
direction (e)
Run no.
Run date
Run time
Ml/2-22-82
1850-1920
M2/2-23-82
1650-1720
M3/2-24-82
1400-1430
M4/3-17-82
1535-1605
M5/3-18-82
1230-1300
M6/3-19-82
1148-1218
M7/7-7-82
1620-1650
M8/7-6-82
1353-1423
M9/7-9-82
1331-1401
M 10/7-1 1-82
1530-1600
Ml 1/7-14-82
1350-1420
M12/7-15-82
1114-1144
M13/7-16-82
1025-1055
Ml 4/8-3-82
1327-1357
Ml 5/8 -5-82
1211-1241
Ml 6/8-6 -82
1210-1240
Ml 7/8-23-82
2030-2100
Ml 8/8-25-82
2125-2155
M19/8-26-82
1422-1452
M20/8-27-82
1320-1350
Air temp
At run
location
16
26
10
24
24
17
29
31
32
28
31
28
27
33
34
31
24
22
32
35
(°C)
EWS a
19.5
29
12.5
26.5
26
19
30
33
35
32
32
30
29
33
32
33
28
25
35
35
At run
location
(2 m)
160
200
35
145
155
240
85
120
160
50
150
155
170
155
185
235
120
115
b
200
EWS
(10 m)
170
205
40
155
160
255
110
140
160
65
180
185
180
210
170
240
120
140
220
235
Wind speed (m/sec)
At run
location
(2 m)
9.6
6.7
10.3
2.4
7.9
6.6
11.4
6.9
4.2
7.7
6.4
8.0
7.3
4.3
4.9
3.3
0.9
0.6
b
2.5
EWS
(10 m)
5.5
7.0
11.5
2.5
9.0
8.0
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1.25
3.0
2.5
Humidity
at run
location
46
50
49
34
66
21
59
54
29
51
40
51
54
37
24
39
59
77
51
31
Radiation at run location
Dewpolnt
at^EWS
-12
-4
-17
-5
-7
-12.5
-4
-2
-1
-3
-2.5
-4
-4.5
-1.5
-2.5
-1.5
-6
-9
0
0
Cloud
cover
(8ths)
3
<}
7
6
4
"'
4
<}
<]
2
2
<]
0
0
0
0
<]
3
1
<'
Cloud
height
NA
-
High
High
High
-
Middle
—
-
High
High
-
-
-
—
-
-
High
Middle
& High
—
Solar radiation
gcal/cm2/mln
0
0.73
0.90
0.93
0.95
1.23
0.51
1.25
1.26
0.15
1.35
1.10
1.05
1.20
1.15
1.17
After sunset
After sunset
0.63
1.14
NA - not aval(able
a Meteorological data collected from ClImatron Ics Electronic Weather Station (EWS) at research plot
b Field met system malfunction
-------
TABLE A-13. SUMMARY OF METEOROLOGICAL CONDITIONS—QUALITY ASSURANCE RUNS—OPERATIONAL YEAR 1982
Mean wind
direction (°)
Run no.
Run date
Run time
Q 1/3-1 5-82
1543-1613
02/7-13-82
1359-1429
Air temp CO
At run
location EWSa
19 11
29 30
At run
. location
(2 m)
230
170
EWS
(10 m)
250
190
Wind speed
At run
location
(2 m)
9.4
3.8
(m/sec)
EWS
(10 m)
11.5
NA
Humidity
at run
location
<*)
30
49
Radiation at run location
Dewpolnt Cloud
at EWS cover Cloud
CO (8ths) height
-10.5 Blowing dust
-4 <1
Solar radiation
gcal/cm2/mln
0.44
1.34
NA - not aval I able
a Meteorological data collected from CI(matronIcs Electronic Weather Station (EWS) at research plot.
vo
en
-------
TABLE A-14. SUMMARY OF METEOROLOGICAL CONDITIONS—VIRUS RUNS—OPERATIONAL YEAR 1982
u>
vo
o\
Run no.
Run date
Run time
V1/3-16-82
1027-1057
1109-1139
1204-1234
1246-1316
1349-1419
V2 78-2-82
1431-1501
1509-1539
1600-1630
1637-1707
1733-1803
1121-1151
1200-1230
1247-1317
1326-1356
1414-1444
V4/8-24-82
1113-1143
1153-1223
1246-1316
1326-1356
1426-1456
Segment
no.
1
2
3
4
5
Avg
1
2
3
4
5
Avg
1
2
3
4
5
Avg
1
2
3
4
5
Avq
Air temp (°C)
At run
location EWSa
14
-
17
19
22
18 18
31
31
31
31
31
31 33.5
29
30
32
32
33
31 32
29
30
31
32
33
31 33
Mean wind
direction (°)
At run
location EWS
(2 m) (10 m)
290
270
215
210
210
239 260
155
155
150
150
155
153 170
150
155
125
150
125
141 170
NA
170
140
155
180
161 180
Wind speed
(m/sec)
At run
location EWS
(2 m) (10 m)
6.0
3.5
4.6
4.5
5.8
4.9 4.0
4.8
5.2
5.1
5.0
5.9
5.2 NA
4.5
4.7
3.4
2.9
4.0
3.9 NA
NA
3.6
4.6
3.1
2.3
3.4 NA
Humidity
at run
location
(*>
41
-
42
40
27
38
51
51
40
42
40
45
53
50
43
52
40
48
41
44
40
44
42
42
Radiation at run location
Dewpolnt Cloud
at EWS cover Cloud Solar radiation
(eC) (8ths) height gcal/cm2/min
0.93
.12
.20
.12
.14
-13 6 High .10
1.24
1.15
1.05
0.95
0.69
-1 <1 High 1.02
.08
.15
.20
.15
.18
-2.5 0 - .15
,-
.02
.09
.15
.12
.10
-2 0 .10
NA - not aval I able
a Meteorological data collected from ClImatronlcs Electronic Weather Station (EWS) at research plot.
-------
TABLE A-15. SUMMARY OF METEOROLOGICAL CONDITIONS—DYE RUNS—OPERATIONAL YEAR 1982
u>
VO
-J
Mean wind
direction (e)
Run no.
Run date
Run time
01 73-18-82
1455-1502
D2/7-11-82
1733-1740
D3/7-11-82
1752-1758
D4/7-13-82
1533-1539
Air temp (°C)
At run
location EWS a
25 28
26 28.5
25 28
30 31.5
At run
location
(2 m)
NA
60
50
155
EWS
(10 m)
160
65
60
180
Wind speed (m/sec)
At run
location
(2 m)
NA
7.9
7.9
3.6
EWS
(10 m)
9.5
NA
NA
NA
Humidity
. at run Dewpolnt
location at EWS
(if) CO
59 -6
63 -5
63 -5.5
50 -2
Radiation at run location
Cloud
cover C 1 oud
(8ths) height
4 High
2 High
2 High
<1
Solar radiation
gcal/cm2/mln
0.55
<0.05
<0.05
1.34
NA - not aval(able
a Meteorological data collected from Climatronlcs Electronic Weather Station (EWS) at research plot.
-------
TABLE A-16. SUMMARY OF METEOROLOGICAL CONDITIONS—PARTICLE SIZE RUNS—OPERATIONAL YEAR 1982
Mean wind
direction C)
Run no.
Run date
Run time
PI 72-23-82
1609-1619
P2/3-16-82
1539-1549
P3/7-8-82
1510-1518
P4/7-14-82
1519-1527
P5/8-25-62
w 1730-1738
00
Air temp (°C)
At run
location EWSa
28 29.5
22 13.5
31 33.5
29 32.5
29 31.5
At run
location
(2 m)
200
180
130
155
100
EWS
(10 m)
210
210
150
185
120
Wind speed
At run
location
(2 m)
7.8
6.7
7.6
6.7
2.5
(m/sec)
EWS
(10 m)
7.2
7.0
NA
NA
NA
Humidity
at run
location
<*)
20
21
46
43
49
Dewpolnt
at EWS
CO
-4
-8
-1.5
-2
-1
Radiation at
Cloud
cover C 1 oud
(8ths) height
<1
6 High
<1
2 High
5 High
run location
Solar radiation
gcal/cm2/min
0.73
0.61
1.21
1.15.
NA
a Meteorological data collected from ClImatronlcs Electronic Weather Station (EWS) at research plot.
-------
TABLE A-17. RECOVERY OF SALMONELLA FROM WASTEWATER SAMPLES
USING TWO PROCEDURES
Sample
Lubbock-LV-7
Lubbock-LV-8
Lubbock-LV-9
Lubbock-LV-12
Lubbock-LV-13
Lubbock LV-14
Standard selenite enrichment
Salmonella Volume enriched
detected mL
200
+ 200
+ 200
+ 100
100
+ 25
Double enrichment B
Salmonella Volume enriched
detected mL
+ 100
+ 100
+ 10
+ 1
+ 10
+ 1
+ 0.1
+ 1
+ 0.1
+ 0.01
+ 0.1
0.01
1
+ 0.1
a Kaper et al. (1977).
399
-------
TABLE A-18.
COMPARISON OF PROCEDURES FOR RECOVERY OF YERSINIA
ENTEROCOLITICA—UNSEEDED SAMPLES
Recovery of
Y. enterocol
quadrant at plating
Enrichment
None
0.067 M PBS
0.067 M PBS with
1% mannitol
0.85% Nad with
potassium
tellurite
(25 ug/mL)
Medium
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
Treatment
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
Di rect
from
sample Zero
Oa
2
0
0
0
1
0
2
0
2
0
0
0
2
0
2
1
0
0
0
1
0
0
0
3
days
0
3
0
2
0
0
0
2
0
2
0
0
2
0
0
0
0
0
7
days
3
2
0
3
0
2
0
2
0
3
0
2
0
0
0
0
0
0
itica
time
14
days
0
2
0
3
0
0
0
3
0
3
0
2
0
0
0
0
0
0
from
21
days
0
2
0
3
0
1
0
3
0
3
0
3
0
0
0
0
0
0
a 0 = none detected
400
-------
TABLE A-19. COMPARISON OF PROCEDURES FOR RECOVERY OF YERSINIA
ENTEROCOLITICA--SEEDED SAMPLES
Recovery of
Y. enterocol
quadrant at plating
Enrichment
None
0.067 M PBS
0.067 M PBS with
1% mannitol
0.85% NaCl with
potassium
tellurite
(25 pg/mL)
Medium
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
Treatment
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
Direct
from
sample Zero
03
0
0
0
0
0
4
3
3
3
2
1
3
3
0
2
3
2
3
0
0
3
0
0
3
days
4
3
0
2
0
1
3
2
3
2
4
1
0
0
0
0
0
0
7
days
0
2
4
3
3
2
3
3
4
2
0
1
0
0
0
0
0
0
itica
time
14
days
3
2
0
3
0
3
0
4
0
4
4
3
0
0
0
0
0
0
from
21
days
0
3
0
2
0
2
0
3
0
0
0
2
0
0
0
0
0
0
a 0 = none detected
401
-------
TABLE A-20. PARALLEL TESTING OF CLOSTRIDRIUM PERFRINGENS ASSAYS:
COMPARISON OF MULTIPLE TUBE INOCULATION AND
MEMBRANE FILTRATION TECHNIQUES
Sample
Lubbock 4
Wilson 4
Lubbock 4
Wilson 4
Lubbock 5
Wilson 5
Lubbock 5
Wilson 5
Lubbock 6
Wilson 6
Lubbock 6
Wilson 6
Heat
treatment3
+A
+A
-A
-A
+A
+A
-A
-A
+A
+A
-A
-A
Clostridrium perfri
Multiple tube
(MPN/100 mL)
7.5 x 104
4.3 x 104
2.1 x 106
7.5 x 104
1.1 x 105
2.4 x 104
2.8 x 105
4.6 x 106
1.5 x 104
2.1 x 104
1.1 x 106
1.1 x 105
ngens enumerated
by
Membrane filtration
(cfu/100 mL)
3.5 x 104
5.0 x 103
5.0 x 104
1.5 x 104
no growth*5
no growth
no growth
no growth
5.9 x 104
6.9 x 104
6.0 x 104
7.6 x 104
Sample heated at 80°C for 30 minutes on multiple tube procedure and 65°C
for 15 minutes on the membrane filtration procedure (A = heat).
Increased volumes of sample tested were also negative for isolated
colonies of C. perfringens.
402
-------
TABLE A-21. VIRAL TYPES RECOVERED FROM WASTEWATER BY
THE BENTONITE ADSORPTION PROCEDURE
Cell line Viruses isolated
HeLa Poliovirus 1, 2. 3
Coxsackievirns Al. A7, A9, A10, A16
Coxsackievirns B3, B4. B5
Echovirus 1. 3. 6. 7, 11, 21, 25
BGM Poliovirns 1, 2, 3
Coxsackievirns B2, B3, B4, B5
Echovirus 11, 25
RD Poliovirus 2, 3
Coxsackieyirns Bl
Echovirus 6. 7. 11. 19. 22. 24. 30. 33
a Isolated from San Antonio, Lubbock, and Wilson
samples; identified by a micronentralization tech-
nique using Lim Benyesh-Melnick typing pools.
403
-------
TABLE A-22. VIRAL ISOLATES RECOVERED FROM THE SAME WASTEWATER SAMPLES
BY VARIOUS ASSAY PROCEDURES
Sample
Type
assay
Cell
line
Viruses isolated3
Lubbock-1
Lubbock-2
Wilson-1
Plaque
Tube
Tube
Plaque
Tube
Tube
Plaque
Tube
Tube
HeLa Poliovirus 1, 2, 3
Coxsackievirus Al, A7, A9, A16
Coxsackievirus B3, B4, B5
Echovirus 1, 3, 6, 11, 21, 25
BGM Coxsackievirus B2, B3, B4, B5
Echovirus 11, 20, 24
RD Poliovirus 1
Coxsackievirus Bl
Echovirus 6, 15, 24, 25, 29, 33
HeLa Poliovirus 2, 3
Coxsackievirus B2, B3, B5
BGM Coxsackievirus B2, B3, B5
RD Echovirus 11, 15, 19, 30
HeLa Poliovirus 1, 3
Coxsackievirus A10
Echovirus 25
BGM Poliovirus 1
Coxsackievirus B2, B5
Echovirus 25
RD Poliovirus 3
Coxsackievirus Bl
Echovirus 24
Identified by a microneutralization technique using Lim Benyesh-Melnick
typing pools.
404
-------
TABLE A-23. ENTEROVIROS ASSAY MATRIX FOR WASTEWATER SAMPLES
Number of 100 mm plates/dilution
Cell line/assay system Undiluted IP"1
He La
HeLa + polio antisera
Rn 4- nnlin ant i
10
10
10
10
0
5
TABLE A-24. VIRAL NEUTRALIZATION BY POLIOVIRDS ANTISERUM*
Ant i serum
Viral antigen
Type
Type
Type
1
2
3
polio
polio
polio
(LSC)
(P-712)
(Leon)
Batch
Date
6-63
7-65
6-65
Viral titer
(pfn/mL) Neutralization
Test virus Tn
Polio
Polio
Polio
1
2
3
(LSC)
(MEF)
(Sabin)
1.
1.
3.
6xl04
8x105
9xl04
Tan' »
<5xlO°
5x100
<5xlO°
(Tsn» »/Tn)
>3.1xlO~4
2.8x10-5
>1.3xlO"4
a Results shown are for 1:100 dilution of rehydrated, heat-inactivated
(56°C, 30 minutes) antiserum supplied as an NIH research reagent.
TABLE A-25. CONCENTRATION EFFICIENCY OF ORGANIC
FLOCCULATION AND TWO-PHASE SEPARATION
Concentration
procedure
Organic flocculation
0% beef extract
1% beef extract
2% beef extract
3% beef extract
Two-phase separation
% Polio la % CB3B
recovered recovered
33 53
41 61
55 77
33 62
50 61
% Echo 6b
recovered
60
79
84
81
43
a Results are an average of four experiments.
b Results are an average of two experiments.
405
-------
TABLE .A-26. LISS HEALTH DATA PROCESSING STATUS REPORT (excluding serology)
-u
o
ON
Data
Collection
Period
Oil
012
013
014
015
016
017
018
019
020
108
109
110
111
112
113
114
MS
116
117
118
119
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
Start
Date
1980
Hay 18
Jun. 1
Jun. IS
Jun. 29
Jul. 13
Jul. 27
Aug. 10
Aug. 24
Sept. 7
Sept. 21
1981
Apr. 5
Apr. 19
Hay 3
Hay 17
Hay 31
Jun. 14
Jun. 28
Jul. 12
Jul. 26
Aug. 9
Aug. 23
Sept. 6
1982
Jan. 3
Jan. 17
Jan. 31
Feb. 14
Feb. 28
Mar. 14
Mar. 28
Apr. 11
Apr. 25
Hay 9
Hay 23
Jun. 6
Jun. 20
Jul. 4
Jul. 18
Aug. 1
Aug. IS
Aug. 29
Sept. 12
Sept. 26
Oct. 10
Oct. 24
Nov. 7
Nov. 21
Dec. 5
Dec. 19
Household
Health
Diary
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
LARKV
Scheduled
Fecal
Bacteriology
ARCKVD
ARCKVD
ARCKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
Clinical
Virology
ARCKVD
ARCKVD
ARCKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
Illness
Sped tie n Electron Activity
Bacteriology Microscopy Diary
AR
AR
ARKP AR
AR
AR
AR
ARKP
AR
AR
ARKP AR
ARKP
ARKP
ARKP AR
ARKP LARCKVD
ARKP AR
ARCKVD
AR
ARKP
ARKP
ARKP
ARKP AR LARCKVD
ARKP
ARKP
ARKP AR
ARKP
ARKP
ARKP
ARKP
ARKP ARCKVD
ARKP
Household/
Participant Polio
Interview Innunlzatlon
ARKVP
ARKP
ARKP
ARKP
ARKP
ARKVP
continued.
June 1984
-------
TABLE A-26. (CONT'D)
Data
Collection
Period
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
Start
Date
1983
Jan. 2
Jan* 16
Jan* 30
Feb. 13
Feb. 27
Mar. 13
Mar. 27
Apr. 10
Apr. 24
May 8
May 22
Jun. 5
Jun. 19
Jul. 3
Jul. 17
Jul. 31
Aug. 14
Aug. 28
Sept. 11
Sept. 25
Oct. 9
Oct. 23
Nov. 6
Nov. 20
Dec. 4
Dec. 18
Household Scheduled
Health Fecal Clinical
Diary BacterloloRv Virology
LARKV
LARKV
LARKV LARKVD LARKP
LARKV
LARKV
LARKV
LARKV
LARKV LARKVD LARKP
LARKV
LARKV
LARKV
LARKV LARKVD LARKP
LARKV
LARKV
LARKV LARKVD LARKP
LARKV
LARKV LARKVD LARKP
LARKV
LARKV
LARKV
Illness Household/
Specimen Electron Activity Participant Polio
Bacteriology Microscopy Diary Interview Immunization
ARKP
ARKP
ARKP AR
ARKP
ARKP
ARKP AR ARCKVD
ARKP
ARKP
ARKP
ARKP AR
ARKP
ARCKVD
ARKP AR
ARKP AR
ARKP
ARKVP
'
Status Codes
L - Labels Generated
S - Samples Stored
A - Activity Conducted
R - Received by Data Manager
C - Coded by Data Processing Group
K - Keypunched
P - Preliminary on Data Base
V - Verified
D - Data Processing Completed
-------
TABLE A-27. SAMPLED MICROORGANISM DENSITIES ON THE QUALITY ASSURANCE AEROSOL RUNS
Quality
assurance
run
number
Qia.b
(75 m from
nozzle line)
Q2
w (50 m from
nozzle line)
Sampler
alignment
(from left
to right)
(Wastewater cone. ,
no./mL)
123
201
210
211
217
219
223
226
227
(Wastewater cone. ,
no./mL)
210
219
226
106
227
123
211
223
217
Microorganism concentration in air
Fecal
col i forms
(cfu/m3)
(51,000)
160,170,160
250
640
TNTC, TNTC, TNTC
TNTC
(50,000)
87,90,80
52 ,
430
180,170,170
200
Fecal
streptococci
(cfu/m3)
(4,800)
120,330,330
260
270
280,210,280
390
(3,600)
70,78,76
120
270
53,60,50
38
Mycobacteria
(cfu/m3)
(20,000)
3.5
Coliphaqe
(pfu/m3)
(1,100)
6.7,8.0,5.9
2.6,5.3,5.3
4.4
4.2
8.2,5.3,4.0
(25,000)
2.0.60°
<0.15,<0.15,0.15
0.30
20.90C
2.0,0.52,0.67
11
12
8.3,10.4,8.
16
(720d)
4.6,d3.8 d9.
4.0d
6.0d
7.0,d6.6 d7.
10Q
3
6d
2d
TNTC - too numerous to count
a Conducted during a dust storm.
b Portions received at laboratory at elevated temperature (9°C).
c A large number of colonies with indistinguishable morphology were present. Since only
representative colonies were examined for acid fastness, reported data are minimal values.
d Possible laboratory contamination due to phage aerosolization.
-------
TABLE A-28. CONSISTENCY OF AEROSOL MEASUREMENT PRECISION OVER DENSITY RANGE
Microorganism group/
sample set
Fecal coliforms (cfu)
Usual detection limit
M1-M20: 6 pairs with C<1
M1-M20: 8 pairs with 110
Q2: 5 samplers
Ql: 3 samplers
AVERAGE OVER ALL SETS
Fecal streptococci (cfu)
Usual detection limit
M1-M20: 14 pairs with C<1
M1-M20: 10 pairs with 11
Ql: 5 samplers
AVERAGE OVER ALL SETS
Clostridium perfringens (cfu)
Usual detection limit
Sporulated: M17-M20: 3 pairs
Vegetative: M2.M17-M20: 7 pairs
AVERAGE OVER BOTH SETS
Coliphage (pfu)
Usual detection limit
M1-M20: 13 pairs with C<1
M1-M20: 9 pairs with 15
AVERAGE OVER ALL SETS
Mean
aerosol
density
(no./nr)
0.1
0.3
3.3
21
190
350
0.3-350
0.1
0.3
3.7
27
75
110
290
0.3-290
0.1
0.3
0.9
2.9
4.5
0.3-4.5
0.3
0.8
1.6
0.1
0.3
3.4
6.7
11.0
11.1
0.3-11
Average coefficient
of variation
for replicate
measurements
0.70
0.60
0.37
0.84
0.82
0.67
0.71
0.52
0.21
0.20
0.90
0.21
0.46
0.75
1.26
1.02
0.20
0.81
0.69
0.74
0.72
0.56
0.20
0.37
0.33
0.71
0.43
409
-------
TABLE A-29. ESTIMATED MAGNITUDE OF SOURCES OF PRECISION VARIATION
Average coefficient of variation (s/x)
Microorganism group/
quality assurance run
Fecal coliforms (cfu)
Q2
Ql
Fecal streptococci (cfu)
Q2
Ql
Mycobacteria (cfu)
Q2
Ql
Coliphage (cfu)
Q2
Ql
Mean
density
in air
(no./nr*)
190
350
110
290
0.88
4.5
6.7
11.0
Measurement
variation
(all
sources)
0.84
0.82
0.90
0.21
1.26
0.20
0.37
0.33
Portion
variation
(shipping and
lab sources)
0.053
0.040
0.085
0.35
1.40
0.41
0.32
0.16
Al iquot
variation
(lab
sources)
0.1
0.06
0.08
0.08
0.4
0.17
0.16
Presumed
shipping
sources3
_b
-
0.04
0.3
1.3
0.3
0
Presumed
field
sources3
0.8
0.8
0.9
-
-
-
0.2
0.3
a Determined by subtraction of variances.
b Subtraction gives negative variance; presumably little variation due to this source.
-------
TABLE A-30. POLIOVIRDS TITER REPRODUCIBILITY: COMPARISON OF
RESULTS REPORTED BY U. OF ILLINOIS AND D. OF IOWA FOR THE SAME SERUM
Serum
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Polio 1
Illinois
>1024
32
4
32
32
64
16
8
8
32
16
8
4
8
32
4
16
256
Iowa
256
16
<8
16
64
128
16
8
16
64
16
8
<8
8
64
<8
128
256
Polio 2
Illinois
32
16
<4
128
256
64
16
256
16
32
32
16
<4
16
64
4
32
32
Iowa
r -
64
8
<8
64
512
128
64
256
32
64
>1024
64
<8
64
8
128
64
128
Polio 3
Illinois
8
4
<4
16
8
8
8
8
<4
<4
32
<4
8
8
8
8
64
8
Iowa
32
<8
<8
8
8
8
16
16
16
8
128
<8
16
8
8
16
64
8
411
-------
TABLE A-31. REPEATABILITY OF CLINICAL BACTERIOLOGY RESULTS:
SPLIT FECAL SPECIMENS
ID number
Reported results
QA results
Agreement3
Period 108
55713
55913
32111
43414
21112
53313
32412
12311
12302
31011°
42613
22712
53913
E. colib
E. coli (M)
E. coli (M)
E. coli (M)
K. oxytoca (L)
E. coli (H)
S. aureus (L)
C. freundii H2S+ (L)
C. freundii H2S" (VL)
E. coli (H)
K. oxytoca (L)
H. alvei (L)
E. cloacae (L)
C. albicans (L)
S. aureus (H)
K. pneumoniae (VL)
K. pneumoniae3
E. sakazakii3
E. coli (H)
S. aureus (L)
K. pneumoniae (VL)
E. cloacae (VL)
E. coli (M)
K. pneumoniae (VL)
E. coli (M)
C. albicans (VL)
E. coli (M)
E. coli (M)
K. oxytoca (VL)
Fl. pseudomonas (VL)
E. coli (H)
S. aureus (L)
E. colib
C. albicans (VL)
E. coli (H)
E. cloacae (VL)
E. coli (H)
E. coli (M)
K. oxytoca (L)
E. coli (H)
S. aureus (L)
C. freundii (L)
K. pneumoniae (VL)
E. coli (H)
K. oxytoca (L)
H. alvei (L)
E. cloacae (L)
C. albicans (L)
S. aureus (H)
K. pneumoniae (VL)
K. pneumoniae3
E. sakazakii3
E. coli (H)
S. aureus (L)
K. pneumoniae (VL)
E. cloacae (VL)
E. coli (M)
C. albicans (VL)
E. coli (M)
C. albicans (VL)
E. coli (M)
C. albicans (VL)
E. coli (M)
E. coli (M)
E.
S.
coli (H)
aureus (L)
continued.
412
-------
TABLE A-31. (CONT'D)
ID number
12202
40812
E.
K.
C.
E.
Fl
Reported results
coli (M)
oxytoca (VL)
freundii (VL)
coli (M)
. pseudomonas (VL)
QA results
c •
E. coli (M)
K. oxytoca (VL)
C. freundii (VL)
C. albicans (VL)
E. coli (M)
Fl . pseudomonas (VL)
Agreement
.;
Period 110
55912
55913
42613
40411
12211
53312
E.
S.
E.
K.
S.
E.
K.
E.
E.
C.
E.
K.
C.
coli (M)
aureus (L)
coli (M)
pneumoniae (VL)
epidermidis (VL)
coli (M)
oxytoca (L)
coli (H)
coli (H)
albicans (M)
coli (H)
pneumoniae (L)
albicans (VL)
E.
S.
E.
E.
K.
S.
E.
K.
E.
E.
C.
E.
K.
C.
coli (M) +
aureus (L)
cloacae (VL)
coli (M) ++
pneumoniae (VL)
epidermidis (VL)
coli (M) ++
oxytoca (L)
coli (H) ++
coli (H) ++
albicans (M)
coli (H) ++
pneumoniae (L)
albicans (VL)
a Degree of agreement:
++ Total agreement (same organisms identified and same level of growth)
on split specimens.
+ The level of growth differed by one quadrant, or organisms were identified
in one specimen at the VL level (1 to 10 colonies on plate) but not in
the respective split specimen. Because of the small numbers of organisms
represented by the VL level of growth, such differences are probably
not significant.
Disagreement in identification of one or more organisms isolated at
the light or greater level, or a two quadrant or greater discrepancy
in level of growth.
b Isolated by enrichment procedures, therefore nonquantitative.
c Sample split into three portions, rather than two.
413
-------
TABLE A-32. ACCURACY OF CLINICAL BACTERIOLOGY RESULTS:
ANALYSIS OF SEEDED UNKNOWN FECAL SPECIMENS
Specimen
1
2
3
4
Identification reported
Klebsiella pneumoniae
Shigella flexneri
Yersinia enterocolitica
Enterobacter cloacae
Salmonella species
Serratia marcescens
Staphylococcus anreus
Klebsiella pneumoniae
Shigella flezneri
Yersinia enterocolitica
Candida albicans
Escherichia coli
Proteus vulgaris
Level8
H
H
H
H
H
H
H
H
H
H
H
H
H
Correct identification
Klebsiella pneumoniae
Shigella flezneri
Yersinia enterocolitica
Enterobacter cloacae
Salmonella typhimurium
Serratia marcescens
Staphylococcus aureus
Klebsiella pneumoniae
Shigella flezneri
Yersinia enterocolitica
Candida albicans
Escherichia coli
Proteus vulgaris
Level
H
H
H
H
H
H
H
H
H
H
H
H
H
a Quant itat ion of growth: H - heavy.
414
-------
TABLE A-33. LEVELS OF GROWTH REPRESENTED BY DIFFERENT CONCENTRATIONS
OF KNOWN ORGANISMS: FECAL SPECIMEN PROCEDURE
Organism
Escherichia coli
Elebsiella pneomoniae
Psendomonas aernginosa
Seeded organism
concentration.
cfn/mL
9 x 101
9 x 102
9 x 103
9 x 104
4.5 x 106
9 x 106
4.5 x 10?
9 x 10?
0
33
3.3 x 103
3.3 x 105
3.3 x 10^
7
700
7.0 x 104
7.0 x 106
7.0 x 10?
Level of quant itat ion from
e clinical
NG
L
L
L
M
M
M
M
NG
VL
NG
L
M
NG
NG
L
M
H
lab reoorta
VL
L
L
L
L
L
M
H
NG
NG
NG
M
H
NG
NG
L
M
H
a Quant itation of growth on duplicate platings of seeded unknowns:
NG - negative
VL - very light
L - light
M - moderate
H - heavy
415
-------
TABLE A-34. LEVELS OF GROWTH REPRESENTED BY DIFFERENT CONCENTRATIONS
OF KNOWN ORGANISMS: THROAT SWAB PROCEDURE
Organism
Escherichia coli
Enterobacter cloacae
Klebsiella pneumoniae
Streptococcus pyogenes
Seeded organism
concentration,
cfu/mL
4.0 x 102
4.0 x 103
4.0 x 104
4.0 x 105
4.0 x 106
4.0 x 107
4.0 x 108
3.1 x 102
3.1 x 103
3.1 x 104
3.1 x 105
3.1 x 106
3.1 x 10?
3.1 x 108
2.7 x 102
2.7 x 103
2.7 x 104
2.7 x 10s
2.7 x 106
2.7 x 10?
2.7 x 108
1.8 x 101
1.8 x 102
1.8 x 103
1.8 x 104
1.8 x 105
1.8 x 10*
1.8 x 10?
Level of quantitation from
, clinical
L
L
H
H
H
H
H
VL
L
H
H
H
H
H
VL
L
M
H
H
H
H
NG
VL
L
M
H
H
H
lab report8
VL
L
L
H
H
H
H
VL
L
L
H
H
M
H
VL
L
H
M
H
H
H
NG
VL
L
H
L
H
H
a Quantitation of growth on duplicate platings of seeded unknowns:
NG - negative
VL - very light
L - light
H - moderate
H - heavy
416
-------
TABLE A-35. REPEATABILITY OF CLINICAL VIROLOGY RESULTS:
SPLIT FECAL SPECIMENS
Period
116
117
and
118
Sample
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
18
16
17
19
20
21
22
23
25
26
27
28
29
Participant
22712b
22712b
12211
12202
10413
55912
55913
32412
23112
32411
53912
53911b
53911b
20211
53313
22512
40311
40312
56211
56202
45114
53312
45113
40312b
40312b
40311b
40311b
40216
12202
12211
55715
32412
53911
55911
22512
40214
43613
Routine -Ana
HeLa
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
0/2
1/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
lysisa QA Analysisa
RD
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
HeLa
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
2/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
RD
0/2
2/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
0/2
0/2
1/2
0/2
0/2
0/2
0/2
0/2
0/2
a Number of tubes showing viral cpe/total number of tubes inoculated for
each cell line listed. Other cells used with negative results were BGM
and primary RhMK.
b Replicate QA sample.
417
-------
TABLE A-36. ENVIRONMENTAL QUALITY ASSURANCE: REPLICATE ANALYSES OF SPLIT WASTEWATER SAMPLES
Analysis
bounce
Sample 1
Sample 2
Mean
oo
Bacteriology3
Fecal coliform
Wilson LV-9
Lubbock LV-9
Wilson LV-10
Total coliform Lubbock LV-9
Fecal streptococci Lubbock LV-9
Virology
Lubbock LV-9
Wilson LV-10
Enteroviruses on
HeLa (unaltered)
4.2 x 106/100 mL
8.8 x 106/100 mL
6.9 x lO^/lOO mL
1.5 x 107/100 mL
4.2 x 10&/100 mL
1.1 x 102 pfu/L
1.6 x 102 pfu/L
3.9 x 106/100 mL
8.4 x 106/100 mL
6.2 x 107/100 mL
1.6 x 107/100 mL
4.8 x 105/100 mL
1.2 x 102 pfu/L
1.7 x 10? pfu/L
4.1 x 106
8.6 x 106
6.6 x 107
1.6 x 107
4.5 x 105
1.2 x 102
1.7 x 102
a Membrane filtration.
-------
TABLE A-37. REPRODUCIBILITY IN SEPARATE LABORATORIES OF BACTERIAL
INDICATOR DENSITIES IN WASTEWATER DURING BASELINE PERIOD
Sample
date
6-4-80
7-29-80
11-4-80
1-20-81
2-17-81
3-10-81
3-24-81
4-21-81
5-5-81
Total col i form
(cfu/100 mL)
LCCIWR
4.3 x 107
5.0 z 107
3.2 x 10?
1.0 x 107
1.5 x 107
2.7 x 107
1.8 x 107
4.0 x 107
2.9 x 107
DTSA
3.5 107
3.8
1.4
6.0
1.1
1.2
107
107
106
107
107
1.6 x 107
5.2 x 107
Not done
Fecal coliform
(cfu/100 mL)
r LCCIWR
Not done
2.5 x 107
1.5 x 107
2.0 x 106
4.6 x 106
4.5 x 10*
4.0 x 106
5.3 x 10^
5.9 x 106
DTSA
8.7 x 1Q6
7.2 x 106
8.8 x 106
1.5 x 106
3.4 x 106
1.6 x 106
8.3 x 106
5.9 x 106
8.6 x 106
419
-------
TABLE A-38. REPRODCCIBILITY IN SEPARATE LABORATORIES OF
FECAL COLIFORM DENSITIES IN WASTEWATER DURING 1982 AND 1983
Fecal coliforms 1
Samplinn date
2-15/16-82
2-15/16-82b
3-1/2-82
3-8/9-82
3-15/16-82
3-22/23-82
3-29/30-82
4-5/6-82
4-19/20-82
4-26/27-82
6-14/15-82
6-29/30-82
7-26/27-82
8-9/10-82
8-30/31-82
9-13/14-82
11-1/2-82
12-13/14-82
2-16/17-83
3-7/8-83
3-21/22-83
4-4/5-83
4-18/19-83
5-16/17-83
6-27/28-83
7-11/12-83
7-25/26-83
8-8/9-83
8-22/23-83
Hancock
UTSA*
520
60
190
390
10
350
UTA
3.5
730
15
4
150
100
440
-
300
150
3
110
30
reservoir
LCCIWR
940 (600)c
200
370
2 (1.7)
700 (490)
2.8
180
10
1.7
90
44
200
-
160
5.5
1
50
1.7
Pipeline
DTSAa
39
11,000
5.600
75,000
79,000
81,000
55,000
84,000
110,000
9,100
66,000
68,000
58.000
35,000
200
65,000
OTA
49.000
31,000
59,000
23,000
6,100
20,000
18,000
-
59,000
53.000
48.000
120,000
90,000
[ colon ies/mf •)
effluent
LCCIWR
30
97,000
30.000
100,000
180,000
50,000
52,000
16,000
55,000
60,000
20,000
(30,000)
41
34,000
90,000
40,000
4,000
18,000
20,000
14,000
10,000
-
39,000
27,000
40,000
40,000
20
Wilson Imhoff
influent
UTA
UTA
130.000d
110.000*
14,000
150.000
76,000
150,000
130.000
350.000
260,000
370,000
240,000
310.000
230.000
LCCIWR
90,000
100,000
40,000
180.000
45,000
(60,000)
51,000
90,000
60,000
54,000
180.000
13.000
90.000
20,000
a mean of triplicate assays
b trickling filter plant effluent
c parenthetical value, when given, is the result of a duplicate analysis
d samples taken as Imhoff tank effluent
420
-------
TABLE A-39. REPRODDCIBILITY IN SEPARATE LABORATORIES OF FECAL
STREPTOCOCCI DENSITIES IN WASTEWATER DURING 1982 AND 1983
_ Fecal streptococci (colonies/mL)
Sampling date
2-15/16-82
3-1/2-82
3-8/9-82
3-15/16-82
3-22/23-82
3-29/30-82
4-5/6-82
4-19/20-82
4-26/27-82
6-14/15-82
6-29/30-82
7-26/27-82
8-9/10-82
8-30/31-82
9-13/14-82
a Mean of triplicate
Hancock
DTSAa
1
20
3
3
6.6
0.3
10
assays.
b Parenthetical value, when
reservoir
LCCIWRb
12.8
10
6.0
1.1
100 (20)
given, is the
Pipeline effluent
UTSAa
120
1,000
5,900
3,500
7,900
5,000
2.800
4,800
1.800
1,000
4.200
2,300
2,500
30
3.500
result of a
LCCIWRb
40
400
5,000
4,000
2,200
2,600
1,400
1,890 (1
1,800
1,000 (2
61
5.100
.500)
.000)
duplicate analysis.
421
-------
TABLE A-40. IDENTIFICATION OF FECAL COLIFORH ISOLATES
K>
Sample Fecal col i form isolates
Source
Hancock
reservoir
Wilson
influent
Wilson
influent
Date Ozidase -
7-25/26-83
E. cloacae
E. coli
K. pneumoniae
Unidentified"
TOTAL ID
7-25/26-83
E. coli
K. ozytoca
ti. pneumoniae
Unidentified
TOTAL ID
8-8/9-83
E. coli
Klebsiella sp.
Citrobacter sp
Total ID
Oxidase +
8 0
2
2
3
1
8
23 0
13
2
6
2
23
27 3
4 A. hydrophila 2
3 TOTAL ID 2
. 1
8
Nonfecal coli form isolates
Ozidase -
ND
ND
10
E. aerogenes 1
H. alvei 1
Klebsiella sp. 3.
Total ID 7
Ozidase +
ND
ND
5
Fl. Pseudomonas 1
NSC* 1
TOTAL ID 4
a Based on carbohydrate utilization, probably Klebsiella sp., but retesting necessary for positive
ID.
b No such code, presumably not common member of Enterobacteriaceae family.
-------
TABLE A-41. REPRODDCIBILITY OF TOTAL ORGANIC CARBON RESULTS
FOR WASTEWATER DURING 1982 AND 1983
TOC (mg/L)
Hancock reservoir Pipeline effluent
Sampling date UTA LCCIWR j_ UTA LCCIWR
11-1/2-82 28 27 54 41
2-16/17-83 19 19 49 48
3-7/8-83 28 25 109 67
3-21/22-83 23 15 83 75
4-4/5-83 26 21 62 52
4-18/19-83 34 23 50 47
6-27/28-83 17 13 42 36
7-11/12-83 21 17 35 26
7-25/26-83 24 21 22 34
8-8/9-83 27 29 28 32
8-22/23-83 33 49 32 26
423
-------
APPENDIX B
INITIAL PERSONAL INTKKVJLKW QDBSTIONNAIKB
425
-------
HH name
HH Q
Phone 9
HH size
University of Illinois
School of Public Health
lubbock Land Treatment Project:
Personal Interview for Health Watch
(Tune In-teAv-cew Began
cum
'.pm
ASSURANCE OF CONFIDENTIALITY - All information that would permit identifica-
tion of individuals will be held in strict confidence, will be used only by
persons engaged in and for the purpose of the survey and will not be disclosed
or released to others for any purpose. The results will be used only when
combined with those of many other people.
427
-------
First, I would like to ask you a few questions about your household.
1. a. Do you have air conditioning in your home?
Yes 1
No (Stop to 2. 2) 0
b. Do you have
central air conditioning or 1
window or wall units or 2
both 3
c. During the summer, do you have the air conditioning on:
all or most of the time 1
some of the time every day 2
only when it is very hot or 3
never 4
2. Do you obtain your drinking water from
a private well, or 1
public water supply 2
3. Do you dispose of sewage through
a septic tank or cespool or 1
city sewage system . 2
Now, I would like to ask you some questions about household members and
their activities.
4. a. Including yourself, how many people live in this household?
b. How many of these people are related to you?
1$ theAe. one, u.nn.e££Vte.d houAtkoid membeM, (KM);
I will be asking you some questions about each of your family members.
I will be talking with unrelated household members separately.
428
-------
5. a. Beginning with yourself, please tell me the first name of each
person now living in the household who is related to JKJU.
b. How is _ ________ _ related to you?
-te£otcoM-i^u.p and
6. In what year (were you/was _ ) born?
7. a. Do you (does _ ) have a job or go to school outside your home or
farm?
Yes 1
No (SfexLp to Q.. g] 0
b. Looking at the map, please show me where (you/ _ ) works or
goes to school. ( Indicate Zone)
Zone 1
Zone 2
Lubbock 3
Other area (excluding Lubbock) 4
8. Approximately how many hours per week (do you/does ) spend
outside the outlined area shown on this map? (Show map)
9. During the non-winter months, how many hours per day (do you/does )
generally spend out of doors, within the outlined area on: (Show map)
a. Weekdays
less than 1 hour/day 1
more than 1; less than 4 hrs./day 2
more than 4; less than 8 hrs./day 3
more than 8 hrs./day 4
b. Weekends
less than 1 hr./day 1
more than 1; less than 4 hrs./day 2
more than 4; less than 8 hrs./day 3
more than 8 hrs./day 4
Aife Qu.uti.oni, 10 through. 14 4.^ hoLLtuhotd -u, (.ocatid on a
(jo/im. S/u.p to Q.. IS -1$ houAe.ho£d not ioc.ate.d on. a faa/un.
10. How many hours per week (do you/does ) spend doing farm work
out of doors?
0 1
less than 10 hrs./week 2
more than 10; less than 20 hrs./week 3
more than 20; less than 40 hrs./week 4
more than 40 hrs./week 5
429
-------
We also need to find out a little bit about your farm, so we can more
accurately judge what types of farm work household members might be
doing.
11. What crops are you producing on your farm this year? Please tell
me each crop which you are growing, and the amount of acreage de-
voted to it. (Checfe oi many cu> appiy)
Crop Acreage
None None (000)
cotton
wheat
other
12. What types of livestock are you raising on your farm this year?
Please tell me each type of livestock and the number of animals.
(Check 04 many 04 appty)
Livestock Number
None None (000)
cattle
hogs
sheep
fowl
other
13. a. Do you currently irrigate your farm land?
Yes 1
No (Sfexp to £. 14) 0
b. What is the source of that water?
Well 1
Other (specify) 2
14. Approximately how many acres of land do you farm, including pastures,
fallow ground and grazing land?
Sfe-ip to g. 16
430
-------
Aife Q.. 15 j.^ kouAihoid -a, not located on a
15. a. Do you or does anyone in your household ever work on a farm within
the outlined area? Udow map)
Yes 1
No (Skip -to i. 16) 0
) work on a farm?
) work on a farm, when
_) generally work on
b. Who is that?
c. How many weeks per year (do you/does
d. How many days per week (do you/does
(you/ ) work(s)?
e. During which season(s) (do you/does
a farm? (Checfe cu> many 06 app-ty)
a. Spring
b. Summer
c. Fall
d. Winter
16. a. Approximately how many times per month (do you/does ) travel
to Lubbock?
b. Approximately how much time (do you/does ) spend in Lubbock
on each visit?
17. a. Do you or does anyone in your household drink bottled water regularly?
Yes 1
No (Skip to 1. 1&] 0
b. Who is that?
c. Do you/does ever drink water from the tap?
Yes 1
No 0
431
-------
Now I would like to find out about any long term or chronic illnesses
or conditions which you or anyone in this household has ever had which
required consultation with a doctor.
18. a. Have you or anyone in this household ever seen a doctor for any of
these respiratory illnesses or conditions? (Show CflAd A)
Yes 1
No (Sfe^p to £. J9) 0
DK (Skip to Q.. 19) 8
b. Who is that?
fan. eacA yu to Q.. l&a.
Which illness or conditions (do you/does
(Checfe out, many ajt, appty)
) have?
How old (were you/was
first appeared?
a. Allergies
b. Chronic bronchitis
c. Emphysema
d. Asthma
e. Tumor of cancer of the lung
f. Tumor of cancer of the mouth
or throat
g. Other (specify)
) when the
aoniitLonI
(fon. each -UULneJ,& oiAc£ed, A.ecoA.d age on adjacent tine.)
What medications and/or treatments, if any, (are you/is
taking for (your/his/her) _^___^ ?
condition)
432
-------
19. a. Have you or has anyone in this household ever seen a doctor for any
of these heart conditions? [Show COAd 8)
Yes 1
No (S(up to Q,. 20) 0
DK (Sfex.p -to Q,. 20) 8
b. Who is that?
Fo/i eac.fi yu to Q. J9a a&k:
c. Which type of heart condition (do you/does ) have?
a. High blood pressure
b. Stroke
c. Heart attack
d. Angina
e. Other (specify)
d. How old (were you/was ) when the _^__^.^^_^__^_ first
occurred? (Hia.d aondLtion)
e. What medications and/or treatments, if any, (are you/is )
taking for (your/her/his) _ ?
comictccm)
433
-------
20. a. Have you or has anyone in this household ever seen a doctor for
any of these stomach or abdominal conditions? [Shou) coAd C)
Yes 1
No (Skx.p to Q.. 21) 0
DK {SkJ.p to Q,. 21) 8
b. Who is that?
fox. each yu to Q. 20a a&k:
What of these conditions (do you/does
Tumor or cancer of the
have?
How old (were you/was_
appeared?
a. Stomach
b. Intestine
c. Colon
d. Esophagus
e. Stomach (peptic) or intestinal
(duodenal) ulcer
f. Ulcer of the colon (ulcerative
colitis)
g. Diverticulosis
h. Gall bladder problems
i. Other (specify)
) when the
\iiad coniLtcoia)
first
e. What medications and/or treatments, if any, (are you/is
rently taking for (your/her/his) _^__^ ?
condition}
) cur-
434
-------
21. a. Have you or has anyone in this household ever seen a doctor for
any of these other types of conditions? (Shou) c.OJid V)
Yes
No (Sfu.p to Q.
DK (Sfex.p to Q..
Who is that?
1
23) 0
23) 8
Fo/i eodi yu to Q_. 2 Jo. (Life:
Which of these conditions (do you/does ) have?
Hov old (were you/was
peared?
a. Skin cancer
b. Leukemia
c. Hodgkin's Disease
d. Other cancers
e. Arthritis
f. Diabetes
g. Anemia
h. Immunological disorder
i. Rheumatic fever
j. Serum hepatitis (Hepatitis B)
k. Infectious Hepatitis (Hepatitis A)
1. Infectious mononucleosis
m. Other chronic conditions
) when the
[lead con<£t£con)
first ap-
What medications and/or treatments, if any, (are you/is
rently taking for the ?
condt£con)
) cur-
22. Speot^ted medication/'tfl.e&tmznti>
435
-------
23. a. Have you or has anyone in this household ever had a blood trans-
fusion?
Yes 1
No (Sfexp to Q.. 24) 0
DK (SlUp to Q.. 24) 8
b. Who is that?
24. a. Have you or has anyone in this household ever been on a kidney machine
or hemodialysis?
Yes 1
No (Sfex.p to Q.. 25) 0
DK (Sfe^.p to £. 25) 8
b. Who is that?
25. a. Have you or has anyone in the household ever been in close contact
with (i.e. lived with or helped care for) a person who had TB (tuber-
culosis)?
Yes 1
No (Sfextp to Q..- 26) 0
DK (Sfexp to 0. 26) 8
b. Who is that?
26. a. Do you or does anyone in this household smoke cigarettes regularly?
Yes 1
No (Skip to Q.. 27) 0
DK [Skip to Q.. 27) 8
b. Who is that?
436
-------
10
fan each HM boin fae^oie 1962, a
-------
11
31. Which household members contribute to the financial support of this
household?
32. a. Considering all of the income from employment, net farm income and
from all other sources, please tell me which category on this card
best describe your total household income before taxes in 1979?
(Show OlAd E}
a. less than 5,000 1
b. 5,000 - 7,999 2
c. 8,000 - 9,999 3
d. 10,000 - 14,999 4
e. 15,000 - 19,999 5
f. 20,000 - 29,999 6
g. 30,000 and over 7
h. VK [cu,k 328) «
(a&k 328) 9
b. Can you tell me if it was:
less than 10,000 or 1
more than 10,000 2
VK. B
9
33. Now, in case the office finds I've missed something what would be
the best time to call you? a.m.
p.m.
************************************************************
438
-------
I
(Respondent)
Male. I
Femo£e 2
19
!Age )
II
Wo£e I
Female 2
79
(Age )
ill
Ma£c 7
Fema£e 2
19
(Age )
f -
IV
JiaJLe. I
Female 2
79
(Age )
V
da£e I
Female 2
19
(Age )
VI
tafo I
Fema£e 2
19
(Age )
34.
35.
Recoil Phone * on ({/ton* 0|$
-tace 0(5
Bia.dk/Ne.gfw
Qtu.intaJL/teJjiui
Lati.no/Mexxcan/PueAyto R^can
37. Voej> the. x.uponde.n£ Live, •in a:
Scngie &ami£y diueJLtinq 7
Biulctcng ^OA. 2 ijamcttei OA duplex 2
noaie (3-4 u/u-ti) 3
noaie (5 01 mo/ie unx^s) 4
3S.
1.4 ihe household LocsiJUid. on a
Wo
ended
a.m.
p.m.
439
-------
APPENDIX C
PERSONAL QUESTIONNAIRE UPDATE IN FEBRUARY 1982
441
-------
ilCUfiC
__.
HU. S. Ql __ "" "5"
0 :• o
Phone S 21 • : 7
IIH Size 20-7.-i
Interviewer
University oS Illinois
School of Public Health
Lubbock Health Effects Study
Personal Interview Update
(Dace of Interview
ASSURANCE OF CONFIDENTIALITY - All information that would nerrr.it identifi-
cation of individuals will be held in strict confidence, will be used only
by persons engaged in and for the purpose' of the survey and will not be
disclosed or released' to otheis for any purpose. The results will be used
orilv when combined rfith those of many other people.
442
-------
HOUSEHOLD ItlFORI-lATION
la.
Have you changed residences since you enrolled in the Health
Watch ?
Yes (Skip to Q. 2)
No
Have you made any of the following changes in your residence
since you enrolled in the Health Watch?
a. Installed air conditioning (Ask Zb-c)
b. Changed water supplies (Ask 3a)
c. Changed waste disposal (Ask 3b)
2a.
b.
Do you now have air conditioning in your home?
Yes
No (Skip to Q. 2)
Do you have central air conditioning or
window or wall units
or both
1
2
1
2
3
Card Columns
30
31
During the summer, do you have the air conditioning on:
All or most of the time 1
Some of the time everyday 2
Only when it is very hot 3
Never 4
3a. Do you now obtain your drinking water from
A private well, or
public water supply
b. Do you now dispose of sewage through:
A septic tank or cesspool
or city sewage system
End of Household
File
443
-------
PARTICIPANT INFORMATION
la Has anyone left your household permanently or temporarily since
you enrolled in the Health Watch?
Yes 1
No (Skip to Q. 2) 2
b. Who was that?
For each "yes" to Q. la-b, ask the following questions.
c. When did leave? (Record month, year)
d. Did _____ leave permanently?
Yes (Sk.ip to Q. 2) 1
No 2
e. When did return? (Record month, year. If EM has not returned
record "NR" and ask If.)
f. When do you expect to return? (Record month, year. Record
"DK" if return not know.)
2a. Have you added any new members, including infants, to your household
;ir.cc j-:u enrolled in-tl-.c "czlth ";tch?
Yes 1
No (Skip to Q. 3) 2
*b. 'What is his or her name? (Record name in column at top of facing page.)
For each new household member, ask the following questions:
*c. How is related to you? (Record in column at top of facing page.)
*d. What is 's sex? (Record in column at top of facing page.)
*e. What is 's age? (Record in column at top of facing page.)
f. When did enter your household? (Record month, year.)
g. How long will be staying with you? (Record "permanently"
for infants and other permanent residents. Otherwise record length
of stay in weeks.)
444
-------
How, I would like to ask you about any long-term or chronic
illnesses which you or anyone in your household may have develoued
sinc'e you enrolled in the.Health Watch. If you are not sure whether
a household member developed a condition before or after enrolling
in the study, please tell me about it anyway and we can check that
later.
3a. Have you or has anyone in your household been newly diagnosed
as having any of these respiratory illnesses or conditions since
you enrolled in the study?
Read li.?i of conditions. Pause after each condition to allnu
respondent to revl'J. For each "yes", ask "who was that?" and
record condition in appropriate co'uirm,
a. Allergies
b. Chronic bronchitis
c. Emphysema
d. Asthma
e. Tumor or cancer of the lung
f. Tumor or cancer of the mouth or throat
g. Other (specify)
Ask Zb. for each condition reported.
b. What medications and/or treatments, if any, (are you/is }
taking for the ? (Record medications.)
(rsad condition)
4a. Have you or has anyone in your household been newly diagnosed
as having any of these cardiovascular conditions since you
" enrolled in the study?
Read list of conditions. Pause after each condition to allow
respondent to reply. For each "yes", ask "who was that?" and
record condition in appropriate column.
a. High blood pressure
b. Stroke
c. Heart attack
d. Angina
e. Other (specify}
Ask 4b. for each condition reported.
b. What medications and/or treatments, if any, (are you/is
taking for the ? (flecord medications.)
(read condition)
445
-------
5a. Have you or has anyone in your household been newly diagnosed as
having any of these stomach or abdominal conditions since you
enrolled in the study?
Read list of conditions. Pause after each conditions to allow
respondent to reply. FOP each "yes", ask "Who is that? and
record condition in appropriate colwm.
Tumor or cancer of the:
a. Stomach
b. Intestine
c. Colon
d. Esophagus
e. Stomach (peptic) or intestinal (duodenal) ulcer
f. Ulcer of the colon (ulcerative colitis)
g. Diverticulosis
h. Gall bladder problems
i. Other (specify)
Ask Sb. for each condition reported.
b. What medications and/or treatments, if any, (are you/is )
taking for the ? (Record all medicationsTJ
6a. Have you or has anyone in your household been newly diagnosed as
having any of these other types of conditions since you enrolled
in the study.?
Read list of conditions. Pause after each condition to allow
respondent to reply. For each "yes", ask "Who is that?" and
record condition in appropriate column.
a. Skin cancer
b. Leukemia
c. Hodgkin's Disease
d. Other cancers
e. Arthritis
f. Diabetes
g. Anemia
h. Immunological disorder
i. Rheumatic fever
j. Serum hepatitis (hepatitis B)
k. Infectious hepatitis (hepatitis A)
1. Infectious mononucleosis
m. Other chronic conditions (specify)
Ask Sb. for each condition reported.
b. What medications and/or treatments, if any, (are you/is )
taking for the ? (Record all medications.)
446
-------
7a. Have you or has anyone in your household started working, stopped
working or changed jobs since you enrolled in the study?
Yes 1
No (Skip to Q. 8) 2
b. Who was that?
Stopped working (Ckip to Q. 3) 1
Started working 2
Changed, jobs 3
c. Wha.t is the name of the place where (you/ ) now work(s)? (F.esorJ ri'lacs)
d. What is (your/ 's) new job title? (Record job title)
Now, I would like to ask you about a couple of other types of health
conditions which are of interest to us. We want to know if you or
anyone in your household has ever seen a doctor for these conditions.
8a. Have you or has anyone in your household ever seen a doctor for a
goiter or other thyroid condition?
Yes ' 1
No (Skip to Q. 9) 2
DK (Skip to Q. 9) 8
b. Who is that?
c. Please tell me what the doctor called the thyroid condition, if
you know. (Record condition if known. Enter "DK" if not knoun.)
d. How old (were you/was ) when the thyroid condition first
occurred? (Record acs.)
e. Bo you/does still have the thyroid condition?
Yes 1
No 2
f. What medications or treatments have yofl/foas ^ ever received
for the" thyroid condition? (Record all medications and trec.imsr.ts.)
g. Which of those medications or treatments, if any, (are you/
is ) currently taking for the thyroid condition?
(Record all current medications and treatments.)
447
-------
9a, Have you or has anyone in your household ever s'een a doctor for
pneumonia?
Yes 1
No (Skip to Q. 10., -if applicable) 2
DK (Skip to Q. 10,- if applicable ) 8
b. Who is that? (Record condition in appropriate column.)
c. HCw many times have you/has had pneumonia? (Record if times.)
d. How old (were you/was ) the last time that the pneumonia
occurred? (Record age.)
e. Were you/was ever hospitalized for pneumonia?
Yes 1
No 2
DK 8
f. Approximately how long did the pneumonia last the last time
that it occurred? (Record duration in weeks.)
This question is to be asked only for children IB years of age or less.
Ask at/pruyi'iutii questions for uye uj~ auuh u'n-i'id.
lOa. Where (did/does _ ) go to grammar school? (Record all schools
attended and location of school. )
b. Where (did/does _ ) to to junior high- or middle school? (Record all
schools attended and location of school. )
c. Where (did/does ) go to high school? (Record all schools attended
and location of scnool. )
d. Did _ ever receive a polio immunization at school?
Yes 1
No (End of interview) 2
DK (End of interview) 8
e. Could you please tell me which school that was? (Record name and
location of school. )
448
-------
APPENDIX D
PERSONAL QUESTIONNAIRE UPDATE IN OCTOBER 1983
449
-------
HH#
Name
Phone I
Current HH Size
Interviewer
Date of Interview
University of Illinois
School of Public Health
LUBBOCK HEALTH EFFECTS STUDY
1983 PERSONAL QUESTIONNAIRE UPDATE
ASSURANCE OF CONFIDENTIALITY — All information that would permit identification
of individuals will be held in strict confidence, will be used only by persons en-
gaged in and for the purpose of the survey and will not be disclosed or released to
others for any purpose. The results will be used only when combined with those of
many other people
451
-------
Household File
1. Has your household moved since January 1982?
When did you move? (Record month and year)
Where are you now living? (Record approximate location)
2. a. Do you have air conditioning in your home?
YES 1
NO (Skip of Q. 3) 0
ACOND
b. Do you have:
Central air conditioning - refrigeration 1
Central air conditioning - evaporative cooler 2
Window or wall units -- refrigeration 3
Window or wall units — evaporative cooler 4
c. During the summer, do you have the air conditioning on:
All or most of the time 1
Some of the time every day 2
Only when it is very hot 3
Never 4
ACNAME
ACUSE
3. a. Do you obtain your drinking water from:
A private well (go to b.)
Public water supply (go to d.)
b. Do you chlorinate your well water?
YES
NO
2 (Go to Q. 4.;
DWATER
WCHLOR
452
-------
c. How frequently is chlorine added to your water? (Choose best answer)
Continually (automatic chlorinator) 1
Dally 2
Weekly 3
Monthly 4
Only when well is known to be contaminated 5
FCHLOR
(GO TO Q. 3)
d. Is your water supplied by:
City of Wilson 1
Canadian River 2
PWATER
4. Do you dispose of sewage through:
A septic tank or cesspool 1
City sewage system 2
SEWAGE
5.
What 1s the highest level of education achieved by any member of
the.household? (Include children who have left home)
None
Elementary
High school
College
0
1234
9 10 11 12
13 14 15 16
Some graduate or professional school
Graduate or professional degree
ASK THE FOLLOWING QUESTIONS FOR HOUSEHOLDS THAT FARM:
17
18
HEDUC
6. Approximately how many acres of land do members of your household farm?
(Include fallow ground, pastures and grazing land)
Acres
453
-------
7. What crops are you producing on your farm this year? Please tell me
the amount of acreage and if any acreage usually used for that crop
is fallow due to the payment in kind program.
CROP ACRES PLANTED PAYMENT IN KIND
ACREAGE
Cotton C.OTTOV3
Wheat \jf4EA-T3
Oats OATS 3
Ml-coJ
8. What types of livestock are you raising this year?
LIVESTOCK NUMBER
Cattle _
Hogs _
Sheep _ SH-SBC3
Fowl
9. Do you currently irrigate your farmland?
YES 1
IRRIG3
NO 2
10. What is the source of that water and approximately how many acres
are irrigated by that source?
I of acres
Well IWELL
Wastewater IWASTE
454
-------
Participant information
1. Enter Participant ID, Name and birthdate on opposite page.
2. a. Has anyone left your household permanently or temporarily since
January 1982?
YES 1
NO (Skip to Q. 3.) 0
b. Who was that? (Record names)
(FOR EACH "YES" TO Q. 2 a.-b., ASK THE FOLLOWING QUESTION)
c. When did leave? (Record month, year)
Now I would like to ask you about any longterm or chronic illnesses which
you or anyone in your household may have developed since January 1982. If you
are not sure whether a household member developed a condition before or after
January 1982, please tell me about 1t anyway and we can check that later.
3. a. Have you or has anyone in your household been newly diagnosed as having
any of these respiratory illnesses or conditions since January 1982?
(READ LIST OF CONDITIONS. PAUSE AFTER EACH CONDITION TO ALLOW
RESPONDENT TO REPLY. FOR EACH "YES", ASK "WHO WAS THAT?" AND
RECORD CONDITION IN APPROPRIATE COLUMN.)
a. Allergies
b. Chronic bronchitis
c. Emphysema
d. Asthma RESP
e. Tumor or cancer of the lung
f. Tumor or cancer of the mouth or throat
g. Other (specify)
(ASK 3.b. FOR EACH CONDITION REPORTED)
b. What medications and/or treatments, if any, are you/is taking
for the ? (RECORD MEDICATIONS)
(read condition)
455
-------
4. a. Have you or has anyone In your household been newly diagnosed as
having any of these cardiovascular conditions since January 1982?
(READ LIST OF CONDITIONS. PAUSE AFTER EACH CONDITION TO ALLOW
RESPONDENT TO REPLY. FOR EACH "YES", ASK "WHO WAS THAT?" AND
RECORD CONDITION IN APPROPRIATE COLUMN.)
a. High blood pressure
b. Stroke
c. Heart attack HEART
d. Angina
e. Other Cspecify)
(ASK 4.b. FOR EACH CONDITION REPORTED)
b. What medications and/or treatment, if any, are you/is
taking for the ?
(.read condition) (RECORD MEDICATIONS)
5. a. Have you or has anyone in your household been newly diagnosed as
having any of these stomach or abdominal conditions since January 1982?
(.READ LIST OF CONDITIONS. PAUSE AFTER EACH CONDITION TO ALLOW
RESPONDENT TO REPLY. FOR EACH "YES", ASK "WHO IS THAT?" AND
RECORD CONDITION IN APPROPRIATE COLUMN.)
Tumor or cancer of the:
a. Stomach
b. Intestine
c. Colon
d. Esophagus
e. Stomach (peptic) or intestinal (duodenal) ulcer ABOOM
f. Ulcer of the colon (ulcerative colitis)
g. Oiverticulosis
h. Gall bladder problems
i. Other (specify)
(ASK S.b. FOR EACH CONDITION REPORTED)
B. What medications and/or treatments, if any, are you/is
taking for the ?
(read condition) (RECQRD MEDICATIONS)
456
-------
6. a. Have you or has anyone in your household been newly diagnosed as
having any of these other types of conditions since January 1982?
(READ LIST OF CONDITIONS. PAUSE AFTER EACH CONDITION TO ALLOW
RESPONDENT TO REPLY. FOR EACH "YES", ASK "WHO IS THAT?" AND
RECORD CONDITION IN APPROPRIATE COLUMN.)
a. Skin cancer
b. Leukemia
c. Hodgkin's Disease
d. Other cancers
e. Arthritis
f. Diabetes OTHERO
g. Anemia
h. Inmunologlcal disorder
1. Rheumatic fever
j. Serum hepatitis (Hepatitis B)
k. Infectious hepatitis (Hepatitis A)
1. Infectious mononucleosis
m. Other chronic conditions (specify)
(ASK 6.b. FOR EACH CONDITION REPORTED)
b. What medications and/or treatments, 1f any, are you/1s
taking for the ?
(read condition)
(RECORD ALL MEDICATIONS)
7. a. Old you or anyone in your household see a doctor for a goiter or other
thyroid condition during 1982 or 1983?
NO (Skip to Q. 8) 0
YES 1
b. Who is that?
c. Please tell me what the doctor called the thyroid condition if you know.
(.RECORD CONDITION IF KNOWN. ENTER "OK" IF NOT KNOWN.)
d. What medications or treatments have you/has ever received for
the thyroid condition?
(RECORD ALL MEDICATIONS AND TREATMENTS.)
457
-------
8. a. Did
d you or anyone in your household see a doctor for pneumonia
during 1982 or 1983?
NO
YES
OK
b. Who is that? (RECORD CONDITION IN APPROPRIATE COLUMN)
PNEU
c. How old were you/ was
occurred? (RECORD AST)
at the time that the pneumonia
d. Were you/was
NO
YES
OK
hospitalized?
0
1
8
PNEUAGC
PNEUHOS-
e. Approximately how long did the pneumonia last the last time
that it occurred? (RECORD DURATION IN WEEKS.)
PNEUDUR
9. a. Do you or does anyone in your household drink bottled water
regularly?
YES 1
NO (Skip to Q. 10) 0
b. Who is that?
BOTTLED3
c. Do you/does
ever drink water from the tap?
YES 1
NO (Skip to Q. 11) 0
TAP WATER3
10. Compared to other people in your/_^ 's age group, how much tap water
do you/ drink? (Include beverages made with tapwater, i.e.
coffee, tea, Kool-Aid.)
Less than average 1
Average 2
More than average 3
WCONSM
458
-------
11. a. Do you or does anyone in this household smoke cigarettes regularly?
YES 1
NO 2 SMOKEJ
DK 3
b. Who is that?
c. How much do you/does smoke in a day?
One half pack or less per day 1
One half to one pack per day 2
More than one pack per day 3
PACKDAY
12. a. Does anyone in this household chew tobacco on a regular basis?
NO 0
YES 1
DK 8
b. Who is that?
TCHEW
13. a. Have you or has anyone 1n your household started working, stopped
working or changed jobs since January 1982?
NO 0
YES 1 WORKS 3
b. Who was that? (RECORD NAME AND STATUS)
Stopped working (also ask 13. c.) 1
Started working (also ask 13. d.) 2
Changed jobs (also ask 13. d.) 3
c. Are you/ is : (READ CATEGORIES)
Usually employed, but just out of work temporarily
Retired
Homemaker CSIdp to Q. 14)
Disabled or handicapped (Skip to Q. 14)
Not usually employed (Skip to Q. 14)
Student (Skip to Q. 14)
Other (specify)
EMPSTAT3
459
-------
d. What 1s the name of the place where you/
(RECORD PLACE)
What 1s your/ 's new job title?
(RECORD JOB TITLE)
now work(s)?
14. How many occasions a week do you/does
large groups of people (large * 10 or more people)?
have contact with
Less than once a week
One to 5 times a week
6-10 times a week
11 - 15 times a week
15 or more times
(INCLUDES SCHOOL ATTENDENCE, CHURCH MEETINGS, SOCIAL OCCASIONS,
CONGREGATIONS AT THE COTTON GIN, tTC.).
OCCUPJ
CONTACT
15. Does your family, or your spouse's family, have a history of cancer?
YES ]
NO 0
Would you mind giving us some Information about these relatives?
(Include spouse, 1f deceased, children, grandparents, siblings and
aunts or uncles)
NAME
HCANCER
RELATIONSHIP
(to respondent or
'respondent's spouse) TYPE OF CANCER
» YEARS LIVED
YEAR (DIAGNOSED IN LYNN COUNTY
OR DIED) (if none, enter Q)
460
-------
APPENDIX B
INFORMED AND PARENTAL CONSENT FORMS
461
-------
ADULT* S CONSENT FOR PARTICIPATION IN A HEALTH
(ESEARCH PROJECT
FORM CA
I, / state that I Am over twenty one(21)
(NAME OF PARTICIPANT)
years of age and wish to participate in an infectious disease study being
conducted by the School of Public Health at the University of Illinois under
the direction of Doctor Robert I~ Northrop.
The, purpose- of the research is to ascertain the number and types of
infections and other illnesses I will have during the next three (3) years to
evaluate the wealth effects, if any, of aerosols emitted from nearby
irrigation tigs spraying wastewater.
This project involves my allowing you to obtain from 03 six (6) blood
samples and t.iree (3) tuberculin teats in the next three (3) years.
I understand that there are no experimantal procedures to be performed
on me in this research and that there are no personal risks involved.
I acknowledge that I have been informed that this research is designed to
assist in maintaining or improving my personal health and will benefit me
personnally if causes for my infections are found.
I understand that in the event of physical injury resulting from this
research therj is no compensation and/or payment for radical treatment from
The University of Illinois at' the Medical Center Cor such injury except as
may be required of the University by law.
I acknowledge that Doctor Horthrop, or his representative, has Cully
explained to .-» the need for the research; has informed me that I may withdraw
from participation at any tine and has offered to answer any inquiries which I
may make concjrning the procedures to be followec.
I freely and voluntarily consent to my participation in this research
project.
(SIOJATQRE OF VOLUNTEER)
(Witness to Scplanation)
(Not to Sigiature)
(Date)
463
-------
MINOR'S CONSENT FOR PARTICIPATION
IN A HEALTH RESEARCH PROJECT
FORM CM
I, _ , state that I am _ years of age
WAME OF PARTICIPANT)
and wish to participate in a health watch program being conducted by the
School of Public Health at the University of Illinois under t±ie direction
of Doctor Jtobert L. Northrop.
The purpose of the research is to ascertain the number and types of
infections I will have during the next three (3', years to evaluate the
health effects, i:: any, of aerosols emitted from nearby irrigation rigs
spraying
This project involves my allowing you to obtain from toe six (6) blood
samples and three (3) tuberculin tests in the next three (3) years.
I understand that there are no experimental procedures to be performed
on ne in this research and that there are no personal risks involved.
I acknowledge that I have been informed that this research is designed
to assist in maintaining or improving my personal health and will benefit ne
personally if causes for my infections are found.
I understand that in the event of physical injury resulting from this
research there is no condensation and/or payment for medical treatment from
The University of Illinois at the Medical Center for such injury except as
may be required o : the University by law .
I acknowledge that Doctor Northrop or his representative has fully
explained to me tlie need for the research, has informed me that I may
withdraw from participation at any time and has offered to answer any
inquiries which I may make concerning die procedures to be followed. I
freely and voluntirily consent to my participation in this research project.
(SIS1ATURE OF MINOR)
Date
464
-------
PARENTAL CONSENT
FORM CM
We, parents or guardians of the above minor volunteer, agree to the
participation of :he above minor in the research project set out above.
We have been informed of the need for the research, the benefits to be
derived from it, and the risks involved. He have been informed that the
research cannot bo conducted with adults only because of the nature of the
research.
We also understand that in the event of physical injury resulting from
this research the.re is no compensation and/or payment for medical treatment
from The University of Illinois at the Medical Center for such injury except
as may be require 1 of the University by law.
Being aware >f the necessity for the partisipation of minors in this
research project and being informed that the procedures will also benefit
the above-named minor personally by reporting ts me/us, the parents or
guardians, and to his or her physician, test results which may assist in
diagnosis of an infectious illness the minor may have during this study, we
consent to the minor's participation.
(SIGNATUPE OF PARENTS OR GUARDIANS)
(SIGNATURE OF PARENTS OR GUARDIANS)
(WITNESS TO EXPLANATION)
(NOT TO SIGNATURE)
(DATE)
465
-------
IMPORTANT INFORMATION
ABOUT POLIO AND INACTIVATED POLIO VACCINE
Please read this carefully
IP 10/1/80
WHAT IS POLIO? Polio is a virus disease that often
causes permanent crippling (paralysis). One person out
of every 10 who get polio disease dies from it. There
used to be thousands of cases and hundreds of deaths
from polio every year in the United States. Since polio
vaccine became available in the mid 1950's. polio has
nearly been eliminated. In the last five years, fewer than
25 cases have been reported each year. It's hard to say
exactly what the risk is of getting polio at the present.
Even for someone who is not vaccinated, the risk is very
low. However, if we do not keep our children protected
by vaccination the risk of polio will go back up again.
INACTIVATED POLIO VACCINE (IPV): Immuniza-
tion with inactivated polio vaccine is effective in
preventing polio and has successfully controlled polio in
several countries. The vaccine is given by injection.
Several doses are needed to provide good protection.
Young children should get three doses in the first year of
life, each separated by 1 to 2 months, and another dose
6 to 12 months later, at about 18 months of age. A
booster dose is needed every 3 to 5 years, especially
when children enter school or when there is a high risk of
polio, for example, during an epidemic or when travel-
ing to a place where polio is common. The vaccine is ef-
fective in providing protection to over 90% of people
who receive it.
POSSIBLE SIDE EFFECTS FROM THE VACCINE:
Inactivated polio vaccine is not known to produce any
side effects.
PREGNANCY: Polio vaccine experts do not think inac-
tivated polio vaccine can cause special problems for
pregnant women on their unborn babies. However.
doctors usually avoid giving any drugs or vaccines to
pregnant women unless there is a specific need. Preg-
nant women should check with a doctor before taking in-
activated polio vaccine.
WARNING — SOME PERSONS SHOULD NOT
TAKE INACTIVATED POLIO VACCINE
WITHOUT CHECKING WITH A DOCTOR:
— Those who are sick right now with something more
serious than a cold.
— Those with allergies to antibiotics called neomydn
or streptomycin
— Pregnant women
NOTE ON ORAL POLIO VACCINE: Besides the inac-
tivated polio vaccine, there is also an oral polio vaccine
which is given by mouth and which after several doses
protects against polio for a long time, probably for life.
Many polio experts feel that the oral vaccine is more ef-
fective for preventing the spread of polio and for con-
trolling polio in the United States. However, it should
not be given to persons who have a low resistance to in-
fection or who live with persons with low resistance to
infections. It has been associated very rarely with
paralysis in persons who receive the vaccine or who are
in close contact with those recently vaccinated. Oral
polio vaccine is widely used in this country. It can be
given alone or in combination with IPV. If you would like
to know more about oral polio vaccine or combinations
of oral and inactivated vaccine, please ask us.
QUESTIONS: If you have any questions about polio or
polio vaccination, please ask us now or call your doctor
or health department before you sign this form.
REACTIONS: If the person who received the vaccine
gets sick and visits a doctor, hospital, or clinic in the 4
weeks after vaccination, please report It to:
BXAS DEPARTMENT Of HEALTH
KURSING DIVISION 797-4331
PLEASE KEEP THIS PART OF THE INFORMATION SHEET FOR YOUR RECORDS
I have read the information OH this form about potto and the inactivated vaccine. I nave nod a chance to earn questions which were answered to my satisfaction. I Mieve I understand the benefits
and risks of inactivated polio vaccine and request tnat it be given to me or to the person named below for whom f am authorized to make this request. |p to/1/80
INFORMATION ON PERSON TO RECEIVE VACCINE
(Please print first three llnesl
Name
Address
City
X
llastl iHrstl imiddlel Blrthdate
State
Signature of person to receive vaccine or person authorized to make the request
Age
County
Zip Code
Date
FOR CLINIC USE
Clinic Ident.
Date Vaccinated
Manufacturer and Lot No.
Site of administration
466
-------
INFORMACION IMPORTANTE ACERCA DE
LA POLIOMIELITIS Y LA VACUNA ANT1POLIQ ATENUADA
Favor de leer cuidadosamente
iOUE ES LA POLIOMIELITIS? La poliomielitis
Ipoliol es una enfermedad causada por un virus y que
muchas veces results en paralisls permanente. Muere
aproximadamente I de cada 10 personas que se
contagian de ella. Antes ocurian miles de casos de polio y
centenares de muertes causadas por esta enfermedad
todos los artos en los Estados Unidos. Desde que se hizo
disponible la vacuna antipolio a mediados de la decada
de los cincuentas. la poliomielitis ha sido casi totalmente
eliminada. En los ultimos 5 aftos. se han reportado
menos de 25 casos en cada aflo. Es diftcil seftalar con
exactitud el riesgo aaual de contagiarse de polio. Aun
para las personas no vacunadas. el riesgo es muy
reducido. Sin embargo, si no mantenemos la proteccibn
de nuestros hiios por medio de la vacunacibn regular, el
riesgo de contraer polio volvera a aumentar.
LA VACONA ANTIPOLIO ATENDADA (IPV): La
inmunizaci6n por medio de la vacuna antipolio
atenuada sirve efectlvamente para prevenir la
poliomielitis. y ha logrado controlar la enfermedad en
varios paises. La vacuna se administra en forma de
inyeccibn. Se requieren varias dosis para lograr una
protecci6n satisfactory. Los bebes deben recibir 3
dosis en su primer afio de vida. con una separacibn de 1
o 2 meses entre cada dosis. y deben recibir otra dosis
entre 6 y 12 meses despues. a los 18 meses de edad
aproximadamente. Se requiere una dosis de refuerzo
cada 3 o 5 afios. particularmente cuando los niflos
entren a la escuela o cuando haya un alto riesgo de
contraer polio, como por ejemplo durance una
epidemia. o durante viajes a lugares donde la
poliomielitis es una enfermedad comun. La vacuna
protege eficazmente a mas del 90% de las personas que
la reciben.
EFECTOS SECUNDARIOS DE LA VACDNA: Por lo
que se sepa. la vacuna antipolio atenuada no produce
efecto secundario alguno.
IMOIERES EMBARAZAOAS: Los expertos en
vacunas antipolio no creen que la vacuna antipolio
atenuada cause problemas para mujeres embarazadas.
ni para sus niftos adn no nacidos. Sin embargo, los
medicos generalmente se abstlenen de recetar drogas o
vacunas para mujeres embarazadas. a menos que haya
alguna necesidad especiflca de ello. Las mujeres
IP10/1/80
embarazadas deben consultar con un medico antes de
tomar la vacuna antipolio atenuada.
PRECAOCION — ALGDNAS PERSONAS NO
DEBEN RECIBIR LA VACONA ANTIPOLIO
ATENOADA SIN CONSULTAR PRIMERO CON
UN MEDICO:
— Las personas que sufren actualmente de
cualquiera enfermedad mi seria que un catarro.
— Las personas que padezcan alergias a los
antibi6ticos conocidos como Neomicina y
Estreptomicina.
— Las mujeres embarazadas.
NOTA SOBRE LA VACUNA ANTIPOLIO DE
ADMINISTRACION ORAL: Ademas de la vacuna
antipolio atenuada. existe tambien una vacuna antipolio
de administracibn oral, que se toma por la boca. y que.
despues de varias dosis. ofrece protecci6n contra la
poliomielitis por un tiempo largo, probablemente por
toda la vida. Algunos expertos creen que la vacuna oral
es mas eficaz para prevenir la propagacibn de polio y
para controlar esta enfermedad en los Estados Unidos.
Sin embargo, la vacuna oral no se debe administrar a
personas que tengan una baja resistencia a infecciones.
ni a las que vivan con otras personas que tengan una
baja resistencia a infecciones. En ciertas ocasiones
raras. esta vacuna ha sido asodada con la parallsis en
personas que han recibido la vacuna o que han estado
en contacto intimo con otras personas recien
vacunadas. La vacuna antipolio oral se usa ampliamente
en este pais. Puede ser administrada sola o junto con la
IPV (vacuna antipolio atenuada|. Si usted desea saber
mas acerca de la vacuna antipolio oral, o acerca de las
combinaciones de vacuna atenuada y oral, por favor
consultenos.
PREGUNTAS: Si tiene usted alguna pregunta acerca
de la poliomielitis o la vacunaci6n antipolio. por favor
hagala ahora mismo. a llame a su medico o su
Departamento de Saiud antes de firmar esta forma.
REACCIONES: Si una persona que recibe la vacuna se
enferma y visita a un medico, algiin hospital o alguna
cllnica en las primeras 4 semanas despues de la
vacunaci6n. por favor repbrtelo a:
FAVOR DE GOARDAR ESTA PARTE DE LA HO|A PARA SU INFORMACION
Hr Irido la information out contiette etta forma acerat de \a poliotnielitis if la vacuna attnuada. Hr tenido la orjortunidad de kacer preauntas. uestos fueron conusladas tatistactoriamenU. Crro
we eittiendo los beneticim if l(K riesgos de la vacuna antifotio atenuada. if soficitoaue se me administre amioa la persona abato mencioiiada. a favor de wien tengo la autondad de fiacer rtM
toliutud. IP 10/1/80
INFORMACION SOBRE LA PERSONA A QUE RECIBIRA LA VACUNA
(Por favor uu letra de imprenti en In primeras tm lirteas)
Membra (apellido)
(primer)
ttegundol
Fecha de
nacirriiento
Direcct6n
Condado de residencia
Ciudad
X
Estado
Zip Code
Firma da la persona qua recibira la vacuna o de la
persona autorizada pare solicitarla.
Fecha
PARA EL USO DE LA
CL1NICA
Idemidad de la clmica
Fecha de vacunacidn
Fabricante y n*de lote
Lugar de le inveccion
467
-------
APPENDIX F
HOUSEHOLD HEALTH DIARY BOOKLET (1980)
469
-------
Household Number
Starting date
Ending date
471
-------
GENERAL INSTRUCTIONS
FOR KEEPING THIS DIARY
1. Information Irom this diary can help to determine
the health levels of people in your community. Since
it is so important, we appreciate your doing your
best to make the data as complete as possible
Always make entries in the diary at the time that the
event happens so you won't forget.
2. Be sure to include all household members—adults,
children and babies. Do not include short-time
visitors.
3. Record any notes on page 8.
4. If you have any questions about how to report
something in this diary please call:
Telephone Number
or consult the examples which appear below.
N>
SAMPLE
Dale
Illneii
began
F&l
Dale
ol
recovery
fee, it
Who in Ihe family?
(first name)
Whal was his/her Illness?
DIRECTIONS
1.
2.
3.
4.
List all illnesses and injuries during these two weeks for
all household members. Even the slightest cold, cough
or cramps should be reported.
II the same person gets sick, stays home for two or
three days, feels better and returns to work, then stays
home again, you would record this illness twice.
If anyone in your household visits a doctor, note that in
the appropriate box in the diary. Then, on the back cover
of the diary, please indicate that doctor's name and the
town in which he Is located.
If a household member plans to be out of the study area
for longer than 5 days, note this on the back cover of the
diary.
How many da
Feel III but
do usual
laski?
^
-
D
Call or
*I»M a
doctor?
^
t/
d they . . . (choc
Take any
over the
counter drugs?
^
L/
<^
k all thai app
Take any
prescription
medicine?
L^
- •
ly)
Become
hoipllal-
Ized? •
-------
Date
Illness
began
Dale
ol
recovery
Who In the family?
(Mrsl name)
Whal was his/her Illness?
How many da
Feel III bul
do usual
tasks?
E:
y 5 did he/the
Miss work
or
school?
-—
:::
o
Call or
>lsll a
doctor?
:.
. ._ .
d ihey . . . (chec
Take any
over the
counter drugs?
.
k all lhal app
Take any
prescription
medicine?
y)
Become
hospilsl-
Ized?
*-*
-------
APPENDIX 0
HEALTH DIARY FOBMS AN) 1EEKLY ILLNESS SURVEILLANCE
SONAR! (1982 AND 1983)
475
-------
A. First aeek of data collection period
1. Since I last called you, has anyone in the household had a cold, sore throat, flu or any other respiratory illness?
1. Yes (Enter information beloa) 2. No
2. Since I last called you, has anyone in the household had any stomach or abdominal illness?
1. Yes (Enter information beloa) 2. No
3. Since I last called you, has anyone in the household had any skin conditions?
1. Yes (Enter information beloa) 2. No
4. Since 1 last called you, has anyone in the household had any eye or ear conditions?
1. Yes (Enter information beloa) 2. No
5. Since I last called you, has anyone in the household had any other kinds of illnesses or conditions?
1. Yes (Enter information beloa) 2. No
6. Since 1 last called you, has anyone in the household heen away from the area for more than two days, or returned home after an extended absence?
1. Yes (Enter information below) 2. No
list Contact Attempt_
I D
Dal*
Illneu
begin
Dal*
of
recovery
Who In lh« lamlly?
(llrtl name)
Whit w» hli/her Illneu?
Code
How many da
FM! Ill but
do usual
tasks?
ps did he/she
Miss work
or
school?
0
Can or
vlill a
doctor?
d they . . . (chec
Take any
ovar lha
counter drugs?
k all lhal epp
Take any
prescription
medicine?
y)
Become
hospital-
lied?
2nd Contact Attempt_
3rd Contact Attempt_
Respondent
Interviewer
-------
00
B. Second week of data collection period
1. Since I last called you, has anyone in the household had a cold, sore throat, flu or any other respiratory illness?
1. Yes (Enter information below) 2. No
2. Since I last called you, has anyone in the household had any stomach or ahdominal illness?
1. Yes (Enter information below) 2. No
3. Since I last called you, has anyone in the household had any skin conditions?
1. Yes (Enter information below) 2. No
4. Since 1 last called you, has anyone in the household had any eye or ear conditions?
1. Yes (Enter infonnation below) 2. No
5. Since I last called you, has anyone in the household had any other kinds of illnesses or conditions?
1. Yes (Enter information below) 2. No
6. Since I last called you, has anyone in the household been away from the area for more than two days, or returned home after an extended absence?
1. Yes (Enter information below) 2. No
i n
Dale
Illness
began
Date
at
recovery
Who In the family?
(llril name)
What was Mil/her Illneil?
Code
How many da
Feel III bat
do usual
tasks?
ys did he/she
Miss work
or
school?
O
Call or
visit a
doctor?
d they . . . (chec
Take any
over the
counter drugs?
k all lhat app
Take any
prescription
medicine?
y)
Become
hospital-
ized?
-
1st Contact Attempt
2nd Contact Attempt^
3rd Contact Attempt_
Respondent
Interviewer
-------
A. First aeek of data collection period
I am calling to get health watch information
from you (your household) for last week,
beginning Sunday (data) and ending Saturday
(date). During that time have you (or any
member of your family) had any of the following
illnesses?
1. Cold, sorethroat. flu or any other respiratory illness?
AFFIX LABEL HERE
!•« contact attopt
2nd contact
3rd contact
ISS (Enter information beloti)
Z. Any stomach or abdominal Illnesses?
(Snter •information belou)
3. Any skin conditions?
ISS (Enter -information belomj
4. Any eye or ear conditions?
IBS (Snter information belott)
5. Any other kinds of Illnesses or conditions?
XBS (Enter infoxnation belouj
6. Since I last talked with you, has anyone in the household been away froa the area
for more than two days or returned home after an extended absence?
HO
90
90
9O
90
ZES (Enter information belavj 90
(Aak only of household* net located on tit* Sonoook POrmJ
Have you (or any member of your household) spent more than 30 minutes
on the Hancock farm this week?
90
7.
TES (Enter information on
Sxpoeure Sheet)
3. (Aak all ganaoak ftaw reeidtnte and nan-reeidtntt unto antuered ISS to Question #7.
A. Old anyone in the household have direct contact with the wastewater?
ISS (Enter information an 90
Vamteaater Sxpoetofe Sheet)
3. Was anyone exposed to the Mst or the aerosol fro* an operating spray rig?
Ollf
Mlnt»»
btqiin
Out
01
rocovcy
Who in Iht iMMy?
(ilrsl n«m«|
WKM «m* MMMr IUMM7
^^^Bt^m^^^jmft
ff^^imem9ff/tmt
f^tmtxt
\mtmr
fttfrtlWMlM
MlMIMrt
flr _
O
CM or
**•_
4 Itl^f . . . (dMC
T«n«iy
OMTIfW
OfunttKfuqtl
kiMitut «pp
Titeiny
nMtfetiw?
r»
B0conM
ho*iMl«.
it*d?
479
-------
B. Second week of data collection period
1.
2.
3.
4.
5.
Since I last called you, has anyone in the
household had a cold, sore throat, flu or
any other respiratory illness?
Yes (Entar information belou)
HO
Since I last called you, has anyone in the
household had any stomach or
illness?
AFFIX LABEL HERE
1st contact attempt
2nd contact attempt
3rd contact attempt
Respondent
Interviewer
Yes (Enter information belou)
No
Since I last called you, has anyone in the household had any skin conditions?
Yes (Enter information balau) NO
Since I last called you, has anyone in the household had any eye or ear conditions?
Yes (Enter information balau) Mo
Since I last called you, has anyone in the household had any other kinds of illnesses
or conditions?
Yes (Entar information balau)
NO
Since I last called you, has anyone in the household been away from the area for
more than two days, or returned hos» after an extended absence?
Yes (Enter information balau)
NO
Since I last called you, has anyone in the household had any contact whatsoever
with wastewater on the Hancock farm (i.e., wastewater on shoes; clothes; skin or
hair; eyes or mouth) ? If any uaateuater contact ia reported, record type of
contact and brief explanation of. hau contact occurred.
Yes (Enter indentation belou) Ho
Dlti
Illnesa
begin
Oat*
ol
raco»ary
Who In In* family?
(Ural name)
What «•§ Mt/ntr UMM?
Maaiaapjaaj
FMlMbul
douwtf
inks?
ytdHlMMi*
MlMwork
or
tchoolT
0
CaN or
visit •
doctor?
M tlwy . . . (choc
Tito any
orartlw
count* dnigt?
k mm* ape
TO* any
prescription
mMlcina?
>r>
Bacon^
hotoUil-
IM07
480
-------
oo
NUMBER OF HEM ACUTE ILLNESSES REPORTED IN STUDY POPULATION BY WEEK - 1982 *
(BASED ON PHONE INFORMATION FROM FIELD REPRESENTATIVES)
DCP
201
201
202
3)2
203
203
201
m
205
205
206
206
207
207
STARTING
DATE
1-3
1-10
1-17
1-21
1-31
2-7
2-11
2-21
2-28
3-7
3-11
3-21
3-28
1-1
ft OF
PARTICIPANTS
REPORTING
392 (51)
380 (51)
389 (51)
387 (51)
382 (51)
379 (51)
379 (53)
379 (53)
377 (53)
371 (53)
367 (18)
351 (19)
371 (50)
367 (50)
RESPIRATORV
26 (0)
17 (1)
11 (0)
16 (3)
18 (1)
6 (2)
15 (3)
12 (3)
3 (1)
18 (0)
10 (0)
9 (0)
12 (0)
15 (6)
Gl
__
1 (0)
1 (0)
5 (C)
7 (2)
2 (0)
5 (0)
8 (0)
1 (0)
~
2 (0)
6 (0)
—
1 (0)
EYE 8 EAR
„
—
—
1 (0)
—
~
1 (0)
1 (0)
—
—
2 (0)
—
~
—
SKIN
^__
—
1 (0)
—
—
2 (0)
2 (0)
—
1 (0)
--
~
3 (0)
~
—
OTHER
ACUTE
..
—
--
2 (C)
—
—
1 (1)
1 (0)
1 (0)
2 (0)
2 (0)
~
—
—
OTHER
CONDITIONS
3 (0)
2 (0)
—
1 (0)
~
2 (0)
2 (C)
3 (0)
1 (0)
1 (0)
—
—
1 (0)
2 (0)
* NUMBERS IN PARENTHESIS ( ) INDICATE ILLNESSES OCCURRING IN ZONE I
-------
APPENDIX H
ACTIVITY DIARIES AND HAPS
483
-------
UNIVERSITY OF ILLINOIS A.T THE MEDICAL CENTER,, CHICA.O*
March 11, 1982
Dear Study Participant:
In order for us to get a better understanding of the relationship
between all of the environmental and health data which are being
collected, it is necessary for us to know how much time; individuals
spend in various parts of the study area. Obviously, it would be
impossible for you to keep track of your whereabouts every day that
we are collecting health information, sc we "nave developed an "activity
diary" which we would like study participants to keep for one week.
We hope that this week will be representative of people's normal
activities at this time of year.
We are asking that each member cf your household complete an
activity diary for the week of March 21 - 2"/. Each person should
fill out the activity diary with his cr her name on it each day
for the one week period. (Mothers should fill cut the diary tor
young children.)
Included with the diary is a map of the study ar'-a with diiferent
colored sections on it. This map should b*-; used when answering
qxaestion 1. If you live or spend time within the city oi Wilson, /on
may also need to use the enlarged map of Wilson in order to distinguish
exactly where the boundaries between the orange and white areas are.
When answering question 1, try to record as accurately as possible the
number of hours spent in the various areas each day. For example, if
you live in the orange ?jrea and spend only 10 minutes driving through
the blue area on a particular day, it would not be neces; ;.ry to record
the 10 minutes spent in the blue area. If, however, you spend half an
hour cr more in any of the areas, that time should be recorded.
Question 2 requests nore specific information as to how much time
is spent in Lubbock or at home. "At ho??.e": in this case, means that
you are either in your house, yard, or barnyard area. For both
questions, if you do not spend any'tine in a certain area, please
mark a "0" in the column, instead of leaving it blank.
If there are college students or other family r.smbers in your
household who normally spend most of their tirr.e away from the area,
an activity diary shculd still bo completed for them during the week
of March 21st. The tiir.e during which- they are r.w.ay from home would
simply be recorded as "hours outside map area". If there is-scnaone
in your household who is usually at home, but just happens to be
gone all or most of the vaek of the 21st, that person should complete
the activity diary the first week that he or she returns hone.
485
-------
The activity diaries should be returned to the University of
Illinois in the enclosed stamped, self-addressed envelope as soon
as they are completed.
We hope that filling out the activity diary will not be too
much of an inconvenience. The information which the diary will
provide is crucial to the health study, and we greatly appreciate
your efforts in completing it.
Sincerely yours,
Robert Northrop, Ph.D.
Associate Professor
Epidemiology-Biometry Program
RN/cb
486
-------
ACTIVITY DIARY
A. Basic Data
1. Name:
, First
2. Reporting week dates:
Last
B. Activity Information
Please record the number of hours per day which you.spend within each area
(column) listed below. Use the reference maps to locate the areas for question
1; question 2 refers to specified locations familiar to you. This should be done
onch day. for one week. If you are out-of-town during the entire week, please
complete this diary the first week that you return home.
Don't forget to include sleeping hours when you record daily activities.
The number of hours for each day should total 24 hours.
Question 1: Kow many hours per day did you spend in the following areas?
HOURS PER DAY
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
Blue Map
Area (Hancock
farm)
Orange
Map Area
White
Map Area
Outside
Map Area
Daily Total
(24 hrs.)
In addition to the above, we would also like more detailed
information as to how many hours per day you spent in the following
specific locations.
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
HOURS PER DAY
In
Lubbock
At
Home
487
-------
488
-------
00
vo
A<
.x
' OF
WILSON
/
/
/"'"' "
// ...,
f
104
'M ! F
Mi
^2-J
ISO 1
' >- -
»
<
/ s
-'I1
IS
•3
,1
/^'HS'
/f
^'.- »>»^p
168 !
cvtwri
ColMKc
167
Cl^^
ill,:
ffuffl
1¥
W v
fOUSIO
•J'i!;
jTh
,A*L
VANM
' hM«T
M*«Q*
fflp
, —
s
M
T
, v.
= -191.4-
B-kUJ
iritri
M3
H
1 »r(
' ' X
f,
\
'j.t|'f
'ih I li
(23
ffl
Ifl
1
I
iiB :L, i_.
••"-* '^Tirij'11'1' ^
UES
!
-------
c/ Pn'niic tlttil'li
Oin ILIjIlsroiS AT THE MHUDIOAXj CENTER,, CHICAGO
.'/<.• I/I'M;: .l:!i:r,-is: P.O. SPX ::y^i • i.itif.i^c. I'.'.iito
July 27, 1982
Dear Study Participant:
1 believe you are familiar with the procedure for keeping the activity
diary, so 1 will not restate all the directions we have given to you pre-
viously. There are 3 important points about this diary:
1. Please keep the diary for the week of August 1st through 7th;
2. The completed activity diaries will be collected when fecal
specimens are collected during the week of August 9 through
August 13- The diaries can either be brought to the Wilson
Mercantile Building or arrangements can be made to pick up
these diaries at your home by calling Pearl Davidson (628-2961);
3« Please be sure to use the enclosed maps when you refer to times
spent in the colored areas. These maps are different from
previous activity diary maps.
If you have difficulty in keeping this diary, Parrie Graham will be
glad to answer your questions when she is at the Wilson MercantiVe Build-
fng (628-2621) during the week of August 9 " 13-
This diary is particularly important to us since your activities may
be very different from previous times, particularly those of you who
would have been doing more farming than has been possible this year.
We really do appreciate your time in doing this task for us.
Sincere
Robert Northrop, Ph.D.
Associate Professor
Epidemiology-Biometry Program
490
-------
491
-------
C.'TY OF WILSON
-------
APPENDIX I
WILSON EATING ESTABLISHMENT SURVEY FORM
493
-------
NAME
Did you/
eat any food which was prepared at any of the establishments In
Wilson during 1982 or 1983 ?
Which
0 NO
1 YES
Frequency 1n the
summer compared
Establishment? Year
1982 0
1
Restaurant A
1983 0
1
1982 0
1
Restaurant B
1983 0
1
1982 0
1
Restaurant C
1983 0
1
1982 0
1
Restaurant D
1983 0
1
no
yes
no
yes
no
yes
no
yes
no
yes
no
yes
no
yes
no
yes
to rest of year
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
more
same
less
never
more
same
less
never
more
same
less
never
more
same
less
never
more
same
less
never
more
same
less
never
more
same
less
never
more
same
less
never
495
Summer
Frequency
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1+ times/week
I/week to I/month
1 to 3 tlmes/sumner
1+ times/week
I/week to I/month
1 to 3 tlmes/sumner
1+ times/week
I/week to I/month
1 to 3 times/summer
!•*• times/week
I/week to I/month
1 to 3 times/summer
1+ times/week
I/week to I/month
1 to 3 times/summer
1+ times/week
I/week to I/month
1 to 3 times/summer
1+ times/week
I/week to I/month
1 to 3 times/summer
1+ times/week
I/week to I/month
1 to 3 times/ summer
-------
APPENDIX J
PROCEDURE FOR WASTEWATER SAMPLE COLLECTION.
LUBBOCK SOUTHEAST WATER RECLAMATION PLANT
497
-------
PROCEDURE FOR WASTEWATER SAMPLE COLLECTION
Operational Year - 1981
Trickling Filter Effluent - Lubbock Southeast Water Reclamation Plant
SwRI Project 01-6001
Purpose -
The purpose for collection of this sample is to determine relative
densities of a wide range of indigenous enteric bacteria and viruses
prevalent in the wastewater to be land applied at the Hancock
site. To accomplish this purpose a 24-hour flow-weighted composite
is derived by collecting three eight-hour time-weighted samples
from the Trickling Filter Plant (TFP) effluent followed by compositing
based on plant flow data for each eight-hour period.
Equipment Required -
Sample Collection -
I SCO Model 1580 Sampler with Nicad battery
109 ft. (3 m) of 3/8" 0.0. x 1/4" I.D. Tygon tubing
Weighted stainer
3 clean 3-gallon polyethylene containers (for ISCO)
10 to 20 Ibs. cracked or cube ice (function of ambient conditions)
Sample Compositing -
5-gallon Nalgene (or requivalent) polypropylene carboy with
lid (sterile)
1-liter Nalgene (or equivalent) graduated cylinder (sterile)
1-liter Nalgene polypropylene bottles (sterile)
Sample Shipment
1 frozen Kool-Pac per six 1-liter sample bottles
1 insulated shipping container, labeled, with means of lid
attachment
1 counter-to-counter shipping ticket (Southwest or Braniff
Airlines)
Procedure
Preparation
1. Charge two Nicad batteries for 24 hours prior to sample
collection.
2. Check equipment for completeness including new Tygon
tubing with weighted strainer securely attached.
3. Place Kool-Pacs in freezer at least 24 hours prior to
sample compositing.
4. Sterilize equipment for compositing as appropriate.
499
-------
Sample Collection -
1. Locate sample adjacent to combined channel from the
secondary clarifiers of the TFP.
2. Place a 3-gallon container in the Sample Container Tub
with the false bottom open end up. Carefully add crushed
or cube ice to the tub without disturbing the position
of the container.
3. Replace the Pump and Controls Section and latch securely
making sure that the Stop Float Mechanism is free.
Attach the battery to the sampler and securely connect
the battery cable to the "12 VDC" socket on the side
of the control box. Attach the Tygon tubing to the
pump inlet, tape to secure, and lower weighted strainer
into the effluent channel. Tape tubing to side of sampler
to reduce strain on pump inlet connection.
4. Set the Control Panel as follows:
Mode Switch - Time
Time Interval Multiplier Control - 1.0
Suction Line Length Switch - 14 2/3' (1/4" I.D.)
Sample Rate Switch - 10 min.
Volume Selector Switch - 268 mL/sample (81 head)
Pump Switch - Auto
5. Turn Sample Rate Switch to the Manual Cycle position,
then return it to the 10 min. Time Inverval Position.
The pump should be automatically activated, first for
a brief period in the reverse mode to purge any liquid
in the line followed by a forward pumping action of
sufficient time to collect approximately 268 mL of sample.
This cycle is completed by a second reverse pumping
opertaion to again purge the sample line. If all functions
operate correctly in this test cycle, confirm the position
of all control switches, especially that the Pump Switch
is in the Auto Mode, then place and latch both the protective
lid over the Control Panel and the cover over the Pump
and Controls Section. Refer to the instruction manual
should problems be encountered.
6. Check the TFP Flow meter in the treatment plant office
for operation, and if necessary, mark the chart for
start of sample collection.
7. At the end of each 8-hour sampling period, turn the
Pump Switch to Off, remove the 3-gallon sample container,
label it, and place a clean sample container in the
tub. Turn the Pump Switch to the Auto position and
repeat Step 5. Renew the ice bath as required to maintain
the collected sample at 4°C. Store the collected sample
at 4°C until composited. At the conclusion of the 24-
hour sampling period, remove all equipment from the
sampling site.
500
-------
8. Prior to leaving the treatment plant obtain information
on TFP flows as follows:
(a) Remove the TFP flow chart recorder from the
instrument panel after disconnecting the multi-
lead sockets at the back.
(b) On a clean work table carefully unroll sufficient
chart paper from the take-up chart spool to
correspond to the 24-hour collection period.
Mark the 8-hour intervals, and using a transparent
straight edge, pencil a horizontal line through
a visually estimated average for each 8-hour
segment. Record average flows for each segment.
(c) Rewind the chart paper on the take-up spool
to the correct time, replace the recorder
in the instrument panel, and connect the multi-
lead sockets. Confirm that the recorder is
functional.
Sample Compositing
1. Based on total flow through the TFP during the 24-hour
composite period (E of the average flow for each 8-hour
sample segment), determine the fraction of total flow
for each segment.
2. Knowing the final volume of composite desired (18 L
max for 5-gal. jug), determine the amount of sample
needed for each segment based on the fraction of total
flow for that segment (final volume desired X fraction
of total flow).
3. Add appropriate amounts of each sample to the sterile
composite container using a sterile 1-L graduate. Cap
and shake to mix.
4. Apply sample labels to sterile, 1-L polypropylene bottles
and cover with a complete circle of clear protection
tape.
5. Transfer composite sample to 1-L bottles and cap tightly.
Sample Shipment
1. Samples should be shipped at 4°C. If samples are not
at this temperature and the shipping schedule permits,
place samples in a 4°C environment (refrigerator or
ice bath) prior to packing.
2. Pack a shipping container with the sample bottles.
Add a frozen Kool-Pac to the container insulating the
sample containers where necessary to prevent direct
contact between container and Kool Pac.
501
-------
3. Close container and strap securely. Check address label
for legibility.
4. Present shipping container with completed shipping ticket
at the passenger check-in counter or freight counter
of designated airline (Braniff or Southwest) at lease
45 minutes prior to scheduled departure. Shipment is
to be prepaid.
1/15/81
01-6001
J. Harding
bz:2H
502
-------
APPENDIX K
PROCEDURE FOR WASTBWATBR SAMPLE COLLECTION,
WILSON IMHOFF TANK EFFLUENT
503
-------
PROCEDURE>FOR WASTEWATER SAMPLE COLLECTION
Operation Year - 1981
Wilson, Texas Imhoff Tank Effluent
SwRI Project 01-6001
Purpose -
The purpose for collection of this sample is to determine relative
densities of a wide range of indigenous enteric bacteria and viruses
prevalent in the wastewater from the Wilson community, the most
densely populated area adjacent to the Hancock Site. To accomplish
this purpose a 24-hour time-weighted composite sample is collected
by utilizing a self-contained automatic sampler.
Equipment Required -
Sample Collection -
• ISCO Model 1580 Sampler with Nicad battery
6 ft. (2m) of 3/8" O.D. x 1/4" I.D. Tygon tubing
Short length of pipe for tubing weight
1 clean 3-gallon polyethylene container for ISCO
10 to 20 Ibs cracked or cube ice (function of ambient conditions)
Sample Shipment -
1 frozen Kool-Pac per 6 (six) 1-liter sample bottles
1 insulated shipping container, labeled, with means of lid attachment
1 counter-to-counter shipping ticket (Southwest or Braniff Airlines)
Procedure -
Preparation -
1. Charge two Nicad batterys for 24 hours prior to sample collection.
If this sample is collected simultaneously with the Trickling
Filter Effluent from the Lubbock Southeast Reclamation Plant,
only one extra. Nicad battery needs to be charged.
2. Check equipment for completeness including new Tygon tubing
with weight attached to end.
3. Place Kool-Pacs in freezer at least 24 hours prior to sample
shipment.
Sample Collection -
1. Locate sampler adjacent to Imhoff tank effluent drain.
2. Place a 3-gallon container in the Sample Container Tub with
the false bottom open and up. Carefully add crushed or cube
ice to the tube without disturbing the position of the
container.
3. Replace the Pump and Controls Section and latch securely making
sure that the Stop Float Mechanism is free. Attach the battery
to the sampler and securely connect the battery cable to the
505
-------
-2-
»
"12 VDC" socket on the socket on the side of the control box.
Attach the Tygon tubing to the pump inlet, tape to secure, and
lower weighted end into the Imhoff tank drin. Tape tubing to
side of sampler to reduce strain on pump inlet connection.
4, Set the Control Panel as follows:
Mode Switch - Time
Time Internal Multiplier Control - 1.0
Suction Line Length Switch - 7 1/3' (1/4" I.D.)
Sample Rate Switch - 136 mL/sample (8' head)
Pump Switch - Auto
5. Turn Sample Rate Switch to the Manual Cycle position, then
return it to the 10 min. Time Internal position. The pump
should be automatically activated, first for a brief period
in the reverse mode to purge any liquid in the line followed
by a forward pumping action of sufficient time to collect
approximately 136 mL of sample. This cycle is completed by a
second reverse pumping operation to again purge the sample line.
If all functions operate correctly in this test cycle, confirm
the position of all control switches, especially that the Pump
Switch is in the Auto Mode, then place and latch both the
protective lid over the Control Panel and the cover over the
Pump and Controls Section. Refer to the instruction manual
should problems be encountered.
6. At the end of the 24-hour sampling period, remove the 3-gallon
sample container from the Sample Container Tub, cap and label it,
and remove all equipment from the sampling site.
Sample Shipment-
1. After thoroughly mixing the sample, fill the appropriate number
of 1-1 bottles and cap tightly.
2. Samples should be shipped at 4°C. If samples are not at this
temperature and the shipping schedule permits, place samples in
a 4°C environment (refrigerator or ice bath) prior to packing.
3. Pack a shipping container with the sample bottles. Add a
frozen Kool-Pac to the container insulating the sample
containers where necessary to prevent direct contact between
container and Kool-Pac.
4. Close container and strap securely. Check address label for
legibility.
5. Present shipping container with completed shipping ticket at
the passenger check-in counter or freight counter of designated
airline (Braniff or Southwest) at least 45 minutes prior to
scheduled departure. Shipment is to be prepaid.
2/02/81
01-6001
J. Harding
506
-------
APPENDIX L
DESCRIPTION OF LTTTON MODEL M HIGH VOLUME AEKOSOL SAMPLER
507
-------
APPENDIX L
DESCRIPTION OF LITTON MODEL M HIGH VOLUME AEROSOL SAMPLER
"The Model M Sampler is designed to continuously collect particulate
matter from a large volumetric flow rate of air (approximately 1000 liters/
minute) and deposite it into a small amount of liquid (flow rate of 2
mL/min). This effects a volumetric concentration factor on the order of 5
x 105. Basically, the sampler is an electrostatic precipitator of a rather
unusual configuration. With reference to the schematic diagram, Figure
L-l, and an interior view, Figure [_-2, aerosol is drawn into the unit
through a converging nozzle and passes through the center of the high-
voltage area. It then flows radially between this plate and a lower
rotating collection disc. An electric potential of 15,000 volts, which is
maintained across a 11/16-inch spacing between the plate and disc, creates
two effects: 1) A corona is emitted from a ring of 60 needles that is
located concentric to the air inlet. Particles, exposed to air ions
created from the corona, acquire an electrical charge. 2) The electric
field provides the driving force to precipitate charged particles onto the
lower disc.
"Liquid is pumped onto the center of the collection disc and, because
of the centrifugal force, forms a thin moving film over the entire disc
surface. Particles collected on the film are transported to a rotating
collection ring where the liquid is removed by the pickup. Subsequently,
the liquid drips into the collection funnel where it is pumped to a
receiver located outside the sampler.
"To accommodate a broad range of sampling situations, several variable
features are incorporated into the unit. These are:
Air Flow Rate 400 to 1200 liters/minute
Liquid Flow Rate 0 to 8 mL/minute
Disc Speed 0 to 45 rpm
High Voltage 0 to 20 kilovolts
"When the sampler is in operation, the air flow rate is read directly
from a calibrated meter on the front panel and is adjusted with a blower
control potentiometer (see Figure L-3). Both disc speed and pump flow rate
509
-------
in
M
o
Corona Needles
High-Voltage Plate
Collection Ring
Pickup
Aerosol
Inlet
v rrr/
Liquid Output
To Pump
and Receiver
Liquid Inlet Tube
Collection Disc
D
Liquid
Input
From
Pump
Air Discharge
Figure L-l.. Schematic Diagram of Large-Volume Air Sampler System
-------
Hinged Top*
Strobelight
Ozone-Re sis tint
Casket Material
High-Voltage Plate
Ring Motor
Air Exhaust 7aa
Strobelight
Circuitry
Fluid Supply Tank
Removable
Side Panel
Collection Disc
Speed Control
Air Inlet
Ceramic Insulator
Pickup Assembly
Collection Ring
Collection Disc
Disc Motor
Peristaltic
.Pump Motor
Removable Side Panel
High- Voltage
Power Supply
Pump Speed Control
Electrical Connector
Figure L-2. Interior View of Large-Volume Air Sampler
511
-------
10
Air Flow
Rate Gauge
Control
tentiometers'^V.
High-Voltage
Voltmeter
High-Voltage
Milliamme ter
High-Voltage
Control
Potentiometer
High-Voltage
Circuit Breaker
and ON-OFF
Switch
Figure L-3.Instrument Panel of Model M Large-Volume Air Sampler
-------
are controlled by high and low range toggle switches, together with
potentiometers. Although no direct readouts are provided for these two
variables, calibrations are easily obtained so the arbitrary scales on the
potentiometers"can be converted to actual speed or flow rates. The high-
voltage systemtis set with the aid of a potentiometer and is provided with
the meter to show voltage and current."*
To facilitate visual observation of the surface condition of the disc
in operation, the operator made observations through the windows with the
aid of a flashlight. The air flow rate was set at 1000 liters/minute.
1. Litton Model M Large-Volume Air Sampler: Instruction Manual, Report
3028. Minneapolis, Minnesota, 1966.
513
-------
APPENDIX M
DECONTAMINATION PROCEDURE FOR MODEL M SAMPLERS
515
-------
APPENDIX M
DECONTAMINATION PROCEDURE FOR LITTON MODEL M
SOLUTIONS:
1% Clorox
Buffers--KH2P04 (71 g/L) 50 mL \ „ nT
Na2HP04 (115 g/L) 50 mL / /L DI
Autoclave 50 mL of the buffer in 2-oz bottles.
Add 1 mL of 5% Clorox prior to use.
1% sodium thiosulfate
10 g NaThio/L DI ^0
Sterile water
Autoclave 100 mL in 4-oz bottles prior to use.
PROCEDURE:
1. Calibrate air flow meter for 1000 1pm.
2. Disconnect electrical supply and remove side plate from unit.
3. Using Kimwipes dipped in 70% ethyl alcohol, wipe the inside top half
sides and all upper section parts.
4. Run disk (but not blower) and pump 1% Clorox solution through all
tubes. Hold Clorox solution in sampler tubing for a minimum of 30
minutes. The pump may be started periodically to move cleaning
solution through the tubing.
5. After decontamination with Clorox solution, flush the system with the
contents of a sodium thiosulfate bottle.
6. Rinse the system with the contents of a sterile water bottle. After
most of the liquid has been pumped out of the system, attach a
microfilter to the sampler inlet and run the blower until the disk is
dry.
7. Wipe the ends of the tubes with a Kimwipe saturated with 70% ethyl
alcohol. Place the ends of the tubes in a clean plastic bag and tape
shut. Seal the sampler inlet and exhaust ports with decontaminated
plastic caps.
01-6001-313
HJH 2/82
517
-------
APPENDIX N
COLLECTION EFFICIENCY OF LITTON MODEL M LARGE VOLUME SAMPLERS
519
-------
APPENDIX N
COLLECTION EFFICIENCY OF LITTON MODEL M LARGE VOLUME SAMPLERS
The Litton Model M large volume sampler (LVS), used to collect aerosol
data, is an electrostatic precipitator. During operation of an LVS, an
electrical potential of approximately 15,000 volts (15 kV) is maintained
across an 11/16-inch spacing between the plate and disk. This creates two
effects: 1) a corona is emitted from a ring of 60 needles thereby giving
the microorganism particles a charge and 2) the resultant electric field
attracts the charged particles to the collecting disk.
Collection efficiencies for electrostatic precipitators depend on the
operating high voltage producing the internal charging corona and electric
field. Sufficient voltage must be supplied to the corona source to charge
the particles suspended in air; the greater the voltage, the greater the
driving force to effect particle separation from air.
Electrostatic precipitators are usually operated at the highest
voltage possible without sparking (arcing). Sparking disrupts the
operation of the electrical equipment and lowers collection efficiency by
reducing the applied voltage, redispersing the collected particles, and
promoting current channeling (effectively reducing particle charging and
collection to localized areas).
Very high dust loadings increase the potential difference required for
the production of a corona and reduce the current due to the space charge
of the particles. This tends to reduce the average particle charge and
reduces collection efficiency. Compensation can be obtained by increasing
the potential difference when high dust loadings are involved.
The collection efficiency of an LVS is affected by many other factors
than simply the operating voltage and dust loading. The performance will
change according to intake air velocity, particle size distribution,
particle concentration in air, and environmental conditions (e.g., wind
gusts, wind speed, direction, and relative humidity).
Data obtained from field operation of the LVS are used in LHES to
calculate microbial concentrations in air as discussed in the second annual
LHES report (Calculation of Microorganism Density in Air section). The
resultant microbial concentrations assist the interpretation of the degree
521
-------
of aerosol exposure an individual would receive based upon the time of day
and distance from an operating rig whose source is either reservoir or
pipeline wastewater. Thus, it is important to correct all LVS sampling
data to a reference set of operating conditions to obtain internally
consistent da,ta. For example, an LVS may measure 20 cfu/m3 of air with
operating conditions which result in a relative collection efficiency of
40% and its paired sampler may measure 40 cfu/m3 of air with different
operating conditions which have a relative collection efficiency of 80%.
If only the raw data were used to calculate microbial concentrations
without regards to operating conditions (i.e., collection efficiency), then
one would incorrectly conclude that the second sampler observed microbial
concentrations twice as great as the first sampler. If the reference set
of operating conditions had an effective collection efficiency of 100%,
then both samplers would be recorded as having measured 50 cfu/m3 of air.
To determine correction factors for operating conditions, rigorous
experimentation is required in a controlled environment. A few
environmental conditions can be reconstructed in the laboratory to evaluate
their effect on collection efficiency. However, certain factors such as
microbial concentrations, particle size, and wind gusts cannot be
evaluated. Thus, the calculated microbial concentrations will be subjected
to indeterminate errors; the magnitude of these errors cannot be estimated.
Some factors (e.g., operating voltage) are known to affect the collection
efficiency and since these can be evaluated, it is necessary to adjust the
raw data for these factors.
The Naval Biosciences Laboratory (NBL) in Oakland, California
conducted experiments on three separate occasions (1976, July 1980 and
October 1982) to develop a collection efficiency data base from which to
calculate correction factors. In all of the NBL studies, data were
obtained for relative collection efficiencies of LVS to all-glass impingers
(AGI) samplers in a controlled environment (an atomizer created a specified
amount of aerosol in an enclosed wind tunnel). In these studies the AGI
samplers had a high degree of precision (for November 1982 NBL data the
average s/x was 6.70%), but their accuracy was not evaluated. On the other
hand, the LVS performed with less precision, as is demonstrated by the
average s/x of 11.7% for operating voltages greater than 12 kV (precision
decreases for smaller operating voltages).
The experimental procedures employed by NBL to study the LVS
collection efficiencies are thoroughly documented in their three final
reports; a capsule summary of these reports follows.
Disinfecting procedures prior to a sampling period were identical for
all three NBL studies and SwRI field operations. Operating time for the
samplers varied in each study, but discrepancies among the reported results
should not be caused by this procedural change since the results are
522
-------
reported as relative collection efficiencies (relative to AGI samplers
operating simultaneously with the LVS in the same wind tunnel).
Bacil1 us subtil is var. Mi ger replaced Flayobacterium as the test
organism for the November 1982 NBL study. Bacillus' subtil is var. Niger is
a hardy spore', but in spite of this, no problems of residual contamination
carryover were encountered.
Before the 1976 study, the samplers were completely overhauled;
defective and worn parts were either replaced or repaired. For the other
two NBL studies, the samplers were not overhauled; however, routine
preventative maintenance was continued. It is unknown whether the 1976
overhaul affected collection efficiencies differently than the routine
maintenance procedures.
The collection fluid (BHI) circulation rate varied among all three NBL
studies. In July 1980, NBL reported that no collection efficiency
differences were observed for a BHI rate greater than 8 mL/min; only data
obtained with BHI rates greater than this were used for correction factor
evaluations. The air intake sampling rates were approximately 1.0 m3/min.
LVS operating voltages in the three NBL studies ranged from 8 to 18
kV. The 1976 study reported two LVS sampler responses at various operating
voltages (8 to 14 kV) were obtained by NBL for LVS samplers operated at the
highest voltage attainable without producing excessive arcing; these data
are reported as relative collection efficiencies at 15+ kV. The October
1982 data is reported on raw data sheets as relative collection
efficiencies at the actual LVS operating voltage.
During the effort to identify operating variables that influence the
LVS collection efficiency, NBL studied relative humidity and temperature
effects in the October 1982 study. According to NBL no strong effect of
relative humidity was observed for the range tested (relative humidities
from 51 to 81), and the rather narrow temperature range (unqualified) of
the tests showed no collection efficiency effects. These conclusions from
NBL are most likely incomplete for two reasons:
1) When relative humidity is plotted versus collection efficiency a
negative correlation between collection efficiency and relative
humidity for voltages greater than 12 kV is suggested (see Figure
N.I). This correlation is less apparent for operating voltages
of 12 kV. Insufficient data makes it impossible to evaluate the
effect of relative humidity at lower operating voltages.
2) NBL does not report the operating temperatures, but it seems
unlikely that the wide temperature range in the field (10 to
35°C) was adequately studied.
523
-------
u
i i
! I
i I i !
I 1 I
i I i
444
50
60 70
Relative Humidity (%)
80
90
Figure N.I.
Relative humidity versus relative collection efficiency
(LVS/AGI ratio) (1982 NBL data)
524
-------
Nevertheless, correction factors for temperature and relative humidity are
not applied to field data since insufficient data exist to formulate
accurate correction factors.
Only the October 1982 NBL report included data'that could be used to
develop an air'flow rate correction factor Cf. Each sampler will require a
unique Cf, however, NBL only reported one data value for each LVS. Since
neither reproducibility nor accuracy was demonstrated by NBL for Cf, it is
not recommended to use the "correction factors" to adjust field data; it is
presumed that exclusion of Cf will not result in severe deficiencies in the
final evaluation since none of these corrections changed the raw data by
more than 20%.
It would be possible to obtain sufficient data to derive a reasonable
correction factor by repeating these tests for each sampler set at the 1000
L/min mark. However, these data experiments are unwarranted, since during
field sampling wind gusts alter the sampling air flow rate making it
impossible to achieve the same laboratory precision in determining the air
flow rate.
In October 1982 NBL measured the voltage supplied to the corona source
at four different high voltage settings on nine different LVS samplers.
Calibration curves were drawn for each sampler by plotting the indicated
versus the measured voltage. Each calibration curve was a straight line
with a slope of approximately one but with various y-intercepts.
Repetition of the voltage measurements was not reported, so it is unknown
whether these results are reproducible. Consequently, the voltage
correction factor uses the recorded operating voltage as the independent
variable, not the actual measured voltage.
To determine whether each LVS should have an individual sampler
correction factor, the 1982 NBL data was analyzed at SwRI on a Cyber
170/171 with the SPSS package and the ANOVA subroutine. No consistent
differences were observed among samplers both at the 12 kV and at greater
than 12 kV (15+ kV). It appeared that the actual run numbers had greater
significance than individual samplers. The significance may be partly due
to relative humidity values; other operating variables (e.g., temperature)
may also contribute to the difference observed between runs.
In the 1976 NBL study, no effects from operating at voltages greater
than 12 kV were observed. However, in the October 1982 study, large
variations of collection efficiencies occur for LVS operating at voltages
greater than 12 kV. At this time there is no explanation for these
conflicting results.
The raw data from the October 1982 NBL study are plotted on a semilog
plot in Figure N.2 (operating voltage versus relative collection
efficiencies of LVS to AGI samplers). From these data, four different
correction factor curves could be drawn.
525
-------
1 -1
n Q -
0.8 -
0.7 -
0.6 -
0.5 -
.4
0.3 '
>>
y n 9
C U. L.
0)
•r—
U
•*~
H-
\ i I
c
Q
£ 0.1 -
£ 0.09 -
;= o.os -
3 0.07 -]
% 0.06 -
% 0.05 -
-------
In the field, several measurements were made with paired samplers.
These paired field samplers may help to identify the most valid correction
factor, i.e., the correction factor that minimizes the difference between
the reported micYobial concentrations for all microorganisms for all paired
samplers.
The four possible correction factor curves are plotted in Figure W.3.
Curve A represents no correction factor. Curve B is modeled after the 1976
data where data below 12 kV are corrected as an average between reported
values and above 12 kV no correction is made. The third method (Curve C)
was calculated from all averages at various voltages from the 1982 NBL
data. Curve D is a minimum correction factor.
The physical interpretation for Curve A is that an LVS sampler
operates similarly at all voltages. From the preceding discussion, it is
known that this is unrealistic.
Curve B assumes that once the operating voltage reaches 12 kV no
effect on collection efficiency is observed as long as operation occurs
below sparking. In addition, this correction factor has no minimum
asymptote for operating voltages below 12 kV.
The third correction factor (Curve C) demonstrates the same low
voltage correction as Curve B. High voltage operation distinguishes
between these two methods. In Curve C the NBL 1982 data is corrected to 12
kV. Since the data peaks at 14 kV, an inflection point is observed at 11.5
kV, a maximum at 14 kV, and then an asymptote at 14.5 kV. No minimum
asymptote exists. A physical interpretation could be the following: at
low voltages, the collection efficiency increases proportionately with the
operating voltage. At 11.5 kV all of the particles are charged. Greater
voltages affect a greater driving force for separating the charged
particles from the air. Above 14 kV visually undetectable sparking occurs
that reduces the effective voltage until it reaches an asymptote in which
the increased sparking is counteracted by the increased driving force from
the high voltage.
Curve D has a minimum asymptote that implies that under certain field
conditions, a low voltage will always be able to charge a few particles and
will be able to collect these. Moving from the asymptote, at higher
operating voltages, proportionately more particles are charged and
consequently collected. At 11.5 kV, an inflection point occurs that
implies that a different mechanism is responsible for greater collection
efficiencies. It is hypothesized that at 11.5 kV all particles are charged
but that the collecting electric field determines the percentage of
particles that are collected. Thus, an increase in operating voltage above
11.5 kV increases the electric field which in turn increases the collecting
527
-------
u
-------
driving force. A maximum asymptote is then observed where an increase in
operating voltage increases the sparking phenomenon which reduces the
effective electric field. If operation had occurred while excessive
sparking occurred, it is predicted that Curve D would show a decrease in
collection efficiency beyond the maximum asymptote.
Differences in Curves C and D are a result of the calculational basis
of correction. Curve C was calculated from average efficiencies at various
operating voltages; Curve D was calculated from the highest efficiency
observed for operating voltages below 12 kV and lowest efficiencies
observed for voltages above 12 kV. The latter produces a conservative
correction that adjusts all data to the minimal degree expected. Thus, the
corrected data may be required to be adjusted further, but it will never be
overcorrected.
Curve D seems to be more realistic than Curves A and B because the
data suggest that some correction is required in both the high and low
voltage regions. It also seems to be more realistic than Curve C because
it will not result in overcorrections. This latter is an important
consideration since at low voltages (9 kV) an order of magnitude range was
observed in the experimental data (see Figure N.2).
All of the field data are corrected using Curve D (minimum
corrections) and are presented in the aerosol data results section along
with a table of all of the operating field voltages. The correction
factors employed are in Table 4.15 of the Calculation of Microorganism
Density in Air section. With these data, the interested reader can develop
his own correction factor method and test it against the field data (paired
samples).
529
-------
APPENDIX 0
EHTEROVIRUS SEROLOGI QUALITY CONTROL:
TTTER KBPKODUCIBILI1T (TR) FROM REPLICATE TESTING
531
-------
VIRUS: Adenovirus 3
FREQUENCY DISTRIBUTION
HigTT
Intennediate
SOURCE OF CONTROL SERA:
High titer fins..
Intennediate titer 804
6
12
TOTAL
GEOMETRIC
MEAN
TITER
TR
18
91
0.79
18
32
1.00
RUN
#
I
2
3
DATE
7-11-83
11-16-83
2-02-84
VIRUS
DOSE
(TCID50)
41
316
147
GEOM. MEAN
High Titer
141
50
56
TR
High Titer
1.00
1.00
1.00
GEOM. MEAN
Interm. Titer
32
32
32
TR
Interm. Titer
1.00
1.00
1.00
533
-------
VIRUS: Adenovirus 5
TITER
FREQUENCY DISTRIBUTION
HignIntermediate
SOURCE OF CONTROL SERA:
High titer sna
Intermediate titer ?oa
1
15
7
TOTAL
TR
21
GEOMETRIC
MEAN
TITER 145
0.71
23
50
0.97
RUN
#
1
2
3
4
DATE
7-06-83
11-17-83
2-02-84
3-01-84
VIRUS
DOSE
(TCID5Q)
261
178
178
178
GEOM. MEAN
High Titer
40
224
125
202
TR
High Titer
1.00
0.78
1.00 '
1.00
GEOM. MEAN
Interm. Titer
40
50
56
45
TR
Interm. Titer
1.00
1.00
1.00
0.88
544
-------
VIRUS: Adenovirus 7
FREQUENCY DISTRIBUTION
SOURCE OF CONTROL SERA:
TITER
< 10
10
20
40
80
160
320
>.640
TOTAL
HlSh intermediate H-gh mer ^
7 Intermediate titer 704
1 11
21 19
16 1
38 38
GEOMETRIC
MEAN
TIiER 26 13
TR
RUN
#
1
2
3
4
5
6
0.97 0.89
VIRUS
DOSE GEOM. MEAN TR GEOM. MEAN
DATE (TCIDso) Hi9h Titer H1gh T1ter Interm. T1ter
10-06-82 178 18 1.00 9
10-07-82 178 22 1.00 13
10-19-82 178 22 1.00 18
11-17-83 316 28 1.00 5
2-02-84 100 40 1.00 22
3-01-84 215 36 1.00 18
TR
Interm. Titer
1.00
1.00
1.00
1.00
1.00
1.00
535
-------
VIRUS: Coxsackie 82
TITER
< 10
10
20
40
80
160
320
> 640
TOTAL
FREQUENCY DISTRIBUTION
High Intermediate
1
_16_
17
GEOMETRIC
MEAN
TITER 614
TR
1.00
2
12
5
19
39
0.94
SOURCE OF CONTROL SERA:
High titer 802
Intermediate titer 610
RUN
#
1
2
3
DATE
5-18-83
5-20-83
2-28-84
VIRUS
DOSE
(TCID50)
40
26
41
GEOM. MEAN
High Titer
640
640
557
TR
High Titer
1.00
1.00
1.00
GEOM. MEAN
Interm. Titer
100
112
65
TR
Interm. Titer
1.00
1.00
1.00
536
-------
VIRUS: Coxsackie 84
FREQUENCY DISTRIBUTION
TITER
< 10
10
20
40
80
160
320
> 640
TOTAL
GEOMETRIC
MEAN
TITER
.High
1
6
10
1
18
31
Intermediate
5
7
4
2
18
22
SOURCE OF CONTROL SERA:
High titer
Intermediate titer aoo
TR
0.95
0.86
RUN
#
1
2
3
DATE
8-01-83
8-02-83
1-19-84
VIRUS
DOSE
(TCID50)
100
164
83
GEOM. MEAN
High Titer
32
20
45
TR
High Titer
1.00
0.95
0.83
GEOM. MEAN
Interm. Titer
20
13
45
TR
Interm. Titer
0.95
1.00
0.89
537
-------
VIRUS: Coxsackie B5
FREQUENCY DISTRIBUTION
TITER
< 10
10
20
40
80
160
320
> 640
High
14
18
7
5
Intermediate
4
12
26
7
1
TOTAL
GEOMETRIC
MEAN
TITER
TR
44
168
0.87
50
17
0.82
SOURCE OF CONTROL SERA:
High titer 904
Intermediate titer 713
RUN
I
1
2
3
4
5
6
7
DATE
2-03-82
2-04-82
10-05-82
5-04-83
11-02-83
1-17-84
2-28-84
VIRUS
DOSE
(TCID50)
316
144
68
56
32
178
121
GEOM. MEAN
High Titer
224
457
132
174
141
100
141
TR
High Titer
0.56
0.78
0.88
0.81
1.00
1.00
0.89
GEOM. MEAN
Interm. Titer
16
13
18
22
14
18
45
TR
Interm. Titer
0.83
0.83
1.00
1.00
1.00
1.00
1.00
538
-------
VIRUS: Echovirus 1
FREQUENCY DISTRIBUTION
TITER
< 10
10
20
40
80
160
320
> 640
TOTAL
GEOMETRIC
MEAN
TITER
High
6
6
12
113
Intermediate
1
3
7
1
12
16
SOURCE OF CONTROL SERA:
High titer 42502
Intermediate titer 32401
TR
1.00
0.92
RUN
#
DATE
VIRUS
DOSE
(TCID50)
GEOM. MEAN
High Titer
TR
High Titer
GEOM. MEAN
Interm. Titer
TR
Interm. Titer
11-18-82
11-19-82
10-27-83
1-31-84
32
32
24
122
112
112
1.00
1.00
13
20
0.83
0.94
539
-------
VIRUS: Echovirus-3
TITER
< 10
10
20
40
80
160
320
> 640
2
6
13
9
5
TOTAL
GEOMETRIC
MEAN
TITER
TR
FREQUENCY DISTRIBUTION
High Intermediate
10
18
5
7
35
96
0.71
40
23
0.69
SOURCE OF CONTROL SERA:
High titer 802
Intermediate titer 601
RUN
#
1
2
3
4
5
6
DATE
5-25-83
5-27-83
11-12-83
1-18-84
2-16-84
2-21-34
VIRUS
DOSE
(TCID50)
242
242
32
48
10
1 10
GEOM. MEAN
High Titer
45
71
105
200
141
cont.
TR
High Titer
0.67
1.00
0.68
0.83
0.89
cont.
GEOM. MEAN
Interm. Titer
11
18
14
22
56
50
TR
Interra. Titer
1.00
1.00
1.00
1.00
0.78
0.83
cont.
2-23-84
32
79
0.77
25
0.83
540
-------
VIRUS: Echovlrus 5
TITER
< 10
10
20
40
80
160
320
>. 640
TOTAL
GEOMETRIC
MEAN
TITER
TR
TREQUENCY DISTRIBUTION
High Intermediate
1
4
2
5
7
1
20
0.56
19
46
0.81
SOURCE OF CONTROL SERA:
High titer 42702
Intermediate titer 41001
RUN
#
1
2
3
4
5
DATE
3-10-82
3-11-82
10-04-82
10-27-83
2-24-84
VIRUS
DOSE
(TCID5Q)
100
215
261
64
32
GEOM. MEAN
High Titer
20*a
32*a
209
40
282
TR
High Titer
1.00*
l.OO*3
0.88
0.94
0.89
GEOM. MEAN
Interm. Titer
5*a
5*a
74
25
46
TR
Interra. Titer
1.00*a
1.00*
1.00
0.78
0.88
a Due to the low liters in staff sera, study participant sera was used as controls
in runs 3-5; the staff titers were not included in the frequency distribution.
541
-------
VIRUS: Echovirus 9
TITER
< 10
10
20
40
80
160
320
>.640
TOTAL
GEOMETRIC
MEAN
TITER
TR
.FREQUENCY DISTRIBUTION
HlqJiIntermediate
4
17
15
5
18
13
3
39
103
0.82
36
12
0.91
SOURCE OF CONTROL SERA:
High titer 702
Intermediate titer 709
RUN
#
1
2
3
4
5
DATE
3-03-82
3-04-82
10-21-82
11-03-83
2-02-82
VIRUS
DOSE
(TCID50)
68
56
178
32
100
GEOM. MEAN
High Titer
200
100
74
126
112
TR
High Titer
1.00
0.83
1.00
0.67
1.00
GEOM. MEAN
Interm. Titer
18
13
9
18
13
TR
Inter™. Titer
1.00
0.83
1.00
1.00
1.00
542
-------
VIRUS: Echovirus 11
TITER
FREQUENCY DISTRIBUTION
lgliIntermediate
2
12
17
4
2
9
19
6
2
SOURCE OF CONTROL SERA:
High titer 3211L
Intermediate titer 22411
TOTAL
GEOMETRIC
MEAN
TITER
TR
37
69
0.78
36
20
0.83
RUN
#
1
2
3
4
5
6
7
8
9
DATE
3-10-82
3-11-82
10-20-82
11-03-83
1-12-84
2-09-84
2-10-84
2-14-84
2-28-84
VIRUS
DOSE
(TCID50)
178
147
316
83
68
75
100
53
100
GEOM. MEAN
High Titer
10*d
13*a
79
56
63
40
*b
143
56
TR
High Titer
1.00
1.00
0.97
1.00
0.61
0.92
0.67
1.00
GEOM. MEAN
Interm. Titer
5"
5
15
14
45
14
*b
28
28
TR
Interm. Titer
1.00
1.00
1.00
1.00
0.83
1.00
1.00
1.00
a Due to low titers in the staff sera, study participant sera was used as controls in
runs 3-9.
Control titers for this run were misplaced.
543
-------
VIRUS: Echovirus 17
TITER
< 10
10
20
40
80
160
320
>. 640
TOTAL
GEOMETRIC
MEAN
TITER
TR
FREQUENCY DISTRIBUTION
High Intermediate
2
8
18
2
11
9
8
1
29
39
0'.87
SOURCE OF CONTROL SERA:
High titer 800
Intermediate titer 614
30
13
0.38
RUN
#
1
2
3
4
5
DATE
6-01-83
6-03-83
11-09-83
1-19-84
2-28-84
VIRUS
DOSE
(TCID50)
241
562
242
133
56
GEOM. MEAN
High Titer
25
28
25
89
50
TR
High Titer
1.00
1.00
1.00'
1.00
0.83
GEOM. MEAN
Interm. Titer
11
16
14
25
16
TR
Interm. Titer
0.88
1.00
0.78
1.00
1.00
544
-------
VIRUS: Echovirus 19
TITER
' FREQUENCY DISTRIBUTION
H i gh Intermediate
SOURCE OF CONTROL SERA:
High titer 702_
Intermediate titer
704
TOTAL
TR
16
GEOMETRIC
MEAN
TITER 31
0.74
17
11
0.86
RUN
»
I
2
3
DATE
7-05-83
11-10-83
1-17-84
VIRUS
DOSE
(TCID5Q)
61"
100
130
GEOM. MEAN
High Titer
45
13
50
TR
High Titer
1.00
1.00
1.00
GEOM. MEAN
Interm. Titer
19
5
14
TR
Interm. Titer
1.00
1.00
1.00
545
-------
VIRUS: Echovirus 20
TOTAL
FREQUENCY DISTRIBUTION
TITER
< 10
10
20
40
80
160
320
> 640
High
5
11
4
3
Intermediate
2
5
11
4
23
GEOMETRIC
MEAN
TITER 23
TR
0.87
22
13
0.39
SOURCE OF CONTROL SERA:
High titer 702
Intermediate titer 614
RUN
#
1
2
3
4
DATE
8-09-83
8-10-83
11-10-83
2-28-84
VIRUS
DOSE
(TCID50)
83
130
100
56
GEOM. MEAN
High Titer
20
16
18
56
TR
High Titer
0.94
0.83
1.00
1.00
GEOM. MEAN
Interm. Titer
22
16
7
25
TR
Interm. Titer
0.89
1.00
1.00
1.00
546
-------
VIRUS: Echovirus 24
FREQUENCY DISTRIBUTION
SOURCE OF CONTROL SERA:
TITER
< 10
10
20
40
80
160
320
>.640
TOTAL
High
1
2
5
7
5
1
21
GEOMETRIC
MEAN
TITER 135
TR
RUN
#
1
2
3
4
0.58
DATE
7-26-83
7-28-83
11-09-83
1-31-84
Inten"edlate High titer 904
a Intermediate titer 702
6
3
2
4
1
24
15
0.58
VIRUS
DOSE GEOM. MEAN TR GEOM. MEAN
(TCIDgg) High Titer High Titer Interm. Titer
75 112 1.00 8
90 112 1.00 14
56 63 0.72 6
32 320 0.89 80
TR
Interm. Titer
0.83
0.72
1.00
0.89
547
-------
APPENDIX P
SUPPLEMENTAL TABLES FOR SECTION 5 (RESULTS)
549
-------
TABLE P-1. MICROORGANISM CONCENTRATIONS IN LUBBOCK WASTENATER
SURD 11 no date
24-Hour conposlte
sarnies analyzed
Bacteria [cTu/mL]
Standard plate count
Total conforms
Fecal collforas
Feoal streptococci
Mycobacterla sp.
Clostrldlua perfrlngens*
- vegetative
- aporulated
Staphylococcus aureus
SalMonella ep.
Shi gel IB ap.
Yeralnla enterocolltlce
Cavpylobacter Jejunl
Candida alb leans
Fluoreecent PseudoMonas sp.
Klebslalla ep.
VtnMOT (pfu/nL)
Bacterlophage
En tero viruses
HaLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Pollovlrus concentration
efficiency (X)
PbfslMl AMlya** lmg/L]
Tote I organic carbon
Total suspended solids
Totel volatile suspended solids
DH
Jun 3-4
3,600,000
350,000
87,000
4,700
1,200
7,500
930
<33
<0.004
<0.004
<0.002
10,000
<33,000
1,400
0.78
38
83
88
85
6.5
1880
Jul 28-28
5,700,000
380,000
72,000
2,000
170,000
110,000
430
<3
XJ.002
<0.002
<0.004
6,300
130,000
3,200
1.2
42
40
78
52
6.8
Nov 3-4
3,400,000
140,000
88,000
5,100
1,100
2,400
830
<3
<0.002
<0.002
<0.004
3,100
53,000
2,800
0.73
38
215
135
7.2
Jen 18-20
60,000
15,000
880
0.086
87
115
184
130
7.0
1881
Feb 16-17 Mar 8-10 Mar 23-24
110,000 120,000 160,000
34,000 16,000 83,000
I
<10
<0.01b
X).01
100
66
<0.3
130,000
0.054 0.058 0.046
78 28 105
133 141 81
151 234 88
120 178 '" 74
7.3 7.0 7.1
continued.
-------
TABLE P-1. (CONT'D)
Sam Ling date
24-Hour composite
sacDles analyzed
•Mtorl* (cfu/ML)
Standard plate count
Total conforms
Fecal collfoma
Fecal streptococci
Mycobecterla ep.
CloatHdltm perfrlngens"
- vegetative
- speculated
Stephy lococcus aureus
Salmonella sp.
Shlgella sp.
Yerslnla enterocolltlce
Cempylobecter Jejuni
Candida alb leans
Fluorescent Paeudoaonas sp.
Klebslella sp.
VI HMO* (pfu/aL)
Bacterlophege
Enterovl ruses
HeLat 6 day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
PollovlruB concentration
efficiency (X)
PlqnBlml AawlyMS [«g/LJ
Tote I organic carbon
Total suspended solids
Total volatile suspended solids
DH
Aor 80-21
9 1 600 (000
520,000
59,000
8,900
400,000
110,000
460
3
>0.005
XI.008
70.005
0
220,000
230,000
1,600
0.057
0.018
0.008
69
237
200
147
7.5
Mav 4-5
88,000
<3
>0.005
XI
>0.005
<3
<3
2,600
0.11
0.006
0.033
95
104
115
92
7.6
Jun 15-16
360,000
110,000
1,100
<3
<0.01
<0.01
<0.01
<3
<3
200,000
0.1
0.065
0.15
78
47
47
44
8.5
1981
Jun 29-30
120,000
50,000
8,700
<3
<0.01
<0.01
<0.01
>200C
<3
30,000
0.085
0.055
0.1
77
100
51
38
7.6
Jul 20-21
3,000,000
380,000
100,000
2,400
14,000
230
210
<10
>10
<0.007
<0.007
<10
<10
23,000
88,000
2,100
0.085
0.02
0.093
85
100
43
33
7.2
Aua 17-18
81,000
<3
>10
<0.008
<0.008
<0.1
<3
50,000
0.045
0.005
0.42
34
78
68
49
8.4
Nov 17-18
60,000
3
>10
<0.01
<0.01
<3
<3
130,000
0.055
0.0013
0.13
80
100
118
87
7.3
continued..,
-------
TABLE P-1. (COMT'D)
Ul
U)
Saw> Una date
24-Hour composite
saw lee analyzed
Bacteria (cfu/«L)
Standard plate count
Total collfoms
Fecal co 11 fonts
Fecal streptococci
Nycobactarla sp.
ClostrldluM perfrlngens8
- vegetative
- sporulated
Staphylococcus eureue
Salnonella sp.
Shi gel la ep.
Yerslnla enterocolltlca
Canpylobactar jejunl
Candida alb leans
Fluorescent Pseudononas sp.
Klebslalla sp.
VI ruses Ipfu/mL]
Bactsrlophage
Enterovl ruses
HeLa, 5 day (uncorrected]
He La, polio-neutralized
RD» polio-neutralized
PoUovlrus concentration
efficiency (X)
Physical *Mly»*s lug/L)
Tote I organic carbon
Total suspended so I Ids
Totel volatile suspended solids
oH
. , 19
Feb IB-IB*1 Fob Iff1'" Mar 1-2
150
240
C
tar 8-8" Mar 15-18 Mar 22-23
57,000
11,000 38 5,600 75,000 79,000 81,000
11,000 120 1,000
5,900 3,500 7,900
1,000 28,000 53,000 30,000 13,000
210
28
<3 2.5
<0.04 X).04
<0.01 <0.01
<0.01 <0.01
<0.01 <3
<3 <3
30
!
<3 0
100
<0.01 <0.01
<0.01 <0.01
40 <3
<3 <3
260,000
Mar 29-30
50,000
5,000
10,000
130,000 180 50,000 68,000 50,000
900 750 1,000
0.037 0.033 0.07
<0.003 <0.005 0.034
<0.003 <0.002 <0.002
227 50
138 103 88
111 143 150
86 80 113
7.1 8.8 7.1
1 ,800 780 1 ,500
0.11 0.11 0.063
0.022 0.017 0.004
<0.002 0.010
86 f 63
116 85 151
178 92 269
153 82 170
7.1 7.4 7.3
69
0.012
0.002
0.034
125
205
165
7.1
continued...
-------
TABLE P-1. (CONT'DJ
24-Hour conposlte
analyzed
Sarollnfl date
Apr 5-6
Aor 19-20
Apr 26-27B
1882
Jun 14-16
Jun 29-30
Jut 26-27
Aun 9-10
en
iris (cfu/*L)
Standard plate count
Total conforms
Fecal co11 forms 84,000
Fecal streptococci 2,800
Nycobaoterla sp. 20,000
Clostrldlua perfrlngens"
- vegetative
- speculated
Staphy lococcus aureus <3
Salmnella sp. 0.01
Shigel la sp. <0.01
Yeralnla antarocolltlce 1,000
Canpylobacter jejunl <3
Candida albleans <3
Fluorescent PseudoMnas sp.
Klebslella sp. 1,000
VlrasM (pfu/«L)
Bacterlophage 380
En terovl ruses
HeLa, 5 day (uncorrected) 0.017
He La, polio-neutralized 0.004
RD, polio-neutralized 0.044
PoUovlrus concentration
efficiency (X) 77
110,000
4,800
6,000
<0.01
100
10
40
130,000
830
0.042
0.016
0.010
54
8,100
1,800
8,500
66,000
1,000
13,000
68,000
4,200
43,000
220
0.028
0.008
0.004
68
840
0.026
0.026
<0.002
64
<0.01
<0.01
».
8,000n
100,000
840
0.48
0.39
0.056
68
1,300,000
120,000
58,000
2,300
13,000 ,
750
9
<3
-------
TABLE PH. (CONT'D)
Saapllnn date
Ut
wi
Ul
24-Hour
sa no lea
conpoalte
analyzed
Aua
30-31*
4
Sec 13-1
982
4
Nov 1-2
Dae
13-14
Fab 18-17
1883
Mar 7-8
Mar 21-22
BMtcria (cfu/*L)
Standard plate count
Total coll forms
Fecal collforas
Fecal streptococci
Nycobactarla ap.
Clostrldlun perfrlngensJ
- vegetative
- eporulated
Staphylococcus aureue
Selnonelle ap.
Shlgelle ep."
Yerelnla entarocolltlca*
Canpylobacter Jejunl
Candida albleans
Fluorescent Peeudoaonas ep.n
Klebelelle ep.
VlraM* (pfu/ML)
Bacterlophage
Enterovlruaee
HeLa, 5 day (unoorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Pollovlrue concentration
efficiency (X)
(•g/L)
200
30
780
<0.01
<0.01
<0.01
<3
<3
30,000
300
1
1
0.018
47
65,000
3,500
1,400
>0.01
<0.01
<0.01
X10
<300
2,000
40,000
0.022
0.008
0.84
42
210,000
48,000
2,100
170,000
31,000
800
140,000
58,000
3,000
230,000
23,000
8,000
0.11
0.082
0.52
0.082
0.018
0.082
0.044
0.020
0.028
18
0.11
0.012
0.072
82
330,000
6,100
4,000
k
10
<0.01
>0.01
<0.33
2,000
5,500
0.031
0.016
0.024
88
Total
Total
Total
oH
organic carbon
suspended solids
volatile suspended
solids
52
51
38
7.3
58
50
42
7.8
54
81
68
7.3
o
o
o
7.7
48
78
55
7
.7
108
126
102
7
.6
83
83
68
7.5
continued..
-------
TABLE P-1. ICOMT'D)
24-Hour composite
analyzed
Saapllnn date
Apr 4-6
Apr 18-18
Jun 27-28
1883
11-12
Jul 25-26
Aug. 8-9
AUQ 22-23
ri« (cfu/nL)
Standard plate count
Total co 11 forms
Fecal coltfonu
Fecal streptococci
Hycobacterla ap.
Cloetrldlun perfrlngeneJ
- vegetative
- eporulated
Staphylococcue aureue
Salmonella eg.
Shlgella op."
Yerelnle enterocolltlca"
Canpylobacter jejunl
Candida alb leans
Fluorescent Pseudononee ep.
Klebalelle ep.
VfrwM (pfu/«L)
Bacterlophege
Enterovlruses
HeLef 5 day (uncorrected)
HeLa, polio-neutralized
HD, polio-neutralized
Pollovlrue concentration
efficiency (X)
Physical AMlysn (ng/L)
180(080
20,000
3,100
140,000
18,000
4,000
58,000
1,200
0.12
0.044
0.080
47
.
<5.0l
<0.01
<0.01
400
100
4,000
0.10
<0.004
<0.004
88
0.27
0.14
0.34
72
53,000
1,200
48,000
500
0.28
0.30
0.68
44
0.28
0.12
0.16
42
120,000
1,000
0.12
0.13
0.18
30
80,000
0.2
0.24
0.38
0.20
61
Total organic cerbon
Total suspended so I Ids
Total volatile suspended solids
DH
62
58
51
7.6
50
41
31
7.6
42
35
25
7.6
35
23
16
7.7
22
28
23
7.6
28
44
34
7.1
32
17
14
7.8
continued.
-------
TABLE PH. ICONT'D)
24-Hour composite
sendee analyzed
Semolina date
1983
Sap 18-13
Bacteria [cfu/mL)
Standard plate count
Total conforms
Fecal co 11 fo me
Fecal streptococci
Mycobecterla ep.
Clostrldlun parfrlngens-1
- vegetative
- eporulated
Staphylococcue eureus
SalnoneUa ep.
Shi ge lie ep.*
Yerelnle enterocolltlca*
Cempylobecter jejunl
Candida alb leans
Fluoraecent Pseudomonas sp.n
Klebslella ep.
VlraM* (pfu/»L)
Bacterlophege
En tero viruses
HeLa, 5 dey (uncorrected]
HeLe, polio-neutralized
HD, polio-neutralized
PollovlruB concentration
efficiency (X)
(ng/L)
210,000
1,000
Total organic carbon
Totel suspended eollds
Total volatile suspended solids
pH
0.056
0.18
0.12
74
37
25
17
7.3
e. Most probable number (MPN)/nL.
b. A new procedure «as used for detection of Salmonella spp. (Kaper et
al., App. Environ. Nlcroblol., 83iB28-35, 1977) beginning In March
1881.
c. Value calculated from representative colonies Identified as C.
jejunl, ectual nunber nay be higher. * ;
d. On February 16, 1982 the eanple source «as changed fron the trickling
filter to the pipeline; the first set of data on February 16 *ae
sampled fro* the trickling filter *h1le the second set was collected
fro* the pipeline.
e. ChloHnatlon of itasteaater at treatment plant.
f. Lost.
g. Chlorlnatlon In Lubbock of a portion of the sampled «aste«ater.
h. Beginning n1th eanples collected on June 29-30, 1982 fluorescent
PseudoMnas ep. was substituted for Staphylococcus eureus as part of
Halted bacterial screen.
1. HeLa eel IB used for the assay were contaminated; results could not be
obtained.
j. Membrane filtration technique.
k. Contaminated.
1. Fungal contamination at lover dilutions.
m. Enrichment procedure (for samples after November 1, 1982).
n. Assayed on Cetrlnlde egar (for samples after November 1, 1982).
o. Analysis not performed.
+ Presence of Salmonella (>1 colony/100 nL)
Salmonella not detected (<1 colony/100 mL)
-------
TABLE P-2. MICROORGANISM CONCENTRATIONS IN HANCOCK RESERVOIR
00
Sampl inq date
Source reservoir
Sample type3
Bacteria (cfu/mL)
Standard plate count
Total collforms
Fecal collforms
Fecal streptococci
Mycobacterla sp.
Clostrldium perf r lngensb
- vegetat 1 ve
- sporu 1 ated
Staphylococcus aureus
Salmonel la sp.
S hlgel la sp.
Yers 1 n 1 a enteroco 1 1 1 1 ca
Campylobacter jejunl
Candida alb leans
Fluorescent Pseudomonas sp.
Klebslel la sp.
Viruses (pfu/mL)
Bacter lop hag e
Enterovl ruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, po 1 1 o-neutra 1 1 zed
Pollovlrus concentration
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
PH
Jun 14-15
1
C
520
20
4,000
14
0.002
0.005
<0.002
81
33
218
50
7.6
Jun 29-30
1
C
60
3
200
<0.01
<0.01
O.01
<10
<10
230C
10
19
0.014
0.056
0.017
71
21
67
28
7.9
1982
Jul 26-27 Aug 9-10
1 1
C C
36,000
500
190
3
<10
430
4
<3
0.01
O.01
O.01
<3
<3
13
30
0.9
O.002
O.002
0.004
100
14
21
20
8.0
390
6.6
O.01
O.01
O.01
<3
<3
16
130
0.002
0.004
0.004
87
27
24
21
8.1
Aug 30-31
1
C
to
0.3
1,000
O.01
0.01
O.01
<3
<3
2,000
<50
0.8
d
d
O.002
61
23
24
19
7.9
Sep 13-14
1
C
350
10
550
O.01
O.01
O.01
<10
<10
250
1,000
O.002
0.002
0.008
27,-
28
44
34
8.4
continued.
-------
TABLE P-2. (CONT'D)
tjt
vo
Sampl Ing date
1982
Nov Dec Feb
1-2 13-14 16-17
Source reservoir 1,3 1 1
Sample type3 G G C
Bacteria (cfu/mL)
Standard plate count
Total collforms 1,000 10,000 500
Fecal collforms 3.5 730 15
Fecal streptococci 0.1 23 14
Mycobacterla sp.
Clostrldlum perfrlngens6
- vegetative
- sporulated
Staphylococcus aureus
Salmonel la sp.
Shlgel la sp.9
Yerslnla sp.g
Campy lobacter jejunl
Candida alblcans
Fluorescent Pseudomonas sp.n
Klebslel la sp.
Viruses (pfu/mL)
Bacterlophage
Enterovl ruses
HeLa, 5 day (uncorrected) O.004 0.020 0.002
HeLa, polio-neutralized O.004 0.008 O.004
RD, polio-neutralized <0.004 0.012 O.004
Pollovlrus concentration 1 1 76
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon 28 1 19
Total suspended solids 50 1 16
Total volatile suspended solids 42 1 12
pH 9.0 7.9 8.4
1983
Mar Mar Apr
7-8 21-22 4-5
1 1,2 1
C G C
100 2, 100 2, 100
4 100 100
2 19 29
0.60
<1 .0
-
<0.01
<0.01
10
400
13
O.004 O.004 O.004
f <0.004 O.004
0.004 0.004 O.004
83 50 45
28 23 26
34 31 30
25 18 17
8.5 8.5 8.4
Apr
18-19
1.2
G
•
20,000
440
30,000
<5.0f
<5.0f
-
<0.01
0.01
50
400
65
0.004
O.004
0.004
'"94
34
43
32
8.6
continued...
-------
TABLE P-2. (CONT'D)
Source reservoir
Sample type3
Bacteria (cfu/mL)
Standard plate count
Total collforms
Fecal collforms
Fecal streptococci
Sampl Ing date
1983
Jun Jul Jut Aug
27-28 11-12 25-26 8-9
1 1 1,2 1,3
C C G G
300 150 3.0 110
10 2.0 1.9 1.8
Aug
22-23
1
G
30
4.0
Sep
12-13
1
C
15
0.9
Mycobacterla sp.
Clostrldlum perfrlngens9
- vegetative
- sporulated
Staphylococcus aureus
Salmonella sp.
Shlgel la sp.g
Yerslnla sp.9
Campylobacter jejunl
Candida alblcans
Fluorescent Pseudomonas sp.h
Klebslella sp.
Viruses (pfu/mL)
Bacterlophage
Enteroviruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, po 11 o-neutra 11 zed
Po11ovIrus concentratI on
efficiency (%)
Physical Analyses (mg/L)
0.004
<0.004
O.004
57
O.004
I
O.004
41
I
O.004
I
0.008
42
O.004
I
O.004
25
O.004
I
O.004
24
O.004
I
O.004
70
Total organic carbon
Total suspended sol Ids
Total volatile suspended solids
pH
17
11
6
8.2
21
13
8
8.2
24
18
17
8.9
27
23
17
8.2
33
54
46
9.8
23
54
33
9.5
a G -Composite of grab samples from source reservoir; C - 24-hour composite of source reservoir.
b Most probable number (MPN)/mL.
c Beginning with samples collected on June 29-30, Fluorescent Pseudomonas sp. was substituted for Staphylococcus aureus as part
of the limited bacterial screen.
d HeLa cells used for the assay were contaminated; results could not be obtained.
e Membrane filtration technique.
f Fungal contamination at lower dilutions.
g Enrichment procedure.
h Assayed on Cetrlmlde agar.
I Analysis not performed.
+ Presence of Salmonella (>1 colony/100 mL)
- Salmonella not detected (<1 colony/100 mL)
-------
TABLE P-3. MICROORGANISM CONCENTRATIONS IN WILSON WASTEWATER
24-Hour composite
samples analyzed
Bacteria (cfu/mL)
Standard plate count
Total collforms
Fecal collforms
Fecal streptococci
Mycobacterla sp.
Clostrldlum perfrlngens3
- vegetative
- sporulated
Staphylococcus aureus
Salmonel la sp.
Shlgel la sp.
Yerslnla enterocol Itlca
Campy lobacter jejunl
Candida alblcans
Fluorescent Pseudomonas sp.
Klebsfel la sp.
£ Viruses (pfu/mL)
Bacterlophage
Enterovl ruses
HeLa, 5 day (uncorrected)
HeLa, pol lo-neutral Ized
RD, polio-neutralized
Pollovlrus concentration
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
pH
Jun
3-4
1,600,000
270,000
100,000
6,800
1,400
11,000
1,500
33
O.004
O.004
O.002
8,300
100,000
410
0.047
56
87
68
39
6.5
1980
Jul
28-29
3,300,000
160,000
30,000
2,300
1,900
24,000
240
<3.3
O.002
0.002
O.004
1,500
70,000
3,300
15
47
64
45
29
6.6
Sampl
Jan
19-20
390,000 52,
64,000 15,
3,100
O.0009
55
90
64
54
7.0
Ing date
Feb
16-17
000
000
0.22
69
159
97
77
7.3
1981
Mar
9-10
98,000
44,000
0.002
46
96
73
58
7.0
Mar Apr
23-24 20-21
.
98,000 :
19,000 80,000
0.001 0.003
<0.001
0.002
42 ,,76
87 200
70 151
51 89
7.2 7.7
continued...
-------
TABLE P-3. (CONT'D)
Samp I Ing date
24-Hour composite
samp Ies analyzed
May
4-5
May
18-19
Jun
1-2
1981
Jun
15-16
Jun
29-30
Jul
20-21
Aug
17-18
to
Bacteria (cfu/mL)
Standard plate count
Total collforms
Fecal collforms
Fecal streptococci
Mycobacterla sp.
Clostrldlum perfrlngens3
- vegetative
- sporulated
Staphylococcus aureus
Salmons I la sp.
S hlgella sp.
Yerslnla enterocolItlca
Campylobacter jejunl
Candida alb I cans
Fluorescent Pseudomonas sp.
Klebslella sp.
Viruses (pfu/mL)
Bacterlophage
Enteroviruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralI zed
Pollovlrus concentration
efficiency (%)
Physical Analyses (mg/L)
41,000
0.025
O .001
1.5
32
66,000
0.17
0.004
0.14
110,000
110,000 36,000 54,000 53,000
>D.01d
O.007
0.007
56,000
0.078
0.0015
b
74
<0.001
O.014
0.075
55
0.99
0.008
0.058
42
0.006
0.002
0.053
53
O. 1
O.008
O.008
0.1
<3
20,000
0.013
0.001
1.5
,-63
Total
Total
Total
pH
organic carbon
suspended
vo 1 at 1 1 e
solids
suspended sol Ids
92
75
60
7.8
108
80
59
6.
5
57
44
36
6.4
56
30
26
6.5
97
26
22
7.6
101
57
42
7.3
80
30
23
6.
9
contInued.
-------
TABLE P-3. (CONT'D)
24-Hour composite
samples analyzed
Samp I Ing date
1981
Sep
14-15
1982
Nov
17-18
Feb
15-16
Mar
1-2
Mar
8-9
Mar
22-23
Apr
5-6
Bacteria (cfu/mL)
Standard plate count
Total collforms
Fecal collforms 8,700
Fecal streptococci
Mycobacterla sp.
Clostrldlum perfrlngens3
- vegetative
- sporulated
Staphylococcus aureus
Salmonella sp.
S hlgel la sp.
Yerslnla enterocolIt lea
Campylobacter jejunl
Candida alblcans
Fluorescent Pseudomonas sp.
Klebslella sp. 7,500
Viruses (pfu/mL)
Bacterlophage
Enterovlruses
44,000 17,000 130,000 140,000 81,000
<3 <3 10,000 <3
£0.006 il 0.01 0.01
O.006 O.005 O.01 O.01
<0.006 O.005 O.01 O.01
<3 <3 <3 <3
<3 <3 <3 <3
130,000 50,000 100,000
110,000
<3
0.01
O.01
O.01
<3
<3
1,000
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Po 1 1 ov 1 rus concentrat 1 on
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
pH
0.001
O.001
1.0
50
75
57
7.4
0.06
O.002
0.15
96
72
60
50
7.4
O.0007
O.001
O.003
233
102
82
73
7.5
O.0008
e
87
92
98
74
7.2
0.12
0.012
74
103
82
76
7.2
0.11
O.002
86
87
70
67
7.3
1.5
0.085
b
,- 77
89
72
59
7.7
continued..
-------
TABLE P-3. (CONT'D)
Samp I Ing date
1982
24-Hour composite
samples analyzed
Apr
19-20
May
3-4
May
17-18
Jun
14-15
Jun
29-30
Jul
19-20
Bacteria (cfu/tnL)
Standard plate count
Total col I forms
Fecal collforms
Fecal streptococci
Mycobacteria sp.
Clostrldium perfrlngens3
- vegetative
- sporulated
Stap hyl ococcus aureus
Sal mono I la sp.
S hlgella sp.
Yersinia enterocolItica
Campylobacter jejuni
Candida albicans
Fluorescent Pseudomonas sp.
Klebslella sp.
Viruses (pfu/mL)
Bacteriophage
Enteroviruses
270,000
37,000
140,000
150,000
8,200
85,000
6,500
120,000
1,300
20.01
<0.01
O.01
11,000f
16,000
1,500
20.01
O.01
<3
<3
9,300
35,000
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Po 1 1 ov 1 rus concentrat I on
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
PH
0.27
0.003
0.0045
58
92
74
65
7.6
0.70
0.008
0.008
72
68
89
69
7.5
0.0076
O.002
O.003
72
81
60
50
7.5
O.002
O.002
<0.002
61
75
67
56
7.2
0.034
0.036
0.036
58
69
70
61
7.0
0.44
0.004
0.004
170
76
44
41
7.5
continued.
-------
TABLE P-3. (CONT'D)
24-Hour composite
samples analyzed
Samp I Ing date
1982
Aug Aug Sep Sep Oct
9-10 30-31 13-14 27-28 11-12
o\
Bacteria (cfu/mL)
Standard plate count
Total collforms
Fecal collforms
Fecal streptococci
Mycobacterla sp.
Clostrldlum perfringens3
- vegetative
- sporulated
Staphylococcus aureus
Salmonella sp.
S hlgel la sp.
Yerslnla enterocolItlca
Yerslnla Intermedia
Campylobacter jejunl
Candida alblcans
Fluorescent Pseudomonas sp.
Klebslella sp.
Viruses (pfu/mL)
Bacter I op hag e
EnterovI ruses
HeLa, 5 day (uncorrected)
HeLa, poI Io-neutra11 zed
RD, polio-neutralized
Pollovlrus concentration
efficiency (%)
Physical Analyses (mg/L)
130,000 120,000 81,000
<0.01
<0.01
O.01
O.01
^0.01
O.01
O.01
<3
<3
9,700
36,000
<3
<3
11,000
26,000
<300
9,500
30,000
0.058
0.012
0.007
117
g
9
0.016
47
0.61
0.85
0.013
33
18,000 51,000
20.1
0.1
O.01
0.1
30,000
350,000
O.01
O.01
O.01
£1,000
750
40,000
0.043
0.045
0.036
0.008
O.002
0.052
92 ,
Total
Total
Total
pH
organic carbon
suspended sol Ids
volatile suspended solids
83
59
49
7.5
93
66
55
7.3
81
54
48
7.5
81
123
70
7.5
89
27
25
7.6
continued.
-------
TABLE P-3. (CONT'D)
ON
ON
24-Hour composite
samples analyzed
Bacteria (cfu/mL)
Standard plate count
Total collforms
Fecal collforms
Fecal streptococci
Mycobacterla sp.
Clostridlum perfrlngens
- vegetative
- sporulated
Staphylococcus aureus
Salmonel la sp.
S hlgel la sp.
Yerslnla enter ocol It ica
Campy lobacter jejunl
Candida alblcans
Fluorescent Pseudomonas sp.
Klebslel la sp.
Viruses (pfu/mL)
Bacterlophage
Enterovl ruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Pollovlrus concentration
efficiency (%)
Physical Analyses (mg/L)
Total organic carbon
Total suspended sol Ids
Total volatile suspended solids
pH
1982
Nov
1-2
670,000
130,000
12,000
-
0.012
O.004
<0.004
j
80
51
48
7.5
Dec
13-14h
710,000
110,000
1,600
-
0.096
0.056
0.020
J
j
J
7.6
Feb
16-171
220,000
14,000
9,000
-
0.004b
j
j
12
84
40
33
7.6
Sampl Ing
1983
Mar
7-8
date
Mar
21-22
750,000 430,000
150,000
100,000
-
0.004
O.004
0.004
86
119
153
130
7.8
76,000
2,800
+
0.160
0.049
O.004
59
88
118
99
7.5
Apr
4-5
710,000
150,000
25,000
-
0.028
0.040
O.004
89
205
721
504
7.4
Apr
18-19
730,000 ,
130,000
5,000
-
0.190
0.031
0.044
24
84
185
148
7.6
May
16-17
440,000
350,000
10,000
-
0.096
0.004
0.004
89
95
167
132
7.4
continued..
-------
TABLE P-3. (CONT'D)
Sampl Ing date
24-Hour composite
samples analyzed
Jun
27-28
Jul
11-12
Jul
25-26
1983
Aug
8-9
Aug
22-23
Sep
12-13
Sep
27-28
Bacteria (cfu/mL)
Standard plate count
Total colIforms
Fecal colIforms
Fecal streptococci
Mycobacterla sp.
Clostrldlum perfrlngens
- vegetative
- sporulated
Staphylococcus aureus
Salmonella sp.
S hlgel la sp.
Yerslnla enterocolIt lea
Campylobacter jejunl
Candida alblcans
Fluorescent Pseudcxnonas sp.
Klebslella sp.
Viruses (pfu/mL)
Bacter lophage
Enterovlruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Pollovlrus concentration
efficiency (jt)
Physical Analyses (mg/L)
260,000 370,000 240,000 310,000 230,000 530,000 260,000
34,000 250 5,100 7,000 9,000 12,000 20,000
3.8
5.0
<0.004
62
0.40
0.52
0.008
42
0.44
0.15
0.004
44
0.29
0.15
0.016
36
0.15
0.26
0.10
78
0.30
0.028
O.004
65
0.032
0.11
O.004
43
Total organic carbon 71
Total suspended sol ids 170
Total volatile suspended solids 126
£H 7.6
78
95
75
7.5
71
126
100
7.5
67 84
123 186
94 130
7.8 7.6
41 82
26 139
22 '" 101
7.1 7.5
a Most probable number 1 colony/100 mL)
- Salmonella not detected (<1 colony/100 mL)
-------
TABLE P-4. BACTERIAL SCREENS4—WILSON, TEXAS
Sampling date
Organism
Jnn 3-4. 1980
Jul 28-19. 1980
ENTEROBACTERIACEAE (103 cfu/mL)
Citrobacter diversus
Citrobacter frenndii
Citrobacter sp., other
Enterobacter agglomerans
Enterobacter cloacae
Enterobacter sakazakii
Escherichia coli
Hafnia alvei
Klebsiella oxytoca
Klebsiella ozaenae
Klebsiella pnenmoniae
Serratia liqnefaciens
Serratia rnbidaea
Yersinia enterocolitica
NON-ENTEROBACTERIACEAE (103 cfn/mL)
5
30
5
20
30
5
40
5
55
5
5
10
5
5
10
30
30
90
10
10
Achromobacter sp.
Achromobacter zylosoxidans
Aeromonas hydrophila
Alcaligenes sp.
CDC Group II K-2
Eikenella corrodens
Morgenella morgani
Pastenrella mnltocida
Psendomonas cepacia
Psendomonas floorescens
Psendomonas pntida
Psendomonas pntrefaciens
Psendomonas sp., other
5
-
150
5
5
20
5
5
15
15
15
25
45
—
20
120
20
-
—
-
10
-
-
50
—
500
Highest levels observed on either HacConkey agar or brilliant green agar
and identified by API 20E biochemical tests.
568
-------
TABLE P-5. VIRUSES ISOLATED FROM LUBBOCK EFFLUENT DURING BASELINE YEARS3
ON
VO
Assay
HeLa (unaltered concentrate)
Concentration (pfu/L)
Virus type
Polio 1
Polio 2
Polio 3
Coxsackle At
Coxsackle A7
Coxsackle A16
Coxsackle B1
Coxsackle B3
Coxsackle B4
Coxsackle 85
Echo 1
Echo 3
Echo 6
Echo 11
Echo 14
Echo 21
Echo 24
Echo 25
Echo 30
Unidentified
TOTAL SAMPLED
HeLa (polio-neutralized)
Concentration (pfu/L)
Virus Type
Polio 2
Coxsackle B3
Coxsackle 85
Echo 14
Unidentified
TOTAL SAMPLED
RD (polio-neutralized)
Concentration (pfu/L)
Virus type
Coxsackle A16
Coxsackle B4
Echo 5
Echo 7
Echo 11
Echo 12
Echo 13
Echo 15
Echo 19
Echo 20
Echo 24
Echo 27
Echo 31
Unidentified
TOTAL SAMPLED
Jun 3-4
780
2
20
3
19
1
1
1
4
2
1
1
21
81
Sampl Ing Date
1980
Jul 28-29 Nov 3-4 Apr 20-21
1.200 730 57
1
16
7
14 16
2 4
1
2
18 20 25
300 18
19
4
2
19 6
8
2
2
4
198
Jun 15-16
100 b
1
1
3
4
1
25
1
1
1
4
42
65"
150
1
1
3
1
5
11
1
Jul 20-21
65
6
11
4
21
20
11
11
93
1
6
4
1
3
1
2
18
Aug 17-18
45
3
4
4
1
1
13
5.3
1
1
1
1
4
420
5
2
1
1
1
1
1
1
3
16
a Plaque forming units on cell mono I avers, .
6 Labeling error precluded separating neutral I zed/unaltered viruses.
-------
TABLE P-6. VIRUSES ISOLATED FROM WILSON EFFLUENT DURING BASELINE YEARS*
Sampling date
Assay
1980
1981
Jun
3-4
Jul
28-29
Jun
15-16
Aug
17-18
HeLa (ualtered'eoaceatrate)
Concentration (pfu/L)
Virus type
Polio 1
Polio 2
Polio 3
Cozsackie A10
Coxsackie B3
Echo 2
Echo 25
Unidentified
TOTAL SAMPLED
HeLa (polio-neatxalized)
Concentration (pfu/L)
Virus type
KD (polio-memtxalixed)
47
2
16
1
1
5
25
15,000
12
15
13
_2
12
1.0
Concentration (pfu/L)
Virus type
Polio 2
Cozsackie A9
Echo 5
Echo 31
Unidentified
TOTAL SAMPLED
75
1
5
1
_1
9
1500
1
6
7
a Plaque forming units on cell monolayers.
570
-------
TABLE P-7. VIRUSES ISOLATED FROM WILSON EFFLDENT DURING 1982
Sampling date
Mar
Assay 8-9
HeLa (unaltered. concentrate)
Concentration (pfu/L) 120
Virus type
Polio 1 1
Polio 2 10
Polio 3 8
Cozsackie B5
Echo 11
Echo 24
Unidentified
TOTAL SAMPLED 19
HeLa (polio-neutralized)
Concentration (pfu/L) <2
Virus type
Polio 2
Cozsackie B4
Cozsackie B5
Echo 11
Unidentified
TOTAL SAMPLED
KD (polio-neutralized)
Concentration (pfn/L) 12
Virus type
Echo 13
Unidentified
TOTAL SAMPLED
Apr Jun
5-6 29-30
"
1500 34
1 1
23
1
10
1
25 12
85 36
4
2
10
1 _1
5 13
a 36
3
_3
6
Ang
9-10
58
8
3
6
3
1
2
1
24
12
2
1
_ - -
3
6.6
LA
1
Sep
13-14
610
1
20
4
25
850
14
, _, -
14
13
a Tozic sample.
571
-------
TABLE P-8. VIRUSES ISOLATED FROM WILSON INFLUENT DURING 1983
Assay
HeLa (unaltered concentrate)
Concentration (pfu/L)
Virus type
Polio 1
Polio 2
Polio 3
Coxsackle A13
Coxsackle B2
Coxsackle B5
Echo 7
Echo 25
Echo 26
Echo 27
Echo 29
Unidentified
TOTAL SAMPLED
HeLa (neutralized)
Concentration (pfu/L)
Virus Type
Coxsackle 82
Coxsackle B3
Coxsackle B5
Unidentified
TOTAL SAMPLED
Feb Mar
16-17 21-22
4 160
5
8
1
1
1
1 3
1 19
<6 49
2
9
11
Apr
18-19
190
5
6
1
2
3
1
18
31
2
4
1
7
Sampl Ing Date
1983
May Jul
16-17 11-12
96 400
9
1
6
1 27
3
20 27
4 520
1
1
Aug
8-9
290
6
5
1
1
7
1
1
22
150
16
16
Sep Sep
12-13 26-27
300 ' 32 ,
24
1
1 8
26 8
28 110
6 12
1
7 12
-------
TABLE P-S. GEOMETRIC MEAN OF MICROORGANISM CONCENTRATIONS IN WILSON WASTEWATER
Ul
-»j
to
Sampling period
Number of samples
Bseterla (cfu/mL)
Standard plate count
Total co 11 forms
Fecal co 11 forme
Fecal streptococci
Virus** [pfu/mL]
Bacterlophage
Enterovlrusee
HeLa, 5-day (unoorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Physical Analyses [mg/L]
Total organic carbon
Total suspended solids
Total volatile suspended solids
PH
May/Jun 80
6-3
1
1,600,000
270,000
100,000
6,800
410
0.047
87
68
39
6.5
Summer 80
7-88
1
3,300,000
160,000
30,000
8,300
3,300
15
64
45
29
6.6
Fall-Kin 80 Spring 81
1-19 2-16/4-80
1 4
390,000 79,000
64,000 38,000
3,100
<0.0009 0.006
<0.001
0.002
90 128
64 93
54 67
7.0 7.3
May/Jun 81
5-V6H5
4
76,000
0.068
0.001
0.25
75
53
43
6.8
Summer 81
6-29/9^14
4
1
31 ,000
0.017
0.003
0.26
92
43
33
7i.3
Fall-Win 81
11-17/2-15
2
87,000
0.03
<0.002
0.08
86
70
60
7.4
continued...
-------
TABLE P-9. (CONT'D)
Sampling period
Number of samples
Spring 62
3-1/4-19
5
Hay/Jun 82
5-3/7-19
5
Summer 82
8-9/9-13
3
Fall-Win 82
9-27/12-13
4
Spring 83
2-16/4-18
5
Hay/Jun 83
5-16/6-27
2
Summer 83
7-11/9-27
6
Bacteria (cfu/mL)
Standard plate count
Total conforms
Fecal collforms
Fecal streptococci
Vi
(pfu/mL)
130,000
Bacteriophage
En terovi ruses
HeLa, 5-day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Physical Analyse* (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
pH
95,000
7,300B
1,4QOa
110,000
690,000
60,000
4,400°
520,000
79,000
13,000
440,000
300,000
18,000
310,000
5,200
0.40
0.016
0.007
92
79
68
7.4
0.094
0.010
0.005
74
64
55
7.3
0.188
0.10
0.011
86
60
51
7.4
0.025
0.025
0.010
83
55
44
7.5
0.027
0.030
0.011
109
157
126
7.6
0.60
0.14
<0.004
82
168
129
7.5
0.20
0.15
0.021
69
100
77
7.5
a Based on two samples.
-------
TABLE P-10. WASTEWATER SAMPLES COLLECTED DURING 1982 AEROSOL MONITORING (30 MINUTE COMPOSITES)
WASTEWATER FROM PIPELINE DURING SPRING IRRIGATION PERIOD
Ul
Sampling date/aerosol run
Parameter
Bacteria (cfu/mL)
Fecal collforms
Fecal streptococci
Clostrldlum perfrlngens3
Vegetative
Sporulated
Mycobacterla sp.
Viruses (pfu/mL)
Bacter lop hag e
Enterovl ruses (uncorrected)
HeLa, 5 day
HeLa, polio-neutralized
RD, polio-neutralized
Pollovlrus concentration
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended
solids
Sample conditions
PH
Temperature (°C)
Feb 22
Ml
100,000
4,400
16,000
1,200
0.054
0.015
0.051
57
135
147
121
6.9
2
Feb 23
M2
1,000,000
7,200
360
360
18,000
1,500
0.093
0.024
0.012
49
161
182
152
6.8
3
Feb 24
M3
110,000
6,300
45,000
1,400
0.047
76
168
217
176
7.0
8
Mar 15
01
51,000
4,800
13,000
1,100
0.067
0.0084
0.034
60
92
87
74
7.2
9
Mar 16
VI
81,000
4,500
20,000
840
0.16
0.035
0.030
100
101
90
7.4
9
Mar 17
M4
39,000
1,900
29,000
530
0.11
0.022
0.067
64
158
245
207
7.0
4
Mar 18
M5
•
57,000
5,800
13,000
1,100
0.12
0.047
49
128
92
90
7.1
2
Mar
M6
68,000
16,000
15,000
940
0.
0.
63
164
185
160
'""
7
5
19
,028
,0023
.1
a Membrane filtration procedure used to enumerate C. perfringens In aerosol-related samples.
-------
TABLE P-11. WASTEWATER SAMPLES COLLECTED DURING 1982 AEROSOL MONITORING (30 MINUTE COMPOSITES)
WASTEWATER FROM PIPELINE DURING SUMMER IRRIGATION PERIOD
Parameter
Bacteria
Fecal col
(cfu/mL)
1 forms
Fecal streptococci
Jul 7
M7a
44,000
4,200
Jul 8
MS
31,000
3,200
Sampl Ing
Jul 13
02
50,000
3,600
date/aerosol
13
4
Jul 14
Mil
,000
,600
run
76
5
Jul 15
M12
,000
,600
Aug 2
V2
180,000
2,000
Aug
M14
.
1
37,000
4,900
3
Vegetative
Sporulated
Mycobacterla sp.
Viruses (pfu/mL)
Bacterlophage
Enterovlruses (uncorrected)
100,000
1,700
550,000
930
25,000
720
11,000
16
10,000
1,100
4,000
880
5,300
1,900
HeLa, 5 day
HeLa, polio-neutralized
RD, po 1 1 o-neutra 1 1 zed
Po 1 1 ov 1 rus concentrat I on
efficiency (%)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended
solids
Sample conditions
pH
Temperature ( °C )
0.54
0.47
0.17
64
128
307
213
7.0
1
0.51
0.55
0.26
57
92
213
161
7.3
10
0.14
0.067
0.020
39
76
82
66
7.4
3
0.013
0.002
0.016
50
51
67
54
7.6
3
0.078
0.097
0.018
49
80
170
119
7.6
5
0.10
0.10
0.004
80
52
79
62
7.4
2
1.5
0.10
0.011
214
71
86
68
,-,
7.5
2
continued...
-------
TABLE P-11. (CONT'D)
Sampling date/aerosol run
Parameter
Bacteria (cfu/mL)
Fecal col (forms
Fecal streptococci
Clostridlum pert r ingens"
Vegetative
S populated
Mycobacterla sp.
Aug 4
V3
5,600
2,600
2,300
Aug 5
M15
30,000
2,700
6,000
Aug 23
M17C
16,000
300
460
200
Aug 24
V4C
93
<5.0
5.0
Aug 25
M18
29,000
830
360
190
Aug 27
M20C
360
10
93
230
Viruses (pfu/mL)
Bacterlophage
Enterovi ruses (uncorrected)
HeLa, 5 day
HeLa, polio-neutralized
RD, polio-neutral Ized
Pollovirus concentration
efficiency (%)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended
solids
Sample conditions
pH
Temperature (°C)
1,600
2.2
0.060
0.020
350
66
93
75
7.5
3
1,200
0.21
0.080
0.022
94
65
69
57
7.8
2
820
0.39
0.34
0.34
58
62
49
7.2
2
150
0.066
0.051
0.008
85
61
58
47
7.3
3
2,100
0.10
0.11
0.28
46
48
36
7.4
3
140
0.044
0.13
0.28
63
49
41
r->
7.3
3
a Presumed pipeline source based on mlcroblal parameters.
b Membrane filtration procedure used to enumerate C. perfrlngens In aerosol-related samples.
c Chlorinated.
-------
TABLE P-12. WASTEWATER SAMPLES COLLECTED DURING 1982 AEROSOL MONITORING (30 MINUTE COMPOSITE)
WASTEWATER FROM RESERVOIR DURING SUMMER CROP IRRIGATION
-a
oo
Parameter
Bacteria (cfu/mL)
Fecal collforms
Fecal streptococci
Jul 9
M9
230
30
Sampl
Jul 11
M10
40
13
Ing date/aerosol run
Jul 16 Aug 6
M13 M16
1, 100 450
53 3.0
Aug 26
M19
750
3.0
Clostrldlum perfrlngens3
Vegetative
Sporulated
Mycobacterla sp.
Viruses (pfu/mL)
430
100
230
10
3.0
Bacterlophage
Enterovi ruses (uncorrected)
HeLa, 5 day
HeLa, polio-neutralized
RD, polio-neutralized
Pollovlrus concentration
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended
solids
Sample conditions
pH
Temperature (°C)
1.2
0.034
0.002
<0.002
61
19
26
26
8.2
5
0.40
0.002
O.002
O.002
71
16
27
24
8.0
1
15
0.004
0.013
0.002
52
16
21
19
7.8
8
2.4
0.12
0.008
0.002
108
43
35
35
8.5
2
5.3
8.7
0.006
O.002
17
12
12
'•>
7.9
2
a Membrane filtration procedure used to enumerate C. perfrlngens on aerosol-related samples.
-------
TABLE P-13. SOURCE STRENGTH OF RHODAMINE IN WASTEWATER DURING DYE RUNS
Dye
Run
Dl
D2
D3
D4
Rhodamine concentration in wastewater
Min 0 Min- 1
96 126
53 94
112 119
95 111
TABLE P-14.
Min 2 Min 3 Min 4
183 95 10
93 91 91
118 108 110
109 112 115
Min 5 ,
12
91
102
113
RHODAMINE AEROSOL CONCENTRATION
Rhodamine concentration
Dye
run
Dl
D2
D3
D4
Tower
3
5
6
4
6
4
5
3
Near pairs
(Dist) L
(31 m) 22
(40 m) 1.1
(25 m) 80
(25 m) 1.9
(25 m) 2.3
(25 m) 3.7
(40 m) 3.7
(40 m) 2.5
sample, mg/L
Min 6
25
87
99
105
DURING
Min 7
88
6.7
DYE RUNS
Min 8
9.0
in air, 10~6 yg/rn^
Far pairs
R
4.5
0.89
0.46
7.5
9.7
0.47
6.3
2.4
(Dist)
(81 m)
(115 m)
(75 m)
(75 m)
(75 m)
(75 m)
(80 m)
(80 m)
L
0.38
1.1
0.67
2.3
0.71
1.9
1.3
1.0
R
1.5
0.96
0.87
1.3
0.50
0.79
2.4
1.8
579
-------
TABLE P-15. SAMPLED STANDARD PLATE COUNT IN AIR BY PARTICLE SIZE
8
Run no. Andersen Range of
Run date sampler particle
Run time stage sizes (y)
PI
2-23-82
1609-1619
P2b
3-16-82
1539-1549
P3
7-8-82
1510-1518
P4
7-14-82
1519-1527
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
>7.0
4.7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
>7.0
4.7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
>7.0
4.7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
>7.0
4.~7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
Standard plate count concentration in air by particle size, cfu/m3
Upwi nd
L
81
47
68
72
60
TNTC
260
60
22
78
95
22
16
27
38
38
11
32
110
5
5
<1
5
16
R
170
65
86
65
60
TNTC
94
64
64
47
210
43
370
330
400
180
290
37
_c
<1
37
>540
37
11
L
36
260
130
240
300
180
82
33
1700
1200
1300
390
190
29
20
1080
1200
340
120
95
5
35
1200
660
TNTC
290
46
10
Downwind
R
m
240
190
540
140
140
100
m
2300
2500
1500
650
100
20
m
TNTC
1300
650
87
130
<1
m
520
550
550
150
45
110
of irrigation nozzle line
L
61
200
140
280
TNTCa
220
70
58
210
210
960
130
43
TNTC
45
TNTC
380
240
68
15
10
60
410
390
300
83
15
29
R
m
280
150
70
280
250
90
m
1700
540
1200
920
180
78
m
1500
590
200
87
110
<1
m
390
690
370
150
31
CS
L
75
290 •
110
240
170
TNTC
74
83
290
920
450
190
120
TNTC
70
350
180
59
74
53
48
85
280
64
160
43
37
64
R
m
,140
150
180
170
170
TNTC
m
110
140
180
120
TNTC
TNTC
m
528
169
- 77
67
56
10
m
640
180
360
72
82
5
continued.
-------
TABLE P-15. (CONT'D)
00
Run no.
Run date
Run time
P5
8-25-82
1730-1738
Andersen Range of
sampler
stage
1
2
3
4
5
6
particle
sizes (p)
>7.0
4.7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
Standard plate
count concentration in air by particle size, cfu/m3
Upwind
L
1000
160
TNTC
150
100
59
R
410
540
11
150
11
160
L
35
TNTC
TNTC
1420
880
310
26
Downwind
R
m
TNTC
TNTC
TNTC
490
150
5
of irrigation nozzle line
L
60
600
400
640
190
130
180
R
m
880
1000
250
160
87
26
L
85 m
640 •
520
370
140
140
43
R
490
630
270
250
110
31
CS - fungal contamination
a TNTC - either too numerous to count (>2500 cfu/m3 for PI to P3; >1500 cfu/m3 for P4 and P5) or
fungal contamination. For data summary, it was assumed that TNTC = 3000 cfu/m3 for PI to P3 and
TNTC = 2000 for P4 and P5 when values from paired sampler and/or adjoining stages were large.
When these neighboring values were low, presumed fungal contamination TNTC was assumed to equal
the value of the same stage for the paired sampler or the average of the adjoining stages.
b Standard plate count of wastewater = 5.1 x 10^ cfu/mL
c Sample lost.
-------
TABLE P-16. MICROORGANISM DENSITIES IN AIR ON BACKGROUND AIR RUNS9
Back-
ground
run
no.
Sampler
Wilson 'Wilson Wilson
ABC
Effluent
pond
D
location^ <.
Rural
E
Rural
F
Rural
G
Rural
H
Rural
I
Standard Plate Count (cfu/m3)
Bl
82
83
84
Fecal
Bl
B2
83
84
Fecal
Bl
B2
83
84
1150 260
530 680 CS
1050 CS 500
CS 430 630
Col i forms (cfu/m3)
<0.4 <0.1
<0.1 <0.1 <0.4
<0.1 <0.2 <0.1
<0.1 <0.1 <0.2
Streptococci (cfu/m3)
0.5 8.0
0.9 0.3 2.1
0.7 <0.1 0.3
11 0.6 0.3
1900
430
370
73
<0.
<0.
<0.
<0.
<0.
0.
0.
0.
2
1
3
1
1
3
2
3
2800
1220
280
65
<0.
<0.
<0.
<0.
<0.
0.
0.
0.
3
2
1
1
2
2
2
3
CS
990
1030
130
<0.1
<0.3
0.3
<0.1
1.1
1.3
2.3
1.5
390
CS
_
60
<0.
<0.
-
<0.
0.
0.
.
0.
1
2
1
1
3
2
190
3500
200
CS
<0.
<0.
<0.
<0.
<0.
0.
<0.
0.
2
2
I
2
1
3
1
8
CS
450
260
500
<0.4
<0.1
<0.4
<0.1
0.3
0.1
2.4
0.2
Mycobacteria (cfu/m3)
Bl
82
83
84
<0.2 0.1
<0.1 0.1 <0.3
<0.1 <0.1 0.1
<0.1 <0.1 <0.1
<0.
<0.
<0.
3.
1
1
2
4c
<0.
0.
<0.
<0.
2
1
1
1
<0.1
<0.2
<0.1
<0.1
0.
<0.
_
0.
1
1
1
0.
<0.
<0.
<0.
1
1
I
1
0.3
<0.1
0.5
<0.1
Coliphage (pfu/m3)
Bl
B2
B3
B4
<0.4 <0.1
<0.2 <0.1 <0.4
<0.1 <0.2 <0.1
<0.1 <0.1 <0.2
<0.
<0.
<0.
<0.
2
1
3
2
<0.
<0.
<0.
<0.
3
2
1
1
<0.2
<0.4
<0.1
<0.1
<0.
<0.
-
<0.
1
2
1
<0.
<0.
<0.
<0.
2
2
1
2
<0.4
<0.1
<0.4
<0.1
- - No sample collected.
CS - Contaminated sampler (presumed).
a Conducted August 5-8, 1980.
b Sampler locations shown in Figure 8.
c Cows grazing approximately 300 to 500 m upwind from sampling site.
582
-------
TABLE P-17. SAMPLED FECAL COL I FORM DENSITIES ON THE MICROORGANISM AEROSOL RUNS
00
Fecal
col Iform Fecal col Iform concentration in air (cfu/tn' of air)
Aerosol concentration Upwind of
run In wastewater irrigation
number (cfu/mL) rig 20-39 m 40-59 m
WASTEWATER FROM PIPELINE— SPRING 1982 IRRIGATION
Ml 100,000 O.2 O.2 >250
M2 1,000,000 O.I CS 150
M3 110,000 O.2 O.4 190
M4 39,000 O.I O.I 0.2a
M5 57,000 0.1 0.1
M6 68,000 O.2 O.I 120
WASTEWATER FROM PIPELINE— SIMMBR 1982 IRRIGATION
M7b 44,000 O.3 O.7 140
M8 31,000 0.2 0.3 900
M11 13,000 0.3 0.3
M12 76,000 O.3 CS
M14 37,000 CS O.I
M15 30,000 O.I O.I
Ml7<=»d 16,000 O.2 O.2
M18d 29,000 O.2 O.2
M20C 360 CS 0.2
WASTEWATER FROM RESERVOIR— SIMMER 1982 IRRIGATION
M9 230 O.3 O.3 <3.3
M10 40 O.3 O.3 CS
M13 1,100 CS O.4f
M16 450 O.I CS 1.2
M19 750 0.3 0.3 15 CS 5.7 0.3
CS - contaminated sample.
250 21 15
110 2.3 2.1
330 36 26
133a O.3 O.I
120 CS
49 7.7 4.3
83 14 10
137 0.3 1.2
CS
37
O.4
0.2
0.5
0.4
0.1
CS 0.4 0.3
1.2 0.1 0.3
CS
0.2 CS 0.3
0.5 0.2
nozzle line
150-249 m 250-349 m
3.2
O.I O.I
40 13
0.1 0.1
16 15 3.5 3.8
CS 0.1
3.7 3.2
O.3 O.3
0.3 0.4 0.6
70 0.1 O.3
0.2 O.I O.I
0.2 0.1 CS
2.7 3.5 CS
0.3 27e
0.1 0.1 0.3
0.3 0.3
0.1 0.3
1.5 O.3 O.3
2.2 2.0
350-409 m
;
O.2 O.3
0.6 0.2
O.2 O.2
O.I O.3
1.6 4.8
0.6 4.8
O.I O.I
O.4 O.3
-------
TABLE P-18. SAMPLED FECAL STREPTOCOCCUS DENSITIES ON THE MICROORGANISM AEROSOL RUNS
Fecal
streptococcus
Aerosol concentration Upwind of
run In wastewater Irrigation
number (cfu/mL) rig
WASTEWATER FROM PIPELINE— SPRING 1982
Ml 4,400 0. 0.1
M2 7,200 0. CS
M3 6,300 O. 0.3
M4 1,900 O. O.I
M5 5,800 0. 0.1
M6 16,000 O.2 0.2
WASTEWATER FROM PIPELINE— SUMMER 1982
M7b 4,200 0.1 0.7
M8 3,200 0.1° 2.7C
Mil 4,600 O.3 O.3
M12 5,600 O.3 CS
M14 4,900 O.I O.I
M15 2,700 1.3 1.0
M17d«e 300 O.2 O.2
M18e 830 0.2 0.2
M20d 10 CS O.2
WASTEWATER FROM RESERVOIR— SUMMER 198
M9 30 O.3 O.3
M10 13 O.3 O.3
M13 53 CS O.4
M16 3 9.6* 0.1
M19 3 O.3 O.3
CS - contaminated sample.
-------
TABLE P-19. SAMPLED MYCOBACTERIA DENSITIES ON THE MICROORGANISM AEROSOL RUNS
Mycobacterla
Aerosol concentration
run In wastewater
number (cfu/mL)
WASTEWATER
Ml
M3
M4
M5
M6
WASTEWATER
M7a
M8
Mil
M12
M14
M15
Ul
°o WASTEWATER
M9
M10
M13
M16
Mycobacteria concentration In air (cfu/itP of air)
Upwind of
Irrigation
Downwind of Irrigation nozzle
rig 20-39 m 40-59 m
60-89 m
90-149
m
150-249
line
m 250-349 m
350-409 m
FROM PIPELINE— SPRING 1982 IRRIGATION ' :
16,000
45,000
29,000
13,000
15,000
1.3 1.3 7.0
0.1 0.3
O.I O.I
CS CS
O.I O.I
3.6
7.0
20
11
0.
7.
9.
32
4
9
0
2.5 6
1.7 3
2.0 O
CS
2.0 3
.5
.0
.1
.7
6
0
4
O
5.6
.7 0.
.1 0.
.7 1.
.2 0.
1
1
5 4.1 CS
1
FROM PIPELINE— SUMMER 1982 IRRIGATION
100,000
550,000
11,000
10,000
5,300
6,000
0.1 0.3
0.1 0.2
0.1 0.1
0.2 0.2
0.1 0.7
CS 0.1
0.3
1.4
0.
0.
2
2
0.3 0
0.2 O
0.2
0.5
0.7
5.0
.3
.1
O
.3 0.
2
0.2 O.2
0.2
0.2
0.2
0.2
O.2 O.3
0.4 0.6
0.1 0.1
O.I 0.4
O.I O.I
CS 0.2
1.0 O.2
O.I O.3
FROM RESERVOIR— SUMMER 1982 IRRIGATION
430
100
230
10
0.1 0.2
0.1 0.1
0.2 0.2
O.I CS
0.2
CS
0.2
0.
CS
-------
TABLE P-20. SAMPLED CLOSTRIDIUM PERFRINGENS DENSITIES ON THE MICROORGANISM AEROSOL RUNS
Aerosol
run
number
M2-Plpel Ine
Vegetative
M17-Plpellnea'b
Vegetative
S porulated
M18-Pipelineb
Vegetative
Sporul ated
M19-Reservolr
Vegetative
Sporul ated
M20-Plpellnea
Vegetative
en Sporul ated
ff. _____________
C lostrldlum
perfr Ingens
concentration
In wastewater
(cfu/mL)
360
460
200
360
190
3
<1.0
93
230
C lostrldlum perfr ingens concentration In
Upwind of
Irrigation
rig 20-39 m
0.1 0.2 8.2
O.3 O.3
0.3 0.3
O.3 O.3
O.3 O.3
0.3 0.3 0.2 0.3
O.3 O.3 O.2 O.2
0.2 0.5
O.2 O.2
Downwind of Irrigation
air (cfu/m^ of air)
nozzle 1
40-59 m 60-89 m 90-149 m 150-249 m
9.3 2.8 1.7
9.8
1.8
1.3
0.3
O.3 O.3 O.3 O.3
0.3 0.3 0.3 0.3
0.1
0.2
1.6 1.7
6.7
0.9
4.1
5.4
0.1
0.1
Ine
250-349 m
3.9 1.4
1.6 0.8
2.7
0.3
0.1 0.3
0.1 0.3
350-409 m
0.5 2.6
1.4 0.3
2.3 3.2
0.5 0.5
O.I O.I
O.I O.I
-------
TABLE P-21. SAMPLED COLIPHAGE DENSITIES ON THE MICROORGANISM AEROSOL RUNS
Col Iphage Col Iphage
Aerosol concentration Upwind of
run In wastewater Irrigation
concentration In air
Downwind
number (pfu/mL) rig 20-39 m 40-59 m 60-89 m
WASTEWATER FROM PIPELINE— SPRING 1982 IRRIGATION
Ml 1,200 O. O.I 38
M2 1,500 0. 0.3 4.0
M3 1,400 O. O.3 5.7
M4 530 O. O.I 8.2
M5 1,100 O. O.I
M6 940 O. O.1 23
WASTEWATER FROM PIPELINE— SUMMER 1982 IRRIGATION
M7a 1,700 O.I O.3 10
M8 930 3.5b 5.7b 8.3
M11 16 CS CS
M12 1,100 0.1 0.1C
M14 1,900 O.1 O.I
M15 1,200 O.I O.I
M17d»e 820 O.5 O.I
Ml8e 2,100 0.1 0.1
M20d 140 O.I O.I
WASTEWATER FROM RESERVOIR— SUMMER 1982 IRRIGATION
M9 1.2 O.I O.I O.I
M10 0.4 O.I O.I O.I
M13 15 O.I O.2
M16 2.4 O.I O.I O.I
M19 5.3 O.I O.I 0.1 O.I 0.1 O.I
CS - contaminated sample.
-------
TABLE P-22. DISTRIBUTION OF PARTICIPANT EXPOSURE MEASURES XAEREM,
XDIREM AND FHRSEM IN 1983 IRRIGATION PERIODS
. Irrigation period
Spring .1983
Index of Ext ens ire Aerosol Exposures
Minimum
Maximum
XAEREM Levels (XAEREL)
# None (XAEREM=0)
# Low (O.liXAEREMaO)
# High (XAEREM>10)
IDHEM, Index of Ext ens ire Direct Waste-
water Contacts
Minimum
Maximum
XDIREM Levels (XDIREL)
# None (XDIREM=0)
# Low (O.lODIREMaO)
# High (XDIREMMO)
FHKSEM, Average Homxs per Week on
Hancock Far*
Minimum
Maximum
FHRSEM Levels (FHRSEL)
# None (FHRSEM=0)
# Low (0.11FHRSEM120)
# High (FHRSEM>20)
0
157.4
293(87%)
22(7%)
20(6%)
0
303.7
316(94%)
8(2%)
11(3%)
0
160.8
272(81%)
37(12%)
26(8%)
Summer 1983
0
288.0
276(88%)
21(7%)
18(6%)
0
438.5
290(92%)
10(3%)
15(5%)
0
158.8
240(76%)
49(16%)
26(8%)
TABLE P-23.
CORRELATION COEFFICIENTS r AMONG LOGARITHMICALLY
TRANSFORMED* EXPOSURE MEASURES
Season
AEI
XAEREM
XDIREM
FHRSEM
XAEREM
XDIREM
FHRSEM
TLUBOCKb
Spring 1983
Summer 1983
Spring 1983
Summer 1983
Spring 1983
Summer 1983
Spring 1983
Summer 1983
0.508
0.610
0.365
0.536
0.445
0.579
-0.058
0.005
0.767
0.901
0.807
0.755
0.139
0.067
0.593
0.630
0.058
0.024
0.167
0.162
Natural logarithm (exposure measure + detection limit/10) used to improve
the symmetry of each marginal distribution, especially for AEI.
TLUBOCK = hours per week spent in Lnbbock; weighted average of activity
diary values.
588
-------
TABLE P-24. DEMOGRAPHIC CHARACTERISTICS OF PARTICIPATING HOUSEHOLDS BASED ON RESPONSES
TO THE INITIAL (MAY 1980) AND FINAL (OCTOBER 1983) QUESTIONNAIRE
oo
VO
Household location by sampling zone
1980
1980
1983
1983
1980
1980
1983
1983
1980
1980
1983
1983
#
%
#
%
#
%
#
%
#
%
#
%
Rural
0-0.5
mile
28
17
19
18
Cauca-
sian
133
82
91
85
1
34
21
24
22
Wilson
0-0.5
mile
36
22
21
20
Race
His-
Danic
30
18
16
15
2
56
34
40
37
Education
1980
1980
1983
1983
#
%
#
%
MR
4
2
0-8
53
33
34
32
Rural
0.5-1
mile
14
9
8
7
Total
163
100
107
100
3
26
16
11
10
Wilson
0.5-1
mile
40
25
30
28
Number of
4
21
13
14
13
category of head
9-11
20
12
11
10
12
52
32
36
34
Rural
1-2+
miles
42
26
27
25
Workers
>2
miles
3
2
2
2
Total
163
100
107
100
.
'
household members
5
13
8
9
8
6
6
4
5
5
7 9
4 1
2 1
3
3
10 Total
2 163
1 100
1 107
100
of household
Some
college
(13-15)
18
11
14
13
College
grad
(16-18)
16
10
12
11
Total
163
100
107
100
continued.
-------
TABLE P-24. (CONT'D)
Ol
VO
o
1983 #
1983 %
1980 #
1980 %
1983 #
1983 %
Most
0-11
16
15
MR
1
1
educated member of
12
34
32
<5000
21
13
13
12
Some
college
(13-15)
21
20
5000-
7999
25
15
17
16
household (1983 only)
College
grad
(16-18) Total
36 107
34 100
Total household income in 1979
8000- 10000- 15000- 20000- Don't
9999 14999 19999 29999 >30000 know
14 21 22 24 31 1
9 13 13 15 19 1
9 14 12 17 23 2
8 13 11 16 21 2
•
Refused Total
3 163
2 100
107
100
Location of households
1980 #
1980 %
1983 ft
1983 %
Rural
86
53
55
52
Wilson
76
47
51
48
Classification
1980 #
1980 %
1983 #
1983 %
bv
Child
<5
97
60
69
64
presence
Child
6-17
42
26
26
24
TotaJ
162
100
106
100
r*\
of households
of children
No
chil-
dren
24
15
12
11
Total
163
100
107
100
continued...
-------
TABLE P-24. (CONT'D)
1980 #
1980 %
1983 #
1983 %
1980 #
1980 %
1983 #
1983 %
1980 #
1980 %
1983 #
1983 %
NR
3
2
NR
1
1
1
1
NR
1
1
Air
None
18
11
13
12
Source
Wilson
72
44
49
46
Sewaee
Septic
tank
91
56
59
55
conditioning system
Refrig-
eration
52
32
52
49
Evapor
cooler
46
28
42
39
Type
unknown Total
44 163
27 100
107
100
of drinking water
Canad.
river
4
2
3
3
disposal
City
system
71
44
48
45
Private
well
86
53
53
50
Total
163
100
107
100
Tot a;
163
100
107
100
-------
TABLE P-25. DEMOGRAPHIC CHARACTERISTICS OF STUDY PARTICIPANTS BASED
ON RESPONSES TO THE INITIAL8 (MAY 1980) AND FINAL
(OCTOBER 1983) QUESTIONNAIRES
Race
Caucasian Hispanic
1980 #
1980 %
1983 #
1983 %
337
70
221
72
' 145
30
85
28
Total
482*
100
306
100
f
Household location
1980 #
1980 %
1983 #
1983 %
1980 #
1980 %
1983 #
1983 %
1980 #
1980 %
1983 #
1983 %
1980 #
1980 %
1983 #
1983 %
Rural
0 to 0.5
mile
68
14
44
14
Other
5
1
1
0
NR
1
0
Male
237
49
143
47
Wilson
0 to 0.5
mile
117
24
71
23
Dwe 1 1 ins
Rural
240
50
148
48
0-5
34
7
21
7
Sex
Female
245
51
163
53
Rural
0.5 to 1
mile
47
10
27
9
location
Wilson
237
49
155
52
Aee grout)
6-17
118
24
79
26
Total
482
100
306
100
Wilson Rural Workers
0.5 to 1 1 to 2+ >2
mile miles miles
120 122 8
25 25 2
84 74 6
27 24 2
Total
482
100
306
100
(as of June 30. 1982)
18-44 45-64 65+
173 94 62
36 20 13
86 79 41
28 26 13
Total
482
100
306
100
Total
482
100
306
100
continued.. .
592
-------
TABLE P-25. (CONT'D)
1980 #
1980 %
1983 #
1983 %
1980 #
1980 %
1983 #
1983 %
1980 #
1980 %
1980 #
1980 %
1983 #
1983 %
1983 #
1983 %
Drinks
NR
3
1
2
1
Smoke s
NR
1
0
3
1
Trios
NR
7
1
NR
27
5
Tap water
NR
4
1
Contacts
NR
3
1
bottled water regularly
No Tes
416 63
86 13
248 56
81 18
Total
482
100
306
100
cigarettes regularly
No Yes
413 68
86 14
265 38
87 12
to Lubbock per month
0-5 6-10
292 81
61 17
Hours in Lubbock
0-5 6-15
358 89
74 18
consumed vs. others
Less than
Average Average
51 208
17 68
per week with 10 or
Less than
once 1-5
10 124
3 41
Chews tobacco regularly (1983
1983 #
1983 %
NR
3
1
No Yes
281 22
92 7
Total
482
100
306
100
(1980 onlv)
11+ Total
102 482
21 100
per trip (1980 only)
16-25 26-100 >100
521
100
your age (1983 only)
More than
Average Total
43 306
14 100
more people (1983 only)
More than
6-10 11-15 15
92 43 34
30 14 11
only)
Total
306
100
Total
482
100
Total
306
100
a Includes four individuals who only provided an initial blood sample.
593
-------
TABLE P-26.
vo
CROSSTABDLATION OF SELECTED HODSEHOLD VARIABLES BY
OVERALL AEROSOL EXPOSURE INDEX LEVEL
Household AEI
level for
1982 and 1983
Dropped
Low exp
Med ezp
Hi ezp
TOTAL
Household AEI
level for
1982 and 1983
Dropped
Low ezp
Med ezp
Hi ezp
TOTAL
Grouped household size
1 2-4 >5
NR person people people
9 34 7
2 2 18 4
19 32 15
4 15 2
2 34 99 28
Head of household occupation
Prof +
mgr Farmer
NR (Ior2) (9orlO) Other
3 4 17 26
4 14 8
14 11 41
2 17 2
3 24 59 77
Total
50
26
66
21 '
163
group
Total
50
26
66
21
163
Education catenorv of head of household
Household AEI
level for
1982 and 1983
Dropped
Low ezp
Med ezp
Hi ezp
TOTAL
NR 0-8 9-11 12
4 16 9 15
829
25 9 16
4 12
4 53 20 52
Some College
college grad
(13-15) (16-18) Total
3 3 50
4 3 26
9 7 66
2 3 21
18 16 163
continued.
-------
TABLE P-26. (CONT'D)
Household AEI
level for
1982 and 1983
Dropped
Low exp
Hed exp
Hi exp
TOTAL
Household AEI
level for
1982 and 1983
Dropped
Low exp
Hed exp
Hi exp
o. TOTAL
Total household income in 1979
NR <5000
1 7
1
12
1
1 21
Air
NR None
3 4
3
7
4
3 18
5000-
7999
8
3
10
4
25
8000-
9999
4
3
5
2
14
10000-
14999
5
6
7
3
21
15000-
19999
10
1
8
3
22
20000- Don't
29999 >30000 know
581
4 8
11 11
4 4
24 31 1
Refused Total
1 50
26
2 66
21
{
3 163
conditioning system
Refrig-
eration
17
29
6
52
Evapor
cooler
1
6
28
11
46
Type
unknown
42
2
44
Total
50
26
66
21
163
«•>
-------
TABLE P-27. CROSSTABDLATION OF SELECTED PARTICIPANT4 VARIABLES BY
OVERALL AEROSOL EXPOSURE INDEX LEVEL
Aerosol
exposure
level
Dropped
Low exp
Hed exp
Hi exp
Total
Dropped
Low exp
Hed exp
Hi exp
Total
Age group (as of June 30, 1982)
NR -
1
1
Race
Cauca-
sian
112
71
120
34
337
0-5
18
5
8
3
34
6-17
36
25
49
8
118
18-44 45-64
86 16
25 31
48 38
14 9
173 94
65+ Total
21 178
11 97
27 170
3 37
62 482*
of respondent
His-
panic
66
26
50
3
145
Chews tobacco
Dropped
Low exp
Hed exp
Hi exp
Total
Dropped
Low exp
Hed exp
Hi exp
Total
Dropped
Low exp
Hed exp
Hi exp
Total
NR
166
1
10
1
178
History
No
100
42
74
15
231
Hale
92
49
75
21
237
No
11
94
149
28
282
of chronic
Yes
78
55
96
22
251
Female
86
48
95
16
245
Total
178
97
170
37
482
regularly
Yes
1
2
11
8
22
illness
Total
178
97
170
37
482
Total
178
97
170
37
482
Total
178
97
170
37
482
continued.
596
-------
TABLE P-27. (CONT'D)
Total
Education category of head of household
(used as index of socioeconomic status)
Aerosol
exposure
level NR
Dropped 4
Low ezp
Med ezp
Hi ezp
0-8
70
37
58
10
9-11
27
4
19
12
57
34
45
15
Some
college
(13-15)
7
13
29
6
College
grad
(16-18)
13
9
19
6
Total
178
97
170
37
175
50
151
55
47
482*
Dropped
Low ezp
Med ezp
Hi ezp
Total
Recommended for
polio immunization
No
136
64
91
18
309
Yes
42
33
79
19
173
Total
178
97
170
37
482
a Includes four individuals who only provided an initial blood sample.
59?
-------
TABLE P-28. CROSSTABULATION OF SELECTED DEMOGRAPHIC VARIABLES"
NR
0-5
Age group
6-17
(as of June 30. 1982)
18-44 45-64
65+
Total
Recommended
for polio
immunization
Yes
No
Total
Sex
Male
Female
Total
Dwelling
location
Other
Rural
Wilson
Total
Race of
iesjpondent
Caucasian
Hispanic
Total
Dwe 1 1 ing
location
Other
Rural
Wilson
Total
Dwelling
location
Other
Rural
Wilson
Total
1
1
1
1
1
1
Cauca-
sian
5
214
118
337
History
No
2
99
130
231
8
26
34
18
16
34
19
15
34
21
13
34
Race
His-
panic
26
119
145
of chronic
Yes
3
141
107
251
55
63
118
60
58
118
2
45
71
118
61
57
118
Total
5
240
237
482
illness
Total
5
240
237
482
54 36
119 58
173 94
88 42
85 52
173 94
2
88 54
83 40
173 94
122 75
51 19
173 94
20
42
62
28
34
62
34
28
62
57
5
62
173
308
481
237
245
482»
5
240
237
482
337
145
482
a Includes four individuals who only provided an initial blood sample.
598
-------
TABLE P-29. HEALTH HISTORY OF STUDY PARTICIPANTS4
ARC at onset
Condition 0-5
Chronic respiratory conditions
Allergies ' 32
Chronic bronchitis 4
Emphysema
Asthma 14
Tumor or cancer
of the long
Tumor or cancer
of the mouth
or throat
Other
Chronic abdominal conditions in
Tumor or cancer of
Stomach
Intestine
Colon
Esophagus
Peptic or 1
duodenal ulcer
Ulcerative colitis
Divert iculit is
Gall bladder
Other 1
6-11 12-17 18-30
in study population by
22 8 11
112
224
1
31-50
51+
Total
aee at onset
5
5
2
1
2
13
3
5
4
1
1
3
91
16
7
27
1
1
6
study population by age at onset
1
3 3 10
1
3
4
Chronic cardiovascular conditions in study population
High blood
pressure
Stroke
Heart attack
Angina
Other
8
1 1
Other chronic conditions in studv copulation bv age at
Skin cancer
Leukemia
Hodgkins
Other cancers
Arthritis 1
Diabetes
Anemia 2
Immuno logic
disorder
Rheumatic fever
3
1
2
119
2 1
1 3
3
1
11
1
3
12
8
by age at
27
1
2
2
3
onset
7
2
22
1
7
1
6
10
7
onset
40
4
3
3
6
10
5
44
8
3
1
1
1
0
0
1
35
3
9
25
20
75
5
5
5
11
20
1
0
9
78
12
9
1
4
continued..
589a
-------
TABLE P-29. (CONT'D)
Age at onset
Condition
Infectious
hepatitis
Serum hepatitis '
Mononnecleosis
Other chronic
Blood transfusion
1980 number
1980 percent
Hemodialysis (1980
1980 number
1980 percent
0-5
2
1
1
6
(1980 only)
NR
1
0
only)
NR
1
0
Close contact of person with
1980 number
1980 percent
NR
1
0
6-11
4
1
3
No
435
90
No
479
99
12-17
3
1
Tes
42
9
Tes
2
0
18-30 31-50 51+
2 , 3
1 1
1
3 6 12
Don't
know Total
4 482«
1 100
Total
482
100
Total
14
4
3
30
tuberculosis (1980 only)
No
467
97
History of pneumonia (asked only in
1982 number
1982 percent
History of cancer
1983 number
1983 percent
NR
86
18
No
362
75
in blood relatives
No
106
56
Yes
83
44
Yes
14
3
1982)
Yes
34
7
Total
482
100
Total
482
100
of household adults (1983 only)
Total
186
100
a Includes four individuals who only provided an initial blood sample,
599
-------
TABLE P-30. CROPS AND LIVESTOCK
1980
1983
1980
1983
Total
acres
farmed -
38045
29623
Cattle
297
121
Cotton
23885
14023
HOBS
886
100
Crop
Wheat
993
1105
L
Sheen
175
51
types (in
Oats
NR
339
livestock
Fowl
227
124
acres)
Hilo Other
NR 2344
2607 1192
Horses Other
0 0
NR 8
Payment
in kind
NR
2320
Total
1585
404
Farmland irrigation
No
Yes
Total
farms
1980
1983
4
11
67
25
71
36
TABLE P-31. COMPARISON OF CHARACTERISTICS:
CAUCASIAN PARTICIPANTS VS. HISPANIC PARTICIPANTS
Variable
ACOND
ACSYS
ABDOM
AGEGRP
BOTTLED3
CHRONIC
DWATER-B
GHSIZE
GINCOME
HCBILD
HEART
HOHEDGR
HOHOCC
LOCATE
OTHERO
RESP
SEX
SMOKE
n
161
116
477
477
303
478
477
468
158
475
477
474
160
478
477
477
478
302
p valve
0.03
0.001
0.001
<0.001
0.06
<0.001
<0.001
<0.001
0.005
<0.001
<0.001
<0.001
0.026
<0.001
<0.001
<0.001
Comment
higher proportion of ''yes'' in Caucasian HHs
higher proportion of Caucasians report "refrig-
eration"
higher proportion of Caucasians report ''yes''
higher proportion of hispanics age 17 or
less; Caucasians 65+
higher proportion of Caucasians report ''yes''
higher proportion of Caucasians report ''yes''
higher proportion of hispanics drink "public''
water
higher proportion of hispanics live in HH
with 5 + ; higher proportion of Caucasians
live in HH of 1
higher proportion of Caucasians report ilO,000+
higher proportion of hispanics live in HHs
with children
higher proportion of Caucasians report ''yes''
higher proportion of Caucasians report "college"
higher proportion of Caucasian HHs headed
by ''prof, or manager''
higher proportion of hispanics live in Wilson
higher proportion of Caucasians report ''yes''
higher proportion of Caucasians report ''yes''
600
-------
TABLE P-32. COMPARISON OF CHARACTERISTICS:
RDRAL PARTICIPANTS VS. WILSON PARTICIPANTS
Variable
ACOND
ACSTS
ABDOM
AGEGRP
BOTTLED3
CHRONIC
DWATER-B
GHSIZE
GINCOME
HCHILD
HEART
HOHEDGR
HOHOCC
OTHERO
RACE
RESP
SEX
p value
161
116
477'
477
303
478
477
468
163
478
477
474
477
477
478
477
478
0.002
<0.001
0.007
0.007
0.004
<0.001
<0.001
<0.001
higher proportion of ''yes'* in rural
"public" in Wilson, "private" in rural
higher proportion of single and 5+ HHs in
Wilson
higher proportion of high income HHs in rural
area
higher proportion of HHs with children in
Wilson
higher level of education in rural
higher proportion of farmers in rural
higher proportion of "hispanic" in Wilson
601
-------
TABLE P-33. COMPARISON OF STUDY PARTICIPANT CHARACTERISITICS
BY SAMPLING ZONE
Variable
ACOND
ACSYS
ABDOM
AGEGRP
BOTTLED3
CHRONIC
DWATER-B
GHSIZE
GINCOME
HCHILD
HEART
HOHEDGR
HOHOCC
OTHERO
RACE
RESP
SEX
SMOKE
n
163 -
116
477'
477
475
478
477
468
163
478
477
474
160
477
477
477
436
477
p value
0.074
<0.001
<0.001
0.038
0.008
0.011
<0.001
<0.001
<0.001
0.016
0.082
Comment
c
higher proportion of ''yes'' in Zones 1 and 3
higher proportion of ''yes'' in Zone 3; lowest
in Zone 4
"public'' in Zones 2 and 4; "private"
in Zones 1, 3 and 5
higher proportion of single member HHs in
Zones 1 and 4
higher proportion of $30,000 in Zones 1 and 5
higher proportion of HHs without children
in Zone 1
higher proportion of ''college'' in Zone 1
higher proportion of "farmer" in Zones
1 and 3
higher proportion of "hispanic" in Zones
2 and 4 (Wilson)
highest proportion of ''yes'' in Zone 3; lowest
in Zone 4
highest proportion of ''yes'1 in Zone 2; lowest
in Zone 3
602
-------
TABLE P-34. COMPARISON OF CHARACTERISTICS:
PARTICIPANTS WHO PROVIDED ALL REQUESTED BLOOD SAMPLES VS. THOSE
WHO PROVIDED EITHER SOME (4-7) OR FEW (1-3) OF THE REQUESTED SAMPLES
Variable
ACOND
ACSYS
ABDOM
AGEGRP
BOTTLED3
CHRONIC
DWATER-B
GHSIZE
G INCOME
HCHILD
HEART
HOHEDGR
HOHOCC
LOCATE
OTHERO
RACE
RESP
SEX
SMOKE
ZONE
n
433
317
429
435
291
436
435
429
426
436
435
432
433
436
435
436
435
436
290
436
D value
<0.001
0.046
0.001
<0.001
0.016
0.041
<0.001
0.01
0.005
0.002
<0.001
0.006
<0.001
Comment
higher proportion of ''yes'' provided all
samples
higher proportion of ' 'refrigeration" provided
all samples
higher proportion of ages 45+ provided all
samples
higher proportion of ''yes'' provided all
samples
higher proportion of "bottled" and "public"
provided all samples
higher proportion of $20,000+ provided all
samples
higher proportion of HHs without children
provided all samples
higher proportion of ''yes'' provided all
samples
higher proportion of ''college education"
provided all samples
higher proportion of ''prof, or manager''
provided all samples
higher proportion of ''Wilson'' provided
4-8 samples
higher proportion of ''yes'' provided all
samples
higher proportion of ''Caucasian" provided
all samples
603
-------
TABLE P-35. COMPARISON OF CHARACTERISTICS:
SENTINEL POPULATION VS. GENERAL STUDY POPULATION
Variable
ACOND
ACSYS
ABDOM
AGEGRP
BOTTLED3
CHRONIC
DWATER-B
GHSIZE
G INCOME
HCHILD
HEART
HOHEDGR
HOHOCC
LOCATE
OTHERO
RACE
RESP
SEX
SMOKE
ZONE
n
472 -
472
•
472
472
472
472
472
472
472
472
472
472
472
472
472
472
472
472
302
472
TABLE P-36.
P value
0.093
0.095
0.005
<0.001
0.078
<0.001
<0.001
<0.001
<0.001
0.026
0.019
<0.001
Comment
c
higher proportion of ''refrigeration''
sentinel
higher proportion of ''yes'' in sentinel
higher proportion of ''yes'' in sentinel
higher proportion of ''private well''
sentinel
higher income in sentinel
higher education level in sentinel
higher proportion of ''prof or manage''
sentinel
higher proportion of ''rural1' in sentinel
in
in
in
higher proportion of ''Caucasian'' in sentinel
higher proportion of ''yes'' in sentinel
higher proportion of ''no'' in sentinel
higher proportion of ''Zone 1'' in sentinel
DEMOGRAPHIC DIFFERENCES BETWEEN FECAL DONORS AND
NONDONORS DURING SUMMER 1982
Variable
GINCOME
HCHILD
SEX
n
478
478
478
D value
0.045
0.014
0.063
Comment
higher proportion of fecal donors from
income households
lower proportion of HHs with children
6-17 were fecal donors
low
age
higher proportion of fecal donors were female
604
-------
TABLE P-37. DEMOGRAPHIC DIFFERENCES BETWEEN FECAL DONORS AND
NONDONORS DURING SPRING 1983
Variable
CHRONIC
GHSIZE
HCHILD
RACE
SMOKES
n p value Comment
478 - 0.042 higher proportion o£ fecal donors reported
"yes"
468' 0.001 higher proportion of fecal donors from single
member HHs
478 0.081 higher proportion of fecal donors from HHs
without children
478 0.015 higher proportion
302 0.048 higher proportion
of fecal donors were Caucasian
of fecal donors were nonsmokers?
TABLE P-38. DEMOGRAPHIC DIFFERENCES BETWEEN FECAL DONORS AND
NONDONORS DURING SUMMER 1983
Variable
CHRONIC
GHSIZE
GINCOME
HCHILD
RACE
SMOKES
n
478
478
478
478
478
302
p value
0.042
0.001
0.073
0.072
0.033
0.011
Comment
higher proportion of fecal donors reported
"yes"
higher proportion of fecal donors from single
member HHs
higher proportion of fecal donors reported
low income
higher proportion of fecal donors from HHs
without children
higher proportion of fecal donors were Caucasian
higher proportion of fecal donors were nonsmokers
605
-------
TABLE P-39. DEMOGRAPHIC DIFFERENCES OBSERVED BETWEEN EXPOSURE GRODP
SDBPOPULATIONS AND BETWEEN EXPOSURE LEVEL SDBPOPULATIONS DURING SPRING 1982
Exposure group
Variable
ACOND
ACSYS
n
377
314
p-valne
0.016
Comment
higher proportion of
hi exp
n
374
314
Exposure
p-valne
0
0
.076
.001
used evaporative cooler
DWATER-B
GHSIZE
GINCOME
HOHEDGR
LOCATE
RACE
o ZONE
ON
377
369
368
374
377
377
377
0.092
0.003
<0.001
0.078
<0.001
higher proportion of
HHs in high exposure
higher proportion of
in low exp
higher proportion of
residents in hi exp
higher proportion of
hispanics in hi exp
higher proportion of
1 and 2 in hi exp
1-4 mem
$20,000+
Wilson
Zones
376
369
368
374
377
377
377
<0
<0
0
<0
<0
<0
.001
.001
.004
.001
.001
.001
higher
in med
higher
level
Comment
proportion
exp
proportion
tive cooler in hi
higher
in med
higher
in low
higher
in hi
higher
in med
proportion
exp
proportion
exp
proportion
exp
proportion
exp
of
of
' 'none' '
evapora-
exp
of
of
of
of
"ptublic"
$20,000+
"college"
Wilson
higher proportion of hispanic
in med
higher
and 4
exp
proportion
in med; Zone
of
1
Zones 2
in hi exp
-------
TABLE P-40. DEMOGRAPHIC DIFFERENCES OBSERVED BETWEEN EXPOSURE GROUP
SUBPOPULATIONS AND BETWEEN EXPOSURE LEVEL SDBPOPULATIONS DURING SUMMER 1982
0\
o
Exposure group
Variable
ACSYS
DWATER-B
GHSIZE
GINCOME
HCHILD
HOHEDGR
LOCATE
RACE
ZONE
n p-value
317 0.024
363
354 0.046
355
364 0.012
363
364
364
364 <0.001
Comment
higher proportion of
''evaporative1' in hi exp
lower proportion of 5+
HHs in hi exp
lower proportion of HHs
with children in hi exp
higher proportion of Zones
1 and 2 in hi exp
n
317
364
364
364
364
361
364
364
364
Exposure level
p-value
0.008
0.011
0.057
0.037
<0.001
0.003
<0.001
Comment
higher proportion of ''public''
in med ezp
higher proportion of ''2-4''
in hi exp
higher proportion of |20,000+
and $10,000 in low exp
higher proportion of "college''
in hi exp
higher proportion of Wilson
in med exp
higher proportion of hispanic
in med exp
higher proportion of Zones 2
and 4 in med; Zone 1 in hi exp
-------
TABLE P-41. DEMOGRAPHIC DIFFERENCES OBSERVED BETWEEN EXPOSURE GROUP
SUBPOPULATIONS AND BETWEEN EXPOSURE LEVEL SUBPOPULATIONS DURING SPRING 1983
o\
o
oo
Exposure group
Variable n p-value
ACOND 331
ACSYS 309 <0.001
DWATER-B 333
GHSIZE 323 <0.001
333
333 0.025
332
GINCOME
BCHILD
HOHOCC
LOCATE
RACE
ZONE
higher proportion of
''evaporative'' in hi ezp
higher proportion of 1-4
HH members in hi exp
0.022 higher proportion of $20,000+
in low exp
higher proportion of HHs
with no children in hi ezp
333 <0.001
333
333 <0.001
higher proportion of Wilson
in hi ezp
higher proportion of Zones
1 and 2 in hi ezp
Exposure level
j-value
331
309
333
323
325
333
332
333
333
333
0.048 higher proportion of "yes"
in hi ezp
<0.001 higher proportion of
''evaporative" in hi ezp
<0.001 higher proportion of "public"
in med ezp
0.022 higher proportion of 1-4
member HHs in hi ezp
0.001 higher proportion of i20,000+
in lo ezp
0.083 higher proportion of HHs
with no children in hi ezp
0.037 lower proportion of "farmer"
in med ezp
<0.001 higher proportion of Wilson
in med ezp
<0.001 higher proportion of hispanic
in med ezp
<0.001 higher proportion of Zones 2
and 4 in med; Zone 1 in hi ezp
-------
TABLE P-42. DEMOGRAPHIC DIFFERENCES OBSERVED BETWEEN EXPOSURE GROUP
SUBPOPULATIONS AND BETWEEN EXPOSURE LEVEL SUBPOPULATIONS DURING SUMMER 1983
Exposure group
Exposore level
Variable
p-value
Comment
p-value
Comment
ACSTS 308 <0.001 higher proportion of
''evaporative'' in hi ezp
DWATER-B 313
GHSIZE 303 0.056 higher proportion of 1-4
HH members in hi ezp
LOCATE 313
ZONE 313 <0.001 higher proportion of Zones
1 and 2 in hi ezp
308 <0.001 higher proportion of
''evaporative'' in hi ezp
313 <0.001 higher proportion of "public"
in med ezp
303 ,
313 <0.001 higher proportion of wilson
in med ezp
313 <0.001 higher proportion of Zones 2
and 4 in med; Zone 1 in hi ezp
0\
o
-------
TABLE P-43. DEMOGRAPHIC DIFFERENCES OBSERVED BETWEEN EXPOSURE GROUP
SDBPOPULATIONS AND BETWEEN EXPOSURE LEVEL SUBPOPULATIONS DURING 1982
Exposure group
Variable
ACSTS
CONTACT
DWATER
GHSIZE
GINCOME
HCHILD
HOBEDGR
HOHOCC
M LOCATE
o
RACE
SHOKE3
ZONE
n p-valne
312 0.005
293 0.037
358
359 0.057
350
359 0.53
356
357 0.014
359
359
293
385 <0.001
Comment
higher proportion of
' 'evaporative " in hi ezp
higher proportion of 6+
contacts in low ezp
higher proportion of 5+
in low ezp
higher proportion of HB's
with no children in hi ezp
higher proportion of farmer
in hi ezp
n
312
293
358
359
350
359
356
357
359
359
293
359
Ezposure level
p- value
0.018
<0.001
0.003
0.037
0.039
<0.001
<0.001
0.004
0.91
<0.001
Comment
higher proportion of ''evapora-
tive ' ' in hi ezp
higher proportion of- ''public''
in med ezp
higher proportion of ''2-4'' in
hi ezp
higher proportion of 120,000+
in low ezp
higher proportion of hi ezp
reported college education
higher proportion of farmers
in hi, low ezp
higher proportion of Wilson
in med ezp
lower proportion of hispanic
in hi ezp
higher proportion of smokers
in med ezp
higher proportion of -Zones 1
and 2 in hi ezp; Zones 4 and
5 in low ezp
-------
TABLE P-44. DEMOGRAPHIC DIFFERENCES OBSERVED BETWEEN EXPOSURE GROUP
SUBPOPULATIONS AND BETWEEN EXPOSURE LEVEL SUBPOPULATIONS DURING 1983
Exposure group
Variable
ACSYS
n p— value
308 <0.001
higher
Comment
proportion
of
n
309
Exposure
p-valne
<0.001
"evaporative" in hi exp
CHRONIC
DWATER
GHSIZE
HOHOCC
LOCATE
SMOKE3
ZONE
313 0.089
313
313 0.085
313 <0.001
313
301
313 <0.001
higher
in hi
higher
in low
higher
in hi
higher
1 and
and 5
proportion
exp
proportion
exp
proportion
exp
proportion
2 in hi exp
in low
of "yes"
of 5+
of farmer
of Zones
; Zones 4
314
314
314
314
314
301
314
<0.001
<0.001
<0.001
0.066
<0.001
higher
tive"
in low
higher
in med
higher
in hi
higher
in med
higher
in med
highe
and 2
in low
level
Comment
proportion
in hi exp; '
proportion
exp
proport ion
exp
proportion
exp
proportion
exp
of
' 'evapora-
'refrigeration' '
of
of
of
of
r proportion
"public'
farmer
Wilson
smokers
of Zones
hi exp; Zones 4 and
exp
'
1
5
-------
TABLE P-45. FREQUENCY DISTRIBUTION OF TITERS BY AGENT AND COLLECTION PERIOD
Agent
Titer
Jan 80
(012-
016)
Dec 80
(025-
111)
-
Jan 81
(112-
120)
Jan 82
(201-
206)
Jun 82
(212-
218)
Dec 82
(225-
305)
r
Jun 83
(312-
314)
Oct 83
(320-
323)
Adeaoviru* 3
<10
10
20
40
80
160
320
640
57%
14%
13%
10%
5%
1%
0%
0%
N=214
48%
19%
14%
13%
3%
1%
1%
0%
N=69
30%
15%
20%
5%
30%
0%
0%
0%
N=20
51%
14%
17%
9%
5%
3%
1%
0%
N=276
52%
30%
7%
11%
0%
0%
0%
0%
N=27
51%
15%
14%
11%
7%
2%
1%
0%
N=303
48%
12%
16%
4%
8%
8%
4%
0%
N=26
50%
20%
15%
9%
5%
0%
0%
0%
N=266
Adenorins 5
<10
10
20
40
80
160
320
640
53%
9%
16%
13%
8%
3%
0%
0%
N=216
40%
18%
19%
6%
12%
3%
1%
0%
N=68
38%
13%
6%
19%
19%
6%
0%
0%
N=16
46%
12%
17%
11%
9%
4%
0%
0%
N=279
30%
10%
7%
17%
20%
10%
5%
0%
N=40
46%
11%
17%
15%
6%
3%
1%
0%
N=302
27%
5%
11%
22%
16%
16%
3%
0%
N=37
44%
11%
18%
15%
8%
4%
0%
0%
N=266
AdenoYirms 7
<10
10
20
40
80
78%
17%
5%
0%
0%
N=236
CozsackieTirus
<10
10
20
40
80
160
320
640
43%
9%
15%
15%
11%
6%
0%
0%
N=245
Coxsackierirus
<10
10
20
40
21%
11%
20%
23%
77%
15%
6%
1%
0%
N=79
A9
17%
14%
18%
24%
4%
4%
4%
0%
N=50
B2
21%
19%
13%
24%
50%
25%
19%
6%
0%
N=16
33%
18%
18%
9%
18%
9%
5%
0%
N=ll
26%
11%
21%
5%
72%
17%
9%
3%
0%
N=305
27%
14%
12%
16%
13%
5%
2%
0%
N=306
23%
10%
20%
20%
70%
21%
8%
2%
0%
N=304
24%
9%
9%
9%
33%
67%
0%
0%
0%
N=3
25%
10%
17%
20%
81%
19%
0%
0%
0%
N=21
100%
0%
0%
0%
86%
11%
3%
0%
0%
N=266
0%
0%
0%
0%
continued. . .
612
-------
TABLE P-45 (CONT'D)
Ageat
liter
Jun 80
(012-
016)
Coxsackievirvs
80
160
320
640
15%
6%
3%
1%
N=219
CoxsackieTirms
<10
10
20
40
80
160
320
640
1%
3%
16%
25%
27%
20%
4%
5%
N=113
Coxsackierirvs
<10
10
20
40
80
160
320
640
27%
14%
22%
15%
16%
4%
2%
0%
N=220
CoxsackieYirws
<10
10
20
40
80
160
320
640
68%
16%
7%
8%
1%
0%
0%
0%
N=238
Dec 80
(025-
111)
B2 (Cont
' 13%
11%
0%
0%
N=72
B3
0%
0%
16%
26%
29%
16%
3%
11%
N=38
B4
24%
14%
17%
29%
9%
6%
2%
0%
N=66
B5
49%
26%
14%
9%
1%
0%
0%
0%
N=69
Jan 81
(112-
120)
fd)
26%
5%
5%
0%
N=19
0%
0%
0%
0%
50%
0%
50%
0%
N=2
15%
15%
8%
23%
23%
15%
0%
0%
N=13
47%
18%
12%
0%
12%
12%
0%
0%
N=17
Jan 82
(201-
206)
16%
10%
2%
0%
N=284
1%
3%
10%
23%
32%
19%
6%
7%
N=153
22%
11%
19%
24%
14%
8%
2%
0%
N=284
64%
14%
15%
6%
2%
0%
0%
0%
N=307
Jan 82
(212-
218)
35%
12%
3%
0%
N=34
41%
3%
13%
10%
23%
8%
3%
0%
N=39
51%
19%
16%
9%
4%
0%
0%
0%
N=303
Dec 82
(225-
305)
c
17%
8%
3%
1%
N=303
25%
12%
14%
18%
20%
8%
3%
0%
N=303
61%
13%
13%
9%
4%
0%
0%
0%
N=303
Jan 83
(312-
314)
0%
0%
0%
0%
N=l
100%
0%
0%
0%
0%
0%
0%
0%
N=l
46%
14%
14%
9%
11%
3%
3%
0%
N=35
Oct 83
(320-
323)
100%
0%
0%
0%
N=l
50%
0%
0%
0%
0%
50%
0%
0%
N=2
52%
18%
14%
9%
2%
2%
. 1%
0%
N=266
Bchovirms 1
<10
10
20
40
80
160
90%
8%
2%
0%
0%
0%
N=236
86%
11%
3%
0%
0%
0%
N=75
91%
9%
0%
0%
0%
0%
N=ll
84%
11%
3%
1%
1%
0%
N=307
88%
7%
3%
1%
1%
0%
N=304
100%
0%
0%
0%
0%
0%
N=l
90%
10%
0%
0%
0%
0%
N=21
92%
5%
2%
1%
0%
0%
N=266
continued..
613
-------
TABLE P-45. (CONT'D)
Agent
Titer
Jun 80
(012-
016)
Dec 80
(025-
111)
-
Jan 81
(112-
120)
Jan 82
(201-
206)
Jun 82
(212-
218)
Dec 82
(225-
305)
r
Jnn 83
(312-
314)
Oct 83
(320-
323)
Behovirns 3
<10
10
20
40
80
160
320
640
78%
12%
7%
3%
0%
0%
0%
0%
N=214
' 64%
11%
15%
8%
2%
0%
0%
2%
N=66
38%
13%
6%
25%
13%
0%
6%
0%
N=16
71%
12%
6%
7%
3%
1%
0%
0%
N=276
43%
18%
14%
14%
7%
4%
0%
0%
N=28
70%
12%
7%
5%
4%
2%
0%
0%
N=303
44%
15%
15%
10%
8%
3%
3%
3%
N=39
54%
20%
11%
8%
5%
2%
1%
1%
N=266
BehoTirms 5
<10
10
20
40
80
160
320
72%
13%
9%
4%
2%
1%
0%
N=223
67%
10%
12%
3%
2%
2%
3%
N=58
44%
0%
0%
11%
22%
0%
22%
N=9
69%
13%
10%
3%
4%
1%
1%
N=279
66%
15%
7%
7%
4%
1%
0%
N=302
76%
5%
10%
10%
0%
0%
0%
N=21
81%
11%
5%
1%
1%
0%
0%
N=263
BchoTiras 9
<10
10
20
40
80
160
320
59%
16%
11%
8%
5%
2%
0%
N=237
46%
13%
14%
18%
6%
3%
0%
N=71
39%
33%
6%
6%
0%
6%
11%
N=18
55%
12%
11%
12%
6%
3%
1%
N=306
63%
16%
13%
5%
3%
0%
0%
N=302
75%
25%
0%
0%
0%
0%
0%
N=4
48%
13%
17%
4%
13%
4%
0%
N=23
59%
16%
12%
9%
3%
2%
0%
N=263
BchoYirus 11
<10
10
20
40
80
160
320
64%
20%
8%
5%
2%
1%
0%
N=241
48%
22%
9%
14%
6%
0%
1%
N=69
29%
24%
14%
19%
5%
5%
5%
N=21
59%
17%
12%
7%
3%
1%
1%
N=309
46%
14%
19%
12%
4%
2%
4%
N=57
51%
19%
17%
7%
3%
3%
0%
N=300
51%
20%
17%
6%
6%
0%
0%
N=35
55%
21%
14%
6%
2%
1%
0%
N=269
EckOTims 17
<10
10
20
40
80
160
87%
8%
3%
0%
1%
0%
N=213
74%
13%
7%
2%
5%
0%
N=62
82%
9%
0%
0%
0%
9%
N=ll
83%
10%
5%
2%
1%
0%
N=274
75%
8%
0%
4%
8%
4%
N=25
82%
11%
5%
1%
1%
0%
N=303
74%
9%
0%
0%
9%
9%
N=23
81%
10%
6%
2%
1%
1%
N=266
continued..
614
-------
TABLE P-45. (CONT'D)
Agemt
liter
Jun 80
(012-
016)
-
Dec 80
(025-
111)
Jun 81
(112-
120)
Jan 82
(201-
206)
Jun 82
(212-
218)
Dec 82
(225-
305)
r
Jun 83
(312-
314)
Oct 83
(320-
323)
EchoTirns 19
<10
10
20
40
80
160
320
82%
11%
5%
1%
0%
0%
0%
N=211
81%
14%
3%
2%
0%
0%
0%
N=63
54%
8%
15%
8%
8%
8%
0%
N=13
79%
12%
6%
2%
1%
0%
0%
N=271
52%
26%
17%
4%
0%
0%
0%
N=23
77%
13%
7%
2%
1%
1%
0%
N=303
100%
0%
0%
0%
0%
0%
0%
N=21
91%
6%
2%
0%
0%
0%
0%
N=266
BehoYirms 20
<10
10
20
40
80
160
640
82%
11%
5%
1%
0%
0%
0%
N=217
84%
13%
2%
0%
0%
0%
2%
N=64
77%
0%
15%
0%
8%
0%
0%
N=13
83%
9%
6%
2%
0%
0%
0%
N=277
67%
15%
7%
7%
4%
0%
0%
N=27
79%
13%
4%
4%
0%
0%
0%
N=303
53%
31%
16%
0%
0%
0%
0%
N=32
67%
20%
10%
3%
1%
0%
0%
N=266
Bcnovirvs 24
<10
10
20
40
80
160
320
640
89%
5%
5%
1%
0%
0%
0%
0%
N=213
81%
14%
2%
2%
2%
0%
0%
0%
N=64
64%
7%
14%
0%
0%
7%
7%
0%
N=14
79%
13%
4%
2%
2%
0%
0%
0%
N=272
52%
12%
20%
4%
12%
0%
0%
0%
N=25
74%
16%
6%
4%
1%
0%
0%
0%
N=303
72%
7%
10%
3%
7%
0%
0%
0%
N=29
84%
7%
5%
2%
1%
1%
0%
0%
N=266
B. histolytica
<64
64
128
99%
1%
0%
N=189
99%
1%
1%
N=189
99%
0%
1%
N=189
Hepatitis A Virus
neg
pos
58%
42%
N=313
72%
28%
N=275
95%
5%
N=169
88%
12%
N=198
94%
6%
N=174
92%
8%
N=178
90%
10%
N=165
89%
11%
N=160
Influenza A
<4
4
8
16
14%
30%
33%
15%
8%
25%
35%
22%
13%
29%
30%
21%
16%
20%
26%
25%
continued. . .
615
-------
TABLE P-45. (CONT'D)
Agent Jan 80
(012-
Titer 016)
-
Influenza A (Cent
32 6%
64 1%
N=194
Leg lone 1 la
<64
64
128
256
Nexvalk Virms
<50 11%
50 16%
100 16%
200 16%
400 11%
800 11%
1600 11%
3200 0%
6400 11%
N=19
Poliorirvs 1
<4 10%
4 19%
8 23%
16 20%
32 15%
64 9%
128 2%
256 2%
N=204
Poliovirns 2
<4 9%
4 16%
8 26%
16 25%
32 12%
64 8%
128 3%
256 0%
N=210
Dec 80
(025-
111)
•d)
16%
17%
26%
18%
12%
7%
3%
1%
N=311
13%
16%
27%
22%
12%
6%
2%
1%
N=312
Jun 81
(112-
120)
6%
4%
N=251
47%
16%
15%
22%
N=269
0%
10%
20%
40%
10%
10%
0%
10%
N=10
9%
18%
27%
18%
9%
9%
9%
0%
N=ll
Jan 82
(201-
206)
14%
5%
23%
18%
14%
9%
9%
0%
5%
N=21
6%
12%
21%
22%
16%
13%
4%
4%
N=253
7%
16%
17%
20%
18%
11%
6%
3%
N=250
Jon 82
(212-
218)
6%
0%
N=278
47%
15%
15%
23%
N=297
7%
13%
19%
21%
23%
8%
4%
3%
N=307
9%
12%
20%
25%
17%
9%
4%
4%
N=306
Dec 82
(225-
305)
r
17%
33%
17%
33%
0%
0%
0%
0%
N=6
0%
17%
33%
17%
33%
0%
0%
0%
N=6
Jun 83
(312-
314)
9%
4%
N=257
47%
17%
20%
17%
N=266
51%
11%
6%
6%
14%
3%
6%
3%
3%
N=36
Oct 83
(320-
323)
continued...
616
-------
TABLE P-45. (CONT'D)
Agomt
liter
Jnn 80
(012-
016)
Dec 80
(025-
111)
Jun 81
(112-
120)
Jan 82
(201-
206)
Jun 82
(212-
218)
Dec 82
(225-
305)
Jun 83
(312-
314)
Oct 83
(320-
323)
c
Polioviras 3
<4
4
8
16
32
64
128
256
37%
26%
17%
8%
9%
2%
1%
0%
N=211
41%
24%
14%
10%
7%
2%
0%
0%
N=311
40%
10%
10%
20%
10%
0%
10%
0%
N=10
20%
22%
21%
12%
10%
7%
5%
3%
N=249
27%
21%
17%
16%
9%
5%
4%
2%
N=306
16%
50%
17%
17%
0%
0%
0%
0%
N=6
ReoTirng 1
<8
8
16
32
64
128
256
512
70%
11%
6%
6%
4%
1%
0%
1%
N=235
47%
22%
7%
5%
12%
3%
1%
3%
N=74
50%
0%
25%
8%
17%
0%
0%
0%
N=12
58%
12%
7%
10%
6%
3%
1%
2%
N=307
65%
11%
7%
7%
6%
3%
1%
0%
N=308
52%
18%
14%
10%
3%
2%
1%
0%
N=300
63%
17%
7%
7%
4%
1%
1%
0%
N=251
Reoviras 2
<8
8
16
32
64
128
256
512
42%
23%
12%
14%
5%
1%
1%
0%
N=236
44%
19%
14%
14%
7%
3%
0%
0%
N=73
33%
25%
25%
8%
8%
0%
0%
0%
N=12
33%
17%
18%
17%
9%
5%
1%
0%
N=307
41%
15%
16%
17%
8%
3%
0%
0%
N=308
38%
22%
18%
15%
7%
0%
0%
0%
N=299
45%
20%
18%
11%
6%
0%
0%
0%
N-251
Rotaviras
<4
4
8
16
32
64
128
256
512
11%
7%
4%
7%
18%
29%
21%
4%
0%
N=28
5%
0%
3%
10%
28%
21%
23%
3%
8%
N=39
3%
6%
6%
15%
18%
33%
12%
6%
0%
N=33
2%
5%
0%
14%
23%
30%
14%
12%
0%
N=43
12%
10%
4%
13%
17%
19%
19%
6%
0%
N=52
9%
6%
4%
15%
23%
32%
8%
4%
0%
N=53
13%
2%
4%
17%
28%
21%
9%
6%
0%
N=47
4%
2%
7%
24%
29%
18%
11%
4%
0%
N=45
617
-------
TABLE P-46. PREVALENCE OF ANTIBODY BY AGENT AND AGE GROUP
Presence of
antibody
Adeaovim* 3
Positive
Negative
Total tested
Adeaoviras 5
Positive
Negative
Total tested
Adeaovirvs 7
Positive
Negative
Total tested
Coxttckievirm* B2
Positive
Negative
Total tested
Cox»«ckievirm* B4
Positive
Negative
Total tested
Coxsackievirms B5
Positive
Negative
Total tested
Bchovirms 1
Positive
Negative
Total tested
Bcaovirms 3
Positive
Negative
Total tested
Bcaovirvs 5
Positive
Negative
Total tested
Eeaovirms 9
Positive
Negative
Total tested
0-5
31%
69%
13
46%
54%
13
8%
92%
13
27%
73%
11
17%
83%
12
13%
87%
15
0%
100%
13
38%
62%
13
0%
100%
12
25%
75%
12
6-17
32%
68%
81
54%
46%
81
7%
93%
84
67%
33%
83
66%
34%
85
42%
58%
91
2%
98%
86
35%
65%
80
17%
83%
87
44%
56%
87
Age group
18-44
53%
47%
93
42%
58%
92
29%
71%
106
77%
23%
94
74%
26%
96
43%
57%
111
9%
91%
108
26%
74%
94
30%
70%
105
46%
54%
107
Total
45-64
f
53%
47%
77
52%
48%
75
29%
71%
79
90%
10%
78
81%
19%
77
23%
77%
78
15%
85%
78
21%
79%
76
35%
65%
78
41%
59%
79
65+
36%
64%
42
57%
43%
42%
27%
73%
48
90%
10%
42
77%
23%
43
31%
69%
49
26%
74%
47
24%
76%
42
36%
64%
47
39%
61%
49
N
135
171
306
152
151
303
74
256
330
239
69
308
224
89
313
121
223
344
36
296
332
83
222
305
91
238
329
141
193
334
%
44%
56%
50%
50%
22%
78%
78%
22%
72%
28%
35%
65%
11%
89%
27%
73%
28%
72%
42%
58%
continued...
618
-------
.TABLE P-46. (CONT'D)
Presence of
antibody
Eckovirns 11
Positive
Negative
Total tested
Bekovirms 17
Positive
Negative
Total tested
Eckorirvs 19
Positive
Negative
Total tested
Bekorirws 20
Positive
Negative
Total tested
Bekovirms 24
Positive
Negative
Total tested
Hepatitis A
Positive
Negative
Total tested
Imf Imenxa A
Positive
Negative
Total tested
Legiomells
Positive
Negative
Total tested
PolioTirms 1
Positive
Negative
Total tested
Poliorims 2
Positive
Negative
Total tested
0-5
-
31%
69%
13
8%
92%
13
15%
85%
13
8%
92%
13
23%
77%
13
0%
100%
23
14%
86%
7
46%
54%
13
88%
12%
17
100%
0%
17
6-17
40%
60%
90
7%
93%
82
4%
96%
81
5%
95%
81
9%
91%
80
15%
85%
104
68%
32%
56
56%
44%
73
86%
14%
103
93%
7%
103
ARC R rout)
18-44
38%
62%
111
12%
88%
91
16%
84%
92
19%
81%
93
13%
87%
92
30%
70%
151
68%
32%
82
62%
38%
77
88%
12%
127
86%
14%
127
Total
45-64
r
42%
58%
79
25%
75%
76
23%
77%
75
22%
78%
74
20%
80%
75
65%
35%
89
65%
35%
68
49%
51%
75
92%
8%
85
89%
11%
85
65+
48%
52%
48
19%
81%
42
47%
52%
40
22%
78%
41
17%
83%
42
98%
2%
59
69%
31%
39
52%
48%
42
85%
15%
55
76%
24%
55
N
138
203
341
45
259
304
56
245
301
48
254
302
44
258
302
178
248
426
166
86
252
154
126
280
341
46
387
340
47
387
%
40%
60%
15%
85%
19%
81%
16%
84%
15%
85%
42%
58%
66%
34%
55%
45%
88%
12%
88%
12%
continued...
619
-------
Presence of
antibody
TABLE P-46. (CONT'D)
Age group
Total
0-5
6-17
18-44
45-64
65+
N
Poliovirns 3
Positive
Negative
Total tested
Raovirws 1
Positive
Negative
Total tested
Beovirus 2
Positive
Negative
Total tested
lotavirws
Positive
Negative
Total tested
59%
41%
17
29%
71%
14
31%
69%
13
57%
43%
14
44%
56%
103
24%
76%
89
47%
53%
89
94%
6%
31
67%
33%
127
40%
60%
109
60%
40%
109
86%
14%
7
73%
27%
85
43%
57%
79
61%
39%
79
100%
0%
3
c
64%
36%
55
35%
65%
48
75%
25%
48
0%
100%
1
237
150
387
120
219
339
195
143
338
46
10
56
61%
39%
35%
65%
58%
42%
82%
18%
620
-------
TABLE P-47. INFECTION INCIDENCE DENSITY RATES FOR WASTEWATER AEROSOL
EXPOSURE LEVELS BY AGENT AND TIME INTERVAL
(Number of infection events indicated in parentheses)
[When different than number of infection events, number
of infected individuals indicated in brackets]
Agent
Interval
Adenovirus 3
0-Baseline
1-Spring 1982
2 -Summer 1982
3-Spring 1983
4-Suomer 1983
5-1982
6-1983
7-Irrigation
AdomoYirns 5
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
Adenovirus 7
0-Baseline
1-Spring 1982
2 -Summer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
Coxsackievirns B2
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrisation
Low exp level
(AEK1)
2.07 (2)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
1,19 (1)
0.00 (0)
0.57 (1)
3.16 (3)
3.53 (1)
2,18 (1)
0.00 (0)
0.00 (0)
2.35 (2)
0.00 (0)
1.15 (2)
0.84 (1)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
7.14 (7)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
Hed exp level
(KAEK5)
11.40 (11)
0.00 (0)
1.27 (1)
1.33 (1)
0.00 (0)
3.68 (6)
0.81 (1)
1.91 (7)
5.27 (5)
2.80 (2)
1.33 (1)
2.75 (2)
0.79 (1)
3.87 (6)
2.55 (3)
2.58 (9)
2.51 (3)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
5.10 (5)
1.34 (1)
1.33 (1)
4.34 (7)
4.51 (7)
High exp level
(AEI>5)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0,00 (0)
0.00 (0)
3.17 (1)
1.17 (1)
3.38 (2)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
3.32 (2)
0.00 (0)
0.00 (0)
5.80 (2)
5.80 (2)
continued.
621
-------
TABLE P-47. (CONT'D)
Agemt
Interval
CoxsackieTirus B4
0-Baseline
1-Spring 1982
2-Sumner 1982
3-Spring 1983
4-Sunmer 1983
5-1982
6-1983
7-Irrigation
Cozsackievirns B5
0-Baseline
1-Spring 1982
2-Sommer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
Rckoviraa 1
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
Eckovirus 3
0-Baseline
1-Spring 1982
2 -Summer 1982
3-Spring 1983
4 -Summer 1983
5-1982
6-1983
7-Irrigation
Behoviros 5
0-Baseline
1-Spring 1982
2-Summer 1982
Low ezp level
(AEK1)
5.07 (5)
0.00 (0)
2.38 (1)
8.22 (7)
7.93 (6)
0.82 (1)
3.45 (1)
0.00 (0)
0.00 (0)
2.76 (2)
1.16 (1)
3.62 (2)
1.67 (3)
0.85 (1)
3.24 (1)
0.00 (0)
0.00 (0)
0.00 (0)
1.16 (1)
0.00 (0)
0.57 (1)
8.29 (8)
0.00 (0)
0.00 (0)
6.79 (2)
1.41 (1)
4.70 (4)
5.61 (3)
3.98 (7)
0.96 (1)
0.00 (0)
0.00 (0)
Med ezp level
(KAEK5)
c
11.15 (11)
2.64 (2)
2.57 (2)
4.83 (8)
5.63 (9)
6.57 (8)
4.08 (3)
2.52 (2)
1.34 (1)
4.59 (6)
2.56 (4)
4.90 (6)
3.62 (13)
5.11 (6)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
4.15 (4)
0.00 (0)
3.81 (3)
1.36 (1)
6.19 (8)
2.51 (4)
9.00 (11)
4.19 (15)
0.96 (1)
1.46 (1)
0.00 (0)
High ezp level
(AEI>5)
0.00 (0)
6.03 (1)
11.86 (2)
14.49 (5)[4]a
13.91 (5)t>
3.44 (2)
0.00 (0)
10.89 (2)
0.00 (0)
0.00 (0)
9.19 (3)
3.21 (1)
2.28 (2)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
1.80 (1)
0.00 (0)
0.00 (0)
0.00 (0)
5.77 (2)
3.15 (1)
12.70 (4)
5.75 (5)
1.82 (1)
0.00 (0)
0.00 (0)
continued. . .
622
-------
TABLE P-47. (CONT'D)
Agemt group
Interval
Bcborins 5 (Coat'd)
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
EcAorins 9
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Snmmer 1983
5-1982
6-1983
7-Irrigation
BckOTirns 11
0-Baseline
1-Spring 1982
2 -Summer 1982
3-Spring 1983
4-Snmmer 1983
5-1982
6-1983
7-Irrigation
Bchovirus 17
0-Baseline
1-Spring 1982
2-Sommer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
Echorirns 19
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
Low ezp level
(AEK1)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
1.64 (2)
0.00 (0)
0.00 (0)
3.37 (1)
1.44 (1)
0.00 (0)
3.84 (2)
1.19 (2)
5.85 (7)
6.56 (2)
4.36 (2)
0.00 (0)
1.44 (1)
5.75 (5)
5.43 (3)
4.48 (8)
1.05 (1)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
Hed exp level
(KAEK5)
r
0.00 (0)
0.00 (0)
0.66 (1)
0.00 (0)
0.28 (1)
4.11 (5)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
6.69 (8)
2.58 (2)
3.80 (3)
0.00 (0)
3.04 (4)
4.85 (8)
4.03 (5)
3.79 (14)
1.05 (1)
1.32 (1)
0.00 (0)
0.00 (0)
0.00 (0)
0.62 (1)
1.63 (2)
0.83 (3)
3.21 (3)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
1.24 (2)
0.82 (1)
0.83 (3)
High ezp level
(AEI>5)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
3.44 (2)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
3.47 (2)
0.00 (0)
12.11 (2)
0.00 (0)
2.81 (1)
18.37 (6)c
6.19 (2)
7.91 (7)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
5.93 (1)
0.00 (0)
0.00 (0)
3.24 (1)
0.00 (0)
1.18 (1)
continued. . .
623
-------
TABLE P-47. (CONT'D)
Ageat
Interval
Ecfcovirus 20
0-Baseline
1-Spring 1982
2-Summer 1982
3 -Spring 1983
4 -Summer 1983
5-1982
6-1983
7-Irrigation
Bekovirvs 24
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
Influenza A
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Summer 1983
7-Irrigation
Legionella
1-Spring 1982
2-Snmmer 1982
7-Irrigation
Poliovirms ld
0-Baseline
1-Spring 1982
PoliOTiru* 2d
0-Baseline
1-Spring 1982
PoliOTirm* 3d
0-Baseline
1-Sorina 1982
Low exp level
(AEK1)
1.05 (1)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
3.81 (2)
1.18 (2)
2.15 (2)
3.81 (1)
0.00 (0)
3.51 (1)
0.00 (0)
5.02 (4)
1.97 (1)
2.98 (5)
3.24 (3)
3.69 (1)
31.10 (9)
10.33 (10)
0.00 (0)
1.18 (1)
0.59 (1)
2.17 (1)
0.00 (0)
0.00 (0)
5.52 (1)
0.00 (0)
0.00 (0)
Hed exp level
(KAEK5)
4.19 (4)
1.33 (1)
1.28 (1)
0.00 (0)
3.14 (4)
1.27 (2)
4.19 (5)
1.97 (7)
6.46 (5)
0.00 (0)
0.00 (0)
2.69 (2)
3.83 (5)
1.25 (2)
6.51 (8)[6]
2.77 (10)
12.96 (12)
8.47 (5)
29.82 (22)
13.29 (27)
0.54 (1)
1.28 (2)
0.87 (3)
4.33 (2)
6.10 (3)
2.24 (1)
0.00 (0)
0.00 (0)
0.00 (0)
High exp level
(AEI>5)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
5.62 (2)
0.00 (0)
6.35 (2)
2.31 (2)
1.80 (1)
6.71 (1)
0.00 (0)
5.27 (1)
5.77 (2)
3.15 (1)
9.77 (3)
4.66 (4)
7.34 (4)
0.00 (0)
21.47 (4)
8.04 (4)
2.41 (1)
0.00 (0)
1.29 (1)
0.00 (0)
30.45 (3)
0.00 (0)
10.15 (1)
0.00 (0)
0.00 (0)
continued. . .
624
-------
TABLE P-47. (CONT'D)
Low exp level
Med ezp level
(KAEK5)
High ezp level
(AEI>5)
Ageat
Interval
(AEK1)
Reovirms 1
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
Reoyirus 2
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Sommer 1983
5-1982
6-1983
7-Irrigation
14.22 (17)
9.58 (3)
0.00 (0)
0.00 (0)
5.14 (4)
0.00 (0)
2.99 (3)
7.53 (9)
9.58 (3)
0.00 (0)
0.00 (0)
2.45 (2)
0.00 (0)
2.93 (3)
12.55 (15)
14.63 (12)
0.00 (0)
1.42 (1)
7.29 (11)
0.89 (1)
5.75 (13)
17.57 (21)
12.19 (10)
0.00 (0)
2.83 (2)
7.12 (11)
1.77 (2)
5.19 (12)
5.07 (3)
6.03 (1)
0.00 (0)
0.00 (0)
3.15 (1)
0.00 (0)
1.94 (1)
11.80 (7)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
0.00 (0)
Rotariras
0-Baseline
1-Spring 1982
2-Summer 1982
3-Spring 1983
4-Summer 1983
5-1982
6-1983
7-Irrigation
0.
0.
19.
0.
0.
13.
31.
8.
00
00
47
00
00
59
06
75
(0)
(0)
(1)
(0)
(0)
(1)
(1)
(1)
151.
16.
0.
13.
12.
10.
19.
10.
24
01
00
38
75
61
11
89
(7)
(2)
(0)
(2)
(3)
(3)
(4)
(7)
23.
21.
46.
13.
19.
24.
30.
23.
50
56
3
66
97
77
25
91
(4)
(1)
(3)
(1)
(3)
(3)
(4)
(8)
The 95% confidence interval for the high-to-intermediate incidence density
ratio does not include the value 1.
The 90% confidence interval for the high-to-intermediate incidence density
ratio does not include the value 1.
The 95% confidence intervals for both the high-to-low level and high-to-
intermediate incidence density ratios do not include the value 1.
Rates include only nonimmunized participants.
625
-------
TABLE P-48. DISTRIBUTION OF SEROLOGIC INFECTIONS BY NUMBER
OF HOUSEHOLD MEMBERS DONATING SPECIMENS
(Entries are number of households having a specified number
of infected members)
Agent
AD3
AD3
ADS
AD5
AD7
CB2
CB2
CB4
CB4
CB4
CBS
CBS
CBS
CBS
CBS
CBS
Seasons
0
5
0
5
0
0
5
0
2
5
0
1
2
4
5
6
No. with
infections
per house-
hold
0
1
2
0
1
0
1
0
1
0
1
0
1
2
0
1
0
1
3
0
1
0
1
2
0
1
3
0
1
2
0
1
0
1
2
0
1
2
0
1
2
No.
1
37
3
44
1
38
1
43
3
38
37
4
37
3
34
2
40
2
38
4
38
1
41
41
37
43
36
r
of household members
2
40
2
41
1
39
3
37
39
4
40
2
40
4
41
2
42
1
41
3
39
4
41
42
2
33
3
38
3
33
4
3
10
2
11
1
11
3
12
2
17
9
2
11
4
6
12
1
10
2
16
1
15
13
1
12
1
13
12
1
4
9
1
12
1
9
11
1
11
6
3
12
1
6
1
12
1
8
4
11
9
8
9
8
10
5
6
6
1
4
3
1
7
1
4
1
8
5
4
4
2
1
5
2
6
1
8
7
1
6
1
6
1
donating
6
1
1
1
3
2
3
7
1
4
1
3
3
3
1
6
5
3
7
6
3
6
3
7
1
2
1
1
2
1
2
1
1
1
2
1
2
1
1
2
1
1
3
1
1
2
1
1
1
specimens
8 Total
104
7
3
1 120
7
105
7
1 115
8
118
6
99
12
1
113
9
93
13
1
118
5
107
17
1
113
8
1
121
2
1
1 122
3
102
4
2
116
5
1 1
101
5
2
continued..
626
-------
TABLE P-48. (CONT'D)
Aeent
E01
E03
E03
E03
EOS
E09
Ell
Ell
Ell
Ell
Ell
Ell
El 9
E20
E20
E20
No. with
infections
per house-
Season8 - hold
0
0
4
5
6
0
0
1
2
4
5
6
5
0
4
6
0
1
0
1
2
0
1
2
0
1
0
1
2
3
0
1
3
0
1
2
0
1
0
1
0
1
2
0
1
2
0
1
2
0
1
0
1
0
1
3
0
1
4
No.
1
41
2
40
3
39
1
45
38
3
42
1
41
3
39
38
41
39
3
38
44
1
37
35
1
35
1
of household members
2
38
1
41
3
1
33
1
40
3
33
2
39
2
37
3
42
1
42
2
34
40
3
34
3
39
1
43
3
38
1
36
1
3
17
6
3
8
2
10
1
8
2
14
14
3
16
1
15
10
12
1
10
11
1
13
10
10
2
4
10
2
9
10
1
11
1
10
1
10
2
8
1
1
10
1
9
2
12
1
7
5
11
1
13
6
8
1
1
8
1
, 5
6
1
9
1
5
2
1
5
1
3
3
7
5
1
1
6
6
1
3
1
4
2
1
3
2
6
7
1
5
5
1
dona tine
6
4
1
2
3
2
5
3
3
2
1
1
1
1
3
1
4
3
1
4
1
3
1
4
1
5
4
4
4
7
1
1
2
1
1
3
1
3
1
1
3
0
1
1
1
3
1
1
2
2
specimens
8 Total
117
7
107
10
1
98
9
1
117
9
95
13
1
1
115
5
1
109
13
2
120
4
116
1 7
105
2
2
108
15
2
101
6
2
121
3
111
5
102
3
1
100
5
1
continued.
627
-------
TABLE P-48. (CONT'D)
Agent
£24
E24
£24
£24
RE1
RE1
RE2
RE2
ROT
ROT
ROT
ROT
ROT
ROT
ROT
Seasona
0
4
5
6
0
1
0
1
0
1
2
3
4
5
6
No. with
infections
per house-
hold
0
1
0
1
2
3
0
1
0
1
3
0
1
2
3
0
1
2
0
1
2
0
1
2
0
1
2
0
1
0
1
2
0
1
0
1
0
1
2
0
1
2
No.
1
36
1
37
41
1
37
34
2
37
2
34
4
37
6
7
14
2
13
1
12
1
12
2
14
2
12
3
of household members dona tine
2
40
5
38
37
6
36
1
32
7
1
40
6
1
30
9
1
42
5
5
2
10
1
12
1
11
1
7
3
8
2
1
5
4
3
11
1
7
1
13
7
2
10
4
3
1
13
9
6
11
2
1
2
1
1
1
1
1
1
1
4,5 6 7
9831
1
10 6 5
1
1
1
12 7 5 2
10 6 5
1
1 1
861
2112
1 1
9654
1 1
2
8322
4 2
1 2
9743
21 1
1
1
2
1
1
1
1
2
1
1
specimens
8 Total
108
8
103
2
1
1
117
7
101
4
2
91
19
6
1
114
10
3
88
25
4
113
11
1
11
9
1
27
3
28
2
1
25
3
21
6
25
5
1
19
7
1
continued..
628
-------
TABLE P-48. (CONT'D)
No. with
infections
per house-
Agent Seasona - hold
LEG 7
INA 0
INA 1
INA 3
0
1
2
0
1
2
0
1
0
1
2
3
No.
1
29
2
28
3
33
3
33
2
of household members
2
40
32
6
1
44
1
28
5
3
3
7
1
6
2
1
11
10
5
1
4
10
5
1
8
3
2
1
, 5
3
1
1
3
3
1
2
3
1
donating
6 7
2
1
1
3
1
3 2
1
1
specimens
8 Total
91
4
1
73
15
2
102
6
81
16
8
1
0 if baseline period, 1 if spring 1982, 2 if summer 1982, 3 if spring
1983, 4 if summer 1983. 5 if 1982, 6 if 1983, 7 if 1981-1983.
629
-------
TABLE P-49. DISTRIBUTION OF BACTERIAL INFECTIONS BY NUMBER
OF HOUSEHOLD MEMBERS DONATING SPECIMENS
(Entries are number of household having a
specified number of infections)
No. with
infections
No. of household members
donating specimens
Agent
KLB-Xb
KLB-W
KLB-X
KLB-W
OOB-X
PBW-X
PBW-f
PBW-X
PBW-W
PBW-X
PBW-W
PBW-X
PBW-W
a 0 if
1983.
b X if
Seasdn*
2
2
4
4
3
1
1
2
2
3
3
4
4
baseline
per
0
1
2
0
1
2
0
1
0
1
2
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
0
1
period,
household 1
39
3
39
9
38
5
38
7
54
1
59
2
59
3
45
3
44
3
51
2
51
2
44
39
5
1 if spring 1982
234
643
1
632
1
1
20
3
20
3
1
22
4
17 3 2
17 3 2
542
542
1
26
26
25 1
21
4
, 2 if summer 1982,
5 Total
52
3
1
50
1 11
1
58
8
58
10
1
76
5
81
2
81
3
2 58
3
2 57
4
77
2
77
2
70
60
9
3 if spring
4 if summer 1983.
onset of
all infection events during
des infection events for
which onset may
irrigation period,
have preceded the
W if inclu-
irrigat ion
period.
630
-------
TABLE P-50. APPROXIMATE POWER8 OF TEST OF TEE NULL HYPOTHESIS pt=p2
AGAINST SPECIFIED ALTERNATIVES OF THE FORM P2>Pl WITH o = 0.05
(The number of individuals in the low exposure group is nj and in the
high exposure group is n2. The observed incidence rate in the low
exposure group is assumed to be equal to PI, and the specified
alternatives are given by P2=pi + A where A = 0.05, 0.07, 0.10, 0.15,
0.20, 0.25. Power less than 0.50 is indicated by a dash.)
Agent
-5.1-
0.05
0.07
0.10
0.15
0.20
0.25
Serologic Agents—Baseline and Control**
AD3
ADS
AD7
CB2
CB4
CB5
E01
E03
E05
E09
Ell
El 7
El 9
E20
E24
RE1
RE2
ROT
INA
INA
INA
LEG
POR
wwv
SNV
Serologic Agents—Spring 1982
164
159
198
156
156
188
194
164
168
177
190
169
171
173
171
186
181
13
132
163
164
133
138
91
87
104
88
87
94
97
95
91
94
95
97
96
97
98
95
96
17
54
72
90
82
70
0.06
0.03
0.01
0.06
0.06
0.03
0.02
0.05
0.01
0.01
0.05
0.01
0.01
0.02
0.03
0.16
0.14
0.31
0.11
0.02
0.15
0.04
0.45
0.65
0.50
0.60
0.55
0.55
0.55
0.50
0.60
0.80
-
-
0.65
0.70
0.50
0.80
0.75
0.55
0.75
0.75
0.70
0.65
-
-
0.70
0.80
0.90
0.70
0.70
0.85
0.90
0.75
0.90
0.90
0.80
0.90
0.90
0.85
0.85
0.55
0.55
0.90
0.95
0.95
0.90
0.90
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.85
0.85
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.60
0.50
0.80
0.55
0.75
H
0.70
0.95
0.80
0.90
0.60
0.90
0.95
0.95
0.95
0.80
0.95
0.95
0.95
0.95
0.95
AD3
AD5
AD7
CB2
CB4
CBS
E01
E03
E05
E09
185
186
198
190
188
197
197
187
189
193
106
101
108
104
108
110
110
101
103
108
0.00
0.02
0.00
0.00
0.01
0.01
0.01
0.00
0.01
0.00
0.75
0.55
0.75
0.75
0.70
0.65
0.70
0.75
0.65
0.75
0.85
0.75
0.90
0.85
0.85
0.80
0.85
0.85
0.85
0.90
0.95
0.90
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
continued...
631
-------
TABLE P-50. (CONT'D)
Agent
Serologic
Ell
E17
El 9
E20
£24
RE1
RE2
ROT
LEG
FOR
wwv
SNV
Serologic
ADS
ADS
AD7
CB2
CB4
CBS
E01
E03
EOS
E09
Ell
El 7
E19
E20
£24
RE1
RE2
ROT
LEG
FOR
WW
SNV
Serologic
ADS
ADS
AD7
CB2
ni
n-j p-i
Agents — Spring 1982
199
190
186
191
182
202
200
24
148
124
146
122
104 0.01
106 0.01
103 0.00
104 0.01
105 0.01
111 0.05
110 0.05
24 0.04
65 0.03
64 0.07
76 0.05
61 0.10
0.05
(Cont'd)
0.65
0.70
0.75
0.65
0.65
-
-
-
-
-
-
—
0.07
0.80
0.85
0.85
0.85
0.80
0.60
0.60
-
0.55
-
-
—
0
0
0
0
0
0
0
0
0
0
0
A
.10
r
.90
.95
.95
.95
.95
.80
.80
-
.75
.55
.65
—
0
0
0
0
0
0
0
0
0
0
0
0
.15
.95
.95
.95
.95
.95
.95
.95
-
.90
.80
.90
.75
0
0
0
0
0
0
0
0
0
0
0
0
.20
.95
.95
.95
.95
.95
.95
.95
-
.95
.95
.95
.90
0.25
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.60
0.95
0.95
0.95
0.95
Agents — Summer 1982
231
228
222
224
223
237
223
229
222
219
235
234
230
234
226
-
-
36
-
164
193
150
69 0.00
66 0.01
55 0.00
65 0.00
66 0.01
71 0.01
56 0.00
69 0.01
54 0.00
54 0.00
68 0.02
70 0.00
68 0.00
67 0.00
70 0.00
- -
- -
18 0.03
- -
44 0.05
57 0.05
40 0.11
0.65
0.55
0.65
0.60
0.50
0.60
0.65
0.55
0.65
0.65
-
0.70
0.70
0.65
0.70
-
-
-
-
-
-
-
0.80
0.75
0.75
0.75
0.85
0.75
0.80
0.70
0.75
0.75
0.65
0.85
0.80
0.80
0.80
-
—
-
-
-
-
-
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.90
.90
.90
.90
.70
.90
.90
.85
.90
.90
.85
.90
.90
.90
.90
-
-
-
-
.55
.65
—
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.95
.95
.95
.95
.85
.95
.95
.95
.95
.95
.95
.95
.95
.95
.95
-
-
-
-
.80
.85
.65
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.95
.95
.95
.95
.95
.95
.95
.95
.95
.95
.95
.95
.95
.95
.95
-
-
.55
-
.90
.95
.80
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
-
-
0.65
-
0.95
0.95
0.90
Agents — Spring 1983
175
173
178
-
97 0.01
93 0.01
99 0.00
-
0.65
0.55
0.70
-
0.80
0.75
0.85
-
0
0
0
.90
.90
.95
-
0
0
0
.95
.95
.95
-
0
0
0
.95
.95
.95
-
0.95
0.95
0.95
-
continued...
632
-------
TABLE P-50. (CONT'D)
Agent,
O.OS 0.07 0.10 0.15
0.20
0.25
Serologic Agents—Spring 1983 (Cont'd)
CB4
CB5
E01
EOS
E05
E09
Ell
E17
E19
E20
E24
RE1
RE2
ROT
LEG
FOR
wwv
SNV
174
177
175
177
175
178
172
172
167
171
159
159
21
100
102
93
99
98
97
97
95
98
98
90
90
27
0.01
0.00
0.02
0.00
0.01
0.00
0.00
0.00
0.00
0.01
0.00
0.01
0.05
0.65
0.70
0.50
0.70
0.65
0.70
0.70
0.70
0.70
0.55
0.65
0.60
-
0.80
0.85
0.70
0.85
0.80
0.85
0.85
0.85
0.85
0.75
0.80
0.75
-
0.90
0.95
0.90
0.95
0.90
0.95
0.95
0.95
0.95
0.90
0.90
0.90
-
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
-
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
-
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.50
137
75
0.07
0.60
0.85
Serologic Agents—Summer 1983
0.95
0.95
ADS
ADS
AD7
CB2
CB4
CBS
E01
E03
EOS
E09
Ell
El 7
E19
E20
E24
RE1
RE2
ROT
LEG
FOR
WWV
SNV
197
191
196
-
-
197
197
194
197
196
196
193
194
192
193
-
-
24
-
-
-
160
59
57
61
-
-
59
61
58
59
59
59
58
57
55
58
-
-
21
-
-
-
49
0.00
0.01
0.00
-
-
0.04
0.00
0.04
0.00
0.01
0.02
0.00
0.00
0.02
0.02
-
-
0.13
-
-
-
0.14
0.65
0.55
0.65
-
-
-
0.65
-
0.65
0.55
-
0.60
0.60
-
-
-
-
-
-
-
-
-
0.75
0.70
0.80
-
-
0.50
0.80
0.50
0.75
0.70
0.65
0.75
0.75
0.55
0.65
-
-
-
-
-
-
-
0.90
0.85
0.90
-
-
0.70
0.90
0.70
0.90
0.85
0.80
0.90
0.90
0.75
0.80
-
-
-
-
-
-
-
0.95
0.95
0.95
-
-
0.90
0.95
0.90
0.95
0.95
0.95
0.95
0.95
0.90
0.95
-
-
-
-
-
-
0.65
0.95
0.95
0.95
-
-
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
-
-
-
-
-
-
0.85
0.95
0.95
0.95
-
-
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
-
—
-
-
-
-
0.95
continued..
633
-------
TABLE P-50. (CONT'D)
Aeent n\
n-> 01 0.05
Fecal Agents — Spring 1982
KLB-X 68
KLB-W 70
OOB-X 71
OOB-W 71
PBW-X 70
PBW-W 71
VIR-X 72
VIR-W 77
WWI-X 65
WWI-W 69
42 0.00
42 0.03
42 0.00
42 0.00
42 0.01
42 0.03
42 0.08
43 0.14
40 0.06
41 0.12
A
0.07 0.10 0.15
c
0.60 0.80
0.70
0.65 0.80
0.65 0.80
0.55 0.75
0.70
0.60
0.50
0.60
0.50
0.20
0.90
0.85
0.90
0.90
0.90
0.85
0.75
0.70
0.80
0.70
0.25
0.95
0.95
0.95
0.95
0.95
0.95
0.90
0.85
0.90
0.85
Fecal Agents — Summer 1982
KLB-X 59
KLB-W 65
OOB-X 65
OOB-W 66
PBW-X 65
PBW-W 65
VIR-X 79
VIR-W 80
WWI-X 59
WWI-W 64
21 0.05
23 0.14
23 0.00
24 0.02
23 0.03
24 0.03
26 0.08
26 0.09
19 0.14
22 0.20
_ _ _
_ _ _
0.50 0.70
0.60
0.55
0.55
0.50
_ _ _
_ _ _
_ _ _
0.60
0.50
0.80
0.75
0.70
0.75
0.70
0.65
-
-
0.75
0.70
0.90
0.85
0.85
0.85
0.80
0.80
0.60
0.60
Fecal Agents — Spring 1983
KLB 60
OOB 60
PBW 60
VIR 62
WWI 59
47 0.00
47 0.03
45 0.03
47 0.00
45 0.03
0.60 0.80
0.70
0.70
0.60 0.80
0.65
0.90
0.85
0.85
0.90
0.85
0.95
0.95
0.95
0.95
0.90
Fecal Agents — Summer 1983
KLB-X 65
KLB-W 67
OOB-X 67
OOB-W 68
PBW-X 62
24 0.05
26 0.07
26 0.01
26 0.03
23 0.00
0.50
_
0.65
0.60
0.65
0.70
0.65
0.80
0.75
0.80
0.80
0.80
0.90
0.85
0.90
continued...
634
-------
TABLE P-50. (CONT'D)
Agent
Jll-
-Bl-
0.05 0.07 0.10
0.1S
0.20
0.25
Fecal Agents—Summer 1983 (Cont'd)
PBW-W
VIR-X
VIR-W
WWI-X
WWI-W
68
69
72
60
69
26
25
25
21
26
a Approximate power
b See Table 112 for
0.
0.
0.
0.
0.
09
01
06
05
17
calculations use
exact periods of
0.
- - 0.
_
_
the method of Fleiss
observation.
65
50
et
0
0
0
0
0
al.
.65
.80
.70
.65
.50
(1980)
0.
0.
0.
0.
0.
•
80
90
80
75
70
635
-------
GLOSSARY
Study Objective
The general objective of the LISS was to identify possible adverse
effects on human health from slow rate (sprinkler) land application of
wastewater which contained potentially pathogenic microorganisms. More
precisely, the objective was to determine the association, if any, between
the occurrence of infectious diseases in residents and workers and their
exposure to the wastewater and aerosols produced by wastewater spray irri-
gation. This objective was accomplished by disease surveillance of the
study population, by description of the distribution of infections, and
principally by evaluation of the incidence of infections for association
with exposure.
Disease Surveillance
Disease surveillance was the continuing scrutiny of all aspects of
occurrence and spread of infectious diseases in the study population.
Included were the systematic collection and evaluation of self-reported
illness information, investigation of cases and outbreaks for source of
illness, isolation and identification of infectious agents from routine
and illness specimens, testing sequential blood samples for evidence of
infection, and other relevant epidemiological data. The primary function
of this activity was the protection of the population from any obvious
untoward effects.
Illness Prevalence Density
The illness prevalence density was defined as the number of person-days
of self-reported illness per 1000 person-days of observation.
Illness Incidence Density
The illness incidence density was defined as the number of new illnesses
reported per 1000 person-days of observation.
Bacterial Infection
A fecal donor was considered to be having a bacterial infection when
an overt or opportunistic bacterial pathogen was isolated from a fecal
specimen at or exceeding a specified semiquantitative level which might
be associated with enteric disease. The levels equated with bacterial
infection were:
Category 1 any isolate of a major enteric bacterial pathogen (i.e.,
Salmonella or Shigella species, Campylobacter i e i un i . or
Yersinia enterocolitica);
636
-------
Category 2 isolation at the heavy level of a possibly significant oppor-
tunistic pathogen (i.e., API Group I, Candida alb ic ans.
Chromobacterium, Citrobacter. Klebsiella. Moreanella. Proteus.
Providencia. Serratia. and Staphylococcus aureus);
r
Category 3 isolation at the moderate or heavy level of selected organisms
found to be uncommon in feces but prominent in the sprayed
wastewater (i.e., Aeromonas hvdrophila and the fluorescent
Pseudomonas group: P. aeruginosa. P. fluorescens. and P.
putida).
Bacterial Infection Event
A bacterially infected fecal donor was considered to have had a bacterial
infection event since donation of the prior fecal specimen in the series
when the level of the organism in the prior specimen had been:
1) negative, for major enteric pathogens,
2) negative to light, for possibly significant opportunistic pathogens,
3) negative to light, for organisms prominent in the wastewater.
The criteria for a bacterial infection event were summarized for all three
bacterial pathogen categories in Table 10.
It was of primary interest to determine the bacterial infection status
of a routine fecal specimen donor in relation to a period of irrigation.
Routine specimens were collected from designated donors in scheduled weeks
before, during and near the end of each irrigation period (see Figure 2),
usually at intervals of about 6 and 4 weeks, respectively. Thus, the onsets
of bacterial infection events could be temporally related to wastewater
irrigation periods. When the change in infection status occurred between
the two specimens donated during an irrigation period, onset occurred in
the interim (i.e., during the irrigation period). When the change in infection
status occurred in consecutive specimens donated before and during the
irrigation period, it was uncertain whether onset occurred after irrigation
commenced. When a bacterial agent was not recovered at a level equated
with infection in either routine fecal specimen provided during an irrigation
period, the donor was considered to have experienced no infection events
by the agent during the observation period preceding and spanning the collection
dates of the consecutive specimens.
Viral Infection Event
A viral infection event was defined as the detection of a specific
virus by laboratory cultivation or by EM examination in the second and
not the first of paired fecal specimens from the same person. Subsequent
recovery of the same virus in a specimen from the same individual would
be a new event if more than 6 weeks elapsed between sequential recoveries.
Detection of a virus in the first of serial specimens was also considered
a viral infection event. Viral infection status was correlated with an
irrigation period in the same manner as bacterial infection status.
637
-------
Serological Antibody Titer
The serological antibody titer was the reciprocal of the highest serum
dilution at which a predefined endpoint of reaction was observed.
~~ r
Serological Infection Event (Serological Conversion)
A serological conversion (''sereconversion' ') was defined as a fourfold
or greater rise in agent-specific antibody titer in successive sera from
one individual that were tested simultaneously. Since successive sera
from 1982 and 1983 spanned an irrigation period and several additional
months (see Figure 2), it was not possible to determine if the onset of
serologically detected infection events was during the irrigation period.
Serological Infection Incidence Density (Seroconversion Incidence Density)
The serological infection incidence density was defined as the number
of serological infection events per hundred person-years of observation.
ID was calculated as:
Number of Serological
ion Events in Til
of Person-days (
During Interval
rr. Infection Events in Time Interval ,-,_ „. , . x /«AA \
ID = -—r 7-7, rr y x (365.25 days/yr) x (100 yr)
Number of Person-days Observed * J *
Infection Episode
An infection episode was defined as the observation in the study population
of a number of similar infection events (either serologically, microbiologi-
cally, or clinically) within a restricted interval of time. The minimum
number of infections which constituted an infection episode was set by
determining the number of infections that would be needed to reject the
null hypothesis (of no association between infection status and wastewater
exposure), assuming that all of the infections occurred in the high exposure
group and no infections occurred in the low exposure group. Infection
episodes were classified as exposure situations when the observation period
corresponded to one or two major irrigation periods and when the causative
agent was found (or could be presumed) to be present in the wastewater
at that time. Infection episodes were classified as control situations
when the causative agent could not survive in wastewater (i.e., influenza
A) or when the episode preceded the start of irrigation. Each exposure
and control infection episode was statistically analyzed for association
with wastewater aerosol exposure.
638
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