&EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
EPA/600/1-90/005b
September 1990
Waterborne Disease
Outbreaks
Selected Reprints of
Articles on Epidemiology,
Surveillance,
Investigation, and
Laboratory Analysis
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EPA/600/1-90/005b
September 1990
Waterborne Disease Outbreaks
Selected Reprints of Articles on
Epidemiology, Surveillance, Investigation,
and Laboratory Analysis
Edited by
Gunther F. Craun
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
-------
Notice
The opinions presented in this document are those of the authors and do not necessarily reflect the
views of the Agency. Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
11
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Preface
This volume is dedicated to the engineers, sanitar-
ians, microbiologists, epidemiologists, and other
health officials who have labored throughout this
century to improve the quality of our drinking water.
Because of their endeavors, we in the United States
and most developed countries now enjoy a quality of
life in which waterborne disease outbreaks are no
longer a constant threat or danger. This status has
been achieved by the tireless efforts of many and
often with little public recognition.
Although the incidence of waterborne disease has
been drastically reduced in the United States, we
must not forget that infectious diseases still remain a
threat for billions of people in the developing world
where water supplies are not as well protected from
sewage and may be inadequately treated. The lessons
we have learned are important for reducing the
worldwide incidence of waterborne diseases. We must
also continue our vigilance because a residual
number of outbreaks still occur in the United States,
and this indicates the vulnerability of our water
supplies.
We have learned that many of our water sources are
inadequately protected and treated to prevent the
transmission of giardiasis. The waterborne trans-
mission of this disease was first reported in the
United States in 1965 and has increasingly been
reported since 1971. Giardia is now the most
commonly identified pathogen in waterborne out-
breaks. Contaminated surface water is a significant
source of infection for giardiasis, and almost half of
the outbreaks of waterborne giardiasis have occurred
in surface water systems where the only treatment
was disinfection. In addition to Giardia, a number of
new etiologic agents have been recognized in recent
years and as laboratory analysis and investigation
procedures improve, more agents are likely to be
identified. Some of the newly recognized waterborne
pathogens include Cryptosporidium, Campylobacter,
Yersinia, hemorrhagic E. coli 0157:H7, rotavirus,
and Norwalk agent. This volume also describes a
chronic diarrhea for which an agent has yet to be
identified despite extensive laboratory analyses.
Only a fraction of the waterborne outbreaks that
occur in the United States are recognized, inves-
tigated, and reported, and in only half of the reported
waterborne outbreaks was an etiologic agent iden-
tified. We must improve the investigation and sur-
veillance of waterborne disease to identify the causes
of these outbreaks and the etiologic agents. The
identification of Giardia as an important waterborne
pathogen stimulated the research which has
identified the types of filtration, operating param-
eters, and disinfection levels necessary for removal
and inactivation of this pathogen. Water treatment
research is now being conducted to obtain similar
information for Cryptosporidium.
The emphasis of this volume is on drinking water
systems and the prevention of infectious waterborne
diseases. We must improve surveillance, reporting,
epidemiologic investigations, and laboratory anal-
yses, and we cannot rest on past accomplishments.
Continuing microbiological challenges are a remind-
er that our task to eliminate waterborne illness is yet
unfinished.
G.F.C.
ui
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Contents
Preface iii
Acknowledgments vi
I. Introduction 1
Epidemiology in Search of Infectious Diseases: Methods in Outbreak Investigation 2
II. Disease Surveillance 7
Waterborne Disease Outbreaks in Colorado:
Three Years' Surveillance and 18 Outbreaks 8
A Three-State Study of Waterborne Disease Surveillance Techniques 12
Routine Coliform Monitoring and Waterborne Disease Outbreaks 14
A Follow-up Study of Gastrointestinal Diseases Related to
Bacteriologically Substandard Drinking Water 18
Risk Factors for Endemic Giardiasis 21
Epidemiologic Surveillance for Endemic Giardia Lamblia Infection in Vermont 24
Cryptosporidiosis and Surface Water 31
III. Investigation of Waterborne Disease Outbreaks 35
Community Health Effects of a Municipal Water Supply
Hyperfluoridation Accident 36
An Epidemic of Gastroenteritis Traced to a
Contaminated Public Water Supply 39
Chronic Diarrhea Associated with Drinking Untreated Water 47
Epidemic Giardiasis Caused by a Contaminated Public Water Supply 54
A Waterborne Outbreak of Cryptosporidiosis in Normal Hosts 59
Large Community Outbreak of Cryptosporidiosis Due to
Contamination of a Filtered Public Water Supply 62
Waterborne Outbreak of Cryptosporidiosis 67
What Happens When the Multiple Barrier Concept Is Neglected 68
Chlorination to Prevent and Control Waterborne Disease 74
IV. Sampling and Analytical Methods 79
Small Round Structured Viruses: The Norwalk Family of Agents 80
DNA Hybridization Probe for Clinical Diagnosis ofEntamoeba histolytica 84
Diagnosis of Giardia lamblia Infections by Detection of Parasite-Specific Antigens 90
Identification of Cryptosporidium Oocysts by Monoclonal Antibody 95
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Acknowledgments
A number of previously published articles have been reprinted in this volume to
supplement information contained in a separate publication, Methods for the Investigation
and Prevention of Waterborne Disease Outbreaks. These articles provide examples of
outbreak investigations and surveillance activities. They have been reprinted to illustrate
various aspects of epidemiologic methods and engineering principles and compiled as a
convenience to those investigators with limited access to library facilities. Articles have
also been included on new clinical procedures for viral and protozoan pathogens as these
are discussed only briefly in the methods volume. The contributions of the authors of the
journal articles are gratefully acknowledged.
Permission to reprint these articles has been provided by: Journal of Epidemiology and
Community Health, American Journal of Public Health, Journal of Environmental
Health, American Journal of Epidemiology, Annuals of Internal Medicine, The New
England Journal of Medicine, Lancet, Journal American Water Works Association, and
Journal of Clinical Microbiology.
This report has been reviewed by the U.S. Environmental Protection Agency and approved
for publication.
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I. Introduction
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Reproduced, with permission, from Journal
of Epidemiology and Community Health.
Journal of Epidemiology and Community Health, 1989, 43, 311-314
Review article: Research methods in epidemiology, III
Epidemiology in search of infectious diseases: methods
in outbreak investigation
S R PALMER
From the Communicable Disease Surveillance Centre (Welsh Unit), Cardiff Royal Infirmary, Newport Road,
Cardiff CF2 1SZ, UK.
The value of epidemiology in the investigation of
incidents of infectious disease in the UK does not
seem to be always readily appreciated within
microbiology' 2 or environmental health.3 Yet
because microbial disease is a result of the interaction
of the agent, the host and environmental factors4
neither laboratory diagnosis nor environmental
inspection are of themselves sufficient for successful
investigation and control. Simply isolating microbes
from the environment, even if appropriate samples are
available, cannot distinguish between a reservoir of an
agent and the outbreak source or cross contamination
from the reservoir or source. Microbiologically
plausible explanations of outbreaks may be wrong,
but it is often difficult to persuade investigators of the
need to test such hypotheses.
On the other hand, whilst epidemiology may have
evolved from the study of infectious disease epidemics,
academic epidemiology seems in the main to have
passed infectious diseases by in the UK. This is
regrettable as there is still the need to consolidate the
foundations of a section of public health medicine
which has refused to disappear.5 This review, in
following the steps taken in an outbreak
investigation,6 draws attention to both the usefulness
of epidemiology and to regularly recurring
methodological problems which may be encountered.
Describing the outbreak
The classical "shoe leather" approach to infectious
disease incidents is the detailed investigation of
individual cases in the light of the well established
microbiological features of the particular infection
and a confidence in the possible routes of
transmission.7 The background incidence of infectious
diseases in the UK is usually well known and is
sufficiently low not to require sophisticated statistical
methods of cluster analysis8 to recognise a local
outbreak. However, with newly recognised infections
or diseases presumed to be caused by an unknown
infectious agent, surveys of disease incidence or
laboratory isolation rates may be necessary to confirm
that an outbreak has occurred. The methodological
problems encountered are similar to those found in
investigating clusters of cancers.8
The cases first reported in an outbreak are usually
only a small and sometimes biased sample which may
mislead, rendering control measures ineffective.
Identification and definition of the exposed
population is necessary to enable thorough case
finding to be carried out. Paradoxically, however, a
single case which does not fit the usual characteristics
of time, place and person may provide the most
valuable evidence of the source of infection.7
Data from cases are characterised by time, place and
person.6 The epidemic curve, a plot of the number of
cases over time of onset of illness, is the most useful
and immediate means of assessing the type of
outbreak. In point source outbreaks in which all cases
are exposed at a given time, onset of symptoms of all
primary cases will cluster within the range of the
incubation period. For each organism there is a
characteristic range within which infecting dose9 and
portal of entry, as well as host factors10 (eg, age,
immunosuppression) give rise to individual
variability. In outbreaks propagated from person to
person the occurrence of cases will be spread over
several incubation periods with peaks at intervals of
the incubation period. In larger community outbreaks
of diseases spread by person to person the epidemic
curve is usually smoother and the peaks at the
generation time of the new cases less obvious. Despite
their almost universal use in outbreak investigations,
there has been little formal evaluation of their use in
practice. Sartwell in 1950 established that the
frequency distribution of incubation periods of
infectious diseases of both long and short incubation
311
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312
period followed a log normal curve.11 lie suggested
(lie use ol the dispersion factor, (he anlilogarilhm ol'
Ihe logarithmic standard deviation, to measure
variation in incubation periods. Such analyses may be
used to distinguish between point source and
continuing source outbreaks, estimate Ihc data ol' a
common exposure, determine time of maximum
infecliousness, help explain pathogenic mechanisms,12
'dcnlily Ihe occurrence of secondary spread, anil
evaluate control measures. However, Armenian and
Lilienfckl have claimed (he initial efforts of Sarlwell
"have not been followed by any systematic review of
the data on incubation periods".13
Developing ii hypothesis
The traditional distinction between descriptive
(hypothesis generating) and analytical (hypothesis
testing) studies has been criticised by Rothman,14 a
criticism which holds true in outbreak investigation.
At the simplest level cpidemiological investigation
may be described in two stages: Instly, generating a
hypothesis of causation from descriptive data
analysed by lime, place and person, and secondly,
lesting thai hypothesis by a case-control or cohort
stuily. liul in almost all outbreaks competing a priori
hypotheses are voiced at Ihe outset by public health
investigators, patients, employers, food producers,
elc. Some ol these hypotheses may be excluded by the
"descriptive" data. These descriptive dala in turn may
stimulate alternative hypotheses. Indeed, as in clinical
investigation of an individual patient, this process of
hypothesis generation anil refutation goes on
continuously as new cases are being interviewed, and
challenges the rigid dichotomy of descriptive versus
analytic. l;or example, in an outbreak ol'.Sulinmii'lla
eating, cases were mainly infants who were discovered
to have been fed on a single brand of milk formula.15
Comparison with a group of well infants showed a
significant difference in use of this brand. Was this
comparison lesting the hypothesis, or could such a
design only firm up the hypothesis—the hypothesis
seemingly was generated on the same set of cases as it
was "tested"? In such outbreaks a priori hypotheses
are formulated on Ihe prior knowledge of the
epidemiology of salmonellas and, in this case, on the
observation of the age distribution of eases. The search
for a common milk formula led to Ihe infants followed
a prediction based on the deduction lhal Ihe mosl
likely vehicle of infection in infants was formula feed.
In some outbreaks the descriptive epidemiology
suggests hypotheses for which there are no biological
explanations'6 '7 (eg, a cluster of three casesof aplasliu
anaemia in children"1) or which go against the
accepted microbiological theory (eg, an outbreak of
shingles'''1 2I). In the practice of infectious disease
control, Ihe framework of proven microbiological
cnuses and modes of transmission enables statistical
data to be interpreted confidently. Once outside
known causal pathways statistical associations are
difficult to interpret and public health action, like Ihe
clinical diagnosis of an individual patient, usually is
taken on the basis of clinical experience and instinct.
Hypothesis testing
When hypotheses are formally tested the
epidemiological techniques usually used are
retrospective cohort and case-control studies. In
certain outbreaks, eg, food poisoning outbreaks in
institutions or following receptions, it is usually
possible to identify retrospectively and follow up the
whole population to identify simultaneously
exposures and'disease slalus. (This itself may lead to
biases which are described below.) Comparison of
attack rates in exposed and unexposed (eg, food
specific allack rales) or exposures in cases and controls
will often implicate the source. Paradoxically, when a
vehicle is universal, eg, lap water, it may be impossible
to demonstrate an association with illness unless ihere
is a dose response (which is less likely if the vehicle of
infection is heavily contaminated). In addition to
universal problems of matching, confounding and
appropriate analysis (here are regularly recurring
problems of design and implementation in outbreak
investigations.
SAMI'I.ING BIAS
Sampling may not be required at all as Ihe si/.e of the
population at risk or the number of cases is usually
small. I lowever (he corollary of this is lhal Ihe power
of possible studies in outbreak investigations cannot
usually be controlled ' by Ihe investigators.
Nevertheless, the speed at which such studies can be
done by experienced investigators, and the relatively
low cosl of Ihe studies, as well as the potential benefits
of demonstrating an association between infection and
a possible source, mean that studies of low power will
usually still be worth carrying out.
Diagnostic suspicion bias"2 in case selection is
always a problem when a possible vehicle of infection
is widely publicised. For example, people who have
eaten a food believed to be the source of an outbreak
of botulism may present with neurological symptoms
unrelated to botulism. When laboratory confirmation
of diagnosis is not possible the only safeguard is to
choose cases which meet the strictest clinical case
definition (and therefore who are at the most severe
end of the disease spectrum).
In case control studies Ihe selection of controls
always poses a problem of practicality versus validity.
Sampling frames commonly used to select controls
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Methods in outbreak investigation
include general practitioner age/sex registers, hotel
and reception guest lists, family members of cases,
neighbours of cases, and persons investigated by the
laboratory but who were negative for the disease in
question. When diagnostic access bias22 is considered
potentially, important (eg, cases of enteric infections
are only recognised in the main if they have general
practitioners who regularly recommend faecal
sampling), it is important to match for neighbourhood
of residence, or use patients investigated by
laboratories but found to be negative, since failure to
do so may lead to a spurious association with, say, a
restaurant in the neighbourhood of the "keen" general
practitioner. However, using general practitioner lists
may result in overmatching. Obtaining controls from
general practitioner registers is laborious and slow,
and in our experience about 5-10% of general
practitioners are reluctant to cooperate.
Increasingly, acquaintances nominated by cases are
used as controls15 although the representativeness of
such groups and possible biases they may introduce
have not been evaluated systematically. It is possible,
for example, that if cases know that a particular
exposure is the source of the outbreak their
nomination of controls will be influenced.
Additionally, the case may know the exposure history
of several acquaintances and reveal this with their
names to the interviewer, so possibly influencing the
interviewer's choice of controls. A protocol to avoid
this may be derived, although in practice there is the
danger of a conflict of priorities when local
investigators, eg, environmental health officers with
their own local responsibilities for outbreak
investigation, are called upon to carry out the national
protocol.
Misclassification of controls can be avoided by
asking all the individuals in the cohort or control
group about symptoms over the appropriate time
period so that those who may be unrecognised cases
can be excluded or reclassified. The feasibility of
serotesting, swabbing or otherwise testing controls to
exclude or reclassify asymptomatic infected persons
should always be considered. However, few well
people are willing to give a sample of faeces, and
obtaining blood samples from well children is difficult
and may be considered by some clinicians to be
unethical.
MEASUREMENT BIASES
In all but the smallest outbreaks data are collected by
questionnaire, but often practicalities require groups
to be interviewed by a combination of telephone, mail,
face to face and proxy interviews. The sensitivity and
specificity of questionnaires is seldom considered. In
one study food consumption recalled by people 2-3
313
days after luncheon was compared with that observed
at the time and recorded on video tape.23 Only four of
32 attendees made no errors in reporting. The
sensitivity of the food consumption histories
questionnaire was 87.6% and the specificity 96.1%.
Thirteen percent of the respondents reported eating
"dummy" food items listed on the questionnaire but
not served at the luncheon. Such errors in recall can be
reduced by providing background details of events,
good questionnaire design, and making use of other
sources to check data such as diaries, menus,
discussion with relatives, etc. Validation of patient
histories is often not possible in practice due to the
nature of the exposures, the lag between exposure and
investigation and the urgency of investigation.
However, random errors in recall are unlikely to give
rise to false associations between illness and, for
example, a food item. Rather they will reduce the
power of a study to identify the true vehicle of
infection. For some types of data, such as history of
immunization, patient or parent recall are particularly
inaccurate and validation of vaccine history from
medical records is required.24
Loss of cases or controls from the study because of
refusal to be interviewed or failure to trace patients is
much less a problem in outbreak investigation since
the affected population is usually eager to know the
cause, and response rates should approach 100%.
Throughout the course of their illness cases will have
gone over in their minds possible exposures and their
recall may be biased by their own preconceptions or by
press and media speculations.
Cases will often have been interviewed on many
occasions (by general practitioners, environmental
health officers, community physicians) before an
analytical study is carried out, and this may have
introduced bias from suggestions made by
interviewers, as well as prompting a more detailed
recall. Family members may have suggested likely
sources of infection to the cases. Patients may have
anticipated the view of the interviewer if the suspected
cause of the outbreak has been learned from local
media reports and they may alter their answers,
accordingly. These biases can often be anticipated and
can sometimes be assessed in analysis. For example, in
a suspected waterborne outbreak of giardiasis in
Reno, Nevada, considerable local publicity occurred
before the case-control study was conducted.25
However, because of the long incubation period of
giardia many new cases became ill several days after
the water supply was treated and so media attention
persisted and water was still considered suspect.
However, increased water consumption was only
associated with cases exposed before the water supply
was treated. Bias from media reports was likely to have
affected later cases also but no association was found.
-------
314
A major methodological problem is (he bias which
may result from the interviewer knowing the disease
status of the person and having his own suspicion or
prejudice as to the source. Selective histories may be
taken in which the patient with salmonella is only
•usked in detail about a particular food. Blinded
interviewing is rarely possible since- resources are
limited (most outbreaks are investigated by only one
.or two people), anil indeed exposure history is usually
obtained at the same lime as disease history.
.Professionalism in the technique of interviewing, and
use of a structured questionnaire are the only
safeguards. Controls, who anyway have no special
reason to recall certain exposures, are usually
interviewed several days after cases, which adds to
problems of accuracy and detail of recall. To help
overcome this problem, questionnaires may include
memory jogging points related to notable events, etc.
It is important that the controls are interviewed as
carefully and thoroughly as the cases were.
Conclusion
Do epidemiological studies in outbreak investigations
have to be "quick and dirty" or can they be "quick and
clean"? They must be quick. There is a need to develop
and evaluate epidemiological methods in outbreak
investigation so that they are both practical and
.sound. Hpidemiological standards for the practice of
the newly re-established discipline in public health
medicine -communicable disease control - must be
developed \so that attributed causes of outbreaks are
not left to be a "matter of opinion".2'1
Utfcrciicc's
1 Anonymous. Stanley Royd: the epidemiological lesson, lir
Med J. 1986; 292: 644 5.
2 Powell OKU. Salmonella cnlerilidis. Lancet 1989; i: 99.
3 North R. .•/ failure <>t government. A report on ilie mA of
/'(Kill puixiHiiiif; from i-jf^.v anil i'KK I'riiiliicls. Bradford:
Toodguard Publications, 1989.
"* Bracliman I'S. The control of infectious diseases. In:
Holland WW, Delels R, Knox Ci, eds. Oxfordlexllwok of
pt/hlic liciillli, Vol 2. Oxford: Oxford University Press,
1985: 8 18.
5 Kviins AS. The eradication of communicable diseases:
myth or reality? Am J KpidcinMl 1985; 122: 199 207.
''Gregg Mil. The priiH'iplcs of an epidemic Held
, investigation. In: Holland WW, Delels R, Knox G, cds.
Oxford lexlhook of pnhtie health. Vol 2. Oxford: Oxford
University Press, '1985: 284 99.
7 Galbraith NS. The application of epidemiological methods
in the investigation and control of an acute episode of
infection. In: Holland WW, Delels R, Knox (i, eds.
Oxford ti'Mhinik tifpnhlic health, Vol 4. Oxford: Oxford
University Press, '1985: 321.
"Openshaw S, Craft AW, Charllon M, Birch JM.
Investigation of leukaemia clusters by use of a
geographical analysis machine. Linnet 1988; i: 272 3.
'' Blaser MJ, Newman I.S. A review of human salmonellosis
I. Infective dose. Rev Infect /)/v 1982; 4: 109ft I Oft.
10 Khoury MJ, Handcrs WO, Greenland S, Adams MJ On
the measurement of susci-plihilily in cpidcmiologic
studies. Am J Lpidemiol 1989; 129: 18.1 90.
" Sartwell PI1'. The tlisliJlnilion of incubation pounds of
infectious disease. Am .1 llyx 1950; 51: .110 18.
12 Sarlwell PK. The incubation period and the dynamics of
infectious disease. Am J lipiilemiol 1966; 83: 204 16.
13 Armenian UK, l.ilienfeld AM. Incubation period of
disease. Kpidemiol Kev 198.1; 5: I 15.
14 Rolhnian KJ. Modern cpiile»ii<>lot;i'. Boston, Toronto:
Little Brown, 1986.
15 Rowe B, Begg NT, I lulcliinson DN, el i//. Salmonella
ealing infections associated with consumption of infant
dried milk. hineet 1987; ii: 900 .V
"'Scliwarl/ RS, Callen JP, Silva J. A cluster of llodgkin's
disease in a small commnnily. Am J K/iitlemiul 1978; 108:
19 26.
17 Ciordis L. Challenges to epidemiology in the next decade.
Am J Kpidemiol 1988; 128: I 9.
111 Morgan GJ, Palmer SR, Onions O, Anderson M,
("artwrighl RA, Benlley DP. A cluster of three cases of
apliistie anaemia in children. Clin l. llucmiiiiil 1988; 10:
29 32.
I>J Palmer SR, Caul HO, Donald Ol-:, Kwantes W, Tillell II.
An outbreak of shingles1.' l.aneel 1985; ii: 1108 II.
20 PeloTKA, (iilks CI-', Juel-Jensen UK. Clusters of shingles.
Laiiert 1985; ii: 14.1.1.
21 Palmer SR, 'lillell II. Shingles clusters. Lancet 1986, i:
27.1 4.
22 Siickelt 1)1.. Bias in analytic lesearch. ./ ('hrnnie /)/'.v 1979;
M: 51 63.
-'Decker MO, Booth AL, Dewey MJ, l-'ricker RS.
llulclieson Rll, SchalTncr W. Validity of food
consumption histories in a I'oodhorne oulbieak
investigation. Am J Kpulemiiil 1986, 12-1, 859 6.1.
24 Orenslein WA, Bernier Rll, Oondero TJ, el ,il. l-'ield
evaluation of vaccine efficacy. Hull H'llO 1985; 63:
1055 68.
25 NavinTRJuranek DO, ford M, Minedew D.I. l.ippy K.C,
Pollard RA. C'nse control study of walei borne giaidiasis
in Reno, Nevada. Am J Kpidemitit 1985; 122: 269 75.
26 I louse of Commons Agriculture Committee. Salmonella in
I'XKS- l''irxl Report, Vol 2 (Minnies of evidence and
Appendices). London: IIMSO", 1989.
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II. Disease Surveillance
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AMERICAN
JOURNAL
OF
Public
Health
Reproduced, with permission,
Journal of Public Health
reprint
American
Waterborne Disease in Colorado:
Three Years' Surveillance and 18 Outbreaks
RICHARD S. HOPKINS, MD, PAMELA SHILLAM, BS, BARRY GASPARD, BS,
LINDA EISNACH, MPA, AND RICHARD J. KARLIN, PE
Abstract: The Colorado Department of Health conducted inten-
sive surveillance for waterborne diseases during the three-year
period July 1, 1980-June 30, 1983. Eighteen outbreaks of water-
borne illness were investigated. Outbreaks involved from 15 to 1,500
ill persons. Giardia lamblia was confirmed or suspected as the agent
in nine outbreaks, rotavirus in one, and no agent could be identified
in eight. Seventeen outbreaks occurred on surface-water systems;
none of these had adequate chemical pretreatment and filtration.
Investigation of water systems exhibiting positive coliform results
during the first year detected no outbreaks. Activities important to
effective surveillance included educational outreach programs to
local health agencies, physicians and the public, and the designation
of one individual to whom all water-related public complaints and
health department inquiries were directed. (Am J Public Health
1985; 75:254-257.)
Introduction
There has been a steady increase in the number of
reported waterborne disease outbreaks occurring in the
United States since the early 1960s. The average annual
number reported for 1971-72 was 32; from 1980-82 the
average was 41.1.1 The actual number of outbreaks that
occur is certainly greater than this.
Over a three-year period prior to June 1980, six water-
borne outbreaks were reported in Colorado but about 20
additional clusters of gastrointestinal illness was suspected
to be waterborne. Energy development, tourism, and popu-
lation growth combine to place stress on water systems that
typically have relied on protected or remote surface sources
of supply with marginal treatment.
Prior to'June 1980, the Colorado Department of Health
had a passive waterborne outbreak surveillance system, and
responsibility for follow-up of water-related complaints was
divided among two Sections of the Water Quality Control
Division and the Communicable Disease Control Section.
From June 1980 through June 1983, a field epidemiolo-
gist was made responsible for improving the detection of
waterborne illness.* The project was housed in the Commu-
nicable Disease Control Section, Division of Disease Control
and Epidemiology, which receives reports of communicable
disease cases from the state's 63 counties.
Methods
In the first year, surveillance was expanded to include a
daily review of coliform results from the required testing of
community and non-community water systems. When posi-
tive samples were received, physicians, pharmacists, public
health nurses, and schools in areas served by the affected
water systems were promptly surveyed by telephone for
: *US Environmental Protection Agency contract #68-03-2927, June 1980-
June 1982.
Address reprint requests to Dr. Richard S. Hopkins, Colorado Depart-
ment of Health. Communicable Disease Control Section, Division of Disease
Control and Epidemiology, 4210 E. 11th Avenue, Denver. CO 80220. Authors
Shillam, Gaspard, and Eisnach are also affiliated with that Division; author
Karlin is with the Drinking Water Section. Water Quality Control Division,
CDH. This paper, submitted to the Journal May 7, 1984, was revised and
accepted for publication October 15, 1984.
© 1985 American Journal of Public Health 0090-0036/85 $1.50
evidence of an outbreak of gastrointestinal disease, without
waiting for check samples.
Throughout the project, engineers in the Water Quality
Control Division supplied information from their inspections
of water systems that indicated a potential hazard, and
investigated water systems when a waterborne outbreak was
suspected. In the second and third years of the project,
questionnaires were mailed to all persons with laboratory-
confirmed giardiasis reported to state or local health depart-
ments. Two or more laboratory-confirmed giardiasis case
reports linked by time and place or two or more independent
citizen reports from the same area of excessive illness
suspected of being waterborne prompted an active surveil-
lance effort as described above.
In May 1981, an education program directed toward
county health officials was instituted; this included formal
presentations to I'O of the 13 largest county health depart-
ments. Public health nurses and sanitarians were encouraged
to report to one another and to the project office unusual
occurrences of disease and problems discovered in local
water systems.
Public awareness about waterborne diseases was
heightened through media coverage of outbreak investiga-
tions. Complaints from the public about illness or poor
quality drinking water were directed from the engineering
sections to the waterborne disease project.
The state health department's bi-weekly Colorado Dis-
ease Bulletin (circulation 4000) was used to inform private
physicians and local health agencies of the project and to
solicit reports of potential waterborne disease problems.
Outbreak investigations were described in the Bulletin to
maintain interest in the project.
Community surveys conducted in the investigation of all
potential waterborne outbreaks sought information about
various symptoms, onset dates, duration, amount of water
consumption prior to illness, and water supply source. The
standard water question was: "How many 8-ounce glasses
of unheated tap water, or drinks made up with unheated tap
water, did you drink on an average day before (date of
outbreak)?' These surveys were usually conducted via
telephone, using modified random digit dialing techniques
when a water system's distribution area approximated an
area served by all or part of one telephone exchange.
Samples were randomized and consisted of at least 40
households. In several investigations dealing with persons
who had dispersed from a common site, mailed question-
naires were used.
254
AJPH March 1985, Vol. 75, No. 3
-------
WATERBORNE DISEASE OUTBREAKS IN COLORADO
An outbreak was considered to be waterborne when a
positive dose-response relationship between amount of wa-
ter consumed and attack rate could be demonstrated and
when there was a significant difference in attack rate be-
tween persons drinking from the implicated supply and those
drinking no water from the implicated supply. The project
protocol required a minimum of six stools from ill persons
for examination and ideatification of pathogenic agents
including bacteria (Salmonella, Shigella, Campylobacter
and Yersinia), intestinal parasites, and viruses and the same
number from well controls. When a viral outbreak was
indicated by symptom profile, the protocol also required
collection of paired sera from a minimum of six ill and six
well individuals. Viral specimens were frozen and sent to the
Health Effects Research Laboratory, US Environmental
Protection Agency (EPA), Cincinnati, Ohio. Bacteriological
and parasitological laboratory analyses were conducted at
the Colorado State Laboratory by standard methods. Be-
cause of late notification of outbreaks, not all indicated
specimens could be collected in all outbreaks.
In suspected waterborne outbreaks, water samples were
collected and examined for total coliforms and fecal coli-
forms. In outbreaks of suspected giardiasis, 1,000 gallons
each of treated and untreated water were filtered through a
one micron filter and the filter examined for the presence of
Giardia cysts by Dr. Charles Hibler of Colorado State
University.
Results
Eighteen outbreaks of gastrointestinal disease were
shown to be waterborne. An additional 10 outbreaks were
strongly suspected of being waterborne. Twelve reports of
outbreaks were sufficiently suspicious to warrant significant
further investigation, but most were shown to be due to
person-to-person spread, not waterborne.
Etiology of Outbreaks
Nine of the 18 waterborne outbreaks are considered to
have been caused by Giardia lamblia, although confirmation
of the responsible agent is not equally good in all nine (Table
1). One was associated with rotavirus.2 No agent was
identified for the remaining eight outbreaks. In four of these
outbreaks (#1, 2, 3, 6) which resembled Norwalk virus
gastroenteritis clinically, epidemiologic investigation began
at a time when no new cases were occurring and a decision
was made not to collect stool or serum samples. In the four
others (#13, 16-18), the symptom profile suggested bacterial
or parasitic disease but laboratory tests on ill persons for
bacteria and parasites were negative.
In one of the presumed giardiasis outbreaks (#9), initial
reports of numerous laboratory confirmed giardiasis cases
dissuaded us from aggressive pursuit of stool specimens only
to find that, in fact, physicians' diagnoses were based on
clinical picture. In another (#7), all the symptomatic cases
had already been treated by the time we were able to collect
stool specimens. The only stool positive for Giardia lamblia
was from a well, untreated camper.
Source of Notification of Outbreaks
Eleven of the 18 waterborne outbreaks were brought to
the attention of local health departments by citizens com-
plaining by telephone of illness in themselves and their
neighbors; four were brought to state health department
attention directly by such citizen phone calls. Three out-
breaks were picked up by local health departments in the
course of routine active disease surveillance, as clusters of
illness were reported by doctors' offices when the local
health department made its regular weekly telephone con-
tact. Two outbreaks were reported by physicians, and two
outbreaks came to our attention through a combination of
direct contacts by ill persons and ongoing review of positive
Giardia lamblia results in the state laboratory.
No outbreaks were found through 12 months of daily
review of positive coliform test results from state water
systems although an average of two unsafe coliform results
per week were followed up. This activity was therefore
discontinued in subsequent years.
Epidemiologic Data
In all 18 outbreaks, a statistically significant association
was found between self-reported water consumption and
illness (Table 1). In 11 of the 18 outbreaks, a control group
on a different water supply was interviewed and had a
significantly lower attack rate. Community attack rates
ranged from 10 per cent to 64.4 per cent. Outbreaks occurred
in all seasons, but 11 of 18 occurred between June 1 and
September 30.
Type of Water System
Community water systems** accounted for 15 of the 18
confirmed and seven of the 10 suspected waterborne out-
breaks. One confirmed outbreak (#2) was related to a well
not intended as a drinking-water supply, and the remainder
(two confirmed, three suspected) were on non-community
systems. There are 715 community water systems and 1,250
non-community systems registered with the Colorado De-
partment of Health. The largest system with a community-
wide problem served approximately 3,500 persons. One
outbreak (#1) occurred on a supply serving approximately
50,000 people, but only a few hundred were affected by the
cross-connection.
All but one of the outbreaks (#1) occurred on systems
using surface water as the source of supply. In three
outbreaks (#11, 16, 17), the sources were shallow wells in
glacial alluvium immediately adjacent to a river; pollen
grains and other particles characteristic of surface water
were found when the "well" water was sampled through a
one micron filter.
Coliform Counts and Chlorine Residual
Coliform counts were elevated at or near the time of the
outbreak in seven outbreaks, normal in nine, and unknown
in two. Chlorine residuals were inadequate in six, adequate
in eight, and unknown in four outbreaks. In only one of the
nine documented or suspected Giardia lamblia outbreaks
were there elevated coliforms and absent disinfectant at the
time of the outbreak. In one Giardia outbreak (#9), coliform
counts indicated safe water on two occasions during the
outbreak but were elevated two weeks after the outbreak
stopped.
Deficiency Responsible for Outbreak
One outbreak (#1) was caused by a contaminated well
and another (#2) by a cross-connection. The remaining
outbreaks occurred on systems without adequate chemical
pretreatment, filtration, and chlorination. In the nine water-
borne outbreaks attributed to Giardia lamblia, there was
either no filtration at all (six outbreaks), a malfunctioning
filter and decreased chlorine contract time (one outbreak), or
"Defined as 15 or more service connections and/or 25 or more residents
year-round.
AJPH March 1985, Vol. 75, No. 3
255
-------
HOPKINS, ET AL.
TABLE 1—Epidemlologlc Features of 18 Waterborne Disease Outbreaks, Colorado, 1980-83
Out-
break
No.
1.
2.
3.
4.
5.
6.
7.
B.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Date
6/80
7/80
8/80
3/81
81
9/81
781
11/81
11/81
12/81
1/82
7/82
8/82
9/82
2/83
5/83
6/83
6/83
Estimated
# of
cases
15
34
38
1500
100
600
29
40
85
135
unk
28
600'
28
50
80
300
370
Per
Cent
Attack
Rate
28.3
70.2
64.4
32.0
24.0
22.0
28.0
18.6
29.2
10.0
15.1
35.0
27.3
53.8
17.2
10.0
34.9
. 11.4
Relative Risk1
(90% C.I.)
4.5(1.21,6.7)
8.46(3,4,21.06)
1.56(1.13, 2.16)
2.7(1.70, 5.38)
3.09(1.52,6.27)
2.86(1.51,5.4)
2.22(1.17, 4.20)
4.21 (1.09,
16.28)
4.0(1.71,9.33)
8.12 (2.86,
23.03)
2.3 (.98, 5.42)
2.92(1.32,6.48)
3.34(1.01,
1 1 .02)
2.71 (1.59,4.63)
6.4(1.73,23.65)
_
4.54 (.74, 27.7)
2.18 (.66, 7.2)
6.00 (.71, 51.1)
Syn-
drome2
C.D.V
D
D,V
D,C,V
D,C
D,V
0
D
D
D
D,C.
C,D
D,V
D,C
D.C.V
D.C
D,C
D,V,C
Duration
1-2 days
3-5 days
2-3 days
3-4 days
3-1 4 days
2-3 days
7-10 days
7-14 days
4-30 days
(mean
8.5)
7-14 days
5-30 days
(mean
15.5)
7-30 days
2 days
5-30 days
3-20 days
3-30 days
1-5 days
1-10 days
Agent
Implicated
unknown
unknown
unknown
Rotavirus
Giardia
lamblia
unknown
Giardia
lamblia
Giardia
lamblia
Giardia
lamblia
Giardia
lamblia
Giardia
lamblia
Giardia
lamblia
unknown
Giardia
lamblia
Giardia
lamblia
unknown
unknown
unknown
Evidence for Agent
—
—
Positive serology &
stools in cases
clinical picture
Positive stools in
cases, clinical
picture, water filter
showed large
particles passing
through
—
Clinical picture in
cases, positive
water filter
Positive stools in
cases, negative
stools in controls,
clinical picture,
positive filter
Clinical picture,
engineering
evaluation
Positive stools
Positive stools in
cases, clinical
picture, positive
filter
Stools syndrome
—
Stools syndrome,
positive filter
Stools filter
—
—
—
Agents Cause of
Ruled Out3 Outbreak
— Cross-connection
SS.C Laundry well used
for drinking
water
SS,C Chlorinator failure,
unfiltered
supply
SS.C.EEC Chlorinator failure,
filtration
inadequate
— Unfiltered surface
source
— Unfiltered surface
source
— Filter off line
SS.C Shortened chlorine
contact time,
filter inadequate
— Unfiltered pond
water backed
up into spring
box
SS No pretreatment
before filtration
No pretreatment
before filtration
SS
C Unfiltered surface
source, heavy
runoff
S,C virus Unfiltered surface
source, heavy
runoff
unknown Unfiltered surface
source
— Filtration with
inadequate
pretreatment
G.C.SS Filter
overwhelmed
by heavy runoff
G Filter
overwhelmed,
heavy runoff,
inadequate
pretreatment
G,C No filtration, heavy
runoff
1. For persons drinking more than the median amount of water versus those drinking less than the median
2. C - cramps, D = diarrhea, V = vomiting
3. SS = Salmonella/Shigella. C = campylobacter, G = Giardia lamblia, EEC = Enteropalhogenic E. coli
filtration without any chemical pretreatment (two out-
breaks). In the four suspected or confirmed viral outbreaks,
effective filtration was also absent, but the precipitating
event was a Chlorinator failure (two outbreaks), bypass of a
Chlorinator (one outbreak), or very high raw water turbidity
leading to low free chlorine residuals (one outbreak). Out-
breaks #16, 17, and 18 were associated with very high raw
and finished water turbidity during spring runoff, when
filters were overwhelmed or bypassed.
Specific precipitating events were found for only two of
the suspected or confirmed giardiasis outbreaks: backup of
beaver pond water into a spring house in #9 decreased
chlorine contact time compared to the normal condition in
#8.
256
AJPH March 1985, Vol. 75, No. 3
10
-------
WATERBORNE DISEASE OUTBREAKS IN COLORADO
Discussion
Some ol'lhe lessons we have learned from Ihis surveil-
lance experience will be summarized briefly. Daily monitor-
ing and follow-up of colilbrm tests results failed to uncover
any gastroenteritis outbreaks in the communities involved.
Increased communication with county health personnel,
sanitarians, public health nurses, and physicians apparently
paid oil', although we were still hampered by delay between
the occurrence and reporting of illness. The presence of a
single person who reviewed all complaints and all communi-
cable disease reports and initiated investigations was impor-
tant.
Resort areas may pose special problems in identifying
outbreaks. Unsolicited reports from out-of-state visitors
alerted us to four of the waterborne outbreaks eventually
attributed to Giardia. In the Winter Park outbreak (#11),
local physicians had seen no increase ia diagnosed cases of
giardiasis in residents, but letters from visiting skiers con-
vinced us that a problem existed. A survey of out-of-state
visitors to the area showed an attack rate of 15.1 per cent for
visitors drinking from (he town's main water system during
the first two weeks of February. Most residents are not
served by the town water but most visitors stay in hotels and
condominium units served by that water. Long-term resi-
dents may have lower attack rates than residents even when
comparably exposed.4
The decision to classify outbreaks in this study as
waterborne was based on demonstrations of a dose-response
relationship between water consumption and illness. As long
as over- and underestimation of water consumption are not
associated with illness status, the magnitude of any observed
association between illness and amount of water consumed
should be an underestimate.
Ill persons may selectively recall higher water consump-
tion than well persons. Interviewer bias is also possible. In
the Eagle-Vail outbreak (#4), however, there was no associ-
ation between reported water consumption and gastrointesti-
nal illness during the two weeks prior to the outbreak, even
though the respondents clearly thought these illnesses were
.also waterborne.2 In Aspen Highlands (#8), a relationship
was seen'between illness and amount of water consumption
in short-term but not long-term residents."
Analysis of the data from these two outbreaks using the
method described by Marshall et a IS shows that 30 to 40 per
cent of ill persons would have had to overestimate their
water consumption by two or more glasses per day (while no
well persons overestimated their water consumption) in
order to show a dose-response relationship significant at the
.05 level when none really existed. Differential misclassifica-
tion would have to be considerably more common than this
to cause a spurious relationship at the levels seen in most of
the outbreaks reported here.
The attribution of outbreaks to particular agents was the
most difficult and frustrating part of these investigations.
Only in one instance. Aspen Highlands,4 were these major
criteria for agent clearly met:
• The agent should be found in ill persons significantly
more often than in well persons from the affected community
or from an unaffected control community;
• The agent should be found in the epidemiologically
incriminated source, the water; or there should be an appro-
priately timed event that would have allowed the presumed
.agent to pass through the treatment system.
A positive water sample from a sampling filter is not by
itself sufficient evidence that a waterborne outbreak is
caused by Giardia lamhliu. We have been able to find
morphologically intact organisms in finished water from
communities with no current outbreak.*** Nonetheless we
have classified two outbreaks (#7, 9) as presumed giardiasis
when there was an appropriate clinical syndrome for giardia-
sis, evidence of walerborne spread of disease, and evidence
by filtration sampling for Giardia la/nblia in the water, even
in the absence of positive stools in ill persons.
The patterns seen in this waterborne disease surveil-
lance effort are consistent with the efficacy of the multiple
barrier concept in preventing watciborne disease. None of
the 17 surface-water outbreaks occurred on the many sys-
tems which have coagulation, flocculation, sedimentation,
filtration, and disinfection.
Bacteriological standard violations were associated with
only one of the Giardia outbreaks (#14). Giardia lamblia is
relatively resistant to chlorine disinfection. The large num-
ber of coliform organisms relative to Giardia lamblia in
wastes has not allowed the presence of coliforms to predict
the presence of Giardia.
None ol'lhe Ginrd'ui outbreaks had turbidity violations.
Turbidity is a poor measure of the microbiological quality of
water in areas (such as Colorado) where raw water turbidi-
ties are low. Systems with raw water turbidities below the
1.0 NTU standard often discontinue chemical addition ahead
of filtration since it is not needed to meet the standard.
Filtration without chemical pretreatment has been shown to
be ineffective in removing Giardia-tized particles.h
Although beaver and other animals have been implicat-
ed as Giardia carriers, ubiquitous human and domestic
animal activity has led us to the conclusion that all surface-
water supplies are at risk for Giardia lamblia.'Since 1977.
Colorado has required that all surface water be completely
treated, including coagulation, flocculation, sedimentation,
filtration, and chlorination steps. This requirement is sup-
ported by the absence of reported outbreaks of all types
(Giardia and viral) where full treatment is employed. Such
systems are expensive for small communities. Effective
inexpensive surface water treatment methods could substan-
tially reduce the risk of waterborne disease.
"*L.apham SC. Hopkins RS. White M, Blair JR. Smadcs RH, Bissel RC.
Simpson (il..1 Giardiasis in travelers 10 Colorado. A prospective .study of 484
visitors. (Manuscript submitted Tor publication.)
REFERENCES
I Water-related Disease Outbreaks. 1982 Surveillance Summary. Atlanta:
Centers for Disease Control, August 1983.
2. Hopkins RS, Uaspard GB, Williams KP. Karlin RJ. Cukor G, Bladlow NR:
A community walerborne gastroenteritis outbreak: evidence for rotavirus
as the agent. Am J Public Health 1984: 74:263-26?.
3. Rothman K I, Boicc JD' Hpidemiologic Analysis with a Programmable
Calculator. NIH Pub. No. 79-1649:36-37. Washington, DC: Govt Printing
Ofliec, 1979.
4. Istre GR. Dunlop TS. Gaspare! GB. Hopkins RS: Waterborne giardiasis at
a mountain resort: evidence tor acquired immunity. Am J Public Health
1984: 74:602-604.
5. Marshall JR. Priore R. Graham S. Brasure J: On the distortion of risk
estimates in multiple exposure level case-control studies. Am J Epidemiol
1981: I 1.3:404-473
6. I.ogsdon GS. L.tppy t:C: 1 he role of filtration in preventing waterborne
disease. J Am Water Works Assoc 1982; 74:649-655.
This rescaich snppi
Contract #68-0.3-2927.
ACKNOWLEDGMENT
•ted in pail by Hnviiomncnlal Protection Agency
AJPH March 1985, Vol. 75, No. 3
257
11
-------
AMERICAN
JOURNAL
OP
Public
Reproduced, with permissions
Journal of Public Health
reprint
American
A Three-state Study of Waterborne Disease Surveillance Techniques
LUCY HARTER, FLOYD FROST, PHD, RICHARD VOGT, MD, ANN A. LITTLE, RN,
RICHARD HOPKINS, MD, BARRY GASPARD, AND EDWIN C. LIPPY, MS, PE
Abstract: For a two-year period, the states of Colorado, Ver-
mont and Washington tested the effectiveness of ten surveillance
methods for identifying waterborne disease. Nine were active sur-
veillance methods, soliciting illness reports; one was passive, rely-
ing on voluntary disease reporting. One waterborne disease out-
break was identified through use of the nine active methods, while
14 were reported through the passive surveillance method. The
presence of coliform bacteria during routine water testing was not
related to illness in the community. (Am J Public Health 1985;
75:1327-1328.)
Introduction
Since the early 1960s, there has been a steady increase
in the reported number of waterborne disease outbreaks in
the United States.' An average of 41 outbreaks were re-
ported annually, 1979-83. The increased number of reported
outbreaks may reflect more complete reporting rather than
an actual increased risk.
To examine the methods for improving surveillance of
waterborne disease, the US Environmental Protection
Agency (EPA) funded three two-year projects by the State
Health Departments of Colorado (1980-82), Vermont
(1980-82), and Washington (1981-83). Although each project
differed somewhat in approach, all were designed to test
alternative surveillance techniques and to improve reporting
of what would have been previously unreported waterborne
disease. Detailed individual reports are available from each
state.2"1
Methods
Before the implementation of the special surveillance
projects, Colorado, Vermont, and Washington detected
enteric diseases and disease outbreaks through passive sur-
veillance systems, based upon voluntary reporting with little
or no effort devoted to soliciting specific illness reports
related to contaminated drinking water. Disease reports
from physicians, citizens, and health officials were investi-
gated when there was evidence of an outbreak. Reportable
diseases which are caused by potentially waterborne patho-
gens were reviewed and investigated when drinking water
appeared to be a possible source of infection. Salmonellosis
and shigellosis are reportable in Colorado, Vermont, and
Washington; as of 1984, Colorado and Vermont report
giardiasis and campylobacteriosis.
During the project period, each of the three states
expanded waterborne disease surveillance activities. A full-
time project epidemiologist was employed in each state;
Address reprint requests to Lucy Harter. Division of Health, Washington
Department of Social and Health Services. Mail Stop ET-I3, Olympia. WA
98504. Dr. Frost is affiliated with the same Department; Dr. Vogt and Ms.
Little are with the Vermont Department of Health, Epidemiology Office,
Burlington; Dr. Hopkins and Mr. Gaspard are with the Colorado Department
of Health. Epidemiology and Disease Control Division. Denver; Mr. Lippy is
with the US Environmental Protection Agency, Health Effects Research
Laboratory, Cincinnati. This paper, submitted to the Journal December 21.
1984. was revised and accepted for publication May 13. 1985.
-------
PUBLIC HEALTH BRIEFS
TABLE 1—Active Waterborne Disease Surveillance Methods In Colorado, Vermont, and Washington
Method
Daily Review of Mandatory Collform Tests
Weekly Telephone Surveillance of Schools, Industries, Camps
One-Time Customer Surveys of 12 Water Systems with Water
Quality Problems
Twice Weekly Additional Coliform Tests of Selected Water Systems
Pathogen Testing of 60 Water Systems with Water Quality
Problems.
Surveillance of Laboratory Confirmed Giardiasis Cases
Surveillance of Laboratory Confirmed Yersiniosis and
Campylobacteriosis Cases
Surveillance of Private Laboratories for Increased Enteric Work
Requests
Surveillance of Camps, Parks, Ski Programs, Recreational
Organizations
Passive Surveillance Enhanced with Staff Epidemiologist and Public
Education
Time Period (State)
6/81-5/82 (CO)
9/81-12/81 (VT)
12/80-9/81 (VT)
4/81-3/83 (WA)
2/82-1 2/82 (VT)
4/81-3/83 (WA)
4/81-3/83 (WA)
6/81-5/82 (CO)
4/81-3/83 (WA)
4/81-3/83 (WA)
4/81-3/82 (WA)
6/80-5/82 (CO)
12/80-12/82 (VT)
4/81-3/83 (WA)
Total Months
of Observation
15
9
—
10
—
36
24
12
12
72
toring of laboratory confirmed infections, the passive sur-
veillance system provides the most timely reports. There is
an inherent delay in laboratory based surveillance. Stool
sample collection, sample delivery to the laboratory, and
specimen analysis take one to three weeks, whereas, a
physician can become aware of illness in a community and
report it within days of the onset.
Under the Safe Drinking Water Act, coliform bacteria is
the criterion used to judge the microbiological safety of
drinking water. In this study, routine and intensive surveil-
lance of water systems with coliform violations did not
reveal illness among water users. Coliform bacteria were
elevated in only 10 of the 15 outbreaks which occurred
during the project period. The presence of coliforms failed to
predict illness in the community. The absence of coliforms
did not assure safe drinking water. Although coliform bac-
teria can be indicative of water contamination, it appears
less reliable as an indicator of health risk.
Except in Colorado, the surveillance activities that were
undertaken in this study did not result in more reported
outbreaks of waterborne disease. The methods used in
Vermont and Washington were either too insensitive to pick
up additional outbreaks or the current passive surveillance
systems employed by the states are adequate. The special
surveillance methods described in this paper may be more
useful for other local and state public health agencies if
current surveillance is inadequate.
REFERENCES
1. CDC: Water related disease outbreaks in the US 1980. MMWR 1982;
30:623.
2. Hopkins RS, Shillam P, Gaspard B, Eisnach L, Karlin RJ: Waterborne
disease in Colorado: three years surveillance and 18 waterborne outbreaks.
Am J Public Health 1985; 75:254-257.
3. Vogt RL, Little AA: Vermont Waterborne Disease Project. Final Report
on EPA contact #68-03-2967. Vermont Department of Health, Burlington,
VT 05402.
4. Harter LC. Frost FJ, Perkins-Jones K, Holman R: Washington State
Waterborne Disease Surveillance Study. Final Report on EPA Contract
#68-03-3059. Washington Department of Social and Health Services.
Olympia, WA 98504.
1328
13
AJPH November 1985, Vol. 75, No.11
-------
Reporduced, with permission, from Journal
of Environmental Health.
Routine Coliform Monitoring and
Waterborne Disease Outbreaks
Odette Batik
Gunther F. Craun
Wesley O. Pipes
Abstract
To provide protection against the transmission of waterborne disease
all public drinking water systems are required to monitor for coliform
bacteria at a prescribed frequency. However, waterborne outbreaks have
previously been documented in public water systems which have not ex-
ceeded these coliform regulations. A comparison of coliform monitoring
results in a sample of both community and non-community water
systems showed no statistical differences in coliform results between
systems in which an outbreak had occurred and systems in which no out-
breaks had been reported. Although the results of this study must be
interpreted cautiously, the authors feel that public health officials should
reevaluate the importance of routine coliform monitoring in their
surveillance program for the prevention of waterborne disease.
All public water systems, which in-
clude both community systems ser-
ving at least 25 year-round residents
or 15 service connections and non-
community systems serving transients
or temporary residents, are required
by the U.S. Environmental Protec-
tion Agency (EPA) to routinely moni-
tor for coliform bacteria(6). The fre-
quency of coliform monitoring in-
creases as the size of the population
served increases, with systems of 1000
persons or less required to collect
only one sample per month. It is not
until a system serves a population of
25,001 that 30 samples per month are
required. There is no requirement
that samples be collected daily, and
no rationale is provided in the EPA
regulations to support the frequency
of monitoring.
Journal of Environmental Health, V. 45 (5)
227-230.
Odette Batik, Columbia University,
School of Medicine, New York, NY 10027;
Gunther F. Craun, U.S. Environmental
Protection Agency, Cincinnati, Ohio
45268; and Wesley O. Pipes, Department of
Biological Sciences, Drexel University,
Philadelphia, Penn. 19104.
March/April, 1983
The coliform group has been used
as an indicator of pollution for some
70 years because they are present in
the normal intestinal flora of humans
and warm-blooded animals and are
eliminated in large numbers in fecal
wastes. The absence of coliform or-
ganisms in a public water supply is
generally interpreted as evidence of a
safe drinking water free of water-
borne pathogens(6). Implicitly, rou-
tine coliform monitoring of drinking
water is to provide protection against
transmission of waterborne disease.
Outbreaks of waterborne disease
have occurred in water systems where
the maximum contaminant level
(MCL) for coliforms has not ex-
ceeded EPA regulations(3,4,5). Case
studies have suggested that the cur-
rent EPA regulations for coliform
monitoring frequency, especially for
small systems, are inadequate to pro-
tect against transmission of water-
borne disease and that it is inappro-
priate to use coliforms for monitoring
surface water systems where disinfec-
tion is the only treatment(3). Anec-
dotal information indicates it is also
possible to detect coliforms in water
systems that have not reported out-
breaks.
14
Journal of Environmental Health
A study was conducted to compare
coliform monitoring results between
water systems experiencing a water-
borne outbreak and systems in which
an outbreak had not been reported
and to determine if reported water-
borne outbreaks could have been pre-
dicted on the basis of violations of the
coliform MCL.
The study consisted of two separate
but related investigations, one of
community water systems and one of
non-community water systems. In
both cases only the data from existing
reports of outbreaks and records of
coliform monitoring results were uti-
lized.
Community Systems
Waterborne-disease outbreaks
were reported in 47 community water
supplies in the United States during
the period between 1971-1977. Water
supply officials from states in which
these outbreaks occurred were con-
tacted, and coliform records for three
years (the year preceding, following,
and during the time the outbreak oc-
curred) were requested. Records for
an identical period were also re-
quested for comparison communities
in which an outbreak had not been
reported.
-------
Two groups of comparison com-
munities were selected. The first
group was selected with the aid of the
state water supply officials: a com-
munity of about the same size, with
the same type of water source, and
that employed the same type of water
treatment procedures as the outbreak
community. To minimize differences
in outbreak reporting and water qual-
ity surveillance practices (e.g. number
of coliform samples collected) be-
tween the comparison and outbreak
community, the comparison commu-
nity was selected from the same gen-
eral geographic area (health district)
as the outbreak community. The
second group of comparison commu-
nities was selected at random from an
inventory of water supplies in each
county in which an outbreak occurred
after excluding systems not within
15% of the same size as the outbreak
system. No statistical differences in
demographic characteristics were
found between the two groups of
comparison communities.
These specific questions were
examined: (a) Were positive coliform
results more likely to occur in the out-
break community during the three
months prior to the outbreak? (b)
Over the total three-year period, was
the coliform standard exceeded more
often for those communities experi-
encing outbreaks? (c) When positive
results were obtained over the three-
year period, were there more coliform
organisms per sample in the outbreak
communities?
Unfortunately, much of the data
requested was not made available: no
monitoring results existed for 10 wa-
ter systems in communities experienc-
ing an outbreak; four systems had
been monitored, but the records had
been discarded; for 16 systems the
records requested were not made
available by the state health officials.
Records were provided for 17 com-
munity water systems experiencing an
outbreak but not all could be used
either because the recording methods
prevented determination of all re-
quired variables or because the rec-
ords for the comparison community
had been discarded. Only 32% of the
originally selected data set remained
for analysis. Thirteen pairs of com-
munities selected with the aid of state
officials and 10 pairs from the ran-
dom selection procedure had suffi-
cient information for comparison.
228
Regardless of the manner in which
the comparison community was se-
lected, there were eight instances
where the community experiencing an
outbreak and its comparison commu-
nity had similar coliform results in
the three months prior to the out-
break: in four instances both commu-
nities had positive coliform results,
and in four instances both had nega-
tive coliform results.
Because of the small number of
pairs of communities with different
coliform results, Cox's(2) exact test
for discordant pairs was used to
evaluate the first question (Table 1).
The second and third questions were
evaluated by paired comparison pro-
cedures using Students' t test (Table
2). The outbreak and comparison
communities were not found to
differ in regard to positive coliform
results within the three months prior
to the outbreak (Table 1). It is reasur-
ing, however, that when different
coliform results were obtained be-
tween the community experiencing an
outbreak and its comparison commu-
nity, the trend was toward detection
of coliforms in the outbreak commu-
nity arid negative coliform tests for
the comparison community during
the three prior months. With respect
to the number of times the coliform
standard was exceeded per number of
samples collected and mean number
of coliform organisms in the water
system during the entire three year
period, no statistical difference was
observed between outbreak and com-
parison communities (Table 2).
Non-Community Systems
In this study, coliform monitoring
results for non-community water sys-
tems experiencing an outbreak were
Table 1
Community Water Systems
Comparison of Coliform Monitoring Results Three Months Prior to Outbreak
A. Comparison Community Selected by State
(matched on size, water source, water
treatment, location)
Comparison Community
Outbreak Community
Positive Test
Negative Test
Total Pairs
P(1) = 0.188
Comparison Community Selected at Random
(matched on size, location)
Positive Negative
Test Test
4 4
1 4
8
Total
Pairs
8
5
13
Outbreak Community
Positive Test
Negative Test
Total Pairs
P(1) = 0.250
4
0
4
2
4
6
6
4
10
Table 2
Community Water Systems
Comparison of Coliform Monitoring Results During 3-Year Period
Mean Coliform Levels
Multiplied by
Number of Times Number of Times
Standard was Exceeded per Standard was Exceeded per
Number of Samples During Number of Samples During
•' 3-year Period 3-year Period
Outbreak Communities vs.
Comparison Communities
Selected by States
(n = 1 5 pairs)
t = 0.27
P(1) = 0.395
t = 1.07
P(1) = 0.151
Outbreak Communities vs.
Comparison Communities
Selected at Random
(n = 1 5 pairs)
t = 1.72
P(1) = 0.054
t = 1.26
P(1) = 0.1 14
15
Journal of Environmental Health
Vol. 45, No. 5
-------
compared to non-community systems
not reporting an outbreak in Pennsyl-
vania during 1973-1978. Pennsylvan-
ia was chosen because it reported the
largest number of outbreaks during
this period, reflecting a dedication to
identifying and investigating water-
borne disease outbreaks. Thirty-nine
waterborne disease outbreaks in non-
community systems were reported
from Pennsylvania in this six year
period.
Data on coliform monitoring of
non-community water systems were
obtained from county offices. During
1973-1978, non-community water
systems in Pennsylvania were in-
spected at least once a year by a sani-
tarian and were sampled for coliform
analysis if they were considered un-
satisfactory with regard to protection
and construction or if the disinfection
device was not functioning properly.
Data were obtained on 34 outbreaks
occurring in 18 different counties.
There were five instances where two
outbreaks of waterborne disease were
reported for the same system. The 29
non-community systems with re-
ported outbreaks included 12 restau-
rants, 12 campgrounds, two recrea-
tional areas, one church, one apart-
ment complex, and one fire house.
However, no coliform monitoring
data were available (apparently no
samples had been taken) for 13 of the
29 establishments. This eliminated
the church, the apartment complex,
the fire house, the two recreational
areas, two campgrounds, and six
restaurants from the analysis. Coli-
form monitoring data were also ob-
tained for the remaining 798 non-
community water systems for which
no waterborne disease outbreaks had
been reported in these 18 counties.
These included 715 restaurants, 81
campgrounds, and two recreational
areas.
The Chi-square test for comparison
of two proportions in an independent
sample was used to determine if coli-
forms had been found in a larger pro-
portion of the establishments with re-
ported outbreaks than of the estab-
lishments with no reported out-
breaks. Discriminant analysis was
also employed in an attempt to iden-
tify which of several variables were
the best predictors of outbreaks of
waterborne disease in non-commu-
nity systems.
This analysis also showed it is poss-
ible to find coliforms in non-commu-
nity systems for which there are no re-
ported outbreaks and to have out-
breaks in non-community systems for
which there are no positive coliform
monitoring results. No difference in
positive coliform results was found
between non-community systems ex-
periencing an outbreak and those not
experiencing an outbreak (Table 3).
For the discriminant analysis, the
dependent variable considered was
waterborne outbreaks (yes or no) and
the independent variables were estab-
lishment type, type of water treat-
ment, total number of coliform sam-
ples collected over the five year per-
iod, and the average coliform density.
The only variable showing a statisti-
cal association was the total number
of coliform samples collected over the
five year period, inferring that non-
community water supplies with out-
breaks are sampled more frequently
than those without outbreaks. The
frequency of sampling is controlled
by a sanitarian who has responsibility
for inspecting and sampling non-
Table 3
Non-Community Water Systems
Comparison of Coliform Monitoring Results Three Months Prior to Outbreak
Coliform Results
Positive Negative
Result Result Total
Non-Community System
Experiencing an Outbreak
Non-Community System
Total
8
343
351
8
455
463
16
798
814
P(1) = 0.288
March/April, 1983
16
Journal of Environmental Health
community water supplies. Appar-
ently, the sanitarians are able to iden-
tify by sanitary surveys those water
supplies which are most likely to have
outbreaks and concentrate the sam-
pling where they think a problem
exists. However, the sampling of sys-
tems with identified problems did not
always cause an outbreak to be
avoided. These data provide no evi-
dence that coliform monitoring re-
sults provide any prediction of out-
breaks of waterborne disease in non-
community systems.
Discussion
These studies suggest that the data
obtained from routine coliform
monitoring required by EPA regula-
tions may be of little help to public
health officials in preventing the
transmission of waterborne disease in
public water systems; however, these
results must be cautiously interpreted
for several reasons. The study of
community water systems included
only 32% of the communities which
experienced an outbreak during the
study period. This provided a rather
small sample which may not be repre-
sentative of communities experi-
encing outbreaks. There is also in-
complete reporting of waterborne
outbreaks and additional outbreaks
which were not investigated and re-
ported might have occurred during
this period. This would result in unre-
ported outbreaks which were not in-
cluded in the study, and it is also
possible that unreported outbreaks
occurred in one or more of the com-
parison communities. It appears un-
likely that comparison communities
used in this study experienced a
waterborne outbreak that was neither
investigated nor reported during the
time selected, as outbreak and com-
parison communities were matched
on both geographic location (same
local health district) and time to mini-
mize possible differential disease sur-
veillance. The water quality data for
both the community and non-com-
munity studies were collected from a
period before the EPA regulations
were applied and may represent in-
complete reporting and inadequate
monitoring conditions which no long-
er exist. It is not known to what ex-
tent these conditions could influence
results, but it is felt that differential
monitoring or reporting did not oc
cur. These data also represent results
-------
from a number of different labora-
tories, but this should not have an in-
fluence on interpretation of results,
as laboratory procedures are gener-
ally standardized for coliform moni-
toring.
The fact that no differences in coli-
form monitoring results could be
found between the systems with and
without reported outbreaks in this
study might be explained by several
other factors, which were observed
during analysis. Of the community
systems in the study, the median
population was 1,950 and half of the
communities in the study were re-
quired to sample only two times a
month or less. Most non-community
systems were sampled only once a
year. In instances involving sharp
upward fluctuations in contamina-
tion immediately prior to the time of
outbreak, routine sampling could
miss the period of contamination by
several days to several months. This
situation has previously been docu-
mented in several waterborne out-
breaks(3). Almost half of the out-
breaks in community systems were
caused by Giardia lamblia. Data from
waterborne outbreaks of giardiasis
have shown that Giardia cysts can be
recovered from drinking water meet-
ing the coliform standard, especially
for systems providing chlorination as
the only treatment, and that water-
borne giardiasis outbreaks can occur
in water systems meeting the coliform
standard(4,5).
There are several possible explana-
tions why the use of routine coliform
monitoring may be ineffective in pre-
venting transmission of waterborne
diseases. It is possible that the coli-
form test may neither be sensitive nor
specific enough to be useful in alert-
ing officials of the necessity to take
action to prevent the occurrence of an
outbreak. In addition, routine moni-
toring may not include enough sam-
ples to provide a good probability of
detecting the presence of coliforms or
an outbreak occurs so quickly after
contamination that it is highly un-
likely that coliforms would be de-
tected before the outbreak by means
other than continuous monitoring.
The elapsed time between sample col-
lection and transmittal of results to
operating personnel under current
regulations, even if contamination is
detected, may also be too lengthy to
permit corrective action to be taken in
230
a timely manner. A related factor
concerns the type of corrective action
generally stimulated by a positive
coliform result. Too often the initial
reaction to a positive result is the col-
lection of a check sample, as it is as-
sumed the result is due to a sampling
or laboratory error. Many times
check samples continue to be taken
until a negative coliform result is ob-
tained, and no positive action is ever
taken to determine how the contami-
nation occurred or if there was a
treatment or distribution deficiency.
Although the results of this study
must be interpreted cautiously and it
is recommended that additional stud-
ies of this type be conducted, it is,
nevertheless, felt that regulatory
agencies should carefully examine the
relative importance they have as-
signed to routine coliform monitoring
of tap water in their surveillance pro-
gram. It should be realized that the
primary reason for the establishment
of such surveillance programs is the
prevention of waterborne disease and
other kinds of surveillance activities
may be more effective and cost effi-
cient in this regard.
The American Water Works Asso-
ciation Committee on the Status of
Waterborne Disease in the United
States and Canada has just released a
report in which they recommend that
surveillance activities be structured to
help prevent waterborne disease(l).
Their conclusions on the usefulness
of the coliform test in preventing
waterborne disease are of interest:
"Routine coliform surveil-
lance of treated water is
mandated by the Safe Drinking
Water Act; however, the
committee feels that its
importance is to provide a
historical record of operation
rather than to prevent water-
borne outbreaks."
"The committee feels that it
is more important to know the
quality of the source water and
potential sources of contamina-
tion so that source protection
and treatment can be provided.
A water supply surveillance pro-
gram should emphasize fre-
quent engineering evaluation
and sanitary surveys to identify
and correct potential deficien-
cies. Microbiological resources
are better applied to assessing
17
Journal of Environmental Health
raw water quality, identifying
sources of contamination, and
evaluating the efficiency of
treatment than the routine sur-
veillance of water quality in the
distribution system. In some in-
stances, alternative microbi-
ological or chemical indicators,
such as chlorine residual analy-
sis, may be more useful in pre-
venting outbreaks than coliform
surveillance of the distribution
system."
References
1. Committee Report (1981) Waterborne Di-
sease in the United States and Canada, J.
Amer. Water Works Assoc. 73:528-529.
2. Cox, D.R. (1978) Analysis of Binary Data,
Methuen and Co. LTD, London, p. 56-58.
3. Craun, G.F. (1978) Impact of the coliform
standard on the transmission of disease,
Evaluation of the Microbiology Standards
for Drinking Water, C.W. Hendricks,
Ed., USEPA 570/9-78-006, USGPO,
Washington, D.C.
4. Craun, G.F. (1979) Waterborne giardiasis
in the United States: a review, Amer. J.
Publ. Health 69:817.
5. Craun, G.F. (1979) Waterborne disease
outbreaks in the United States, J. Environ.
Health 41:259-265.
6. U.S. Environmental Protection Agency
(1976) National Interim Primary Drinking
Water Regulations, USEPA 570/9-76-003,
USGPO, Washington, D.C.
Vol. 45, No. 5
-------
AMERICAN
JOURNAL
OF
Public
Reproduced, with permission, American
Journal of Public Health
reprint
A Follow-up Study of Gastro-lntestinal Diseases
Related to Bacteriologically Substandard Drinking Water
D. ZMIROU, MD, MPH, J.P. FERLEY, MD, J.F. COLLIN, PD, M. CHARREL, AND J. BERLIN, MS
Abstract: In a prospective follow-up study conducted in 52
French alpine villages, one weekly water sample was taken in each
village provided with untreated ground water and analyzed as to the
presence of four indicator bacteria: total plate count, total coliforms,
thermotolerant (fecal) coliforms, and fecal streptococci. Cases of
acute gastro-intestinal disease (AGIO) occurring among 29,272
inhabitants were reported through physicians, pharmacists, and
primary school teachers. A loglinear model identified fecal strepto-
coccus (FS) as the best predictor; the presence of fecal coliforms
enhanced the effect of FS. The total bacteria count and the total
coliforms had no independent contributions. A threshold analysis
suggested that any level of indicator bacteria above zero was
associated with an excess of AGID. (Am J Public Health 1987;
77:582-584.)
Introduction
Microbiology standards for drinking water are periodi-
cally evaluated.' The indicator bacteria used in the European
community to assess whether a 100 ml water sample is
potable are fecal coliforms (FC) and fecal streptococci (FS).2
Total coliforms are also used as indicator bacteria in many
countries, such as the United States.3 The dramatic decline
of "classical" waterborne diseases in developed countries
has lessened the concern of local authorities with these
issues. The frequency of substandard water samples is
relatively common in the alpine regions, but water-related
disease outbreaks are rather rare.4 Alpine health authorities
are highly concerned, however, about the increasing costs
and constraints of the water policies.
This study had the following objectives: to assess the
risks related to the consumption of drinking water that does
not meet current bacteriology standards; to identify the
indicator bacteria that best predict this risk; to determine
whether there is an indicator bacteria threshold other than
zero above which water should be declared "non-potable".
Methods
The study design is presented in detail elsewhere.5 Two
parallel surveys were conducted: 1) a weekly water sample
was taken in each of 52 villages and analyzed the same day
in a single central laboratory; and 2) a day-to-day count of the
acute gastro-intestinal diseases occurring among inhabitants
of the study villages and identified through 119 physicians, 52
pharmacists, and 118 primary school teachers (for children
aged 7 to 10 years). Physicians and pharmacists were located
in the villages or within a 10 km surrounding area. None of
these sources was informed of the findings of the weekly
water samples.
Villages were chosen to meet several criteria: population
between 100 and 3,500; non-tourist areas (to prevent wide
variations in village population across seasons); public water
systems with untreated ground water; sufficiently far from
large cities so that the cases of AGID seeking help could be
Address reprint requests to D. Zmirou, MD, MPH, Centre Alpin de
Recherche Epidemiologique et de Prevention Sanitaire, Centre Hospitaller
Universitaire, Pavilion D. B.P. 217 X, 38043 Grenoble CEDEX, France.
Ferley and Charrel are also affiliated with the Centre; Collin is with the Medical
School, Nancy, France; Berlin is with the Department of Biostatistics,
Harvard School of Public Health. This paper, submitted to the Journal
November 5, 1985, was revised and accepted for publication September 4,
1986.
1987 American Journal of Public Health 0090-0036/87$!.50
assumed to be completely enumerated by all the participating
professionals.
The limits of the public water system in each village were
known to the survey coordinators. Any case reported for an
individual not using the municipal water system was excluded
from the analysis. The village population was restricted
accordingly to users of the public water system.
Although 64 weekly samples were available for most
villages, because of technical problems, only 58 samples
could be analyzed for some villages. Of the 52 villages
available for study, two were excluded prior to examination
of the data because of especially large numbers (>14) of
missing observations, and two villages were excluded be-
cause the ground water they provided was subjected to
treatment half-way through the survey, leaving 48 villages in
the study group.
The bacteriologic assays conducted to ascertain the
presence of indicator bacteria were as follows: 1) "standard
plate count" (aerobic bacteria): 1 ml water cultivated on
standard medium (PCA Institut Pasteur Production), with a
24-hours incubation at 37° ± 1°C; 2) "total coliforms": 100 ml
filtered on cellulose membrane and cultivated on Tergitol
TTC during 24 hours at 37° ± 1°C; 3) "fecal coliforms"
(thermotolerant coliforms): same technique as for total
coliforms but incubated at 42° ± 1°C for 24 hours; 4) "fecal
streptococci: 100 ml filtered through cellulose membrane and
cultivated 48 hours at 37° ± 1°C on Slanetz and Bartley
medium (Institut Pasteur Production).
The 48 villages were followed for 64 weeks and contrib-
uted 3,072 "village-weeks". There were 1,807 cases of
gastro-intestinal disease reported during the study period.
This relatively small number of cases precluded analyzing
each village separately across weeks. We assumed that each
village-week constituted an independent observation. To
avoid being misled by errors in this assumption (i.e., in a
given village the water quality was liable to be correlated
across weeks), we used a variable that indicated whether
cases had occurred the previous week in the same village.
This variable was included in the multivariate model to adjust
for the experience of the previous week. The unit of obser-
vation was the village-week: the cases occurring during a
given week in a given village were related to the population
of the village (person-week) exposed to the water quality
indexed by the weekly sample.
Because of the sparseness of AGID and of the large
denominator (each week, 29,272 person-weeks were accrued
in the whole set of villages), the events were modeled using
a Poisson distribution.
582
18
AJPH May 1987, Vol. 77 No. 5
-------
GASTROINTESTINAL DISEASE AND SUBSTANDARD DRINKING WATER
TABLE 1—Risk of Acute Gastro-intestinal Diseases According to the
Fecal Conforms Exposure, in Absence of Fecal Streptococci,
by Village Size
Relative Risk*
Village Fecal Person-
Population Coliforms Cases weeks
Point 95% Confidence
Estimate Interval
<400
»400
Present
Absent
Present
Absent
3
127
17
221
20594
237 188
52438
777 252
27
1 14
[.14; .51]
[.67; 1.92]
*RR = rate-ratio
Log-linear analysis can be used with Poisson models and
accommodate categorical covariates6"8 yielding estimates of
relative risk. Further details on methods and data analysis are
discussed elsewhere.* The following covariates were includ-
ed as potential predictors or confounders of the AGIO
incidence rate: FS count had four levels (0, 1-5, 6-10, 11 and
over, per 100 ml); FC was a binary variable (absent, present);
so were total coliforms, total plate count, and village size
(under or over 400 inhabitants); and indicator of at least one
case the previous week.
The data were restricted to the cases occurring within a
three-day period centered on the water sampling day. This
restriction, which removed 60 per cent of the total cases from
the analysis was implemented to assess and control for a
possible misclassification of exposure. Cases far from the
sampling day, say the day before the next week or just after
the previous week, were liable to result from water pollution
as indexed by the water sample of the next week (or the
previous week).
The confidence intervals of the relative risks should be
computed using the covariances between various pairs of
variables. Unfortunately, the Loglin package* used on a Vax
11/780 computer does not generate covariance matrices.
Therefore, the confidence intervals of the relative risks
presented below were approximated using the appropriate
crude (i.e., unadjusted) data tables and the usual formulas for
the variance of rate ratios.9
Results
The total plate count and total coliforms had no inde-
pendent predictive value. When the water met the standards
(no FC nor FS), the incidence rate was 3.44 per 10,000
person-weeks when at least one germ was observed on the
total plate count; it was 3.43 per 10,000 person-weeks when
no germ was present. The corresponding incidence rates in
the presence of at least one coliform was 3.74 per 10,000
person-weeks, and 3.32 per 10,000 person-week when no
coliform was observed.
On the other hand, when both FS and FC were absent,
354 cases occurred, out of a total of 698 (51 per cent). Out of
these, 198 cases (53 per cent) occurred when the total plate
count was positive, the other half of the cases occurring while
no germ was found.
Table 1 presents the raw data for the effect of FC, in
absence of FS. This effect is somewhat unclear, and depends
on the village size category: in large villages, FC are not
TABLE 2—Relative Risk (and 95% Cl) due to Increasing Concentration of
Fecal Streptococci, by Level of Fecal Coliforms and Village
Size
Fecal Streptococci Count/100 ml
Village
Fecal
Population Coliforms 0*
1-5
6-10
11 +
<400
5*400
Present
Absent
Present
Absent
1 4.25 (2
1 1.40(1
1 2.72 (1
.1-8.6)
.0-2.0)
.6-^.1)
1 .89 (.7-1.2)
4.74 (1.6-9.6)
2.46
1.83
.71
(.8-7.7)
(.9-3.8)
(.3-1.6)
5.05
2.27
3.21
1.44
(2.8-9.0)
(.8-6.7)
(1.8-5.6)
(.6-3.2)
'Level 0 ol FS is the referent category; Relative Risk = 1
related to AGIO risk, whereas they seem protective in small
villages.
The incidence rates when no FS were found served as
baseline risk in further analysis. The absolute risk is rather
small; however, it is underestimated by a factor of 2.5
because only the cases occurring within the three-day period
about the sampling day were considered. One can infer from
Table 2 that the risk due to a given level of FS contamination
is higher when FC are also present; the risk due to a given
joint FC and FS exposure is higher in small villages than in
larger villages; in larger villages, no risk seems associated
with FS alone (i.e., when no FC are present), whereas they
suffice to predict a significant risk (although weak) in small
villages.
These data do not show a clear trend, but it is notable
that the highest concentration of FS corresponds to the
highest relative risk. The Mantel extension for the analysis of
trend10 was used on the crude data (i.e., unadjusted for the
previous week experience) and yielded a significant positive
trend only in small villages, in presence of FC [ slope = 6.0
x 10~5 cases per person-week per unit of FS increase; xi2
(slope) = 22.6; \22 = 4.6, testing departure from linearity].
This test for trend was done using the program written by
Rothman and Boice for HP.41, applied to incidence rate
data."
In almost all strata, the lowest positive level of FS (1 to
5) is associated with a point estimate of the relative risk
greater than 1. This class may be too large to assess whether
there is a threshold. Another categorization can be adopted,
where all concentrations greater than 5 bacteria/100 ml were
merged (thus any inference from this highly heterogeneous
class would be misleading). Table 3 shows how the relative
risk changes with small increases of FS concentration,
according to whether some cases had been reported the week
before or not. The risk is systematically enhanced when cases
had occurred previously. As to the threshold issue, no cut-off
point other than 0/1 can be clearly identified.
TABLE 3—Relative Risk (and 95% Cl) due to Increasing Concentrations
of Fecal Streptococci with Special Focus on Low Levels
(adjusting for the previous week experience)
Fecal Streptococci Count/100 ml
Cases Week Before 0
1-2
3-4
5 +
'Berlin J, Zmirou D: Poisson regression for the analysis of relative risk
(Unpublished manuscript), Harvard School of Public Health, 1985.
None
1 +
1 .79 (.6-1.1)
1 1.47(1.0-2.1)
1.56 (1.0-2.4)
2.45 (1.1-5.5)
1.40(1.0-1.9)
2.44 (1.8-3.3)
AJPH May 1987, Vol. 77 No. 5
19
583
-------
ZMIROU, ET AL
Discussion
The categorization of villages into "small" (<400) and
"large" (>400) populations split the total set of villages into
two groups of 24 villages each. Biologic and socioeconomic
considerations may contribute to the understanding of the
effect of the village size. In small villages, economic activity
(agriculture is more oriented toward cattle breeding) and/or
general hygiene and wealth may facilitate the spread of a
contamination, and make more dramatic the effect of a given
waterborne hazard, through direct interpersonal contamina-
tion, for example. This hypothesis is consistent with the
multiplicative model of risk that underlies the analysis.9
Although some work has been done on these issues, there is
still no unequivocal evidence as to the relationship between
indicator bacteria and pathogens, in drinking12 or non-
drinking13'14 water. In 1981, 44 per cent of the water-related
disease outbreaks reported to the Centers for Disease Control
(CDC) remained of unknown origin.15
This study showed that the four classes of indicator
bacteria had very different predictive value with respect to
disease. Total bacteria and total coliforms had no predictive
value; however, half of the cases of gastro-intestinal disease
occurred when the water met the standards. Half of these
were observed while only the total plate count was positive.
Those who are primarily interested in identifying which
indicator germ best predicts the risk of disease should be
concerned with FS and FC (as modifying the effect of FS).
Geldreich claims that the FC/FS ratio provides a useful
indicator as to the origin of the fecal pollution, human or
animal.16 In our study, the average FC/FS ratio was 4.0 and
was different in large villages (5.2) and in small villages (1.3).
This tends to confirm that the origin of the contamination in
small villages was more related to animals than in larger
villages. Those who want to set standards to decide whether
a water is safe or not might also be interested in the total plate
count. Finally the "background risk" (when all indicator
germs are absent) amounts to about one-fourth of total cases;
however, this figure may well depend on the specific condi-
tions of this study such as the frequency of the water samples
taken in the villages (one per week).
FS was the most predictive indicator and the validity of
the standard threshold (0 bacteria per 100 ml) cannot be
disputed with these data. We must recognize that our findings
depend upon the ecological context and may not hold, for
instance, in warm waters or treated waters. Surprisingly,
most of the cases of gastro-intestinal disease were sporadic;
throughout the 18 months of the survey, only one outbreak
was observed (with about 50 cases), even though 42 per cent
of the total water samples did not meet the bacteriology
standards. Hence, this study shed special light on endemic
water-related hazard, which may well be overlooked by
health authorities. Although modest, this risk persists, and a
lot remains to be done in order to provide a safe and
wholesome drinking water to the consumers.
ACKNOWLEDGMENTS
This study was supported by the French Ministry of Health, the Ministry
of Agriculture, and the Water Agency Rh6ne-M£diterranee-Corse. The Min-
istry of External Affairs also contributed to this work through a scholarship for
one of the authors. We acknowledge the invaluable help of N. Laird and J.
Ware (on the statistical analysis), and N. Gutensohn and G. Hutchison (on the
epidemiological interpretation), from the Harvard School of Public Health. The
support of J. Vial, president of the water section of the Council of Public
Hygiene of France, is also acknowledged.
REFERENCES
1. Hendricks CW (ed). Evaluation of the Microbiology Standards for Drink-
ing Water. USEPA 570/9-78-OOC; 1979.
2. Official Journal of the European Communities—No L229/11—Council
Directive No 80/778/EEC 15 July 1980 relating to the quality of water
intended for human consumption.
3. Federal Register: EPA-Water Programs. National Interim Primary Drink-
ing Water Regulations, 1982.
4. Collin JF, Foliguet JM: A propos de quelques epidemics d'origine
hydrique survenues en France. Microbiol Alim Nut 1983; 1:27-33.
5. Ferley JP, Zmirou D, Collin JF, Charrel M: Etude longitudinale des risques
li£s a la consommation d'eaux non conformes aux normes bacten-
ologiques. Revue Epidem et Sante Publ 1986; 34:89-99.
6. Bishop VMM, Fienberg SE, Holland PW: Discrete Multivariate Analysis:
theory and practice. Cambridge: MIT Press, 1975.
7. Laird N.Oliver D: Covariance analysis of censored survival data using
log-linear analysis techniques. J Am Stat Assoc 1981; 76:374, 231-240.
8. Frome EL, Checkoway H: Use of Poisson regression models in estimating
incidence rates and ratios. Am J Epidemiol 1985; 121:309-323.
9. Kleinbaum DG, Kupper LL, Morgenstern H: Epidemiologic Research.
Principles and Quantitative Methods. Lifetime Learning Publications
1982.
10. Mantel N; Chi-square tests with one degree of freedom; extensions of the
Mantel-Haenszel procedure. J Am Stat Assoc 1963; 58:590-700.
11. Rothman KJ, Boice JD: Epidemiologic Analysis with a Programmable
Calculator. Boston: Epidemiology Resources, Inc, 1982.
12. Petersen NJ, Hines VD: The relation of summertime gastro-intestinal
illness to the sanitary quality of the water supplies in six rocky mountain
communities. Am J Hyg 1960; 71:314-320.
13. Robertson WJ, Tobin AS: The relationship between three potential
pathogens and pollution indicator organisms in Nova Scotian coastal
waters. Can J of Microbiol 1983; 29:1261-1269.
14. Cabelli VJ: Health effects criteria for marine recreational waters, USEPA,
1983,600/1-80-031.
15. Harris JR, Cohen ML, Lippy EC: Water-related disease outbreaks in the
United States in 1981. J Infect Dis 1983; 148:759-762.
16. Geldreich EE: Fecal coliforms and fecal streptococcus density relation-
ship in waste discharges and receiving wastes. Crit Rev Environ Control
1976; 6:349-369.
584
20
AJPH May 1987, Vol. 77 No. 5
-------
Reproduced, with permission, American
Journal of Public Health
Risk Factors for Endemic Giardiasis
CHRISTOPHER G. CHUTE, MD, MPH, ROBERT P. SMITH, MD, MPH, AND JOHN A. BARON, MD, MS, MSc
Abstract: In a mail survey, 171 Hitchcock Clinic patients with
giardiasis were compared with an age- and sex-matched control
group of 684 clinic patients with respect to potential risk factors.
Households with shallow well or surface water sources had an odds
ratio (OR) for giardiasis of 2.1 (95% confidence interval (95%CI)
1.3-3.2) compared with households with drilled well or municipal
water supply. Other observed risks include family member in day
care program (OR 2.2, 95%CI 1.3-3.7) and family member with
diagnosed giardiasis (OR 17, 95%CI 7.4-37). Previously reported
risks such as travel out of country (OR 3.2, 95%CI 1.5-7.2) and
camping (OR 1.7, 95%CI 0.9-3.2) were also observed. Virtually no
giardiasis risk was observed associated with report of dog or
barnyard animal proximity. Control for confounding and adjustment
for recall and non-response bias does not materially alter the risk
estimates. We suggest that shallow well or surface household water
source is an important and previously unrecognized giardiasis risk
factor. (Am J Public Health 1987; 77:585-587.)
Introduction
Giardiasis is traditionally considered an epidemic dis-
ease, often occurring in waterborne community outbreaks. '~3
Recently, however, increasing numbers of isolated case
reports in New Hampshire and Vermont4-5 suggest multiple
sources of endemic infection.
Relatively little has been written about endemic or
sporadic giardiasis. Probable risk factors include mountain
camping,6 day care exposure,7 person to person contact,8'9
and travel abroad.10''' We evaluated these and other potential
endemic risk factors such as shallow well household water
sources and animal exposure.
Shallow or dug well household water sources were of
particular interest to us. Often old and deteriorating, these
shallow water sources are frequently open to surface con-
tamination, and are common in northern New England (20
per cent among our controls overall). Several animal species
have been implicated as potential giardiasis reservoirs.12"14
We were interested in the giardia risk conferred by household
animal contact and whether such contact interacted with the
risk conferred by shallow household water sources.
Methods
We used the Dartmouth-Hitchcock Medical Center lab-
qptory log to identify 190 giardiasis cases seen between
January 1, 1977 and June 1, 1984. Nineteen were Dartmouth
College students, a highly mobile population, and were not
considered in the analysis. The endemic nature of the
remaining 171 cases was demonstrated by their failure to
cluster by date of diagnosis or zip code; contact with the New
Hampshire and Vermont Health Departments confirmed our
impression that there had been no epidemic outbreaks of
giardiasis in our region during the study period.
Control patients were selected from among the 500,000
Hitchcock Clinic patients in the clinic computer registry (a
cumulative data base maintained since 1969). Four age- and
sex-matched controls for each case, excluding Dartmouth
College students, were randomly drawn from the computer
registry. We did not have access to patient medical records,
precluding knoweldge of medical diagnosis or vital status.
From the Department of Medicine, Dartmouth-Hitchcock Medical Cen-
ter, Hanover, NH 03756. Address reprint requests to Dr. Robert P. Smith at
that address. Dr. Chute is currently with the Department of Epidemiology,
Harvard School of Public Health, Boston. This paper, submitted to the Journal
March 29, 1986, was revised and accepted for publication October 20, 1986.
We mailed a one-page questionnaire to cases and con-
trols asking about place of residence and work, domestic
water supply, child day care utilization, animal contacts,
foreign travel, household exposure to giardiasis, diarrhea
history, and outdoor activities. The questionnaire specified
that answers should refer to the year and month of diagnosis
for the cases, or the year and month of the case for which a
given control was matched. A second mailing was sent to all
nonresponders. Finally, we attempted to interview by tele-
phone all the cases and one-third of the controls who did not
respond to both mailings (see Table 1).
Standard contingency tables and unconditional logistic
regression15'16 were used to analyze the data. The matching
variables (age, sex, year of diagnosis) were included in the
logistic models. Recall bias was minimized by also including
the year of diagnosis. Response bias was evaluated by
weighting the telephone subset to simulate the entire group of
non-responding cases and controls in a regression model, and
comparing the weighted and unweighted risk estimates. We
report exposure frequencies, etiologic fractions,17 and crude
and adjusted odds ratios.15
Results
Table 1 summarizes the response patterns for both
mailings and the telephone follow-up. The matching criteria
are distributed similarly for responding cases and controls
(Table 2), while crude exposure rates differ (Table 3).
Table 3 outlines crude and adjusted giardiasis odds ratio
(OR) estimates with their 95 per cent confidence intervals
(95%CI) and etiologic fractions. Adjustment for matching
factors does not materially alter the point estimates. We
confirm the influence in our area of previously established
risk factors, although the camping risk estimate is imprecise.
Shallow well or surface water sources, compared with all
other water sources (drilled well or municipal) confers the
hypothesized risk for giardiasis (OR 2.1, 95%CI 1.3-3.2).
TABLE 1—Response Rates
Cases
Controls
Source
© 1987 American Journal of Public Health 0090-0036/87$ 1.50
Identified
1st Mailing
2nd Mailing
Phone subset
TOTAL
171
101
14
15
130
59
8
9
76
684
246
75
50
371
36
11
/
54
AJPH May 1987, Vol. 77, No. 5
585
21
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CHUTE, ET AL.
TABLE 2—Characteristics of Respondents
Characteristic
Mean Age (years)
Oldest
Youngest
Male
Female
Year of Diagnosis
1977
1978
1979
1980
1981
1982
1983
1984
Case
(130)
31
76
2
54%
46%
14%
8%
9%
13%
15%
15%
21%
5%
Control
(371)
29
76
1
51%
49%
14%
10%
14%
12%
15%
15%
16%
4%
"Year of Diagnosis" for Controls refers to that of the matched case.
Compared with municipal water sources, the OR estimate is
even larger (OR 2.6, 95%CI 1.6-4.3). Adjustment for match-
ing factors, travel history, age of well, or animal exposure
does not substantially change the estimate. Animal exposure
is not clearly associated with disease, with the possible
exception of cats. Similar results are found when the analysis
is limited to households with shallow wells.
Discussion
This case-control study of non-epidemic giardiasis iden-
tifies several risk factors of importance in rural northern New
England. We confirm the importance of some established risk
factors (foreign travel, day care center exposure, and house-
hold case contact) and calculate a positive but unstable risk
estimate for camping (Table 3). We also find a previously
unrecognized giardiasis risk for the household use of shallow
water sources, while domestic and barnyard animal contact
does not seem to be important.
Shallow wells are a common household water source in
northern New England, and their association with giardiasis
may be an important public health consideration. Inadequate
construction or maintenance may be responsible for surface
or septic contamination. Among households with shallow
wells, those with pets or livestock did not have an increased
giardiasis risk, perhaps reflecting a lack of importance of
domestic animal reservoirs.13'14 We believe our questionnaire
did not reliably ascertain exposure to wild animals since the
reported prevalence was unexpectedly low for this region.
TABLE 3—Risk Measures for Giardiasis
Risk estimates for wild animal proximity are extremely
imprecise and are excluded from this report.
The risk associated with household contact may operate
through contaminated water, but this may simply represent
person to person contact or common>exposure to a different
risk factor. Large urban day care centers are recognized as an
important source of giardiasis7; we observed increased risk
among those exposed to children attending small rural day
care centers. Camping and domestic animal exposure in
northern New England appear to be less important giardiasis
risk factors than in other parts of the United States.6'12 The
etiologic fraction (14 per cent) for cat exposure incorporates
an unstable odds ratio estimate and should be interpreted
with caution.
Several potential biases are possible in this retrospective
case-control study. Recall bias was evaluated using logistic
models that included year of diagnosis as a covariate. This
adjustment did not materially affect the risk estimates. The
Hitchcock Clinic records from which the controls were
selected included patients seen since 1969, although cases
occurred only since 1977. To evaluate the potential impact of
this difference in time frames, the analysis was repeated using
the subset of controls with patient billing activity since 1980
(a list through 1977 was unavailable). This subset analysis
generated risk estimates similar to those reported for the
entire sample.
Response bias was controlled by weighted logistic re-
gression in which the cases and controls interviewed by
telephone were weighted to represent all non-respondents to
the mailings. Crude and weighted odds ratios appear in Table
4. The point estimates differ substantially only for "travel
abroad" and "household contact",.but these are based on
small numbers of exposed cases and are therefore unstable.
Overall, we do not think nonresponse greatly distorted our
findings. Detection bias was possible if physicians tested for
giardiasis more commonly among returning travelers or
household contacts of known cases. We believe this may
have inflated our risk estimates for these exposures.
Information bias may misclassify water source informa-
tion. We did not ask if municipal water sources were
filtered—a water treatment known to reduce water supply
giardiasis risk. However, in 1981 fewer than 20 per cent of the
community water sources in our region were filtered. Since
we do not know the addresses of controls at the time orcase
diagnosis, we are unable to establish the filtering status of
municipal water sources in-our data. However, failure to
control for this would tend to bias conservatively our esti-
mate of the giardiasis risk associated with shallow wells. Our
information regarding onsite water sources is probably ac-
% Exposure
Risk Factor
Foreign Travel
Camping
Day Care
Household Case
Shallow Well
Household Dog
Household Cat
Farm Animals
Case
11
14
23
31
34
56
48
14
Control
4
8
12
12
20
57
41
12
Fraction
8
5
12
29
18
0
14
2
Crude OR
OR
3.3
1.7
2.2
16.5
2.1
1.0
1.4
1.2
95% Cl
(1.5-7.2)
(0.9-3.2)
(1.3-3.7)
(7.4-37)
(1 .3-3.2)
(0.7-1.5)
(0.9-2.0)
(0.7-2.2)
Adjusted OR*
OR
4.2
1.6
2.2
21.0
2.1
0.9
1.3
1.1
95% Cl
(1.8-9.5)
(0.6-3.1)
(1 .2-4.0)
(8.8-50)
(1.3-3.2)
(0.6-1.4)
(0.9-2.0)
(0.6-2.1)
•Adjusted for matching factors: age, sex, year of diagnosis.
586
AJPH May 1987, Vol. 77, No. 5
22
-------
RISK FACTORS FOR ENDEMIC GIARDIASIS
TABLE 4—Impact of Potential Response Bias
Factor
Mailing OR
Weighted OR
Shallow Well
Day Care
Travel
Household Contact
2.2
2.2
3.0
15
1.9
2.1
5.4
27
curate: rural homeowners are aware of household water
source, since municipal or drilled water sources can substan-
tially improve the market value of a home.
It is possible that some of our controls may have had
giardiasis. Among controls, 3 per cent indicated a past
diagnosis of the condition, while 7 per cent reported a
prolonged episode of diarrhea. Because we could not verify
the giardiasis status of controls, we did not exclude respon-
dents on the basis of self-reported disease. This misclassifica-
tion would tend to bias observed risks toward the null; we
believe this conservative approach justifiable in the absence
of systematic disease ascertainment on all controls.
We found no evidence of important confounding by the
matching variables (age, sex, and year of diagnosis) using
unconditional logistic regression (Table 3). Adjustment for
camping and foreign travel did not alter the risk estimates for
shallow well household water source. We cannot exclude the
possibility of residual confounding by unknown factors. In
particular, socioeconomic factors that may influence the type
of water source might also influence other giardiasis risks.
In summary, our study confirms the previously reported
endemic or sporadic giardiasis risks of foreign travel, day
care exposure, and household case contacts. We suggest that
shallow well household water sources are an important and
previously unreported risk for endemic giardiasis in northern
New England. The 18 per cent etiologic fraction attributable
to this exposure suggests that it has a significant impact on
endemic giardiasis.
ACKNOWLEDGMENTS
We gratefully acknowledge the Hitchcock Foundation for funding the initial
phase of this project. Dr. Baron was partially supported by the Milbank Memorial
Fund (Milbank Scholar Program). We thank Dr. E. Robert Greenberg for his
critica! review. This study was initially presented at the Society for Epidemiologic
Research national meeting in Chapel Hill, NC, June 1985.
REFERENCES
1. Shaw PK, Brodsky RE, Lyman EO, el al: A community wide outbreak of
Giardisis with evidence of transmission by a municipal water supply. Ann
Intern Med 1977; 87:426-432.
2. Lippy EC: Tracing a giardiasis outbreak at Berlin, New Hampshire. J Am
Water Works Assoc 1978; 70:512-520.
3. Lopez CE. Dykes AC, Juranek DD, Sinclair SP, Conn JM, Christie RW,
Lippy EC, Schultz MG, Mires MH: Waterbome Giardiasis: A com-
munitywide outbreak of disease and a high rate of asymptomatic infection.
Am J Epidemiol 1980; 112-495-507
4. Vermont Department of Health, Epidemiology Division: Vermont Disease
Control Bulletin 1985; 2.
5. New Hampshire Division of Public Health Services: Giardiasis Study
Planned. Communicable Disease Rep Lab News 1983.
6. Wright RA, Spencer HC, Brodsky RE, Vernon TM: Giardiasis in Colo-
rado: An epidemiologic study. Am J Epidemiol 1977; 105:330-336.
7. Pickering LK, Woodward WE. DuPont HL, Sullivan P: Occurrence of
Giardia lambliain children in day care centers. J Pediatr 1984; 104:522-526.
8. Meyer EA, Jarroll EL: Reviews and commentary: Giardiasis. Am J
Epidemiol 1980; 111:1-12.
9. Schmerin NJ, Jones TC, Klein IJ: Giardiasis associated with homosexual-
lity. Ann Intern Med 1978; 88:801-803.
10. FiumaraN: Giardiasis in travelers to the Soviet Union. NEngl JMed 1973:
288:1410-1411.
11. Brodsky RE, Spencer HC, Schultz MG: Giardiasis in American travelers
to the Soviet Union. J Infect Dis 1974; 130:319-323.
12. Davies RB, Hibler CF: Animal reservoirs and cross-species transmission
of Giardia. In: Jakubowski W, and Hoff JC: EPA Symposium on
Waterbome Transmission of Giardiasis. EPA Pub. No. 600.9-79-001.
Cincinnati: EPA, 1979.
13. Kirkpatrick C, Green G: Susceptibility of domestic cats to infections with
Giardia lamlia cysts from human sources. J Clin Microbiol 1985;
21:678-679.
14. Hewlett E. Andrews J. Ruffier J, et al: Experimental infection of mongrel dogs
with giarrdia lamblia cysts and trophozoites. J Infect Dis 1982; 145:89-93.
15. Breslow N, Day N: Statistical Methods in Cancer Research: Vol 1-The
Analysis of Case-Control Studies. Lyon: Internationl Agency for Research
on Cancer, 1980.
16. SAS Institute: SAS User's Guide. Cary. NC: SAS Institute. 1982.
17. Kleinbaum D, Kupper L, Morgenstern H: Epidemiologic Research.
Belmont. CA: Lifetime Learning Publications. 1982.
AJPH May 1987, Vol. 77, No. 5
587
23
-------
Reproduced, with permission, from American
Journal of Epidemiology
AMERICAN JOURNAL OF EPIDEMIOLOOY Vo1- 129. N°- 4
Copyright © 1989 by The Johns Hopkins University School of Hygiene and Public Health Printed in U.S.A.
All rights reserved
EPIDEMIOLOGIC SURVEILLANCE FOR ENDEMIC GIARDIA LAMBLIA
INFECTION IN VERMONT
THE ROLES OF WATERBORNE AND PERSON-TO-PERSON TRANSMISSION
GUTHRIE BIRKHEAD1 -:1 AND RICHARD L. VOGT1
Birkhead, G., and R. L. Vogt (Vermont Dept. of Health, Burlington, VT 05402).
Epidemiologic surveillance for endemic Giardia lamblia infection in Vermont:
the roles of waterborne and person-to-person transmission. Am J Epidemiol
1989;129:762-8.
The authors studied 1,211 laboratory-confirmed, non-outbreak-related cases
of giardiasis in Vermont residents reported through Vermont's laboratory-based,
active surveillance system between 1983 and 1986. Giardiasis was the most
common reportable disease in the state, with an average annual incidence rate
of 45.9 cases per 100,000 population per year. This rate is higher than that in
other states reporting giardiasis incidence. Morbidity from giardiasis was also
significant in that 30% of cases reported symptoms lasting four or more weeks.
Waterborne transmission was suggested to be an important cause of non-
outbreak-related cases because rates of infection were highest in persons
receiving nonfiltered municipal or nonmunicipal residential drinking water. Rates
were also higher at higher elevations, where water supplies may be difficult to
protect from contamination. In addition, the pattern of age-specific incidence
rates and the high estimated incidence of infection in children attending day care
suggested that person-to-person transmission also played a role in causing non-
outbreak-related cases. Routine surveillance data can serve to indicate likely
important routes of transmission of giardiasis in the community.
Giardia; giardiasis; water supply
Giardia lamblia has been recognized as a demies in the United States in terms of
human pathogen since outbreaks of giar- both the number of outbreaks and the num-
diasis were first described in the 1960s and ber of persons affected (3). In addition,
1970s (1, 2). It is the most commonly de- there have been numerous reports of the
termined cause of waterborne disease epi- high prevalence of giardiasis in child day-
care settings due to person-to-person trans-
T> • j c ui- *• r. u ™ ,,«,„ ,. mission (4). However, the extent of en-
Received for publication February 29, 1988, and in , . .
final form August 23,1988. demic, or non-outbreak-related, giardiasis
' Epidemiology Division, Vermont Department of and the risk factors for endemic transmis-
Heaith, Burlington VT. sion in the United States are less well de-
-Division of Field Services, Epidemiology Program .,
Office, Centers for Disease Control, Atlanta, GA. scribed, partly because this infection has
' Current address: New York State Department of only recently been made reportable in many
Altany NY™" Of Communicable Disease Contro1' states and few population-based data are
Reprint requests to .Dr. Richard L. Vogt, Epide- available.
miology Division, Vermont Department of Health, Epidemiologic surveillance systems are
Box 70, 60 Main Street, Burlington, VT 05402. one source of information used to deter-
The authors thank Dr. Robert A. Gunn, Division , j • , ,, ,, ,.
of Field Services, Centers for Disease Control, for his mme the Predominant patterns of disease
comments on the earlier versions of the manuscript. transmission in a community. Giardiasis
762
24
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SURVEILLANCE FOR (UARDIA INKECTION
763
has been subject to epidemiologic surveil-
lance in Vermont since it was made a re-
portable disease on July 1, 1979. We report
on a review of Vermont giardiasis surveil-
lance data for 1983 to 1986 conducted to
better define the epidemiology of endemic
giardiasis in the state.
MATERIALS AND METHODS
The Vermont Department of Health con-
ducts active laboratory surveillance for re-
portable diseases and has previously re-
ported on the attributes of this system (5,
6). Briefly, all 18 licensed clinical labora-
tories in the state are required to contact
the Epidemiology Division weekly by tele-
phone or by mail whether or not they have
cases to report. Laboratories failing to re-
port regularly are contacted by the health
department. Physicians are also required to
report cases.
A public health nurse interviews each
case using a standard form recording de-
mographic information, the date of onset
and nature of symptoms, and possible risk
factors for infection. The risk factors in-
quired about are residential drinking water
source, child day-care attendance, drinking
of untreated water while away from home
(e.g., while hiking), and history of foreign
travel in the month before illness. The
nurse offers free laboratory stool testing to
asymptomatic members of case households
and recommends that those with symptoms
be treated by a physician.
For this study, a case of giardiasis was
defined as a Vermont resident with G. lam-
blia cysts or trophozoites in the stool who
either had onset of symptoms of giardiasis
(diarrhea or cramping) beginning in the
period January 1, 1983 through December
31, 1986, or, if asymptomatic, submitted
the laboratory specimen during this time.
Laboratory reports were examined through
March 31, 1987, to ensure complete ascer-
tainment of 1986 cases. Reports were not
included in the study if the case was part
of a recognized community outbreak or
child day-care center outbreak (two or more
cases with the same probable source of in-
formation and not living in the same house-
hold), was a recently arrived refugee, or had
previously reported giardiasis. Symptom
and risk factor information was reviewed
in detail for 1985 cases.
Only symptomatic cases were included in
incidence calculations because we assumed
that a larger and more constant proportion
of symptomatic than asymptomatic cases
come to medical attention. We felt that
information on symptomatic cases would
then be more representative of all sympto-
matic cases and less subject to biases re-
lated to ease of attaining medical care or
other factors. In addition, the date of infec-
tion for incidence calculations could be
known with certainty only for symptomatic
cases. Finally, including asymptomatic
cases identified in the case-household in-
vestigations could bias incidence rates
downward in groups less completely
screened, such as adults (compared with
children) or persons in areas of the state
served by a less vigorous public health
nurse.
Age- and sex-specific population esti-
mates for Vermont for 1983-1986 were used
to calculate rates (7). Incidence rates by
town were calculated from the median
number of cases per town during the four-
year period.
The rates of symptomatic G. lamblia in-
fection were calculated for persons with
different sources of residential drinking
water. For this analysis, cases were ex-
cluded if they reported other possible risk
factors for infection such as day-care at-
tendance, contact with a day-care attendee,
drinking of untreated water while away
from home, or a history of recent foreign
travel. Cases with a household member who'
had G. lamblia infection beginning more
than three days before their own illness
were also excluded to eliminate cases that
were possibly due to person-to-person
transmission in the home. Residential
water sources were categorized as munici-
pal or private. Municipal systems were fur-
ther categorized by type of source (surface
25
-------
764
BIRKHEAD AND VOGT
or well) and by type of treatment for surface
sources (filtered or not filtered). The types
of water systems serving each town and
estimates of the population served in 1985
were obtained from the municipal water
system inventory of the Environmental
Health Division, Vermont Department of
Health. Since we could not determine the
type of water system supplying the homes
of giardiasis cases in towns with multiple
municipal water systems, we assumed that
cases were on the system serving the largest
population in the town. We calculated the
total population receiving drinking water
from nonmunicipal water systems by sub-
tracting the number of persons on munici-
pal systems from the total state population.
We determined the risk of G. lamblia infec-
tion by elevation of town of residence using
each town's elevation above sea level (8).
The rate of infection was also calculated
for children attending day care. An esti-
mate of the number of children under age
five years attending licensed or registered
day-care centers in the state in 1985 was
obtained from the Department of Social
and Rehabilitative Services, State of Ver-
mont.
RESULTS
A total of 1,406 laboratory-confirmed
cases of giardiasis in Vermont residents
were reported to the Vermont Department
of Health between 1983 and 1986 (table 1).
Of these, 1,211 (86 per cent) cases were not
outbreak-related. These form the basis of
the study. Case investigation forms were
completed for 1,202 (99 per cent) cases.
Symptomatic cases accounted for 977 (81
per cent) and asymptomatic cases for 234
(19 per cent) of the cases. A total of 127
outbreak-related cases were identified as
being part of one community outbreak or
more than 20 day-care outbreaks. Refugees,
predominantly from southeast Asia, ac-
counted for the remainder of the cases.
The mean annual incidence rate for
symptomatic cases was 45.9 cases per
100,000 population per year (table 1). Giar-
diasis was the most common reportable
enteric disease in Vermont during this
time. The occurrence of symptomatic cases
peaked during the months of July through
November (figure 1). This pattern was ev-
ident regardless of residential drinking
water source or whether drinking of un-
treated water outside the home was re-
ported (data not shown). The rate of symp-
tomatic G. lamblia seemed to be highest in
towns located in the mountainous northern
and central regions of the state (figure 2).
The mean annual age- and sex-specific
incidence rates for symptomatic cases for
1983 to 1986 are shown in figure 3. The
highest rates are in children aged one to
four years. In this age group, males had 1.5
times the rate of symptomatic giardiasis of
females (95 per cent confidence interval
(CI) 1.1-2.0). The rate was also high for
persons, especially women, aged 20 to 39
TABLE 1
Laboratory-confirmed cases of giardiasis reported to the Vermont Department of Health, 1983-1986: Vermont
Department of Health giardiasis surveillance data
Year
1983
1984
1985
1986
Non -outbreak -related
Symptomatic
cases
163
306
252
256
Rate/100,000
population*
31.0
57.7
47.1
47.4
Asymptomatic
cases
39
73
57
65
Outbreak-related
Community
outbreak
0
0
0
24
Day-care
outbreak
22
66
9
6
Refugees
26
26
7
9
Total reported
cases
250
471
325
360
Total
977
45.9
234
24
103
68
1,406
* Population estimates by the Vermont Department of Health (7).
26
-------
SURVEILLANCE FOR GIARDIA INFECTION
765
Jon Fob Mar Apr May Jun Jul Aug Sop Oct Nov Dec
FIGURE 1. Number of symptomatic G. lamblia in-
fections, by month, in Vermont, 1983-1986. Vermont
Department of Health giardiasis surveillance data.
years. Women in this age group were 40 per
cent more likely to have symptomatic giar-
diasis than men (relative risk (RR) = 1.4,
95 per cent CI 1.1-1.6).
Diarrhea was the most commonly re-
ported symptom (96 per cent), followed by
cramps (64 per cent) and weight loss (48
per cent). Thirty per cent of symptomatic
cases reported having symptoms lasting
more than four weeks. The most commonly
reported risk factor for infection was his-
tory of contact with a person with diarrhea
in the last month (38 per cent), followed by
drinking of untreated water while away
from home (13 per cent), attendance at day
care (12 per cent), contact with a day-care
attendee (8 per cent), and foreign travel (6
per cent).
Information on the source of residential
water supply was available for 243 (96 per
cent) symptomatic 1985 cases, of whom 118
received their drinking water from munic-
ipal water systems and 125 obtained it from
nonmunicipal water sources. The health
department's water systems inventory
showed that an estimated 56 per cent
(300,273) of the state's population was
served by municipal water systems in 1985.
Using these data, we estimated that state
residents with nonmunicipal residential
water systems have approximately a 30 per
cent excess risk of developing symptomatic
giardiasis compared with those on munici-
pal water systems (RR = 1.3, 95 per cent
Rates per 10,000 per year
<5/IO,000/Year
>5 to IO/IO,000/Year
VERMONT
Scale of Kllomatan
) 20 40
>--
FIGURE 2. Median yearly incidence rate of symp-
tomatic G. lamblia infection, by town, in Vermont,
1983-1986. Rales are given per 10,000 population per
year. Vermont Department of Health giardiasis sur-
veillance data.
CI 1.0-1.7). The rates of giardiasis varied
by type of water system (table 2). Nonmun-
icipal water system users had 2.2 times the
rate of giardiasis of those on filtered, sur-
face water municipal systems. Municipal
surface water systems without filtration
had almost twice the rate of giardiasis of
those with filtration. Rates were elevated
for users of municipal well water systems.
The risk of infection also increased with
increasing elevation of town of residence
(table 2).
Among 92 cases less than five years of
age, 36 (39 per cent) attended day care. The
Department of Social and Rehabilitative
Services estimated that 12,000 positions
were available in licensed or registered day-
care centers in 1985, which meant that 29.4
per cent of children in the state under age
five years were in officially sanctioned day
27
-------
766
BIRKHEAD AND VOGT
E3 Male
ESS Female
<1 1-4 5-9 10-14 15-19 20-2930-3940-4950-5960-69 70+
AGE GROUP (years)
FIGURE 3. Incidence rates of symptomatic G. lamblia infection, by age and by sex, in Vermont, 1983-1986.
Vermont Department of Health giardiasis surveillance data.
TABLE 2
Risk factors for development of G. lamblia infection in Vermont, 1985: Vermont Department of Health giardiasis
surveillance data
Risk factor
Population at Rate/100,000
risk population
RR*
95% CI*
Residential water supplyt
Municipal
Surface water, filtered
Surface water, not filtered
Well water
Private
Total
Child day-care attendance^:
Attendee
Nonattendee
Total
Elevation of town of residence (m)§
0-99
100-199
200+
Total
20
30
17
77
144
36
56
92
190
394
389
973
132,178
104,732
63,363
234,727
535,000
12,000
28,762
40,762
135,817
222,399
173,598
531,814
15.1
28.6
26.8
32.8
300.0
194.7
35.0
44.3
56.0
1.0
1.9
1.8
2.2
1.0
1.5
1.0
1.3
1.6
1.1-3.3
0.9-3.4
1.3-3.6
1.0-2.3
1.1-1.5
1.4-1.9
* RR, relative risk; CI, confidence interval.
t Cases without other risk factors for giardiasis.
$ Cases aged less than five years.
§ Case data for 1983-1986, missing elevation for five towns and missing town of residence for four cases.
Rates of G. lamblia infection in this section are calculated per 100,000 population per year.
28
-------
SURVEILLANCE FOR GIARDIA INFECTION
767
care, assuming full enrollment. The relative
risk of day-care attendees compared with
that of nonattendees is shown in table 2.
DISCUSSION
Our review of surveillance data showed
that G. lamblia was the most common re-
portable enteric disease in Vermont during
the period 1983-1986. The incidence rate
of symptomatic giardiasis (45.7 per 100,000
population) in Vermont is much greater
than rates reported previously by other
states. In Minnesota, the incidence rate was
9.8 per 100,000 population based on data
from the state laboratory for 1971 through
1975 (9). A survey of most laboratories in
Colorado in 1972 and 1973 found a giardi-
asis incidence rate of 11.6 per 100,000 pop-
ulation per year (10). This had increased
by 1981-1982 to approximately 16 per
100,000 population per year based on state
laboratory reports and active case finding
among most primary-care providers (11).
These reports did not exclude asympto-
matic, outbreak-related cases or cases in
refugees.
One explanation for the higher giardiasis
incidence rate in Vermont is that the active,
laboratory-based reporting and active case
investigations may detect a greater propor-
tion of cases than do other state surveil-
lance systems. In addition, physician rec-
ognition and diagnosis of giardiasis may
have improved since the previous reports.
Finally, the incidence of giardiasis may
truly be higher in Vermont than in other
states, perhaps partly because of the rural
nature of Vermont and the possibly higher
frequency of contact with sources of infec-
tion in the environment, particularly water.
The true giardiasis incidence in Vermont is
probably higher than that reported here
because some state residents may leave the
state to be diagnosed. This is a threat to
the sensitivity of most surveillance sys-
tems. We have no information on the num-
ber of these cases, but we suspect that they
are probably few.
Our data suggest that waterborne trans-
mission is an important source of non-
outbreak-related G. lamblia infection in
Vermont. Persons receiving drinking water
from nonmunicipal residential drinking
water systems, which often have an unpro-
tected water source and may not be filtered,
had the highest rate of symptomatic giar-
diasis of any group. Municipal water sys-
tems with filtration had the lowest rates of
giardiasis, which is in accord with the
knowledge that filtration is necessary for
adequate treatment of water to remove
Giardia cysts (3, 12). Shallow, poorly con-
structed wells have been shown by others
to be a risk factor for giardiasis (13). The
increased risk of infection among users of
municipal well water in Vermont suggests
that some of these systems are not adequate
to prevent giardiasis.
The increase in the rate of infection with
elevation also suggests that waterborne
transmission is occurring. Outbreaks of
giardiasis in mountainous areas have been
traced to drinking contaminated water (14).
Rates of illness are higher in these areas
where easily contaminated surface waters
may be the predominant source of drinking
water and where low water temperature
may prolong the survival of Giardia cysts
(14). A final point in favor of the possibility
that waterborne transmission is important
in endemic giardiasis is that more cases had
onset in the summer months when outdoor
activities bring more people into contact
with possibly contaminated water. It is un-
clear why the peak in the number of cases
continued into the fall.
Our data are limited by lack of knowledge
of the exact municipal water source for
cases in towns with multiple water systems
and by the crude nature of the estimates of
the population served. It is possible that
other factors associated with having unfil-
tered municipal or nonmunicipal residen-
tial drinking water, such as lower socioeco-
nomic status, poorer access to medical care,
or crowding, may have a role in the in-
creased risk of infection in this group. The
variable sensitivity of the giardiasis sur-
veillance system in different regions of the
state may also contribute to some of the
29
-------
768
BIRKHEAD AND VOGT
differences in rates. These factors are un-
likely to significantly affect our results be-
cause of the consistency of relative risks
associated with different types of water
supply and elevation and because the more
rural parts of the state, where access to
medical care and reporting might be ex-
pected to be the poorest, had the highest
rates of giardiasis.
Our data also indicate that person-to-
person transmission may play a role in non-
outbreak-related giardiasis. Children aged
one to four years had the highest rates of
symptomatic infection of any age group.
This pattern has been described in other
enteric infections (15-18) and may be due
to poorly developed immunity or increased
fecal-oral contact among children. Child
day-care centers are a known site of person-
to-person transmission among children (4).
The higher rates observed for male children
are unexplained, but a similar pattern has
been observed by others with both Giardia
and Campylobacter infections (18).
The data we have presented indicate that
G. lamblia may be a more common pathogen
than has been previously recognized from
reports of outbreak investigations. Surveil-
lance data are limited in identifying caus-
ative factors by their descriptive nature,
but where data are available on the popu-
lations at risk, they can serve to confirm
known risk factors and to estimate their
importance in disease transmission in the
community.
REFERENCES
1. Moore. GT, Cross WM, McGuire D, et al. Epi-
demic giardiasis at a ski resort. N Engl J Med
1969;281:402-7.
2. Shaw PK, Brodsky RE, Lyman DO, et al. A
community-wide outbreak of giardiasis with evi-
dence of transmission by a municipal water sup-
ply. Ann Intern Med 1977;87:426-,12.
3. Centers for Disease Control. Water-related dis-
ease outbreaks: annual summary, 1983. Atlanta,
GA: Centers for Disease Control, September 1984.
4. Pickering LK, Woodward WE, DuPont HL, et al.
Occurrence of Giardia lamblia in children in day-
care centers. J Pediatr 1984;104:522-6.
5. Vogt RL, Clark SW, Kappel S. Evaluation of the
state surveillance system using hospital discharge
diagnoses, 1982-1983. Am J Epidemiol 1986;123:
197-8.
6. Harter L, Frost F, Vogt RL, et al. A three-state
study of waterborne disease surveillance tech-
niques. Am J Public Health 1985;75:1327-8.
7. Vermont Department of Health. 1985 estimates
of the population and housing of Vermont coun-
ties and towns. (Vermont public health statistics
bulletin). Burlington, VT: Vermont Department
of Health, March 1986.
8. Hennenberger JD, ed. 1982 Vermont year book.
Chester, VT: The National Survey, 1982:135-507.
9. Weiss HB, Winegar DA, Levy BS, et al. Giardiasis
in Minnesota 1971-1975. Minn Med 1977;60:815-
20.
Wright RA, Spencer HC, Brodsky RE, et al. Giar-
diasis in Colorado: an epidemiologic study. Am J
Epidemiol 1977; 105:330-6.
11. Hopkins RS, Shillam P, Gaspard B, et al. Water-
borne disease in Colorado: three years surveillance
and 18 outbreaks. Am J Public Health 1985;
75:254-7.
12. Logsdon GS, Symons JM, Hoye LH. Water filtra-
tion techniques for removal of cysts and cyst
models. In: Jakubowski W, Hoff JC, eds. Water-
borne transmission of giardiasis. Cincinnati, OH:
US Environmental Protection Agency, 1979:240-
56. (Publication no. EPA-GOO 9-79-001).
13. Chute CG, Smith RP, Baron JA. Risk factors for
endemic giardiasis. Am J Public Health 1987;
77:585-7.
14. Meyer EA, Jarroll EL. Giardiasis. Am J Epidemiol
10.
15. Ryder RW, Merson MH, Gangarosa EJ. Salmo-
nellosis in the United States, 1968-1974. J Infect
Dis 1976;133:483-6.
16. Blaser MJ, Pollard RA, Feldman RA. Shigella
infections in the United States, 1974-1980. J In-
fect Dis 1983;147:771-5.
17. Tauxe RV, Pegiies DA, Hargrett-Bean N. Cam-
pylobacter infections: the emerging national pat-
tern. Am J Public Health 1987;77: 12 19-21.
18. Hopkins RS, Olmstead RN. Campylobacter jejuni
infections in Colorado: unexplained excess of
cases in males. Public Health Rep 1985; 100:
333-6.
30
-------
AMERICAN
JOURNAL
OF
Public
Health
Reproduced, with permission,
from American Journal of
Public Health
reprint
Cryptosporidiosis and Surface Water
MARGARET M. GALLAHER, MD, JOY L. HERNDON, MS, LINDA JEAN NIMS, MS, CHARLES R. STERLING, PuD,
DEBRA J. GRABOWSKI, MS, RS, AND HARRY F. HULL, MD
Abstract: In the period July through October, 1986, 78 labora-
tory-confirmed cases of cryptosporidiosis were identified in New
Mexico. To determine possible risk factors for development of this
disease, we conducted a case-control study; 24 case-patients and 46
neighborhood controls were interviewed. Seventeen (71 per cent) of
the 24 case-patients were females, seven (29%) were males; their
ages ranged from 4 months to 44 years, median 3 years. There was
a strong association between drinking surface water and illness: five
of the 24 case-patients, but none of the 46 controls drank untreated
surface water. Among children, illness was also associated with
attending a day care center where other children were ill (odds ratio
= 13.1). (Am J Public Health 1989; 79:39^2.)
Introduction
Ciyptosporidiitm, first recognized as a cause of diarrhea
in humans in 1976.' has become known as a common cause
of diarrhea worldwide.2'3 No effective therapy is known; the
diarrhea is often prolonged and accompanied by weight loss.
In immunocompromised persons, cryptosporidiosis can
cause a life-threatening dehydration.
Cryptosporidium can be transmitted through contact
with animals, especially cattle and sheep,4 through person-
to-person contact, especially in day care centers,5 and
through a contaminated community water source.6 In several
reported outbreaks, the source of the infection was
unknown.7'8
In 1984, 24 cases of cryptosporidiosis were reported to
the New Mexico Office of Epidemiology, 13 of them were
associated with two day care centers. In 1985, 24 cases were
reported. In July 1986, a cluster of 20 cases of cryptospori-
diosis was recognized in Bernalillo County (the city of
Albuquerque). Because these cases did not appear to be
related and the source of infection was unclear, a case-
control study to evaluate potential risk factors for illness was
conducted.
Methods
Since February 1984, all stool samples submitted to the
New Mexico Health and Environment Department's Scien-
tific Laboratory Division for ova and parasite testing have
been examined for Cryptosporidium. Stool samples are
concentrated using a formalin-ethyl acetate technique. A
heat-fixed fecal smear is stained with Kinyoun modified
acid-fast stain9 and counterstained with Loeffler methylene
blue. The specimen is then examined under a light micro-
scope for the presence of Cryptosporidium oocysts.
In August 1986, the Office of Epidemiology sent a letter
to primary care physicians in Bernalillo County, alerting
them to the cluster of cases, reviewing the symptoms and
diagnosis of cryptosporidiosis, and requesting that stool
samples be sent if cryptosporidiosis was suspected.
A case-patient was defined as a Bernalillo County
resident with laboratory-confirmed cryptosporidiosis report-
ed to the Office of Epidemiology between July 1 and October
1, 1986. If more than one person with laboratory-confirmed
cryptosporidiosis was identified within a household or day
care group, only the one with the earliest onset of symptoms
was included in the study.
Controls were matched for age, sex, and neighborhood
using a reverse telephone directory. The home of the case-
patient was identified in the directory. The homes listed on
either side of the case-patient were contacted by telephone
alternately until suitable controls were identified. Case-
patients 10 years old or younger were matched within two
years of age, and those over 10 years were matched within
five years of age. A control could not have had diarrhea in the
four weeks before the onset of disease in the case-patient to
which he/she was matched. Twenty-two of the 24 index cases
were matched with two controls each. Two index cases were
matched with only one control.
A standard questionnaire, administered via telephone to
both case-patients and controls, requested information on
size of household, day care attendance or contact, travel,
surface water exposure, pets and domestic animal exposure
during the four weeks before onset of illness in the case-
patient.
Two surface water samples were collected on October
27, 1986 from a lake in southern New Mexico and a lake in
Arizona where three of the case-patients had been exposed to
surface water. These samples were tested for Cryptospori-
dium and Giardia using a fluorescent labeled monoclonal
antibody technique.10'11
Using the matched triplets or pairs, we calculated
Mantel-Haentzel chi-square, odds ratios (OR), and 95%
confidence intervals (CI) and fitted a conditional logistic
regression to determine which variables were independently
associated with infection with Cryptosporidium.
Address reprint requests to Margaret M. Gallaher, MD, Office of Epi-
demiology, New Mexico Health and Environment Department, P.O. Box 968,
Santa Fe, NM 87504-0968. Ms. Nims and Dr. Hull are also with the NM Health
and Environment Department in Santa Fe. Ms. Herndon is with the Epidemi-
ology Program Office, Centers for Disease Control, Atlanta; Dr. Gallaher is on
assignment to New Mexico from the CDC Epidemiology Program Office. Dr.
Sterling is with the Department of Veterinary Sciences, University of Arizona,
Tucson; Ms. Grabowski is with the Albuquerque (NM) Environmental Health
Department. This paper, submitted to the Journal December 24, 1987, was
revised and accepted for publication April 22, 1988.
© 1989 American Journal of Public Health 0090-0036/89$!.50
Results
Of the 56 laboratory-confirmed cases of cryptosporidi-
osis in Bernalillo County reported between July 1 and
October 1, 26 met the definition of a case-patient. Two (8
percent) of the cases were lost to follow up (a 1-year-old
female who attended a day care center and a 43-year-old
female). Of the 30 cases excluded from the study, two were
patients with acquired immune deficiency syndrome who
died during the course of the study, 10 were household
AJPH January 1989, Vol. 79, No. 1
39
31
-------
GALLAHER, ET AL.
H Male
:;::: Female
TABLE 1—Frequency of Reported Symptoms of Cryptosporidiosis, New
Mexico, 1986(N = 24)
Symptom
No
Diarrhea
Flatulence
Abdominal Pain
Nausea
Fever
Vomiting
Abdominal distention
23
20
17
15
13
12
12
96
79
67
58
54
46
46
15-19 20-2J 25-29 30-34 35-39 40-44
AGE GROUP (Years)
FIGURE 1—Cryptosporidiosis, by Age Group and Sex, New Mexico, 1986
contacts of case-patients, and 18 were day care contacts of
case-patients.
The 24 remaining case-patients ranged in age from 4
months to 44 years with a median age of 3 years. Figure 1
shows the age and sex distribution of the index cases.
Seventeen of the cases were 10 years of age or younger; 13
of whom were females; 17 of all 24 cases were female.
Thirteen (54 per cent) of the case-patients were White and 11
(46 per cent) were Hispanic. Controls showed similar demo-
graphic characteristics.
Case-patients had onset of symptoms from May 28 to
September 2, 1986 (Figure 2). The duration of symptoms
ranged from five to 60 days with a median of 21 days. The
most frequent symptom (Table 1) was diarrhea followed by
flatulence and abdominal pain.
Matched set analysis identified two possible sources of
infection: surface water (rivers, lakes and streams) and day
care centers (Table 2). Five case-patients drank untreated
surface water, two in Mexico, and three in southern Colorado
and New Mexico; none of the controls drank surface water.
Case-patients reported more swimming in surface water in
6 —
the four weeks prior to illness. The time between exposure to
surface water (swimming or drinking) and onset of illness
ranged from four to 21 days with a median of seven days.
Fourteen (82 per cent) of 17 household members with
exposures to surface water similar to the case-patients also
became ill with diarrhea. These household members became
ill within two to seven days of the onset of illness in the
case-patient.
Case-patients were substantially more likely to attend a
day care center where other children were ill with diarrhea
than were their matched controls (Table 2). Attendance at
day care centers where no other children were ill was not
related to risk.
The logistic regression model for multivariate analysis
included all variables except drinking surface water, which
did not vary. Infection was associated with swimming in
surface water, attending a day care center, camping, and
having a pet which was less than six months of age or was ill,
with odds ratios all greater than 3.0. Having a pet over six
months of age was associated with a decreased risk of
disease. All of these estimates were unstable, with 95%
confidence limits including 1.0.
Among the 24 case-patients, eight were exposed to
surface water, eight were day care center attendees or
employees, six were household contacts of a day care center
attendee, and eight had young and/or ill pets. Four had none
of these exposures.
The surface water samples from New Mexico and
Arizona where cases had been exposed did not yield Cryp-
tospondium or Giardia on testing.
Two of the 24 index cases were coinfected with Giardia.
ili
I
Hi
1-1
28 1 5 9 13 17 21 25 29 3 7 11 15 19 23 27 31 4
12 16 20 24 28 1
MAY JUNE JULY AUQ
FIGURE 2—Cryptosporidiosis, by Date of Onset, New Mexico, May 28-September 4, 1986
SEP
40
32
AJPH January 1989, Vol. 79, No. 1
-------
CRYPTOSPORIDIOSIS AND SURFACE WATER
TABLE 2—Exposures Associated with Cryptosporidlosis
Exposure
Drink surface water
Swim in surface water
Attend a day care center
where other children
were ill
Household contact of
day care center
attendee
No. of Cases
Exposed
0
1
0
1
0
1
0
1
No.
0
17(2)
5
14
4(2)
15(1)
6(1)
12(1)
5(1)
of controls
1
0
0
3
1
1
0
4
1
exposed
2
0
0
0
0
0
0
0
0
95% Confidence
Odds Ratio Interval
00 2.44, 00
3.7 1.02,13.5
13.1 2.58, 66.4
3.0 0.91, 9.85
Numbers in parentheses represent cases with only one control.
One of them attended a day care center. The other had no
apparent risk factors for cryptosporidiosis or giardiasis.
Discussion
Person-to-person transmission of cryptosporidiosis is
well recognized, especially in day care centers.5'12 Lack of
hygiene and frequent mouthing of objects among diapered
babies and toddlers can lead to the rapid spread of this and
other enteric infections in day care centers and homes.13 This
study confirmed the association between attending a day care
center, especially where other children are ill, and crypto-
sporidiosis.
Cryptosporidiosis has also been found to be a cause of
traveler's diarrhea, particularly in people traveling to less
developed countries.2'14 In this study, two (8 per cent) of the
index cases had recently traveled to Mexico; both of these
case-patients also drank untreated surface water in Mexico.
Although puppies and kittens have been shown to
excrete Cryptosporidium,* these pets have not previously
been demonstrated to transmit cryptosporidiosis to humans.
Contact with a young and/or ill pet was associated with an
increased risk of disease in this study, but the numbers are
too small to be convincing. Nevertheless, this observation is
in keeping with what is known of cryptosporidiosis in
domestic animals: young animals contract cryptosporidiosis
more often than older animals, and the severity and duration
of illness is inversely related to the age of the animal.1'15
This study suggests for the first time that drinking surface
water may be a mode of transmission of cryptosporidiosis in
the United States. This is feasible since waterborne crypto-
sporidiosis has been previously demonstrated in a commu-
nity in Texas due to a contaminated artesian well.6 The
plausibility of waterborne transmission is increased by the
time relationship between exposure to surface water and
onset of illness. The median incubation period of seven days
in those case-patients exposed to surface water is consistent
with what is known of the incubation period for
cryptosporidiosis.l6
Cryptosporidium sp. has been found in a broad variety of
both domestic and wild animals. Cattle and sheep appear to
be important hosts, but mice, snakes, rabbits, and deer have
also been found to excrete the organism.4'15 Much of the
recreational water in New Mexico and other western states
drains land which has multiple uses including the grazing of
livestock. Livestock could contaminate surface water with
Cryptosporidium by direct deposit of feces in the water or via
surface runoff washing animal feces into the water. Similarly,
contamination of the water by wild animal feces might also
play an important role. New Mexico received heavier than
normal rainfall during the summer months of 1986, a factor
which may have promoted contamination of surface water.
The finding of an elevated risk of cryptosporidiosis
among persons who had recreational contact with, but did not
drink surface water, supports waterborne transmission of the
disease. Recreational surface water contact, specifically
swimming, has been shown to be a risk factor for other
enteric infections.17'18 Swimmers become infected by swal-
lowing small amounts of contaminated water, especially
following submersion. The infective dose of Cryptosporidium
is unknown, but is probably small, because of the organism's
ability to complete its life cycle in a single host and to
reinitiate the developmental cycle in that host.4 A small
infective dose for cryptosporidiosis would facilitate trans-
mission by water contact without frank water consumption.
Our studies of surface water in New Mexico were limited
in scope, but failed to demonstrate the presence of Crypto-
sporidium. These studies were done two months after the
outbreak and probably represent a failure to detect intermit-
tent, low-level, or seasonal contamination. Additionally, the
samples were taken from a large lake and it was difficult to
determine the exact site of exposure. Studies of surface water
conducted in Arizona showed that there was widespread
contamination of streams with Cryptosporidium during the
summer of 1986," but that most of the water sources did not
contain Cryptosporidium during the fall months.*
Cryptosporidiosis occurs more commonly in summer
and fall in these and other data.3'4 Increased outdoor activity
during the summer and early fall with drinking of or swim-
ming in surface water may partially explain the seasonal
variation.
Coinfection with Giardia lamblia has been found in 2 per
cent to 23 per cent of cryptosporidiosis cases in previously
described outbreaks19"21 and 8 per cent in this study. The
coincidental occurrence of two parasitic infections suggests
a common mode of transmission. Person-to-person transmis-
sion of both giardiasis and cryptosporidiosis in day care
centers has been well described. Waterborne transmission
of giardiasis from surface water is also well known.22'23 Our
* Unpublished data, CR Sterling, University of Arizona, 1986.
AJPH January 1989, Vol. 79, No.
33
41
-------
GALLAHER, ET AL.
study suggests that waterborne transmission of cryptospori-
diosis may explain coinfection with these two parasites, even
though neither of the coinfections in this study occurred in
persons who had exposure to surface water.
The median duration of diarrhea in the study population
was longer than the three to 14 days described by others,' •'7-25
perhaps because patients who had stool samples submitted for
ova and parasite examination had diarrhea that lasted longer
than three to five days and did not respond to usual symptomatic
treatment.
The predominance of young children found here has
been seen in other studies, and follows the same pattern seen
in domestic animals. In addition, young children are more
likely to come to medical attention earlier in the course of a
diarrheal illness than adults and more likely to be tested for
the cause of their diarrhea. The explanation for the predom-
inance of females seen in this group of children is not clear
and may reflect only incomplete case ascertainment.
Further study of the role of surface water in transmission
of cryptosporidiosis is clearly needed. Meanwhile, boiling of
surface water rather than chemical disinfection may be
prudent, since Cryptosporidium is resistant to most common-
ly used chemical disinfectants including chlorine and
iodine,15'24 but oocysts are destroyed by heating above 65°C
for 30 minutes.24 Boiling also destroys other organisms,
including Giardia, that may be in the water. We also
recommend that physicians consider cryptosporidiosis in the
differential diagnosis of persons with diarrhea and a history
of surface water contact.
ACKNOWLEDGMENTS
The authors wish to thank Kathy Powers. RN, and Judy Knott, RN, for
their assistance in this study. A portion of the data described in this paper was
accepted for presentation at the annual EIS Conference. Centers for Disease
Control, April 6-10. 1987. in Atlanta, Georgia.
REFERENCES
1. Navin TR, Juranek DD: Cryptosporidiosis: Clinical, epidemiologic, and
parasitologic review. Rev Infect Dis 1984; 6:313-327.
2. Navin TR: Cryptosporidiosis in humans: Review of recent epidemiologic
studies. EurJ Epidemiol 1985; 1:77-83.
3. Mata L: Cryptosporidium and other protozoa in diarrheal disease in less
developed countries. Pediatr Infect Dis 1986; 5:8117-8130.
4. Current WL: Cryptosporidiosis. J Am Vet Med Assoc 1985; 187:1334-
1338.
5. Centers for Disease Control: Cryptosporidiosis among children attending
daycare centers—Georgia, Pennsylvania, Michigan. California, New
Mexico. MMWR 1984: 33:559-601.
6. D'Antonio RG, Winn RE, Taylor JP, el al: A waterborne outbreak of
cryptosporidiosis in normal hosts. Ann Intern Med 1985:103:886-888.
7. Hunt DA. Shannon R. Palmer SR, Jephcott AE: Cryptosporidiosis in an
urban community. Br Med J 1984; 289:814-816.
8. Wolfson JS. Richter JM, Waldron MA, Weber DJ, McCarthy DM,
Hopkins CC: Cryptosporidiosis in immunocompetent patients. N Engl J
Med 1985; 312:1278-1282.
9. Ma P, Soave R: Three-step stool examination for cryptosporidiosis in 10
homosexual men with protracted watery diarrhea. J Infect Dis 1983; 147:
824-828.
10. Garcia LS, Brewer TC, Bruckner DA: Fluorescent detection of Crypto-
sporidium oocysts in human fecal specimens using monoclonal antibodies.
J Clin Microbiol 1987; 25:119-121.
11. Madore MS. Rose JB, Gerba CP, Arrowood MJ, Sterling CR: Occurrence
of Cryptosporidium oocysts in sewage effluents and selected surface
waters. J Parasit 1987; 73:702-705.
12. Pickering LK, Bartlett AV, Woodward WE: Acute infectious diarrhea
among children in day care: Epidemiology and control. Rev Infect Dis
1986; 8:539-547.
13. Pickering LK. Evans DG, DuPont HL, Vollet JJ, Evans DJ: Diarrhea
caused by Shigella, rotavirus and Giardia in day-care centers: Prospective
study. J Pediatr 1981; 99:51-56.
14. Sterling CR. Seegar K. Sinclair NA: Cryptosporidium as a causative agent
of traveler's diarrhea. J Infect Dis 1986; 153:380-381.
15. Tzipori S: Cryptosporidiosis in animals and humans. Microbiol Rev 1983;
47:84-96.
16. Jokipii L, Jokipii AMM: Timing of symptoms and oocyst excretion in
human cryptosporidiosis. N Engl J Med 1986; 315:1643-1647.
17. Centers for Disease Control: CDC Shigella surveillance. Atlanta: CDC,
1976: 37:4.
18. Makintubee S. Mallonee J. Istre G: Shigellosis outbreak associated with
swimming. Am J Public Health 1987; 77:166-168.
19. Skeels MR, Sokolow R, Hubbard CV, Foster LR: Screening for coinfec-
tion with Crypwsporidium and Giardia in Oregon public health clinic
patients. Am J Public Health 1986; 76:270-273.
20. Wolfson JS, Hopkins CC, Weber DJ, el al: An association between
Crypiosporidium and Giardia in stool. N Engl J Med 1984; 310:788.
21. Jokipii L, Pohjola S, Jokipii AMM: Cryptosporidium: A frequent finding
in patients with gastrointestinal symptoms. Lancet 1983: 2:358-361.
22. Dykes AC, Juranek DD, Lorenz RA, el al: Municipal waterborne
giardiasis: An epidemiologic investigation. Beavers implicated as a pos-
sible reservoir. Ann Intern Med 1980; 92:165-170.
23. Lopez CE, Dykes AC, Juranek DD, el al: Waterborne giardiasis: A
communitywide outbreak of disease and a high rate of asymptomatic
infection. Am J Epidemiol 1980; 112:495-507.
24. Soave R, Armstrong D: Cryptosporidium and cryptosporidiosis. Rev
Infect Dis 1986; 8:1012-1023.
25. Current WL, Reese NC, Ernst JV, Bailey WS, Heyman MB, Weinstein
WM: Human cryptosporidiosis in immunocompetent and immunodeficient
persons: Studies of an outbreak and experimental transmission. N Engl J
Med 1983; 308:1252-1257.
42
AJPH January 1989, Vol. 79, No. 1
34
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III. Investigation of Waterborne Disease Outbreaks
35
-------
AMERICAN
JOURNAL
OF
Public
Reproduced, with permission,
from American Journal of
Public Health
reprint
Community Health Effects of a Municipal Water Supply
Hyperfluoridation Accident
LYLE R. PETERSEN, MD, DIANE DENIS, MPH, DAVID BROWN, DSc, JAMES L. HADLER, MD, MPH,
AND STEVEN D. HELGERSON, MD, MPH
Abstract: For 12 hours, excess hydrofluorosilicic acid was
diverted to a 127-home community water supply. Fluoride levels
peaked at 51 parts per million (ppm). Water acidification caused
copper to leach from the domestic plumbing; raising copper levels to
25-41 ppm. Fifty-two (33 per cent) of those who drank hyper-fluorid-
ated water developed mild gastroenteritis. Vomiting was uncommon
and symptom onsets usually occurred >30 minutes after drinking
water; suggesting that fluoride, rather than copper, caused illness.
Skin contact with hyperfluoridated water caused itching and skin
rashes. (Am J Public Health 1988; 78:711-713.)
Introduction
Hyperfluoridation accidents in public water supplies are
uncommon1"5 and accidents in municipal water supplies have
been reported only twice previously.4-5 This report describes
the public health effects of a third municipal water supply
hyperfluoridation accident that occurred in a residential
Connecticut community.
Background
The community has 127 homes. Its water source is a
nearby treatment plant which also supplies water to three
distant larger metropolitan areas. The community is on a
"dead-end" portion of the water distribution system. The
street water mains are cement lined while feeder lines from
the street to each house are copper as is the piping within the
houses.
At the treatment plant, hydrofluorosilicic acid (H2SiF6)
is injected into the water supply. At approximately 3:00 pm
on March 11, 1986, an inadvertently opened valve began to
divert hydrofluorosilicic acid that normally would have been
injected into water supplying the community and distant
metropolitan areas, solely into the community's water sup-
ply. Hyperfluoridated water would have reached the domes-
tic taps at approximately 6:00 pm (beginning of exposure
period, time = 0 hours). At +1 hours (7:00 pm) residents
began notifying water company personnel that the water
tasted abnormal and turned blue on contact with soap, and of
itching and gastrointestinal symptoms. At +1 and +4 hours
(7:00 and 10:00 pm), household tap water samples revealed
fluoride and copper concentrations >40 times normal (fluo-
ride 42-51 ppm [normal 1.0 ppm], copper 25-41 ppm [normal
0.03 ppm]).
At +10 hours (4:00 am), a sample of water from a water
main had fluoride and copper concentrations of 50 ppm and
0.03 ppm, respectively. The water mains were then flushed.
Beginning at +12 hours (6:00 am), residents were told not to
From the Division of Field Services, Epidemiology Program Office,
Centers for Disease Control (Dr. Petersen); the Preventable Diseases Division,
State of Connecticut Department of Health Services (Ms. Denis, Dr. Brown,
Dr. Hadler); Office of the Director, Centers for Disease Control (Dr.
Helgerson); and the Yale University School of Medicine, Department of
Epidemiology and Public Health (Dr. Helgerson).
Address reprint requests to James L. Hadler, MD, MPH, Epidemiology
Section, State of Connecticut Department of Health Services, 150 Washington
St., Hartford, CT 06106. This paper, submitted to the Journal August 31, 1987,
was revised and accepted for publication December 14, 1987.
drink or bathe in the water and to discard ice or beverages
made with tap water.
Methods
Epidemiologic Investigation
On March 15, 1986, a door-to-door survey of the 127
community households was conducted. Data were obtained
from at least one adult (>16 years in age) to determine each
household member's water consumption, dermal exposure to
water, and symptoms.
The exposure period was considered to have been +0
hours to +12 hours. The quantity of ingested hyperfluorid-
ated water was estimated as the number of glasses of tap
water consumed during the exposure period plus the number
of glasses of beverages made from tap water during that
period. The latent interval was defined as the time from
consumption of the last glass of hyperfluoridated water to the
illness onset. A person was considered dermally exposed to
the hyperfluoridated water if that person bathed or showered
at home during the exposure period.
The outbreak period was defined from 0 to +54 hours. A
case of gastrointestinal irritation was a resident with onset
during this period of any of the following symptoms: diarrhea
(£2 watery stools in a 24-hr period), abdominal cramping,
severe nausea, or vomiting. A case of skin irritation was a
resident with onset of unusual itching during the outbreak
period.
Statistical relations between water consumption or der-
mal exposure and illness were determined by the method of
related ratios to control for household.'-7 The dose-response
relation of water consumption and gastroenteritis was as-
sessed by the Mantel Chi square test for trend8; other
differences were assessed by the Chi square test. The
effective dose of hyperfluoridated water that would be
expected to cause .gastroenteritis in 50 per cent of a popula-
tion (ED50) was calculated by the log probit method.9
Results
Information concerning 321 persons was gathered from
86 (68 per cent) of the 127 households. Representatives from
two households refused interviews and in 39 households no
adult was available for interview on the survey day. Abnor-
mal taste or color of water was reported in 62 per cent (53/86)
of households.
Gastrointestinal Illness
Gastrointestinal symptom histories were obtained for
312 persons, 55 (18 per cent) of whom were cases; symptoms
included: abdominal cramping (66 per cent), nausea (62 per
cent), headache (49 per cent), diarrhea (42 per cent), vomiting
(13 per cent), diaphoresis (12 per cent), and fever (4 per cent).
The onset of illness for the 46 persons with known time of
symptom onset is depicted in Figure 1. The median duration
of gastroenteritis symptoms was 5.5 hours with a range of 1
to 60 hours. No person sought medical evaluation for
gastroenteritis symptoms.
AJPH June 1988, Vol. 78, No. 6
36
711
-------
PUBLIC HEALTH BRIEFS
1 Exposure Period
TABLE 1—Cases of Skin Irritation by Bathing or Showering Status,
Connecticut Community, March 11-12, 1986
6P 10P 2 A
March 11
6P 10P 2 A
6A 10A 2P
March 13
6P
6 A 10A 2P
March 12
Time ot Symptom Onset
FIGURE 1—Gastroenteritis, by Time of Symptom Onset, Connecticut Commu
nity, March 11-13, 1986
Water consumption and symptom histories were avail-
able for 301 persons. Of the 160 persons who drank water, 52
(33 per cent) had gastroenteritis compared to only two (1.4
per cent) of the 141 persons who did not drink water (relative
risk = 23; 95 per cent confidence intervals = 5.7, 92.4).
Information about latent interval was available for 37 persons
and, for those persons, the median latent interval was two
hours (Figure 2). Only four persons (11 per cent) had
symptom onsets <30 minutes after drinking water. Attack
rates were similar by gender, age, and location of residence.
Attack rates by quantity of tap water consumed for 160
persons for whom data were available were: 29 per cent
(33/114), 1-2 glasses; 31 per cent (11/36), 3-4 glasses; and 80
per cent (8/10), >5 glasses. The calculated ED50 was 2.7
glasses of water (95 per cent CI = 1.8, 4.1). This correspond-
ed to 33.8 mg of fluoride and 20.2 mg of copper if an average
glass of water was 250 cc and contained 50 ppm of fluoride
and 30 ppm of copper.
Skin Irritation
Of the 300 persons whose skin irritation histories were
obtained, 30 (10 per cent) reported unusual itching, with a
duration of 2 to 62 hours. Of these 30 persons, 12 reported
skin rash compared to only two of 270 persons who did not
report itching. No person had a skin rash on the survey day.
9 10 11 12 13 14
Latent Interval (Hours)
FIGURE 2 Interval from Water Consumption to Onset of Gastroenteritis,
Connecticut Community, March 11-13, 1986
Water
Exposure
Bath
Shower
Neither
Unknown
Totals
Cases
5
13
11
1
30
Total*
19
88
176
17
300
Attack
Rate %
26
15
6
Rate
Ratio
4.2
2.4
1.0
95%
Confidence
Intervals
1.7-10.7
1.1-5.0
°21 persons had unknown skin irritation histories.
Persons dermally exposed to hyperfluoridated water in a
shower or bath (Table 1) were 2.7 times as likely to have
reported itching than unexposed persons (95 per cent CI = 4,
5.3). Persons who reported itching, but who had not bathed
or showered, had other water exposures such as dishwash-
ing, and their itching was localized to the area of water
contact.
Discussion
This hyperfluoridation accident was caused by diversion
of excess hydrofluorosilicic acid into a community water
supply. The resultant water acidification substantially ele-
vated water copper levels by solubilizing copper compounds
coating the domestic copper service lines, as demonstrated
by elevated copper levels in tap water and normal levels in
water mam water. In the previous municipal water supply
outbreak'caused by excess hydrofluorosilicic acid, high iron
levels resulted from contact with iron pipes.4
Although one-third of those who drank the water became
ill, symptoms were mild and of short duration. The EDso of
2.7 glasses of water in this outbreak corresponded to symp-
tom-producing doses of copper and fluoride observed in other
studies. Copper ingestion causes vomiting usually within 10
minutes,10 and outbreaks have occurred in water supplies
containing from 4-430 ppm of copper10"13 and with as little as
a 5 mg ingested dose.10
Low-dose fluoride ingestion causes nausea, vomiting,
abdominal cramping, and diarrhea,''2'4'14 and outbreaks have
occurred in water supplies with levels from 30 to > 1,000
ppm.1"5 Symptoms occur with a 5 mg ingested dose.15 Both
copper and fluoride may have had additive effects in this
outbreak; however, the infrequency of vomiting and symp-
toms occurrences <30 minutes after water ingestion suggest-
ed fluoride toxicity.
Skin irritation symptoms with a hyperfluoridation acci-
dent have not been reported and, in this outbreak, could have
been caused by fluoride, copper, or the water's acidity.
Although hydrofluorosilicic acid produces burns in high
concentrations,16 its cutaneous effects in low concentrations
are unknown. Dermally applied copper salts can produce
itching and skin rash17 but the cutaneous effects of copper at
levels recorded in this outbreak are unclear.
ACKNOWLEDGMENTS
The authors thank Loraine Good for editorial assistance with manuscript
preparation and Dr. Robert Gunn for his advice during all phases of the
investigation.
712
37
AJPH June 1988, Vol. 78, No. 6
-------
PUBLIC HEALTH BRIEFS
REFERENCES
1. Centers for Disease Control: Acute fluoride poisoning—North Carolina.
MMWR1974;23:199.
2. Hoffman R, Mann J, Calderone J, Trumbull J, Burkhart M: Acute fluoride
poisoning in a New Mexico elementary school. Pediatrics 1980;
65:897-900.
3. Craun GF: Waterborne outbreaks in the United States, 1971-78. Am
Water Works Assoc 1980 Annual Conference Proceedings. Denver: Am
Water Works Assoc, 1980.
4. Leland DE, Powell KE, Anderson RS: A fluoride overfeed incident at
Harbor Springs, Michigan. J Am Water Works Assoc 1980; 72:238-243.
5. Centers for Disease Control: Fluoride intoxication in a dialysis unit—
Maryland. MMWR 1980; 29:134-136.
6. Cochran WG: Sampling Techniques. New York: John Wiley, 1977.
7. Mendenhall W, Ott L, Schaeffer RL: Elementary Survey Sampling. North
Scituate, MA: Duxbury Press, 1979.
8. Mantel N: Chi-square tests with one degree of freedom: Extensions of the
Mantel-Haenzel procedure. J Am Stat Assoc 1963; 58:690-700.
9. Gad SC, Weil CS: Statistics for lexicologists. In: Hayes AW (ed): Principles
and Methods of Toxicology. Vol 2. New York: Raven Press, 1984: 273-320.
10. Gosselin RE, Hodge HC, Smith RP, Gleason MN (eds): Clinical Toxicol-
ogy of Commercial Products, 5th Ed. Baltimore: Williams and Williams,
1984.
11. Wyllie J: Copper poisoning at a cocktail party. Am J Public Health 1957;
47:617.
12. Nicholas PO: Food-poisoning due to copper in the morning tea. Lancet
1968; 2:40-42.
13. Hopper SH. Adams, HS: Copper poisoning from vending machines. Public
Health Rep 1958; 1:910-914.
14. Semple AB, Parry WH, Phillips DE: Acute copper poisoning: an outbreak
traced to contaminated water from a corroded geyser. Lancet 1960;
2:700-701.
15. Spoerke DG, Bennett DL, Gullekson D: Toxicity related to acute low dose
sodium fluoride ingestions. J Fam Pract 1980; 10:139-140.
16. Burke WJ, Hoegg VR, Phillips RE: Systemic fluoride poisoning resulting
from a fluoride skin burn. JOM 1976; 15:39-41.
17. Clayton GD, Clayton FE (eds): Patty's Industrial Hygiene and Toxicology.
2nd Ed., Vol II. New York: Wiley and Sons, 1963.
AJPH June 1988, Vol. 78, No. 6
38
713
-------
Reproduced, with permission, from American
Journal of Epidemiology
AMERICAN JOURNAL OF EPIDEMIOLOGY Vnl 10:! No -1
Copyright© 1976 by The Johns Hopkins University School of Hygiene and Public Health Printed iii'il.S.A.
AN EPIDEMIC OF GASTROENTERITIS TRACED TO A CONTAMINATED
PUBLIC WATER SUPPLY
JACK B. WEISSMAN.1 GUNTHER F. CRAUN,2 DALE N. LAWRENCE,' ROBERT A. POLLARD,1
MILTON S. SASLAW AND EUGENE J. GANGAROSA1
Weissman, J. B., G. F. Craun, D. N. Lawrence, R. A. Pollard. M. S. Saslaw
and E. J. Gangarosa (CDC, Atlanta, GA 30333). An epidemic of gastroen-
teritis traced to a contaminated public water supply. Am J Epidemiol 103:
391-398, 1976.
Between January 1 and March 15, 1974, approximately 1200 cases of
acute gastrointestinal illness occurred in Richmond Heights, Florida, a resi-
dential community of 6500 in south Dade County. Over one-third of all fam-
ilies in the area had at least one member affected. The findings of 10 culture-
proven cases of shigellosis among those who became ill and clinical signs
and symptoms in the others suggest that most of the other cases that were
not cultured may have been shigellosis also. Epidemiologic investigation
showed that consumption of tap water was associated with illness in the ini-
tial cases of affected families. Evaluation of the Richmond Heights public
water supply disclosed numerous inadequacies in both design and operation.
One of the wells providing water to the community was continuously con-
taminated with excessive levels of fecal coliforms from a nearby septic tank,
and a breakdown in chlorination on January 14-15 caused approximately 1
million gallons of inadequately chlorinated water from the contaminated
well to be distributed to the community 48 hours before the epidemic began.
Correction of deficiencies in the water plant was undertaken by the utility
company; the residents of Richmond Heights were instructed to boil their
drinking water or to use commercially bottled water pending completion of
corrective measures. A full scale study is planned for all similar public water
supplies in Dade County.
disease outbreaks; gastroenteritis; Shigella infections; water pollution
INTRODUCTION Miami in south Dade County, indicated
In March 1974, routine investigation of that UP to 1000 clinical cases of an illness
10 cases of shigellosis in Richmond characterized by diarrhea and abdominal
Heights, Florida, a residential suburb of Pain had occurred in residents between
January 1 and March 15, 1974. Subsequent
Received for publication May 21, 1975. and in final studieg developed evidence that illness re-
form October 1, 1975. * .
•Bacterial Diseases Division, Bureau of Epidemi- suited from massive contamination ot the
ology. Center for Disease Control, Atlanta, GA non:!.'!. area's public water supply. This report
• Water Supply Research Laboratory, Environmen- describes the results on these investiga-
tal Protection Agency, Cincinnati, OH. .
3 Field Services Division, Bureau of Epidemiology, lions.
Center for Disease Control, Atlanta.
'Dade County Department of Public Health, EPIDEMIOLOGIC METHODS
MThe'auU,o« gratefully acknowledge the assistance To evaluate the true extent of disease in
of Robert Quick, Margaret Pearson. R.N., and .John Richmond Heights, we reviewed county
Black, D.V.M., Dade County Department of Public records from 1970-1974 for cases of shigel-
Health, Prof. David Hertzig, Department of Mathe- , , , ,, j , i
matics, University of Miami, and the staffs of the losis and subsequently undertook a survey
Perrine and North Miami Health Units. of 75 households in the area (approxi-
391
39
-------
392
WEISSMAN. CRAUN. LAWRENCE, POLLARD. SASLAW AND GANGAROSA
mately 5 per cent of all households) chosen
by a two-stage cluster sampling technique
(1).
A survey form was developed for use by
public health nurses in the field. The
name, age, sex, and school or occupation of
everyone in the household was listed. The
respondent was asked about illness, de-
fined as diarrhea and abdominal pain since
January 1, 1974, in each person. For every-
one who had been ill, the date of onset,
duration of symptoms, symptom complex,
and details of medical treatment were
obtained. Each family member, ill or well,
was asked to estimate his daily water
consumption, indicating whether he drank
tap or bottled water.
Each respondent furnished additional
information: whether a private well was
present on the property, and if so, if it was
used only for irrigation or as a source of
potable water, and whether there_4nad been
recent septic tank malfunction. A variety
of questions were asked to uncover possible
common exposures, such as the source of
the household's meats and vegetables, res-
tuarants frequented, church and commu-
nity activities, and contacts with people
who had similar illnesses. Respondents
with ill family members were also asked
how they thought illness was introduced
into the home.
Since the endemic level of diarrheal
diseases in Richmond Heights was un-
known and could not be determined relia-
bly due to time lapse and unusual inter-
vening events, we selected a nearby com-
munity to serve as a control. After review-
ing a variety of socioeconomic characteris-
tics of approximately 2 dozen communities
in Dade County, we chose Bunche Park
about 15 miles (24 km) north of Richmond
Heights, because its social, racial, and
economic profile most closely approx-
imated that of Richmond Heights. A sur-
vey similar to the one performed in Rich-
mond Heights was conducted in Bunche
Park.
Florida Water and Utilities, Inc., the
company operating the Richmond Utilities
Company, provided records of water usage,
termination of services to consumers be-
cause of non-payment of bills, blueprints of
the water distribution system, and daily
operational records. The Dade County De-
partment of Public Health provided rec-
ords of coliform counts on routine water
samples obtained by health department
sanitarians.
LABORATORY METHODS
Rectal swabs and stool specimens were
processed by the Epidemiologic Services
Laboratory Branch, Center for Disease
Control (CDC). Shigella sonnei isolates
from Dade County were forwarded to CDC
for biochemical confirmation, antibiotic
sensitivity testing, and colicin typing.
Water samples collected during the inves-
tigation were analyzed by the State's
Miami Regional Laboratory, at CDC and
at the Water Supply Research Laboratory,
Environmental Protection Agency (EPA).
Water samples were analyzed for total and
fecal coliforms by the membrane filter
technique; standard plate counts were per-
formed on several samples to provide an
estimate of total bacterial population.
Fluorescein was used in dye studies, and
fluorescence was measured spectro-
photometrically.
RESULTS
Epidemiologic investigations in Dade
County
Review of county records of culture-
proven cases of Shigella sonnei infection for
the five-year period 1970-1974 disclosed
that a statistically significant excess num-
ber of isolates were reported in the first 10
weeks of 1974 as compared with the four
preceding years (p < .05) (table 1). With-
out the 10 cases reported from Richmond
Heights, however, the total in the first 10
weeks of 1974 was within 2 SD of the mean
40
-------
GASTROENTERITIS EPIDEMIC TRACK!.) TO PUUUC WATER SUPPLY
393
TARI.F, 1
Coses of shigellnais, Dadp County, Florida 1970-1974
Year
1970
1971
1972
1971.1
1974
Annufll
li.tiil
108
76
103
75
148
First
in weeks
17
12
15
10
29
Mean, 1970-1973
90.5
13.5
for the same time period during the four
preceding years. Of the 29 reported cases,
those in Richmond Heights occurred signif-
icantly earlier than cases elsewhere in
Dade County. Seven of 10 cases in Rich-
mond Heights occurred in the first five
weeks of the 10-week period, compared
with none of eight cases in communities
adjacent to Richmond Heights (p •= .004)
or with three of 16 cases elsewhere in Dade
County (p = .006).
Shigella sonnei isolates from Richmond
Heights differed in their antibiotic resist-
ance and colicin-typing patterns from all
other isolates in Dade County during the
period January-March 1974. All those from
Richmond Heights patients had an untyp-
able colicin pattern and were sensitive to
all antibiotics tested. Four patients from
elsewhere in Dade County had untypable
isolates that were antibiotic resistant. Four
additional colicin patterns (none were the
pattern of an established colicin "type")
were observed among six of the Dade
County isolates, but none was an isolate
from a patient in Richmond Heights. There
were no shigella isolates from Bunche Park
during the time period studied.
Interviewers .succeeded in contacting 73
of 75 households in Richmond Heights and
70 of 75 in Bunche Park. In Richmond
Heights, data were obtained for 353 per-
sons, and in Bunche Park for 292. Twenty-
seven of 72 households (37.5 per cent) in
Richmond Heights had members who were
ill, compared with 11 of 70 (15.7 per cent)
in Bunche Park (x2 = 7.52, p < .01).
Sixty-seven of 353 people (19.0 per cent)
in Richmond Heights were ill, compared
with 16 of 292 (5.5 per cent) in Bunche
Park (p < .01, t test). Thus, the incidence
of diarrheal disease in Richmond Heights
was significantly greater than in a control
community during the time period exam-
ined. Extrapolation of these data to the
community as a whole would indicate that
1256 (±450) cases of diarrheal illness oc-
curred in Richmond Heights in the first 10
weeks of 1974 (95 per cent confidence
interval).
Analysis of the distribution of cases in
(he Richmond Heights survey population
disclosed a mean age of patients of 17.2
years, close to the mean of 20.9 years of
household members who were not ill; at-
tack rates were distributed with relative
uniformity throughout all age groups (table
2). The age distribution of cases was also
slightly different from that of shigellosis in
the United Slates (2).
Cases of diarrheal disease in the survey
population began to occur in epidemic
numbers on January 17, 1974, peaking
about one month later and rapidly falling
off afterward (figure 1). The lime course of
cases in the survey population paralleled
that of the culture-proven cases of S.
ftonnei in Richmond Heights. Illness in
both survey populations was characterized
by fever, nausea, vomiting, and diarrhea;
overall illness in Richmond Heights was
characterized by a greater number of spe-
cific symptoms than in either Bunche Park
or among the culture-proven cases (table
3). Symptoms of patients in the Richmond
Heights survey population differed signifi-
cantly from those in Bunche Park patients
in that there was a significantly higher
incidence of tenesmus, lever, and vomit-
ing, but did not differ significantly from
the culture-proven cases. In none of the
symptoms noted was the incidence higher
for patients in the Bunche Park survey
41
-------
394
WEISSMAN. CRAUN, LAWRENCE, POLLARD. SASLAW AND GANOAROSA
TABLE 2
Age-specific illness in Dade County survey populations and in households of patients with culture-positive
shigelloais, 1974
0-4
5-9
10-19
20-29
30-39
40-49
50-59
60-69
70 +
Unknown
Total
111
(No.)
0
5
4
1
2
0
1
1
2
0
16
Not
ill
(No.)
1
2
11
6
4
5
0
4
0
0
33
Attack
rate
(%)
.00
.71
.27
.14
.33
.00
1.0
.20
1.0
.00
.33
Survey populations
Richmond Heights
(No.)
7
16
18
5
8
2
7
4
0
0
67
Not
ill
(No.)
15
31
102
32
30
35
18
17
2
4
286
Attack
rate
(%)
.32
.34
.15
.14
.21
.05
.28
.19
.00
.00
.19
(N, .)
0
1
4
3
2
4
2
0
0
0
16
Bunche Park
Not
ill
(No.)
31
24
75
21
15
46
28
23
11
2
276
Attack
rate
(%)
.00
.04
.05
.13
.12
.08
.07
.00
.00
.00
.05
20
ie-
16-
u
U
3 10 17
MAR.
23 30 I 6 S3 20 27 3 10 17 24
DEC- JAN. FEB
1973 1974
FIGURE 1. Gastroenteritis by week of onset. Rich-
mond Heights survey population, January-March
1974.
population than in the Richmond Heights
survey group.
There were no significant correlations
between illness in the Richmond Heights
survey population and attendance at spe-
cific schools, churches, or clubs, particular
sources of meats or vegetables, specific
TABLE 3
Symptom complex in survey populations, by per cent,
Dade County, 1974
Symptom
Fever
Tenesmus
Nausea
Vomiting
Headache
Bloody diarrhea
Culture-
proven
srugellos.s
80.0
53.3
50.0
46.7
38.5
23.1
Survey populations
Richmond
Heights
70.2
78.6
76.9
63.2
66.7
11.1
Bunche
Park
33.3
38.5
53.8
28.6
53.8
6.7
restaurants, or the presence of a private
well; 23.9 per cent of the survey households
had private wells, generally for use with
sprinkler systems and not as a source of
potable water. Sixty-nine per cent of
households with illness knew of other
households in which illness had occurred;
only 9.3 per cent of households without
illness knew of families with illness (x2 =
25.1, p < .0001). Many people volunteered
that their drinking water had been inter-
mittently discolored, "fishy" smelling or
tasting, and had contained particulate
matter. In the sample survey population as
a whole, illness was not associated with
water consumption. To eliminate the am-
42
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GASTROENTERITIS EPIDEMIC TRACED TO PUKI.IC WATER Sl'PPLY
395
biguity caused by households with multi-
ple cases where some cases may have
-occurred by person-to-person spread as
well as by a common source such as drink-
ing water, the analysis of water-specific
attack rates was recalculated, this time
comparing water consumption of the initial
cases only with water consumption by the
non-ill members of the same households.
Eighteen of 49 (36.7 per cent) who drank
more than four glasses of water daily be-
came ill; this incidence was significantly
greater than 15 of 82 (19.5 per cent) who
drank four glasses of water or less daily (x2
= 4.60, p < .05). If seven households who
drank only bottled water are also removed
from analysis, the association of illness
with water consumption increases further
in statistical significance (x2 = 5.44, p <
.03). Similar analyses relating to illness in
Bunche Park did not identify water as
being associated with the low level of
illness there.
Wafer system evaluation
At the time of the outbreak, Richmond
Heights was served by a single public water
system operated for the Richmond Utilities
Company by Florida Water and Utilities.
Inc. (figure 2). The average quantity of
water pumped for the time in question was
slightly more than 3790 kl per day. Water
was pumped from several wells on the
premises of the utility company and came
from a relatively shallow aquifer located
less than 15.2 meters below the surface.
The principal well in use was a well, 6,
drilled to a depth of 7.3 meters with a
casing extending clown to 6.1 meters. On
January 31, a new and deeper well, 7, was
put into service and, together with well 6,
supplied the needs of the Richmond
Heights residents.
Water from both wells was mixed, chlo-
rinated and distributed. A 379 kl elevated
storage tank was connected to the system
in such a manner that during periods of
high usage, water was pumped from the
wells, and after passing the point of chlo-
rine addition, was distributed directly
throughout the community. As the de-
mand in the distribution system slackened,
the water surging from the wells under the
[P~] PUMP ® CHECK VALVE
'•#fy, PIPELINE AND DIRECTION OF FLOW
^±
GAS CHLOR1NATOR
FIGURE 2. Richmond Heights water treatment plant.
43
-------
396
WRISSMAN. CRAUN, LAWRENCE. POLLARD, SASLAVV AND GANGAROSA
influence of the pumps was intermittently
diverted into the storage tank. Then, dur-
ing periods of low usage, the demand was
satisfied entirely by water in storage. Only
when the water level in the elevated tank
dropped to a predetermined level did the
pumps automatically reactivate.
During the investigation, bacteriologic
testing of untreated water from wells 6 and
7 revealed consistently elevated total coli-
form counts (>50 per ml) in samples from
well 6. Fluorescein dye introduced into the
flush toilet at a neighboring nursery school,
which had a septic tank located approxi-
mately 38 meters from well 6, was identi-
fied in water from (he well after only nine
hours. Due to the porosity of the soil, the
cone of inlluencc of the pumps had been
estimated to have a radius of perhaps up lo
400 meters.
The method of chlorination in use con-
sisted of the addition of chlorine from a
68 kg cylinder through a booster pump.
The average amount of chlorine used per
day during January. 1974, was 11.3 kg.
This was successful in achieving recorded
chlorine levels at the pumping station of
between 1.3 ppm and 3 ppm. Similar read-
ings were recorded for preceding and subse-
quent months. However, review of pump-
ing station records revealed that on Janu-
ary 15, the plant operator arrived to find
the chlorinator booster pump malfunction-
ing and only 2.3 kg of chlorine used during
the preceding 24 hours. The chlorine resid-
ual recorded at the plant effluent on that
day was 0. Thus, it was estimated that
approximately 3790 kl of unchlorinated or,
at best, inadequately chlorinated water
had been distributed to the community 48
hours before the epidemic began.
CONTROL MEASURES
With bacteriologic evidence of continu-
ing fecal contamination of the Richmond
Heights raw water supply and epidemio-
logic evidence linking gastroenteritis in the
community with consumption of water, the
Dade County Department of Public Health
issued a "boil-water emergency" alert on
March 29, 1974. Announcements in news-
papers and on radio and television in-
structed residents to boil drinking water.
Public water fountains in schools and
churches in the areas were closed. Person-
nel at the health department offices were
made available to answer public inquiries.
The health department also made recom-
mendations to assure the safety of the
water supply after several water system
deficiencies were designated. These in-
cluded the recommendations that coliform
counts in raw waters to be treated exclu-
sively by disinfection should not exceed 20
fecal or 100 total coliforms per 100 ml as
measured by monthly arithmetic means
(in accordance with EPA guidelines), that
provisions be made to insure continuous
chlorination of the raw water source, and
that, a chlorine contact time of at least 30
minutes be assured.
DISCUSSION
Our investigation indicated that over
1200 individuals in Richmond Heights,
about 20 per cent of the area's population,
became ill in January and February 1974.
A substantial body of evidence supports
the association of water consumption from
the public water supply with diarrheal
disease, and we have concluded that the
infectious agent responsible for this epi-
demic was waterborne.
No samples of raw water from well 6, the
well supplying most of the community's
water in January, were obtained during
that time. However, multiple samples ob-
tained on several days during our investi-
gation in March were invariably contami-
nated with excessive numbers of fecal coli-
forms.
Moreover, the recovery of fluorescein dye
from well 6 water shortly after introduc-
tion of the dye into a nearby sewerage
system suggests that well 6 was continu-
ously contaminated by sewage. Conse-
44
-------
GASTROENTERITIS EPIDEMIC TRACED TO PUBLIC WATER SUPPLY
397
quently, any interruption in disinfection
would inevitably have resulted in the dis-
tribution of contaminated water to the
community. On January 14, two days be-
fore the epidemic began, a malfunction in
the chlorinator booster pump resulted in
the distribution of inadequately treated
water for an entire day. Numerous poten-
tial troUblespots, none of which was spe-
cifically implicated in this outbreak, in-
cluded pumping raw water when chlorine
cylinders were being changed, inadequate
chlorine contact times, substantial pres-
sure drops in certain areas of the distribu-
tion system, and evidence of substantial
water loss through leaks or illegal connec-
tions; these factors afforded the possibility
of contamination of the system in its distri-
bution as well as its source.
Ten cases of Shigella sonnei infection
provided the initial stimulus for this inves-
tigation. Culturing the survey population
would have been both impractical and
unrewarding, given the elapsed time be-
tween their illness and our investigation;
symptomatology was used instead as the
primary means of differential diagnosis.
We cannot prove, therefore, that the clini-
cal cases were shigellosis; however, the
concordance of symptoms in the Richmond
Heights survey population with the symp-
toms in the culture-proven cases (rather
than with non-shigellosis cases in Bunche
Park) and the high incidence of tenesmus,
thought to be a relatively specific indicator
of shigella infection, suggest that most
community illness was shigellosis rather
than another acute intestinal illness. Nu-
merous reports in the literature of sewage
poisoning from contaminated water have
depicted a syndrome characterized by
shorter duration and milder symptoms
than we observed here (3-5). The possi-
bility that some of the diarrheal illnesses
may have been caused by etiologic agents
other than shigella cannot be ruled out.
One year prior to this investigation CDC,
EPA, and Bade County Health Depart-
ment workers investigated another epi-
demic of waterborne disease in Dade
County. In February and March 1973, 210
confirmed cases of typhoid fever occurred
among 1400 residents at the South Dade
Migrant Farm Labor Camp, in Home-
stead, Florida (6). Epidemiologic investi-
gation at that time disclosed a highly
significant correlation between water con-
sumption and illness. Bacteriologic exami-
nation of the camp's well water supply
showed the well to have been intermit-
tently contaminated. The water was chlo-
rinated before distribution; however, an
engineering evaluation of the system's op-
eration revealed that the single chlorine
cylinder had run out of chlorine and un-
treated water was distributed prior to the
outbreak.
Deficiencies in the water system that, led
to the Dade County waterborne typhoid
epidemic of 1973 were duplicated in the
Dade County waterborne gastroenteritis
epidemic of 1974: contamination of a raw
water source without, a reliable system for
preventing its distribution in the absence
of adequate disinfection, and an unsatis-
factory surveillance system to pinpoint
long-standing potential hazards before
their translation into real disease.
The morbidity which may occur from the
waterborne transmission of enteric infec-
tion is difficult to overestimate. Less than
10 years ago, contamination of a public
water system in California caused over
16,000 cases of salmonellosis in a three-
week period (7). This and other outbreaks
(4-6, 8, 9) underscore that contaminated
public water is by no means a problem
exclusively of underdeveloped or economi-
cally disadvantagecl societies but rather a
significant problem of contemporary urban
life.
REFERENCES
]. Serfling R, Sherman I: Attribute Sampling
Methods for Local Health Departments. PHS
Publication No 1230, 1965, pp 46-53
2. Center for Disease Control: Shigella Surveillance
45
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398
WEISSMAN. CRAUN, LAWRENCE, POLLARD, SASLAW AND (5ANGAROSA
Report No 36, First and Second Quarters 1974.
issued February 1975
3. Lobel HO, Bisno AL, Goldfield M, et al: A water-
borne epidemic of gastroenteritis with secondary
person-to-person spread. Am J Epidemiol
89:384-392, 1969
4. Werner SB, Jones PH, McCormack WM, et al:
Gastroenteritis following ingest ion of sewage-pol-
luted water: An outbreak at a logging camp on the
Olympic Peninsula. Am J Epidemiol 89:277-285,
1969
5. Gastroenteritis—Illinois. Center for Disease Con-
trol: Morbidity and Mortality Weekly Report
21(23):198-199, June 10, 1972
6. Feldman KK, Maine WB, Nilzkin JI,, et al: Epide-
miology of Salmiinclla typhi infection in a migrant
labor camp in Dade County, Florida. .J Infect Ois
130:334 342, 1974
7. A wat.erborne epidemic of salmonellosis in River-
side, California, 196.r>. A collaborative report. Am J
Epidemiol 93:3:! 48, 1971
8. Gorman AE, Wolman A: Waterborne outbreaks in
the United States and Canada and their signifi-
cance. J Am Water Works Assn 31:225-37;), 1969
9. Weibel SR. Uixon Fll, Weidner RB, et al: Water-
borne disease outbreaks 1946-1960. J Am Water
Works Assn 56:947-958, 1964
46
-------
Reproduced, with permission, from Annals
of Internal Medicine
Chronic Diarrhea Associated with Drinking
Untreated Water
Julie Parsonnet, MD; Susan C. Trock, DVM; Cheryl A. Bopp, MS; Cynthia J. Wood, BA;
David G. Addiss, MD; Frank Alai, MPH; Leo Gorelkin, MD; Nancy Hargrett-Bean, PhD;
Robert A. Gunn, MD, MPH; and Robert V. Tauxe, MD, MPH
Purpose: To determine the cause of an outbreak of chron-
ic diarrhea and to define the clinical profile of the illness.
Design: A case series of patients with chronic diarrhea
and case-control and cohort studies to determine the vehicle
and cause of the illness.
Setting: Rural Henderson County, Illinois.
Patients: Seventy-two patients who had onset of chronic
diarrheal illness between May and August 1987. Controls
were local residents and eating companions who did not
have diarrheal illness. A cohort study included 80 truck
drivers from a local firm.
Methods and Measurements: Nonbloody diarrhea was
characterized by extreme frequency (median, 12 stools/d),
marked urgency, fecal incontinence, and weight loss (mean,
4.5 kg). The median incubation period was 10 days. Nine
patients were hospitalized; none died. Diarrhea persisted in
87% of patients after 6 months. Antimicrobial therapy pro-
duced no clinical improvement. No bacterial, mycobacterial,
viral, or parasitic agents known to be enteropathogenic were
detected in stools or implicated water. Three of five small-bow-
el biopsies showed mild inflammatory changes. Mild inflam-
mation was also seen in two of nine colonic biopsies. Case-con-
trol studies implicated a local restaurant (P = 0.0001, odds
ratio =19) and subsequently the untreated well water
served in the restaurant (P = 0.04, odds ratio = 9.3) as the
vehicle of transmission.
Conclusions: This is the first outbreak of chronic diarrhea
linked to drinking untreated water. The causative agent and
pathophysiologic mechanism of the illness remain elusive.
In 1984 and 1985, outbreaks of a distinctive chronic
diarrheal illness occurred in Brainerd, Minnesota and
San Antonio, Texas (1, 2). The illnesses were charac-
terized by dramatic, urgent, watery diarrhea persisting
for many months in the absence of systemic symp-
toms. Although epidemiologic evidence strongly im-
plicated unpasteurized milk as the vehicle in the
Brainerd outbreak, the San Antonio case-patients were
not exposed to unpasteurized milk products. Extensive
clinical and microbiologic studies revealed no likely
pathogens in either episode, and the causative agent
for "Brainerd diarrhea" remains unknown.
Physicians throughout the United States have re-
ported sporadic cases of chronic diarrheal illness clini-
cally similar to those seen in the Minnesota and Texas
outbreaks (3), and other smaller clusters have been
recognized (1). Medical evaluation of these patients is
often frustrating. Although some chronic diarrhea cas-
es have been linked with two newly denned clinicopa-
thologic entities, collagenous colitis (4-7) and micro-
scopic colitis (8, 9), the vehicles of transmission and
causative agents are obscure.
Henderson County, Illinois is a corn and soybean
farming community of 9000 people located on the
eastern bank of the Mississippi River. In July 1987, a
physician reported 12 cases of a distinct chronic diar-
rheal illness to the Henderson County Health Depart-
ment. By the middle of August, the health department
had accumulated information on approximately 50 pa-
tients with chronic diarrhea. Patients had been evalu-
ated by physicians at several local hospitals; no causa-
tive agent was identified, but a local restaurant was
suspected as the source. This large outbreak provided
an opportunity to further delineate the clinical and
pathologic profile of "Brainerd diarrhea" and to define
a new vehicle of transmission, water.
Annals of Internal Medicine. 1989;110:985-991.
From the Centers for Disease Control, Atlanta, Georgia,
and the Illinois Department of Public Health, Springfield,
Illinois. For current author addresses, see end of text.
Methods
A case was defined as diarrhea (three or more loose stools
per day) of unknown cause lasting 4 or more weeks, with
onset after 1 May 1987, in a person who had been in Hen-
derson County after 1 April 1987. A case was considered to
be restaurant-associated if the case-patient had visited the
implicated restaurant during the 6 weeks before onset of ill-
ness. Cases were identified by direct patient reports to the
Henderson County Health Department, by calls to local
physicians and hospital infection control administrators, and
by referral from known case-patients. The Illinois Depart-
ment of Public Health solicited case reports from region-
al and county health departments. Requests were made
through the newspaper and television media for persons
with chronic diarrhea to notify the county health depart-
ment.
15 June 1989 • Annals of Internal Medicine
47
Volume 110 • Number 12 985
-------
Table 1. Symptoms of Chronic Diarrhea! Illness in 67 Case-
Patients in Henderson County, Illinois, 1987
Symptom
Frequency, n(%)
Urgency
Borborygmi
Fecal incontinence
Flatulence
Cramps
Bloating
Nausea
Feverishness
Vomiting
67(100)
53 (79)
43 (64)
42 (63)
30 (45)
24 (36)
16 (24)
8 (12)
5 (8)
Clinical Studies
Sixty-seven case-patients were interviewed regarding symp-
toms and treatment of their diarrhea! illness. Available med-
ical records, laboratory results, and pathology specimens
from 51 case-patients were reviewed. Six months after the
initial investigation a follow-up questionnaire was sent to all
case-patients to assess the duration of illness.
Thirty-two case-patients submitted stool specimens; all
specimens were submitted at least 4 weeks after the onset of
illness. Ten specimens were cultured at the Centers for
Disease Control (CDC) for Salmonella species, Shigella spe-
cies, Campylobacter jejuni/coli, Yersinia enterocolitica,
pathogenic Vibrio species, Aeromonas species, Plesiomonas
species, Bacillus cereus, and Staphylococcus aureus. Ten col-
onies selected from the MacConkey agar were identified, ser-
otyped (if identified as E. coif), and tested by enzyme-linked
immunosorbent assay for E. coli enterotoxins (heat-labile
and heat-stable) (10, 11). Shiga-like toxins I and II, local-
ized and diffuse adherence, and invasiveness genes were
sought with specific DNA probes (12-15). Ten bacterial col-
onies were also selected from blood agar plates and identi-
fied. The National Veterinary Services Laboratories cultured
ten case and eight control stools for mycobacterial species,
including Mycobacterium paratuberculosis (16). Ten stools
were cultured for yeast. Ova and parasite examination, in-
cluding examination for Cryptosporidia organisms, Isospora
belli, and Blastocystis hominis (ten stools), and electronmi-
croscopy for viral agents (five stools) were also done. Twen-
ty-two case-patients and twenty-one asymptomatic controls
were skin-tested for Mycobacterium avium-intracellulare,
Mycobacterium kansasii, Mycobacterium scrofulaceum,
Mycobacterium tuberculosis, Candida albicans, and Tricho-
phyton species. Pathologists at CDC and the Baylor Univer-
sity Medical Center reviewed biopsy slides obtained from
local hospitals.
Epidemiologic and Environmental Studies
To determine risk factors for chronic diarrheal illness in the
community, we conducted a case-control study (case-con-
trol study 1) and a survey. Case-control study 1 included
case-patients who were residents of Henderson County
(n = 23) or the neighboring town of Kirkwood in Warren
County (n = 5) and had consulted a physician for their
diarrheal illness. One age- and neighborhood-matched con-
trol, with no diarrheal illness lasting longer than 1 week in
the previous 3 months, was systematically selected for each
case-patient. Truck drivers employed by an Iowa-based na-
tional retail company who frequently passed through Hen-
derson County and had reported cases of chronic diarrheal
illness to the health department were surveyed. In both the
case-control study and the survey, data gathered included
foods and beverages purchased (particularly foods and dairy
products purchased from farms or produce stands), restau-
rants and food stores visited, domestic water supply used,
exposure to animals, history of previous gastrointestinal ill-
ness or medication use, and illness among household con-
tacts.
After these two studies implicated the restaurant, a sec-
ond case-control study and a cohort study were done. In
case-control study 2, 30 case-patients who named 65 meal-
companion controls were analyzed in a matched design with
a variable number of controls per case. The cohort study
included all truck drivers identified in the survey who had
eaten at the restaurant. Information asked included frequen-
cy, date and time of restaurant visits, specific food and bev-
erages consumed in the restaurant, seating and parking pref-
erences, restroom use, cigarette and alcohol use, history of
gastrointestinal illnesses, and use of medications. All avail-
able restaurant employees were interviewed.
The restaurant was inspected by local and state sanitari-
ans, and water quality reports from the restaurant and local
community were reviewed. Water from the implicated well
was analyzed for pesticides, herbicides, volatile organic com-
pounds, and heavy metals. The Environmental Protection
Agency cultured water for mycobacterial species (17). Viral
cultures of water, human enteroviral gene probe of a water
filter, and examination of water for ova and parasites were
done at the University of Arizona (18-20).
The statistical analyses were done using the Wilcoxon
rank sum test, the Fisher exact two-tail test, the McNemar
test, and conditional logistic regression.
Results
Clinical Studies
Seventy-two case-patients were identified; 36 (50%)
case-patients were male. The mean and median age of
case-patients was 56 years (range, 9 to 83 years). Ill-
ness was characterized by sudden onset of severe
watery diarrhea; in the initial week of illness, some
case-patients described more than 20 stools per day
(median, 12 stools/day). Urgency was reported by
100% of case-patients and was severe, often associated
with fecal incontinence (65%). Weight loss was re-
ported by 62% at 2 months (mean loss, 4.5 kg). Fever
was uncommon. The frequency of symptoms is shown
in Table 1.
Six months after the initial investigation, 62 (87%)
of 71 case-patients who responded to the follow-up
questionnaire continued to have diarrhea; 52 (73%)
reported having diarrhea every day (mean duration of
illness, 261 days). Frequency of bowel movements in
those with daily diarrhea had decreased to a mean of
six bowel movements per day. Complete resolution of
diarrhea occurred in 7 case-patients (mean time until
resolution, 183 days); these case-patients were young-
er than those who remained ill (median age, 37 com-
pared with 56, P = 0.01).
A median incubation period of 10 days (range, 2 to
40 days) was estimated from 15 case-patients who vis-
ited the restaurant on only 1 day. Of 150 case-patient
household members at risk for secondary transmis-
sion, none developed diarrhea. One Henderson Coun-
ty case-patient frequently visited her adult daughter in
Iowa; that daughter developed chronic diarrhea 3
months after the mother's diarrhea onset; however, be-
cause sporadic cases of chronic diarrhea occur and
because contact was difficult to quantify, it was not
possible to definitely ascribe this case to secondary
spread.
Of the 72 case-patients, 60 (83%) consulted a phy-
sician. Nine persons, eight of them women, were hos-
pitalized; none died. Laboratory results are outlined in
986 15 June 1989 • Annals of Internal Medicine
Volume 110 • Number 12
48
-------
Table 2. Clinical Data for 67- Case-Patients in Henderson County, Illinois Compared with 78 Case-Patients in Brainerd,
Minnesota with Chronic Diarrhea*
Laboratory Test
Henderson County
Brainerd
Results
n(%) (n)
Stool culture for Shigella,
Salmonella, Yersinia,
and Campylobacter
Stool for ova and parasites
Fecal leukocytes
Barium enema
Small-bowel series
Sigmoidoscopy
Colonoscopy
Large-bowel biopsy
Small-bowel biopsy and
aspirate
Hematocrit
Leukocyte cell count
37(55)
39(58)
9(13)
16(24)
8(12)
6 (9)
6 (9)
9(13)
5 (7)
17(25)
18(26)
43(55)
43(55)
13(17)
31(40)
12(15)
28(36)
8(10)
9(12)
5 (6)
19(24)
19(24)
No pathogens (77) , Salmonella (2),
Campylobacter jejuni ( 1 )
No pathogens (79), Blastocystis hominis (2),
Giardia lamblia ( 1 )
None (4), few (16), moderate (1), many (1)
Normal (22), diverticuli (18), spasm (2),
possible colitis (2)
Normal (17), duodenal diverticulum (1),
rapid transit ( 1 ) , gastric bypass ( 1 )
Normal (29), proctitis (2), erythema (1),
edema (1), punctate hemorrhages (1)
Normal (5), nonspecific patchy erythema
(4), inflammation (3), punctate
hemorrhage (2)
Normal (4), nonspecific mild, chronic
inflammation (13), prominent lymphoid
aggregates (1)
Normal (8), increased histiocytes (1), mild
villous blunting (1), no Giardia (7)
Normal (35), low (1)
Normal (33), low (2), high (2)
* Other tests performed on fewer than 10 case-patients (n) in Henderson County. All results were normal except where indicated: serum albumin (9;
3 low), thyroid function tests (6), vasoactive inhibitory peptide (2), gastrin (3; 1 high), 5-hydroxyindole acetic acid (3), serum B12 (3), serum folate
(3), serum carotene (3; 1 high), qualitative fecal fats (2), quantitative fecal fats (3; 1 high).
Table 2; no consistent abnormalities to suggest inflam-
matory bowel disease or malabsorption were observed.
Therapy included use of numerous antimicrobial and
antidiarrheal agents (Table 3); none cured the illness.
Opioid analogs provided subjective improvement in
half of those receiving them.
All bacterial and mycobacterial stool cultures were
negative for known pathogens, and no viral agents
were seen on electronmicroscopic examination of stool
specimens. Giardia lamblia was identified for one case-
patient in the first of several stool specimens submitted
for parasite examination. Diarrhea in this case-patient
temporarily improved with metronidazole therapy but
worsened several days later. A course of quinacrine
hydrochloride also produced temporary improvement.
Later examinations for stool parasites and a small-
bowel aspirate did riot show evidence of Giardia spe-
cies although the case-patient continued to have five to
six stools per day. Two case-patients had B. hominis
noted in isolated stool samples. Both had continued
symptoms despite later ova and parasite examinations
that revealed no B. hominis organisms. Skin-test re-
sponse to mycobacterial antigens was similar for case-
patients and controls.
Twelve patients had sigmoidoscopy or colonoscopy;
no gross abnormalities were seen. The mean interval
from date of diarrhea onset to date of biopsy was 43
days. Local pathologists reported mild, nonspecific in-
flammatory changes in eight of the nine case-patients
who had either colonic or rectal biopsies. All slides
were reviewed at CDC and the Baylor University
Medical Center, Dallas. The degree of inflammatory
infiltrate was believed to fall within the normal range
for colonic mucosa in all but two biopsies. Specifically,
there was no evidence for either microscopic colitis or
collagenous colitis.
Five case-patients had small-bowel biopsies. No par-
asitic infestation in any specimen and no significant
abnormalities were noted by local pathologists. The
four biopsy specimens reviewed at Baylor University
Medical Center were believed to show a mild increase
in plasma cells and eosinophils within the lamina
propria. Similarly, the CDC pathologist reported in-
creased plasma cells and eosinophils in three of five
biopsies. In all cases, changes were concluded to be
mild and nonspecific.
Epidemiologic and Environmental Studies
Attack rates among Henderson County residents were
as follows: 0 in the group 0 to 4 years, 1.0/1000 in the
group 5 to 17 years, 3.2/1000 in the group 18 to 64
Table 3. Medications Used to Treat Chronic Diarrhea! Ill-
ness in 67 Case-Patients in Henderson County, Illinois, 1987
Medication*
Number
Treated
Number
Improved
n(%)
metronidazole
sulfonamide
neomycin
tetracycline
bismuth salicylate
loperimide
diphenoxylate
Metamucil
cholestyramine
19
14
10
7
35
32
28
15
7
3(18)
2(15)
2(17)
2(29)
2 (6)
18(64)
13(50)
1 (9)
2(29)
* Other medications used by more than one case-patient include van-
comycin (5), erythromycin (3), sulfasalazine (2), cimetidine (2), and
Donnagel (3).
15 June 1989 • Annals of Internal Medicine • Volume 110 • Number 12 987
49
-------
18-
16-
14-
12-
8 io-
CO
(8 o
o 8
6
4
a
a
8 152229 5 121926 3 10172431|7 14
May Jun Jul Aug
Date of Onset
Figure 1. Number of cases of chronic diarrhea by week of onset, in
Henderson County, Illinois, May through August 1987. The date of
closure (7/31) of the implicated restaurant is indicated by the arrow.
years, and 9.2/1000 in group 65 years and older. The
earliest onset dates were in early May, with a peak in
mid-July, and a precipitous drop in the beginning of
August (Figure 1). The last case-patient had an onset
on 7 August, 1 week after the restaurant was closed.
Thirty (42%) of the case-patients resided in Hender-
son County.
Eating at the restaurant in the 4 weeks before onset
of illness was strongly associated with illness in case-
control study 1. Twenty-five (89%) of twenty-eight
case-patients visited the restaurant, and 6 (21%) of 28
neighborhood controls visited the restaurant (odds ra-
tio = 19.0, P < 0.001).
Sixty-three (79%) of eighty truck drivers respond-
ed to the survey questionnaire and nine cases (14%)
were identified. Three truckers described diarrheal ill-
ness but did not supply enough information to catego-
rize them as case-patients and they were excluded
from further analysis. One trucker did not respond to
questions concerning restaurants. Nine (20%) of for-
ty-five truckers who ate at the restaurant and none of
14 truckers who did not eat at the restaurant were
case-patients (relative risk = indeterminate, P = 0.1,
Fisher exact two-tail test).
The results of case-control study 2 (study of restau-
rant-associated risk factors for illness) are outlined in
Table 4. Case-patients were older than controls (me-
dian age of 60.5 years for case-patients and 52 years
for controls, P — 0.02), and visited the restaurant
more frequently than controls (median visits in June
and July, four for case-patients and two for controls,
p = 0.5). The only exposure that increased the risk of
developing illness was drinking water at the restau-
rant. Twenty-nine (97%) of thirty case-patients re-
ported drinking water at the restaurant, compared
with 40 (71%) of 56 controls (P = 0.04; 9 controls
who could not recall whether they drank water were
excluded from analysis). Water remained a risk factor
for illness when controlled for number of visits to the
restaurant (P = 0.03) and age (P = 0.06). Drinking
coffee prepared with water heated to 65 °C did not
increase the risk of illness (odds ratio = 1.0). The res-
taurant did not serve unpasteurized milk products.
Total restaurant water consumption was estimated
by multiplying the average number of glasses con-
sumed per visit times the estimated number of visits in
the 3-month period from May to July. Case-patients
drank more water at the restaurant than did matched
controls (median, 4.0 glasses compared with 1.8 glass-
es; P = 0.3). When the one case-patient and 16 con-
trols who drank no water were excluded from analysis,
this difference in total water consumption became less
pronounced (median, 3.0 glasses compared with 2.0
glasses). No trend to suggest a dose-response was ob-
served even when controlled for age.
Case-patients and controls were similar in their
use of antibiotics (Table 4), antacids, and histamine
blockers.
Twenty-four (53%) of the forty-five truckers who
had visited the restaurant participated in the truck-
driver cohort study. The chronic diarrhea attack rate
was higher among the older age group (Table 5). A
high frequency of restaurant visits also increased risk
Table 4. Risk Factors for Diarrhea] Illness in the Restaurant Matched Case-Control Study, Henderson County, Illinois
Matched Risk Factor
Univariate
Female
Mean age
Median number of restaurant visits,
June to July
Drank water
Drank coffee
Previous antibiotic use
Multivariate
Drank water
Visit frequency
Drank water
Age
Drank water
Drank coffee
Case-Patients
50%
60. 5y
4
97%
63%
7%
Controls
57%
52.0y
2
71%
63%
10%
Odds
Ratio
0.8
NAf
NA
9.3
1.0
1.5
10.2
7.5
10.0
1.3
95% Confidence
Interval
0.3 to 1.9
NA
NA
1.2 to 73.8
0.4 to 2.6
0.3 to 8.4
1.3 to 82.0
0.9 to 61.5
1.2 to 81. 2
0.3 to 3.7
P Value*
0.6
0.02
0.5
0.04
1.0
0.6
0.03
0.3
0.06
0.05
0.03
0.7
* Statistics done using conditional logistic regression.
t Not applicable.
988 15 June 1989 • Annals of Internal Medicine
Volume 110 • Number 12
50
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Table 5. Risk Factor for Chronic Diarrhea Cohort Study of 24 Truck Drivers in Henderson County, Illinois, 1987
Variable
Age,y
< 40
> 40
Visits to the restaurant, n
< 15
> 15
Glasses of water drunk, J n
1-10
11-30
> 30
Total
11
13
12
12
8
8
7
Number of
Case-Patients
1
8
1
8
1
3
4
Attack
Rate*
9.1
61.5
8.3
66.7
12.5
37.5
57.1
Relative
Risk
1.0
6.8
1.0
8.0
1.0
3.0
4.6
95% Confidence
Interval
1.0 to 46.0
1.2 to 54.5
0.4 to 23.1
0.7 to 31.9
P Valuef
0.01
0.009
0.6
0.1
0.07§
* Attack rate in case-patients per 100 persons exposed.
t Fisher exact test, two-tailed.
| Excludes one person who did not drink water.
§ Chi-square due to linear trend.
of illness as did drinking a greater total volume of
water (Table 5).
The restaurant employed 11 people, 8 of whom
were interviewed. Three employees met the case-pa-
tient definition. Two additional employees, including
the cook-manager, described prolonged diarrheal ill-
ness but did not meet the case-patient definition. The
cook-manager had G. lamblia in a stool sample and
reported cure with metronidazole therapy. The other
employee described having two to three loose stools
per day only intermittently over the summer months.
Henderson County rests on limestone through
which water drains from east to west into the Missis-
sippi River. Although there are scattered beef cattle,
there are no dairies within the county. Municipal wa-
ter is available within the county's towns, but county
residents who live in more rural areas rely on either
private shallow sandpoint wells or deep wells that pen-
etrate into the limestone aquifer.
The restaurant obtained untreated water from a 4-
inch drilled well, 125 feet deep, which also supplied a
neighboring, vacant house. Routine water samples col-
lected between September 1981 and May 1987 showed
satisfactory results for total coliforms (15 samples)
and total nitrate (16 samples). In June 1987, three
power failures and two mild earthquakes occurred at
or near the well. The restaurant continued using water
during these power failures. On 30 July, i day before
the restaurant was closed, a water sample collected
from the restaurant's combination soft drink and wa-
ter dispenser showed a high total coliform count
(> 16/100 mL, tube dilution method). On 1 August,
the well was hyperchlorinated.
The well had been inspected in April 1987, and the-
owners were put under notice to correct several con-
struction deficiencies. At a May reinspection, deficien-
cies remained that could have permitted water from
contaminated sources to siphon back into the well,
particularly from within the vacated house and from
abandoned, blind pipes that branched from the well
into a surrounding field. In the 2 months after chlorin-
ation, 2 of 20 water samples taken from the well or its
attached plumbing showed a mild elevation of fecal
coliforms. One sample, collected on 9 September from
the vacant house, revealed a high fecal coliform count
(2300 organisms/100 mL of water). Several nearby
wells of varying depths tested satisfactorily.
Microscopic examination of water collected on 9
September revealed no parasites. A water filter was
nonspecifically positive by gene probe for a human en-
terovirus; however, viral cultures of the water grew no
organisms. No pesticides, herbicides, heavy metals,
or volatile organic agents were detected from well wa-
ter collected on 9 September. The restaurant owner
drilled a new well, replaced the restaurant's plumbing
system, and reopened on 8 October. No subsequent
illness has been reported.
Discussion
This is the first reported outbreak of chronic diarrhea
associated with drinking untreated well water. The
dramatically severe symptoms in this and the two pre-
viously described outbreaks suggest a distinct illness
with a common pathophysiologic mechanism and cau-
sative agent. Because an environmental exposure ap-
pears to cause illness, a standard case definition is
needed so that responsible vehicles can be identified
and transmission interrupted. We suggest a case defi-
nition for "Brainerd diarrhea" that includes the fol-
lowing criteria: nonbloody diarrhea lasting longer
than 1 month with negative stool examination for
known bacterial and parasitic pathogens; acute onset
of illness; fecal urgency; absence of fever; eight or
more stools per day throughout the first week of ill-
ness; normal stool fecal fat or D-xylose test, or both;
and normal or mild, chronic inflammatory changes on
colon biopsy. The proposed case definition, though
untested, would distinguish this chronic diarrheal
syndrome from mild diarrheal illness that may be het-
erogeneous in cause and from inflammatory bowel dis-
eases, malabsorptive diseases, and collagenous or mi-
croscopic colitis.
The causative agent of outbreak chronic diarrhea
remains obscure. Heating water to 65 °C may have
prevented transmission of illness, however, suggesting
either a heat-labile toxin or a microorganism. In the
Minnesota outbreak, a possible case of person-to-per-
15 June 1989 • Annals of Internal Medicine
51
Volume 110 • Number 12 989
-------
son transmission suggested an infectious pathogen. We
were unable to confirm this finding in Henderson
County. Blastocystis hominis organisms were found in
the stools of two case-patients; each of these case-pa-
tients later had negative stool examinations despite
continuing symptoms. Furthermore, the clinical pic-
ture ascribed to Blastocystis infection—recurrent ab-
dominal cramps, fatigue, and anorexia with only mild
diarrhea—is not consistent with the outbreak illness
(21, 22). We believe B. hominis infection to be an
incidental finding in these two case-patients, and no
other known pathogens were identified.
Microscopic colitis and collagenous colitis, two
newly defined pathologic entities, both cause watery,
urgent, chronic diarrhea (4-8). Although these symp-
toms are compatible with the outbreak illness, the
mild pathologic abnormalities noted on Henderson
County biopsy specimens are not consistent with ei-
ther microscopic or collagenous colitis. In microscopic
colitis, mucosal goblet cell depletion, excess neutro-
phils, round cells within the lamina propria, and pene-
tration of neutrophils into the surface epithelium are
typically seen (9). In collagenous colitis, the sub-
epithelial collagen layer is thickened, and lympho-
cytes and plasma cells infiltrate the lamina propria (7,
23). Neither the acute inflammation of microscopic
colitis nor the collagen banding of collagenous colitis
was evident in the Henderson County biopsies. How-
ever, the time from onset of illness to colon biopsy was
comparatively brief (mean, 43 days). Collagenous co-
litis and microscopic colitis are generally diagnosed
histopathologically after more than 1 year of symp-
toms. It is conceivable that the three clinical entities
represent different stages of the same disease. In some
studies, progression from microscopic colitis to col-
lagenous colitis has been demonstrated (24, 25). Out-
break diarrhea may be the forme fruste. Sequential
colonic biopsies of Henderson County patients are
planned to assess this possible association.
Subtle changes in small-bowel mucosal biopsies
were also present; however, clinical studies by local
physicians do not evince malabsorption. Three pa-
tients from the Minnesota outbreak had abnormal
small-bowel motility, but again, abnormalities were in-
constant and nonspecific (26). Understanding these
mild changes would be enhanced by follow-up studies.
Although recall of water consumption by case-
patients and controls was subject to considerable bias,
the association between the restaurant's water and ill-
ness was both strong and plausible. Unlike food items
that had rapid turnover in the restaurant, well-water
usage continued uninterrupted throughout the out-
break period. Some restaurant patrons reported that
the water appeared filmy or foamy, or smelled foul.
Inspections of the restaurant and the well revealed
plumbing deficiencies that could have permitted water
contamination; a sample taken from the water dis-
penser 1 day before the restaurant closed was contami-
nated with coliforms. Several points along the plumb-
ing system (for example, filters, the water softener, or
the combination water-soft-drink dispenser) could
have been a source for continuous or sporadic water
contamination. Furthermore, the restaurant continued
to use water during power failures, enhancing the
opportunity of contamination by back-siphonage. The
unlikely possibility remains however, that water di-
rectly from the limestone aquifer could have been re-
sponsible.
A substantial proportion of sporadic, chronic diar-
rheal illness may be caused by exposure to an as yet
unidentified environmental agent. Because no diagnos-
tic test for this illness exists, it is important that both
primary-care physicians and specialists recognize the
syndrome clinically and inquire about case clusters
and possible exposures to untreated water or unpas-
teurized milk. Improved national awareness, early
case recognition, and continued clinical and epidemic-
logic investigation of both sporadic and outbreak-
related chronic diarrhea are fundamental to further
definition of this debilitating disease.
Acknowledgments: The authors thank Ms. Joan Ray and Ms. Judy Kir-
berg of the Henderson County Health Department for their assistance in
the epidemiologic investigation; Drs. Doris D. Vendrell, Joan B. Rose,
Gary C. duMoulin, Roger I. M. Glass, and Mr. Jerald Jarnagin for their
laboratory support; and Drs. Mitchell L. Cohen, Paul A. Blake, and
John S. Fordtran for their reviews of the manuscript.
Use of trade names is for identification only and does not imply endorse-
ment by the Public Health Service or by the U.S. Department of Health
and Human Services.
Requests for Reprints: Julie Personnel, MD, EDB/DBD/CID, Room 1-
5428, Centers for Disease Control, Atlanta, GA 30333.
Current Author Addresses: Drs. Parsonnet, Addiss, Hargrett-Bean, and
Tauxe, and Ms. Bopp: Enteric Diseases Branch, Division of Bacterial
Diseases, Center for Infectious Diseases, Centers for Disease Control,
Atlanta, GA 30333.
Drs. Trock and Gunn: Division of Field Services, Epidemiology Pro-
gram Office, Centers for Disease Control, Atlanta, GA 30333.
Ms. Wood and Mr. Alai: Illinois Department of Public Health, 535 West
Jefferson Street, Springfield, IL 62761.
Dr. Gorelkin: Division of Host Factors, Center for Infectious Diseases,
Centers for Disease Control, Atlanta, GA 30333.
References
1. Osterholm MT, MacDonald KL, White KE, et al. An outbreak of a
newly recognized chronic diarrhea syndrome associated with raw
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2. Martin DL, Hoberman LJ. A point source outbreak of chronic diar-
rhea in Texas: no known exposure to raw milk [Letter]. JAMA.
1986;256:469.
3. Read NW, Krejs GJ, Read MG, Santa Ana CA, Morawski SG,
Fordtran JS. Chronic diarrhea of unknown origin. Gastroenterolo-
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4. Rams H, Rogers AI, Ghandur-Mnaymneh L. Collagenous colitis.
Ann Intern Med. 1987;106:108-13.
5. Wang KK, Perrault J, Carpenter HA, Schroeder KW, Tremalne
WJ. Collagenous colitis: a clinicopathologic correlation. Mayo Clin
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6. Kingham JG, Levison DA, Morson BC, Dawson AM. Collagenous
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7. Jessurun J, Yardley JH, Giardiello FM, Hamilton SR, Bayless
TM. Chronic colitis with thickening of the subepithelial collagen
layer. Hum Pathol. 1987;18:839-48.
8. Kingham JG, Levison DA, Ball J, Dawson AM. Microscopic coli-
tis—a cause of chronic watery diarrhoea. BrMedJ. 1982;285:1601-
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9. Bo-Linn GW, Vendrell DD, Lee E, Fordtran JS. An evaluation of
the significance of microscopic colitis in patients with chronic diar-
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11. Lockwood DE, Robertson DC. Development of a competitive en-
zyme-linked immunosorbent assay (ELISA) for Escherichia coli
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12. Nataro JP, Baldini MM, Kaper JB, Black RE, Bravo N, Levine
MM. Detection of an adherence factor of enteropathogenic Esche-
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Mycobacteria. Ames, Iowa: U.S. Department of Agriculture; 1985.
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the decontamination of water for culture of mycobacteria. Appl En-
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18. Gerba CP, Margolin A, Trumper B, Rose JB, Zhang C. Low cost
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15 June 1989 • Annals of Internal Medicine • Volume 110 • Number 12 991
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AMERICAN
JOURNAL
OF
Public
Jtl6£L.Lull reprint
Reproduced, with permission,
from American Journal of
Public Health
Epidemic Giardiasis Caused by a Contaminated Public Water Supply
GEORGE P. KENT, MD, JOEL R. GREENSPAN, MD, MPH, JOY L. HERNDON, MS, LYNNE M. MOFENSON, MD,
JO-ANN S. HARRIS, MD, THOMAS R. ENG, VMD, MPH, AND HETTY A. WASKIN, MD, MSPH
Abstract: In the period November 1, 1985 to January 31, 1986,
703 cases of giardiasis were reported in Pittsfield, Massachusetts
(population 50,265). The community obtained its water from two
main reservoirs (A and B) and an auxiliary reservoir (C). Potable
water was chlorinated but not filtered. The incidence of illness
peaked approximately two weeks after the city began obtaining a
major portion of its water from reservoir C, which had not been used
for three years. The attack rate of giardiasis for residents of areas
supplied by reservoir C was 14.3/1000, compared with 7.0/1000 in
areas that received no water from reservoir C. A case-control study
showed that persons with giardiasis were more likely to be older and
to have drunk more municipal water than household controls. A
community telephone survey indicated that over 3,800 people could
have had diarrhea that might have been caused by Giardia. and 95 per
cent of households were either using alternate sources of drinking
water or boiling municipal water. Environmental studies identified
Giardia cysts in the water of reservoir C. Cysts were also detected
in the two other reservoirs supplying the city, but at lower concen-
trations. This investigation highlights the risk of giardiasis associated
with unfiltered surface water systems. (Am J Public Health 1988:
78:139-143.)
Introduction
Waterborne giardiasis is becoming an important public
health problem in the United States: during the period from
1965-84, 90 outbreaks and 23,776 cases of giardiasis were
reported. Sixty-nine per cent of outbreaks and 74 per cent of
cases related to contamination of public water supplies, the
majority of which were surface water systems with inade-
quate filtration or chlorination.' We describe one of the
largest waterborne outbreaks of giardiasis ever reported in
the United States and highlight the risk of giardiasis in
communities that are supplied by unfiltered surface water
systems.
Background
The outbreak occurred in the western Massachusetts
city of Pittsfield (population, 50,265, 1985 census). The
problem was first recognized during the first week of Decem-
ber J 985, when the health department of Pittsfield received 70
reports of laboratory-confirmed giardiasis. Only 53 positive
stool samples for Giardia had been submitted during the
entire one-year prior to November 1, 1985. Interviews of the
70 case-patients revealed that they had negative stool cul-
tures for bacterial enteric pathogens, no common personal
contact, and they were widely distributed throughout the
city. This information, coupled with the knowledge that a
new reservoir had been added to the community water supply
in November, led city and state health officials to suspect a
waterborne outbreak of giardiasis.
Water Supply
Prior to November 1985, the community obtained its
drinking water from two surface reservoirs, A and B. Water
was chlorinated but not filtered. However, during November
1985, alterations were made to the water system to facilitate
the construction of a filtration system for water originating
Address reprint requests to Division of Field Services. Epidemiology
Program Office. Centers for Disease Control, Atlanta, GA 30333. Dr. Kent is
affiliated with the Division of Field Services at CDC; Dr. Greenspan is with the
Division of Field Services at CDC, currently assigned to the Division of
Communicable Disease Control. Massachusetts Department of Public Health.
Boston; Ms. Herndon is with the Division of Surveillance and Epidemiologic
Studies. EPO. CDC; Drs. Mofenson. Harris and Eng are with the Division of
Communicable Disease Control, Massachusetts Department of Public Health,
Boston: Dr. Waskin is with the Division of Parasitic Diseases. Center for
Infectious Diseases. CDC. This paper, submited to the Journal June 16. 1987,
was revised and accepted for publication August 17, 1987.
Editor's Note: See also related editorial p 123 this issue.
from reservoir A. Specifically, on November 5, an auxiliary
reservoir C was brought on line for the first time in over three
years to replace water from reservoir A which was to be
phased out while the filtration system was being installed.
This was expected to be a temporary yet necessary step in
order to meet the usual demand for water, as installation of
filtration equipment for reservoir A required decreasing the
flow of reservoir A water into the city's water system during
the construction period. On November 14, as construction
proceeded, the flow from reservoir C was increased and the
flow from reservoir A was correspondingly increased. On this
day, the water department received complaints of turbid
water from many areas of the city; the increased turbidity was
attributed to reversals in water flow which suspended sedi-
ment in the water mains. After approximately two days, the
turbidity resolved spontaneously. Reservoir C supplied wa-
ter to the city until December 21, when it was replaced by
reservoir A.
Methods
Epidemiologic Investigation
Daily reports of positive Giardia isolates were obtained
from the two clinical laboratories in the region. The investi-
gators attempted to reach every reported case-patient by
telephone; patients were asked to report demographic infor-
mation and the onset date of symptoms. Local physicians
were sent letters encouraging them to obtain stool analyses
from patients with symptoms clinically consistent with
giardiasis, and were urged to report positive cases to the local
health department.
Laboratory analysis of stool samples were performed
locally by the major clinical laboratory in the area. Stool
samples were cultured for Salmonella, Shigella, and Cam-
pylobacter. Analysis for ova and parasites was performed
using standard direct saline and formalin-ethyl acetate meth-
ods.2
A confirmed case of outbreak-associated giardiasis was
defined as a Pittsfield resident with a stool sample positive for
Giardia lamblia submitted after November 1, 1985. A suspect
case of giardiasis was anyone interviewed in the community
surveys who reported having had diarrhea (>3 loose stools
per 24-hour period) of undetermined etiology of >5 days
duration with onset since November I, 1985.
Information obtained from the city water engineer con-
AJPH February 1988, Vol. 78, No. 2
54
139
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KENT, ET AL.
cerning placement of values in the municipal water system
was used to construct a water distribution map depicting the
reservoir origin of drinking water and the distribution pattern
throughout the city between November 1, 1985 and January
31, 1986 (Figure 1). We estimated the population in each of
these regions using census tract data obtained in March 1985;
populations in areas that were included in more than one
census tract data were divided in proportion to the length of
city streets in each area. Attack rates were then determined
for each water distribution area by counting the number of
reported case-patients residing in each region divided by the
estimated population.
Case-Household Telephone Survey
Questionnaires were administered by telephone to each
of the first 139 reported case-patients during the first week of
the investigation (December 13-19); each of these case-
patients was asked to supply information about all household
members. Whenever possible, all household members were
interviewed individually. They were asked to describe their
principal source of drinking water and the number of glasses
of beverages containing both unboiled and boiled municipal
water they customarily drank per day, as well as any
gastrointestinal symptoms they may have experienced.
A case-control study was performed using the survey
data to identify risk factors for giardiasis. Cases were defined
as persons with laboratory-confirmed giardiasis; controls
were defined as persons who did not have positive stool
samples and had no diarrhea. Cases and controls were
matched within households, and conditional logistic regres-
sion3 was used to determine whether age and water con-
sumption were independently associated with giardiasis.
Case Telephone Survey
Questionnaires similar to those used in the case-house-
hold survey were administered to the next 100 reported
case-patients. Additional questions were included for these
100 persons specifically describing their consumption of
municipal water at home and away from home. Information
about household members was not obtained.
FIGURE 1—Water Distribution by Reservoir Source, Pittsfield, Massachusetts,
November 1 to December 21, 1985
Community Telephone Survey
A community telephone survey of 220 households ran-
domly selected from the telephone directory was conducted
on January 16, 1986. The purpose of the survey was to
determine whether laboratory reports of positive stool sam-
ples were true indicators of the occurrence of disease in the
community. By this time, physicians may have been treating
an increasing number of symptomatic patients empirically
without having obtained stool samples because of the wide-
spread publicity associating diarrhea with giardiasis. Each
household member was to be interviewed concerning water
consumption, gastrointestinal symptoms, diagnosis, and
treatment. Heads of households were also asked about
compliance with control measures. The systematic sampling
technique was performed using methods described by Kish,4
and 95% confidence intervals (CI) of estimated numbers of
cases were determined using a method described by
Mendenhall.5
Environmental Investigation
We reviewed all available water quality testing results
indicating turbidity, coliform, and chlorine levels since Jan-
uary 1, 1985. Personnel from city and state environmental
engineering agencies inspected the water distribution system
for cross-connections with sewage lines.
Chlorinated water from reservoir C immediately distal to
the chlorination point was sampled for Giardia cysts on
December 12; untreated water in all three reservoirs was
sampled on December 16; additional samples were obtained
from three sites in the distal portion of the distribution system
on four occasions in late December and early January.
Sampling for Giardia cysts was performed using volume-
sampling orlon yarn filters supplied by the US Environmental
Protection Agency (EPA).6 Analysis of water samples for
Giardia was also performed by the EPA using an im-
munofluorescent antibody technique.7 Giardia cysts extract-
ed from the water samples obtained from each of the three
reservoirs were tested for infectivity by inoculation in ger-
bils.8 Giardia trophozoites obtained from these animals were
then examined at the University of Minnesota by scanning
electron microscopy for morphological characteristics in
order to determine whether they were from human or animal
origin.
Inspectors surveyed the watershed areas of the three
reservoirs for signs of sewage contamination and beaver
activity in December 1985; beavers obtained during the
survey were examined for Giardia. During the summer of
1986, muskrats were obtained from the watershed areas of all
three reservoirs and examined for infection with Giardia.
Eleven outbreak-related human fecal samples were sent
to the University of Minnesota for use in a cross-species
transmission experiment. Beavers and muskrats were admin-
istered Giardia cysts from these samples and examined for
infection. Giardia trophozoites obtained from these animals
were also studied for morphological characteristics.
Results
From November 1, 1985 to January 31,1986,703 persons
were identified with positive stool samples for Giardia.
During this same period, approximately 2,600 persons sub-
mitted stool samples, yielding a Giardia-positive rate of 27
per cent. Onset dates of illness were obtained from 604 (86 per
cent) of the 703 confirmed case-patients, and residential
address was confirmed for 635 persons (90 per cent). The
greatest number of persons reported onset of symptoms on
140
AJPH February 1988, Vol. 78, No. 2
55
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WATERBORNE GIARDIASIS EPIDEMIC
Reservoir C
off
Boil water order
rescinded
7 13 19 25
NOVEMBER
13 19 25
DECEMBER
1985
T" i i~
12 18 24
JANUARY
1986
ONSET
•604 stool-positive cases. Date of onset unknown for 99 cases.
FIGURE 2—Laboratory-confirmed Cases of Giardiasls, by Date of Onset, Pittsfield, Massachusetts, November 1,1985
to January 31, 1986
November 28, two weeks after the flow from reservoir C was
increased and the episode of turbid water occurred (Figure 2).
When attack rates of laboratory-confirmed giardiasis were
calculated by place of residence, the highest attack rates were
found for areas of the city served by reservoir C (Table 1).
Case-Household and Case Telephone Surveys
In the case-household survey of 139 households, infor-
mation was obtained about all 437 household members. Eight
additional persons with positive stool samples, who had not
yet been reported, were identified, yielding 147 confirmed
case-patients. Of the next 100 case-patients who were inter-
viewed in the case telephone survey, seven had been inter-
viewed before in the case-household survey; thus, a total of
240 confirmed case-patients were interviewed in detail con-
cerning their symptoms. Among these 240, 236 (98 per cent)
had diarrhea. The mean duration of diarrhea was 11.3 days,
TABLE 1—Attack Rates by Residential Water Source, Laboratory-con-
firmed Cases of Giardiasls, Pittsfield, Massachusetts, Novem-
ber 1,1985 to January 31,1986
Water Source
Reservoir A
Reservoir B
Reservoir C
Mixed (A, B, and C)
TOTAL
Residential water
from reservoir C
Yes
No
TOTAL
Population
9405
2309
4200
34351
50265
Population
38551
11714
50265
No.
Cases
68
14
126
427
635
No.
Cases
553
82
635
Attack Rate
(per 1000)
7.2
6.1
30.0
12.4
12.6
Attack Rate
(per 1000)
14.3
7.0
12.6
with a range of 1-34 days. Additional gastrointestinal symp-
toms were frequent (Table 2).
Among the 139 households interviewed, we identified
147 persons with laboratory-confirmed giardiasis (cases) and
239 persons who had neither diarrhea nor positive stool
samples for Giardia (controls). Seventy-three per cent
(108/147) of cases reported drinking two or more glasses of
municipal water per day, compared to 36 per cent (86/239) of
controls (OR = 4.9 95% CI = 3.1-8.0). Eighty-eight per cent
(130/147) of cases were age 20 years or older, compared with
52 per cent (124/239) of controls (OR = 7.1 95% CI =
3.9-13.3). Conditional logistic regression analysis, used to
match cases and controls by household and to control for age
and water consumption, indicated that drinking two or more
glasses of unboiled municipal water per day and being age 20
years or older were independently associated with giardiasis
[OR (water) = 9.2 95% CI = 4.2-19.9; OR (age) = 3.3 95%
CI = 1.7-6.2].
TABLE 2—Frequency of Symptoms, Laboratory-confirmed Cases of
Giardiasls, Case-Household and Case-Telephone Surveys,
Pittsfield, Massachusetts, November-December 1985
Symptom
Persons with Symptom (%)
Diarrhea (any duration)
(a 5 days)
Flatulence
Fatigue
Abdominal cramps
Loss of appetite
Bloating
Nausea
Weight loss
Vomiting
Fever (undocumented)
Bloody diarrhea
(N=240)
236 (98)
190 (79)
214 (89)
207 (86)
204(85)
197 (82)
180 (75)
178 (74)
165 (69)
87(36)
37 (15)
7 (3)
AJPH February 1988, Vol. 78, No. 2
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KENT, ET AL.
Among 100 case-patients interviewed concerning the
number of glasses of unboiled municipal water consumed at
home or away from home, 74 reported that all or most of their
water consumption occurred at home. They reported drink-
ing a mean of 3.2 glasses per day at home, and 0.9 glasses per
day away from home.
Community Telephone Survey
Of the 220 households selected, 200 were contacted; we
obtained information on all 527 persons living in these
households. Fourteen persons (2.6 per cent) had positive
stool samples for Giardia since November 1 (confirmed
cases); all of these persons had had diarrhea for a 5 days
before receiving treatment. Twenty-six persons (5 per cent)
had diarrhea of undetermined etiology with > 5 days duration
(suspect cases). Projections from this survey indicated that
between November 1, 1985 and January 15, 1986, 1,335
people (95% CI = 598-2072) had confirmed giardiasis and
2,480 additional persons (95% CI = 1426-3534) were suspect
cases. The onset date of illness in suspect cases resembled
that of the confirmed cases, suggesting that many of these
suspect cases could have been outbreak-associated. Rela-
tively few cases were reported with onset in January, which
was also consistent with reports from laboratory-confirmed
cases.
Of the 200 households interviewed, 189 (95 per cent)
reported continuous compliance with a boil-water order that
had been issued on December 12; 78 (39 per cent) were boiling
municipal water, and 111 (56 per cent) were either using
commercial bottled water or were obtaining water from local
springs or wells. All respondents indicated awareness of the
outbreak and the boil-water order.
Environmental Investigation
Daily records of free residual chlorine levels in water
entering the distribution system immediately distal to the
chlorination point indicated that chlorine levels in water from
reservoir C were between 0 and 0.5 mg/1 during the entire
month of November due to a malfunction in the chlorinating
machinery which was not successfully repaired until Novem-
ber 30. During December, chlorine levels in the distribution
system water from reservoir C averaged 2 mg/1 with approx-
imately 15 minutes minimum contact time before water
reached the first households. Chlorine levels in water enter-
ing the distribution system from reservoirs A and B imme-
diately distal to the chlorination point during both months
averaged 3 mg/1 and 2 mg/1, respectively, with 15-minute
minimum contact times. Water temperature in all three
reservoirs was approximately 40°F, measured turbidity levels
were within acceptable limits, and pH ranged from 7.0 to 7.4.
During October 1985, all bacterial cultures of water
samples from various points in the distribution system
contained levels of coliform bacteria below the acceptable
limit of 4 colony forming units (CFU)/100 ml; no bacteria
were detectable in 71 of 80 samples. However, on November
19, five days after the episode of turbid water (observed but
not measured), five of 17 samples had > 5 CFU/100 ml. All
of these five samples were taken from areas of the city that
were at least partially supplied by water from reservoir C.
Sampling was performed daily for the next seven days; after
two days, all samples were negative for coliform bacteria
except for a single site located in the region served exclu-
sively by reservoir C, which continued yielding elevated
coliform counts (8^41 CFU per 100 ml) until December 1,
when the defective chlorinator was repaired.
Microscopic examination of water samples revealed that
chlorinated water from reservoir C obtained immediately
distal to the chlorination point contained 80 Giardia cysts/100
gallons; untreated water obtained directly from reservoir C
contained 28 Giardia cysts/100 gallons. The samples obtained
on December 16 directly from untreated water in reservoirs
A and B contained seven cysts and nine cysts/100 gallons,
respectively. Subsequent water samples taken from distal
points in the distribution system in December and January
yielded no Giardia cysts. G. lamblia cysts and trophozoites
were detected in the laboratory animals inoculated with the
extract from untreated reservoir C water; animals receiving
portions of water samples obtained from reservoirs A and B
were negative. Morphological studies suggested that the
trophozoites obtained from reservoir C did not resemble
those of beaver origin, but instead more resembled
trophozoites found in humans. Giardia cysts that were
isolated from the outbreak-related human fecal samples and
inoculated in beavers and muskrats produced infection in
both types of animals and also resembled Giardia typically of
human origin.
The original source of contamination for reservoir C was
not identified. Inspectors found no evidence of sewage
contamination in any of the water mains or in any of the
watershed areas of the three reservoirs. However, they
encountered signs of human activity (graffiti and empty
beverage containers) near the shoreline of reservoir C in
officially restricted areas; they also noted many signs of
recent beaver activity (fresh decorticated wood cuts) near
reservoir C, and near a small pond that ultimately drained
into reservoir B, but no evidence of beavers near reservoir A.
Nine beavers were found during the December 1985 survey
of the three watershed areas; one of three beavers found near
reservoir C yielded stool samples positive for Giardia,
compared to none of six beavers found in the watershed area
of reservoir B. All of seven muskrats obtained during summer
1986 from watershed areas of all three reservoirs were
infected with Giardia. Thus, the water and animal studies
suggested that beavers and muskrats in the area contributed
to contamination of the water supply and may have originally
been infected from a human source, but they did not
determine whether the reservoir water had also been directly
contaminated by human sewage.
Control measures for the outbreak included the follow-
ing:
• A boil-water order was issued on December 12, 1985.
Pittsfield residents were asked to either boil municipal
water or use alternative sources of drinking water.
• Reservoir C water was removed from the municipal
water supply on December 21, 1985.
• Water in storage tanks and in the distribution system
was hyperchlorinated and flushed in order to kill any
remaining Giardia cysts that may have settled in the water
mains. On December 21, free residual chlorine levels were
increased to 3 mg/1, and minimum contact times with
chlorine were increased to one hour. The entire distribution
system was then flushed over a nine-day period.
• The boil-water order was removed after the following
conditions were met: a) laboratory reports showed no
evidence of significant increase in new illness after two
incubation periods (four weeks) following completion of
the hyperchlorination and flushing; b) results of the second
community survey over the same two incubation periods
were consistent with the data from confirmed cases, show-
ing no evidence of continuing infection; c) adequate chlo-
rination and contact times were maintained; d) water
142
AJPH February 1988, Vol. 78, No. 2
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WATERBORNE GIARDIASIS EPIDEMIC
sampling during January in the distribution system revealed
no Giardia cysts.
• Continued weekly reporting of laboratory-confirmed
cases of giardiasis was required for at least one month after
removing the boil-water order.
The boil-water order was rescinded on January 24,1986,
six weeks after it was issued. As of April 1, 1986, only one
case of giardiasis had been reported with onset of symptoms
after late January.
Discussion
It is difficult to maintain appropriate concentrations of
chlorine that prevent waterborne giardiasis; thus, communi-
ties should not rely upon chlorination alone to protect public
water supplies. Proposed additions or alterations to munic-
ipal water supplies which lack filtration should be thoroughly
evaluated to ensure that contamination levels do not exceed
the capacity of the chlorination system. Untreated water
should be examined for the presence of fecal coliforms,
turbidity and, if possible, Giardia cysts. This outbreak was
facilitated by a malfunction in the chlorinating machinery
which left very low levels of chlorine in water from reservoir
C, but even if chlorine levels had been maintained at 2 mg/1
for 15 minutes as intended, viable Giardia cysts might still
have been present; water at 40°F and pH 7.0 probably
requires more than 30 minutes of contact with 2 mg/1 of
chlorine to adequately kill Gardia cysts.9
The dramatically increased incidence of illness noted
during the last few days of November suggests that a
relatively high dose of Giardia cysts was present in drinking
water during a short period of time. Assuming a mean
incubation period of two weeks, as is commonly described,
the time course of infection implicates the episode of turbid
water on November 14,1985 as being potentially responsible
for the increased number of cases. Giardia cysts that had
settled to the bottom of water mains could have been
resuspended by the increased turbulence in water flow. The
result could have been a bolus of higher concentrations of
Giardia cysts causing more infections. Additionally, the
increased turbidity probably reduced the effect of chlorine
which was already at inadequate levels. The increased
concentrations of bacteria in water samples during the week
following the episode of turbid water could have occurred as
a result of inadequate chlorination.
The validity of calculating attack rates by place of
residence to implicate a specific water source depends upon
the assumption that most water consumption occurs at home;
our survey supports this assumption. John Snow, the famous
British epidemiologist, used this technique effectively to
implicate the Southwark and Vauxhall Company as the
source of contaminated water in his classic cholera investi-
gations of 1853.10
This is the largest reported outbreak to date of labora-
tory-confirmed cases of waterborne giardiasis (703 cases).
Previous investigations of waterborne giardiasis outbreaks
have described a larger number of possible cases and com-
munity-wide attack rates of clinical illness ranging from 3.8
per cent to 10.6 per cent.n~15 It is likely that those who
obtained a laboratory diagnosis of their infection in Pittsfield
represented only a portion of the infected population: the
survey indicates 1.9 additional symptomatic suspect cases
for every one laboratory-confirmed case. Additional persons
were likely to have had asymptomatic infection. Estimates of
numbers of infected persons in outbreaks depend upon the
sensitivity and specificity of the case definition and on the
accuracy and validity of telephone surveys.
Community health officials were encouraged by the high
degree of reported compliance with their recommendations
to avoid drinking unboiled municipal water. However, few
people actually boiled tap water; most people were using
alternate sources of drinking water although all were aware
of the boil-water order.
It is ironic that this outbreak occurred as a result of the
attempt to install a filtration system that would probably have
prevented the outbreak. Many small communities lack fil-
tered water supplies; they are costly and require technical
expertise to maintain. In Massachusetts, among 124 surface
water supplies, 76 had either no filtration system or partial
filtration systems in 1985 (personal communication, John
Higgins, Massachusetts Department of Environmental Qual-
ity Engineering). Giardiasis outbreaks may become even
more frequent in the future, as increasing population pressure
on watershed areas increase the risk of Giardia contamina-
tion for many communities that depend on unfiltered surface
water supplies.
ACKNOWLEDGMENTS
The authors acknowledge Louis Bolduc and the Pittsfield Health Depart-
ment staff, Dr. George Douglas of Berkshire Medical Center, Tom Keefe of the
Massachusetts Fish and Game Division, Walter Jakubowski of the Environ-
mental Protection Agency, and Dr. Stanley Erlandsen of the University of
Minnesota for their assistance in this investigation.
REFERENCES
1. Craun GF: Waterborne giardiasis in the United States 1965-1984. Lancet
1986; 2:513-514.
2. Young KH, Bullock SL, Melvin DM, Spruill CL: Ethyl acetate as a
substitute for diethyl ether in the formalin-ether sedimentation technique.
J Clin Microbiol 1979; 10:852-853.
3. Breslow NE, Day NE: In: Statistical Methods in Cancer Research,
Volume I—The Analysis of Case-Control Studies. Lyon: International
Agency for Research on Cancer, 1980; 248-281.
4. Kish L: In: Survey Sampling. New York: John Wiley and Sons, 1965;
113-144.
5. Mendenhall W, Ott L, Scheaffer R: In: Elementary Survey Sampling.
Belmont, California: Duxbury Pres, 1979; 137-139.
6. Jakubowski W, Chang SL, Erickson TH, et al: Large volume sampling of
water supplies for micro-organisms. J Am Water Works Assoc 1978;
70:702-706.
7. Sauch JF: Use of immunofluorescence and phase-contrast microscopy for
detection and identification of Giardia cysts in water samples. Appl
Environ Microbiol 1985; 50:1434-1438.
8. Belosevic M, Faubert GM, MacLean JD, Law C, Croll NA: Giardia
lamblia infections in Mongolian gerbils: an animal model. J Infect Dis 1983;
147:222-226.
9. Jarroll EL, Bingham AK, Meyer EA: Effect of chlorine on Giardia lamblia
cyst viability. Appl Environ Microbiol 1981; 41:483-487.
10. Snow J: Snow on Cholera. London: Oxford University Press, 1936.
11. Veazie L, Brownlee I, Sears JH: An outbreak of gastroenteritis associated
with Giardia lamblia. In: Waterborne Transmission of Giardiasis. Cincin-
nati: US Environmental Protection Agency, 1979; 174-191.
12. Shaw PK, et al: A community-wide outbreak of giardiasis with docu-
mented transmission by municipal water. Ann Intern Med 1977;
87:426-432.
13. Dykes AC, et al: Municipal waterborne giardiasis: an epidemiologic
investigation. Ann Intern Med 1980; 92:165-170.
14. Lopez CE, et al: Waterborne giardiasis: a community-wide outbreak of
disease and high rate of asymptomatic infection. Am J Epidemiol 1980;
112:495-507.
15. Navin TR, et al: Case-control study of waterborne giardiasis in Reno,
Nevada. Am J Epidemiol 1985; 122:269-275.
AJPH February 1988, Vol. 78, No. 2
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Reproduced, with permission, from Annals
of Internal Medicine
A Waterborne Outbreak of Cryptosporidiosis in Normal Hosts
RICHARD G. D'ANTONIO, M.D.; RICHARD E. WIIMN, M.D.; JEFFERY P. TAYLOR, M.P.H.; TRACY L.
GUSTAFSON, M.D.; WILLIAM L. CURRENT, Ph.D.; MAMIE M. RHODES, B.S.; G. WILLIAM GARY, Jr.,
Dr.P.H.; and ROBERT A. ZAJAC, M.D.; Lackland Air Force Base and Austin, Texas; Greenfield,
Indiana; and Atlanta, Georgia
In July 1984, an outbreak of gastroenteritis occurred in a
suburban community in Texas. A random telephone
survey of 100 of 1791 households in the community
identified an attack rate of 34%. The outbreak was traced
to contamination of the community water supply, an
artesian well. Fecal conforms were identified in untreated
drinking water from the well during July. Stool
examinations and serologic tests identified
Cryptosporidium as the etiologic agent. Cryptosporidium
should be added to the list of waterborne organisms
capable of causing outbreaks of gastroenteritis.
CRYPTOSPORIDIUM has been recognized recently as an
important cause of diarrhea in immunocompromised (1)
and immunocompetent persons (2, 3). Most Cryptospori-
dium infections are reported to occur in animal handlers
and patients with the acquired immunodeficiency syn-
drome. In patients with the syndrome, this parasite caus-
es a prolonged and debilitating diarrheal illness (1); in
immunocompetent adults, it produces a self-limited diar-
rheal illness lasting an average of 5 days (2, 3). Two
recent reports described outbreaks of cryptosporidiosis in
children attending day-care centers (4, 5), and in surveys
of patients with diarrhea, prevalence rates as high as 4%
have been reported in Australia and Costa Rica (6, 7).
Infection with this organism may be more common than
previously suspected.
In July 1984, a waterborne outbreak of diarrhea oc-
curred among residents of Braun Station, a suburb of San
Antonio, Texas. The outbreak was caused by Cryptospo-
ridium.
Methods
Stool specimens from Braun Station residents and outpatients
treated at the Wilford Hall Medical Center, Lackland Air
Force Base, in July 1984 were examined for ova and parasites at
the parasitology laboratory of the medical center or at the Bu-
reau of Laboratories, Texas Department of Health. Fresh and
formalin-preserved stool specimens were concentrated by a
modification of Ritchie's formalin-ether concentration method
(8) using ethyl acetate instead of ether. Concentrated speci-
mens were examined by light microscopy with iodine stain. To
identify Cryptosporidium oocysts, air-dried, heat-fixed smears
of fecal concentrates were stained by Truant's fluorescent pro-
cedure (9) and a modified Kinyoun's acid-fast stain (10). Fecal
films of specimens preserved in polyvinyl alcohol were stained
> From the Infectious Diseases Service, Wilford Hall Medical Center, Lackland
Air Force Base, Texas; Bureau of Epidemiology, Texas Department of Health,
Austin, Texas; Animal Health Discovery Research, Eli Lilly Research Laborato-
ry, Greenfield, Indiana; and Viral Gastroenteritis Laboratory, Division of Viral
Diseases, Centers for Disease Control, Atlanta, Georgia.
886 Annals of Internal Medicine. 1985;103(6pt l):886-888.
by Wheatley's trichrome method (11). Stool specimens were
examined for bacterial pathogens including Shigella, Salmonel-
la, Campylobacter, and Yersinia by routine microbiologic tech-
niques. Isolation of viruses was not attempted. Total and fecal
coliform counts in water samples were made by standard proce-
dures at the San Antonio Metropolitan Health District Labora-
tory. Serum specimens from Braun Station residents were tested
for antibody to Norwalk virus by radioimmunoassay (12), for
antibody to Cryptosporidium by an indirect immunofluorescent
technique (13).
A telephone survey of community households was done dur-
ing 25 through 27 July 1984. Pages in the residential telephone
directory for San Antonio were selected from a table of random
numbers. The first number listed on each page with a prefix
corresponding to northwest San Antonio was selected.
Persons contacted by telephone were asked to verify the
street address, provide the number and names of household
members, their ages and sex, occurrence of any illnesses in May
and July, and week of onset of symptoms. The first person in
each household who was ill with diarrhea or vomiting, or if no
one was ill, the person answering the telephone, was asked addi-
tional questions about symptoms; duration of illness; dates of
overnight trips; pets; contact with farm animals; children en-
rolled in day-care centers; recent swimming in pools, lakes, or
rivers; place of employment; recent consumption of raw seafood
or raw milk; source of drinking water; and types of fluids ingest-
ed daily. A total of 100 Braun Station households and 50 house-
holds outside Braun Station were surveyed. In addition, all
Braun Station residents with stool specimens positive for Cryp-
tosporidium were interviewed with this questionnaire. Statisti-
cal analyses were done with the chi-square test and Mantel-
Haenszel chi-square test (14).
Results
Braun Station is a suburban community of approxi-
mately 5900 persons located 20 miles northwest of San
Antonio, Texas. Potable water was supplied to all house-
holds from the same artesian well. Water was not filtered
before use but it was chlorinated with gas shortly before
distribution. We surveyed 100 of 1791 households in
Braun Station for a total of 346 persons. Our investiga-
tion showed two distinct outbreaks of gastroenteritis in
the community between 1 May and 30 July 1984.
The first outbreak in May was identified retrospective-
ly. We found that 251 persons (72%) in 85 households
had an acute gastrointestinal illness. Only 2.5% of the
120 persons living in the 50 households outside Braun
Station had diarrhea or vomiting in May (p < 0.00001,
chi-square test). The dates of onset of symptoms for the
first ill persons in 74 households are shown in Figure 1.
Dates of onset were unknown for 11 persons. Only six
serum specimens had been collected in May at the time of
59
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Figure 1. Dates of onset of symp-
toms in the first ill family members
in 100 surveyed households in
Braun Station, Texas, May and July
1984.
CO
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
24 26 26 30 2 4 6 6 10 12 14 16 18 20 22 24 26 26 30
MAY JUNE JULY
DATE OF ONSET OF SYMPTOMS
the outbreak, but these as well as convalescent sera were
tested for Norwalk virus antibodies. Four specimens had
fourfold rises in titer to Norwalk virus and the remaining
two had seroconverted from undetectable to low liters.
Because no sera were available from well persons, further
serologic testing was not done on them. Symptoms had
been similar to those in other outbreaks of Norwalk gas-
troenteritis.
The second outbreak was investigated prospectively. In
July 1984, 117 persons (34%) in 60 households had a
gastrointestinal illness. Only 2.5% of persons residing
outside Braun Station were ill in July (p = 0.00001, chi-
square test). No geographic clustering of households
with ill persons was evident, and the ill persons ranged in
age from 1 to 72 years with a mean of 32 years. Symp-
toms began during the week of 1 July in 53% and during
the week of 8 July in 20%. Dates of onset of symptoms
for the first ill persons are presented in Figure 1.
The symptoms of persons in the 60 Braun Station
households are presented in Table 1. Most had diarrhea
and abdominal cramps that lasted from 1 to 25 days, with
a mean of 6 days. No one was immunocompromised.
No association was evident between pet ownership, ex-
posure to farm animals, or swimming in the community
pool and occurrence of diarrhea in July. There was, how-
ever, an inverse association between the occurrence of
diarrheal illness in July and taking a vacation between 1
and 11 July. Twenty-one percent of persons who took a
vacation in this period developed a diarrheal illness com-
pared with 72% of those who remained in Braun Station
(p = 0.00006, chi-square test). Analysis for each day
during this interval indicated that absence from the com-
munity on July 2 was most protective (p = 0.01, chi-
square test). A linear relation was seen between the pro-
portion of total daily fluid intake of tap water or tap
water equivalents (reconstituted frozen juices, instant
iced tea, and powdered drinks) and the occurrence of
diarrhea in July, the greater the proportion of daily fluid
intake of tapwater, the higher the attack rates of diarrhea
(p = 0.002, Mantel-Haenszel chi-square test for trend)
(data not presented).
Cryptosporidium oocysts were identified in the stools
of 47 of 79 residents who became ill in July. Forty of
these forty-seven persons had diarrhea, 12 had vomiting,
12 had fever, and 2 were asymptomatic. One was hospi-
talized. Only 12 of 194 stool specimens submitted by out-
patients who did not reside in Braun Station but who
were treated for gastroenteritis at the Wilford Hall Medi-
cal Center contained Cryptosporidium oocysts. Four of
these persons had had person-to-person contact or drank
tap water in Braun Station during July. Twenty-three
stool specimens that contained Cryptosporidium oocysts
were cultured for bacterial pathogens. Salmonella enteri-
ditis was identified in 1 and Giardia lamblia cysts were
identified in 5. Only 3 of the 32 Braun Station families
with cryptosporidiosis had children in a day-care center.
Forty-three persons with a stool positive for Crypto-
sporidium submitted at least one additional stool. Nine-
teen passed oocysts for at least 5 days and 9 passed oo-
cysts for at least 20 days. One person continued to pass
oocysts 30 days after his first positive specimen was sub-
mitted. Paired serum specimens were obtained from 12
residents with positive stool. None showed detectable li-
ters to Cryptosporidium in acute specimens, but 9 devel-
oped liters of 1:40 or greater in convalescenl specimens.
Unlrealed waler samples from wells in this part of
Texas are nol usually tested. Pretreatment water samples,
however, were examined beginning in mid-June because
D'Antonioet al, • Water-borne Cryptosporidiosis
887
60
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Table 1. Symptoms and Duration of Illness in a Cryptosporidiosis
Outbreak in July 1984 at Braun Station, Texas
111 Persons
Reporting
Symptoms
Symptoms, %
Diarrhea 100
Abdominal cramps 76
Nausea 45
Vomiting 19
Fever 14
Headache 29
Muscle aches 13
Duration of illness, d
Mean 6
Range 1-25
of community complaints. Although chlorinated water
samples collected from residences remained negative for
coliforms, nine samples of untreated well water collected
between 2 and 11 July contained fecal coliforms. Samples
collected on 5 and 6 July contained 2600 and 1300
counts/100 mL, respectively. An order to boil water be-
fore drinking was issued on 11 July and remained in ef-
fect until the community was provided with an alternate
water supply later that month. Subsequent water tests
showed that dye introduced into the community's sewage
system appeared in the well water. Attempts to identify
the exact site of surface water or sewage contamination
were unsuccessful.
Discussion
Two outbreaks of gastroenteritis occurred in a single
suburban community in Texas in the summer of 1984,
and evidence shows that both were caused by contamina-
tion (probably intermittent) of the community's water
supply. In both outbreaks, the incidence of diarrheal ill-
ness in Braun Station residents was at least 12 times as
high as that in neighboring communities with a different
water supply.
In the July outbreak, the wide age range of ill persons,
the lack of geographic clustering, the strong protection
afforded to persons on vacation during the first week in
July, and analysis of tap water consumption indicate that
the outbreak was transmitted through the community
water supply. Coliform tests of unchlorinated water con-
firm that the water supply was contaminated between 2
and 11 July. Limited laboratory data indicate that the
major pathogen in the first outbreak was Norwalk virus,
whereas the major pathogen in the second outbreak was
Cryptosporidium, indicating intermittent contamination
of the water supply.
The typical clinical manifestations produced by Cryp-
tosporidium in immunocompetent adults are not well de-
fined. Preliminary evidence from outbreaks in children at
day-care centers shows that it can produce a short-lived
diarrheal illness accompanied by very little vomiting or
fever (4, 5). The symptoms of children in the July out-
break were generally milder than those of young adults.
Diarrhea was the most prominent symptom, as well as
the longest lasting, in adults. Significant volume depletion
occurred in two persons, one of whom was hospitalized
for fluid therapy. Because of the prolonged nature of the
diarrhea, weight loss occurred in 41% of the ill adults. A
proper estimate of the incidence of asymptomatic cryp-
tosporidiosis could not be calculated, but asymptomatic
Cryptosporidiosis clearly occurred in some family mem-
bers of households with proven cases. In some persons
the fecal shedding of the organism was prolonged. Sixty-
nine percent of persons tested had stool specimens posi-
tive for Cryptosporidium after their clinical symptoms
had resolved.
Our report describes the largest common-source out-
break of Cryptosporidiosis to date. The epidemiologic
data show that the source was fecally contaminated wa-
ter. The results also suggest that Cryptosporidium, like
Giardia, may be resistant to the usual levels of chlorina-
tion of potable water supplies. Cryptosporidium should
be added to the list of waterborne microorganisms capa-
ble of causing gastroenteritis.
ACKNOWLEDGMENTS: The authors thank James Perdue, of the Texas
Department of Health, for assisting in the telephone survey; Kathleen Can-
non and Judy Schwitzer for editorial and secretarial support; the staff of the
San Antonio Metropolitan Health District for laboratory support and assist-
ance in the telephone survey; and Alan Rebischke for technical assistance.
> Requests for reprints should be addressed to Richard E. Winn, M.D.;
SGHMMI, Wilford Hall Medical Center; Lackland Air Force Base, TX
78236-5300.
References
1. SOAVE R, DANNER RL, HONIG CL, et al. Cryptosporidiosis in homo-
sexual men. Ann Intern Med. 1984;100:504-11.
2. CURRENT WL, REESE NC, ERNST JV, BAILEY WS, HEYMAN MB,
WEINSTEIN WM. Human Cryptosporidiosis in immunocompetent and
immunodeficient persons: studies of an outbreak and experimental trans-
mission. N EnglJ Med. 1983;308:1252-7.
3. WOLPSON JS, HOPKINS CC, WEBER DJ, RICHTER JM, WALDRON MA,
MCCARTHY DM. An association between Cryptosporidium and giardia
in stools [Letter]. NEnglJMed. 1984;310:788.
4. ALPERT G, BELL LM, KIRKPATRICK CE, et al. Cryptosporidiosis in a
day-care center [Letter]. N EnglJ Med. 1984;311:860-1.
5. CENTERS FOR DISEASE CONTROL. Cryptosporidiosis among children
attending day-care centers—Georgia, Pennsylvania, Michigan, Califor-
nia, New Mexico. MMWR. 1984;33:599-601.
6. TZIPORI S, SMITH M, BIRCH C, BARNES G, BISHOP R. Cryptosporidios-
is in hospital patients with gastroenteritis. Am J Trop Med Hyg.
1983;32:931-4.
7. MATA L, BOLANOS H, PIZARRO D, VIVES M. Cryptosporidiosis in chil-
dren from some highland Costa Rican rural and urban areas. Am J Trop
Med Hyg. 1984;33:24-9.
8. RITCHIE LS. An ether sedimentation technique for routine stool exami-
nations. Bull US Army Med Dept. 1948;8:326.
9. TRUANT JP, BRETT WA, THOMAS W, Fluorescence microscopy of tu-
bercle bacilli stained with auramine and rhodamine. Henry Ford Hosp
Med Bull. 1962;10:287.
10. PAIK G, SUGG MT. Reagents, stains, and miscellaneous test procedures.
In: LENNETTE, EH, SPAULDING EH, TRUANT JP, eds. Manual of Clini-
cal Microbiology. 2nd ed, Washington D.C.: American Society for Mi-
crobiology; 1974:930-50.
11. WHEATLEY WB. A rapid staining procedure for intestinal amoebae and
flagellates. Am J Clin Pathol. 1951;21:990-2.
12. GREENBERG HB, WYATT RG, VALDESUSO J, et al. Solid-phase microti-
ter radioimmunoassay for detection of the Norwalk strain of acute non-
bacterial, epidemic gastroenteritis virus and its antibodies. / Med Virol.
1978;2:97-108.
13. CAMPBELL PN, CURRENT WL. Demonstration of serum antibodies to
Cryptosporidium sp. in normal and immunodeficient humans with con-
firmed infections. J Clin Microbiol. 1983;18:165-9.
14. SCHLESSELMAN JJ, Case-Control Studies. New York: Oxford University
Press; 1982.
888 December 1985 • Annals of Internal Medicine • Volume 103 • Number 6 (Part 1)
61
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1372
THE NEW ENGLAND JOURNAL OF MEDICINE
May 25, 1989
Reproduced, with permission, from the
New England Journal of Medicine
LARGE COMMUNITY OUTBREAK OF CRYPTOSPORIDIOSIS DUE TO CONTAMINATION OF
A FILTERED PUBLIC WATER SUPPLY
EDWARD B. HAYES, M.D., THOMAS D. MATTE, M.D., M.P.H., THOMAS R. O'BRIEN, M.D., M.P.H.,
THOMAS W. McKiNLEY, M.P.H., GARY S. LOGSDON, D.Sc., P.E., JOAN B. ROSE, PH.D.,
BETH L.P. UNGAR, M.D., DAVID M. WORD, P.E., PAUL F. PINSKY, M.P.H., MICHAEL L. CUMMINGS, M.D.,
MARGARET A. WILSON, M.D., M.P.H., EARL G. LONG, PH.D., EUGENE S. HURWITZ, M.D.,
AND DENNIS D. JURANEK, D.V.M., M.Sc.
Abstract Between January 12 and February 7,1987, an
outbreak of gastroenteritis affected an estimated 13,000
people in a county of 64,900 residents in western Georgia.
Cryptosporidium oocysts were identified in the stools of 58
of 147 patients with gastroenteritis (39 percent) tested dur-
ing the outbreak. Studies for bacterial, viral, and other
parasitic pathogens failed to implicate any other agent. In
a random telephone survey, 299 of 489 household mem-
bers exposed to the public water supply (61 percent) re-
ported gastrointestinal illness, as compared with 64 of 322
(20 percent) who were not exposed (relative risk, 3.1; 95
percent confidence interval, 2.4 to 3.9). The prevalence of
IgG to cryptosporidium was significantly higher among ex-
posed respondents to the survey who had become ill than
among nonresident controls.
HUMAN cryptosporidiosis was first described in
1976 in a three-year-old child with no obvious
immunodeficiency.1 It was subsequently recognized
as a cause of severe diarrhea in patients with the ac-
quired immunodeficiency syndrome and found to be a
common cause of sporadic and epidemic gastroenter-
itis in immunocompetent persons.2"13 Transmission
from calves to humans, from person to person, and by
a common source have all been described.4'8'9'12 Out-
breaks have occurred in day-care centers,10'11 among
international travelers,3'13 and in a community sup-
plied with unfiltered drinking water from a contami-
nated municipal well.9
We report the contamination of a filtered public
From the Centers for Disease Control (E.B.H., T.D.M., T.R.O., P.P.P.,
M.L.C., E.G.L., E.S.H., D.D.J.) and the Georgia Department of Human Re-
sources (T.W.M.), Atlanta; the Environmental Protection Agency, Cincinnati
(G.S.L.); the University of Arizona, Tucson, Ariz. (J.B.R.); the Uniformed
Services University of the Health Sciences, Bethesda, Md. (B.L.P.U.); and the
Georgia Department of Natural Resources (D.M.W.) and the Morehouse School
of Medicine (M.A.W.), Atlanta. Address reprint requests to Dr. Juranek at the
Centers for Disease Control, Division of Parasitic Diseases (F-12), Atlanta, GA
30333.
Cryptosporidium oocysts were identified in samples of
treated public water with use of a monoclonal-antibody
test. Although the sand-filtered and chlorinated water
system met all regulatory-agency quality standards, sub-
optimal flocculation and filtration probably allowed the
parasite to pass into the drinking-water supply. Low-
level cryptosporidium infection in cattle in the watershed
and a sewage overflow were considered as possible
contributors to the contamination of the surface-water
supply.
We conclude that current standards for the treatment
of public water supplies may not prevent the contamina-
tion of drinking water by cryptosporidium, with consequent
outbreaks of cryptosporidiosis. (N Engl J Med 1989;
320:1372-6.)
water supply by cryptosporidium that occurred even
though the treated water met federal and state stand-
ards for drinking water and that resulted in the largest
outbreak of cryptosporidiosis described to date.
BACKGROUND
In mid-January 1987 a college physician informed
health authorities of a dramatic increase in gastro-
enteritis among students at West Georgia College in
Carrollton, Georgia. Carrollton is the principal city in
Carroll County (population, 64,900), located in west-
ern Georgia.
An initial investigation by state and federal epide-
miologists indicated that the outbreak involved the
entire community. The laboratory director at Carroll-
ton's hospital reported that four stool specimens ob-
tained from patients with acute gastroenteritis were
positive for cryptosporidium. The first positive report
had been on January 12.
An initial case-control study of patients presenting
with gastroenteritis to the hospital emergency room
and a comparison of gastroenteritis rates at nursing
62
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Vol.320 No. 21 CRYPTOSPORIDIOSIS AND CONTAMINATION OF PUBLIC WATER — HAYES ET AL. 1373
homes connected to the public water system with rates
at homes independent of the system implicated the
water supply as a possible source of the outbreak. An
advisory to boil water was issued on January 30.
METHODS
Surveillance and Laboratory Studies
To define the course of the epidemic, we reviewed emergency-
room logs at the Carrollton hospital and recorded the number of
visits of patients who presented each week with acute gastrointesti-
nal illness from December 1986 through March 1987.
From January 20 through March 10, stool specimens for ova and
parasite examination were obtained from 147 patients with gastro-
enteritis who presented to outpatient facilities. Seventy-six of these
patients completed questionnaires on their symptoms at the time
of the stool collection. Two aliquots of each stool were preserved
separately in 10 percent formalin and in polyvinyl alcohol. The
formalin-preserved stool was examined unstained, after iodine
staining, and (after concentration by formalin—ethyl acetate sedi-
mentation) after modified acid-fast and auramine—rhodamine stain-
ing.14 Stool preserved with polyvinyl alcohol was stained with tri-
chrome and examined by light microscopy. All stool samples
were examined at the Parasitic Disease Laboratory of the Cen-
ters for Disease Control (CDC).
To evaluate the role of bacterial or viral pathogens, we reviewed
the results of all bacterial stool cultures performed at the hospital
laboratory during January, and we collected stool samples from five
acutely ill college students for examination by immune electron
microscopy for viral particles. In addition, paired serum samples
obtained six weeks apart — during the acute phase of the illness and
during convalescence — from 10 college students were tested for
seroconversion to rotavirus by IgG and IgA enzyme-linked immu-
nosorbent assay (ELISA). Seven of these paired serum samples
were also analyzed for the presence of antibody to Norwalk virus
with use of the avidin—biotin test. Viral studies were done at the
Gastroenteritis Laboratory of the CDC.
Telephone Survey
From January 31 to February 2, the Carroll County Health De-
partment surveyed by telephone 304 households systematically
sampled from the county telephone directory. Carrollton house-
holds were oversampled to ensure an adequate sample of persons
who had been exposed to the public water supply.
Adult respondents (over 18 years of age) were asked about the
source of their home water, their age, their sex, the place of employ-
ment of all household members, and whether any household mem-
ber had been ill with abdominal pain or diarrhea since January 1.
They were also asked about their consumption of specific foods,
their use of restaurants, the amount of tap water they consumed,
and their exposure to children in day-care centers and to farm ani-
mals. Data were obtained on all 304 respondents and 507 additional
household members. County engineers determined the water supply
for given work sites and schools. People whose home, school, or
work site was supplied with public water were considered to have
been exposed to the public water supply.
From March 7 through 12, we were able to obtain serum samples
from 86 of the 159 telephone-survey respondents who were living in
Carrollton and had been exposed to the public water supply. These
samples and randomly selected, banked control serum samples from
20 CDC employees who had not traveled outside the United States
were tested for the presence of IgG and IgM to cryptosporidium by
ELISA15 at the Uniformed Services University of the Health Sci-
ences in Bethesda, Maryland.
Investigation of the Water System
During the outbreak, federal and local engineers evaluated the
water-treatment plant in accordance with published procedures.16
Samples of raw and treated water from various points in the water
system were examined for cryptosporidium and giardia by passing
379 to 3785 liters (100 to 1000 gal) of water through a l-/nm poly-
propylene filter. The eluted sediment was examined directly for
giardia, and a fluorescein-tagged monoclonal antibody (Meridian
Diagnostics) was used to detect cryptosporidium in the sediment at
the Parasitic Disease Laboratory of the CDC, according to methods
described by Jakubauski," and at the Department of Microbiology
and Immunology of the University of Arizona, according to meth-
ods described elsewhere.18'19
To evaluate the means of contamination of the water supply, we
reviewed data on sewage and water maintenance and on rainfall,
and examined fresh stool samples for cryptosporidium from 67 of
the 226 cattle (including 3 calves) pastured in the watershed.
RESULTS
Surveillance and Laboratory Studies
The number of visits of patients with gastroenteritis
to the hospital emergency room increased in the sec-
ond week of January, peaked from the third week
of January through the first week of February, and
then declined to preoutbreak levels by tne last week of
February (Fig. 1).
Gryptosporidium was identified in the stools of 58
of the 147 patients with gastroenteritis (39 percent)
tested during the outbreak. Giardia was detected in
stool from only one subject, a two-year-old child who
also had cryptosporidiosis.
Of the 70 stool samples cultured for bacteria at the
hospital laboratory during January, only 2 were posi-
tive: 1 for shigella and the other for campylobacter.
The results of all viral studies in the 10 college stu-
dents who became ill were negative.
Information on signs and symptoms was obtained
from 30 outpatients with cryptosporidium-positive
stools. These patients ranged in age from less than 1
year to 46 years (mean age, 20). Diarrhea (defined as
three or more loose bowel movements per day) was
reported by 87 percent, stomach pain by 80 percent,
nausea by 67 percent, vomiting by 33 percent, fever by
30 percent, and muscle aches by 20 percent.
Telephone Survey
Of the 811 household members surveyed by tele-
phone, 363 had been ill with diarrhea or abdominal
pain since January 1. After adjustment for the over-
sampling of the city of Carrollton, where the attack
rate was 54 percent, the overall estimated attack rate
for the county was 40 percent.
Of the 489 household members who were exposed
to the public water supply, 299 (61 percent) had been
ill, as compared with 64 of the 322 unexposed subjects
(20 percent; relative risk, 3.1; 95 percent confidence
interval, 2.4 to 3.9; Taylor series approximation).20
When we used logistic regression models to control for
the exposure to the public water supply, we found
no strong or significant association between illness
and any other risk factor studied (i.e., exposure to day-
care centers, farm animals, or particular restaurants;
household size; or consumption of chicken or beef).
Among the 489 exposed household members, the
attack rate was 67 percent (170 of 254) in female
and 55 percent (128 of 232) in male household mem-
bers (the sex of 3 subjects was not recorded) (relative
risk, 1.2; 95 percent confidence interval, 1.0 to 1.4).
63
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1374
THE NEW ENGLAND JOURNAL OF MEDICINE
May 25, 1989
30-,
i— Advisory to Boil Water
Week Ending
March
Figure 1. Numbers of Visits of Patients with Gastroenteritis to the
Hospital Emergency Room in Carroll County, Georgia, According
to the Week of the Visit.
Among the exposed respondents, the higher attack
rate in female members remained significant when
we controlled for age and water consumption. The
attack rates according to age group among all 489
exposed household members ranged from 52 to 72 per-
cent and were highest among those 20 to 29 years
of age, but these differences were not statistically
significant (P>0.05 by the chi-square test for inde-
pendence).
The telephone survey indicated that approximately
50 percent of Carroll County residents, or 32,450 peo-
ple, were exposed to the public water supply at home
or at work. The risk of illness attributable to this expo-
sure was 41 percent (61 percent of exposed members
who became ill minus 20 percent of unexposed mem-
bers who became ill). We therefore estimate that ap-
proximately 13,000 persons had an illness attributable
to public-water exposure during the outbreak.
For the 86 exposed respondents to the telephone
survey who provided serum samples, the prevalence
of detectable IgG to cryptosporidium was higher
among the 68 respondents who had become ill than
among the 18 who had not become ill (76 percent vs.
56 percent; P = 0.08 by the chi-square test), and it
was significantly higher among respondents who had
become ill than among the 20 CDC employees (76
percent vs. 35 percent; P = 0.001 by the chi-square
test). The seroprevalence of cryptosporidium-specific
IgM with IgG was 12 percent in respondents who
became ill, 6 percent in those who had not become ill,
and 5 percent in the CDC controls (P>0.50, by Fish-
er's exact test for each comparison).
Investigation of the Water System
Roughly 7900 of the 19,000 households in the coun-
ty receive water from the Carrollton water system.
The remainder have wells or are connected to separate
municipal water systems. The Carrollton water sys-
tem draws water from a small river that runs through
surrounding pasture land. The water is treated by the
addition of alum, lime, and chlorine, rapid mixing,
mechanical flocculation (a process to promote the ag-
gregation of particulates), sedimentation, and rapid
sand filtration.
Samples obtained from the municipal water system
on January 5, 22, and 23 were tested for coliform bac-
teria by standard methods21 and were all negative. All
routine measurements of treated water were within
the limits set by the Environmental Protection Agency
and the State of Georgia for turbidity, coliform bacte-
ria, and residual free chlorine. Nevertheless, on Janu-
ary 28, samples of treated water contained particles as
large as 100 pm (cryptosporidium oocysts are 4 to 6
fjim in size).
Cryptosporidium oocysts were ultimately identified
in samples of treated water taken from the water-
treatment plant on January 28, February 4, and Feb-
ruary 5 and from four dead-end water mains, includ-
ing one at the college, between February 4 and 6. Two
samples of raw water obtained from streams that
drain into the river above the treatment plant were
positive for cryptosporidium, but five samples taken
from the river itself, upstream from the treatment
plant, were all negative. Three samples of treated wa-
ter taken as controls from a nearby town with a sepa-
rate water source were also negative.
No outbreaks of diarrheal illness were reported
among cattle pastured in the county. Cryptosporid-
ium was found in low numbers in stool samples ob-
tained from 3 of the 56 cattle tested from pastures
along the river upstream from the water plant (2 of the
positive stool samples were from calves), but it was
absent in the stool samples from 11 cattle tested from
pastures along the streams in which cryptosporidium
was found. There were no major rainfalls in early Jan-
uary, but more than 25 cm (10 in) of rain and snow fell
from January 15 to 22.
Although no serious breaks in the water or sewage
systems were reported during January, a sewage over-
flow caused by a blocked major sewer line was discov-
ered in mid-February in a wooded area above the
water-treatment plant. When dye was released at the
overflow point, it reached the river and the water-
treatment plant in approximately six hours. We could
not determine when the sewage pipe had begun to
overflow. An analysis of the surface-water supply for
coliform bacteria after the sewage spill had been
cleaned up found no indication of ongoing discharge
of raw sewage.
We attributed the passage of particulates through
the treatment plant to three factors. First, mechanical
agitators that were scheduled for replacement had
been removed from the flocculation basins in Decem-
ber in anticipation of the arrival of new agitators. This
reduced the efficiency of the flocculation step and im-
paired particulate removal. Second, the efficiency of
filtration was impaired by the equipment and proce-
dures used to control the flow of water through the
filters and to monitor turbidity. Finally, the filters
were sometimes restarted without first being back-
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Vol. 320 No. 21
CRYPTOSPORIDIOSIS AND CONTAMINATION OF PUBLIC WATER — HAYES ET AL.
1375
washed. This could have discharged dirt, flocculent
particles, and microorganisms from the filter beds into
the treated water. The Environmental Protection
Agency has set a turbidity limit for water entering the
distribution system of 1 nephelometric turbidity unit
(NTU) or less, measured at least once daily. The Car-
rollton water system met this regulation; however, tur-
bidity measurements of water obtained from individ-
ual filters on February 3 showed that three properly
backwashed and restarted filters were producing wa-
ter with a turbidity of 0.07 to 0.18 NTU, whereas
three filters restarted without being backwashed pro-
duced water with a turbidity ranging from 0.2 to 3.2
NTU in the first three hours after they were restarted.
During the first week of January the number of filters
restarted without undergoing backwashing increased
to 38 from a usual weekly average of 22, because water
use increased after the holidays.
The water-treatment process was improved in Feb-
ruary so that turbidity of continuously monitored
treated water was 0.2 NTU or less — a value consis-
tent with the removal of particles larger than approxi-
mately 1 /u,m. A sample of treated water taken on
February 11 showed no oocysts. The consistently low
turbidity readings resulted in the lifting of the adviso-
ry to boil water on March 2. Clinical and laboratory
surveillance through the end of May showed no recur-
rence of cryptosporidiosis in Carroll County.
DISCUSSION
To our knowledge, this is the first reported contami-
nation of a filtered water system by cryptosporidium.
The sudden onset of widespread gastroenteritis affect-
ing persons of all ages is typical of a waterborne out-
break. There was a strong association of illness with
exposure to the public water supply among the county
residents surveyed by telephone and a high prev-
alence of cryptosporidium-specific IgG in exposed
respondents to the survey who had been ill. Crypto-
sporidium oocysts were identified in the stools of pa-
tients during the outbreak as well as in the treated
public water. Neither laboratory examination of pa-
tients nor evaluation of the water system yielded any
evidence that other enteric pathogens contributed to
the outbreak.
Although there was adequate chlorine in the water
to inactivate most known bacteria and viruses,22 cryp-
tosporidium oocysts are highly resistant to chlorine.23
The efficacy of boiling water to inactivate cryptospo-
ridium is uncertain, but temperatures above 100°C kill
all other protozoa that have been tested, and inactiva-
tion of cryptosporidium oocysts by exposure to a moist
heat (55°C) for 20 minutes has been demonstrated.
Although we have no information on the residents'
compliance with the advisory to boil water, it appears,
in conjunction with the improvements in water-treat-
ment procedures, to have controlled the outbreak.
Exposed persons were defined as county residents
with access to the public water supply at home, school,
or work. Since Carrollton is the commercial center of
the county, it is likely that a number of persons who
were considered to be unexposed in our study were
actually exposed to the public water supply during
recreational or commercial visits to Carrollton. This
may explain the illness rate of 20 percent among those
considered unexposed in the study.
The higher attack rate among female residents in
the survey remains unexplained. Surveys in settings
with no outbreak have found a higher rate of self-
reported diarrheal illness in women than in men (Ho
MS: unpublished data), but our data do not allow us
to determine whether the sex-based difference we ob-
served was due to a difference in reporting behavior or
susceptibility to symptomatic infection.
Cryptosporidiosis is an illness of variable severity.
Many patients reported mild, nonspecific symptoms
during the outbreak in Carroll County. Of those with
cryptosporidiosis confirmed by stool culture, 13 per-
cent did not have diarrhea, as defined by three or more
loose stools per day; therefore, the diagnosis of crypto-
sporidiosis should not be excluded on the basis of the
absence of diarrhea.
The prevalence of cryptosporidium-specific IgG
was higher among exposed survey respondents who
had not been ill than among CDC employees (56 per-
cent vs. 35 percent) and among a group of Peace
Corps volunteers tested before placement overseas
(of 75 subjects, 24, or 32 percent, had IgG to crypto-
sporidium).24 Although not statistically significant,
these differences suggest that before the outbreak,
the background exposure to cryptosporidium among
residents of Carroll County was higher than the typi-
cal background exposure among U.S. residents, or
that many residents had asymptomatic infection
during the outbreak. Since the stool samples examined
for the presence of ova and parasites from the Car-
rollton area were not routinely examined for cryp-
tosporidium before November 1986, previous cases
of cryptosporidiosis were not likely to have been
detected.
Infected cattle in the watershed could cause long-
term low-level contamination of the surface water;
however, we could not conclude that the outbreak was
caused directly by cryptosporidiosis in cattle, since the
level of infection in cattle was low and the distribution
of cattle that tested positive did not match the distri-
bution of positive water samples.
Regardless of the source of contamination of the
untreated surface-water supply, we believe that the
changes in water treatment around the holiday season
allowed oocysts to pass into the treated water in suffi-
cient numbers to cause widespread illness. In addi-
tion, the sewage spill above the treatment plant and
the increase in rainfall just after the onset of the out-
break may have increased the load of cryptosporidium
reaching the plant, but this remains speculative.
Contamination of rivers and streams by cryptospo-
ridium has been reported in several states.25 The re-
sults of our investigation demonstrate that cryptospo-
ridium can contaminate filtered public water systems
65
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1376
THE NEW ENGLAND JOURNAL OF MEDICINE
May 25, 1989
— even when the water quality is within regulatory
limits for coliform bacteria, chlorine, and turbidity —
causing large epidemics of gastroenteritis in otherwise
healthy persons. Further study is needed to assess the
prevalence and the effect on public health of crypto-
sporidium in surface-water supplies and to evaluate
methods of preventing future contamination of public
drinking water by this hardy parasite.
We are indebted to Mr. Olan Ledford for assistance in coordinat-
ing and organizing work involving the Carroll County Health De-
partment; to Dr. Mary Miles for bringing the outbreak to our atten-
tion and for assistance in collecting specimens and epidemiologic
data; to Dr. Charles Gerba and the laboratory staff at the Universi-
ty of Arizona for the application of the most recent water-sampling
technology; to Dr. Henry Mathews, Dr. Govinda Visvesvara, and
Ms. Jennifer Dickerson, CDC, for examining the stool and water
specimens; to Dr. Roger Glass and Dr. Mei-Shang Ho, CDC, for
viral studies; to Dr. Bernard Nahlen, CDC, for assisting the investi-
gation; to Mr. Lewis Mason and Mr. Jim Baxley, Carrollton City
and County Water Utilities, for assistance in evaluating the water
system; to Mr. Ted Mikalsen, Georgia Department of Natural Re-
sources, for testing streams in the watershed for coliform bacteria; to
Ms. Cheri Toni for help in case finding and surveillance; to Ms.
Shirley Johnson for administrative assistance; to Ms. Robbie Green
for notifying us of the sewage overflow; to Dr. Keith Sikes, Dr.
Jeffrey Sacks, and Mr. David Smith, Georgia Department of Hu-
man Resources, for assisting the investigation; to the staffs of the
college, the hospital, and the Carroll County Health Department for
their assistance; and to Ms. Voughn Trader for assistance in the
preparation of the manuscript.
REFERENCES
1 Nime FA, Burek JD, Page DL, Holscher MA, Yardley JH. Acute enteroco-
litis in a human being infected with the protozoan Cryptosporidium. Gasfro-
enterology 1976; 70:592-8.
2. Holley HP Jr, Dover C. Cryptosporidium: a common cause of parasit-
ic diarrhea in otherwise healthy individuals. J Infect Dis 1986; 153:365-
8.
Jokipii L, Jokipii AMM. Timing of symptoms and oocyst excretion in
human cryptosporidiosis. N Engl J Med 1986; 315:1643-7.
Current WL, Reese NC, Emst JV, Bailey WS, Heyman MB, Weinstein
WM. Human cryptosporidiosis in immunocompetent and immunodeficient
persons: studies of an outbreak and experimental transmission. N Engl J
Med 1983; 308:1252-7.
3.
4.
5. Navin TR, Juranek DD. Cryptosporidiosis: clinical, epidemiologic, and
parasitologic review. Rev Infect Dis 1984; 6:313-27.
6. Soave R, Armstrong D. Cryptosporidium and cryptosporidiosis. Rev Infect
Dis 1986; 8:1012-23.
7. Payer R, Ungar BL. Cryptosporidium spp. and cryptosporidiosis. Microbiol
Rev 1986; 50:458-83.
8. Human cryptosporidiosis — Alabama. MMWR 1982; 31:252-4.
9. D'Antonio RG, Winn RE, Taylor JP, et al. A waterborne outbreak of
cryptosporidiosis in normal hosts. Ann Intern Med 1985; 103:886-8.
10. Alpert G, Bell LM, Kirkpatrick CE, et al. Outbreak of cryptosporidiosis in a
day-care center. Pediatrics 1986; 77:152-7.
11. Cryptosporidiosis among children attending day-care centers — Georgia,
Pennsylvania, Michigan, California, New Mexico. MMWR 1984; 33:599-
601.
12. Koch KL, Phillips DJ, Aber RC, Current WL. Cryptosporidiosis in hospital
personnel: evidence for person-to-person transmission. Ann Intern Med
1985; 102:593-6.
13. Jokipii LrPohjola S, Jokipii AM. Cryptosporidiosis associated with travel-
ing and giardiasis. Gastroenterology 1985; 89:838-42.
14. Ma P, Soave R. Three-step stool examination for cryptosporidiosis in 10
homosexual men with protracted watery diarrhea. J Infect Dis 1983;
147:824-8.
15. Ungar BL, Soave R, Payer R, Nash TE. Enzyme immunoassay detection of
immunoglobulin M and G antibodies to Cryptosporidium in immunocompe-
tent and immunocompromised persons. J Infect Dis 1986; 153:570-8.
16. Logsdon GS. Evaluating treatment plants for paniculate contaminant re-
moval. J Am Water Works Assoc 1987; 79(9):82-92.
17. Jakubauski W. Detection ofGiardia cysts in drinking water: state of the art.
In: Erlandsen SL, Mayer EA, eds. Giardia and giardiasis: biology, patho-
genesis and epidemiology. New York: Plenum Press, 1984:263-85.
18. Rose JB. Detection of Cryptosporidium from wastewater and freshwater
environments. Water Sci Technol 1986; 18:233.
19. Musial CE, Arrowood MJ, Sterling CR, Gerba CP. Detection of Cryptospo-
ridium in water by using polypropylene cartridge filters. Appl Environ Mi-
crobiol 1987; 53:687-92.
20. Kleinbaum DO, Kupper LL, Morgenstem H. Epidemiologic research: prin-
ciples and quantitative methods. Belmont, Calif.: Lifetime Learning Publi-
cations, 1982:296-9.
21. American Public Health Association, American Water Works Association,
Water Pollution Control Federation, Georgia Department of Natural Re-
sources. Standard methods for the examination of water and wastewater.
16th ed. New York: American Public Health Association, 1985.
22. Laubusch EJ. Chlorination and other disinfection processes. In: American
Water Works Association. Water quality and treatment: a handbook of
public water supplies. 3rd ed. New York: McGraw-Hill, 1971:201.
23. Campbell I, Tzipori AS, Hutchison G, Angus KW. Effect of disinfectants
on survival of Cryptosporidium oocysts. Vet Rec 1982; 11:414-5.
24. Ungar BLP, Mulligan M, Nutman TB. Serologic evidence of Cryptosporid-
ium infection in US volunteers before and during Peace Corps service in
Africa. Arch Intern Med 1989; 149:894-7.
25. Rose JB. Occurrence and significance of Cryptosporidium in water. J Am
Water Works Assoc 1988; 80(2):53-8.
66
-------
Reproduced, with permission, from the Lancet
1484
THE LANCET,DECEMBER24/31,1988
of free chlorine following booster chlorination procedures are not
oocysticidal.5 The waterborne route should be considered when
clusters of cryptosporidiosis associated with potable water occur,
even in the absence of bacterial or viral contamination.
WATERBORNE OUTBREAK OF
CRYPTOSPORIDIOSIS
SIR,—Despite the fact that Cryptosporidium spp have animal
reservoir hosts and are transmitted by the faecal-oral route,
Cryptosporidium, unlike Giardia duodenalis, has not yet been
acknowledged widely as a significant cause of waterborne
gastroenteritis. In 1985, D'Antonio et al1 described the first
waterborne outbreak, in Texas. Rush et al2 reported
Cryptosporidiian spp oocysts in raw drinking water samples and
postulated waterborne spread to account for a peak in cases of
cryptosporidiosis in the Sheffield area in 1986. Hayes et al3
described a waterborne outbreak in Georgia, USA, affecting 13 000
people exposed to a sand-filtered, chlorinated public water supply.
During April, 1988, several cases of cryptosporidiosis were
reported to the community medicine specialist in Ayrshire.
Extensive epidemiological investigations failed to implicate a
common source of food or milk or consistent history of animal
contact. Mapping of residences of the affected cases revealed that
many of the households concerned received the same public potable
water supply. 27 patients either resident in the area served by that
supply or who consumed that water were diagnosed as
Cryptosporidium spp positive (age range 4 months to 93 years), of
whom 12 were admitted to hospital (11 required intravenous fluid
replacement).
Following notification of the possible involvement of the water
supply and the introduction of booster chlorination procedures, the
water supply system was thoroughly examined. Twice during the
outbreak Esclierichia coli, coliforms, and faecal streptococci were
isolatedj from a service tank at the treatment works, but all other
samples proved negative bacteriologically. Cryptosporidium spp
oocysts were detected by Wright-Giemsa, modified Ziehl-Neelsen,
saffranin/methylene-blue, phenol-auramine, and monoclonal
antibody staining in the treated, chlorinated water supply system.
Oocysts were present in sludge samples from the treatment works,
in the final water leaving one of the service tanks, and in a
break-pressure tank delivering chlorinated final water to the supply
(range 0-13 to 1000 oocysts/1). Reinspection of the break-pressure
tank revealed an old 50 cm fireclay pipe discharging into it; the pipe
collecting run-off from the surrounding area. Water samples from
the pipe and a nearby stream showed evidence of faecal
contamination (244 E coli, 403 coliforms, 58 faecal streptococci per
100ml, and 107 Ecoli, 152 coliforms, 34 faecal streptococci, per 100
ml, respectively). Samples from the stream and soil and grass
adjacent to the fireclay pipe were positive for oocysts (0-13 oocyst/1
and 32 oocysts/g, respectively). Oocysts were also detected in
samples of the treated supply in the absence of faecal bacterial
indicators. Following the isolation, drainage and disinfection of the
contaminated storage tanks, extensive mains flushing, and
emergency booster chlorination, no new cases of waterborne
cryptosporidiosis have been reported, and bacteriological and
parasitological analyses of subsequent water samples have proved
negative.
Oocysts appear to have been introduced via the fireclay pipe into
the break-pressure tank which contained final water for distribution
rather than by failure of the water treatment process to retain them.
There was evidence that cattle slurry had been sprayed on land in
the vicinity of the fireclay pipe before the outbreak. Cryptosporidiian
spp oocysts can remain viable for at least 12 months at 4°C5 and
oocyst contamination of final waters in the absence of
bacteriological contamination has been reported.1 Attainable levels
Scottish Parasite Diagnostic Laboratory,
Department of Bacteriology,
Stobhill General Hospital,
Glasgow G21 3UW
Community Medicine Department,
Ayrshire Central Hospital, Irvine
Area Laboratory,
Crosshouse Hospital, Kilmamock
Srrathclyde Water Department,
Glasgow
Environmental Health Department,
Cunninghame District Council, Irvine
Communicable Diseases (Scotland) Unit,
Ruchill Hospital, Glasgow
Scottish Home and Health Department,
Edinburgh
H. V. SMITH
R. W. A. GIRDWOOD
W. ]. PATTERSON
R. HARDIE
L. A GREEN
C.BENTON
W. TULLOCH
]. C. M. SHARP
G. I. FORBES
I. D'Antonio RG, Winn RE, Taylor JP, et al. A waterborne outbreak of
cryptosporidiosis in normal hosts. Aim Intern Med 1985; 103:886-88.
2. Rush BA, Chapman PA, Ineson RW. Crvplosporidiitin and drinking water. Lancet
1987; ii: 632.
3. Hayes EB, Matte TD, O'Brien TR, et al. Contamination of a conventionally treated
filtered public water supply by Cryptesporiditttn associated with a large community
outbreak of cryptosporidiosis. N Eiigl J Mcd (in press).
4. Anon. The bacteriological examination of drinking water supplies. London: HM
Stationery Office, 1983.
5. Smith AL, Smith HV, Girdwood RWA, Carrington E. The effect of free chlorine on
the viability of Cryptosporidium spp oocysts. WRc Publ PRU 2023-M.
Medmeacham, Bucks: Water Research Centre, 1988.
67
-------
Reprinted from Journal AWWA, Vol. 73,
No. 1, by permission. Copyright 1981,
American Water Works Association.
What Happens When the Multiple Barrier
Concept is Neglected
Waterborne disease: occurrence is on
the upswing
Edwin C. Lippy
Three of the waterborne disease outbreaks in 1979 typified problems common to
many water systems. Investigation of the outbreaks identified as causative factors
deficiencies in water treatment practices and plant operation and a breakdown in
surveillance activities designed to protect public health. Preliminary information
indicates that a total of 45 outbreaks occurred during 1979, increasing the annual
average of outbreaks to 36 for the four-year period from 1976 to 1979. This
represents a 50 percent increase over the 1971-75 average of 24. The five-year
averages have steadily increased from an annual average of ten during 1951-55.
Before that period the trend was declining.
The disturbing trend toward more fre-
quent occurrence of outbreaks of water-
borne disease should serve as a warning
to all who share in the responsibility for
the delivery of a safe and potable water.
The "multiple barrier" concept that
relies on placing protective systems
between the water consumer and actual
as well as potential sources of contami-
nation should be emphasized, with
appropriate consideration for natural
features (distance, dilution, geologic fac-
tors), man-made facilities (adequate
waste treatment, water treatment, opera-
tional considerations), and conscientious
surveillance by regulatory agencies
JANUARY 1981
(monitoring, inspection, certification).
Investigation of three of the outbreaks
that occurred in 1979 illustrates what can
happen when these barriers are ne-
glected. The outbreaks occurred in Brad-
ford, Pa.; a state park near Lake Havasu
City, Ariz., and a church camp near
Brevard, N.C.
Bradford, Pa.
Bradford is located in northwestern
Pennsylvania about 210 km (130 mi)
northeast of Pittsburgh and some 97 km
(60 mi) south of Buffalo, N.Y. It is situ-
ated in the midst of the Allegheny oil
region and is an industrial center for
63
producing and refining crude oil.
The water system serves a population
of approximately 18 000. Three im-
poundments located within a few miles
of the city serve as sources of supply.
The impoundments and watersheds are
wholly owned and protected by the city,
and recreational use is prohibited. Chlo-
rination is the only treatment provided;
gas injection facilities are located imme-
diately downstream from each impound-
ment. Transmission mains convey water
from the impoundments to the distribu-
tion system, where the supplies con-
verge. A number of service connections
draw off each transmission main prior to
their convergence.
An open finished water reservoir of
13-ML (3.5-mil gal) capacity provides
storage and regulates pressure in the
distribution system. Chlorination is not
provided at this reservoir. Data for each
raw water impoundment are given in
Table 1.
The outbreak begins. A waterborne out-
break was suspected when fifteen resi-
dents reported within one week to the
E.C. LIPPY 57
-------
Bradford Hospital for treatment of diar-
rheal illness. When tested, ten of the
fifteen were stool-positive for Giardia
cysts. Newspaper reports implicated the
water supply and advised residents to
boil their water. The advice was based
upon reporters' discussions with state
and city health officials and the Center
for Disease Control (CDC) in Atlanta, Ga.
The suspicion that the illness was related
to the water supply probably arose from
recent water quality problems.
Heavy rain in the Bradford area in July
and August contributed to turbidity
problems and resulted in numerous citi-
zens' complaints of discolored and mud-
dy water. This was compounded by pub-
lic notification of failure to comply with
the coliform maximum contaminant lev-
el (MCL) in August and September. The
city was not required to report violations
of the turbidity MCL because it had been
granted a waiver by the Environmental
Protection Agency (EPA). Records indi-
cated that the reservoir at Marilla Brook
had turbidity values in excess of 10 NTU
on numerous occasions.
The bacteriological record appeared
acceptable until August when three of
twenty samples collected were reported
to be positive for coliform bacteria (Ta-
ble 2). Closer inspection of the record,
however, showed that extensive time
had elapsed between sample collection
and analysis, casting doubt on the valid-
ity of al! the results prior to the use of
local laboratories in September.
Determining the cause. The Meadville
regional office of the Pennsylvania
Department of Environmental Resources
invited EPA to assist in the investigation
of the water system to isolate Giardia
cysts from the water, identify the source
of contamination, and propose correc-
tive measures. Also, a team of epidemio-
logical investigators was dispatched by
the Pennsylvania Department of Health
and was soon joined by a team from the
Center for Disease Control.
Within ten days after the investigation
was begun, cysts were isolated from the
water system. A beaver trapped from a
large lodge in the Gilbert Run reservoir
was sacrificed for necropsy and a sample
from the colon showed an abundance of
Giardia cysts. At this point in the inves-
tigation the number of confirmed cases
of giardiasis at the Bradford Hospital
had increased to 180.
Precautionary measures taken at the
outset of the investigation included a
recommendation that consumers boil
water for one minute and that plant
operators increase chlorination to a con-
centration of sufficient strength to de-
stroy Giardia cysts. Chlorine contact
times were calculated for each source
based on the flow in each main and the
distance from the point of chlorine injec-
tion to the first service connection. These
times were confirmed by increasing
58 RESEARCH AND TECHNOLOGY
TABLE 1
Characteristics of reservoir facilities in Bradford, Pa., 1979
Impoundment
Reservoir 2 (Gilbert
Run)
Reservoir 3 (Marilla
Brook)
Reservoir 5 (Hazelton
Mill)
Drainage
Area
km"-
533
sq mi
206
Storage
Capcity
ML
765
454
2574
mil gal
202
120
680
Average
Output
ML/doy
5.3
4.1
14.4
mgd
1.4
1.1
3.8
Date
1913
1900
1955
TABLE 2
Coliform sample results for Bradford, Pa., 1979
Month
January
February
March
April
May
June
July
August
September*
October
November
December
Number of
Samples
25
20
20
25
20
20
25
20
26t
24}
19
20
Number of
Positive
Samples
0
0
0
0
0
0
0
3
5
2
0
0
Number of Samples and Time
Between Collection and Labora
ory
Analysis
Same
Day
21
24
19
20
2
Days
4
5
3
Days
25
20
20
25
15
5
5
4
Days
5
11
15
15
5
Days
5
5
'Began using local laboratory
tlncludes six check samples
^Includes two check samples
chlorine feed and clocking the amount of
time it took for the increase to appear at
the first service connection. The com-
puted and observed times were in rea-
sonable agreement, and chlorine doses
were adjusted according to the most
current information available for de-
struction of Giardia Cysts.
The chlorine contact time at the Gil-
bert Run reservoir was most critical at
about 20 min, which required a chlorine
dosage that would give a residual of 2.5
mg/L at the first service connection.
Contact time at the Marilla Brook reser-
voir was 30 min, requiring a residual of
2 mg/L. The Hazelton Mill reservoir pro-
vided a contact time of more than 90 min,
which permitted the use of a more rea-
sonable chlorine dose. Feeding the entire
system from Hazelton Mill was consid-
ered as a possibility but was rejected
because inadequate pressure would pro-
hibit service to all customers. However,
after beaver lodges were found in both
the Gilbert Run and Marilla Brooks res-
ervoirs, the output from these sources
was reduced and the deficit sustained by
Hazelton Mill. Common problems noted
at all of the chlorination facilities
included antiquated equipment (a single
chlorine cylinder was connected to the
chlorinator, causing lapses in feed when
empty cylinders were replaced so that if
a 15-min change time at each facility
occurred on the same day 250 kL [66 000
gal] of nonchlorinated water would be
69
delivered to the system); inadequate
chlorinator capacity; and an incorrect
procedure for determining chlorine dose
to achieve a residual in the distribution
system. Chlorine residuals had been
determined by collecting samples from a
utility tap located a short distance from
the injection point and adjusting the feed
rate to attain 1 mg/L. This resulted in no
residual in the system.
The city provided new chlorine feed
equipment, which included manifolded
cylinders with automatic switchover
devices to improve the reliability of chlo-
rination. Chlorination was the only pro-
tective barrier that could be employed in
this situation, so uninterrupted, contin-
uous disinfection was very important.
Even though consumers had been
instructed to boil water, this could not be
considered an effective barrier because it
was not known how diligently the boil
water order was being followed in the
community.
The Pennsylvania Game Commission
estimated that six to eight beavers inhab-
ited the lodge in Gilbert Run reservoir. It
was suggested that they be trapped and
moved out of the watershed; however,
the commisson was concerned that the
beavers would not survive in new sur-
roundings that late in the year. Holding
the beavers in captivity for release in the
spring was then considered.- Suitable
holding facilities were not available and
other problems associated with this
JOURNAL AWWA
-------
Hillside Spray
Irrigation System )L~ ~jk- } (.
Colorado River
Figure 1. Water and sewage facilities for the Sand Point marina and campground
alternative made it unworkable. Even-
tually all the beaver in the Gilbert Run
and Marilla Brook reservoirs and a few
from the watershed of Hazelton Mill
reservoir were trapped and sacrificed.
Two beavers in Gilbert Run, one in
Marilla Brook, and one in Hazelton Mill
tested positive for Giardia cysts.
The number of giardiasis cases deter-
mined by positive stool examination at
the Bradford Hospital eventually in-
creased to 407. However, some local phy-
sicians began prescribing medication
without the benefit of stool examination.
Sales of drugs normally prescribed for
giardiasis were recorded by city pharma-
cies as numbering 3487 during the six-
week period beginning with the start of
the investigation. The epidemiological
study conducted by CDC and the state
health department included a random
household survey to identify occurrence
and distribution of illness and collection
of stool specimens to determine infectiv-
ity rate in residents of Bradford as well
as a control community. The control
community had little interaction with
Bradford and used groundwater as a
water supply source. The survey results
showed that 5.4 percent of the Bradford
residents had giardiasis-like illness (diar-
rhea for > 10 days) with only 0.6 percent
of the control community experiencing
the same symptoms. Examination of
stool specimens showed an infection rate
of 16 percent for Bradford respondents
JANUARY 1981
(seventeen of 106 stools positive) and all
40 stools negative for the control commu-
nity.1 The stool infectivity rate applied to
the Bradford population amounted to
2900 people affected.
The water quality problems that
occurred before the outbreak generated
numerous complaints, and public sup-
port was building for improvement of
the water system. The additional incon-
venience of boiling water combined with
the noticeable odor created by increased
chlorination fostered additional public
pressure for action. The city directed a
consulting engineering firm to begin a
feasibility study for the provision of ade-
quate treatment only eleven days after
investigation of the outbreak began.
Rebuilding protective barriers. The Brad-
ford water system is typical of many US
systems faced with making improve-
ments to comply with the National Inter-
im Primary Drinking Water Regulations.
In Pennsylvania alone there are 113 sim-
ilar systems that rely on surface water as
a source but do not have the facilities to
reduce turbidity. From an energy conser-
vation aspect, the Bradford system is
very efficient, relying on raw water
sources situated at sufficient elevation to
maintain pressure throughout the distri-
bution system without pumping. Howev-
er, nearly two thirds of the system is
unmetered, which is reflected in an aver-
age per capita demand of 1365 L/day (350
gpd) and in the size and cost of sewage
70
collection and treatment facilities.
Breakdown occurred in all compo-
nents of the protective systems in the
multiple barrier concept. With informa-
tion accumulating from waterborne out-
breaks of giardiasis in California,1 Wash-
ington,2-3 Oregon,1 and New Hamp-
shire,--'3 and other studies" implicating
beaver as an intermediate host of the
cyst that is transmitted to man, protec-
tive public health measures should be
incorporated into the multiple barrier
concept for certain water systems. Sys-
tems using surface water as a source of
supply, with disinfection as the only
means of treatment, should consider con-
trol of the beaver population in the
watershed. Control measures may in-
clude periodic surveys to determine
where lodges are being established, trap-
ping during periods of the year when the
beaver can be safely moved, and forestry
practices that replace deciduous trees
with coniferous species, thereby limiting
the food supply along waterways and
around reservoirs to discourage beaver
habitation. These precautionary mea-
sures could be associated with natural
and surveillance barriers, with both util-
ity and regulatory agencies sharing the
responsibility for implementation.
Other breakdowns that occurred were
inadequate chlorination (man-made bar-
rier) and ineffective microbiological
monitoring (surveillance) which cast
doubt on the validity of coliform results.
The turbidity values prior to September
were also questionable because the for-
mazin standard was leaking. It is ironic
that a waiver from compliance with the
turbidity MCL was granted by EPA in
this situation: such a waiver is predi-
cated on qualifiers including demonstra-
tion that turbidity does not interfere with
disinfection, prevent maintenance of an
effective residual throughout the distri-
bution system, or interfere with microbi-
ological determinations.
State park—Lake Havasu City, Ariz.
Cattail Cove state park is located on
the eastern bank of the Colorado River
north of Parker Dam and south of Lake
Havasu City. A recreational area located
within the park-is divided into two facil-
ities, one operated by the state and an
adjacent facility operated by concession-
aires (Sand Point marina and camp-
ground). Sand Point, the larger of the two
facilities, includes a store, comfort sta-
tion, marina, and 170 spaces for campers
and recreational vehicles.
The water system serving the recrea-
tional area is simple in design and oper-
ation. The source is a shallow well con-
structed in a diked area adjacent to the
Colorado River, using bank storage or
infiltrated river water. After chlorina-
tion, water is pumped to a hillside stor-
age tank overlooking the two facilities.
The Cattail Cove and Sand Point distri-
E.C. LIPPY 59
-------
bution systems are fed by separate mains
off a wye connection near the tank (Fig-
ure 1), Each facility has a sewage system
consisting of a package plant with
effluent disposal by irrigation.
Cause lor concern. Disease control offi-
cials of the Arizona Department of
Health Services requested assistance
when they suspected a waterborne out-
break related either to swimming or
water supply at the recreational area.
Reports of illness began in June and
continued through September. Most of
the reports originated in California,
which caused some problems in follow-
up of cases. Of 74 cases investigated, four
were stool positive for Giardia cysts.
Information gathered by questionnaire
suggested that drinking water at Sand
Point was associated with illness.
Coliform sample results from the Sand
Point water system showed two of two
samples positive (TNTC and confluent
growth) in June with followup check
samples negative thirteen days later.
(Two consecutive negative check sam-
ples were initiated on only one sampling
point.) One of four samples collected in
July was unsatisfactory (confluent
growth) with no followup check sample,
and one negative sample was collected in
August. Samples from the state facility
distribution system were all negative.
Samples from the swimming areas
showed coliform and fecal coliform
results in excess of the single sample
recommendation (200 fecal/1000 total) in
August and September. After the Labor
Day weekend beaches were posted with
the warning that water quality did not
meet standards and that swimming was
therefore not recommended. While the
preliminary epidemiological information
implicated Sand Point drinking water,
bacteriological data for the swimming
areas also cast suspicion on water-
related recreational activities. Suspicion
increased when it was learned that the
sewage plant serving Sand 'Point over-
flowed into the marina during a power
failure in July. Overflow facilities had
not been incorporated in the plant
design.
Erratic evolution plagues system. The
water and wastewater systems at Sand
Point evolved over a period of six years
under four different owners. An impor-
tant feature unique to the Sand Point
facility was the design of the sewage
disposal system. The system was initially
designed to dispose of the total sewage
plant effluent through the use of subsur-
face bubbler irrigators. The design was
based on 150 trailer or camper spaces
utilizing 380 L/day/space (100 gpd/
space) and 57 L/day/bubbler (15 gpd/
bubbler), which required 1000 subsur-
face bubblers to irrigate an equal num-
ber of trees and shrubs. A surprising
feature of the design was the use of the
same type and color of pipe for the
60 RESEARCH AND TECHNOLOGY
effluent irrigation system as for the pota-
ble water distribution system.
At some stage in the process of system
development and changes in ownership,
the installation of bubblers was halted
and spray irrigation was added on an
on-site hillside to aid in the disposal of
effluent. Tree and shrub irrigation in the
camping areas was then accomplished
by drip irrigation using potable water
with aboveground drip fittings sub-
merged in pits formed around the bases
of trees and shrubs. As the facility
changed ownership and was expanded
to accommodate additional trailer
spaces, records or sketches were not kept
to indicate where plumbing changes
were made, and the extent of the bubbler
system was not known. (The changes
were made without the required approv-
al by the health department.)
Conditions were optimized for cre-
ating a direct cross connection between
the water system and the subsurface
bubble-irrigation system. Each drip fit-
ting submerged in a tree or shrub pit
provided an entry point for contami-
nated water if subatmospheric pressure
occurred in the water system.
To complicate the situation further,
the location chosen for spray irrigation
was on the side of a hill directly above
the transmission main between the well
and storage tank and the feed line to the
campground. The feed lines to the camp-
ground were located in a gully at the
base of the hill. Because the vegetation
and soil on the hillside could not absorb
all the effluent being applied, effluent
ran off into the gully. To prevent drain-
age into the marina and beaches, small
catchment areas were fashioned to retain
the runoff in pools. These pools were
located almost directly on top of the line
carrying potable water. This presented
another unusual situation.
When the well was developed, it was
constructed in fill material placed to
extend the bank into the river and form
an infiltration gallery. Sufficient fill was
excavated to set the well at a depth of 4.5
m (15 ft) and the material packed around
the screen was to be selected and placed
by specifications referenced on the engi-
neering plan. It is assumed that this
material was intended to be carefully
selected, washed, and graded sand and
gravel. However, local material was used
in backfilling the gallery.
A raw water quality problem subse-
quently developed which is thought to be
related to the use of material excavated
from a local site that contains manga-
nese. After chlorination, the water
pumped from the well turned black in
the transmission main to the storage
tank, indicating the oxidation of manga-
nese. The transmission main, rising
about 45 m (150 ft) and running a length
of approximately 300 m (1000 ft), served
as an inclined settling tube that permit-
71
ted an accumulation of black water
which had to be bled off every three or
four days and before weekends through
a discharge port at the well and into the
swimming area. This process discolored
the river for about 30 min before the
black water was diffused.
The unique situation thus presented
was the possibility that while the trans-
mission main to the storage tank was
being bled, siphonage of sewage or
drainage from the irrigated area could
occur by a venturi effect through a crack
or bad joint in the main, causing contam-
ination of the beaches or affecting the
water being infiltrated into the well.
Standard bacteriological samples were
collected as a result of the concern with
problems of the water and sewage sys-
tems and the swimming areas. A larger
volume sample of raw water was col-
lected from the well, using a filtering
device containing three membrane pads
in series with pore sizes of 5, 2, and 0.45
/mi. Nineteen litres, or 190 times the
volume of a standard bacteriological
sample, were filtered through the pads
for analysis of coliform and fecal coli-
form bacteria. (The pads were halved at
the laboratory and processed separately
for the analysis.) The large-volume sam-
pling technique was used to detect low-
level and, possibly, intermittent contami-
nation.
Sample results showed no coliforms in
the large-volume sample of raw water
and no occurrence of contamination dur-
ing bleed-off of the transmission main
from the well to the storage tank. (The
latter finding was also confirmed by a
dye test.) Positive coliform and fecal
coliform results were obtained on sam-
ples from the Sand Point distribution
system in an area that served campers
who had reported illnesses.
Discussion of the results with the
departments of health services and state
parks led to the agreement to close Sand
Point marina and campground. By plac-
ing dye in the effluent pumping well of
the sewage treatment plant, the northern
regional office of the Department of
Health Services in Flagstaff proved that
a cross connection existed between the
water system and the subsurface bubbler
irrigation system. After dye was ob-
served coming from the camp faucets,
excavation located the cross connection.
Extensive corrections were required to
improve the water and sewage systems.
Improvements also were required in sur-
veillance activities, including prompt
followup on positive coliform samples,
timely reporting of results, and a review
of the state regulations permitting instal-
lation of wastewater irrigation systems.
Particular emphasis was placed on the
necessity of color coding or use of dis-
similar materials for water and waste-
water piping.
Determination of the occurrence and
JOURNAL AWWA
-------
distribution of illness associated with
this outbreak was difficult and time-
consuming. Epidemiologists from the
Department of Health Services were
hampered in their investigative efforts
by a number of factors, including (1)
difficulty in locating visitors because of
incomplete or erroneous identification
entered on the camp register; (2) commu-
nication across state boundaries; (3) con-
tact of visitors who might have been
exposed five or six months before and
would not remember; (4) selection of an
appropriate sample population; (5) defin-
ing an incubation period where exposure
to multiple pathogens could have oc-
curred; (6) complications related to the
possibility that the agent of hepatitis
might have been involved; and (7) limited
investigative resources.
Investigators eventually chose to inter-
view a sample of the population that
visited one of the two facilities on Labor
Day weekend. A case was defined as a
person who experienced a diarrheal ill-
ness during or within a three-week peri-
od after the visit. Results based on a
telephone survey of 44 of 179 groups
visiting Sand Point marina and camp-
ground and 14 of 42 groups who regis-
tered at the state facility showed that 57
(35.2 percent) of 162 campers from Sand
Point were ill compared to only 6 (8.0
percent) of the 75 state park campers.
The attack rate was significantly higher
for Sand Point campers who consumed
campsite water than for those who did
not, and the incidence of illness was
greatest in the vicinity of the cross con-
nection. Approximately 6800 persons
camped at Sand Point from June 1
through September. If water contamina-
tion occurred in this period, it is esti-
mated that as many as 1900 persons may
have become ill in this outbreak.7
Hazards lurk in noncommunity systems.
The water supply serving Sand Point
marina and campground is classified as a
noncommunity system under the Nation-
al Interim Primary Drinking Water Regu-
lations. Current estimates place the total
number of noncommunity systems at
250000. Generally, this type of system
has not received the same degree of
attention from regulatory agencies as
have the community water systems. The
59 000 community water systems have
been given priority consideration in
implementation of the Safe Drinking
Water Act, which is not reassuring since
they also suffer from lack of effective
surveillance, as exemplified by the situa-
tion in Bradford, Pa. If such problems as
those found at Sand Point marina and
campground are common, regulatory
surveillance will require substantial
•changes to control a potentially serious
public health situation.
There was clearly a breakdown in
regulatory surveillance for this system.
Design criteria for reuse systems were
JANUARY 1981
38-kL(10000-gal)
Storage Tank
Toxaway Creek
Figure 2. Water and sewage facilities at The Wilds camp
not adequate, making review of plans
and specifications ineffective. Proce-
dures for inspections during construc-
tion and for changes after the system was
in operation also were inadequate, per-
mitting hazardous conditions to develop.
Microbiological monitoring was not ef-
fective in followup procedures for posi-
tive samples.
The problems discussed that pertain to
investigation of the outbreak do not fully
describe the hazards observed in this
situation. More than 100 potential cross
connections existed with the drip irriga-
tion system; drains for surface water
could be mistaken for sewage hookups,
allowing contamination of bathing areas;
spray from the effluent irrigation system
could drift over the camping area; valves
on the water system were installed by
bringing plumbing runs to the soil sur-
face without using vaults or boxes; and
potable water lines at the sewage dump
station (for spaces without sewage hook-
ups) were not properly protected.
Perhaps a more important concern is
that noncommunity systems get the least
regulatory attention and need the most.
These water systems are frequently oper-
ated and maintained by untrained people
and are beset with design, construction,
and operational problems.
Church camp-Brevard, N.C.
A church camp named "The Wilds" is
located in Pisgah National Forest in
southwestern North Carolina. Religious
groups use the camp for retreats held
throughout the week during the summer
months and over weekends during other
seasons. Camp facilities include dormi-
tories, assembly hall, cafeteria, and
extensive recreational facilities.
The water system consists of two wells
72
that supply untreated water to a storage
tank from which it is pumped to the
distribution system. The major water-
using facilities discharge wastewater to a
package sewage treatment plant. Septic
tanks serve the few buildings inaccessi-
ble to the collection system (Figure 2).
Large number of illnesses closes camp.
An epidemiologist with the North Caro-
lina Department of Human Resources
visited the camp the first weekend in
November, after receiving reports of ill-
ness. It was determined that illnesses had
been occuring in groups visiting the
camp since August. A survey was con-
ducted during the investigator's visit,
with questionnaire results showing that
146 of 186 visitors were affected with
vomiting and diarrhea within 24 hours of
arrival. Stool and acute serum specimens
were collected and arrangements made
for collection of convalescent serum
after a four-week interval. The camp was
closed while the investigation evaluated
food preparation and handling, general
hygienic practices, and water supply and
waste disposal facilities.
EPA participated in the investigation
and evaluated water supply and waste
disposal facilities. A large-volume water
sample (1900 L [500 gal]) was collected
for viral assay and 19-L (5-gal) samples
for coliform and fecal coliform analyses
were collected for each well using the
filtering technique described in connec-
tion with the Arizona investigation.
(Two standard bacteriological samples
collected by the county sanitarian from
the cafeteria kitchen the last two week-
ends in October were positive for coli-
forms.) Collection of 19-L (5-gal) bacteri-
ological samples from each well on mem-
brane filters showed coliform and fecal
coliform contamination of well 1 but no
E.G. LIPPY 61
-------
coliform colonies present on the mem-
brane filter for well 2.
Sources of contamination that could
conceivably affect the wells included the
septic tanks and the effluent line from
the sewage treatment plant. Well 1 was
located about 15 m (50 ft) from the plant
and the effluent line passed within 7.5 m
(25 ft) of the well on its path to a
discharge point in Toxaway Creek (Fig-
ure 2). The effluent line crossed a drain-
age ditch and was found to be damaged
and leaking sewage in September. The
area is underlain by limestone which
can become fractured, making it possible
for the well to bo infiltrated by sewage
effluent. (It was reported that an earth
tremor occurred in the area in late July.)
In addition, a short distance upstream
from the location of the gutter crossing, a
septic tank had been damaged. A truck
had been driven over the site, rupturing
the fiberglass tank. The tile field, which
was located at the base of a steep slope,
was eroded from surface drainage, and
deep gullies had formed that drained into
the gutter. It was therefore possible for
sewage to enter the gutter from the rup-
tured septic tank or the gullied tile field.
This, coupled with a water line crossing
the drainage ditch and passing within
immediate proximity of the well, was
another possibility for contamination.
Sewage flowing down the gutter could
follow the path of the water line in the
excavated and back-filled trench.
Both possibilities were tested by dam-
ming the gutter downstream from the
water line and effluent line crossings and
dosing the pool with fluorescein dye. The
well was sampled unsuccessfully for flu-
orescein for three weeks. While fluores-
cein was apparently not charged into the
well, it is conceivable that solution chan-
nels in the limestone strata became
clogged and were sealed, preventing
entry of the dye. Possibly other factors
intervened. One septic tank that could
have remotely influenced well 2 was also
dye-tested with negative results.
The camp was permitted to reopen
pending improvements which included
installation of chlorination equipment
on both wells, flushing and disinfection
of the well system, and repairs to sewage
disposal facilities. Well 1 was kept out of
service during the opening weekend and
no illness was noted among visitors.
Epidemiological survey data, rein-
forced by laboratory tests of medical
specimens, determined that the agent
responsible for this outbreak was a "Nor-
walk" type virus. Serological studies
conducted by the Center for Disease
Control showed that thirteen of fourteen
symptomatic cases were seropositive for
antibody titers against the Norwalk
agent. Immune electron microscopy
studies of stools from 35 ill volunteers
failed to defect virus.8 Detection of
Norwalk agent in stool specimens col-
62 RESEARCH AND TECHNOLOGY
lected during outbreaks has been very
difficult. Volunteer studies under labora-
tory conditions have shown that about
one half of those exposed to Norwalk
agent excrete the particles in sufficient
concentration to be detected by immune
electron microscopy. Particles are not
detected prior to onset of symptoms and
are found in less than 20 percent of stools
collected 72 hours or longer after onset of
illness.9
Untreated groundwater supplies spell trou-
ble. This outbreak is discussed here
because it typifies many of those
occuring in systems using groundwater
as a source of supply without treatment.
A review of the causes of waterborne
outbreaks shows that for the period from
1971 through 1978, 107 of 224 reported
outbreaks were caused by use of ground-
water that was not chlorinated or not
adequately treated. The outbreaks were
responsible for a total of 17 391 illnesses.
An etiologic agent was identified in few-
er than half of the outbreaks, the remain-
der being characterized as acute gas-
trointestinal illnesses that were presum-
ably caused by bacterial pathogens or
enteric virus for which isolation and
identification techniques were not avail-
able. It is not unusual for laboratories to
culture specimens for only two bacterial
pathogens, Salmonella and ShigeJJa, and
if they are not present, the causative
agent is never determined.
There has been an improvement in the
capability of some laboratories to detect
other bacterial and viral pathogens that
can be cultured. However, nonculturable
viruses, which can be detected in stool
specimens only by electron microscopy
or through the use of stool material from
infected individuals reacted with acute
and convalescent serum to produce spe-
cific rises in antibody titer, have become
increasingly associated with waterborne
outbreaks in groundwater systems. It is
typical of these outbreaks that investiga-
tors detect sources of sewage contamina-
tion in close proximity to wells but
usually cannot demonstrate either
through dye-testing or by sampling for
microbiological indicators that contami-
nation occurred. Viruses identified with
these outbreaks are extremely small in
size (in the range of 30-70 nm), which
probably enables them to penetrate
water-bearing aquifers whereas coliform
organisms are trapped or die. Many fac-
tors interfere with dye-testing in ground-
water situations, and the inability to
demonstrate a positive test is not proof
that the "likely event" did not occur.
Overall summary
A study of three waterborne disease
outbreaks that occurred in 1979 shows
that problems contributing to the occur-
rence of outbreaks can be identified and
related to a breakdown of protective
systems in the multiple barrier concept.
73
Designers,- utility personnel, and regula-
tory agencies share a responsibility for
maintaining the integrity of the protec-
tive systems necessary to ensure the
delivery of a safe and potable water.
Acknowledgments
The author acknowledges the coopera-
tion of the following persons: Isabel
Guerrero, CDC, Atlanta, Ga., James Erb
and John Mead, both of the bureau of
water quality control, Pennsylvania De-
partment of Environmental Resources,
Meadville, Pa.; Patrick Nuzzo, Bradford,
Pa. Karen Starko, bureau of disease con-
trol, Arizona Department of Health Ser-
vices and William Shafer, both of the
bureau of water quality control, Arizona
Department of Health Services, Phoenix,
Ariz.; Robert Anderson, Mojave County
Health Department, Lake Havasu City,
Ariz.; Douglas Collup, Lake Havasu State
Park, Ariz.; Charles Finch, Department
of Human Resources, Raleigh, N.C.; Jack
McGuiness, Transylvania County Health
Department, Brevard, N.C.; and David
Cleary, The Wilds Camp, N.C.
The author also acknowledges fellow
employees S.C. Waltrip, F.W. Schaefer,
T.H. Ericksen, and S.C. Underwood.
References
1. Waterborne Giardiasis—California, Col-
orado, Oregon, Pennsylvania. Morbidity
and Mortality Weekly Rep., 29:11. Center
for Disease Control, PHS, DHEW, Atlan-
ta, Ga. (Mar. 21, 1980).
2. Waterborne Giardiasis Outbreaks—Wash-
ington, New Hampshire. Morbidity and
Mortality Weekly Rep., 26:21 Center for
Disease Control, PHS, DHEW, Atlanta,
Ga (May 27, 1977).
3. KIRNER, J.C.; LITTLER, J.D.; & ANGELO, L.A.
A Waterborne Outbreak of Giardiasis in
Camas, Wash. Jour. AWWA, 70:1:35 (Jan.
1978).
4. LIPPY, E.G. Tracing a Giardiasis Outbreak
at Berlin, New Hampshire. Jour. AWWA,
70:9:512. (Sep. 1978).
5. LOPEZ, CARLOS ET AL. Waterborne Giardia-
sis—A Community-Wide Outbreak of
Disease and a High Rate of Asymptomat-
ic Infection. Am. Jour. Epidemiol. (in
press).
6. DAVIES, R.B. & HIBLER, C. P. Animal Reser-
voirs and Cross Species Transmission of
Giardia. Waterborne Transmission of
Giardiasis Symposium, Natl. Tech. Infor-
mation Service, Springfield, Va., EPA-
600/9-79-001 (June 1979).
7. STARKO, K. ET AL. Diarrhea in Campers (in
preparation).
8. FINCH, C. ET AL. Recurrent Gastroenteritis
at a Camp in North Carolina (in prepara-
tion).
9. BLACKLOW, N.R.; SCHREIBER, D.S.; & TRIER,
J.S. Viral Enteritis-Seminars in Infec-
tious Disease. Stratton Intercontinental
Medical Book Corp. (1978).
Edwin C. Lippy (Active Member, AWWA) is a
sanitary engineer with the acute disease
branch, health effects research laboratory,
US EPA, Cincinnati, Ohio 45268.
JOURNAL AWWA
-------
Reprinted from Journal, AWWA, Vol. 78,
No. 1, by permission. Copyright 1986.
American Water Works Association
Chlorination to Prevent and Control
Waterborne Diseases
Edwin C. Lippy
An analysis of statistics compiled on outbreaks of waterborne disease from 1946 to 1980 and
from 1965 to 1982 showed that disinfection was not provided where it was needed and was not
properly operated where it was provided. Chlorine concentration, contact time, pH,
temperature, and interfering substances all must be evaluated to determine disinfection
requirements. Use of reliable chlorinatioh facilities and proper application of current tech-
nology can control and prevent waterborne diseases.
Two recent publications identified
shortcomings in disinfection contribut-
ing to waterborne disease outbreaks.
Statistics compiled for the period from
1946 to 1980 showed glaring deficiencies
in disinfection practices.1 The primary
problems were that disinfection was not
provided where it was needed and it was
not properly operated where it was
provided, both of which caused or con-
tributed to nearly half of the outbreaks
of waterborne disease in public water
systems. The same shortcomings were
identified when the 63 giardiasis out-
breaks occurring in the period 1965-82
were analyzed.2 Two-thirds, or 42, of
these outbreaks were related to a failure
to provide adequate or reliable treatment,
with chlorination deficiencies identified
as a primary cause.
This article reviews the factors that
influence chlorine's effectiveness in in-
activating bacteria, viruses, and proto-
zoans for the purpose of preventing and
controlling waterborne disease out-
breaks. The factors reviewed are: (1)
chlorine concentration, (2) contact time,
(3) pH, (4) temperature, and (5) inter-
fering substances. Each of these must be
evaluated to determine disinfection
capability under current operating con-
ditions and to determine the adj ustments
necessary for coping with the challenges
of pathogens.
Chlorine concentration and contact time
The product of chlorine concentration
C (in milligrams per millilitre) and
contact time T (in minutes) produces a
CT relationship that is used in deter-
JANUARY 1986
mining the requirements for inactivation
of bacteria, viruses, and protozoans.
Because CT relationships are developed
through laboratory research under care-
fully controlled conditions, applying the
data to a real world situation requires
judgment. The CTvalues listed in Table
1 for viruses were developed by White3
and also can be applied for inactivation
of bacterial agents. The viral CTvalues
are based on inactivation of Coxsackie
A2 viruses, which are more resistant
than poliovirus or any pathogenic vege-
tative bacteria implicated as causative
agents in waterborne disease outbreaks.
Values for protozoan CTwere interpreted
from research conducted by White,3
Jarroll et al,4 and Rice et al.5 A rule of
thumb can be derived from the CT
relationships at pH values of 7-8, which
are common to most water systems.
Systems using groundwater as a source
of supply should apply a minimum CTof
15-30 to provide for the inactivation of
viruses and bacteria, which are generally
the contaminants of concern; i.e., the
size of viral particles permits easier
penetration of strata overlying aquifers
whereas protozoan cysts are trapped
and retained (protozoan cysts are about
1000 times larger than virus particles),
and bacteria are capable of reproducing
in the environment whereas protozoan
cysts are not. Systems using surface
supplies, especially those located in areas
where source water temperatures can be
as low as 41°F (5°C), should apply a
minimum CT of 100-150 for protozoan
inactivation. At temperatures lower than
41°F (5°C), the CT requirements will
increase. Figure 1 illustrates the CT
requirements included in Table 1.
Once the CT value is known, the ac-
tual contact time is determined through
evaluation of the treatment facility, and
the required chlorine concentration is
determined mathematically. Because
several factors must be considered,
determination of the actual contact time
should be done carefully. A common
mistake is using hydraulic displacement
calculations to compute contact time.
For example, a 1-mil gal (3.8-ML) rec-
tangular storage tank, with inflow and
outflow at opposite ends, provides 2.4 h
of contact time when calculations con-
sider the average daily plant output of 10
TABLE 1
CT relationships for 99-percent inactivation of pathogenic agents
PH
6
7
7.0-7.5
7.5-8.0
8
8.0-8.5
8.5-9.0
Viral CT
32-41°F
(O-5°C)
12
20
30
35
50°F
(10°C)
8
15
20
22
Protozoan CT
41°F
<5°C)
80
100
150
59°F
(15°C)
25
35
50
77°F
<25°C)
15
15
15
74
F.nwiN r T IPPV AQ
-------
Disinfection problems
caused or contributed
to more than half
of the outbreaks
ofwaterborne disease.
mgd (38 ML/A), but the contact time
actually provided may be one fourth of
the calculated value.6 With an actual
contact time of 36 min, a chlorine con-
centration based on the 2.4-h contact
time would be insufficient. This differ-
ence in the calculated displacement or
theoretical retention (contact) time and
the actual retention (contact) time stems
from short-circuiting and dead spaces
common to both rectangular and circular
storage tanks. The use of the average
daily plant output can also lead to errors.
Plant output varies with customer de-
mands. Peak daily demands for some
systems can be twice the average daily
plant output. If this is the case, the
contact time is underestimated by an
additional 50 percent. If the chlorinator
is not paced to respond to changes in
plant output, fluctuation in demand must
be taken into consideration when eval-
uating contact time and chlorine dosage
requirements.
The preferred method of determining
contact time is to conduct a tracer test.
One simple and quick method is to
increase chlorine feed and determine
chlorine residual concentrations in the
basin effluent. The initial increase in
concentration signals the flow-through
time for a fraction of the plant output,
and subsequent samples will reflect a
steady rise in chlorine residual until the
incremental dosage has been accounted
for. The mixed-flow and dead space
components of basin hydraulics can also
be estimated through application of the
tracer methods discussed by Hudson.7
Another simple and quick method for
tracer tests is to cease fluoride feed and
read the decrease in fluoride concen-
tration over time. The initial decrease in
fluoride concentration signals the flow-
through time for a fraction of plant
output and represents the contact time
provided for a portion of the basin
volume. The advantage of using chlorine
and fluoride as tracers is that there is no
need to introduce other dyes, salts, or
chemicals into the water system. Use of
other tracers may require special per-
mission because of concern about toxic-
ity. Decreasing the concentration of
fluoride or increasing the concentration
of chlorine for a short time should not
cause objections to conducting the test.
The elapsed time between the increase
or decrease of chemical dosage and the
initial corresponding change in the basin
effluent represents the contact time and
should be used in the CT relationship to
calculate the concentration of chlorine
that will be required to achieve a micro-
biocidal residual. The CT relationships
are based on maintaining the chlorine
o ViralcratSO'F 10°C
• ViralCrat32-41°F 0-5°C
D Protozoan CTal 59° F 15° C
• Protozoan CTal 41° F 5°
20 40
80
CT
Figure 1. CT relationships for 99-percent inactivation of pathogenic agents
50 MANAGEMENT AND OPERATIONS
75
concentration for the entire contact time;
therefore, the dosage must be adjusted
accordingly to compensate for chlorine
demand and dissipation.
Tracer tests are normally not required
where transmission or distribution
mains are depended on for contact time.
The mains are pressurized and, under
full-flow conditions, the hydraulic char-
acteristics approximate plug flow. With
plug flow the theoretical and actual
contact times are equivalent. However,
they can be easily checked by increasing
the chlorine dosage and measuring for
the increase in residual at an appropriate
point downstream. Table 2 is a ready
reference for computing contact time in
transmission and distribution mains.
By dividing the pipe capacity (adjusted
for available length) by the plant output
or flow, the contact time can be determ-
ined. For example, at an output of 1 mgd
(3.8 ML/d) with 1000 ft (300 m) of 24-in.
(600-mm) pipe, contact time = 34 min.
pH and temperature
The effectiveness of chlorine as a
disinfectant is influenced by the pH and
temperature of the water being dosed.
The pH determines the amount of
hypochlorous acid (HOC1) and hypochlo-
rite ion (OC1~) in solution. HOC1 is the
predominant form at lower pHs (6-7.5)
and OC1" is the predominant form at
higher pH values of (8-10). At pH 7 and
32°F (0°C), the dissociation of chlorine
produces 87.04 percent HOC1 and 12.96
percent OC1"; at pH 9, it produces 6.29
percent HOC1 and 93.71 percent OC1~.8
It is important to know the dissociated
forms of chlorine present at various pH
values because HOC1 is the form that
possesses the destructive power as a
microbiocide. According to White, "the
germicidal efficiency of HOC1 is due to
the relative ease with which it can
penetrate cell walls. This penetration is
comparable to that of water, and can be
attributed to both its modest size (low
molecular weight) and its electrical
neutrality (absence of an electrical
charge)." White states that the hypo-
chlorite ion "is a relatively poor disin-
fectant because of its inability to diffuse
through the cell wall of microorganisms
due to the negative electrical charge."
Research has shown that HOC1 is 80
times more effective than OC1" in the
inactivation of Escherichia coli and about
JOURNAL AWWA
-------
HOCl is the form
of chlorine
that possesses the
destructive power
as a microbiocide.
150 times more effective for cysts of
Entamoeba histolytica.3
The key point to remember in the
evaluation of chlorination is that the CT
relationship increases with pH. Contact
time, or the T portion of the CTrelation-
ship, is controlled by structural facilities
such as contact basins, storage tanks, or
transmission mains whose size cannot
be readily adjusted to compensate for a
needed increase in CT. Therefore, the
only immediate alternative is to increase
chlorine concentration. If pH is such
that OC1" is the predominant form, it
may be necessary to lower the pH so that
HOCl is the predominant form. Adjust-
ment of pH should be considered when
the chlorine requirement becomes ex-
cessive and, perhaps, objectionable.
In water, chlorine hydrolyzes rapidly—
occurring in tenths of a second at 65°F
(18°C) and in a few seconds at 32°F (0°C).
Although temperature has little effect
on chlorine's solubility, chlorine's reac-
tion with microorganisms is affected.
CTrelationships are inversely related to
water temperature, and as it decreases,
the CT increases (Table 1). Therefore,
temperature must be considered when
selecting a bactericidal, viricidal, or
cysticidal chlorine dosage.
Interferences
Chemical interference. When chlorine
is introduced into water, it not only
reacts to form microbiocides but also
reacts with other substances to form
organic and inorganic compounds that
have little or no disinfection capability.
The reaction of chlorine with organic
and inorganic substances constitutes
chemical interference with disinfection
through consuming a part of, or all of,
the applied chlorine—leaving little or no
chlorine to react as a disinfectant.
Organic and inorganic forms of nitrogen
are among the compounds that signifi-
cantly interfere with the chlorination of
surface water. Hydrogen sulfide, iron,
and manganese are commonly found in
groundwaters and interfere with disin-
fection by combining with chlorine to
form sulfates, ferric hydroxide, and
manganese dioxide, respectively. The
significance of this interference is ex-
emplified by the requirement of 8.32
mg/L of chlorine to oxidize 1 mg/L of
hydrogen sulfide.3 In fact, the ability of
chlorine to oxidize hydrogen sulfide, iron,
JANUARY 1986
and manganese is used to advantage as a
treatment technique and is employed at
some water plants for removal of these
substances.
Physical interference. This type of
interference occurs when microorgan-
isms become entrapped within the matrix
of particulate matter and are shielded
from the effects of disinfection. Re-
search9-14 indicates that the protective
effect of shielding by inorganic particu-
lates is minimal and that inorganic
particulates are of little consequence as
physical interfering substances. Organic
particulates such as those associated
with sewage effluent solids do, however,
afford protection and permit survival of
microorganisms after long chlorine con-
tact periods (60 min). A recent study by
LeChevallier et al15 on the survival of
coliform organisms in the presence of
turbidity indicated that after 1 h of
contact time there was a 99.5-percent
reduction in the number of organisms at
a concentration of 6 ntu or less and an
approximately 20-percent reduction at a
concentration of 8-13 ntu. The study
showed that turbidity control, to at least
the 1 and 5 ntu concentrations as re-
quired by US Environmental Protection
Agency regulations, is justified. Although
more research is needed to specifically
define the degree of physical interference
resulting from turbidity and particulates,
common sense dictates that where sew-
age discharges affect raw water quality,
turbidity should be reduced by appro-
priate treatment (e.g., mixing, coagula-
tion, flocculation, settling, filtration) for
chlorination to be effective. If chlorina-
tion is the only treatment, it must be
adjusted to compensate for the physical
interference of turbidity.
Example of optimizing chlorination
An example is provided to illustrate
the procedure that can be used in
optimizing chlorination to cope with a
pathogen challenge. Assume that a
community uses an impoundment on a
mountain stream as a source of supply.
There are no sources of human contam-
ination in the watershed. Chlorination
is provided at the impoundment, with
contact time occurring in 5000 ft (1500
m) of 24-in. (600-mm) transmission main.
The average daily demand is 2 mgd (7.6
ML/d), which is increased by 50 percent
during peak periods. The water temper-
ature is 41°F (5°C), pH is 7, and turbidity
is 1 ntu. Research data indicate the CTis
100 to offset a particular pathogen chal-
lenge. The chlorine dosage requirement
is calculated for peak output, and it has
been assumed that the chlorinator feeds
in proportion to flow.
Peak output: 2 mgd X 150% = 3 mgd
Contact time: (2350 gal*/100 ft)
x (5000 ft/3 mgd)
= 56 min
Chlorine demand: test with chlorine comparator
kit yields a demand of 0.5 mg/L
at 56 min
•From Table 2
TABLE 2
Fluid capacity of pipes
Pipe Diameter— d
in.
3
4
6
8
10
12
14
16
18
20
24
30
36
42
48
mm
80
100
150
200
250
300
350
400
450
500
600
750
900
1050
1200
Capacity*
gal/ 100 ft
37
65
147
261
408
588
800
1044
1322
1632
2350
3672
5288
7197
9400
L/30m
140
246
556
988
1544
2226
3028
3952
5004
6178
8896
13899
20017
27244
35583
*4.08d2
76
EDWIN C.LIPPY 51
-------
Chlorination,
properly applied,
could have
prevented
the outbreaks.
Chlorine dosage
requirement: (CT/T) + demand
(100/56) + 0.5
=1.8 + 0.5
= 2.3 mg/L
The calculations account for all factors
affecting chlorination, except for extrap-
olating the CT relationship from labora-
tory research data to applied use. An
increased dosage of 0.5-1.0 mg/L would
not he unreasonable. For this example, a
dosage of 3 mg/L might be chosen to
account for unknown variables in ex-
trapolation. Increasing the dosage to 3
mg/L has the effect of adjusting the CT
relationship by 40 percent (i.e., 1.8 + 0.7
X 56 = 140). The free chlorine residual
that is produced, 2.5 mg/L after 56 min
contact time, should be confirmed by
monitoring. Microbiological monitoring
for the pathogenic agent may be per-
formed to confirm the efficacy of chlo-
rination and account for extrapolation.
If sampling or analytical methods are
not available for pathogen monitoring,
chlorination efficacy could be gauged by
using methods that determine inactiva-
tion of the total bacterial population.
The above example assumes a flow-
proportioning chlorinator that automat-
ically adjusts dosage in response to
changes in plant output. If a constant-
feed chlorinator is used, several adjust-
ments of dosage are required throughout
the day. For example, between the hours
of 10:00 p.m. and 6:00 a.m. customer
demands decrease, and plant output
responds accordingly. If demand de-
creases to 50 percent of the average, the
chlorine residual is going to increase to
objectionable concentrations unless the
dosage is adjusted. With a 50-percent
decrease in average output, or 1 mgd (3.8
ML/d), and the chlorinator adjusted to
maintain the 3-mg/L dosage calculated
for 3 mgd (11 ML/d), the chlorine residual
will increase dramatically during low-
demand hours to 8.5 mg/L. In this
example, the resulting chlorine residual
would likely cause widespread consumer
complaints about tastes and odors.
Equally as important as providing the
correct dosage is assuring that reliable
chlorination is in operation 24 hours a
day. Reliability demands dual cylinder
feed, auxiliary power, standby equip-
ment and spare parts, and maintenance
of records documenting operation. Dual
cylinder feed is necessary to ensure
52 MANAGEMENT AND OPERATIONS
continuous dosage so that if one cylinder
becomes depleted, the fully-charged one
automatically comes on-line. A dual
cylinder hookup also is needed to ensure
continuous feed during cylinder replace-
ment. Changing an empty cylinder for a
full cylinder takes about 30 min, and if a
dual hookup is not provided, unchlo-
rinated water will enter the distribution
network. This means that for a plant
output of 1 mgd (3.8 ML/d), 21 000 gal
(79 493 L) of unchlorinated water would
be distributed. Auxiliary power is neces-
sary to sustain operation during power
outages. A standby chlorinator and spare
parts are needed for uninterrupted opera-
tion during maintenance and repairs.
Records to substantiate reliability in
operation, including pounds of chemical
used, plant output on a daily basis, and
chlorine residual measurements, are also
required. Current technology to ensure
reliability in chlorination utilizes loop-
controlled feed with remote sensing and
automatic adjustment of dosage to a
predetermined residual. The residual is
continuously analyzed and recorded on a
circular or strip chart, and an alarm
system can be installed to warn of low
residuals. The continuous record sub-
stantiates chlorination, or lack of it, and
is a very important factor in verification
of proper disinfection.
The factors that influence the effec-
tiveness of chlorination were evaluated
and the required CT successfully ap-
plied, halting transmission of waterborne
disease. If adequate chlorination facilities
had been in place and current technology
properly applied to cope with the chal-
lenge of'pathogenic agents, the outbreaks
could have been prevented.
References
1. LIPPY, E.C. & WALTRIP, S.C. Waterborne
Disease Outbreaks, 1946-1980: A Thirty-
Five Year Perspective. Jour. AWWA,
76:2:60 (Feb. 1984).
2. LIPPY, E.C. & LOGSDON, G.S. Where Does
Waterborne Giardiasis Occur, and Why?
Proc. 1984 Specialty Conf. on Envir.
Engrg. ASCE, New York (1984).
3. WHITE, G.C. Handbook of Chlorination,
Van Nostrand Reinhold Co., New York
(1972).
4. JARROLL, E.L.; BINGHAM, A.K.; & MEYER,
E.A. Effect of Chlorine on Giardia lamb-
lia Cyst Viability. Appl. & Environ.
Microbiol., 41:483 (1981).
5. RICE, E.W.; HOFF, J.C.; & SCHAEFER, F.W.
Inactivation of Giardia Cysts by Chlo-
77
rine. Appl. & Environ. Microbiol, 41:250
(1982).
6. LIPPY, E.C. Tracing a Giardiasis Outbreak
at Berlin, N.H. Jour. AWWA, 70:9:512
(Sept. 1978).
7. HUDSON, H.E. JR. Water Clarification
Processes: Practical Design and Evalua-
tion. Van Nostrand Reinhold Co., New
York (1981).
8. WHITE, G.C. Disinfection of Wastewater
and Water for Reuse. Van Nostrand
Reinhold Co., New York (1978).
9. BOARDMAN, G.D. Protection of Waterborne
Viruses by Virtue of Their Affiliation
With Paniculate Matter. Doctoral disser-
tation, Univ. of Maine (1976).
10. HOFF.J.C. The Relationship of Turbidity
to Disinfection of Potable Water. Evalua-
tion of the Microbiology Standards for
Drinking Water(C.W. Hendricks, editor).
USEPA-570/9-78-006 (1978).
11. BOYCE, D.S.; SPROUL, O.J.; & BUCK, C.E.
The Effect of Bentonite Clay on Ozone
Disinfection of Bacteria and Viruses in
Water. Water Res., 759 (1981).
12. WALSH, D.S.; BUCK, C.E.; & SPROUL, OJ.
Ozone Inactivation of Floe Associated
Viruses and Bacteria. Jour. Envir. Engrg.
Div.—ASCE, 106:761 (1980).
13. STAGG, C.H.; WALLIS, C.; & WARD, C.J.
Inactivation of Clay-Associated Bacterio-
phage MS-2 by Chlorine. Appl. & Environ.
Microbiol., 33:385 (1977).
14. SCARPING, P.W. Effect of Particulates on.
Disinfection of Enteroviruses in Water
by Chlorine Dioxide. USEPA 600/2-79-
054 (1979).
15. LECHEVALLIER, M.W.; EVANS, T.M.; &
SEIDLER, R.J. Effect of Turbidity on
Chlorination Efficiency and Bacterial
Persistence in Drinking Water. Appl. &
Environ. Microbiol., 42:159(1981).
JOURNAL AWWA
-------
IV. Sampling and Analytical Methods
79
-------
Reprinted by the
U S DEPARTMENT OF HEALTH AND HUMAN SERVICES
PUBLIC HEALTH SERVICE
CENTERS FOR DISEASE CONTROL
From
VIRUSES AND THE GUT
(Proceedings of the Ninth BSG -SK&F International Workshop 1988)
October 2-4,1988,pp. 87-90
SMALL ROUND STRUCTURED VIRUSES:
THE NORWALK FAMILY OF AGENTS
Roger I. Glass, Stephan S. Monroe, Sarah Stine, Paul Madore,
David Lewis, David Cubitt, Gary Grohmann and Charles Ashley
Viral Gastroenteritis Unit, Centers for Disease Control, Atlanta, Georgia, USA; Division of Infectious
Diseases, University of Rochester, Rochester, Neic York, USA; Public Health Laboratory Service, Taunton,
United Kingdom; PHLS, Central Middlesex Hospital, London, United Kingdom; Westmead Hospital, Sydney,
Australia; PHLS, Bristol, United Kingdom
SRSVs of the Norwalk family have remained
among the most difficult viral agents to detect,
characterise and understand. Members of this
family share an asymmetrical structured
morphology when visualised directly in the
electron microscope, but they are antigenically
distinct when examined using immune
electromicroscopy (IEM). None of these agents
has been successfully cultivated, few have been
adequately purified so that either polyclonal
antisera or monoclonal antibodies can be
prepared, few are visible with ease by EM, and'
nearly all assays developed to detect the
antigen of these agents require human sera and
stool as basic reagents. Since exposure to these
agents is common, and since many of these
agents may be antigenically related,1 antibody
responses in humans may not be restricted to
the infecting particle. Consequently, relatively
few of the convalescent human antisera
obtained, even from volunteers, can be used as
specific reference reagents.
The literature on the Norwalk family of
agents is filled with a colourful variety of names
of particles found in a single epidemic. These
agents, of which nearly a dozen have been
identified, are often unsuitable as references
because samples from the key outbreaks have
been exhausted in characterising the new
particle against other reference agents. Only
where volunteer studies have been conducted
to amplify the volume of these reagents has the
particle been further characterised and assays
set up for its detection.2"1
87
80
-------
In recent years, there have been more
reviews written about these novel agents of
viral gastroenteritis than substantive advances
in our understanding of these agents.^"
Consequently, rather than review the history of
each of these particles again, I present an
approach used at CDC to assess where we
would like to go in the future in combining
epidemiological and laboratory studies. In the
past, epidemiological investigations have been
key to identifying novel diarrhoea agents and
establishing their role as pathogens in man.
Improvements in the investigation of future
outbreaks—paying special attention to more
voluminous specimen collection, more basic
virology and extended laboratory collabor-
ations— may help improve our understanding
of the epidemiology of disease caused by these
agents and provide the basic diagnostic
reagents required to achieve major break-
throughs in understanding these viruses.
Epidemiological investigations as a
key to discovery of new agents
In 1986, we reviewed the activities of the viral
gastroenteritis group at CDC to asess the role of
epidemic investigations in discovering new
viral agents and in establishing their causal role
in gastroenteritis. Two of the SRSVs recognised
to be causes of diarrhoea—the Norwalk agent12
and the Snow Mountain agent13—were each
identified during the investigation of outbreaks
of diarrhoea of undetermined aetiology by CDC
epidemiologists. Human specimens from each
of these outbreaks were subsequently used to
establish the identity and causative role of the
virus using techniques of immune electron-
microscopy and to prepare inocula for both
primate and human volunteer challenges. Had
either of these agents been seen in a single
individual, establishing their role as the cause
of diarrhoea would have been impossible.
However, the coincidence of finding particles
in many of the outbreak victims but not in
controls, corrfbined with the determination of
seroconversion to the particle by IEM among
victims but not controls, helped link the
agent in the chain of causality. Specimens from
volunteer studies with the Norwalk agent were
used by Greenberg and Kapikian to develop an
RIA to detect antigen and a blocking assay to
measure antibody litre.1 Use of this assay with
specimens from the CDC collection of unsolved
88
outbreaks led to the recognition that the
Norwalk agent was a prime cause of about 25%
of the outbreaks of non-bacterial gastroenteritis
in the USA. This RIA assay was adapted by
Gary for use with biotin-avidin, leading to a
sensitive and non-radioactive method to detect
antigen and seroconversion to the Norwalk
agent.3'14 These assays have remained the key
diagnostic tests in our laboratory. At the same
time, the infrequency of detecting SRSVs by
direct EM led over the years to the near
abandonment of the use of electronmicroscopic
methods in our laboratory. Further
seroepidemiological studies with the Norwalk
agent established that Bangladeshi children
and American adults had a high prevalence of
antibody, indicating that the Norwalk virus
could be a cause of sporadic diarrhoea as well.l3
Investigations by other groups have led to
the identification of a variety of other particles,
including the Hawaii agent, Montgomery
County agent and Marin County agent in the
USA; the Taunton, Moorcroft and Barnett
agents in the UK; the Paramatta agent in
Australia; the Sapporo and Otofuke agents in
Japan as well as a group of other similar
particles differentiated by IEM in Japan.16 The
wide antigenic diversity of this group, the
inability to compare agents and to refine
nomenclature, and the difficulty in obtaining
quantities of the virus from outbreak specimens
or passage in animals or cell culture have
slowed progress in the field.
Review of outbreak investigations
We have reviewed results from the last 100
outbreaks investigated by CDC staff where
stools and paired sera have been collected and
assayed using the biotin-avidin test for the
Norwalk agent (Fig. 1). Our intent was to
confirm previous observations on the
prevalence of Norwalk-associated outbreaks in
this collection while looking at outbreaks that
were still without an aetiological diagnosis to
see if the causative agent could be found. An
outbreak was defined as being associated with
the Norwalk agent if, from six or more paired
sera collected from victims of the outbreak,
50% or more demonstrated seroconversion
defined as a four-fold rise in litre in the biotin-
avidin assay. We found thai the 20-30%
positive rate for Norwalk-associated outbreaks
observed in earlier sludies17'18 has been
81
-------
maintained recently. Frequency distribution
was not made up of two clear and distinct
peaks of Norwalk-positive and Norwalk-
negative outbreaks. Rather, rates of sero-
conversion represented a continuum with
definite positive and negative outbreaks as well
as a clear middle ground distinguished by
outbreaks having a low percentage of
seroconversions (10-49%) in which
convalescent litres rose only to intermediate
values (e.g. 800 or 1600). The intermediate
group might represent infection with a virus
that antigenically cross-reacted with the
Norwalk agent, that produced an anamnestic
response to a related antigen, or was ill-defined
or non-specific due to an insensitive assay
svstem.
10 20 30 40 50 60 70 80 90100
Seroconversions to Norwalk (%)
Figure 1: Serological analysis of outbreaks of
Norwalk virus infection (n = 100), 1985-1988.
Selected outbreaks in the intermediate group
(Group II) have been examined carefully and in
collaboration with other investigators in an
effort to identify other viral agents. Panels of
specimens from 10-20 outbreaks have been
shared with other investigators including Dolin
and Madore (Rochester) to look for Snow
Mountain agent, Blacklow and Herrmann
(Worcester) to assay for astrovirus, Cubitt
(London) to look for SRSVs using an antigen
detection system still being developed, and
Lewis (Taunton) to look by IEM for three
specific Norwalk-like particles such as the
Taunton agent. Some of our initial results are
presented below.
Taunton agent
Paired sera from 10 outbreaks from the inter-
mediate group were tested using an immune
capture assay to look for a serological rise to the
Taunton agent.14 Paired sera from one outbreak
in a nursing home in Florida proved positive for
the Taunton particle, so 13 additional paired
sera from this outbreak were examined. Of
these, 11 showed a significant and diagnostic
rise to the Taunton agent, identifying this to be
the first such outbreak identified in the USA.
These paired sera represent a reference
resource to look further for the Taunton particle
in future outbreaks.
Snow Mountain agent
In January 1988, we investigated an outbreak of
diarrhoea aboard a British cruise ship that had
come into US territorial waters. A small particle
had been seen in an outbreak on a previous
cruise. More than 20% of the passengers
developed diarrhoea in the two weeks before
the investigation. Paired sera and stools were
negative for the Norwalk agent by both
antibody and antigen detection assays. The
sera did respond to the Snow Mountain agent
with four of 19 passengers showing
seroconversion and 11 of 19 demonstrating
high acute titres. One faecal specimen had
a visible particle which was negative in an assay
for Norwalk or Snow Mountain antigen. This
outbreak appears to be due to a small particle
antigenically related to the Snow Mountain
agent.
Calicivirus
In February 1988, an outbreak of gastroenteritis
occurred among children in an day care centre
in Sydney, Australia. Caliciviruses were seen in
small quantity from several stool specimens
taken from the children in the outbreak. These
stool specimens and paired sera from patients
were negative for the Norwalk antigen by
biotin-avidin assays. They were examined in an
enzyme immunoassay for Calicivirus2" and
found to be positive, making this the first
outbreak involving Calicivirus identified in
Australia.
Detection of SRSV antigen
Faecal specimens from 23 outbreaks in the CDC
collection were tested for SRSV antigen using a
biotin-avidin assay developed by Cubitt. This
89
82
-------
assay recognises a variety of SRSVs, including
the Norwalk agent, although the .exact
specificities of the capture and detection
antibodies are unknown. The assay was able to
determine between outbreaks where many
specimens were positive and other outbreaks
where all specimens were negative, a clustering
which could not occur by chance alone. While
the assay remains to be further characterised,
incorporation of biotinylated human
convalescent sera from other non-Norwalk
outbreaks into assays similar to the biotin-
avidin test for Norwalk antigen may provide a
novel way to detect other SRSVs.
The new challenge in epidemic
investigations
Our experience suggests that the investigation
of new outbreaks of undiagnosed diarrhoea
may be particularly valuable for identifying
new agents and collecting reference reagents
when the outbreaks are recurrent, ongoing, or
located in enclosed environments such as
aboard cruise ships, in nursing homes, or in
institutional settings. Investigations in these
settings permit collection of large volume stool.s
repeatedly from patients who are acutely ill and
where both acute and convalescent sera can be
drawn in large volume. Convalescent sera
which have particularly high titre have
encouraged us to go back to patients and collect
a one unit bleed to use for future reference
work. On the basis of our experience with both
the Snow Mountain agent and Norwalk agent
we believe that outbreak investigations can
provide adequate enriched specimens suitable
for virus purification, preparation of inocula
for challenge, experiments with cultivation and
attempts at blind cloning. A major advance in
understanding the virology of these organisms
will be essential to advance our understanding
of the true importance of these agents. In the
interim, enzyme immunoassays appear to
provide a method to screen outbreak specimens
for the Norwalk agent, caliciviruses, Snow
Mountain agent and other unspecified SRSV
antigens.
References
1 Cubitt VVD, Blacklow XR, Herrmann JE, \owak \'A.
\akata S, Chiba S (1987) Antigenic relationships
between human caliciviruses and Xorwalk virus. J Infect
Dis 156: 806-814.
2. Dolin R, Levy AG, Wyatt RG, Thornhill TS, Gardner JD
(1975) Viral gastroenteritis induced by the Hawaii agent:
jejunal histopathology and seroresponse. Am J Med 59:
761-769.
3. Greenberg HB, Wyatt RC, Valdesuso J ct al. (1978) Solid-
phase microtiter radioimmunoassay for detection of the
Xorwalk strain of acute nonbactenal, epidemic gastro-
enteritis virus and its antibodies. J Med Virol 2: 97-108.
4. Dolin R, Roessner KD, Treanor JT c'l al. (1986) Radio-
immunoassay for detection of the Snow Mountain agent.
J Med Virol 19: 11-18.
5. Blacklow \R, Gukor G (1981) Viral gastroenteritis. N
Engl J Med 304: 397-406.
6. Caul EO. Small round human fecal viruses. In: Pattison
JR (ed) Parvoviruses and Human Disease, pp.140-153.
Boca Raton: CRC Press.
7. Ci'ba Foundation. Novel diarrhoea viruses. Chichester:
John Wiley, 1987. (Symposium no. 128).
8. Cukor G,' Blacklow NR (1984) Human viral gastro-
enteritis. Microbiol Rev 48: 157-179.
9. Dolin R, Treanor JJ, Madore HP (1987) Novel agents of
viral enteritis in humans. J Infect Dis 155: 365-376.
10. Kapikian AZ, Chanock RM (1985) Norwalk group of
viruses. In: Fields BN (ed) Virology, pp.1495-1517. New-
York: Raven Press.
11. Madeley CR (1987) Viruses associated with acute
diarrhoeal disease. In: Zuckerman AJ, Banatvala JE,
Pattison JR (ed) Principles and Practice ol Clinical
Virology, pp. 159-196. New York: John Wiley.
12. Adler I, Zickl R (1969) Winter vomiting disease. J Infect
Dis 119: 668-673.
13. Morens DM, Zweighaft RM, Vernon TM el al. (1979) A
waterborne outbreak of gastroenteritis with secondary
person-to-person spread: association with a viral agent.
Lancet I: 964-966.
14. Gary GW, Kaplan JE, Stine SE, Anderson LJ (1985)
Detection of Norwalk virus antibodies and antigen with a
biotin-avidin immunoassay. j Clin Microbiol 22: 274-278.
15. Black RE, Greenberg HB, Kapikian AZ f( al. (1982)
Acquisition of serum antibody to Norwalk virus and
rotavirus and relation to diarrhea in a longitudinal study
of young children in rural Bangladesh. J Infect Dis 145:
483-489.
16. Ando T, Sekine S, Hayaski Y ct al. (1987) Role of small
round viruses in foodborne gastroenteritis occurring in
winter: Outbreaks in- metropolitan Tokyo and
epidemiologic research. (JaPl- Jap J Food Microbiol 4:
103-134.
17. Greenberg HB, Valdesuso J, Yolken RH cl al. (1979) Role
of Norwalk virus in outbreaks of nonbacterial gastro-
enteritis. J Infect Dis 139: 564-568.
18. Kaplan JE, Gary GW, Baron RC c'l al. (1982)
Epidemiology ol Norwalk gastroenteritis and the role of
Norwalk virus in outbreaks of acute nonbacterial
gastroenteritis. Ann Intern Med %: 756-761.
19. Lewis DC, Lightfoot NF, Pether JVS (1988) Solid phase
immune electron microscopy with human immuno-
globulm M for serotyping of Norwalk-like viruses. J Clin
Microbii.l 26' 938-942.
20. Nakata S, Estes MK, Chiba S (1988) Detection of human
calicivirus antigen and antibody by enyzme-linked
immunosorbenl assays. ) Clin Microbiol 26: 2001-2005.
90
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JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1989, p. 671-676
0095-1137/89/040671-06$02.00/0
Copyright © 1989, American Society for Microbiology
Reproduced, with permission, from the
Journal of Clinical Microbiology
Vol. 27, No.
DNA Hybridization Probe for Clinical Diagnosis of
Entamoeba histolytica
JOHN SAMUELSON,1'2 RODOLFO ACUNA-SOTO,1 SHARON REED,3 FRANCISCO BIAGI,4
AND DYANN WIRTH1*
Department of Tropical Public Health, Harvard School of Public Health,1 and Department of Pathology, Brigham and
Women's Hospital,2 Boston, Massachusetts 02115; Division of Infectious Diseases, University of California, San Diego,
Medical Center, San Diego, California 920933; and Laboratorio de Parasitologia, -Colonia Napoles, Mexico City, Mexico4'
Received 17 August 1988/Accepted 15 December 1988
As an alternative to microscopic identification of Entamoeba histolytica parasites isolated from stool, a
sensitive and species-specific DNA hybridization probe was made for rapid diagnosis of E. histolytica parasites
in clinical samples directly applied to nylon membranes. The DNA hybridization probe was made by screening
a genomic library of a virulent HM-1:IMSS strain of E. histolytica to detect recombinant plasmids containing
highly repeated parasite DNA sequences. Four plasmid clones that reacted across Entamoeba species coded for
highly repeated rRNA genes of E. histolytica. Four other plasmid clones were/?, histolytica specific in that they
bound to four axenized and nine xenic strains of E. histolytica but did not recognize closely related E.
histolytica-\ike Laredo, Entamoeba moshkovskii, or Entamoeba invadens parasites. The diagnostic clones
detected as few as eight cultured amoebae and did not distinguish between pathogenic and nonpathogenic
zymodemes of E. histolytica. The diagnostic clones were sequenced and contained 145-base-pair sequences
which appear to be tandemly repeated in the genome. No stable transcript which is homologous to the
diagnostic DNA was detected. In a study of stool samples from Mexico City shown by microscopy to contain
E. histolytica, Entamoeba coli, Giardia lamblia, Endolimax nana, Trichuris trichiuria, and Chilomastix mesntti
parasites, the DNA hybridization probe demonstrated a sensitivity of 1.0 and a specificity of 0.93. We conclude
that the DNA hybridization probe can be used for rapid and accurate diagnosis of E. histolytica parasites.
Entamoeba histolytica is an enteric protozoan parasite
which causes amoebic dysentery in humans (12, 26). The
most common infection with E. histolytica is asymptomatic,
in which the amoeboid forms of the parasite, called tropho-
zoites, reside in the lumen of the colon and transform to
cysts, which are passed in the feces. Such asymptomatic
infections with E. histolytica are frequent among homosex-
ual men in Western countries (1). However, E. histolytica is
a major cause of morbidity and mortality in developing
countries such as Mexico and India, because the trophozo-
ites may invade the colonic mucosa and cause dysentery or
liver abscesses or both.
Presently, infections with E. histolytica are diagnosed by
light microscopy identification of trophozoites or of cysts
separated from stool by dilution, filtration, and flotation
during centrifugation (5). These methods are time consuming
and require extensive experience to assure accuracy of
morphological identification. Past or present invasive amoe-
biasis may be suggested by the identification of antiamoebic
antibodies in patient serum (24).
In this paper, recombinant DNA methodologies previ-
ously used to create sensitive and specific diagnostic probes
for Plasmodium falciparum and Onchocerca volvulus para-
sites (3, 21) are applied to E. histolytica. We show that the
DNA hybridization probe binds to highly repeated and
species-specific DNA sequences in E. histolytica and that
the probe can be used to identify parasites directly in patient
stool samples.
* Corresponding author.
MATERIALS AND METHODS
Entamoeba strains. Trophozoites of E. histolytica strains
(Table 1), E. histolytica-like Laredo, and Entamoeba mosh-
kovskii were obtained from L. Diamond, National Institutes
of Health, Bethesda, Md., and grown axenically in TYI-S-33
medium (8). Entamoeba invadens trophozoites were ob-
tained from the American Type Culture Collection, Gaith-
ersburg, Md., and cultured axenically. Clinical isolates of E.
histolytica (Table 1) obtained from stools or liver abscess
fluids submitted to the Microbiology Laboratory of the
University of California, San Diego, Medical Center were
cultured in Robinson medium (18) and subsequently trans-
ferred to TYSGM medium containing Escherichia coli 0111
(7). For zymodeme identification, parasites were lysed and
electrophoresed on thin-layer starch gels, and the relative
mobilities of glucose-phosphate isomerase, NADP+ oxido-
reductase, phosphoglucomutase, and hexokinase were de-
termined (20). Other zymodeme-characterized strains (Table
1) were from P. Sargeaunt, London School of Hygiene and
Tropical Medicine, London, England.
Preparation of E. histolytica nucleic acids. For DNA, tro-
phozoites of E. histolytica growing in axenic cultures were
washed in phosphate-buffered saline, lysed in 10 volumes of
lysis buffer (1% N-lauroyl sarcosyl [sarcosyl], 8 M urea, 0.16
M sodium phosphate buffer [pH 6.8]), and extracted with
phenol-chloroform. DNA in the lysis solution was bound to
a hydroxylapatite column, which was washed in 8 M urea
and 0.16 M phosphate and then in 0.19 M phosphate to
remove carbohydrates and RNA, respectively. DNA was
eluted with 0.48 M phosphate, dialyzed exhaustively in 10
mM Tris and 1 mM EDTA, pH 8, and concentrated by
ethanol precipitation. RNA was prepared by lysing tropho-
zoites in 5.7 M guanidinium isothiocyanate, 1% sarcosyl,
and 5% (3-mercaptoethanol and centrifuging them through a
671
84
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672 SAMUELSON ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Strains of Entamoeba species and hybridization results
Species
Strain
Culture
Zymodeme"
Source
Hybridization with:
pEH2
PEH6
E. histolytica
E. histolytica
E. histolytica
E. histolytica
E. histolytica
E. histolytica
E. histolytica
E. histolytica
E. histolytica
E. histolytica
E. histolytica
E. histolytica
E. histolytica
E. moshkovskii
E. invadens
HM-1:IHSS
CDC:0784:4
HK-9
200-NIH
53
1519
1453
1704
1721
100
98
1749
Laredo
Axenic
Axenic
Axenic
Axenic
Xenic
Xenic
Xenic
Xenic
Xenic
Xenic
Xenic
Xenic
Axenic
Axenic
Axenic
II ((P)
II (P)
II (P)
II (P)
II (P)
XIV (P)
XIV (P)
XIX (P)
XIX (P)
I(NP)
III (NP)
II (NP)
Diamond + +
Diamond + +
Diamond + +
Diamond + +
Reed + +
Sargeaunt + +
Sargeaunt + +
Sargeaunt + +
Sargeaunt + +
Reed + +
Reed + +
Sargeaunt + +
Diamond - +
Diamond - +
ATCC - +
" Zymodemes as defined by Sargeaunt et al. (20). P, Pathogenic; NP, nonpathogenic.
* Hybridization with pEH2, an E. histolytica-specific probe, or pEH6, an rRNA gene probe which hybridizes across Entamoeba species.
cesium chloride cushion (14). RNA was radiolabeled for
Southern hybridization by a kinase reaction (14).
Screening of an E. histolytica genomic DNA library for
highly repeated DNA sequences. Genomic DNA of the HM-
1:IMSS strain of E. histolytica was partially cut with the
restriction enzyme Sau3A to produce fragments ranging in
mplecular size from 300 to 5,000 base pairs (bp) on 1%
agarose gels. The genomic DNA fragments were ligated into
the plasmid pUC18, which was cut with BamHl and treated
with calf intestinal phosphatase, and were transformed into
JM109 cells. Some 400 colonies were lifted onto nitrocellu-
lose and hybridized with nick-translated (14) genomic DNA
of E. histolytica.
Characterization of recombinant DNA clones. Twelve
recombinant plasmid clones (pEHl through pEH12) were
selected, purified on cesium chloride gradients (14), radiola-
beled, and hybridized to (i) dot blots of Entamoeba tropho-
zoites (as prepared below), (ii) Southern blots of E. histolyt-
ica DNA which was restriction cut, separated on 1 or 2%
agarose gels (1 p,g per lane), and transferred to nitrocellulose
or Genescreen Plus nylon membranes (Dupont, NEN Re-
search Products, Boston, Mass.), or (iii) Northern (RNA)
blots of E. histolytica RNA which was separated on dena-
turing gels containing 1% agarose and 6% formaldehyde or
1% agarose and 1.2% methyl mercury (14) and transferred to
nitrocellulose. For nitrocellulose, hybridizations were per-
formed at 42°C overnight in 50% formamide, 10 x Denhardt
solution, 5x SSC (Ix SSC is 0.15 M Nad plus 0.015 M
sodium citrate), and 500 ^g of herring sperm DNA per ml
(22), followed by three 30-min washes in 0.5% sodium
dodecyl sulfate (SDS) and 0.1% SSC. For Genescreen Plus,
hybridizations were performed at 42°C overnight in 50%
formamide, 1 M NaCl, 1% SDS, 10% dextran, and herring
sperm DNA, followed by two 30-min washes in 2x SSC at
22°C, 2x SSC and 1% SDS at 65°C, and 0.1 x SSC at 22°C.
The size of the insert DNA in recombinant DNA clones was
determined by cutting with restriction enzymes and by
agarose gel electrophoresis.
DNA sequence analysis. pEH5 and pEHll, two E. histolyt-
j'ca-specific recombinant DNA clones containing 145-bp in-
serts in the opposite orientation within the polylinker, and
pEH3, pEH4, pEH6, and pEHIO, four recombinant clones
coding for rRNA gene sequences, were subcloned into
M13mpl8 and M13mpl9. Single-stranded DNA was pre-
pared from recombinant phage, and the DNA sequence was
determined with the dideoxy chain termination method of
Sanger et al. (19). Both pEH5 and pEHll were sequenced
completely in both directions, while partial sequences of the
rRNA clones were compared with homologous sequences
for the small-subunit rRNA sequence of Dictyostelium dis-
coideum (15) to locate the clones on the rRNA gene. To
determine whether there were any significant DNA sequence
homologies between the 145-bp E. histolytica-specific diag-
nostic sequence and other known DNA sequences, the
entire GenBank was searched by using a Lipman-Pearson
FASTN algorithm with a k-tuple of 2.
Dot blots. E. histolytica DNA was diluted in 10 mM Tris
and 1 mM EDTA, pH 8, denatured in 0.25 N NaOH, and
spotted onto nylon membranes by using a 96-spot minifold
apparatus (Schleicher & Schuell, Inc., Keene, N.H.). For
determination of copy number, inserts of pEH5 and pEH6
were electroeluted from agarose gels, diluted, and spotted
onto nylon membranes and hybridized in parallel with tro-
phozoites. Trophozoites of axenized amoebae were washed
in phosphate-buffered saline, counted with a hemacytome-
ter, diluted, lysed in 10 mM Tris, 100 mM EDTA, 0.1%
Triton X-100, and 100 |o,g of proteinase K per ml for 60 min
at 50°C (3), denatured with NaOH, and spotted. Xenic
cultures of trophozoites were separated from most bacteria
by means of a Percoll gradient (17), extensively washed in
phosphate-buffered saline, frozen in 10 mM Tris and 100 mM
EDTA, and shipped from San Diego to Boston. Frozen xenic
cultures were thawed, diluted, and lysed before application
to nylon membranes.
Determination of the best conditions for detecting E. his-
tolytica parasites in stool. Optimal conditions for preparing
parasite DNA from stool samples were determined by trial
and error experiments in which cultured E. histolytica
trophozoites were mixed with uninfected stools, dot blotted,
and hybridized with the E. histolytica-speciftc. clone pEH12.
Samples containing 1,000 to 5,000 E. histolytica trophozoites
and 200 (jil of stool were diluted into 800 |o,l of 10 mM Tris
buffer and then incubated for 1 h at 37°C with either 0 to 500
mM EDTA, 0 to 1% SDS, or 0.5 N NaOH. Subsequently,
stool bacteria and debris were removed by centrifugation for
1 min in a microcentrifuge. Then, 100 ^,1 of each supernatant
was spotted in duplicate onto nylon membranes, denatured
with 0.5 N NaOH if this had not been done already, and
85
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VOL. 27,1989
DNA HYBRIDIZATION PROBE FOR E. HISTOLYTICA 673
hybridized with radiolabeled pEH12. Alternatively, the
1,400-bp insert of pEH12 was cut out of the plasmid,
separated on an agarose gel, and radiolabeled to reduce
background hybridization.
Because it is not possible to make E. histolytica cysts in
vitro, E. invadens cysts prepared by removing glucose from
the medium and incubating for 4 days (25) were used to
determine the best way to release DNA from cysts. Contam-
inating E. invade ns trophozoites were removed from the cyst
preparations by lysis in 1% Nonidet P-40, which did not
disrupt the cysts. E. invadens cysts (1,000 to 5,000/ml) were
washed three times by centrifugation in phosphate-buffered
saline and then incubated for 1 h at 37°C either in 1% SDS,
5.7 M guanidinium isothiocyanate, and 0.5 N NaOH in 0.14
M NaCl or in 100 ng of proteinase K per ml. Alternatively,
cysts were frozen in dry ice and ethanol and thawed in a 37°C
water bath three times, boiled for 5 min in 10% SDS, or
sonicated for 1 min with a probe sonicator. Cyst walls were
pelleted with a microcentrifuge, and samples of the super-
natants were spotted onto nylon filters and hybridized with
radiolabeled genomic E. invadens DNA, because the E.
histolytica-specific probes did not bind to them.
Preparation of patient stool samples. Stool samples (123)
came from patients referred by their physicians to the
Laboratorio de Parasitologia in Mexico City for identifica-
tion of intestinal parasites. Cysts and eggs were concen-
trated by flotation from 10 to 50 g of stool (5), and parasites
were identified by morphology by microscopic examination.
Parallel samples contained 1 g of untreated stool directly
frozen or cysts concentrated by flotation. The concentrated
cysts were divided into two samples: one frozen directly and
the other fixed with 10% Formalin before freezing.
To each patient sample, 0.5 ml of 10 mM Tris and 500 mM
EDTA, pH 8, were added upon thawing to prevent degrada-
tion of DNA, and samples were frozen in ethanol-dry ice and
thawed in water at 37°C for three cycles to rupture cysts.
One hundred microliters of each sample was directly applied
to the minifold after a spin (14,000 rpm, 1 min) to remove
unlysed bacteria and stool debris, and the DNA on the
membranes was subsequently denatured with 0.5 N NaOH
and 1.5 M NaCl. To prevent background hybridization of
vector sequences to stool, the 1,400-bp insert of pEH12, a
recombinant clone specific for E. histolytica DNA, was cut
out with EcoRI and Hindlll, electrophoresed in low-melting-
point agarose, and radiolabeled by random-oligomer priming
(10). After all the DNA hybridizations of the stool samples
were completed and positive and negative samples were
identified, the microscopic diagnoses were sent from Mexico
City. The sensitivity of the hybridization probe was calcu-
lated by dividing the number of samples identified as positive
with both microscopy and DNA hybridization by the number
positive with microscopy alone. The specificity of the probe
was calculated by dividing the number of samples identified
as negative with both microscopy and the DNA probe by the
number negative with microscopy alone.
RESULTS
Specificity of the DNA hybridization probe for E. histolytica.
When a genomic library of E. histolytica was screened for
highly repeated DNA sequences, two classes of recombinant
plasmid clones were obtained. In the first class were four
clones (pEH2, pEH5, pEHll, and pEH12) which appeared
specific for E. histolytica DNA sequences. These clones
bound to all the axenized strains of E. histolytica tested but
did not bind to closely related E. histolytica-like Lnredo, E.
E.H.
E.m. EJ.
50
FIG. 1. Specificity of binding of recombinant DNA clones to dot
blotted Entamoeba species. (A) Nick-translated pEH2, one of four
diagnostic hybridization probes, bound to trophozoites of HM-
1:IHSS (lane H), 200-NIH (lane 2), and CDC:0784:4 (lane C) strains
of E. histolytica (E.h) but did not bind to trophozoites of E.
histolytica-\ike Laredo (lane L), E. moshkovskii (E.m.), or E.
invadens (E.i.). (B) In contrast, pEH6, a cloned portion of the
small-subunit rRNA gene of E. histolytica, bound to all Entamoeba
species tested.
moshkovskii, or E. invadens parasites (Fig. 1A and Table 1).
The clones did not bind to human, Giardia, or bacterial DNA
(data not shown). These E. /iwfo/vf/co-specific clones recog-
nized all E. histolytica trophozoites tested, including three
pathogenic (II, XIV, and XIX) and two nonpathogenic (I and
III) zymodemes of E. histolytica growing in xenic cultures
(Fig. 2 and Table 1).
In contrast, a second class of recombinant plasmid clones
(pEH3, pEH4, pEH6, and pEHIO) recognized repeated
DNA sequences which were conserved across Entamoeba
species (Fig. IB and Table 1). These nonspecific clones
appeared to code for portions of the rRNA gene of E.
It of parasites
800 400 200 100 50 25 12
«•••
• •• •
HM-1
1519
100
E. Inv
FIG. 2. Representative hybridization of the diagnostic recombi-
nant DNA clone pEH2 to zymodeme-characterized Entamoeba
species. Radiolabeled pEH2 insert bound to axenically cultured
HM-1:IHSS (HM-1) and xenic cultures of isolates 1519, 100, and
1721 (Table 1) with similar affinities, suggesting about the same
target copy number in each. As a control. pEH2 did not bind to E.
invadens (E. inv) spotted in parallel.
86
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SAMUELSON ET AL.
J. CLIN. MICROBIOL.
0.6
0.3
0.3
FIG. 3. Southern blots of £. histolytica DNA hybridized with the
species-specific clone pEH5. DNAs of HM-1:IHSS (lane H), CDC:
0784:4 (lane C), and 200-NIH (lane 2) were cut with Taql (A) or
Sa«3A (B). The ladder produced by incomplete cutting with Taql
shows that the sequence recognized is a tandem repeat. With
Sa«3A, there was a 1.2-kilobase (Kb)-long restriction fragment
length polymorphism, which distinguished 200-NIH amoebae from
the other two E. histolytica strains.
histolytica, because they hybridized to Northern blots of E.
histolytica RNA and to bands on Southern blots of E.
histolytica DNA, which were also recognized by radiola-
beled E. histolytica RNA (data not shown; 4). Partial DNA
sequencing of the clone pEH6 showed homology with the
small-subunit rRNA gene of D. discoideum (16) beginning at
about 300 bp from the 5' end and continuing to the 3' end of
the gene (data not shown).
Molecular analysis of the E. histolytica-speciRc recombinant
clones. The four E. histolytica-specific recombinant DNA
clones showed nearly identical patterns on Southern blots of
restriction-enzyme-cut E. histolytica DNA and so recog-
nized the same highly repeated sequences within the parasite
genome (Fig. 3). The sequences recognized were tandem
145-bp long repeats, as shown by the ladders formed when
the genomic DNA was partially digested with Taql (Fig. 3A).
A degeneracy in the repeat or a modification of a subset of
the repeat sequences was suggested by the failure to cut
completely with SaulA (Fig. 3B).
When parasite DNA was cut with 5a//3A and hybridized
with the diagnostic clone, there was a 1.2-kilobase band in
the DNA from the 200-NIH strain that was not present in the
DNA of the HM-1:IHSS or CDC:0784:4 strain (Fig. 3B).
This restriction fragment length polymorphism may have
potential value in developing a method to distinguish strains
of E. histolytica, as has been shown for restriction fragment
length polymorphisms defined by an rRNA gene probe (4).
pEH5 and pEHll each contained single 145-bp inserts,
which were in opposite directions within the polylinker of
the plasmids. The DNA sequences of these inserts were
identical, AT-rich, and contained no significant open reading
5' GAATTATTCA AAATGGTCGT CGTCTAGGCC 30
AAAATATTTT TTGACCAATT TACACCGTTG 60
ATTITCGATT TCCTAAGAAC CTCACCATTT 90
TTAATGAAAAGTACTAAATACAAAGTACAA 120
TAATTTCTAACTGGGAAAATCGATC3' 145
FIG. 4. DNA sequence of 145-bp tandemly repeated E. histolyt-
ica-specific sequence. The DNA sequences of the inserts of pEH5
and pEHll were identical. Analysis of the sequence revealed no
significant open reading frames.
frames (Fig. 4). There were no DNA sequences found in a
search of the entire GenBank with significant homology to
the 145-bp E. histolytica diagnostic insert. pEH2 and pEH12
contained 1,800- and 1,400-bp inserts, which were composed
of tandem repeats of 145 bp as determined by partial digests
with Taql of gel-purified inserts (data not shown). The E.
histolytica-speciftc recombinant clones did not bind to
Northern blots of parasite RNA, suggesting that the repeated
DNA sequences were not transcribed or that mRNAs were
too few or too unstable to be detected.
Sensitivity of the diagnostic clones. The E. histolytica-
specific recombinant clones were very sensitive, detecting as
few as eight cultured E. histolytica trophozoites in dot blots
after an overnight exposure (Fig. 5A). Using dilutions of the
145-bp insert spotted in parallel to measure amoeba DNA
content, we estimated that each parasite contains 50 to 100 fg
of the 145-bp repeat (Fig. 5A). If we assume that each cell
A * of Parasites
500 250 125 62 31 16 8 4
40 20 10 5 2.5 1.2 0.6 0.3
pg ol pEHS Insert DNA
B # of Parasites
500 250 125 62 31 16 8 4
40 20 10 5 2.5 1.2 0.6 0.3
pg of pEH6 Insert ONA
FIG. 5. Estimation of copy number of inserts of pEH5 and pEH6
in HM-1:IHSS trophozoites. Cultured parasites were diluted and
spotted in parallel with dilutions of purified inserts of pEH5, which
contains a 145-bp species-specific DNA sequence, and pEH6, which
contains 1,600 bp of the small-subunit rRNA gene of £. histolytica.
Radiolabeled pEH5 (A) and pEH6 (B) inserts both detected as few
as eight parasites, and there appeared to be about 50 to 100 fg of
each target DNA per parasite.
87
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VOL. 27, 1989
DNA HYBRIDIZATION PROBE FOR E. HISTOLYTICA
675
34
* 11 •
76
C
* 45 •
**
S. 32
68 •
91
FIG. 6. Representative hybridization of the diagnostic DNA
probe with stool samples. Clinical samples were frozen and thawed,
centrifuged, spotted in triplicate, and hybridized with a radiolabeled
pEH12 insert. On the left is the number of each stool sample.
Numbers 11, 45, and 68 are positive by our criterion.
contains about 500 fg of DNA (9), the 145-bp repeat com-
prises at least 10% of the amoeba DNA. There appeared to
be 50 to 100 fg of the pEH6 insert DNA per trophozoite (Fig.
5B), suggesting that the rRNA genes may also compose 10%
of the parasite genome.
Conditions for detecting E. histolytica parasites in stool.
Cultured E. histolytica trophozoites were mixed with unin-
fected stools, dot blotted, and probed with the diagnostic
clone pEH12 to determine by trial and error conditions for
preserving target DNA and eliminating background hybrid-
ization. We found that the addition of 500 mM EDTA to
stool samples was necessary to prevent the rapid degrada-
tion of parasite DNA by stool DNases. A brief microcentri-
fuge spin to remove bacteria and paniculate debris greatly
increased the amount of sample that could be applied to the
filters. Background hybridization was reduced when the
1,400-bp insert DNA of the diagnostic sequence pEH12 was
cut out and separated by agarose gel electrophoresis from
the pUC vector before radiolabeling. Comparison of E.
histolytica trophozoites spotted with or without stool sug-
gested that the addition of stool decreases the sensitivity of
the probe by about one order of magnitude.
The best method of releasing DNA from the E. invadens
cysts appeared to be by freezing and thawing three times,
which mechanically ruptured the cyst walls. Sonication also
released parasite DNA from the cysts but was labor inten-
sive and potentially hazardous because of production of
aerosols during the procedure. In contrast, treatment of
cysts with SDS, 0.5 N NaOH, proteinase K, or guanidinium
did not release the parasite DNA. We decided therefore to
dilute each of the patient samples in four parts of buffer
containing 10 mM Tris and 500 mM EDTA, freeze and thaw
the samples three times, and then pellet away bacteria and
debris before applying the samples to the nylon filters.
Detection of E. histolytica parasites in clinical samples. The
E. histolytica-specific probe pEH12 was tested against clin-
ical samples from Mexico City in which parasites had been
identified by microscopy. To perform a blind trial, the
investigators performing the hybridizations did not know the
results of the microscopy until after the hybridizations were
performed and scored. Stool or purified cysts and ova were
frozen and thawed to break open cysts in the presence of
EDTA to prevent DNase activity, dot blotted onto nylon
filters, and hybridized with radiolabeled insert DNA of
pEH12 (Fig. 6). The DNA hybridization probe correctly
identified 25 of 25 stools containing E. histolytica parasites,
for a sensitivity of 1.0. The probe found seven stools to
contain E. histolytica parasites found by microscopy to
contain other parasites, not E. histolytica, for a specificity of
0.93. These so-called false-positives did not appear to be
systematic as they included 4 of 36 Giardia lamblia, 2 of 20
Endolimax nana, 1 of 14 Entamoeba coli, 0 of 8 Chilomastix
mesnili, and 0 of 3 Trichuris trichiuria. Ninety-one samples
were found not to contain E. histolytica parasites by the
DNA hybridization probe. Finally, the probe recognized
equally well parasites from unfractionated stool or from
purified cyst preparations.
DISCUSSION
DNA hybridization probes for diagnosis of E. histolytica
were made by identifying and cloning highly repeated and
species-specific amoeba DNA sequences. In a blind study,
these diagnostic probes accurately identified E. histolytica
parasites in patient samples from Mexico City.
Two types of DNA sequences are highly repeated in E.
histolytica. First, there is the tandemly repeated, slightly
degenerate, AT-rich 145-bp long sequence, which is species
specific. This sequence does not appear to be transcribed
into stable RNA and so is similar to other species-specific
and highly repeated DNA sequences used for diagnosis of
malaria and onchocerciasis (3, 21). The function of these
parasite sequences and similar species-specific repeated
DNA sequences in the mammalian genome is not known
(13). Second, the rRNA genes of E. histolytica are also
highly repeated, presumably to amplify the amount of rRNA
produced by the parasites. Because major portions of the
rRNA genes are conserved across species (15), the complete
rRNA genes were not useful as diagnostic probes. However,
portions of the rRNA genes that appear to be species specific
have recently been identified (4).
The DNA hybridization probe developed here recognized
all E. histolytica parasites tested but did not recognize E.
histolytica-\ike Laredo parasites. These Laredo parasites
likely represent a different species from E. histolytica be-
cause they are nonpathogenic, grow at 25 instead of 37°C, do
not bind monoclonal antibodies which bind to E. histolytica
(6), and fail to hybridize with portions of the E. histolytica
rRNA gene (4).
The DNA hybridization probes did not distinguish be-
tween E. histolytica zymodemes. The potential importance
of zymodeme analysis is that it may distinguish potentially
harmful from harmless parasites (20). A recent report sug-
gests that pathogenic and nonpathogenic zymodemes may be
distinguished by monoclonal antibody binding to cultured
trophozoites (23). However, whether the zymodeme of a
parasite is fixed by its genotype or is a phenotypic trait with
differential expression dependent upon the environment of
the parasite is presently a subject of controversy (16).
When compared with microscopy of cysts isolated from
stool by flotation, which is the gold standard for clinical
diagnosis of E. histolytica, the DNA hybridization probe
performed well. The probe correctly identified 25 of 25
specimens containing E. histolytica and called positive an-
other 7 of 98 specimens called negative by microscopy. The
latter positives likely represent errors of the probe but may
represent errors of the microscopist (samples were not
available for review by microscopy). Fortunately, these
errors did not appear to be systematic in that other common
stool parasites such as G. lamblia, E. nana, T. trichiuria, C.
mesnili, or E. coli were not overrepresented. Similar false-
positives have been reported for an ELISA that uses a
88
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SAMUELSON ET AL.
J. CLIN. MICROBIOL.
monoclonal antibody to detect E. histolytica directly from
stool (6). The ELISA correctly identified all samples con-
taining E. histolytica parasites seen by microscopy but
showed an apparent false-positive rate of 13 of 46 positives
(6). Previous ELISAs detected trophozoites but not cysts in
stool (2, 11).
The advantage of either the DNA hybridization probe or
the ELISA over microscopy is the brevity of specimen
preparation: stool is directly spotted onto nylon filters or is
solubilized and placed in microdilution wells, respectively.
In addition, with the DNA probe as many as 96 specimens
per sheet times 10 to 20 sheets can be hybridized in parallel.
Because DNA hybridization is a simple and potentially
inexpensive method for diagnosing E. histolytica, this tech-
nique might be used to survey large numbers of persons to
estimate accurately the prevalence of E. histolytica infection
and to evaluate the effects of control measures to limit
infection with amoebae. Finally, methods to use the poly-
merase chain reaction to amplify target DNA and increase
the sensitivity of the amoeba probe and to use nonradioac-
tive detection systems to replace 32P-labeled probes are
presently being developed in our lab.
ACKNOWLEDGMENTS
This work was initiated with a grant from the Edna McConnell
Clarke Foundation. Further support came from a grant from the
John D. and Catherine T. MacArthur Foundation. J.S. is a Bur-
roughs-Wellcome Life Science Research Foundation Fellow. D.W.
is a Burroughs-Wellcome Scholar in Molecular Parasitology. R.A.-
S. was supported by a predoctoral training grant from CONACYT
(Mexico). S.R. is a Lucille Markey Foundation Fellow.
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Diamond. 1986. Entamoeba histolytica: effect of growth condi-
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17. Reed, S. L., J. G. Curd, I. Gigli, F. D. Gillin, and A. I. Braude.
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24. Trissl, D. 1982. Immunology of Entamoeba histolytica in human
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Induction of encystation of Entamoeba invadens by removal of
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89
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Reproduced, with permission, from the
Journal of Clinical Microbiology
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1989, p. 431-435
0095-1137/89/030431-05S02.00/0
Copyright © 1989, American Society for Microbiology
Vol. 27, No. 3
Diagnosis of Giardia lamblia Infections by Detection of
Parasite-Specific Antigens
EDWARD N JANOFF.^t* J. CARL CRAFT,3 LARRY K. PICKERING,4 THOMAS NOVOTNY,5
MARTIN J. BLASER,1'2 CATHY V. KNISLEY,4 AND L. EARTH RELLER2
Infectious Disease Section, Veterans Administration Medical Center, Denver, Colorado 802201; Division of Infectious
Diseases, Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 80262 ;
Department of Pediatrics, Tulane University School of Medicine, New Orleans, Louisiana 701123;
Program in Infectious Diseases and Clinical Microbiology, University of Texas Medical School,
Houston, Texas 770304; and Centers for Disease Control, Atlanta, Georgia 30333s
Received 28 September 1988/Accepted 30 November 1988
Antigen detection methods may facilitate diagnosis of Giardia lamblia in stool specimens. As determined by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and immunoblotting, G. lamblia cysts and
trophozoites share several antigens, especially in the 65-kilodalton and 30- to 34-kilodalton regions. By using
blind methods, we compared results obtained by counterimmunoelectrophoresis using cyst-immune rabbit
serum and by enzyme-linked immunosorbent assay (ELISA) using trophozoite-immune rabbit serum with
results obtained by microscopic examination of a preserved, concentrated, and permanently stained stool
specimen. Results were similar when these three methods were used to examine 118 stool specimens from
clinical microbiology laboratories (53 specimens with G. lamblia) and specimens from 239 day-care-center
toddlers (39 specimens with G. lamblia). Compared with microscopy, we found, for counterimmunoelectro-
phoresis and ELISA, respectively: sensitivity, 88 versus 94%; specificity, 97 versus 95%; positive predictive
value, 86 versus 76%; negative predictive value, 98 versus 97%; and concordance, 89%. The false-positive rate
by EUSA was 24% (10 of 42) in day-care-center toddlers but only 3% (1 of 32) in healthy adults (P < 0.04)
as corroborated by microscopy. This discrepancy suggests that the ELISA may be more sensitive than
microscopy, which is considered the reference standard, and that results may be dependent, in part, on the
epidemiology of the infection in the study subjects.
Reliable information on the epidemiology and natural
history of Giardia lamblia infections has been limited be-
cause the sensitivity of microscopic diagnosis of the parasite
in concentrated stool specimens varies from 46 to 95% (1, 6,
12,13). Examination of multiple samples may be required for
diagnosis, particularly from persons with early (9) or chronic
infections (3). Other diagnostic methods, some of which
require sampling of the small bowel, are more expensive,
uncomfortable, and invasive (6,10,13). Stool antigen detec-
tion systems for the diagnosis of G. lamblia infections have
been reported to be highly sensitive and specific (2, 7,14, 23,
24). These methods may permit large numbers of stool
samples to be tested rapidly and may reduce technician time
and bias among observers.
Standard diagnostic methods are designed to detect the
specific stages of the life cycle of G. lamblia, cysts and
trophozoites. Microscopic examination of stool samples
most often detects cysts (4, 21), whereas duodenal aspiration
and biopsy primarily identify trophozoites (6). In contrast,
antigen detection systems, although also designed to detect
the distinct stages of the life cycle, show similar diagnostic
accuracies. We identified the antigens detected by two
antigen detection methods to understand the relationship
between cyst and trophozoite antigens and to better define
and standardize the assay systems. We performed the first
blinded comparison of three noninvasive methods for diag-
nosis of G. lamblia infections from stool samples: micro-
* Corresponding author.
t Present address: Infectious Disease Section 111F. Veterans
Administration Medical Center, 1 Veterans Dr., Minneapolis, MN
55417.
scopic examination, detection of cyst antigens by counter-
immunoelectrophoresis (CIE), and detection of trophozoite
antigens by enzyme-linked immunosorbent assay (ELISA).
(This work was presented in part at the 27th Interscience
Conference on Antimicrobial Agents and Chemotherapy,
New York, N.Y., 4 to 7 October 1987.)
MATERIALS AND METHODS
Antigen preparation. G. lamblia Portland-1 trophozoites,
graciously provided by Ernest Meyer, were grown and
prepared as previously described (8). Purified cysts were
prepared from human fecal specimens, as previously de-
scribed (2), and were kindly provided, in part, by Charles
Hibler. The cyst purity was assessed by microscopy (2), and
the preparations were sonicated, centrifuged at 1,000 x g for
30 min, and preserved at -70°C.
Antigen analysis. Rabbit sera used in the antigen detection
systems were compared by ELISA as previously described
(8), except that the solid-phase antigens were whole frozen
G. lamblia Portland-1 trophozoites (104 organisms per well)
or purified G. lamblia cysts (103 cysts per well). The end-
point was defined as the highest dilution of rabbit serum that
resulted in an optical density (OD) greater than 0.200 at 405
nm. The specificities of the antibodies detected were deter-
mined by adsorption of immune serum four times for 45 min
each time at 37°C on a circular rotator with each of the
following Formalin-fixed organisms (number of organisms
per adsorption): G. lamblia Portland-1 trophozoites (6 X
106); Trichomonas vaginalis cultured from a symptomatic
patient (6 x 106); Cryptosporidium oocysts and freshly
excysted sporozoites (107), provided by Charles Sterling and
90
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432 JANOFF ET AL.
J. CLIN. MICROBIOL.
Michael Arrowood; Campylobacter jejuni (109); enterotoxi-
genic Escherichia coli (109); and Candida albicans (109).
Whole-cell preparations of purified G. lamblia cysts and
trophozoites were resolved by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) as previously
described (8). Immunoblots of trophozoite antigens were
prepared with serum from New Zealand White rabbits
immunized with G. lamblia cysts or trophozoites and used in
the CIE and ELISA systems, respectively (2, 8).
Stool specimens. In Denver, Colo., in 1985, we obtained (i)
single unpreserved stool samples from each of 239 randomly
selected toddlers in day-care centers (T. Novotny, R. S.
Hopkins, P. Shillam, and E. N. Janoff, Abstr. Annu. Conf.
Epidemic Intelligence Service, Centers for Disease Control,
1986) and (ii) 118 samples submitted to the clinical microbi-
ology laboratories of the University of Colorado, Veterans
Administration Medical Center, and Denver General Hospi-
tal from 86 persons with diarrhea and 32 healthy adults with
no history of gastrointestinal symptoms or known G. lamblia
exposure within the past 2 months. Fresh stool specimens
were preserved in 10% Formalin or in poly vinyl alcohol, and
a third fraction was stored within 12 h of collection from test
subjects without preservative at —70°C until processed.
Microscopy. The entire 22-mm2 cover slip from each
Formalin-ether (or Formalin-ethyl acetate) concentrate was
examined at a magnification of x 10, as was each trichrome
stain of polyvinyl alcohol-preserved stool specimen exam-
ined under an oil immersion lens. Each concentrated speci-
men was stained for Cryptosporidium oocysts and sporozo-
ites with the modified Kinyoun acid-fast (cold) technique
(11), and the entire slide was scanned for 5 min at a
magnification of x 40. Positive specimens were confirmed by
examination under an oil immersion lens. Identification of G.
lamblia by microscopic examination of preserved concen-
trate or of a permanently stained smear was considered the
reference standard for the antigen detection systems. The
numbers of G. lamblia organisms detected were graded as
rare to few, moderate, or many in 31 of 39 positive speci-
mens from day-care-center toddlers.
Antigen detection. The CIE assay was performed as pre-
viously described (2), with serum from cyst-immune rabbits.
For the ELISA system, New Zealand White rabbits were
immunized with axenically grown G. lamblia Portland-1
trophozoites. A crude serum immunoglobulin fraction was
precipitated with ammonium sulfate and dialyzed, and a
portion was conjugated to horseradish peroxidase (22).
Wells of microdilution plates (Immulon II; Dynatech Labo-
ratories, Inc., Alexandria, Va.) were coated with unlabeled
rabbit anti-G. lamblia antibody in 100 mM carbonate buffer
(pH 9.0) overnight at 4°C. Freshly thawed fecal specimens
were diluted 1:2 with 50% fetal calf serum-0.01 M phos-
phate-buffered saline (pH 7.2) plus 2 mM phenylmethylsul-
fonyl fluoride (Sigma Chemical Co., St. Louis, Mo.) and
0.1% thimerosal, vortexed, and allowed to settle for 30 min.
The supernatant was incubated for 1 h at 37°C in duplicate
wells. After wells were washed three times by using a
multichannel pipette, peroxidase-conjugated rabbit anti-G.
lamblia antibody was applied, incubated for 1 h at 37°C, and
developed with o-phenylenediamine in 53 mM citrate-94
mM phosphate buffer (pH 5.0) for 15 min. The reaction was
stopped with 2.5 N H2SO4, and the A490 was measured on a
microELISA plate reader (MR580; Dynatech). Positive and
negative controls were included on each plate. Specimens
which produced an OD of less than 0.100 were considered
negative, those with an OD between 0.100 and 0.250 were
considered equivocal, and those which produced an OD
above 0.250 were considered positive on the basis of results
previously obtained from specimens characterized by mi-
croscopy (C. V. Knisley, P. G. Englekirk, L. K. Pickering,
S. West, and E. N. Janoff, Am. J. Clin. Pathol., in press).
Preliminary studies showed that an OD of 0.250 resulted
from the addition of 250 trophozoites (1 ng of protein) to a
test well. We decided in advance to consider any specimens
which produced equivocal results upon CIE or ELISA to be
negative for the purpose of analysis.
We evaluated each method on the basis of the number of
specimens tested by that method; all 357 specimens were
tested by microscopic examination, 327 (92%) were tested
by CIE, and 312 (87%) were tested by ELISA. We compared
the three assays by using results from the 282 (79%) speci-
mens tested by all three methods. To investigate the repro-
ducibility of test results, nine stool samples were tested in a
blind manner in duplicate by CIE, as were six by ELISA.
ODs by ELISA were compared for 20 samples tested twice
on separate days to establish the test-retest reliability.
Statistics. Results were evaluated by the chi-square and
Fisher exact tests as indicated.
RESULTS
Antigen analysis. The trophozoite-immune rabbit serum
used in the ELISA system showed a liter of 1:25,600 with the
trophozoites and a titer of 1:400 with the cysts as the
solid-phase capture antigens. The cyst-immune rabbit serum
used in the CIE system showed a titer of 1:200 with the
trophozoites and a titer of 1:3,200 with the cysts as the
solid-phase capture antigens. Preimmune rabbit serum
showed a titer of <1:100, the lowest dilution tested, with
both antigens. Trophozoite-immune serum showed the
greatest decrease in titer following adsorption with G. lam-
blia trophozoites, from 1:25,600 to 1:3,200. Titers after
adsorption with other protozoal, bacterial, and fungal organ-
isms remained 1:12,800.
Eight distinct bands, with major bands at 88, 60, 55,48, 33,
and 31 kilodaltons (kDa), were resolved from the soluble G.
lamblia cyst preparation (Fig. 1). Silver staining of solubi-
lized trophozoites also produced bands of 189, 88, 75, 68, 55,
47, and 34 kDa (13). Immunoblotting of whole trophozoites
with cyst-immune and trophozoite-immune rabbit sera re-
vealed common trophozoite antigens of 185, 65, and 57 kDa
and produced the strongest reactivity with heavy bands of 33
and 30 kDa (Fig. 2).
Antigen detection in stool samples. The sensitivities and
specificities of the two antigen detection methods (CIE and
ELISA) were similar to results obtained by microscopy with
stool specimens from day-care-center toddlers and with
specimens submitted to clinical laboratories (Table 1). The
numbers of organisms identified by microscopy in 31 of 39
day-care-center samples examined in which G. lamblia was
identified were rare to few in 48%, moderate in 16%, and
many in 35%. Both CIE and ELISA showed negative results
for two specimens in which G. lamblia was detected by
microscopy. Two stool samples taken from patients 2 weeks
after they started therapy for documented G. lamblia infec-
tions were negative by microscopy; one sample was positive
by CIE, as was the one sample tested by ELISA. Thirteen
stool samples with other parasites, including Entamoeba
histolytica (n = 2), Cryptosporidium spp. (n = 7), Blastocys-
tis hominis (n = 3), and others (Entamoeba coli, Entamoeba
nana, Trichuris trichuris) showed negative results by both
methods. Nine samples tested in a blind manner in duplicate
by CIE gave concordant results (seven positive and two
91
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VOL. 27, 1989
ANTIGEN DETECTION FOR GIARDIA LAMBLIA 433
116-
92- —'-
66- 1
45-
31-
*«K»S<
31-
31-
21-
14-
14-
FIG. 1. Silver-stained 12% polyacrylamide SDS-PAGE prepara-
tions of solubilized G. lamblia. Lanes: a, cysts from an infected
patient; b, axenically grown Portland-1 strain trophozoites.
negative), as did five of six samples tested by ELISA (three
of four positive and two negative). The test-retest reliability
of ODs produced with 20 samples tested on two occasions by
ELISA was high (correlation coefficient = 0.953), and the
mean values did not differ by the paired / test. The presence
or absence of symptoms, such as diarrhea and abdominal
pain, in the patients from whom the specimens were ob-
tained did not affect the sensitivities or specificities of the
two methods (data not shown). The concordance rates
between the two antigen detection methods was 92% (196 of
214) for samples without G. lamblia and 81% (55 of 68) for
samples with G. lamblia as corroborated by microscopy.
The predictive value of a positive result was lower in
samples from day-care-center toddlers than from routine
FIG. 2. Immunoblots of G. lamblia Portland-1 trophozoites re-
solved on 12% polyacrylamide SDS-PAGE gels. Nitrocellulose
strips were reacted with serum from rabbits immunized with G.
lamblia cysts (lane a), G. lamblia trophozoites (lane b), and preim-
mune serum (lane c).
samples from clinical microbiology laboratories. When mi-
croscopy results were considered the reference standard,
false-positive rates were higher among day-care-center tod-
dlers, among whom rates of exposure to and carriage of G.
lamblia are often higher (1,16; Novotny et. al., Abstr. Annu.
Conf. Epidemic Intelligence Service, 1986) than among
healthy adults with no known G. lamblia exposure, who are
less likely to be infected (Table 2).
Of the samples tested by CIE, 5% (16 of 327) showed
equivocal results (grey zones); 13% of those tested by
ELISA (41 of 312) showed equivocal results. As part of our
protocol prior to testing, any equivocal result was defined as
a negative test result for the purposes of analysis. In the
clinical setting, these results would have been called equiv-
ocal and a second specimen would have been requested. No
G. lamblia was identified by microscopy in 81% (13 of 16)
TABLE 1. Comparison of antigen detection methods with microscopy for diagnosis of G. lamblia infections
Source of specimens
(diagnostic test)"
No. tested
No. (%) with
G. lamblia
Sensitivity
(%)*
Specificity
(%)*
Positive predictive
value (%)
Negative predictive
value (%)
Day-care-center toddlers
MICRO 239 39 (16) 100 100
CIE 212 35 (17) 88 97
ELISA 218 42(19) 94 95
Clinical microbiology laboratory
MICRO 118 53(45) 100 100
CIE 115 48(42) 96 97
ELISA 94 42(45) 90 91
86
76
96
88
98
99
93
92
" MICRO. Microscopic examination of a single preserved, concentrated, and permanently stained specimen; CIE, CIE assay for cyst antigen; ELISA, ELISA
for trophozoite antigen.
* Microscopic examination is considered the reference standard for this analysis.
92
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434 JANOFF ET AL.
J. CLIN. MICROBIOL.
TABLE 2. Comparison of false-positive rates using antigen
detection methods in two epidemiologically distinct groups
Group
No. false-positive/total no. (%) by":
CIE
ELISA
Day-care-center toddlers 5/35 (14)
Healthy adults 0/32* (0)
10/42 (24)
1/32* (3)
° For the purposes of this analysis, results are considered as false-positives
if the sample produced a positive result by ELISA or CIE but no organisms
were identified by microscopic examination of a single concentrated and
permanently stained sample from each subject.
b P < 0.05, Fisher exact test.
and 90% (37 of 41) of these samples, which were equivocal
by CIE and ELISA, respectively. Exclusion of these sam-
ples from the analysis did not significantly change the
evaluation of the tests.
DISCUSSION
This study establishes that both the CIE and ELISA
systems are reliable diagnostic tests for the diagnosis of G.
lamblia infections on the basis of established criteria (18).
We performed a blinded comparison with a reference stan-
dard of diagnosis on stool samples from patients with a
spectrum of clinical manifestations. We described the clini-
cal setting and exact methods for the study, the precision of
the tests, and variation among observers. The advantage of
antigen detection systems is that they provide rapid, sensi-
tive, and reproducible results for large numbers of samples;
they may be less labor-intensive, less expensive, and subject
to less test-to-test variation than diagnosis of G. lamblia
infections by microscopy. These assays serve as a useful
adjunct to microscopic examination, which is directed to
detect a spectrum of enteric infections.
CIE and ELISA antigen detection systems showed results
similar to those of microscopic examination in a large,
prospective, blinded comparison for the diagnosis of G.
lamblia in stool specimens. Although cysts are usually
identified five times more often than trophozoites in stool by
microscopy (4, 21), these assays, which used rabbit sera
hyperimmune to two distinct phases of the life cycle of G.
lamblia, gave comparable results. The recognition of com-
mon antigens by these sera, especially those bands of 65
kDa, as first described by Rosoff and Stibbs (17) and Stibbs
et al. (19), and those between 30 and 34 kDa, which are also
recognized by humans infected with G. lamblia (8, 20), may
in part explain this similarity. These cross-reactive antigens,
which are recognized by the cyst-immune serum, are prob-
ably derived from trophozoites within the cysts. This cross-
reactivity was not described by another group, perhaps
because of differences in immunization technique or in the
method of antigen preparation (5). The CIE assay may well
detect other protein or nonprotein cyst wall antigens (5, 25).
Antigen detection may be more sensitive, rather than less
specific, than the reference standard, microscopic examina-
tion (14). The specificity of microscopic diagnosis in these
university-affiliated and state laboratories is high in Denver,
probably because of the frequency of diagnosis of the
parasite and the long-term interest in G. lamblia in Colorado.
We demonstrated the specificity of the trophozoite-immune
serum in vitro by adsorption studies and by the lack of
cross-reactivity with other protozoa in clinical samples. The
false-positive rate was higher among day-care-center tod-
dlers, among whom carriage rates of G. lamblia are high (1,
16; Novotny et al., Abstr. Annu. Conf. Epidemic Intelli-
gence Service, 1986) and who may excrete few organisms (3,
9, 15), than among adults, who are at low risk of infection.
False-negative samples were uncommon with both antigen
detection methods. The true sensitivities of these tests could
be determined best during an outbreak by testing acutely ill
persons, with multiple specimens being taken from the same
subjects (14), and by using seeding studies.
We conclude that antigen detection systems provide an
acceptable and convenient adjunct to microscopic examina-
tion for noninvasive method of diagnosis of G. lamblia in
stool specimens. Further identification of parasite-specific
antigens may be useful to standardize and improve the
clinical utility of diagnostic methods and to define the natural
history of these infections.
ACKNOWLEDGMENTS
We thank Paul G. Englekirk (University of Texas Medical School
at Houston), Kurt Albrecht and Larry Briggs (Colorado Department
of Health), Elizabeth Lindsay, Sandy Barber, Sally Dembeck, Stan
Merritt, and Jan Monahan (University of Colorado Clinical Micro-
biology Laboratory), and Ben Miyahara and John Paisley (Denver
General Hospital Microbiology Laboratory) for technical assistance
and support and Charles Sterling and Michael Arrowood (University
of Arizona School of Veterinary Science) for providing Ciyptoxpo-
ridium oocysts.
This research was supported in part by the Veterans Administra-
tion Research Service and by Public Health Service grant HD13021
from the National Institutes of Health.
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VOL. 27. 1989
ANTIGEN DETECTION FOR GIARDIA LAMBLIA
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THE LANCET, JANUARY 3, 1987
51
Reproduced, with
from the Lancet.
permission.
IDENTIFICATION OF CRYPTOSPORIDIUM
OOCYSTS BY MONOCLONAL ANTIBODY
SIR,—The protozoan parasite cryptosporidium can cause self-
limiting gastroenteritis in otherwise normal individuals or a chronic
life-threatening diarrhoea in immunologically compromised
individuals, especially those with AIDS.1 Laboratory diagnosis has
relied on the demonstration of oocysts in faeces by modified
Ziehl-Neelsen or auramine-phenol staining.2 One of us (D. P. C.)
has also described an indirect fluorescent antibody test using human
sera containing anti-cryptosporidium IgA antibodies (HIgA) for
confirmatory purposes.1-1 A monoclonal antibody for diagnostic use
has been reported3A but few data on sensitivity and specificity have
been presented. We report here a rapid and accurate method using a
monoclonal antibody in an indirect immunofluorescent antibody
(IFA) test, which seems to be highly specific and more sensitive
than conventional techniques. It allows the demonstration of
oocysts in hitherto unsuitable samples and their differentiation from
bodies that resemble cryptosporidium morphologically.
Balb/c mice were immunised with an enriched oocyst suspension
(from the faeces of an infected calf) which had been gamma-
irradiated and sonicated before intraperitoneal inoculation at
about 10" oocysts per inoculum four times over 24 days. Spleen cells
taken on the 27th day after the first inoculation were fused with a
drug-sensitive tumour cell line (JK-Ag8653), and antibody-
secreting hybridomas were identified by an indirect IFA test. A
single cell line (MAB Cl) secreted antibody of the IgM isotype. At
first this was used in an indirect assay, but we have since conjugated
the antibody directly with fluorescein isothiocyanate (FITC) and
developed a technique with a total assay time of less than 30 min.
Faecal samples were homogenised and concentrated2 and tested
in parallel with conventional staining and HIgA techniques (table).
MAB Cl proved to be specific: it reacted with all samples
containing cryptosporidium oocysts identified by staining and it did
not react with morphologically similar bodies or related parasites. It
also seems more sensitive than conventional tests, since
cryptosporidium oocysts in follow-up samples from confirmed
cases that did not stain by conventional techniques were detectable
with MAB Cl. The monoclonal antibody was superior to the HIgA
because of reagent reproducibility and higher specificity (the HIgA
contained antibodies to other organisms).
The epidemiology of cryptosporidiosis is incompletely
understood, and this reagent may permit the specific identification
of the oocysts and the exclusion of other morphologically similar
bodies in environmental samples that are not suitable for testing by
conventional staining.
We thank Dr D. Blcwitt and Dr K. Angus, Moredun Research Institute,
Edinburgh; Dr A. Balfour, Public Health Laboratory, Leeds; and Dr V.
McDonald, Houghton Poulry Research Centre for donations of antigens.
Division 01" Microbiological Reagents
and Quality Control,
Central Public Health Laboratory,
London NW9 5HT
Public Healdi Laboratory, Rhy!
Division of Microbiological Reagents
and Quality Control,
Central Public Health Laboratory
Animal Department,
Central Public Health Laboratory
Division of Microbiological Reagents
and Quality Control,
Central Public Health Laboratorv
J. MCLAUGHLIN
D. P. CASEMORE
T. G. HARRISON
P. J. GERSON
D. SAMUEL
A. G. TAYLOR
species a "new" human
I Casemore DP, Sands RL, Curry A. Crv/Jmspi
pathogen. J C/in Pal/ml 1985,38: 1321-36.
2. Casemore DP, Armstrong M, Sands RL. Laboratory diagnosis of cryptosporidiosis. J
C/m Palhal 1985; 38: 1337-41.
3. Sterling CR, Arrowood MJ. Detection of GYv/Ti^yonWntm sp infections using a direct
immunofluorescem assay Palmlr hilea Du 1986,5:5139-42.
4. Sterling CR, Seeger K, Sinclair NA. Ov/>fi)x/>onJiw;/ as a causative agent of traveller's
diarrhoea. 7 Infal Dis 1986; 153: 380-81.
RESULTS OF COMPARISON OF DIFFERENT METHODS FOR THE DEMONSTRATION OF CRYPTOSPORIDIUM OOCYSTS
Assessment of sensitivity (specimens from individuals with cryptosporidiosis)
Human faeces (known positive or follow-up samples)
Enriched oocyst suspensions
Calf faeces
Lamb faeces
Guineapig faeces
Mouse faeces (infected by material from a calf)
Assessment of specificity
Human faeces (non-cryptosporidiosis)
Horse faeces (contains CLB and AFB)
Water filtrates (contains CLB and AFB)
Eimena tenella
Toxoplasma gondii (trophozoites)
No of specimens
35t
5
9
2
1
1
10
5
10
1
1
Number of specimens positive by;
MZN
28
5
9
2
1
1
0
5
10
NT
NT
AP
29
5
2
2
1
1
0
5
10
NT
NT
HIgA
34
5
0
7
I
I
0
0
0
NT
NT
MABC1*
35
5
9
2
1
1
0
0
0
0
0
*Higher numbers of oocysts were detected with MAB Cl than with the MZN and AP techniques in 16 samples.
t7 additional follow-up samples were negative with all methods.
MZN = modified Ziehl-Neelsen; AP = auramine-phenol; HIgA = human anti-cryptosporidium IgA antibody; MAB Cl = ami-cryptosporidium monoclonal antibody; AFB = acid-fast
bodies; CLB = cryptosporidium-like bodies which were similar but morphologically distinct; NT = not tested.
*U.S. GOVERNMENT PRINTING OFFICE: 1990—726-115/D09667
95
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