Umtec States Office of Water EPA 315-8-98-0 r
Environmental Protection (4607) July 1998
Agency ^______
V>EPA ADDENDUM TO DRAFT DRINKING
WATER CRITERIA DOCUMENT FOR
CRYPTOSPORIDIUM
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Addendum to Draft Drinking Water Criteria
Document for Cryptosporidium
U.S. Environmental Protection Agency
Office of Science and Technology
Human and Ecological Health Effects Division
July 30,1998
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ACKNOWLEDGMENTS
This document was prepared for the U.S. Environmental Protection Agency, Office of
Ground Water and Drinking Water (OGWDW) by the Office of Science and Technology (OST)
under contract with Dr. Jon Standridge from the University of Wisconsin (Order No. 8 W-1644-
NASA). Overall planning and management for the preparation of this document was provided by
Lisa Almodovar and Robin Oshiro of OST.
EPA acknowledges the valuable contributions of those who wrote and reviewed this ,
document. They include: Jon Standridge, PH.D., task manager, editor and word processor of the
University of Wisconsin; and Lisa Almodovar, Robin Oshiro, and Crystal Rodgers of the U.S.
EPA. EPA also thanks the following external peer reviewers for their excellent review and
valuable comments on the draft document: Mark Borchardt, Ph.D. (Marshfield Medical Research
and Education foundation); Carrie Hancock (CHDiagnostics and Consulting Service, Inc.); and
Charles Gerba, Ph.D. (University of Arizona).
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EXECUTIVE SUMMARY
The Safe Drinking Water Act requires the U.S. Environmental Protection Agency (EPA)
to publish regulations to control disease-causing organisms (pathogens) and hazardous chemicals
in drinking water. One of the regulations published by EPA to control pathogens is known as the
Surface Water Treatment Rule (54 FR 27486: June 29, 1989). The intent of this rule was to
control Giardia, pathogenic viruses, and Legionella, all of which have caused many outbreaks and
cases of waterbome illness.
Another prominent waterbome pathogen is the protozoan Cryptosporidium. This
organism has caused a number of waterbome disease outbreaks in the U.S. and other countries. In
1994, EPA prepared a literature review of the published data on Cryptosporidium, entitled
"Cryptosporidium Criteria. Document", to establish a basis for a regulation to control this
organism. The following document," Addendum to Draft Drinking Water Criteria Document for
Cryptosporidium", updates the 1994 publication. It includes new information available in the
literature from 1994 to the present, and was prepared to support EPA's Interim Enhanced Surface
Water Treatment Rule, which has as a primary focus the control of Cryptosporidium. The update
provides information on general characteristics of Cryptosporidium, its occurrence in human and
animal populations and in water, the health effects associated with Cryptosporidium infection,
outbreak data, and an assessment of risk. The document also includes information about
analytical methods to enumerate Cryptosporidium in water and the effectiveness of various water
treatment practices.
The document demonstrates that Cryptosporidium oocysts are common and widespread in
ambient water, and can persist for months in this environment The dose that can infect humans is
low, and a number of waterbome disease outbreaks caused by this protozoan have occurred in the
U.S., most notably in Milwaukee, where an estimated 400,000 people became ill. The document
shows that otherwise healthy people recover within several weeks after becoming ill, but illness
may persist and contribute to death in those whose immune systems have been seriously
weakened (e.g., AIDS patients). Drugs effective in preventing or controlling disease are not yet
available. The public health concern is aggravated by the resistance of Cryptosporidium to
commonly used water disinfection practices such as chorine. However, a well-operated water
filtration system is capable of removing at least 99 of 100 Cryptosporidium oocysts in the water.
Monitoring for this organism in water is currently difficult and expensive.
EPA believes that the information in the 1994 document and the following update is
sufficient to conclude that Cryptosporidium may cause a health problem and occurs in public
water supplies at levels which may pose a risk to human health.
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Table of Contents
I. Introduction 1
II. General Information and Properties 2
A. History and Taxonomy 2
1. History 2
2. Taxonomy 3
B. Life Cycle , 6
C. Morphological Features .6
D. Species Transmission '. 6
1. Direct Transmission Between Humans 6
2. Transmission Between Animals and Humans 9
E. Summary 10
HI. Occurrence 11
A. Worldwide Distribution 11
1. Distribution in Animal Populations 11
2. Distribution in Human Populations 13
B. Occurrence in Water 14
1. Surface Water '. 14
2. Ground Water " 15
C. Occurrence in Soil ..15
D. Occurrence in Air 16
E. Occurrence in Food and Beverages .16
F. Specific Disease Outbreaks 17
1. Outbreaks Associated with Drinking Water ... 17
2. Outbreaks Associated with Recreational Waters 21
3. Foodbome Outbreaks 22
4. Outbreaks Among Travelers -. 23
5. Outbreaks at Day-Care Centers 23
6. Sensitive (Immunocompromised) Populations 24
G. Environmental Factors 27
H. Summary 28
IV. Health Effects in Animals 29
A. Symptomatology and Clinical Features .29
B. Therapy 33
C. Epidemiological Data , .. 34
D. Summary .36
V. Health Effects in Humans 37
A. Symptomatology and Clinical Features 37
B. Epidemiological Data 39 ,
C. Treatment: Clinical Laboratory Findings and Therapeutic Management 42
D. Mechanism of Action '. 44
E. Immunity 45
F. Chronic Conditions 47
G. Summary ;.: 47
VI. Risk Assessment 48
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A. Experimental Human Data 49
B. Experimental Animal Data : 50
C. Environmental Factors 50
1. Prevalence in Surface Waters 50
2. Oocyst Survival 51
3. Cryptosporidium in Drinking Water 52
D. Epidemiologic Considerations 53
1. Population at Risk and Survey Methods : 53
E. Risk Assessment Models 54
F. Federal Regulations 57
G. Summary ; 58
VII. Analysis and Treatment : 59"
A. Analysis of Water 59
1. Collection and Concentration of Cryptosporidium from Water 60
2. Detection of Cryptosporidium in Water 67
3. Assessment of Laboratory Testing Capabilities 77
B. Detection in Biological Samples 78
C. Water Treatment Practices .. 84
1. Introduction 84
2. Multi-Barrier Treatment .85
3. Removal of Cryptosporidium , 86
4. Inactivation of Cryptosporidium 91
D. Summary , 94
Vm. Research Requirements 97
A. Data Gaps ,. 97
DC References 99
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I. Introduction
The United States Environmental Protection Agency (USE? A) Office of Water is preparing and
revising the health criteria documents that will support the Phase I Disinfectant/Disinfectant
Byproduct (DBP) Rule, the Interim Enhanced Surface Water Treatment Rule (IESWTR) and the
Groundwater Disinfection Rule (GWDR). As part of the rule making process, the USEPA is
required to compile a complete and current compendium of the information used as criteria to
support creation of the rules. The first step in this process occurred in June of 1994 with the
preparation of the USEPA Draft Drinking Water Criteria Document for Cryptosporidium,
hereafter referred to as the "1994 Cryptosporidium Criteria Document". This addendum
provides an update to supplement (but not duplicate) the 1994 Cryptosporidium Criteria
Document. This addendum uses the same table of contents formatting as the 1994 document to
facilitate cross referencing between the two. Much of the published research since the 1994
document focuses on better methods to detect Cryptosporidium in the environment, and
improvements in water treatment technology. Consequently this addendum includes much new
information regarding improvements in analyses and treatment The overall objective is to
provide a comprehensive Cryptosporidium information resource to Federal, State, and local
health officials responsible for protecting public health and the environment
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II. General Information and Properties
A. History and Taxonomy
1. History
Despite the discovery of Cryptosporidium in a wide variety of animal species, this parasite was
considered medically unimportant for almost seventy years after its initial description by Tyzzer
in 1907 (Soave, 1.995). However, the diagnosis of cryptosporidiosis in humans in 1976 and the
subsequent connection of Cryptosporidium to epidemic waterbome disease have since fostered
worldwide interest in the study of this microorganism. By the time the Centers for Disease
Control and Prevention implemented routine reporting for Cryptosporidium among AIDS
patients in 1982, only 13 cases of human cryptosporidiosis had been documented (Ungar, 1990).
Since 1982, more than 1000 reports of human cryptosporidiosis have been documented in almost
100 countries, reaching all continents with the exception of Antarctica (Payer, 1997). At the time
of this writing, it is estimated that the annual number of cryptosporidiosis cases exceeds several
million worldwide (Casemore et al., 1997).
Cryptosporidium was first recognized as a waterborne pathogen during an outbreak in Braun
Station, Texas, where more than two thousand individuals were afflicted with cryptosporidiosis
(D* Antonio et aL, 1985; Graczyk et al., 1998b). Since that time, outbreaks affecting over a
million individuals have been documented throughout North America and Europe, with the
single largest epidemic occurring in Milwaukee, Wisconsin in 1993 (Mackenzie et al., 1994). A
complete history of the waterbome outbreaks of cryptosporidiosis is provided in section ffi-F.
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2. Taxonomy
Cryptosporidium is one of several protozoan genera in the phylum Apicomplexa which develop
within the gastrointestinal tract of vertebrates throughout their entire life cycles. More than
twenty species have been described based upon the hosts from which they were originally
isolated (a complete list is included in Table II-1,1994 Cryptosporidium Criteria Document).
However, interspecies transmission studies, morphological examination and immunological
analyses have reduced this number to eight valid species (Table 1). A number of species isolated
from amphibians, reptiles, and birds remain unnamed, pending taxonomic classification (Payer,
1997). Moreover, the taxonomy of the genus Cryptosporidium is uncertain and changing
(Tzipori and Griffiths, 1998; Feyer et al, 1997).
Table I Valid Cryptosporidium Species
Cryptosporidium Species Host Species
C. baileyi Gallus gallus (domestic chicken)
C.felis Felts catis (domestic cat)
C. meleagridis Meleagris gallopavo (turkey)
C. muris Mus musculus (house mouse)
C. nasorum Naso literatus (fish)
C.parvum Mus musculus (house mouse)
C. serpentis Elaphe guttata (com snake)
£. suboculahs (rat snake)
Stumnia madagcuarensus (Madagascar boa)
C. wrairi Cavia porcellus (guinea pig)
Source: Adapted from Payer (1997) and Upton (1989)
Although infection caused by Cryptosporidium has been observed in over 70 mammalian and
reptilian hosts, illness in humans is confined primarily to infections associated with
Cryptosporidium parvum; a single case of human cryptosporidiosis attributed to C baileyi in an
immunocompromised individual has been reported (Ditrich, 1991), but this organism was later
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shown to be C. Parvum (Payer, pers. Comm.). Until recently, it has not been possible to assess
variation among strains or to evaluate the possibility of distinct transmission cycles among
different isolates. Studies utilizing restriction fragment length polymorphism (RFLP) analysis,
isoenzyme electrophoresis and arbitrarily primed polymerase chain reaction (AP-PCR) have
helped to characterize subtle differences among individual strains and are described in this
section.
A study harnessing the polymerase chain reaction and restriction mapping suggests that
differences in the genetic sequences within the 18S ribosomal RNA (rRNA) region of
Cryptosporidium can be used to distinguish individual strains (mwis, parvum, and baileyi) and
may assist in the development of taxonomic classification (Awad-El-Kariem et a/., 1994).
Webster (1993) applied a battery of molecular taxonomic methods (flow cytometry, PCR, and
RFLP) to detect and classify Cryptosporidium cocysts from geographically diverse isolates.
Isolates exhibited genetic homogeneity for the most part, however differences in isoelectric
points and restriction maps indicated genetic differences among C, parvum isolates from humans
and bovines.
Isoenzyme electrophoresis studies (O'Donoghue, 1995; Awad-El-Kariem, 1995) have been
applied to characterize animal and human oocyst isolates of Cryptosporidium parvum from
V.
different geographical locations. The discovery of two unique isoenzyme forms indicate the
existence of separate subpopulan'ons within the parvum strain, one which infects primarily
humans and the other which infects animals. Follow-up studies using arbitrary primed
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polymerase chain reaction (AP-PCR) and isoenzyme typing (Carraway et ai. 1994; Awad-El-
Kariem, 1996; Awad-El-Kariem, 1997) have confirmed the two unique 'profiles' among C.
parvum corresponding to the animal and human types; cross-transmission infection studies
performed by Awad-El-Kariem et al. (1996). indicated that most human isolates were not
capable of establishing infection in a murine model, whereas all animal isolates were infectious
in mice, supporting the existence of genetically distinct populations of this strain.
Evidence collected during these studies supports the hypothesis that Cryptosporidium may
belong to a clonal population structure, based on correlations between phenotypic and genotypic
markers, and the widespread occurrence of identical genotypes. The essential concept of the
clonal hypothesis is that different strains of the same species may clone (or propagate) different
forms of the same gene due to geographic isolation. As a consequence of this isolation, mutated
or otherwise modified genetic loci will become perpetuated in a clonal manner within the species
in the absence of sexual reproduction. C. parvum may be clonally related to Cryptosporidium
isolated from Guinea Pigs (C. wain), but it is not yet clear whether they should be considered as
two or more species or subspecies.
Analysis of genetic polymorphism among C. parvum isolates from nine human outbreaks and
from several bovine sources (Peng et at.. 1997) indicates the existence of two genotypes
(genotypes 1 and 2) with genetic differences among adhesion proteins. Genotype 1 was observed
exclusively in human isolates; genotype 2 was observed both in calf isolates and in isolates from
human patients who reported direct or indirect exposure to infected cattle, supporting the
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occurrence of rwo distinct transmission cycles of C. parvum in humans: (I) human to human,
and (2) animal to human. This hypothesis is supported by the work of Carraway et al. (1997),
who conducted RFLP analyses on C parvum oocysts isolated from humans and cows. While all
calf isolates exhibited genetic homogeneity at a specific 2.8-kb fragment, human isolates
exhibited multiple profiles at this locus, one found exclusively in humans and one, which
indicated a superposition of both profiles, indicative of heterogeneity among parasite
populations.
B. Life Cycle
A complete description of the life cycle of Cryptosporidium is provided in the 1994 USEPA
Cryptosporidium Criteria Document (Figure n-1, p. H-5). Cryptosporidium is excreted in the
feces of an infected host in the form of an oocyst, which represents the only exogenous stage of
the life cycle. The oocyst consists of four sporozoites housed within a sturdy, multi-layered wall.
The thick-walled oocyst is the environmentally resistant form of this parasite, resulting from the
fertilization of macrogametes within the host, and is appreciably resistant to natural decay in the
environment as well as to most disinfection processes. The life cycle is repeated when
sporulated oocysts excreted by an infected host are ingested by a new host and the sporozoites
excyst within the small intestine.
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C. Morphological Features
A complete description of the morphological features of each life cycle stage (oocyst, sporozoite,
trophozoite, merozoite, microgametocyte, macrogametocyte) of Cryptosporidium is provided in
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the 1994 Cryptosporidium Criteria Document (p.II-7 - II-9). Studies conducted between 1994
and 1998 indicate that the surface "fold" previously thought to be synonymous with the suture
described in ultrastructural studies may not be the same (Robertson, 1994), suggesting that the
fold should no longer be described as a suture, and that during microscopic examination, objects
matching the size, shape and fluorescence criteria should not be discounted based upon the
absence of a surface fold pattern.
D. Species Transmission
1. Direct Transmission Between Humans
A number of studies show that person-to-person transmission of cryptosporidiosis infection can
occur within families, day care centers, hospitals and in urban environments where population
densities are high (1994 Cryptosporidium Criteria Document). The route of infection follows
one of two paths: direct, through fecal-oral contact, or indirect, through fomites (inanimate
objects).
After visitors who came to Milwaukee during the 1993 outbreak returned to their homes, 74
members of their households who had not accompanied them on the visit tested positive for .
Cryptosporidium (Mackenzie ei aL, 199Sb). Five percent of these infected household members
ultimately developed cryptosporidiosis, thereby meeting the definition of secondary transmission
within the household Osewe et aL (1996) also evaluated data following the Milwaukee outbreak
and concluded that secondary transmission rates to household members during the epidemic were
comparatively low (3-5%). In addition to the person-to-person transmission, there appeared to.
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be a recurrence of infections that were acquired by the Milwaukee visitors during the outbreak.
Transmission to susceptible individuals in the Milwaukee area continued after the massive initial
outbreak, but decreased rapidly within the two months that followed.
Cordell and Addiss (1994) tracked cryptosporidiosis in various child care settings and observed a
12 to 22% rate of secondary spread between children to other household members. Newman et
al. (1994) reported household transmission of C. parvum infection in an urban community in
northeast Brazil. In this study, 18 of 31 (58%) households whose members ranged in age from 5
i
months to 47 years showed at least one secondary case of cryptosporidiosis identified either by
stool examination or serologic testing. Secondary cases involved a total of 30 persons, yielding
an overall transmission rate of 19%. Of the 202 persons included in this study, 94.6% had
evidence of serum IgG or IgM antibodies to Cryptosporidivm^ demonstrating that a significant
rate of person-to-person transmission of Cryptosporidivm may occur.
The rate of transmission between immunocompromised individuals may be high. In group
homes housing HIV-positive individuals, Heald and Bartlett (1994) repotted a high (not
specified) rate of transmission among occupants. Lopez-Velez ef fll. (1995) found an overall
prevalence of intestinal cryptosporidiosis of 15.6% in AIDS patients. However, the rate among
homosexual partners was higher (33.3%) than among intravenous drug users (10.6%), strongly
suggesting person-to-person transmission of cryptosporidiosis. Sears er a/. (1994) concluded that
strict infection control measures must be followed especially in crowded living conditions and
where immunocompromised persons reside.
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Secondary transmission of cryptosporidiosis has been obse-rved among humans whose
occupation places them near primary cases within a confined space. An outbreak occurred
among crew members on a U.S. Coast Guard cutter that had obtained water from the city of
Milwaukee during the 1993 epidemic (Moss et al, 1994). Of 50 crewmembers, 62% exhibited
symptoms of cryptosporidiosis and oocysts were detected in stool samples of 10 individuals
(20%). In such an outbreak, distinguishing between primary infections (i.e., due to ingestion of
contaminated water) and secondary infections (i.e., due to fecal contaminated fomites, food, or
other infected individuals) is difficult because the individuals involved were obviously in a
closed environment where frequent contact between humans occurs. In some instances it may
not be possible to determine whether transmission between humans is the primary cause of
cryptosporidiosis, especially when humans also come into contact with animals through
occupational or recreational activities (Adam et al., 1994).
\ .' *
Adegbola et al. (1994) reported that the transmission rate of Cryptosporidium between children
has seasonal peaks associated with rain and high relative humidity. Factors accounting for the
seasonal distribution may include increased survival of oocysts in a high relative humidity
environment and possible dissemination of oocysts to children during heavy rains as a result of
decreased domestic and environmental hygiene.
2. Transmission Between Animals and Humans
The 1994 USEPA Cryptosporidium Criteria Document cites adequate evidence for the
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transmission of Cryptosporidium from animals, particularly livestock, to humans. Of eight valid
species infecting all vertebrate groups, only one, Cryptosporidium parvum, represents a global
public health problem due to its zoonotic potential (Graczyk, 1998b). Other than a single report
of C. baileyi infection in an immunosuppressed individual (Ditrich, 1991), infections in humans
have been confined to C. parvum.
A number of studies have been conducted to determine if C. parvum can cross species lines to
non-mammalian species. Graczyk et al, (1996a) attempted to infect a variety offish, amphibia
and reptiles with C. parvum without success. Although the oocysts were present in the cloaca of
two fish and one lizard post-exposure, no life-cycle stages were detected in histological sections
taken from any of the inoculated species. From these results, the authors concluded that the
oocysts were unable to establish an infection in the gastrointestinal tract of lower vertebrates
even though the inoculated oocysts were retained by these animals at least 14 days following
ingestion. Although animal excreta has been shown to contaminate watersheds and source
waters linked to outbreaks (see Section O-C), evidence of direct animal transmission of
Cryptosporidium to humans has been limited to a few examples (Adam, 1994; Casemore, 1990).
The search for the source of Cryptosporidium in sporadic cases has been just as elusive. To
assess infectious risks associated with pet ownership, Glaser et al. (1998) conducted a case-
control study of HIV-infeeted individuals with and without cryptosporidiosis. No statistically
significant differences were observed in the rate of overall pet ownership, cat ownership, or bird
ownership between the two groups, and only a slight correlation between dog ownership and
human cryptosporidiosis was noted. The authors concluded mat pets do not represent a major
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risk factor for acquisition of cryptosporidiosis.
Despite the strict host-specificity of a number of mammalian strains of Cryptosporidium, the -
discovery that a C. parvw/n-refractive host (a host which ingests infectious oocysts but is not
susceptible to infection) can excrete intact ingested oocysts has raised the issue that if oocyst
infectivity is retained during travel through the intestinal tract, that host could serve as a
mechanical vector for dissemination of the parasite through the environment (Graczyk, 1998b).
Transport of oocysts through migratory waterfowl has epidemiological implications which
should be considered, especially around reservoirs or in waters where shellfish are harvested and
may be consumed raw (Payer el al., 1997; Smith et a/., 1993).
E. Summary
Cryptosporidium has a complex life cycle which involves numerous developmental stages
culminating in the production of environmentally resistant oocysts. Eight species have been
currently classified. The species known to infect humans, C. parvum, appears to be infectious to
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at least 78 other m>*tntnal^; however, at least one genotype within C parvum appears to be
transmitted exclusively through humans. There is sufficient evidence that secondary
Cryptosporidium infections can occur in humans following primary infection from ingestion of
contaminated drinking water. Human-to-human transmission occurs through the fecal-oral route
and reinfections are common where human density is high or among people living in close
/
quarters. C parvum exhibits species specificity with regard to cryptosporidiosis. Lower
vertebrates such as fish, frogs, and lizards are not susceptible to infection. Other than C. parvum,
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only the avian species C. baileyi has been associated with human cryptosporidiosis. Species that
infect other mammals, such as C. wrairi may be clonally h. .
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C'ryptospondium are currently considered as valid pathogens in birds (C. meleagridis and
C baileyi, See Table 1, section El-A) and infections have been attributed to both, although
neither species has demonstrated host specificity.
Cryptosporidium infections have been reported in over 57 different reptilian species
including 40 species of snakes, 15 species of lizards, and 2 species of tortoises.
Cryptosporidium serpentis is currently the only valid named species of Cryptosporidium
known to cause infections in reptiles. Previous descriptions of other species causing
infections in reptiles (C. crotali, C. lampropeltis, C. ameivae and C. ctenosauris) are
consistent with the morphology of another parasite species (Sacrocystis sp.) and therefore
are considered invalid (Payer, 1997).
Cryptosporidium infections have been detected in nine species offish. Infections have
been observed in both freshwater and marine species, and in cultured, captive, ornamental
and wild-caught fish. The only valid named species of Cryptosporidium causing
infection in fish is C nasorum, named after the first reported infection in a tropical
marine fish (Naso lituratus) (Hoover et al, 1981).
In addition to the mammalian, avian, reptilian and piscine hosts mentioned above,
Cryptosporidium infections have been identified in two amphibian species, Ceratophrys
ornata (Bell's homed frog, Crawshaw and Mehren, 1987, cited in O'Donoghue, 1995)
and Limnodynastes tasmaniensis (spotted grass frog, O'Donoghue and Mirtschin,
unpublished, cited in O'Donoghue, 1995), and in one invertebrate species, Ruditapes
decussatus (Portuguese clam, Azevedo, 1989, cited in O'Donoghue, 1995).
Additional hosts reported in the literature, but not mentioned in the review article, include Fell
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Pony foals (Scholes et al., 1998), muskrat (Petri etal, 1997), African hedgehog (Graczyk et al.,
1998a), dugong (Hill et al., 1997), slow loris, white rhinoceros, Indian elephant and Thorold's
deer (Majewska et al, 1997) and iguana (Fitzgerald et al., 1998).
2. Distribution in Human Populations
The. distribution of Cryptosporidium in human populations is worldwide, occurring in both
developed and under-developed countries, urban and rural areas, and in temperate as well as
tropical climates (Payer, 1997; O'Donoghue, 1995). The 1994 Cryptosporidium Criteria
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Document addresses the worldwide distribution of human cryptosporidiosis in 45 different
countries. A recent review article states that since 1982, human cryptosporidiosis has been
documented in 95 countries and on every continent except Antarctica (Payer. 1997). A list of
these countries including the 45 listed in the 1994 document is given in Table 2.
Table 2 Geographic Distribution of Human Cryptosporidiosis
Africa
Algeria
Burundi
Cameroon
Ethiopia
Gabon
Ghana
Guinea
Guinea-Bissau
Ivory Coast
Kenya
Liberia
Mauritania
Mauritius
Morocco
Nigeria
Rwanda
South Africa
Sudan
Togo
Tunisia
Uganda
Zaire
Zambia
Zimbabwe
Asia
Bangladesh
Belarus
Cambodia
China
India
Japan
Korea
Myanmar Republic
Pakistan
Russia
Sri Lanka
Taiwan
Thailand
T
Central/ South
America
Argentina
Brazil
Colombia
Chile
Costa Rica
Ecuador
El Salvador
Guatemala
Mexico
Panama
Peru
Uruguay
Venezuela
Middle East
Egypt
Iran
Israel
Kuwait
Saudi Arabia
North America
Canada
United States
Europe
Austria
Belgium
f zwhxwlQv8lnt
Denmark
Enalftnd
Finland
France
Germany
Greece
Hungary
Ireland
Italy
T ithnfnifl
Netherlands
Poland
Portugal
Romania
Serbia
Spain
Sweden
Switzerland
Turkey
Wales
Pacific
Australia
Malaysia
New Zealand
Papua-New Guinea
Philippines
Singapore
Caribbean
Cuba
Haiti
Jamaica
Puerto Rico
St Lucia
Tobago
Trinw fld
Virgin [«l«yi«
Source: Payer, 1997
Worldwide prevalence rates of human cryptosporidiosis from the most recent compilations of
coprologic (stool) and serologic (blood) surveys (through 1991) are included in the 1994
Cryptosporidium Criteria Document
B. Occurrence in Water
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I. Surface Water
Since the 1994 criteria document was published, dozens of reports have appeared in the literature
further characterizing the occurrence of Cryptosporidium in surface waters. Several of these
articles adequately summarize this large body of work. Lisle and Rose (1995) reviewed the
literature for more than 25 studies involving outbreaks, occurrence, monitoring, and detection, as
well as regulatory implications. They reported that between 5.6 and 87.1% of source waters
tested, contained 0.003 to 4.74 oocysts/L. They concluded that better methods were needed for
oocyst recovery, detection, and treatment In another major study, LeChevallier and Norton
(1995) reported finding oocysts in 60.2% of surface waters tested in the U.S. and Canada. Three
companion articles appeared in the September 1997 issue of the Journal of the American Water
Works Association summarizing the current state of knowledge of Cryptosporidium occurrence
in watersheds (Crockett and Haas, 1997), rivers (States et at, 1997) and reservoirs (LeChevallier
et al, 1997). All three studies concluded that any surface water is subject to a complex set of
watershed characteristics (buffer zones, slope, tend use, watershed management, storm water
management, sewage treatment practices, sediment types, soil types, vegetation, population
density, pathogen sources, best management practices and recreational uses) and watershed
processes (precipitation, or snow and ice melt derived runoff sediment re-suspension, dumping,
spills, wastcwater treatment plant failures, temperature fluctuations, and algal blooms) that may
lead to the presence of oocysts.
2. Ground Water
According to the 1994 Cryptosporidium Criteria Document, oocysts are found less frequently in
IS
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ground water than in surface water and consequently very few cryptosporidiosis outbreaks have
been traced to ground water contamination. Kramer et al. (1996), in a U.S. national survey for
the presence of Cryptosporidium oocysts in drinking water, showed that, of the five outbreaks
recorded from 1993 to 1994, two outbreaks resulted from untreated well water. Both of the
outbreaks occurred in Washington State, one in 1993, which accounted for 7 cases of human
cryptosporidiosis, and one in 1994 accounting for 104 cases (Rose et a/., 1997). Hancock et al.
(1998) recently reported a study of 199 grouhdwater samples tested for Cryptosporidium. They
found that 5% of vertical wells, 20% of springs, 50% of infiltration galleries, and 45% of
horizontal wells tested were positive for Cryptosporidium oocysts. This new data will force
environmental microbiologists and regulators to reassess previous assumptions that groundwater
is inherently free of parasites.
C. Occurrence in Soil
Most Cryptosporidium research has centered on detecting oocysts in either water or biological
samples. Limited studies have been performed to ascertain the presence or viability of
Cryptosporidium in soiL Transport of Cryptosporidium oocysts to water from fecal
contaminated soil within a watershed during weather events was suggested as the most probable
mechanism of source water contamination in several documented outbreaks (Kramer etal,
1996). Mawdsley et al (1996) reported on the vertical movement of Cryptosporidium oocysts
through intact 30 cm soil cores. Transport of oocysts through the soil cores was greater in silty
loam and clay loam than in loamy sandy soil, exactly the opposite of what the researchers
expected. Research funded by the American Water Works Association Research Foundation,
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aimed at creating a better understanding of watersheds and soil oocyst interactions is ongoing.
A new method was recently reported by Anguish and Ghiorse (1997) for examining
Cryptosporidium oocysts in media other than water. They evaluated a computer-assisted laser-
scanning microscope equipped for confbcal laser scanning and color video microscopy to
examine two agricultural soils, barnyard sediment and a calf fecal sample. The authors
concluded that this technique provides a better approach for oocyst identification and
enumeration, as well as for in situ assessment of cellular activity and viability. This technology
could have great utility for detecting Cryptosporidium in soil.
D. Occurrence in Air
The 1994 Cryptosporidium Criteria Document cited no data to show that Cryptosporidium is
found in ambient air. A review of the current literature indicates that this continues to be an
unresearched area.
E. Occurrence in Food and Beverages
The occurrence of several foodbome outbreaks in recent years has highlighted the role of
Cryptosporidium as a foodbome pathogen. The presence of Cryptosporidium has been
documented in. raw milk (Badenoch et aJ., 1990), unpasteurized apple cider (Millard et a/., 1994),
uncooked meat products (Casemore et aL, 1987), and in eight of eight types of fresh, Costa Rica
grown produce tested (Monge et aL, 1995). Refrigeration does not compromise oocyst viability.
• - *
The influence of temperature on oocyst survival is discussed in section ni.G. Recent studies
17
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evaluating oocyst viability (assessed by vital dye staining) showed greater than 85% of oocysts
present in beer and cola made from intentionally contaminated water lost their viability, while
only 35% of oocysts in reconstituted infant formula and orange juice lost viability (Friedman et
al., 1997). The authors speculate the decreassd pH of the carbonated beverages triggers
premature excystation
F. Specific Disease Outbreaks
1. Outbreaks Associated with Drinking Water
The 1994 Cryptosporidiwn Criteria Document describes recent waterbome disease outbreaks
attributed to Cryptosporidium. The 1990/1991 and 1992/1993 MMWR reports on waterbome
disease outbreaks prepared by the U. S. Center for Disease Control were not included in the 1994
document(Herwaldt et al, 1991 and Moore et al., 1993): Since 1994, numerous papers have
appeared in the literature more completely describing the previous outbreaks and documenting
new outbreaks.
Additional research articles have been published specific to the 1993 Milwaukee outbreak.
Addiss et al. (1996) reported on the effectiveness of point-of-use water treatment devices during
the Milwaukee outbreak. They concluded that using sub-micron point-of-use devices may
•
significantly reduce the risk of waterbome cryptosporidiosis. Rodman et al (1997) studied the
utility of monitoring sales data on non-prescription antidianheal medications to detect enteric
disease outbreaks. The information from this study showed mat, while the technique would have
been useful in detecting the Milwaukee outbreak, the costs incurred in collecting the data and the
18
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absence of increased sales during other outbreaks indicate that this tool will be of limited use in
other cities. Turbidity spikes at the Milwaukee Water Treatment Plant correlated strongly with
hospital visits for gastrointestinal disease prior to 1993 (Morris et al., 1998), indicating that
cryptosporidiosis was occurring in Milwaukee for more than a year before the outbreak.
Eisenberg et al. (1998) confirmed this finding and further concluded that 85% of the outbreak
infections could have been avoided if the smaller outbreak had been detected. Researchers
concluded after studying Milwaukee death certificates from before and after the outbreak that
waterbome outbreaks of cryptosporidiosis can result in significant mortality, particularly among
immunocompromised populations (Hoxie et al, 1997). Fox and Lytle (1996) published a
summary article outlining the results of the investigation of the Milwaukee outbreak by the
USEPA. Moss et al. (1998) reported on an outbreak of Cryptosporidiosis involving more than
half of the crewmembers of a Coast Guard cutter which had filled its water tanks with
Milwaukee water during March of 1993.
Duke et al. (1996) reported an outbreak of cryptosporidiosis in Northumberland, U.K. The
source water was a private untreated water supply that appeared to be contaminated by run-off
slurry from surrounding fields and/or lamb carcasses found in a collection chamber connected to
the water supply. Atherton et al (1995) also reported an outbreak of cryptosporidiosis in
northern England which was attributed to failure of the public drinking water system to remove
oocysts. The outbreak onset was characteristic of a point source infection occurring during a
period of heavy rainfall at the reservoir. Maguire et al (1995) reported on an outbreak in
\ • .
London, U.K., where 44 individuals were confirmed to have Cryptosporidium infections
19
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acquired from drinking tap water. Bridgman et al. (1995) reported on 47 cases of
crytosporidiosis linked to a well water supply in northwestern England.
Leland et al. (1993) wrote an article not cited in the 1994 Cryptosporidium Criteria Document
describing the Jackson County, Oregon outbreak of 1992 from an engineering perspective. In a
Florida outbreak, 77% of the counselors and campers at a day camp were infected from a
municipal drinking water supply (CDC, 1996). Goldstein et al (1996) reported on an outbreak
•of cryptosporidiosis in Las Vegas, Nevada, within the Lake Mead watershed area, where 78.2%
of the cases occurred in immunocompromised persons and 21.8% of the infected individuals
were immunocompetent individuals. The treatment system used state-of-the-art technologies and
chemical treatment Recognition of the outbreak was attributed to surveillance conducted by the
State of Nevada, where cryptosporidiosis is a reportable disease.
A number of overview articles concerning Cryptosporidiian outbreaks have been published since
1994. Rose et al. (1997) briefly described a number of outbreaks associated with drinking water,
and Solo-Gabriele and Meumeister (1996) presented an overview of U.S. outbreaks, supporting
their view that current practices and regulations are inadequate to protect consumers from
waterbome disease. The MMWR Surveillance for Waterbome Disease Outbreaks for 1993-1994
(CDC, 1996) lists 10 of 29 outbreaks associated with the protozoans Giardia and
Cryptosporidium. A national survey over a 2-year test period (1993 and 1994) identified five
outbreaks resulting in 403,271 cases involving Cryptosporidium oocysts in drinking water
(Kramer et al., 1996). Of this total, 403,000 were from the outbreak in Milwaukee, Wisconsin,
20
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103 were from Las Vegas. Nevada, and 27 were from an outbreak at a resort in Minnesota. All
three outbreaks were attributed to surface water as the sou: :e. The remaining two outbreaks
resulted from contaminated ground water: one from a private well in Washington State (resulting
in seven cases), and the other from a community well, also in Washington State (resulting in 104
cases). Notable outbreaks are summarized in Table 3.
Table 3. Outbreaks of Cryptosporidiosis Associated with Drinking Water
Year
1984
1986
1987
1991
1992
1993
1993
1993
1993
1994
1995
State
Texas
New Mexico
Georgia
Pennsylvania
Oregon
Wisconsin
Washington
Minnesota
Nevada
Washington
Florida
Number of
Cases
2006
78
12,960
551
15,000
403,000
7
27
103
104
72
Source
Groundwater(C)
Surface water(C)
River(C)
Groundwater(C)
Spring/river(C)
Lake(C)
Well(I)
Lake(NC)
Lake(C)
Well(C)
Not applicable
Deficiency
Sewage contamination
Untreated
Treatment deficiency
Treatment deficiency
Treatment deficiency
Treatment deficiency
Surface contamination
Unknown
Inadequate filtration
Sewage contamination
Cross connection
**
NC = Non-Community; C = Community; I = Individual
In addition to the outbreaks described in the literature, a newsletter called Cryptosporidium
Capsule has anecdotal information about suspected outbreaks in drinking water in Devon U.K.,
1995, drinking water in Maine, 1995, drinking water in Collingwood Ontario, 1996, and lakes in
Cranbrook and Kelowna BC, 19%, and a groundwater outbreak in the U. K. in 1997.
Two articles have been written about the inadequacies of current surveillance practices in
detecting and preventing Cryptosporidiosis from drinking water. Craun et cd. (1997) point out
that the colifonn test can no longer be used as the sole indicator of a water's microbiological
21
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safety. Frost et al. (1996) focused on the importance of epidemiological surveillance and
collaboration between water purveyors and community pu ic health departments.
2. Outbreaks Associated with Recreational Waters
Fourteen outbreaks of gastroenteritis related to recreational waters were reported by nine states
during 1993 and 1994 (Kramer et al, 1996). Ten of these outbreaks were caused by
Cryptosporidium or Giardia, with five specifically linked to Cryptosporidium. Three of the
Cryptosporidium outbreaks were associated with motel swimming pools; two were associated
with community swimming pools. All five pools were filtered or chlorinated, and one had a
malfunctioning filter. None of the other pools had identifiable treatment deficiencies. The
inability of chlorine levels normally used in swimming pools to kill Cryptosporidium, coupled
with poor pool filtration equipment maintenance practices, have been suggested as the primary
cause of swimming pool related cryptosporidiosis.
Outbreaks associated with recreational waters can be difficult to recognize because the
individuals using such facilities may reside in widely separated geographical areas. Forty-four
individuals contracted cryptosporidiosis after swimming in a Los Angeles pool where an
accidental fecal release occurred (CDC, 1990; Sorvillo et al:, 1992). McAnulty et al. (1994)
reported a community-wide outbreak of cryptosporidiosis in Lane County, Oregon that was
linked to swimming at a wave pool with m
-------
.occurred in a city T5 miles from Milwaukee about 30 days after the massive Milwaukee
outbreak. The authors suggest that increased attention should be given to preventing swimming
. - t
pool related outbreaks following outbreaks of cryptosporidiosis associated with water supplies.
Cryptosporidiosis associated with two pools in Dane County, Wisconsin was reported during the
summer of 1993 (CDC, 1994). Kramer et al (1998) reported on an outbreak with 38 infected
individuals who contracted cryptosporidiosis while swimming in a recreational lake. They
speculated that contamination of the lake came from either infected swimmers or run-off.
• .
3. Foodbome Outbreaks
Foodbome transmission of cryptosporidiosis has only rarely been reported. In October 1993, an
outbreak of cryptosporidiosis occurred among students and staff who consumed contaminated
apple cider while attending an agricultural fair in central Maine. This was the first large outbreak
in which foodborne Cryptosporidium could be identified and documented as the causative agent
(Millard et. al., 1994). A survey, completed for 611 (81%) of the estimated 759 attendees at the
fair, found 160 (26%) had primary cryptosporidiosis. Cryptosporidium oocysts were detected in
the stools of 50 (89%) of the primary and secondary case subjects tested. Oocysts were detected
in the apple cider, on the cider press, and in the stool specimen of a calf on the farm of the
supplier of the apples used to make the cider. The outbreak underscores the need for agricultural
producers to take precautions to avoid contamination of foodstuffs by infectious agents
commonly present in the farm environment The Minnesota Department of Health reported on
cryptosporidiosis in 50 attendees of a social gathering who ate a salad contaminated during
preparation by a day-care worker (CDC, 1996). Laberge et al. (1996) have prepared a review
23
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article in which they list foods associated with Cryptosporidium infections that includes;
unpasteurized milk,-sausage, raw beef, kefir, pelleted feed, silage, powdered milk, raw tripe, and
apple cider. Casemore (1990) referred to sausage, offal, and raw milk contamination by
Cryptosporidium, but did not link these food* to specific outbreaks. Harp et al. (1996) reported
that standard commercial pasteurization techniques kill 100% of C. parvum oocysts. Methods to
detect oocysts in food have not been optimized.
4. Outbreaks Among Travelers
The 1994 Cryptosporidium Criteria Document stated that cryptosporidiosis has emerged as an
important cause of traveler's diarrhea and indicated that illnesses frequently occur in travelers
visiting second and third world countries. However, reports since that time indicate that travelers
within developed countries such as the U.S. have also acquired Cryptosporidium infections.
t
During the Milwaukee outbreak in 1993, Milwaukee visitors became infected with
Cryptosporidium as a consequence of drinking water in that city. In addition, upon returning
home, they transmitted this parasite to members of their households (MacKenzie et a/., 1995b).
5. Outbreaks at Day-Care Centers and Prevalence of Cryptosporidiosis Among Children
The 1994 Cryptosporidium Criteria Document discusses the high prevalence of cryptosporidiosis
in children and notes that the evidence comes primarily from reports of diarrhea in day-care
centers. Cryptosporidiosis is now recognized as a significant disease in childcare settings
(Cordell and Addis, 1994). Additional data on the effectiveness of prevention and control
24
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strategies, as well as the economic impact of these outbreaks on the community, state, and
country, should be collected. There is evidence that cryptosporidiosis affects children in other
countries. In China and. Brazil, 42 to 58% of a cross section of children less than 16 years of age
were serologically positive for Cryptosporidium. Among a similar group in Virginia, less than
17% tested positive. The data from the review by Cordell and Addis (1994) indicate that in
impoverished communities, this parasitic infection is highly endemic and occurs in early
childhood. Single stool samples from 1,000 apparently healthy children (ages 6 through 14) in
Jordan showed that 4% of the children had oocysts and, among these children, 37% were
symptomatic (Nimri and Batchou^ 1994). Brandonisio et al. (1996) reported 7 (1.9%) of 368
children hospitalized (for unspecified reasons) in Italy tested positive for oocysts (359 of these
children were immunocompetent and nine were MTV-infected). Six of the seven children were
immune-compromised. Brannan et al. (1996) found that 12% of the children in Romania were
carrying C. parvum oocysts, although 73% of these children had either IgA or IgG antibodies to
this protozoa! parasite.
6. Sensitive (Immunocompromised) Populations
Immunocompromised populations are at high risk of infection and disease from drinking water
contaminated with Cryptosporidium oocysts. Clayton et aL (1994) studied 41 patients with AIDS
who had become infected with Cryptosporidium and found two significant patterns among these
individuals. In 61% of these patients, Cryptosporidia were present in the proximal small bowel
(i.e., upper small intestine) and they had severe clinical disease characterized by malabsorption
of nutrients. In the remaining 39% who had less severe disease, Cryptosporidium was seen only
25
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in the colon or the stool. An increased susceptibility to cryptosporidiosis in
immunocompromised individuals was reported in Kenya, East Africa where 42 to 44% of the
HIV-positive patients at the Kenyatta National Hospital tested positive for Cryptosporidium,
whereas only 8.6% of the non-AIDS patients carried this pathogen. In Zambia, 25.4% of the
HIV-positive patients who were symptomatic carried oocysts and had increased specific anti-
Cryptosporidium IgG and IgA antibodies, but there was no increase observed in specific IgM
antibodies (Cevallos et a/., 1995). This antibody profile suggests that the infection was of
prolonged duration.
The cryptosporidiosis outbreak in Las Vegas, Nevada, (Rocfer et a/., 1995) was recognized
primarily because of the high rate of incidence among the immunocompromised population and
because of state requirements for reporting of disease from this pathogen. Although the water
supply in this city was processed by a state-of-the-art system, the protection it provided proved
inadequate for the immunocompromised person. Research shows HTV-infected patients who
have cleared oocyst infections have much higher levels of specific secretory IgA levels than
AIDS patients with chronic cryptosporidiosis (Flanigan, 1994). This indicates either that
secretory IgA is involved in recovery from infection, or that it is the only marker for an effective
immune response at the mucosal surface.
In a study on asymptomatic carriage of int^ptjiial Cryptosporidium by immunocompetent and
immunodeficient children, the percentage of carriers among immunodeficient children was 22%
as compared to 6.4% among immunocompetent children (Pettoello-Mantovani et a/., 1995).
26
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However, the percentages of symptomatic children in these groups were more similar, with 4.4%
of the immunocompetent children and 4.8% of the inununodeficient children being positive for
oocysts, respectively.
Greenberg et al. (1996) reported on stool sampling from AIDS patients receiving chemotherapy
and/or radiotherapy. The study objective was to determine the yield of Cryptosporidium oocysts
in stools versus biopsies taken from the upper and lower intestines of these patients. Only 53%
of 106 patients were positive for oocysts when a single stool sample was taken, but detection
increased to 73% positives when multiple stool samples were taken. When sampling was by
terminal ileum biopsy, the number of positives increased to 91%. From these studies, it appears
oocysts may invade the small intestine in immunocompromised individuals with no oocysts
detectable in stool examinations. Thus, stool sampling can be expected to miss a substantial
number of Cryptosporidium infections in immunocompromiseoVAIDS patients.
In addition to gastrointestinal disease, AIDS patients can have other complications from
Cryptosporidium infection such as respiratory cryptosporidiosis (Mifsud et a/., 1994). Clavel et
al. (1996a) reported cases of intestinal cryptosporidiosis with pulmonary involvement in AIDS
patients who had diarrhea and were positive for Cryptosporidium oocysts.
Patients with cancer may be immunocompromised as a side effect of their therapeutic treatment
Therefore, these patients are likely to have an increased susceptibility to cryptosporidiosis.
Tanyuksel et al. (1995) examined 106 cancer patients, all of whom were receiving chemotherapy
27
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and/or radiotherapy and surgery, and found that 17% of these patients who had diarrhea were
positive for Cryptosporidium oocysts. They concluded that individuals who are compromised by
such treatment are a high risk for Cryptosporidium infections.
Logar et al. (1996) evaluated the occurrence of C. pdrvum in Slovenia and reported a higher
incidence of cryptosporidiosis in older patients and in. young children. The authors believed that
the infections in older patients and young children (median age 3 years) were due to lowered
resistance or immune response. In the older patients, Cryptosporidium infections appeared to be
a consequence of other diseases or secondary to irradiation or other immunocompromising
treatments common in this age group. Considerable evidence exists to show that immunological
deficiency is a natural consequence of aging (Miller, 1996).
G. Environmental Factors
Because Cryptosporidium oocysts are remarkably resistant to inactivation in the environment,
the survival of Cryptosporidium under a variety of environmental conditions has been evaluated
by a number of investigators. While the majority of these studies have considered the effects of
physical antagonism (freezing, heating, shearing, pressure, UV exposure, etc.), studies have also
been conducted to consider the role of microbial antagonists (microbial predation), chemical
antagonists (such as disinfection) and aging. At least two studies have considered various
combinations of environmental antagonists (shearing and disinfection, etc.). This section will
focus primarily on aspects of physical antagonists in the environment. The aspects of chemical
disinfectants will be covered under Section 4 - "Inactivation of Cryptosporidium Under Water
28
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Treatment Practices".
Robertson et al. (1992) evaluated the sensitivity of C. parvum oocysts to the freezing conditions
that occur in cold climatic regions or during the winter season. More than 97% of the test
oocysts were inactivated after incubation at -22CC for 18 days, suggesting that the levels of
viable oocysts in surface waters subjected to freezing might be influenced by seasonal
temperature variations. Cryopreservation studies conducted by Payer et al. (1991) and Payer
(1997) indicate that oocyst survival depends upon the temperature and duration of freezing
conditions, implying that C. parvum oocysts are not necessarily rendered noninfectious by being
frozen. Temperature stability studies were also conducted by Sattar et al. (in press, 1998) who
evaluated the freeze/thaw susceptibility of various preparations of oocysts including highly
purified preparations as well as infected calf feces. This study indicated that oocyst stability
under freezing conditions is at least partially dependent upon the surrounding matrix, with fecal
material conferring a cryopreservative effect on oocysts. In the absence of freezing conditions,
colder water temperatures tend to promote the survival of most microorganisms. C. parvum may
survive outside of the mammalian hosts for several months or more depending upon water
temperature (Straub et al., 1994). Under conditions of high temperatures, Payer (1994) indicated
that all evidence of C. parvum infectivity was lost within 60 seconds when temperatures
exceeded 72°C or when temperatures of at least 64°C were maintained for two minutes.
Limited studies have been conducted to evaluate the effects of physical shear on oocyst viability
to assess the potentially abrasive effects of oocyst contact with sand and gravel particles or
29
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through fast-flowing waters^ Parker and Smith (1993) demonstrated rapid inactivation of oocysts
in a mixed sand reactor. Sattar et al. (1998) conducted studies to evaluate synergistic effects of
mixed sand reactors upon disinfection efficiency with chlorine, and observed that shear stress
enhanced chlorine inactivation. Sattar ef al. (1998, in press) also evaluated the effects of
microbial predation upon oocyst survival and observed that oocysts incubated in dialysis
cassettes suspended in natural waters exhibited significantly longer survival times when bacterial
populations were excluded from the suspension water, implying that microbial predation may
play an important role in determining oocyst survival in natural waters.
H. Summary
In summary, cryptosporidiosis is zoonotic, widespread and often associated with surface water,
groundwater, recreational water,and contaminated food and drink. Outbreaks associated with
these sources continue to occur among travelers, children in day-care centers, and
immunocompromised as well as healthy individuals throughout the world. Eight valid species of
Cryptosporidia have been described in 78 mammalian species, 3CH- avian species, 57 reptilian
species, nine species of fish and two amphibians. Until more research is completed, public
health workers can do little more man speculate on the human infectivity of all the
Cryptosporidiwn species. Also, there is also little information currently published on the cross-
reactivity of the nine species to commercially available antibodies used for detection of the
organism. Immuno-compromised individuals such as those with HIV infections or AIDS, very
young children, the elderly, and individuals undergoing therapeutic treatment for cancer are more
likely to acquire an infection, develop cryptosporidiosis, and show more severe clinical
30
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symptoms. Deficiencies in water treatment systems are often cited as a major reason for
mtbreaks, and even the best of systems can be overwhelmed by a high density of oocysts
mtering the source waters over a short period of time. Infected individuals will shed oocysts in
Jieir feces and can be expected to transmit the infection to other family or community members.
[n addition, day care centers for children, because of their high' density of a sensitive population,
are a potential source for secondary spread of cryptosporidiosis from infected children to others
both within and outside of their households. Research on environmental factors confirms
previous work showing that oocysts are highly refractile to environmental stressors.
IV. Health Effects in Animals
A. Symptomatology and Clinical Features
The 1994 Cryptosporidium Criteria Document includes general information on the
symptomology and clinical features of cryptosporidiosis in a limited number of domesticated
mammals such as calves and lambs. Cryptosporidiosis has also been documented in many
additional mammals, both domestic and wild, as well as several species of birds, reptiles, and
fish (see section HI-A). Two recent review articles contain comprehensive information on the
symptomology and clinical" features of cryptosporidiosis in these different groups of animals.
(Payer, 1997; O'Donoghue, 1995).
In general, the development of Cryptosporidium depends on the species, age and immune status
of the host (Payer, 1997). Younger jtqjmafa and animala with less developed or compromised
immune systems are generally more susceptible to severe infection than healthy adult animals.
31
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In many cases, infected, healthy adult animals are asymptomatic or possess mild clinical signs
(O' Donoghue, 1995). A summary of the symptomology a 1 primary clinical features of infected
animals follows.
Mammals: Most clinical cases of cryptosporidiosis in mammals involve infection by C. parvum.
The most common feature of cryptosporidiosis in mammals is profuse watery diarrhea that may
be pale yellow in color and may have a distinct offensive smell. Other clinical signs of infection
include: dehydration, fever, anorexia, weight loss, weakness and progressive loss of condition
(O'Donoghue, 1995). Most animals will recover spontaneously within 1-2 weeks of infection.
Histopathological observations may reveal several lesions in the small intestine including mild to
moderate villous atrophy and loss of epithelial cells. Developmental stages of the parasite are
often seen within the small intestine and occasionally elsewhere (stomach, colon, liver, lungs)
(O'Donoghue, 1995). Payer (1997) contains detailed descriptions of the symptomology and
clinical features of cryptosporidiosis in several species of ruminant and non-ruminant mammals.
Birds: Two Cryptosporiditan species, C. meleagridis and C. baileyi, are known to cause
infection in birds. Avian cryptosporidiosis appears as either respiratory, enteric, or renal disease
(Payer, 1997; O'Donoghue, 1995). Normally, only one condition manifests during an outbreak,
and respiratory infections are more common than both enteric and renal infections (Payer, 1997,
O'Donoghue, 1995). Clinical signs of respiratory infections include rales, coughing, convulsive
sneezing, and dyspnea (O'Donoghue, 1995). Excess mucus may exist in the trachea, sinuses and
nasal passages, and fluid may be present in the air sac (Payer, 1997). Histopathological changes
32
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may include hypertrophy and hyperplasia of the respiratory epithelium with reduced or absent
ciliation (O'Donoghue, 1995). Parasites may be detected throughout the respiratory tract
including the nasopharynx, larynx, trachea and bronchi (O'Donoghue, 1995).
Clinical signs associated with enteric infections caused by cryptosporidiosis in birds include mild
to severe diarrhea, dehydration, depression, weight loss and weakness. Histopathology may ,
•
reveal atrophy and fusion of villi along with epithelial hyperplasia and hypertrophy and other
malformations (O'Donoghue, 1995). Parasites may be found primarily in the gastrointestinal
tract (O'Donoghue, 1995).
Renal infections in birds have been detected at necropsy only (O'Donoghue, 1995; Payer, 1997).
In these cases, the kidneys were pale in color (Payer, 1997) and enlarged (O'Donoghue, 1995).
Hypertrophic and hyperplastic epithelial cells were detected throughout the kidney
(O'Donoghue, 1995; Payer, 1997).
Reptiles: Cryptosporidiwn serpentis is the only valid, named species of Cryptosporidium
causing infection in reptiles, although up to five species may exist based on oocyst morphology
(Payer, 1997). Most reports of infections in reptiles have involved clinical or subclinical
infections in captive reptiles (especially snakes); only subclinical infections have been reported
in wild reptiles (Payer, 1997). Cryptosporidiosis in snakes is characterized by anorexia,
regurgitation, lethargy, firm midbody swelling, weight loss and death (Payer, 1997).
Interestingly, most infections in snakes have been detected in mature animals. In addition, most
33
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infections have been associated with chronic gastric disease as opposed to acute enteritis
common in mammals, mfections and intermittent oocyst shedding in snakes may last for several
months to two years (O'Donoghue, 1995). Histopathological observations have included
inflammation, hyperplasia and hypertrophy of the gastric glands (O'Donoghue, 1995).
Clinical features of cryptosporidiosis in lizards Dave involved primarily subclinical gastric
infections, while cryptosporidiosis in tortoises involved gastritis and regurgitation, or progressive
wasting (O'Donoghue, 1995).
Fish: Except for the original report of cryptosporidiosis in fish (Hoover et a/., 1981), infections
in fish have not been associated with clinical symptoms. The original report described a
progressive illness in a tropical marine fish (Naso lituratus) characterized by anorexia,
emaciation, regurgitation and passage of feces containing undigested food. Although
developmental stages of the parasite were found attached to the intestinal mucosa, no pathogenic
changes were evident in this fish. Since then, parasites have been detected in the intestines or
stomach of other fish, but few histopamological changes have been described (O'Donoghue,
1995).
B. Therapy
Treatment of cryptosporidiosis in animals involves a combination of prophylactic and
chemotherapuetic drugs along with other pfeventative measures. As stated in the 1994
Cryptosporidium Criteria Document, there is no approved effective treatment for
34
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cryptosporidiosis in animals. However, numerous drugs have been tested in studies that focused
both on the treatment of naturally acquired infections, and .• ic treatment or prophylaxis of
experimentally induced infections in animals. The majority of efficacy evaluations of agents
tested in animals have involved prophylactic as opposed to therapeutic drug regimens (Blagbum
andSoave, 1997).
A recent review article lists the numerous anticryptosporidial drugs that have been evaluated in
animals (Blagbum and Soave, 1997). The findings of the review article are summarized below.
Most studies have been conducted in laboratory rodents including mice, rats and
hamsters. Over 30 compounds have proven to be effective against cryptosporidiosis in
rodents including rnaduramicin, alborixin, lasalocid, and salinomycin. In some cases,
efficacies of the drugs tested exceeded 90% compared to control (nontreated) animals.
Several anticryptosporidial drugs have also been tested in ruminants. Among the drugs
demonstrating activity against Cryptosporidium parvum infections are paromomycin,
lasalocid, halo fuginone, and sulfaquinoxaline.
Anticryptosporidial drugs have also been tested in several other types of animals
including several species of snakes, birds, and reptiles, as well as mammals (pigs and
cats). There has been little success in identifying successful drugs in these animals,
however some success was achieved in treating cryptosporidiosis in snakes.
Prevention of cryptosporidiosis in animals is best achieved by eliminating contact with viable
oocysts. This is particularly difficult in settings with large numbers of animals such as farms or
zoos (Blagbum and Soave, 1997). To prevent infections in these types of settings, infected
animals should be quarantined in facilities that can be Cleaned and disinfected, contaminated
articles and clothing of animal care workers should be cleaned thoroughly or discarded, clean
food and water should be provided, and rodents and other wild animals should be restricted
access (Blagbum and Soave, 1997). Prevention can also be enhanced by ensuring that neonatal
35
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mammals receive adequate amounts of colostrum early in life (Blagbum and Soave, 1997).
Treatment of animals suffering from cryptosporidiosis is similar to that in humans. Namely,
rehydration with fluids and electrolytes along with antidiarrheal drugs and attempted
chemotherapy with anticryptosporidial drugs (Blagburn and Soave, 1997).
C. Epidemiological Data
The majority of epidemiologjcal data for cryptosporidiosis in animals are confined to
economically important livestock, especially ruminants (Casemore et al., 1997). Three extensive
reviews exist which describe the epidemiologjcal data available for animals including both
domestic and wild a^imal$. These reviews include information on the prevalence and spread of
cryptosporidiosis in many groups of animals including cattle, sheep, goats, pigs, horses, dogs,
cats, deer, mice and several other small mammals, as well as many species of birds, reptiles, and
fish (Casemore er al, 1997; Payer. 1997; O'Donoghue, 1995).
In general, clinical infections are seen primarily in neonates and immunocompromised animals
Age-related resistance has been documented in several species and the age of an animal upon
infection can greatly alter the severity of die infection, and die prepatent period (Casemore et al,
1997). Adult animals often appear asymptomatic even when shedding small numbers of oocysts
(Casemore et al., 1997; Payer, 1997; O'Donoghue, 1995). Serologic surveys in animals tend to
suggest much higher prevalence rates of cryptosporidiosis, especially in adults (Casemore et al,.
1997; Payer, 1997). This prevalence may be due to cross-reaction of other antibodies present in
36
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the sera rather than from actual infections (Casemore et ai, 1997).
Potential sources of infection in animals include other infected animals of {he same or different
species (e.g. it is believed that rodents can infect calves or cattle with C. parvum), mechanical
carriers such as insects, birds and humans, contaminated feed and water, and other contaminated
fomites (inanimate objects) such as bedding, brushes, shovels, and feed utensils (Payer, 1-997).
Additional epidemiological studies reported in the literature, but not mentioned in the review
articles, describe the prevalence of cryptosporidiosis in the following animals!
• cattle/calves (Payer et al, 1998; Olson et al, 1997; Perez et al, 1998; Pena et al, 1997)
• horses (Scholes et al., 1998; Bray et al.. 1998; Forde et al., 1998; Johnson et al., 1997)
• lambs (Bukhari and Smith, 1997)
• goats (Koudela and Jiri, 1997; Goyena et al, 1997)
• nonhuman primates (Muriuki et al., 1997; Majewska et al, 1997)
• rodents (Bajere/ al, 1997; Bull etal, 1998)
• chickens (Sreter et al, 1996)
• ostriches (Jardine and Verwoerd, 1997)
• pigeons (Rodriguez et al., 1997)
• catfish (Muench and White, 1997)
• muskrat (Petri et al, 1997),
• African hedgehog (Graczyk et al., 1998a),
• dugong (Hill et al, 1997),
« • deer (Majewska et al, 1997),
• slow loris (Majewska et al.. 1997) .
• white rhinoceros (Majewska et al., 1997)
• Indian elephant (Majewska et al., 1997)
• iguana (Fitzgerald et al., 1998).
D. Summary
Cryptosporidium infections have been documented in many different species of mammals, as
well as in several species of birds, reptiles and fish. In general, the severity of the infection
37
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depends on the species, age, and immune status of the host. Clinical infections are primarily
seen in younger animals and animals with compromised immune systems, while infected healthy
adult animals may be asymptomatic or possess mild clinical signs.
Most clinical cases of cryptosporidiosis in mammals involve infections by C. parvum. The most
common features of cryptosporidiosis in mammals are profuse, watery, diarrhea; dehydration;
fever; anorexia and weight loss. Two species of Cryptospdridium, C. meleagridis and C. baileyi,
are known to cause infections in birds. Cryptosporidiosis in birds is characterized by respiratory,
enteric, or renal infections. Respiratory infections, causing rales, coughing, convulsive sneezing
and diarrhea, and enteric infections, causing dehydration, weight loss and weakness, are the most
common. The majority of cryptosporidiosis infections in reptiles have been reported in captive
snakes. These infections, caused by C. serpentis, are characterized by anorexia, post prandial
regurgitation, lethargy and midbody swelling. The only clinical infection described in fish was
caused by C. nasonun, and was characterized by anorexia, emaciation, regurgitation and passage
of feces with undigested food.
Treatment of cryptosporidiosis in animala involves a combination of prophylactic and
chemotherapuetic drugs along with other preventative measures. Although there is no approved,
effective treatment for cryptosporidiosis in animate, several drugs have been tested in rodent and
bovine models and have shown substantial success. Several drugs have also been tested in
reptiles and-birds with limited success. Prevention of cryptosporidiosis in animals is best
achieved by eliminating contact with viable oocysts as much as possible. This involves isolation
38
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of infected animals and disinfection of all articles that come into contact with the infected
animals.
The majority of epidemiological data for cryptosporidiosis in animals are confined to
economically important livestock, especially cattle. There is also information available on cattle,
sheep, goats, pigs, horses, dogs, cats, deer, mice and several: other small mammals. These studies
show that the age of an animal can greatly alter the severity of the infection and the prepatent
period, and that the primary sources of infection for animals are other infected giyrnal^
mechanical carriers, contaminated feed and water, and other contaminated objects.
V. Health Effects in Humans
A. Symptomatology and Clinical Features
The clinical manifestations of cryptosporidiosis in humans are directly related to the
immunocompetence of the host, and may include profuse, nonbloody, watery diarrhea that
generally resolves spontaneously within around 48 hours, however variability in clinical
symptoms is appreciable. Diarrheal symptoms are generally not distinguishable from those
caused by other common enteric pathogens. Other symptoms reported by individuals afflicted
with cryptosporidiosis include abdominal cramps, vomiting, lethargy and general malaise.
Diarrhea results from a combination of enterocyte damage and physical blockage of the intestinal
villi, leading to a disruption in the normal balance of intestinal absorption and secretion (Clark
and Sears, 1996). The incubation period in humans is estimated to vary between 2 to 10 days
(Arrowood, 1997), with a mean incubation of approximately 7-9 days (Juranek, 1998).
39
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Symptoms generally last for 10-14 days in immunocompetent individuals; diarrhea may persist
for several weeks beyond the clearing of other symptoms. Severe protracted illness may result in
immunocompromised individuals, with mortality rates which may exceed 50% (Kramer, 1996).
)
Human volunteer studies have been conducted to assess the infectivity and dose-response of C.
parvum in humans (DuPont et al., 1995). Sixty two percent of subjects who ingested doses of
Cryptosporidium ranging from 30 oocysts to 1 million oocysts acquired infection. The infectious
\
dose causing disease in 50% of the population (ID^) for the Iowa strain of C parvum was
calculated at 132 oocysts in humans compared with an IDjo of 60 oocysts in neonatal mice;
however, the test strain of C. parvum in this case was adapted to a mouse model prior to
challenge studies, which may account for the disparity in EDjo values. The mean and median
incubation periods for cryptosporidiosis in the study were 9.0 and 6.5 days, respectively.
Infected humans developed clinical enteric symptoms that were associated with excretion of
oocysts, although one of the eleven subjects who did not pass oocysts passed a single soft stool
on day 10 and exhibited enteric symptoms on days 23 through 31. Symptoms of clinical illness
included abdominal pains, cramps, and diarrhea in six subjects; six had nausea; one reported
vomiting; and one had moderate dehydration. Table 4 summarizes the dosing data and related
*
infection rates from the study. Note that volunteers exhibiting enteric symptoms (diarrhea, loss
of appetite, etc.) did not in all cases test positive for cryptosporidiosis.
Table 4. Rate of infection and enteric symptoms as a function of Intended dosage
40
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Intended dose No. subjects No. (%)
Infected
30
100
300
500
?1000f
Total
5
8
3
6
7
29
1
3
2
5
7
18
(20)
(37.5)
(66.7)
(83.3)
(100)
-
^nteric symptoms No" (%) with
cryptospondiosi:
0
3
0
3
5
11
(37.5)
(50) '
(71.4)
0
3
0
2
2
. 7
(37.5)
(33.3)
(28.6)
* Linear regression analysis of the data yielded an r2 of 0.983 and an ED50 of 132 oocysts.
Source: DuPont et al. (1995)
Follow-up studies indicate that the number of excreted oocysts and the pattern and duration of
shedding vary widely among immunocompetent individuals (Chappell, et al., 1996). In the
volunteer challenge study, high variability in shedding patterns was observed, and oocysts were
observed intermittently in consecutive stool samples, implying that production of oocysts is not
uniform and may be influenced by unknown factors. These data may in part account for the
observation that fewer than half of the individuals who acquire illness during an epidemic
-, produce stools positive for Cryptosporidium when single samples are submitted for diagnostic
analysis.
B. Epidemiological Data
Although only 13 cases of cryptosporidiosis had been documented by the CDC in 1982, human
cryptosporidiosis has been reported in almost 100 countries spanning the globe since that time
(Ungar, 1990). Because C. parvum is ubiquitous, infects most mammals and is highly infectious,
all human populations are at risk to some degree (Griffiths, 1998). Screening of select human
populations was initiated in the United States during the 1980's, with special emphasis on
41
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children and immune suppressed individuals; however, determining the true prevalence of
cryptosporidiosis has proven challenging due to the fact tru: diagnostic methods exhibit limited
sensitivity, and since the majority of individuals who experience mild to moderate diarrheal
illness do not generally seek the services of a physician (Juranek, 1998).
In 1994, a workshop was organized by the National Center for Infectious Diseases (NCID) and
the United States Environmental Protection Agency (USEPA) to assist the Centers for Disease
Control and Prevention and state public health departments in providing guidance on public
health issues relating to waterborae cryptosporidiosis (Juranek et al., 199S; Addiss et al., 1995).
The workshop, titled "Prevention and Control of Waterbome Cryptosporidiosis: An Emerging
Public Health Threat", addressed the following topics: surveillance systems and epidemiolo1 gical
study designs, public health responses, cryptosporidiosis in immunocompromised individuals,
water sampling methods and interpretation of results. The recommended approaches to
surveillance include the following:
• Making cryptosporidiosis incidents or outbreaks reportable to CDC;
• Monitoring sales of antidiarrheal medication through local pharmacies (also described
by Rodman et al., 1997);
• Monitoring logs maintained by health maintenance organizations (HMOs) and
hospitals for complaints of diarrheal illness;
• Monitoring the incidence of diarrhea in nursing homes (also described by Proctor et
al., 1998);
• Monitoring laboratory data for Cryptosporidiim (also described by Proctor et al.,
1998);
• Evaluating distribution system design in selected cities; and >
• Providing prompt epidemiological assistance during outbreaks.
A cohort approach was recommended to facilitate epidemiological study of outbreak data, in
which blood tests are performed quickly to screen out negative subjects. Cohort analysis would
42
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demonstrate whether the exposure(s) was associated with subsequent infection or disease. Also,
since such research requires large sample sizes, the inclusion of blood tests would make this
approach feasible. Strategies recommended for the improvement of public health responses
included the identification of methods for rapid notification of the risks for waterbome
cryptosporidiosis to agencies, advocacy groups and the public.
The workshop participants emphasized that boiled water advisories are not essential in the
absence of supporting epidemiological information suggesting increases in diarrheal disease in
the community. On the subject of cryptosporidiosis in immunocompromised individuals, the
workshop concluded that immunocompromised individuals are no more likely than
immunocompetent individuals to acquire cryptosporidiosis in an outbreak. However, AIDS
patients, patients receiving treatment for cancer, recipients of organ or bone marrow transplants
and individuals who have congenital immunodeficiencies are at greater risk than
immunocompetent individuals for developing severe, life-threatening cryptosporidiosis if they
become infected.
Additional information on the epidemiotagic aspects of cryptosporidiosis in humans is provided
in the review of Casemore (1990). The distribution of Cryptosporidium in humans from several
countries is broken down by age group, non-human reservoirs, and routes of transmission. This
study found that when testing was performed for suspected infections,. 60% of the positive
findings occurred in children, and 30% in adults less than 45 years old. The disease occurred in
single sporadic cases, small cluster cases, and short clinical series. Cryptosporidium is the third
43
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or fourth most commonly identified pathogen in the world, and the reported rates are higher in
underdeveloped countries, especially in children. Seasonal and temporal trends vary from
country to country and occurrence may indirectly reflect rainfall and farming events such as
lambing. Domestic animals such as calves aud lambs in the U.K. are common zoonotic
reservoirs involved in occupational exposure, indirect zoonotic transmission, and contamination
of food (e.g., sausages, offal, and raw milk). Animals also contribute to environmental
contamination in sources such as watersheds, food crops, and swimming pools. For example, 70
cases of cryptosporidiosis in the U.K. resulted from exposure to a single swimming pool with
water contaminated by animal excreta. Pets, however, have not often been implicated as a source
of infection and are not considered a major risk factor for acquisition of cryptosporidiosis
(Glaser, 1998).
Person-to-person transmission of cryptosporidiosis may be associated with nosocomial (hospital-
acquired patient-to-patient) infections, sexual transmission, and traveler's diarrhea (Casemore,
1990). Transmission is affected by ethnic and dietary differences (e.g., Muslims exhibit a lower
prevalence than many other ethnic groups). Casemore observed that the severity of disease from
infection is greatest among children "**<*«*• 5 years of age apd among immunocompromised
patients (e.g., AIDS or cancer patients), and that the impact is greatest in third world countries.
C. Treatment: Clinical Laboratory Findings and Therapentie Management
Cryptosporidiosis is self-limiting in most immunocompetent patients and also in many
immunocompromised patients. Recommended management of Cryptosporidiwn infected
44
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patients includes careful monitoring of hydration and electrolyte balance with oral or intravenous
hydration and nutrition as necessary. Patients with cryptosporidiosis-induced renal failure, liver
disease, etc. may require treatment with a macrolide antibiotic (Griffiths, 1998). Antimotiliry
agents (i.e. opiates or somatostatin and its analogues) may be helpful to.prevent dehydration.
Patients co-infected with HTV should continue or begin anti-retroviral therapy to suppress viral
replication and boost CD4 cell counts. Patients currently undergoing chemotherapy or
immunosuppressive therapy should be removed from treatment (Griffiths, 1998).
Since the publication of the 1994 Cryptosporidium Criteria Document, several new treatment
strategies have been pursued. However, the search for a consistently effective treatment has
fallen short. A comprehensive review of these efforts was published by Blagburn and Soave
(1997) and O'Donoghue (1995). The use of spiramycin, a macrolide antibiotic, was described in
the 1994 Cryptosporidium Criteria Document as showing limited success in the treatment of
cryptosporidiosis. Other macrolides have been evaluated including clarithromycin (Jordan
1996), erythromycin (Connelly et
-------
1995). Singefungin showed in vitro activity but requires further evaluation in humans (Favennec
et al., 1994). lonophores with anti-coccidiocidal properties .ve also been evaluated for
treatment of cryptosporidiosis. The most promising, maduramycin and alboroxin, have shown
96% and 71% decreases of oocysts in immunodeficient mice respectively (Mead et al., 1995).
Bismuth subsalicylate (BSS), which has shown positive activity with a number of other diarrheal
diseases, was evaluated for treatment and prophylaxis in challenged and infected
immunodeficient mice (Greenfield et al., 1996). Results were encouraging and further study with
human patients was warranted. The use of hyperimmune bovine colostrum is thoroughly
described by Crabb (1998) and shows potential for safe and effective treatment However,
considerable variation in efficacy has been noted (O'Donoghue, 1995). In summary, although
there have been new discoveries of potentially effective therapeutic agents for humans, further
research is required.
D. Mechanism of Action
The 1994 Cryptosporidium Criteria Document reports that the pathogenic mechanisms of
Cryptosporidium are not known. While the pathogenesis remains unclear, more recent work has
helped elucidate the process: Cryptosporidium sporozoites and merozoites invade the absorptive
cells covering the small intestinal villi, damaging and eventually killing the enterocytes. Forney
et al. (1996) suggested proteolytic activity is involved in the infectivity of C parvum based on
the interaction between human alpha-1-antitrypsin (ATT) and parasite subcellular components.
This suggests the use of serine protease inhibitors may be useful as a therapeutic strategy. Riggs
etal. (1996) identified antigens that may have a critical role in sporozoite infectivity and
-------
therefore, may be suitable molecular targets for passive or active immunization against
cryptosporidiosis.
Diarrhea occurs when intestinal absorption is impaired or secretion is increased. When killed
enterocytes are extruded from the intestinal epithelium, crypt cells are signaled to repair the
damage. Additionally, there is infiltration of prostaglandin (PGE) secreting inflammatory cells.
Both crypt cells and PGE are known to stimulate chloride ion secretion; in addition, PGE inhibits
NaCl absorption (Clark and Sears, 199.6). This disruption in the absorption/secretion balance can
lead to diarrhea. Clinical studies in C. parvum infected piglets (Argenzio et a/., 1993) have
suggested that Cryptosporidium-wduccd diarrhea is of a secretory nature. However, Kelly et al.
(1996) conducted perfusion studies to measure water and electrolyte transport in vivo in five
HTV-cryptosporidiosis patients and nine healthy volunteers. There were no differences in net
water, sodium, and chloride movement in the jejunum of the two groups. Neither was there
evidence demonstrating that cryptosporidial diarrhea was due to a secretory state in the proximal
small intestine. Other studies have suggested that Cryptosporidium-mduccd diarrhea may be
caused by a toxin (Guarino et aL, 1994; Guarioo et al., 1995). A thorough review of
cryptosporidial pathogenesis can be found in Clark and Sears (1996).
E. Immunity
The importance of cellular immunity in resolving Cryptosporidium infection is highlighted by
the contrasting ability of immunocompetent and immunocomptomised individuals to resolve
infections. While depletion of CDS cells (Ungar, 1990), NK cells (Rasmussen and Healy, 1992),
47
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tumor necrosis factor (McDonald et al., 1992), mast cells (Harp and Moon, 1991) or interleukin-
2 (Ungar et ai, 1991) did not show enhanced infection in mice, removal of CD4 cells and/or
gamma Interferon caused severe chronic infections (Ungar et al., 1991). Additionally,
Cryptosporidium-infected immunodeficient mice reconstituted with spleen or lymph node cells
from irnrnunocompetent Balb/c mice, were able to recover from infection, but upon depletion of
the CD4 T cells from the donor, the curative effects were abrogated (Kuhls et al., 1996). In
humans, HIV infected patients with CD4 counts of 180 cells/mm3 cleared the infection in 4
weeks while of those with lower counts, 87% developed chronic disease (Flanigan et al., 1992).
Little is known about the gut mucosal response to the parasite. Wyatt et al. (1996) used a bovine
animal model to examine mucosal immunity during cryptospohdiosis, and showed that ileal
intraepithelial T lymphocytes are activated coincident with enteric disease. This suggests the
importance of cell-mediated activity during Cryptosporidium infection.
Specific IgG, IgM, IgA, and IgE antibodies have been detected in patients with confirmed
Cryptosporidium infection (Ungar et a/., 1986; Casemore, 1987; Laxer et a/., 1990; Kassa et al.,
1991). The presence of local and secretory antibodies has also been confirmed (Laxer era/.,
1990); however, the role of these antibodies in combating infection is unclear (O'Donoghue,
1995). Kapel et al. (1993) used a time-resolved immunofluorometric assay to determine the
presence of Cryptosporidium antibodies in 12 UIV-Cryptosporidium-vafiocttd patients. These
patients displayed marked elevation in atid-Cryptosporidium IgA and IgM antibody titers. These
high antibody titers were not correlated with the gravity of infection in terms of oocyst shedding.
Also, there was no evidence of protection even though there was a mucosal immune response.
-------
Studies comparing C. parvum infections in B cell-depleted mice showed that infections were
similar to those in normal mice (Taghi-Kilani et al., 1990).
There is evidence for protective immunity to cryptosporidial infection. Repeat infections in dairy
cattle workers occur but are generally much milder than the first infection (Reese et al 1982).
Permanent residents in areas where cryptospordiosis is common often acquire mild or
asymptomatic infections, however, visitors may become very ill. (Current, 1994).
Okhuysen et al. (1998) reports on the rechallenge of human volunteers previously infected with
Cryptosporidium. Nineteen healthy, immunocompetent adults were challenged with 500 oocysts
one year post primary infection. Fewer subjects shed oocysts after the second exposure (16% vs.
63%). Although the rates of diarrhea were similar, the clinical seventy determined by the number
of unformed stools passed, was less. The number of IgG and IgA seroconversions increased but
the antibody response did hot correlate to the presence or absence of infection.
F. Chronic Conditions
Duration of illness in cryptosporidiosis patients is influenced primarily by the immune response
of the individual, with most immunocompetent individuals overcoming the acute enteritis stage
within two weeks. Immunocompromised individuals generally present with chronic enteritis
which may last as long as the immune impairment Irnmunocompromised populations include
patients undergoing chemotherapy for treatment of neoplasms, persons undergoing immune
suppression treatment to prevent rejection of skin or organ transplants, malnourished individuals,
patients who present with concurrent infectious diseases such as measles, the elderly, and AIDS
49
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patients. Chronic illness may manifest itself as a series of intermittent episodes or may be
persistent. A functional threshold has been established using CD4+ cells to define the
probability that infection will resolve; patients presenting with CD4+ counts exceeding 200/iL
can generally expect to clear the infection, while those with CD4+ counts falling below this level
may suffer chronic infection (Payer, 1997).
G. Summary
The primary symptom of cryptosporidiosis in humans is fulminant watery diarrhea. The limited
data available from human volunteer feeding studies indicate a mean ID^ of 132 oocysts. Recent
research suggests that the pathological response to Cryptosporiditan is initiated when the
sporozoites and merozoites invade and kill the intestinal epithelial cells (enterocytes). The
enterocytes are extruded from the intestinal epithelium signaling epithelial repair and infiltration
of inflammatory cells. The host responds with the production of antibodies as well as
intraepitheleal T lymphocytes. The infection may cause malabsorptive and secretory diarrhea.
i
Management of infected patients includes maintenance of fluid and electrolyte balance. Patients
with unresolved infection may be treated with macrolides and anti-motility agents. A previous
Cryptospohdium infection does not confer resistance to reinfection: although reinfection will
result in fewer episodes of diarrhea. A workshop panel focusing on the application of
epidemiologic information on Cryptosporiditan recommended making surveillance information
available to the appropriate federal agencies, HMOs, hospitals, and others who play roles in
maintaining the public health. Panel recommendations also included performing cohort analysis
of outbreaks using information such as blood tests in populations where exposure to
50
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Crypiospondium is likely. In addition, workshop participants suggested strategies to improve
public health, which included identifying methods for informing agencies, advocacy groups, and
the public risks for waterborae Cryptosporidium transmission, and providing the public with
information on dealing with a known or suspected contamination of drinking water source.
VI. Risk Assessment
The International Life Sciences Institute (TLSI) Risk Science Institute (RSI) Pathogen
Assessment Working Group (1996) defined pathogen risk assessment as a process that evaluates
the likelihood of adverse human health effects following exposure to pathogenic microorganisms
in a medium such as water. Until recently, most formal risk assessments on pathogenic
microorganisms such as Giardia and Cryptosporidium have utilized a conceptual framework that
was developed to assess risks due to chemical exposures; however, it is notable that the .
framework for assessing chemical exposures does not account for a number of microbial
considerations including pathogen-host interactions, secondary spread of microorganisms, short-
and long-term immunity, the carrier state, host animal reservoirs, animal-to-human transmission,
human-to-human transmission, and conditions mat lead to propagation/multiplication of
microorganisms. Although significant data gaps exist in the complete characterization of the
pathogenesis of Cryptosporidium, risk assessment approaches will enable health officials to
communicate with water utilities, interpret water quality surveys, and define the adequacy of
treatment at acceptable public health risks (Rose et al., 1997).
A. Experimental Human Data
51
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The 1994 Cry-ptosporidium Catena Document referred to human volunteer studies by DuPont et
al. (1995) that were in progress at the time of manuscript preparation. The aim of the study was
to determine the infectivity of C. parvum in healthy adults in order to predict the likelihood of
enteric infection following exposure to contaminated drinking water. The results of this study
have since been published and are presented in Table 4 of Section V-A. Among 29 subjects who
were provided 30 or more oocysts, 62 percent became infected, with a range of 20% (30 oocysts
ingested) to 100% (>1000 oocysts ingested). Illness lasted 58 to 87 hours with four to eleven
loose stools produced per day, suggesting that human-to-human transmission of C parvum is
more likely to occur 2.5 to 3.5 days following infection in the primary case. Linear regression of
the dose-response data indicated a human IDX of 132 oocysts. The research team concluded that
a 'low' dose of C. parvum oocysts was sufficient to cause infection in healthy adults with no
serologic evidence of past infection by this parasite. .
Follow-up analysis of the 18 individuals who presented with cryptosporidiosis infections
indicated appreciable variation in the numbers of oocysts excreted and in the duration of
excretion (Chappell et al., 1996). Only 1 of the 7 volunteers who exhibited diarrhea had oocysts
in every stool during the shedding period, and oocyst numbers in consecutive stool samples
collected from individual volunteers varied as much as 30-fold. Hence, production of oocysts
during infection may be intermittent, which may help to explain the observation that fewer than
50% of the individuals acquiring illness during waterbome outbreaks produce Cryptosporidium-
posin've stool samples when only one stool is examined.
B. Experimental Animal Data
52
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^ number of dose-response studies using monkeys, gnotobiotic lambs and several strains of mice
re presented in the 1994 Cryptosporidium Criteria Document. Casemore (1990) reported a 2- to
•-day incubation period for C. parvum and an excretion period of about 8 to 14 days in animals
species not identified). DuPont et al. (1995) reported that the IDj0 for the Iowa strain of C.
mrvum oocysts necessary to infect the neonatal mouse was 60, which is approximately half of
he ED50 required to produce infection in humans (132 oocysts). The relative similarity among
nfectious doses in mice and humans may suggest that the mouse model is potentially useful in
defining certain human risks associated with cryptosporidiosis.
C. Environmental Factors
1. Prevalence in Surface Waters
Cryptosporidium oocysts are more likely to occur in surface waters than in ground water, as
described in Section EH of this document and in the 1994 Cryptosporidium Criteria document.
Since the majority of source waters used for production of drinking water are surface supplies,
and because these waters are more vulnerable to direct contamination from sewage discharges
and runoff, the presumption has been mat Cryptosporidium wilMikely be more common in these
supplies. Walk's et a/. (1996) found Cryptosporidium oocysts in 6.1% of raw sewage samples,
4.5% of raw water samples, and 3.5% of treated water samples in Canada. Analyses of raw
sewage samples indicate that Cryptosporidium was present in more man 50% of samples where
one or more liters was examined (Bukhari et al, 1997; Zuckerman et al., 1997). Ong et al. (19%
a and b) studied the source of parasite contamination in different watersheds to assess the
potential impact upon drinking water sources, and found that water from rivers flowing through
53
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cattle pastures in British Columbia exhibited higher Cryptosporidium counts than did water from
a protected watershed. Lisle and Rose (1995) reviewed 25 monitoring studies and report
Cryptosporidium in as much as 87.1% of the source waters, with levels of oocysts as high as 4.7
per liter. LeChevallier et al. (1995) observed oocysts in 60.2% of surface waters tested in North
America. Crockett and Haas (1995) investigated three water treatment plants located in a major
metropolitan area where watershed monitoring was conducted over one year. They found that
creeks which empty into a river were a primary source of parasites in the urban river-derived
water. Each creek's parasite density was greatly influenced by suburban wastewater discharges,
although the authors did not rule out other sources which might have influenced the microbial
density in the river. The implications of each of these studies should be tempered with the
knowledge that erratic and insensitive oocyst detection methods may contribute to significant
underestimation of the true level of oocyst contamination in surface waters.
2. Oocyst Survival
/
The resistance of oocysts to inactivation is an important factor in determining the extent to which
humans or reservoir/host animals can become infected; however, most detection methods for
Cryptosporidium can not distinguish between viable and nonviable oocysts. Furthermore, some
detection methods may render oocysts nonviable due to chemical antagonism during sample
processing. Additionally, the following practices must be characterized more completely in
order to evaluate the risk of contracting cryptosporidiosis: sewage discharges, watershed
protection, agricultural practices, wildlife management, strain specificity (animal or human) and
oocyst survival under various environmental conditions. The survival of oocysts under various
54
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environmental pressures has been evaluated by several groups and is described in Section III-G
"Environmental Factors". The majority of these survival s-udies have relied upon animal
infectivity or in vitro excystation to assess changes in oocyst viability in natural waters. A
complete discussion on methods for the assessment of oocyst viability is also provided in Section
VII. A.2 - "Detection of Cryptosporidium in Water".
3. Cryptosporidium in Drinking Water
Identification of the specific pathogen and route of infection represent early steps in the risk
assessment process. The primary route of human infection by C. parvum involves ingestion of
contaminated drinking water and food (Casemore, 1990); other routes of transmission are
described hi Section n-D. One of the difficulties in conducting risk assessment of
Cryptosporidium lays in the uncertainty associated with the level of infectious oocysts in
drinking water supplies. There are also viability, infectivity and specific epithet issues. Surveys
such as those described in Section ffl-B have indicated the numbers of ooeysts that may occur in
drinking water, however, the impact of these oocyst levels may be underestimated given the
number of gastrointestinal illnesses that occur each year whose etiology is undetermined.
No cases of cryptosporidiosis were reported in the waterbome disease outbreak survey published
by the CDC from 1989 to 1990, however the large number of cases of acute gastrointestinal
illness (AGF) of unknown etiology may have included illness caused by Cryptosporidia. Of the
total number of AGI cases reported in 1989 and 1990,56% (2,402 of 4,288) were unexplained
(Herwaldt et a/., 1991). In the U.S. surveillance report from 1991 to 1992 (Moore et a/., 1993),
55
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approximately 20% of the total enteric illness cases were caused by Cryptosporidia. Cases of
AGI represented 76.5% of the total enteric illnesses reported from 1991 to 1992, many of which
were likely caused by Cryptosporidia. These observations suggest that efforts to identify
Cryptosporidium as the etiological agent during outbreaks frequently fail.
D. Epidemiologic Considerations
1. Population at Risk and Survey Methods ,
The USEPA estimated in 1993 that approximately 155 million people may be exposed to
Cryptosporidium in contaminated water every year, however, the estimated population at-risk
<. •
cannot be reconciled with the reported numbers of infected individuals even when correction
factors for asymptomatic infections and underestimated environmental levels are included (1994
Cryptosporidium Criteria Document). Factors contributing to the disparity between the
environmental occurrence data and the clinical data are outlined in the 1994 document;
additional factors are described in Section V-B of this document Hence, it is difficult to
generate valid figures to describe the risk of acquiring cryptosporidiosis.
In the United States, the incidence of cryptosporidiosis in the U.S. can be assessed through
surveillance reports and documentation of outbreaks that appear in the published literature. The
•^
• , •
CDC currently maintains an active surveillance system for cryptosporidiosis aimed at collecting
information on both outbreak and non-outbreak related cases. Cases are reported using standard
forms which originate with state and local health departments, but the agency also receives
updates from federal agencies, and occasionally from private physicians. While
cryptosporidiosis is not a reportable disease in all states (CDC, 1994), it is designated as
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notifiable at the national level as of January 1, 1997.
A number of reports describe the severe effects of cryptosporidiosis in children (Molbak, 1994),
particularly malnourished infants, although it is generally difficult to determine if malnourished
children are at higher risk of chronic cryptosporidiosis due to immune suppression, or if
cryptosporidiosis is an independent risk factor for becoming malnourished (Griffiths, 1998).
Reports from the U.K. show the occurrence of cases to be highest among children less than 5
years old (Atherton etal, 1995). Other groups at risk for cryptosporidiosis are secondary
contacts, farm workers (Lengerich et al., 1993), immune-suppressed individuals, those Irving in
institutional settings such as group homes and orphanages (Heald and Bartlett, 1994), and
international travelers who visit regions where cryptosporidiosis is endemic. There is little
evidence that risks differ between genders (Meinhardt et a/., 1996).
E. Risk Assessment Modeb
Since Cryptosporidiwn monitoring does not presently give a true picture of the number of
infectious particles and the efficacy of removal of oocysts from treated drinking water, risk
calculations involve many uncertainties. In order to develop risk estimates for specific pathogens
. *
such as Cryptosporidiwn, reliable dose-response data are required. The human dose-response
data currently available is limited to the studies of DuPont et a/. (1995) and Chappell et al.
(1996); however, an exponential dose response model has been developed based upon this
dataset and a number of assumptions governing the Milwaukee epidemic of cryptosporidiosis
(Haas, 1994). This model describes the probability of infection given exposure (PJ as follows:
Equation 1. P,-!-*1*1
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The values of r and N represent the fraction of ingested oocysts which must survive to establish
infection, and the daily exposure, respectively. A value of r specific for Cryptosporidium has
been derived (r=0.0047). Oocyst concentrations were derived for exposures ranging from I to 30
days and a P, range of 0.14 to O.S2. Exposure values predicted according to this model ranged
from a minimum of 0.16 oocysts per liter (P,=.14) to a maximum value of 79 (P,=0.52).
Cryptosporidium concentrations in the Milwaukee water supply were derived by considering the
numbers of oocysts present in ice produced during the epidemic and correcting for losses
associated with poor analytic recovery efficiencies. According to the exponential model,
Cryptosporidium exposure during the epidemic ranged from 0.6 to 1.3 oocysts per liter. Haas
also applied the risk assessment model to consider data from previous water monitoring studies,
and has calculated that the annual risk of contracting cryptosporidiosis in the United States may
range from 1 in 100,000 to 4 in 1,000.
Perz et al. ( 1 998) applied a risk assessment approach to examine the role of tap water in
waterborne cryptosporidiosis. This model was based upon the assumption that clinical infection
results from exposure to a single oocyst, and utilized a theoretical C parvum density in drinking
water of 1 oocyst per 1 ,000 liters. Uncertainties in the model were analyzed by considering
ranges and distributions among the input variables. The number of annual Cryptosporidium
infections (Ij) was estimated according to the following relationship:
Where C = concentration of C. parvum per liter of water
j — population subgroup (categorized by age and AIDS status)
POPj = number of persons in the exposed subgroup
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Q = annual water intake in liters per year
r = single organism infectivity (infection/organism/person)
The model was applied to derive the median annual risk of infection among immunocompetent
individuals (1 in 1,000 probability using the assurried exposure level of 1 oocyst per 1,000 liters).
The dominant parameter contributing to uncertainties in the risk assessment was oocyst
concentration, suggesting that improvements in Cryptosporidium monitoring techniques in
drinking water will facilitate future risk assessment efforts.
The ELSI workgroup (1996) has developed guidance for an infectivity model that may also be
useful if adequate detection methods are available. Since exposure analysis for humans and
animals is an essential step in risk assessment, developing animal systems that can be used to
estimate infectivity will be essential for modeling because detection of oocysts without
knowledge about their infectivity is inadequate. Varga et al. (1995) reported on a model using C.
baileyi infections in chickens. The authors developed a quantitative method to assess oocyst
* N
shedding which was based on the rather slow sedimentation of oocysts. A threshold of
sensitivity of between 5,000 and: 10,000 oocysts per gram of feces was reported for the
technique. Triplicate assays over a wide range of oocyst concentrations (i.e., 2,500 to 1.25 x 106)
were in good agreement Considering the level of oocysts excreted by infected humans, this
model system appears to have adequate sensitivity for demonstrating infection. This model's
sensitivity threshold may be adequate for assessing stool samples of infected individuals, but is
inadequate for monitoring oocysts in water samples for potential infectivity, hence, further
development of sensitive infectivity models is needed.
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F. Federal Regulations
Cryptosporidium as a primary contaminant is currently not specifically regulated by the federal
government. However, since the 1994 Cryptosporidium Criteria Document was published there
has been a substantial amount of federal regulatory activity associated with Cryptosporidium in
drinking water primarily prompted by the 1996 Amendments to the Safe Drinking Water Act.
USEP A has initiated numerous stakeholder meetings and has several new and pending rules that
are being driven by the threat of Cryptosporidium water-borne disease. Lykins and Clark (1994),
Berger (1996) and Pontius (1998) have published exhaustive review articles summarizing these
activities. The most significant promulgated and pending rules are the Information Collection
Rule and the Enhanced Surface Water Treatment Rule, respectively.
On May 14,1996 the USEPA promulgated the Information Collection Rule (USEPA, I996a).
The rule requires those water utilities serving more than 100,000 people to test source water and
finished water for an eighteen-month period (the process began in July 1997). The monthly
testing includes a variety of analytes including conforms, turbidity, and Cryptosporidium, etc.
The rule is primarily a research effort and the USEPA intends to use the information for the
development of future rules. The USEPA outlined its intended approach for regulation in the
November 3, 1997 Federal Register (USEPA, 1997). In this document, USEPA states that a final
Interim Enhanced Surface Water Treatment Rule is scheduled for November, 1998. The rule
will set a maximum contaminant level goal (MCLG) of zero for Cryptosporidium. While
Cryptosporidium is primarily considered a threat to surface water supplies, recent research with
vertical wells, springs, and infiltration galleries suggest that groundwater sources may also
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harbor the parasite (Hancock et al., 1998). The USEPA is currently convening stakeholder
meetings preliminary to expected promulgation of the Grc andwater Disinfection Rule in early
1999.
G. Summary
Environmental risk assessments have historically relied upon a conceptual framework based
upon exposure to chemical pollutants and are generally considered inadequate for pathogen risk
assessment. Although most human populations are assumed to be at risk for cryptosporidiosis at
least to some degree, it has been difficult to collect accurate figures describing the prevalence of
infection in humans due to limitations in public health reporting systems and due to incomplete
characterization of oocyst speciation and survival under various environmental conditions. Risk
assessment for waterborne cryptosponeuosis has also been limited due to the absence of dose-
response data from volunteer challenge. 3ata from animal infectivity studies suggest that
infectious doses may be lower (IDjo or Ooocysts). Risk models have been developed to
assess the probability of cryptosporidiosis infection based upon assumptions governing the levels
of infectious oocysts in drinking water and upon the data generated from volunteer challenge
studies. The estimated annual risk of waterbome cryptosporidiosis based upon these models
ranges from 1 in 1,000 to 1 in 100,000.
VIL Analysis and Treatment
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A. Analysis of Water
The Information Collection Rule modified American Society of Testing and Materials method
(ASTM ICR), described in the 1994 Cryptosporidium Criteria Document, remains the most
commonly used method for monitoring Cryptosporidium in water. The method, which detects
both Cryptosporidium and Giardia, is tedious and requires high levels of technical expertise.
While reproducible and sensitive for Giardia detection, the method is not reproducible between
laboratories, and suffers from low sensitivity for Cryptosporidium detection (Clancy et al., 1994;
USEPA, 1996b).
i
The accuracy and reproducibility of method development and comparison studies was
questioned by Klonickj et al. (1997). The authors noted that vital information regarding oocyst
source, purification method, age, storage conditions, and enumeration method was inadequately
cited in most publications. Further studies compared three enumeration methods;
hemacytometer, membrane, and well slide, and showed statistically significant differences
among the methods. The authors demonstrated the effect of counting method variability on
recovery values and stressed the importance of standardizing method comparisons and providing
adequate information in publications to allow valid comparison studies.
LeChevallier et al. (1995) investigated numerous methodological variations in the collection,
elution, and concentration portions of the assay to determine their influence on recovery of
Cryptosporidium in seeded tap and Mississippi River water samples. The authors tested
multiple filter types, centrifugation speeds, density gradient modifications, elution buffer pHs,
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and centrifuge bottle types. Although they identified several sources of oocyst loss, their
modifications resulted in variable recovery (7-129%).
Before detection data can be applied in a meaningful way, it must be adjusted to reflect the limits
of the methodology used to collect it. For example, if the method can detect 1,000 oocysts in
100 L of water and a sample contains only 50 oocysts, a result showing 0 oocysts could be
obtained. The results, therefore, must be reported to say less than 1,000 oocysts are present.
Harris (1995) developed a method detection limit for IF A analysis of Cryptosporidium and
Giardia. The MDL^ in this study is defined as the minimum concentration that the procedure
can analyze with 95% confidence that the organism will be detected.
Each water type presents unique problems in collection, concentration, isolation, and staining
techniques. Organic and inorganic particulates present in the water can clog filters or interfere
with other portions of the analysis such as clarification or antibody staining. Additionally, the
ASTMICR immunofluorescent staining methods do not give information regarding viability and
infectivity and speciation which is essential to assessing public health threat The reader is
advised to review Table 3.19 in Frey, e*. a/. (1998) for a complete synthesis of research status of
Cryptosporidium detection methods. Efforts to develop improved Cryptosporidium collection,
concentration, and detection methods are described below.
1. Collection of Cryptosporidium from Water
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Filtration based concentration methods
The 1994 Cryptosporidium Criteria Document describes the most common collection method
using polypropylene wound yarn filter with 1 um porosity. This collection method remains the
most popular and can be used for large volume samples with varying turbidity. LeChevallier et
al. (1995) tested ten cartridge filters varying in composition (polypropylene, nylon, rayon, and
cotton) and porosity (0.5 and 1.0 um) for removal of Cryptosporidium and Giardia-sized
particles. Although retention of 3 and 7 um particles was greater using filters with a 0.5 um
porosity, they tended to clog, limiting the amount of water that could be filtered. The use of
cotton, nylon, and rayon filters led to the most efficient removal of Cryptosporidium and
Giardia-sized particles. To further minimize losses during filtration, the filter housing was
matched with the filter and a screw press was used to wring the filters. Concentration of the
eluate was best performed at centrifuge speeds of 6,700 to 10,000 x g.
/
Also described in the 1994 Cryptosporidium Criteria Document is the use of cellulose acetate
membrane (CAM) filters. Nieminski et al. (1995) compared recovery rates of a method using
CAM filters to the ASTMICR method using wound yam filters. Cryptosporidium and Giardia
were spiked into environmental water samples varying in quality and turbidity prior to filtration
by either method. Cyst and oocyst recoveries decreased with increasing water turbidity
regardless of the filter type. Overall, the cellulose acetate method gave higher recoveries,
however, because die parasites were stained on polycarbonate filters, microscopic confirmation
was not possible. Therefore, the authors recommended the use of the ASTM ICR method for
environmental sample analysis and the cellulose acetate method for spiking studies. Adlom and
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Chagla (1995) modified the CAM method by including an acid dissolution step following
filtration. This modification resulted in a 70.5% recovery of oocysts spiked into 3 liters of
treated municipal water. Graczyk et al. (1997) used cellulose acetate filters, followed by filter
dissolution and ASTMICR method processing to test recovery of Cryptosporidium from spiked
drinking water. The overall mean recovery rate was reported at 77.7%. Further studies by
Graczyk et al. (1997) indicate the acetone dissolution step does not compromise viability or
infectivity.
Envirocheck™ capsules contain a pleated polysulphone membrane with a 1 um porosity. This 6
cm diameter x 21 cm long capsule has a surface area of 1300 cm2. Clancy (1997) compared
throughput and recovery rates of this capsule filter with those of polycarbonate membrane filters,
vortex flow filtration, and cellulose acetate membrane filters which were dissolved post filtration.
All four filters were challenged with 10 liters of municipal raw and finished waters. Cellulose
acetate membranes and polycarbonate membranes blocked at 8 and 2.5 liters respectively, at a
raw nephlometric turbidity unit (NTU) of 5. The polymer vortex flow and Envirocheck™
capsule filters were able to process the entire 10 liters of raw water and gave recovery rates of
11-57% and 8-78% respectively. In finished waters from 5 utilities, the vortex flow recovered
18-69% of the seeded oocysts white the capsule filter recovered 45-117%. The researchers
concluded the capsule filter performed best with the various water matrix conditions tested.
Other membrane filters composed of glass fiber have been evaluated and shown to be of poor
integrity (Whitmore and Carrington, 1993).
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Centnfugation based concentration methods
Vortex flow filtration (VFF) is centrifugation-based filtrat: -,n method in which a water sample is
passed through a cylindrical membrane filter rotating at high speed within a second cylinder. The
rotation sets up forces that scrub the membrane surface and prevent blockage by particulates.
The liquid phase (permeate) crosses the filter while the participate containing phase (retentate) is
continuously recirculated and concentrated. Whitmore and Carhngton (1993) evaluated the
ability of a VFF apparatus with a 0.45um polysulphone membrane cartridge filter, followed by
clarification step using density gradient centrifugation, to concentrate Cryptosporidium from
spiked bore hole or river water samples. The VFF device recovered between 30 and 40% of the
seeded oocysts. Fricker (1997) reported greater than 60% recovery using VFF in seeded river
water samples.
Whitmore and Carrington (1993) evaluated cross-flow filtration for recovery of oocysts in clean
water. This apparatus pumps water across a set of alumina niters in a parallel series. In this
study, the retentate, typically 150-200 ml was collected, centrifuged, resuspended in phosphate
buffered saline (PBS), and counted using a hemacytometer under Nomarski differential
interference contrast (DIG) illumination. The authors note this method is rapid and recovered
70% of the seeded oocysts in small volume samples. Studies using larger volume samples
recovered 40%. The authors suggest more effective cleaning or replacement of the filters
between runs may obtain higher recovery rates. The device produces small volumes of retentate
facilitating further concentration, is compact, and can be sterilized.
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Researchers at Marshiield Clinic in Marshfield Wl have developed a continuous centrifugation
method to concentrate parasites from water (Borchardt and Spencer, 1996). This method uses a
blood cell separator, which operates on the principal of channel-type centrifugation, to
concentrate oocysts from water samples. Recoveries over a range of water turbidities, oocyst
concentrations, and water volumes were reported to be 78-101% .
Concentration usingflocculation
Calcium carbonate flocculation has also been used to concentrate Cryptosporidium in up to 10
liters of water. The method, developed by Vesey et al. (1993b), uses calcium chloride and
sodium bicarbonate in a high pH solution to crystallize organic particles. The crystals are
allowed to settle, the supernatant is discarded, and the calcium carbonate precipitate is dissolved
with sulfamic acid. Vesey et al. (1993b) reported recoveries greater than 68% with this method.
Subsequent analyses of this method by Shepherd and Wyn-Jones (1995 and 1996) were in
agreement and reported calcium carbonate flocculation consistently gave higher recoveries when
compared to cellulose acetate membrane and cartridge filters. However, calcium carbonate
flocculation is not recommended for drinking water analysis when viability is important Studies
by Robertson et al. (1994) showed significant viability reduction, determined by vital dye
staining (See Viability, VQ.A.2) following 4 hour exposure to a pH of 10.
Clarification by density gradient centrifugation
Density gradient floatation methods are commonly used to clarify samples and concentrate
oocysts prior to detection. Centrifuge speed and time, and density of the solution vary among
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laboratories using this method. LeChevallier et al. (1995) reported a Percoll sucrose gradient
with a specific gravity of 1.15 was 67% better than a gradient with a specific gravity of 1.0 for
recovery and concentration of oocysts. A gradient with a specific gravity of 1.3 did not clarify
the sample and interfered with microscopic analyses. This study also indicated that floatation
selects for empty oocysts while live, intact oocysts sink to the bottom of the gradient rather than
floating to the upper fraction (LeChevallier et al., 1995). Shepherd and Wyn-Jones (1995)
reported significant decreases in recovery (>50%) when sucrose flotation techniques were
included in detection methods.
Flow cytometry
A flow cytometer is a laser-based instrument that analyses particles in a liquid suspension on a
particle by particle basis. It can differentiate and physically separate (sort) particles based on
their size, internal complexity, and fluorescence. Flow cytometry with cell sorting (FCCS) is
used routinely in the UK and Australia for detection of Cryptosporidium and Giardia in
environmental samples (Vesey, 1993a,1994). Briefly, a concentrated portion of the sample is
incubated with a fluorochrome conjugated monoclonal antibody that binds a portion of the
oocyst wall causing the organism to Quoresce. The instrument analyses the particles and
electrically charges those selected by the operator based on a signal (eg. fluorescence). The
charged particles are pulled out of the sample stream using oppositely charged electrical plates
and deflected onto a microscope slide. The slides, free of the debris .which can obscure oocysts
on a membrane filter slide, can be read in 5 to 10 minutes as opposed to 90 -120 minutes cited
for membrane analysis. In studies published by Vesey et aL (1994), FCCS detected greater than
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92° o of the Crypwsporidium and Giardia in seeded river and reservoir samples. Additional work
incorporating a calcium carbonate flocculation concentration step prior to FCCS reported 64.1%
and 62.7% recovery of the oocysts seeded in filtered and raw waters, respectively (Veal, 1997).
Studies by Hoffman et al. (1997), using a variety of environmental samples, reported that FCCS
detected three times more Cryptosporidium positive samples than membrane IF A (94.1% vs.
35.3%, respectively) and an equal number of Giardia positives samples. This technique has been
repeatedly shown superior to traditional membrane IF A analysis (Danielson, 1995; Compagnon,
1997). Researchers at Macquarie University in Australia, working with a flow cytometer
manufacturer, have modified the traditional flow cytometer to optimize it for water quality
analysis. This type of flow cytometer is not currently available in the US. The FCCS method
provides increased sensitivity, and requires less time, expense, and experience than the ASTM
IF A. Additionally, this method can, with no extra effort, simultaneously detect Giardia.
Disadvantages of this method include the initial expense of the instrument ($150,000-200,000)
and the level of flow cytometry expertise required with the non-optimized models.
Immunomagnetic separation
Immunomagnetic separation concentrates Cryptosporidium using magnetic beads coated with an
anti-Cryptosporidium antibody. Following elution, the sample is incubated with magnetic beads
that bind the oocysts. The solution is inserted into a magnetic particle concentrator that binds the
magnetic bead-Cryptosporidium complex. After the supernatant is decanted, the beads are
released from the magnet Oocysts are dissociated from the magnetic particles using an acid
wash, neutralized with base, and subjected to analysis. This method was evaluated for
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Cryptospondium by Robertson and Smith (1992); later effor- :o develop a Oiardia detection
system were pursued by Bifulco and Schaeffer (1993) who reported an 82% recovery rate for
Giardia in waters of varying turbidities. Campbell et al. (1997) spiked laboratory grade and
turbid water samples with Cryptosporidium and concentrated them using either ASTM IF A, the
UK Standing Committee of Analysts method (SCA), or IMS. Immunomagnetic separation gave
the highest recoveries in either water type. Clean water recoveries using IMS were 97.4% while
the ASTM IFA and SCA methods recovered 26.9 and 19.3% respectively. Turbid water
recoveries were 65.8% using IMS, 5.4% with the ASTM IFA, and 11.7% with SCA. A five site
evaluation comparing IMS to FCCS and a modified SCA reported the IMS method was superior
to the FCCS and SCA methods in low turbidity waters. However, there was a direct relationship
between turbidity and performance of the IMS method. Flow cytometry showed the greatest
recoveries in higher turbidity waters (Campbell et al, 1997). Pricker et al. (1997) evaluated the
IMS procedure in water samples seeded with 100 oocysts. Recovery rates ranged from 71-120%.
Panning
Panning is a technique originally developed to isolate specific cell types from a mixed cell
population. An antibody raised to the target is attached to a solid substrate and incubated with
the target cell containing suspension. During the incubation period, the target cell or organism is
bound by the antibody and the remaining cells or debris can be washed off. Direct and indirect
panning methods were tested by Stone (1997) to concentrate Cryptosporidiian oocysts during the
clarification stage. The authors recovered 50% of the oocysts seeded into Hanks Balanced Salts
Solution (HBSS) by direct panning, and 20% of the seeded oocysts using indirect panning.
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2. Detection of Cryptosporidium in Water
Indirect Immunofluorescence assay
Direct and indirect immunofluorescent antibody detection methods facilitate the visualization of
oocysts which may be obscured by debris in environmental samples. The indirect
immunofluorescence assay (IFA) (Fout et al., 1996), described in the 1994 Cryptosporidium
Criteria document, remains the most widely used detection method. The test, however, does not
provide information regarding viability, infectivity, or species. The HydroFluor Combo staining
reagents have been shown to cross react with various algal species (Rodgers et a/., 1996) and an
experienced microscopist is essential for accurate and reliable examination.
Well slide staining . .
Well slide staining has been evaluated as an alternative to membrane IFA staining (Frederickson
et al., 1995). In a five site side by side comparison, Cryptosporidium recoveries using the well
slide method were in excess of 50% higher than those obtained using traditional membrane.
Additionally, the well staining procedure took 50% less time to perform. The authors suggested
the physical forces of the vacuum used in membrane IFA staining may result in destruction of
cysts and oocysts. Microscopic examination of the contents of the Hoefer tank accounted for a
78% loss of Cryptosporidium with 2% remaining intact Cryptosporidium have been shown to be
compressible (Li et al., 1995) and may slip through filters with pore sizes smaller (ban their 4-6
diameter.
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Enzyme immunoassays
Traditional enzyme immunoassays can provide rapid detection of oocysts with little tedium.
While these assays have been used clinically, their use in environmental analysis is not common.
Several papers have indicated the ability for their use with environmental samples. (Siddons,
199l;Chapman, 1990; Gracyzk et al., 1996b). In one instance, Siddons (1991) reported a positive
EIA detection of one oocyst. Chapman and Rush (1990) reported EIA sensitivity equal to
microscopic examination in both environmental and human samples, de la Cruz and Sivagansen
(1994) tested five G. lamblia EIA kits and two C. parvum EIA tots for detection of cysts and
oocysts respectively in buffered saline and river water. The authors found some kits were
capable of detecting <10 cysts or oocysts, however, results were variable with fixed and unfixed
organisms. The authors also found that disruption of the parasites increased the kit's sensitivity.
Each EIA kit's performance was influenced turbidity and some kits cross reacted with algae.
Gracyzk et al. (1996b) compared the specificity of the Prospect T™ enzyme-linked
immunosorbent assay (ELIS A) to that of the HydroFluor Combo antibody used in the ASTM
ICR method, and the Merifluor direct stool kit antibody. Their results showed the ELIS A gave a
positive reaction with only 6 of 25 non-C. parvum isolates tested, while the HydroFluor combo
•
and Merifluor direct antibodies each cross reacted with 19.
Molecular methods: Pofymerase Chain Reaction Assays
Several reports describe various applications of the polymerase chain reaction™ for the detection
of Cryptosporidium in drinking water (Rochelle et al., 1997 a and b; Johnson et al., I995b;
Wagner-Wiening and Kimmig, 1995; Filkom et al., 1994; Johnson et al., 1993). These methods
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rely upon in vitro enzyme-mediated amplification of Cryptosporidium-specific nucleic acids in
order to facilitate identification in water samples, which should in theory offer unmatched
endpoint sensitivity and the possibility of distinguishing subtle differences among discrete strains
of parasites. A number of techniques aimed at distinguishing viable and nonviable oocyst
i
populations are also reported. These applications are summarized below; however, it is
important to emphasize that due to the enzymatic nature of the PCR process, these assays may
fail due to inhibition of enzyme activity caused by compounds commonly found in natural
waters. Additionally, the majority of the documented studies have been Limited to evaluations of
seeded water samples rather than actual comparative field trials. Hence, the application of these
techniques toward the detection of Cryptosporidium in environmental water samples should be
considered developmental.
Johnson el al. (1995) reported a PCR protocol to detect Cryptosporidium in environmental
samples, based upon oligonucleotide primers specific to a portion of the small 18S ribosomal
RNA. Detection sensitivities of 1 to 10 oocysts were achieved in purified oocyst preparations,
however, the detection sensitivity in seeded environmental samples was up to 1000-fold lower.
Poor endpoint sensitivity was at least partially offset by concentration by flow cytometryor
immunomagnetic capture and oligoprobe hybridization using a chemiluminescent technique.
These methods were applied to confirm the presence of oocysts in water samples from the
Milwaukee outbreak of cryptosporidiosis, and the results of the PCR assays were comparable to
those observed when immunofluorescence methods were practiced.
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Rochelle (\99~b) evaluated four pairs of previously published primers aimed at the specific
detection of C. parvum. Detection sensitivities ranged frcxi 5 to 50 oocysts in seeded
environmental samples when PCR was followed by oligoprobe hybridization, with some primer
pairs offering species specificity while others were only genus-specific. Successful multiplex
reactions aimed at the simultaneous detection of G. lamblia and C. parvum were evaluated and
demonstrated the utility of PCR for the detection of waterborne parasites; however, ho primer
combinations were identified which exhibited the ideal combination of sensitivity, specificity
and compatibility with multiplex reactions, and several primer sequences reported previously
failed to amplify their targets.
Since PCR is capable of detecting the genetic material of both live and dead microorganisms, a
number of studies have targeted unique sequences to distinguish viable from nonviable oocysts.
Wagner- Wiening and Kimmig (1995) applied PCR to detect Cryptpsporidium targeting a large
DNA fragment specific to C. parvum. In order to differentiate between live and dead oocysts,
this group reported the practice of applying an excystation protocol prior to PCR and targeting
sporozoite DNA to ensure that amplified material was associated only with viable oocysts.
Endpoint sensitivity as low as 100 sporozoites was observed, and was reduced to 10 sporozoites
when nested PCR was practiced. FUkhorn et al. (1994) also evaluated RNA-based measurement
of viable oocysts by practicing excystation prior to PCR. To preclude spurious contamination
caused by the presence of DNA in nonviable parasites, DNAse enzyme was applied to digest free
DNA, leaving only free RNA from viable oocysts.
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Stinnear et al. (1996) describe a reverse transcription PCR (RT-PCR) detection method specific
for C. parvum which can detect single viable oocysts based upon the assumption that only viable
oocysts are metabolically active and will produce messenger RNA (mRNA). Since mRNA
exhibits an extremely short half life in living cells (and perhaps even shorter outside of the
protective environment of the oocyst wall), only the mRNA from viable oocysts will be captured
and amplified.
/
PCR has also been used to track Cryptosporidium reductions during water treatment. Mayer and
Palmer (1996) evaluated several methods to identify Cryptosporidium oocysts in sewage and to
determine reductions during wastewater treatment The nested PCR technique described was
compared to a modified ASTM method to track reductions during treatment, and strong
correlations were observed, with approximately 2 loglo Cryptosporidium reductions observed.
The authors concluded that the PCR method was preferable due to a substantial reduction in
sample collection and processing during analysis.
Molecular methods: Cell culture-Pofymerase Chain Reaction
At least one integrated approach has been reported utilising tissue culture, PCR and in situ PCR
(IS-PCR) to assess seeded natural water concentrates for the presence of infectious oocysts
(Rochelle, 1997a). This technique offers the possibility of screening out noninfectious oocysts
since only the infectious oocysts will develop during the initial phase of amplification in human
adenocarcinoma cells. Preliminary experiments suggest mat IS-PCR may offer quantitative
detection of infectious oocysts in natural water concentrates.
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Molecular methods: Strand displacement amplification
A strand displacement assay for the detection of Cryptosporidium in natural waters samples has
been reported to overcome the comparatively long cycle times associated with PCR (Blassak et
al., 1996). This method is based upon the selective replication of a single DNA strand while the
other parental strand is displaced from the template and colorimetric detection of oocyst DNA
under a microplate format. Low Cryptosporidium densities (~l) were detected in deionized
water; reaction inhibition associated with seeded finished and raw water samples resulted in
lower sensitivity.
\
Laser scanning microscopy
The ChemScan RDI is a laser scanning device linked to an epifluorescent microscope with a
motorized stage. Sample analysis includes a filter staining procedure, followed by automated
scanning with the ChemScan instrument. The instrument digitizes the location of fluorescent
objects allowing for quick confirmation. This instrument has the advantage of being highly
automated and more sensitive than FCCS and IMS methods in samples with colloidal clay
(Reynolds et al., 1997). The recovery rate and minimal detection limit were not specified nor
was the oocyst spike concentration. Disadvantages of this method include the cost of the
instrument ($200,000-300,000) and use of membrane filters which have been shown to
contribute to oocyst loss (Frederickson et al., 1995), and the inability to perform any light
microscopy techniques for visualization of internal cytoplasm or sporozoites.
Miscellaneous methods
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Campbell ei at. (I993a) contrasted flow cytometry and a slow scan cooled charge couple device
(CCD) for detection of Cryptosporidium. The authors discuss the advantages of the CCD,
including its ability to simultaneously assess viability by DAPI staining, however, a comparison
of results is not provided. The need for sophisticated and currently unavailable software is
indicated.
Campbell et al. (1993b) evaluated enhanced chemiluminescence for detection of
Cryptosporidium in 21 environmental samples previously assayed by microscopy. The oocysts
were labeled with a FTTC conjugated adti-Cryptosporidium antibody, followed by a biotin
conjugated anti-FITC antibody and streptavidin-peroxidase. Statistical analysis revealed no
difference between the enhanced chemiluminescence assay and microscopy. The authors report
the chemiluminescence method is faster than the currently used microscopic method, however,
visual confirmation was still necessary.
The electrorotation assay (ERA) relies on the principle that small particles can be induced to
rotate in the presence of a rotating electric field. The rate of rotation is due to the field and
surface charge of the particles. Jakubowski et aL (1996) described a proprietary electrorotation
assay in which oocysts are attached to an antibody-coated magnetic bead and placed in a filter
electrode assembly. The assembly is placed under a microscope, connected to an electric field
generator, and the number of rotating oocysts are enumerated This method may be able to
x
differentiate viable and non viable oocysts based on differences in their rotation rates. Oocyst
recovery rates have been reported from 30-95%, however, the efficiency of this method is
77
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dependent on the type and'characteristics of the water. A major disadvantage to ERA is that
magnification can only be performed up to 400x due to th: thickness of the ERA unit.
Method 162 2
Recognizing the need for an improved Cryptosporidium detection method, the EPA initiated an
effort to identify new and innovative technologies for protozoan monitoring and analysis.
Following a comprehensive evaluation of existing and emerging technologies, the EPA Office of
Water developed an initial draft of Method 1622 in December 1996 (EPA-821 -R-97-023). This
method continues to be modified and is currently being tested in several laboratories across the
US.
A 10 L sample is shipped to the laboratory where it is filtered through a polysulfone capsule
filter. Following filtration, the capsule is filled with eluting solution and shaken on a wrist action
shaker for approximately 5 minutes. The capsule is drained and the elution procedure is
repeated. The combined eluate is concentrated by centrifugation, reconstituted to 10 ml, and
subject to IMS as described previously. The solution is stained with axti-Cryptosporidium
antibodies using the well slide method described previously.
Several options are listed in the EPA Method 1622 draft document including concentration by
vortex flow filtration or membrane disk filtration and an alternative immunomagnetic separation
procedure. Additionally, a draft document outlining flow cytometry coupled with cell sorting as
an alternative to the IMS and well slide analysis has been written.
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liability determinations
The public health significance of Cryptosporidium relates primarily to the ability of this parasite
to initiate infections in humans and animals. Although the gold standard among infectivity
assays remains the animal model, the high costs associated with assays using animals and ethical
considerations precludes their routine use. Additionally, these methods do not offer adequate
sensitivity for testing of environmental samples where the level of oocyst contamination may be
on the order of 1 or 2 oocysts per liter, additionally, differences in pathogenesis among humans
and animals have called into question the applicability of animals in providing an accurate
reflection of cryptosporidiosis in humans. Nonetheless, innovative viability assessment methods
for Cryptosporidium are inevitably compared to animal models, primarily using mice, and these
methods are described below.
In vitro excystation (IVE) estimates infectivity by determining the number of potentially
infectious oocysts by simulating the conditions of the mammalian gastrointestinal tract Oocysts
exposed to an acid pretreatment and bile salts at elevated temperatures will release progeny
sporozoites (excyst), whereas metabolically inactive (nonviable) oocysts will fail to excyst
Excystation is tracked by direct microscopic visualization of treated samples and performing a
comparison to the initial untreated population. Black et al. (1996) and Belosevic et al. (1997)
indicate that IVE assay may significantly overestimate the true infectivity of oocysts treated with
chemical antagonists when compared to results of animal infectivity studies. Although IVE
assay is relatively time-consuming and is generally not applicable toward tracking reductions
during water treatment (which may exceed several orders of magnitude), Vesey et al, (1997) has
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reported an adaptation using flow cytometry to increase the sensitivity and throughput and has
observed good agreement with the microscopic method.
The Cryptosporidium Criteria Document (1994) described the early work of Campbell which
focused on vital dye staining. This group evaluated the application of dyes which are
preferentially absorbed by viable oocysts and compared the technique with in vitro excystation,
where strong correlations were observed. This work was confirmed by Bukhari (1995) who
compared vital dye staining and IVE of oocysts concentrated using several different methods.
Subsequent studies by Black et al. (1996) indicated that vital dye staining correlated well with
IVE but had a tendency to overestimate infectivity of ozone-treated oocysts when compared to
animal infectivity. Jenkins et al. (1997) confirmed that vital dye staining tends to overestimate
infectivity relative to animal infectivity, but supported its use as an economical, user-friendly
method that provides information on the effects of stresses on the surface of oocysts over time.
Belosevic et al. (1997) evaluated 14 nucleic acid stains as indicators of Cryptosporidium
viability and compared the staining to both animal infectivity and IVE assay. Both heat-treated
and chemically inactivated oocysts were consistently stained with novel stains (SYTO-9, SYTO
/
59, and hexadium). This staining correlated well with animal inactivity but not IVE assay. The
authors also developed an IF A-viability assay reiving 6nly on propidium iodide (PI) for viability
assessment; however, extraneous factors such as aldehyde fixation may inhibit uptake of PI and
falsely elevate the numbers of viable oocysts (Campbell et a/., 1993c).
*
Various infectivity assays have been described using tissue culture methods to assess the
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ifectivity of C. parsum (Upton et at., 1994a; Upton etal., 1994b; Upton et at., 1995; Rochelle
t al., 1997a; Slifko et al., 1997). These methods track the development of progressive
^ryptosporidium infections in cell cultures. Evaluation of infected cultures can be facilitated by
onducting enzyme-linked immunosorbent ?.ssays (ELISA) following a 1-2 day incubation, and
aen scoring the extent of infection (and hence number of viable oocysts present in the original
noculum) by spectrophotometry in an automatic plate reader. A detection sensitivity of
pproximately 100 oocysts has been described (Upton et al., 1994a; 1994b). Slifko et al. 1997
lescribe a semi-quantitative method which relies upon staining infected tissue cultures with
Increscent antibody, and then tracking the numbers of infectious foci using epifluorescence and
iifferential interference contrast microscopy. An adaptation of the tissue culture method using
PCR (Rochelle et al., 1997a) is described in Section VH-A-2 "Polymerase Chain Reaction" of,
this document.
3. Assessment of Laboratory Testing Capabilities
Detecting of Cryptosporidiwn and Giardia and distinguishing them from other organisms of
comparable size and appearance is a major problem that presents most commercial, state, and
local laboratories with a difficult challenge. However, it is important to establish whether these
t
laboratories can follow a standard procedure and be successful in recovering and detecting these
pathogenic protozoans in water samples.
\
Sixteen commercial laboratories were enlisted in a survey (Clancy et al., 1994) to assess the
ability of a laboratory to recover and detect Giardia and Cryptosporidiwn using the ASTM
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method. Filters spiked with either Giardia (approximately 740) and Cryptosporidium
(approximately 500) oocysts or with approximately 500 cells of Oocystis minuta (algal cells
measuring 8-18 ^m x 5-15 ^m) were sent to the laboratories for analysis. Of the 12 laboratories
that participated in the exercise, four reported O. minuta samples as positive for either Giardia or
Cryptosporidium, while four others failed to recover Giardia from the cyst-spiked filter and six
failed to recover Cryptosporidium from the oocyst-spiked filter. Giardia cyst recovery ranged
from 0.8 to 22.3 % (with an average of 9.1%) while Cryptosporidium cyst recovery ranged from
1.3 to 5.5% (with an average of 2.8%). It was concluded that not all laboratories strictly followed
f ' '
the ASTM methods, and that the majority of laboratories need to improve in one or more areas:
client response, quality of sampling equipment and directions for use, analytical methods, data
accuracy, and reporting format. Expertise in microbiological identification also appeared to be
lacking as indicated by the relatively high numbers of false positives. The EPA established an
approval process for laboratories analyzing samples for the Information Collection Rule for
Cryptosporidium and Giardia. The EPA required approved laboratories to have trained and
experienced personnel performing Cryptosporidium and Giardia testing and the necessary
processing equipment In addition, initial and continuous passing of performance evaluation
samples and passing of the on-site inspection were required.
B. Detection in Biological Samples
Diagnosis of Cryptosporidium infection is typically performed by examining the feces of an
infected individual. Feces of patients with active cryptosporidiosis normally do not require
concentration since oocysts are shed in great numbers; however, the number of oocysts can
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fluctuate dunng the course of infection (Casemore and Roberts, 1993). This reiterates the
importance of making diagnosis using multiple specimens. Concentration methods are employed
and useful when trying to assess infection in immunocompromised patients with a history of
unexplained diarrhea or in asymptomatic patients. Concentration methods including formalin-
ether and formalin ethyl acetate sedimentation are commonly used in clinical laboratories.
Sheather's sucrose floatation, zinc sulfate flotation, saturated sodium chloride floatation,
discontinuous Percoll gradients, and cesium chloride gradient centrifugation are methods more
common in research laboratories. Evaluations of various concentration techniques have been
published and-the results are summarized below.
Concentration methods
A study by Bukhari and Smith (1995), comparing water-ether, sucrose density gradient, and zinc
sulfate concentration method, showed significantly higher numbers of oocysts were recovered
from bovine feces using water-ether concentration. Resales et a/. (1994) used concentration by
Sheather's solution to obtain greater numbers of oocysts than discontinuous Percoll gradients,
and a commercially manufactured parasite concentrator device. Concentration of oocysts in cat
feces (Mtambo, 1992) was best accomplished using formalin ether sedimentation which
recovered 37% of the original oocysts compared to 11% and 33% for zinc sulfate and sucrose
flotation, respectively. Clavel et a/. (1996b) showed that simply increasing centrifugation times
augmented oocyst recovery when using the standard formalin-ether acetate concentration
method. Finn et al. (1996) demonstrated that straining the feces contributed to the loss of
oocysts. This study showed a nearly four fold overall reduction in the number of oocysts detected
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using a wash procedure.
Traditional staining methods
Traditional staining methods were described in the 1994 Cryptosporidium Criteria Document.
Numerous new staining methods and variations of traditional methods have been employed. A
review is included in Chapter 2 of the book, Cryptosporidium and Cryptosporidiosis (Payer,
1997). Kang and Metham (1996) compared five staining methods for detection of
Cryptosporidium oocysts. The safranin-methylene blue technique, a modified Ziehl-Neelsen
method, was used as the "gold standard" and compared to two methods each using auramine and
mepacrine stains with potassium permanganate and carbol fuschin as counterstains. The authors
concluded that mepacrine and auramine staining procedures were easily performed and stained
K
/
equally, however, they preferred mepacrine to auramine because it is less toxic and can be used
with carbol fuschin without a decolorization step. Work by Ungureanu and Pontu (1992)
supported these results. In the Cryptosporidium Screening Guidelines established by a joint
working group, Casemore and Roberts (1993) recommended an auramine method to be used as a
screening method with confirmation by a modified Ziehl-Neelsen. Acid fast staining methods do
not stain all oocysts. Entrala et oL (1995) showed hydrogen peroxide treatment increased the
percentage of oocysts displaying acid fast characteristics.
Immunofluorescence methods
The use of monoclonal antibody detection assays increases the sensitivity of Cryptosporidium
detection. Garcia et ol. (1987) tested 115 samples using a modified acid fast method and
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•nonoclonal antibody staining. Sensitivity and specificity using the monoclonal antibody were
100%, while the acid fast method failed to detect 7 of 99 positive samples. Subsequent testing
by Baron et al. (1989), Rusnak et ai (1989), Garcia et al. (1992), Tee et ai (1993), Grigoriew et
al. (1994), Alles et al. (1995) and Roberts et al. (1996) confirmed these results. Studies using
monoclonal antibodies for detection of Cryptosporidium in animals have also shown increased
sensitivity (Wee et al, 1995; Mtambo et al., 1992; Xiao and Herd, 1993).
Enzyme Immunoassays
' « •
Commercial EIAs have been developed to replace time consuming microscopic methods. Most
studies have shown EIA methods perform better than (Siddons et al., 1991; Dagan et ai, 1995)
or equal to (Chapman and Rush, 1990;'Rosenblatt and Sloan, 1993; Parisi and Tierno 1994)
conventional microscopic methods; however, work by Newman et al. (1993) indicated EIA
methods were not sensitive enough to be used for patients without diarrhea. Enzyme
immunoassays have been shown to be equal in sensitivity to the immunofluorescence assay
(Siddons et al., 1991; Rosenblatt and Sloan, 1993) and inferior using an experimental EIA
(Anusz et al., 1990) and a commercially available EIA (Ignatius et ai, 1997). Two commercial
EIAs were compared to a direct immunofluorescence assay, the ProSpecT and the ColorVue
(Aaraes et ai, 1994), and found to have differences in performance. The sensitivities were 96%
and 72-76% respectively. Specificities were 97.6-99.5% using the ProSpecT and 100% using
ColorVue.
-------
Molecular methods: Polvmerase Chain Reaction Assays
PCR-based assays are becoming more common and may provide a useful alternative for
detecting and quantifying Cryptosporidium in water, stools, and tissue/organ samples. Awad-El-
Kariem et al. (1994) described a PCR method that identifies Cryptosporidium at the species level
and requires no DNA probes. The procedure is based on use of the published 18S rRNA genes
of C. parvum and C. muris. One of the sequence 3 Mae I endonuclease restriction sites is present
only on the C. parvum gene while others are specific for C. muris and C. baileyi, which allows
screening for human, mouse, and avian species, respectively. The authors suggest the protocol
they developed is adaptable to detection of small numbers of C. parvum oocysts in
environmental samples. Morgan et al. (1996) noted the importance of PCR in processing
clinical as well as environmental specimens suspected of being contaminated with
Cryptosporidium. PCR primers specific for Cryptosporidium have been developed, and random
amplified polymorphic DNA (RAPD) is a simpler approach for developing diagnostic primers
since many of the products generated by RAPD-PCR are frequently species specific. Leng et al.
(1996) developed an assay to identify Cryptosporidium DNA in bovine feces involving
standardization of sample preparation and simplification of the DNA recovery process for PCR
amplification and DNA-hybrid detection. The DNA-recovery/PCR detection procedure can
recover DNA suitable for PCR amplification and can detect 103 to 10* times fewer oocysts in
diluted solutions than the commercial enzyme-linked immunoassay, Color-Vue-
Cryptosporidium, and 102 fewer from diarrhetic feces samples man the commercial Color-Vue
kit.
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Amplification methods have also been described which may assist in future efforts to define the
4
role of livestock in waterbome outbreaks of cryptosporidi' sis. Blassak et at (1996) describe a
rapid assay kit desgined to test fecal samples for live Cryptosporidium using a gene probe
method. Oocyst DNA is released by cyclical freeze/thaw and is then amplified by isothermal
strand displacement using biotinylated primers. The amplification product is detected
colorimetrically in a microwell system in which a complementary capture probe binds to oocyst
DNA and then reacts with a horseradish peroxidase-streptavidin conjugate. This complex emits
a color that can be readily detected with qualitative or semi-quantitative results. The authors
suggest that this method could be applied toward the analysis of genetic similarity among
Cryptosporidium strains isolated from livestock and humans.
Balatbat et al. (1996) developed a nested PCR assay for detection of C. parvum directly from
stool specimens. After extracting DNA from a formaldehyde-treated stool, a 400-bp fragment of
DNA was amplified with two 26-mer primers. The amplicon from the first reaction was then
subjected to a second round of amplification using a second set of primers. With these nested
primers, a 194-bp fragment of DNA was amplified and confirmed as C. parvum DNA by internal
probing with an enzyme-linked chemiluminescence system. The test can detect as few as 500
oocysts per gram of stool, and has the potential to detect asymptomatic infections, monitor
response to therapy, or monitor environmental samples. The preliminary results indicate a
significantly enhanced sensitivity compared with traditional assays.
Kelly et al. (1995) developed a sensitive and specific PCR test to confirm Cryptosporidium
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infections. The test, which'uses previously published primers to detect Cryptosporidium spp. in
distal duodenal biopsies, was used to identify infections in HIV-positive patients in Zambia, and
proved especially useful in identifying those infections that were limited to the distal small
intestine.
Other methods
Serological methods have been used to monitor exposure to Cryptosporidium. There is limited
information regarding the seroprevalence of Cryptosporidium infected individuals. The
sero logic response to Cryptosporidium is discussed in the section V-B and in Lengerich et al.
(1993).
Flow cytometry methods have been described for detection of Cryptosporidium in mice
(Arrowood et al., 1995). More recent studies using seeded human feces showed a four-fold
increase in detection over direct immunofluorescence methods (Valdez et al, 1997).
Chemiluminescence assays have been used by Clavel et al. (1996"b) to detect cultured oocysts
and other fecal parasites. You (1996) showed positive detection of Cryptosporidium in MDCK
cell cultures and its potential for testing chemotherapeutic agents is promising. A reverse passive
hemagglutination (RPH) assay (Farrington era/., 1994) measuring agglutination of anti-oocyst
antibody coated sheep erythrocytes with oocysts in diluted fecal suspensions was compared to
auramine phenol staining for detection of Cryptosporidium and showed equal sensitivity.
C. Water Treatment Practices
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Introduction
Multiple barriers are used in most surface water treatment -plants in an effort to prevent public
Kposure to waterborne pathogens like Cryptosporidium. These barriers include removal of
athogens from water by processes like clarification and filtration, which are generally preceded
y coagulation and flocculation processes. Another type of barrier is inactivation by
isinfectants like ozone and chlorine. The purpose of this section is to summarize the removal
nd/or inactivation of Cryptosporidium through multibarrier systems and through individual
reatment processes. The reader is strongly encouraged to look at referenced studies to gain
letailed information regarding site-specific raw water quality and treatment conditions.
n this section, log removal is given by:
og removal = -log(N/N0) - (Eqn. 2)
vhere N is the concentration of Cryptosporidium oocysts remaining after treatment and N0 is the
:oncentration of Cryptosporidium oocysts prior to treatment Log inactivation is given by a
similar equation, but N and N0 refer to the concentration of infectious Cryptosporidium oocysts
in treated water and in untreated water, respectively.
2. Multi-Barrier Treatment
Several studies have evaluated the occurrence of Cryptosporidium in raw and finished waters for
multibamer treatment facilities (LeChevallier and Norton, 1995). In a survey of 72 North
%
American drinking water plants, Cryptosporidium was present in 51.5 percent of raw water
samples and in 13.4 percent of finished water samples. la an earlier survey of 66 drinking water
plants, Cryptosporidium was observed in 87 percent and 27 percent of raw and finished waters,
89
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respectively. The authors attributed the different occurrence levels between studies to normal
variations in raw water quality and treatment performance. The authors claimed that microscopic
analyses of Cryptosporidium oocysts in the finished waters suggested that most of the oocysts
were nonviable. However, no attempts were made to specifically assess oocyst viability in this
study. These occurrence results suggest that a significant percentage of Cryptosporidium oocysts
are removed by drinking water treatment practices.
Other studies have reported removal of Cryptosporidium oocysts through conventional filter
plants using chlorine as the primary disinfectant. The combination of coagulation, flocculation,
and sedimentation achieved 3.8-log removal of oocysts in a treatment plant near Montreal,
Canada (Payment and Franco, 1993). In the same treatment plant, 4.6-log removal of oocysts
was observed through coagulation, flocculation, sedimentation, and granular media filtration
(also known as conventional treatment). No attempts were made to assess oocyst viability in this
study.
In a more comprehensive study of a conventional treatment plant near Pittsburgh,
Cryptosporidium oocysts were detected in 63 percent of raw water samples, 29 percent of settled
water samples, and 13 percent of filtered water samples (States, et a/., 1997). For those cases
where oocysts were detected in both raw and settled waters, the treatment plant achieved 0.8 to
!.
1.3-log removal of oocysts prior to filtration. For those cases where oocysts were detected in
both raw and filtered waters, the treatment plant achieved 1.7 to 3.6-log removal of oocysts
through filtration. Oocyst viability was not measured in this study, either.
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3. Removal of Cryptosporidium
Introduction
As noted in the previous section, detectable Cryptosporidium concentrations occur infrequently
in treated waters. Because of this, the effectiveness of treatment processes has been evaluated in
*
challenge studies where oocysts are spiked into raw water at a concentration large enough to
detect the oocysts in treated water. This section summarizes challenge study results for those
processes that remove Cryptosporidium oocysts. Challenge study results for those processes that
inactivate Cryptosporidium oocysts are summarized in the next section.
Coagulation, flocculation, and clarification
Several clarification methods are available for drinking water treatment and these are usually
preceded by coagulant addition and flocculation. Currently, sedimentation is the most
commonly practiced method of clarification in the United States. Other options include
dissolved air flotation and sludge blanket clarification. One recent bench-scale study showed
that dissolved air flotation was superior to sedimentation for removal of Cryptosporidium
i
oocysts (Plummer, era/., 1995). With proper coagulation and flocculation conditions, the
combination of coagulation, flocculation, and dissolved air flotation could achieve 2.5 to 3.5-log
removals of Cryptosporidium. .Under similar conditions, the combination of coagulation,
flocculation, and sedimentation could achieve no more than 1.0-log removal of oocysts. Similar
bench-scale studies showed that 1.3 to 2.8-log removals of Cryptosporidium could be achieved
by the combination of coagulation, flocculation, and dissolved air flotation (Hall, et a/., 1995).
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Plummet, et al. (1995) observed relatively weak correlations between log removal of
Cryptosporidium and percent removal of turbidity (r = 0.53), LTV absorbance (r2 = 0.52), and
dissolved organic carbon (r2 = 0.50).
Coagulation, flocculation, sedimentation, and filtration (conventional treatment)
Pilot-scale treatment studies with two waters in the western United States showed that
conventional treatment could obtain 3.0 to 6.2-log removal of Cryptosporidium, with a median of
approximately 4.6-log removal (Patania, et al., 1995). An average of 3.0-log removal was
observed in another pilot-scale study in Utah, with a range of 1.9 to 4.0-log removal (Nieminski
and Ongerth, 1995). Another Utah study was designed to evaluate Cryptosporidium removal in a
full-scale treatment plant that was not delivering water to customers. In this case, removals
ranged from 1.9 to 2.8-logs with an average of 2.3-logs (Nieminski and Ongerth, 1995). Actual
performance depends on numerous factors including source water quality, chemical pretreatment
conditions (e.g., coagulant dose and pH), filtration rate, bed depth, and filter media types. As
noted earlier, the reader is advised to read referenced reports for more detailed information
regarding the results summarized here.
The results to date suggest that the 2.0-log removal credit proposed for Cryptosporidium removal
by conventional treatment is reasonable and, as shown by the above data, may be conservative.
However, studies have only been published for waters from the western United States and need
• ,
to be confirmed with results from other types of water supplies.
92
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After combining data from the two Utah studies, Nieminski and Ongerth (1995) reported an r of
0.79 for the relationship between log removal of Cryptosporidium and log removal of 4-7 ^m
sized particles. The correlation between log removal of Cryptosporidium and log removal of
turbidity was weaker, with an r2 of 0.55. Patania, et al. (1995) reported no significant
correlations between log removal of Cryptosporidium and log removals of turbidity, 1-2 ^m
sized particles, 2-5 /urn sized particles, and 5-15 ^m sized particles. These latter results were
obtained from a broader range of source waters and suggest that correlations between
Cryptosporidium removal and removal of surrogate indicators may be site-specific. Further
work is necessary before definitive conclusions can be reached. These two studies also based
their conclusions on data obtained from different types of filtration practices (e.g., conventional
treatment, direct filtration, in-line filtration). Further analysis is needed to determine whether
their observations depend on the type of filtration practice.
Although no significant correlations were obtained between Cryptosporidium removal and
turbidity removal, Patania, et al. (1995) observed that filter effluent turbidities less than or equal
to 0.1 ntu were required to obtain 5-log Cryptosporidium removals on a reliable basis. Filter
effluent turbidities of 0.2 ntu or less were needed to reliably maintain 4-log Cryptosporidium
removals:
Coagulation, flocculation, flotation, and filtration
The effect of substituting sedimentation with dissolved air flotation on filtered water
Cryptosporidium concentrations has not been directly compared in literature reports. However,
93
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pilot-scale studies in the United Kingdom have shown that the combination of coagulation.
flocculation, flotation, and filtration can achieve 2.9 to 4.--log removal of oocysts (Hall, et al,
1995). These results are consistent with those described above.
Coagulation, flocculation, and filtration (direct filtration)\
The performance of direct filtration was assessed for the same Utah waters described in the
conventional treatment section (Nieminski and Ongerth, 1995). For the pilot-scale system,
Cryptosporidium removals ranged from 1.3 to 3.6-logs with an average of 3.0-logs. The full-
scale system achieved an average oocyst removal of 2.8-logs with a range of 2.6 to 2.9-logs.
Comparable removals were observed in pilot-scale studies with water from Seattle (Ongerth and
Pecoraro, 1995). Results in this study showed 2.7 to 3.1-log removal of Cryptosporidium.
Direct comparisons between direct and conventional nitration were only obtained with the pilot-
scale system in Utah. Results indicate that there was no statistically significant difference
between the two types of filtration. At this time, this conclusion can only be applied to this case
and other studies will be necessary to determine those situations in which direct filtration
achieves performance comparable to conventional treatment
i ' '
Coagulation and filtration (in-line filtration)
i • ,
Using water from the Seattle water supply, pilot-scale treatment studies showed that in-line
filtration could obtain 1.6 to 4.2-log removal of Cryptosporidium. The median value was
approximately 2.8-log removal (Patania, et al., 1995). Unfortunately, a direct comparison
between in-line filtration and conventional treatment has not been made for the same water
94
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supply.
Diatomaceous earth filtration
A recent study with water from Sydney, Australia concluded that diatomaceous earth filtration
could perform significantly better than conventional treatment for removal of Cryptosporidium
oocysts (Ongerth and Button, 1997), In this bench-scale study, removals ranged from 3.6 to 6,7
i
logs. Although these results appear to show superior performance of diatomaceous earth
filtration, they were performed at filtration rates lower than those commonly used in
conventional treatment facilities. Another study has shown comparable Cryptosporidium oocyst
removals by pilot-scale diatomaceous earth filtration with a Pennsylvania water, where removals
ranged from 4.6 to 5.9-logs (Schuler, et al., 1991).
Slow sand filtration
Several studies have been performed to evaluate Cryptosporidium oocyst removal by slow sand
filtration. Removals ranging from 3.9 to 7.1-logs were observed in pilot-scale studies with water
/ • -.
from Pennsylvania (Schuler, et al., 1991). Another study in the United Kingdom showed that
oocyst removal exceeded 4.5-logs (Timms, et al., 1995). Only 03-log oocyst removal was
obtained in a slow sand filter located in British Columbia. However, the filter media in this- filter
did not meet standard specifications for slow sand filters (Fogel, et al., 1993). Therefore, the
results obtained to date suggest that slow sand filtration is an effective barrier to oocyst passage
when the process is properly designed.
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\fembrane processes
In several micro filtration and ultrafiltration experiments, Cryptosporidium oocysts were not
•
detected in treated waters when the membranes were intact (Jacangelo, et al., 1995). This result
was observed with feed oocyst concentrations as high as 9.1 x 104 oocysts/L, suggesting a
removal capability of more than 7.1-logs. This result is expected because the pores within the
membrane skin are smaller than Cryptosporidium oocysts. More recent studies have shown that
Cryptosporidium oocysts could pass through a ceramic microfiltration membrane having a
nominal porosity of 0.2 ^m (Drozd and Schwartzbrod, 1997). In this case, oocyst removals
ranged from 4.3 to 5.5-logs. However, no attempts were made to assess the integrity of the
membrane and its associated equipment in this study.
4. Inactivation of Cryptosporidium
Introduction
The majority of studies performed to date have evaluated Cryptosporidium inactivation in
buffered, demand-free waters and in small, batch reactors. Very little is known about the ability
of alternative disinfectants to inactivate Cryptosporidium oocysts in natural waters or in flow-
through treatment systems. Results depend on the method used to quantify inactivation and, if
mouse infectivity is used to quantify inactivation, results may depend on the type of mouse and
on the strain of Cryptosporidium used in the study. For these reasons, the reader is cautioned
against the extrapolation of information presented in mis section to inactivation of
•
Cryptosporidium in natural waters and to infecn'vity in humans. The purpose of this section is to
summarize the inactivation work performed to date for four disinfectant chemicals - ozone,
-------
prechionnation (Finch et al., 1997; Gyurek, et a/., 1997). Again, published reports are very
^
preliminary and further studies will be needed to determire if this phenomenon is observed in
natural waters and in other laboratories.
Mixed oxidants
A proprietary system that electrochemically produces a mixed oxidant solution was recently
evaluated for its ability to inactivate Cryptosporidium oocysts (Venczel, et al., 1997). This
system was observed to achieve more than 3-log inactivation of Cryptosporidium oocysts with a
5 mg/L oxidant dose and a 4 hour contact time.
Ultraviolet radiation
Recent studies have demonstrated that some ultraviolet irradiation technologies may be .
promising for Cryptosporidium inactivation (Campbell, et al., 1995; Arrowood, et al., 1996).
Because the technologies are not comparable, the results of these studies cannot be compared. In
one case, ultraviolet irradiation produced 2 to 3-log inactivation of Cryptosporidium oocysts at
ultraviolet doses and contact times achievable by commercial equipment (Campbell, et al.,
'4 . -. . -
1995). In the other study, up to 6-logs of inactivation were observed with an alternative piece of
commercial equipment (Arrowood, et al., 1996).
D. Summary
Analysis of Water Samples
Filtration through wound yam filters remains the most common collection method. Variations of
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this collection methodology, including cellulose acetate membrane filters and capsule filters,
could result in improved retention of oocysts, however, interlaboratory comparisons have not
been published. Calcium carbonate flocculation methods, which can concentrate up to 10 liters
of water, have also been shown superior to wound yam filters but may interfere with viability
determinations. Centrifugation-based concentration technologies such as vortex flow filtration,
cross flow filtration, and continuous centrifugation could potentially recover greater numbers of
oocysts than the currently used ASTMICR methods, however the methods require
interlaboratory validation. Immunomagnetic capture and flow cytometry also show considerable
recovery increases using either seeded or environmental samples. However, performance will
be influenced by water turbidity and composition. Laser scanning devices have also performed
well in early studies, however more research is required. Several applications of the polymerase
chain reaction for the detection of Cryptosporidiwn have been described in the literature, some of
which may be able to distinguish viable from nohviable oocysts, however, enzymatic inhibition
remains problematic. Since the determination of Cryptosporidiwn viability is critical in
assessing the public health threat of cryptosporidiosis, a number of viability assays have been
described and compared to animal infectivity models. In general, viability assays have produced
conservative estimates of oocyst viability when compared to animal modeling data, however,
limitations in viability assays have precluded their routine use in environmental samples. The
USEP A has established an approval process for laboratories performing detection of
Cryptosporidiwn and Giardia in water.
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:hiorine dioxide, chlorine, and monochloramine.
Ozone
Cryptosporidium inactivation by ozone has received a significant amount of attention. Ozone
clearly achieves the most significant levels of Cryptosporidium inactivation when compared to
the other disinfectants listed above (Korich, et a/., 1990; Finch et a/., 1997). In order to achieve
3-log inactivation of Cryptosporidium at pH 7, the product of contact time and ozone
concentration (CT) needs to be in the range of 8 to 16 mg*min/L at 7°C and in the range of 3 to
15 mg»min/L at 22 °C (Finch et al., 1993). CT values for 2-log inactivation of Cryptosporidium
by ozone at pH 7 are in the 5 to 10 mgnnin/L range at 7°C and in the 2 to 8 mgnnin/L range at
22°C. It is important to stress that these values are based on mean performance and do not
account for the wide variability observed in test results. Results observed by others appear to be
in general agreement when the variability in data is taken into account (Peeters, et al., 1989;
x
Korich, et al, 1990; Parker, et al., 1993;Owens, et al., 1994a and b; Quinn, et al., 1996; Finch et
al., 1997).
Chlorine Dioxide
Results obtained to date suggest that chlorine dioxide is the second most effective disinfectant on
the above list (Peeters, etal., 1989; Korich, et al., 1990; Finch et al., 1997, Liyanage, et al.,
1997a). CT values needed for Cryptosporidium inactivation by chlorine dioxide are considerably
higher than those needed for ozone. To achieve a 1-log inactivation of Cryptosporidium at pH 7
and 25 ° C, CT values of approximately 60 mg-min/L would be necessary. At pH 8, the required
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CT values appear to be greater than this. Recent results have shown that chlorine dioxide may be
more effective if ozone is applied upstream of the chlorine. Jioxide addition point (Liyanage, et
at., 1997b). However, this practice is not likely to become common in the United States if
currently proposed disinfection byproduct regulations are implemented.
Chlorine
At CT values commonly used in drinking water treatment, chlorine does not achieve more than a
1-log inactivation of Cryptosporidium oocysts (Korich, et al., 1990; Payer, 1995; Finch et al.,
1997; Gyurek, et al., 1997; Venczel, etal., 1997). This is true even in demand-free water at pH 6
and 22 °C, conditions where chlorine can be expected to achieve the best level of performance.
Recent screening studies have suggested that the ability of chlorine to inactivate
Cryptosporidium may be enhanced by preozonation (Finch et al., 1997). At this time, published
reports are very preliminary and further studies will be needed to determine if this phenomenon
is observed in natural waters and in other laboratories.
Monochloramine •
Ever since the passage of the Surface Water Treatment Rule, monochloramine has not been
frequently used as a primary disinfectant in drinking water treatment Like chlorine,
monochloramine is not capable of achieving more than a 1-log inactivation of Cryptosporidium
at CT values commonly encountered in treatment practice (Korich, et al., 1990; Finch et al.,
1997; Gyurek, et al., 1997). Also, preliminary studies have shown mat the ability of
monochloramine to inactivate Cryptosporidium may be enhanced by preozonan'on or by
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Analysis of Biological Samples
The 1994 Cryptosporidium Criteria Document describes the increased sensitivity of
immunofluorescent antibody-based (IFA) procedures. Traditional staining methods such as the
Ziehl-Neelsen stain, however, are still widely used. Enzyme immunoassay (EIA) methods are
fast, inexpensive, easily performed, and show sensitivity approaching that of immunfluorescence
methods. However, a lack of confirmatory analyses may preclude their routine use. Enzyme
immunoassays may be useful for busy hospital laboratories or large scale screening surveys.
• /
Several PCR-based methods capable of distinguishing differences among specific strains have
been described in the literature. As with testing the efficacy of different analysis of water
methods, interlaboratory comparisons require strict adherence to oocyst quality and rigorous
enumeration procedures. Recommendations by Klonicki et al. (1997) should be observed in
future studies.
Summary of removal studies
Of the technologies available to the drinking water industry, membrane processes appear to
provide the most significant levels of Cryptosporidium removal. Conventional treatment
practices appear capable of meeting 2-log removals in most of the cases studied to date.
Although direct filtration and in-line filtration may be expectedto be less effective than
conventional treatment, this has not yet been demonstrated in a conclusive manner. Alternative
technologies like diatomaceous earth filtration and slow sand filtration appear capable of
achieving comparable, if not better, levels of Cryptosporidium removal than conventional
treatment
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Summar,- oj inactivation studies
As noted above, ozone appears to be the best chemical disinfectant for Cryptosporidium
inactivation at this time. The mixed oxidant and ultraviolet light systems appear to be promising,
but have only been tested in minimal fashion when compared with ozone. Also holding some
promise are the sequential disinfection systems of ozone followed by chlorine and ozone
followed by monochloramine. Very few studies have evaluated Cryptosporidium inactivation in
natural waters.
. Research Requirements
A. Data Gaps
Many of the data gaps in our knowledge regarding Cryptosporidium previously identified in the
1 994 Cryptosporidium Criteria Document have been filled, and an enormous amount of
information has become available from research completed during the past 4 years. Many of the
lessons learned from Cryptosporidium will be applicable to the next waterbome pathogen that
emerges.
At a recent workshop on Cryptosporidium Issues related to drinking water, data gaps in four
main areas of Cryptosporidium research, Source Water Occurrence, Analysis, Treatment, and
Health Effects, were identified.
Scarce Water Occurrence: Although some data on the source and occurrence of
Cryptosporidium in watersheds exist, more research is greatly needed. In particular, the fate and
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transport of Crypiosporidium within watersheds is poorly understood. Improvements in
monitoring methods and analytical techniques are greatly needed to increase our understanding
of these issues.
Analysis: Research efforts for recovery of Cryptosporidium oocysts from water samples as well
as from clinical samples have been improved and many of the steps in these processes that
historically have been responsible for oocyst losses have been identified. Studies comparing
methods have been done, and the advantages and disadvantages of various approaches
elucidated. New detection methods are being developed, especially those using molecular
biology approaches (e.g., PCR/gene probe procedures), lasej-based technologies, and computer
microscopy. Using these approaches, methods for determination of oocyst survivability in the
environment and infectivity should improve significantly. Detection methods continue to be
quite variable and the need for a standard method that is accurate, precise, quick and affordable
still exists. Many of the newer technologies are yet unproven with real-world samples and
validation testing must be completed. In addition, not enough is known about the basic cell
biology of Cryptosporidium. Greater knowledge in this area will not only help in the
development of an accurate detection method, but also advance viability, infectivity, and
speciation assays for environmental Cryptosporidium.
Treatment: The need is great to develop, identify, and evaluate new methods for disinfection
processes (e.g., ozonation, UV, unproved filtration). In addition, compounds other than chlorine
should be sought to serve as residual disinfectants in finished drinking water. Complete
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evaluation of treatment tor oocyst removal is dependent on better detection methods and more
ngorous enumeration practices. Other gaps in the data retarding treatment of drinking water
include: the usefulness and efficiency of surrogates to determine success of treatment, an
understanding of the cellular biology and biochemistry involved in the treatment process, and
guidelines or a decision matrix to assist in treatment selection.
Health Effects: Progress has been made in identifying compounds that can be used for human
and animal therapy/treatment, although evaluation, validation, and clinical trials will be required.
These drugs should be subjected to FDA approval after such research and clinical trials are
completed. However, studies to develop new drugs should be continued. There has been very
little progress in elucidating the pathogenic mechanisms involved in cryptosporidiosis, but U.S.
EPA-sponsored human infectivity studies should provide some useful information. In addition
to gaps in drug therapy, more information is needed to better identify and characterize outbreaks,
to assess the risks to susceptible populations, and to identify the infectious dose and virulence
i
across different populations. Finally, better diagnostic serological methods need to be developed
and tested and more serology-based epidemiology studies need to be completed.
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