Cryptosporidium: New developments in cell culture

Studies on Cryptosporidium species have been hampered by the limited amount of parasitic stages available for research. One of the major objectives of many laboratories is to develop a reproducible culture model for this important parasite. Recent research has resulted in long-term culturing of Cryptosporidium in cell culture using pH modification, sub-culturing and gamma irradiation. Further advances in the in vitro culturing of Cryptosporidium revealed that this parasite can complete its life cycle in culture medium overcoming the problem of using the host cells, as host cell overgrowth and aging resulted in the termination of the Cryptosporidium life cycle prior to its completion. Improved methods for visualizing life cycle stages in cell-free culture have also been developed. This review will discuss factors that can influence the success of Cryptosporidium culture in vitro and propose new ideas for the future optimization of the cell-free culture system.     

Evolution of Cryptosporidium in vitro culture

This overview discusses findings from culturing Cryptosporidium spp. in cell and axenic cultures as well as factors limiting the development of this parasite in cultivation systems during recent years. A systematic review is undertaken of findings regarding the life cycle of the parasite, taking into account physiological, biochemical and genetic aspects, in the hope that this attempt will facilitate future approaches to research and developments in the understanding of Cryptosporidium biology.     

Biology of Cryptosporidium from marsupial hosts

The majority of biological data on Cryptosporidium has been collected from humans and domestic animal hosts which creates a bias in knowledge on the biodiversity and evolution of this parasite genus. Further to understanding Cryptosporidium biology are studies encompassing broad hosts that represent diverse taxa sampled across wide geographic ranges. Marsupials represent a group of wildlife hosts from which limited information on Cryptosporidium is available. As marsupial hosts are an ancient mammalian lineage they represent an important group for studying parasite evolution. This review summarises information of the biology, epidemiology and evolution of Cryptosporidium in marsupial hosts, and discusses the importance of further understanding interactions in this parasite-host system.     

Prevalence of Cryptosporidium-Associated Diarrhea in a High Altitude-Community of Saudi Arabia Detected by Conventional and Molecular Methods

Cryptosporidium diarrhea represents a relevant clinical problem in developing countries. In Al-Taif, a city of Saudi Arabia that lies at an altitude of an around 2 km above the sea level, Cryptosporidium infection seems to be undiagnosed in nearly all clinical laboratories. Furthermore, nothing was published regarding Cryptosporidium-associated diarrhea in this area. The objectives of this research were to (1) determine the Cryptosporidium prevalence among patients with diarrhea and (2) to estimate the performances of 3 different diagnostic methods. Total 180 diarrheal fecal samples, 1 sample per patient, were collected between January and August 2013. Samples were screened for Cryptosporidium with modified Zeihl Neelsen (ZN) microscopy, RIDA® Quick lateral flow (LF) immunotest, and a previously published PCR. The Cryptosporidium prevalence rate was 9.4% (17/180), 10% (18/180), and 11.6% (21/180) by microscopy, LF, and PCR test, respectively. Infection was significantly (P=0.004) predominant among children <5 years (22%) followed by children 5-9 years (11.1%). Although infection was higher in males than in females (16.2% males and 8.5% females), the difference was not statistically significant (P=0.11). Compared to PCR, the sensitivity of microscopy and the LF test were 80.9%, 85.7%, respectively. To conclude, high Cryptosporidium-associated diarrhea was found in this area especially in children ≤9 years. The PCR test showed the best performance followed by the LF test and ZN staining microscopy. The primary health care providers in Al-Taif need to be aware of and do testing for this protozoon, particularly for children seen with diarrhea.     

Tracking Host Sources of Cryptosporidium spp. in Raw Water for Improved Health Risk Assessment

Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.     

Assessment of Giardia and Cryptosporidium spp. as a Microbial Source Tracking Tool for Surface Water: Application in a Mixed-Use Watershed

Knowledge of host specificity, combined with genomic sequencing of Giardia and Cryptosporidium spp., has demonstrated a microbial source tracking (MST) utility for these common waterborne microbes. To explore the source attribution potential of these pathogens, water samples were collected in a mixed rural-urban watershed in the Township of Langley, in southwestern British Columbia (BC), Canada, over a 2-year period. Cryptosporidium was detected in 63% of surface water samples at concentrations ranging from no positive detection (NPD) to 20,600 oocysts per 100 liters. Giardia was detected in 86% of surface water samples at concentrations ranging from NPD to 3,800 cysts per 100 liters of water. Sequencing at the 18S rRNA locus revealed that 50% of Cryptosporidium samples and 98% of Giardia samples contained species/genotypes (Cryptosporidium) or assemblages (Giardia) that are capable of infecting humans, based on current knowledge of host specificity and taxonomy. Cryptosporidium genotyping data were more promising for source tracking potential, due to the greater number of host-adapted (i.e., narrow-host-range) species/genotypes compared to Giardia, since 98% of Giardia isolates were zoonotic and the potential host could not be predicted. This report highlights the benefits of parasite genomic sequencing to complement Method 1623 (U.S. Environmental Protection Agency) and shows that Cryptosporidium subtyping for MST purposes is superior to the use of Giardia subtyping, based on better detection limits for Cryptosporidium-positive samples than for Giardia-positive samples and on greater host specificity among Cryptosporidium species. These additional tools could be used for risk assessment in public health and watershed management decisions.     

The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment

Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources.     

Sheep May Not Be an Important Zoonotic Reservoir for Cryptosporidium and Giardia Parasites

Little is known of the prevalence of Cryptosporidium and Giardia parasites in sheep and the genotypes that they harbor, although potentially sheep may contribute significantly to contamination of watersheds. In the present study, conducted in Western Australia, a total of 1,647 sheep fecal samples were screened for the presence of Cryptosporidium and Giardia spp. using microscopy, and a subset (n = 500) were screened by PCR and genotyped. Analysis revealed that although both parasites were detected in a high proportion of samples by PCR (44% and 26% for Giardia and Cryptosporidium spp., respectively), with the exception of one Cryptosporidium hominis isolate, the majority of isolates genotyped are not commonly found in humans. These results suggest that the public health risk of sheep-derived Cryptosporidium and Giardia spp. in catchment areas and effluent may be overestimated and warrant further investigation.     

Comparison of various staining methods for the detection of Cryptosporidium in cell-free culture

The complete development of Cryptosporidium in host cell-free medium first described in 2004, represented a significant advance that can facilitate many aspects of Cryptosporidium research. A current limitation of host cell-free cultivation is the difficulty involved in visualising the life-cycle stages as they are very small in size, morphologically difficult to identify and dispersed throughout the media. This is in contrast to conventional cell culture methods for Cryptosporidium, where it is possible to focus on the host cells and view the foci of infection on the host cells. In the present study, we compared three specific and three non-specific techniques for visualising Cryptosporidium parvum life-cycle stages in cell-free culture; antibody staining using anti-sporozoite and anti-oocyst wall antibodies (Sporo-Glo™ and Crypto Cel), fluorescent in-situ hybridization (FISH) using a Cryptosporidium specific rRNA oligonucleotide probe and the non-specific dyes; Texas Red, carboxyfluorescein diacetate succinimidyl ester (CFSE) and 4,6′ diamino-2-phenylindole dihydrochloride (DAPI). Results revealed that a combination of Sporo-Glo™ and Crypto Cel staining resulted in easy and reliable identification of all life-cycle stages.     

Epidemiology and Molecular Characterization of Cryptosporidium spp. in Humans,Wild Primates,and Domesticated Animals in the Greater Gombe Ecosystem,Tanzania

Cryptosporidium is an important zoonotic parasite globally. Few studies have examined the ecology and epidemiology of this pathogen in rural tropical systems characterized by high rates of overlap among humans, domesticated animals, and wildlife. We investigated risk factors for Cryptosporidium infection and assessed cross-species transmission potential among people, non-human primates, and domestic animals in the Gombe Ecosystem, Kigoma District, Tanzania. A cross-sectional survey was designed to determine the occurrence and risk factors for Cryptosporidium infection in humans, domestic animals and wildlife living in and around Gombe National Park. Diagnostic PCR revealed Cryptosporidium infection rates of 4.3% in humans, 16.0% in non-human primates, and 9.6% in livestock. Local streams sampled were negative. DNA sequencing uncovered a complex epidemiology for Cryptosporidium in this system, with humans, baboons and a subset of chimpanzees infected with C. hominis subtype IfA12G2; another subset of chimpanzees infected with C. suis; and all positive goats and sheep infected with C. xiaoi. For humans, residence location was associated with increased risk of infection in Mwamgongo village compared to one camp (Kasekela), and there was an increased odds for infection when living in a household with another positive person. Fecal consistency and other gastrointestinal signs did not predict Cryptosporidium infection. Despite a high degree of habitat overlap between village people and livestock, our results suggest that there are distinct Cryptosporidium transmission dynamics for humans and livestock in this system. The dominance of C. hominis subtype IfA12G2 among humans and non-human primates suggest cross-species transmission. Interestingly, a subset of chimpanzees was infected with C. suis. We hypothesize that there is cross-species transmission from bush pigs (Potaochoerus larvatus) to chimpanzees in Gombe forest, since domesticated pigs are regionally absent. Our findings demonstrate a complex nature of Cryptosporidium in sympatric primates, including humans, and stress the need for further studies.     

Global Positioning System Data-Loggers: A Tool to Quantify Fine-Scale Movement of Domestic Animals to Evaluate Potential for Zoonotic Transmission to an Endangered Wildlife Population

Domesticated animals are an important source of pathogens to endangered wildlife populations, especially when anthropogenic activities increase their overlap with humans and wildlife. Recent work in Tanzania reports the introduction of Cryptosporidium into wild chimpanzee populations and the increased risk of ape mortality associated with SIVcpz-Cryptosporidium co-infection. Here we describe the application of novel GPS technology to track the mobility of domesticated animals (27 goats, 2 sheep and 8 dogs) with the goal of identifying potential routes for Cryptosporidium introduction into Gombe National Park. Only goats (5/27) and sheep (2/2) were positive for Cryptosporidium. Analysis of GPS tracks indicated that a crop field frequented by both chimpanzees and domesticated animals was a potential hotspot for Cryptosporidium transmission. This study demonstrates the applicability of GPS data-loggers in studies of fine-scale mobility of animals and suggests that domesticated animal–wildlife overlap should be considered beyond protected boundaries for long-term conservation strategies.     

Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (≈375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ≈550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.     

Molecular Characterization of Cryptosporidium Oocysts in Samples of Raw Surface Water and Wastewater

Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate the human-pathogenic Cryptosporidium parasites from those that do not infect humans and to track the source of Cryptosporidium oocyst contamination in the environment. In this study, we used a small-subunit rRNA-based PCR-restriction fragment length polymorphism (RFLP) technique to detect and characterize Cryptosporidium oocysts in 55 samples of raw surface water collected from several areas in the United States and 49 samples of raw wastewater collected from Milwaukee, Wis. Cryptosporidium parasites were detected in 25 surface water samples and 12 raw wastewater samples. C. parvum human and bovine genotypes were the dominant Cryptosporidium parasites in the surface water samples from sites where there was potential contamination by humans and cattle, whereas C. andersoni was the most common parasite in wastewater. There may be geographic differences in the distribution of Cryptosporidium genotypes in surface water. The PCR-RFLP technique can be a useful alternative method for detection and differentiation of Cryptosporidium parasites in water.     

Genetic richness and diversity in Cryptosporidium hominis and C. parvum reveals major knowledge gaps and a need for the application of “next generation” technologies — Research review

Cryptosporidium species (apicomplexan protists) are a major cause of diarrhoeal disease (= cryptosporidiosis) in humans worldwide. The impact of cryptosporidiosis is also compounded by the spread of HIV/AIDS and a lack of cost-effective anti-cryptosporidial chemotherapeutics or vaccines. Mitigation of the impact of cryptosporidiosis in humans needs to focus on prevention and control strategies, built on a sound understanding of the epidemiology of Cryptosporidium species. Refined epidemiological studies rely on the use of molecular tools employing informative genetic markers. Currently, the 60-kDa glycoprotein gene (gp60) is the most suitable and widely used genetic marker for Cryptosporidium species infecting humans. Here, we undertake an analysis of all publicly-available gp60 sequence data and associated literature for C. hominis and C. parvum, and yield useful insights into the richness, diversity and distribution of genetic variants, and link these variants to human cryptosporidiosis. This global analysis reveals that, despite high genetic richness in Cryptosporidium isolates from humans, there is a surprisingly low diversity. It also highlights limited knowledge about the genetics of cryptosporidiosis in developing nations and in many animals that might act as infection sources. Clearly, there is a major need for more comprehensive studies of Cryptosporidium infecting humans and other animals in Africa and Asia. As molecular technologies improve and become affordable, future studies should utilize “next generation” sequencing and bioinformatic platforms to conduct comparative ‘genome sequence surveys’ to test the validity of current genetic classifications based on gp60 data. Complemented by in vitro and in vivo investigations, these biotechnological advances will also assist significantly in the search for new intervention strategies against human cryptosporidiosis.     

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies ¹². The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used^1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocystsand Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously^{1-3,5-8,10,11}. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters⁴. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices^9,13 .This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.     

Comparison of Method 1623 and Cell Culture-PCR for Detection of Cryptosporidium spp. in Source Waters

Analysis of Cryptosporidium occurrence in six watersheds by method 1623 and the integrated cell culture-PCR (CC-PCR) technique provided an opportunity to evaluate these two methods. The average recovery efficiencies were 58.5% for the CC-PCR technique and 72% for method 1623, but the values were not significantly different (P = 0.06). Cryptosporidium oocysts were detected in 60 of 593 samples (10.1%) by method 1623. Infectious oocysts were detected in 22 of 560 samples (3.9%) by the CC-PCR technique. There was 87% agreement between the total numbers of samples positive as determined by method 1623 and CC-PCR for four of the sites. The other two sites had 16.3 and 24% correspondence between the methods. Infectious oocysts were detected in all of the watersheds. Overall, approximately 37% of the Cryptosporidium oocysts detected by the immunofluorescence method were viable and infectious. DNA sequence analysis of the Cryptosporidium parvum isolates detected by CC-PCR showed the presence of both the bovine and human genotypes. More than 90% of the C. parvum isolates were identified as having the bovine or bovine-like genotype. The estimates of the concentrations of infectious Cryptosporidium and the resulting daily and annual risks of infection compared well for the two methods. The results suggest that most surface water systems would require, on average, a 3-log reduction in source water Cryptosporidium levels to meet potable water goals.     

Detection of Infectious Cryptosporidium Oocysts by Cell Culture Immunofluorescence Assay: Applicability to Environmental Samples

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.     

Genotypes of Cryptosporidium Species Infecting Fur-Bearing Mammals Differ from Those of Species Infecting Humans

Of 471 specimens examined from foxes, raccoons, muskrats, otters, and beavers living in wetlands adjacent to the Chesapeake Bay, 36 were positive for five types of Cryptosporidium, including the C. canis dog and fox genotypes, Cryptosporidium muskrat genotypes I and II, and Cryptosporidium skunk genotype. Thus, fur-bearing mammals in watersheds excreted host-adapted Cryptosporidium oocysts that are not known to be of significant public health importance.     

Phylogenetic Analysis of the Hypervariable Region of the 18S rRNA Gene of Cryptosporidium Oocysts in Feces of Canada Geese (Branta canadensis): Evidence for Five Novel Genotypes

To assess genetic diversity in Cryptosporidium oocysts from Canada geese, 161 fecal samples from Canada geese in the United States were analyzed. Eleven (6.8%) were positive for Cryptosporidium spp. following nested PCR amplification of the hypervariable region of the 18S rRNA gene. Nine PCR products from geese were cloned and sequenced, and all nine diverged from previously reported Cryptosporidium 18S rRNA gene sequences. Five sequences were very similar or identical to each other but genetically distinct from that of Cryptosporidium baileyi; two were most closely related to, but genetically distinct from, the first five; and two were distinct from any other sequence analyzed. One additional sequence in the hypervariable region of the 18S rRNA gene isolated from a cormorant was identical to that of C. baileyi. Phylogenetic analysis provided evidence for new genotypes of Cryptosporidium species in Canada geese. Results of this study suggest that the taxonomy of Cryptosporidium species in geese is complex and that a more complete understanding of genetic diversity among these parasites will facilitate our understanding of oocyst sources and species in the environment.     

Validity of the Indicator Organism Paradigm for Pathogen Reduction in Reclaimed Water and Public Health Protection

The validity of using indicator organisms (total and fecal coliforms, enterococci, Clostridium perfringens, and F-specific coliphages) to predict the presence or absence of pathogens (infectious enteric viruses, Cryptosporidium, and Giardia) was tested at six wastewater reclamation facilities. Multiple samplings conducted at each facility over a 1-year period. Larger sample volumes for indicators (0.2 to 0.4 liters) and pathogens (30 to 100 liters) resulted in more sensitive detection limits than are typical of routine monitoring. Microorganisms were detected in disinfected effluent samples at the following frequencies: total coliforms, 63%; fecal coliforms, 27%; enterococci, 27%; C. perfringens, 61%; F-specific coliphages, ~40%; and enteric viruses, 31%. Cryptosporidium oocysts and Giardia cysts were detected in 70% and 80%, respectively, of reclaimed water samples. Viable Cryptosporidium, based on cell culture infectivity assays, was detected in 20% of the reclaimed water samples. No strong correlation was found for any indicator-pathogen combination. When data for all indicators were tested using discriminant analysis, the presence/absence patterns for Giardia cysts, Cryptosporidium oocysts, infectious Cryptosporidium, and infectious enteric viruses were predicted for over 71% of disinfected effluents. The failure of measurements of single indicator organism to correlate with pathogens suggests that public health is not adequately protected by simple monitoring schemes based on detection of a single indicator, particularly at the detection limits routinely employed. Monitoring a suite of indicator organisms in reclaimed effluent is more likely to be predictive of the presence of certain pathogens, and a need for additional pathogen monitoring in reclaimed water in order to protect public health is suggested by this study.