Oocysts In Water Research Articles

Novel strategy to quantify the viability of oocysts of Cryptosporidium parvum and C. hominis, a risk factor of the waterborne protozoan pathogens of public health concern

While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.

Detection of DNA from Toxoplasma gondii oocysts in water for reuse.

The absence of a standardized method for detecting oocysts in water samples makes it difficult to characterize them, including in water for reuse. This study aimed to detect Toxoplasma gondii oocysts using two extraction methods. Using method 1693/2014 USEPA, 30 L of water for reuse from two wastewater treatment plants (WWTPs) in the city of São Paulo, Brazil, was concentrated, totaling 20 samples. The supernatant generated from the immunomagnetic separation (IMS) step was collected for detection of T. gondii oocysts. For DNA extraction, two techniques were used: the commercial kit DNeasy PowerSoil Kit® optimized with the enzyme Zymolyase® and with freeze-thaw steps. DNA quantification was performed with the target sequence of gene B1. From 16 samples submitted to enzymatic extraction, four were positive. In freeze-thaw extraction, no DNA was detected. DNA extraction was the essential step for oocyst detection given the resistant nature of their wall.

Development of cell-imprinted polymer surfaces for Cryptosporidium capture and detection

Cryptosporidium parvum is waterborne parasite that can cause potentially life-threatening gastrointestinal disease and is resistant to conventional water treatment processes, including chlorine disinfection. The current Environmental Protection Agency-approved method for oocyst detection and quantification is expensive, limiting the ability of water utilities to monitor complex watersheds thoroughly to understand the fate and transport of C. parvum oocysts. In this work, whole cell imprinting was used to create selective and sensitive surfaces for the capture of C. parvum oocysts in water. Cell-imprinted Polydimethylsiloxane (PDMS) was manufactured using a modified stamping approach, and sensitivity and selectivity were analyzed using different water chemistries and different surrogate biological and non-biological particles. The overall binding affinity was determined to be less than that of highly specific antibodies, but on par with standard antibodies and immune-enabled technologies. These initial results demonstrate the potential for developing devices using cell-imprinting for use in waterborne pathogen analysis.

Survival and infectivity of Toxoplasma gondii and Cryptosporidium parvum oocysts bioaccumulated by Dreissena polymorpha.

The study was aimed to understand the depuration process of Cryptosporidium parvum and Toxoplasma gondii oocysts by zebra mussel (Dreissena polymorpha), to consider the use of the zebra mussel as a bioremediation tool. Two experiments were performed: (i) individual exposure of mussel to investigate oocyst transfers between bivalves and water and (ii) in vivo exposure to assess the ability of the zebra mussel to degrade oocysts. (i) Our results highlighted a transfer of oocysts from the mussels to the water after 3 and 7days of depuration; however, some oocysts were still bioaccumulated in mussel tissue. (ii) Between 7days of exposure at 1000 or 10000 oocysts/mussel/day and 7days of depuration, the number of bioaccumulated oocysts did not vary but the number of infectious oocysts decreased. Results show that D. polymorpha can release oocysts in water via (pseudo)faeces in depuration period. Oocysts remain bioaccumulated and infectious oocyst number decreases during the depuration period in zebra mussel tissues. Results suggest a degradation of bioaccumulated C. parvum and T. gondii oocysts. This study highlighted the potential use of D. polymorpha as a bioremediation tool to mitigate of protozoan contamination in water resources.

Study Effect of Clarithromycin drug on Cryptosporidium Parvum and Efficiency of ELISA Technique in Diagnosis Comparison with Some Traditional Methods in vitro

This research aimed to study effect of Clarithromycin drug on Cryptosporidium Parvum and efficiency of ELISA technique in diagnosis comparison with some traditional methods in vitro. Samples collected from four different sources water were 400 liters:100 liters tap water provider by Al-Kufa District River,100 liters house tank water,100 liters from sea of Al-Najaf and 100 liters stagnant water during period from October 2018 to April 2019.Examined samples by three laboratory methods: Modified Ziehl-Neelson stain, Flotation by saturated sugar solution method and ELISA to detecting from Cryptosporidium oocysts in the water as used Clarithromycin drug effective against C.parvum in vitro. Capability test was implement impregnation isolated and determination cells of parasite counted when at 37°C/ 48h incubated. Differ concentrations from Clarithromycin were 4, 8, 16 and 32 mg/L respectively compared with control group.Results of the current study showed that oocysts were found in 182 from 400 liters 45.5% as showed that height oocysts in stagnant water then followed Sea of Al-Najaf were 55% and 45% respectively, height percentage of oocysts were in April and March were 69.09% and 66% while decrease percentage of infection in October was 25%. As showed study results presence significant differences in efficiency of ELISA technique was 83.33% at detection from oocysts compared with Flotation by saturated sugar solution and Modified Ziehl-Neelson stain methods were 66.66% and 61.11% respectively. Shown results this study that Clarithromycin with 16 and 32 mg/L lead to reduction in number oocysts was 17.3±3.5 and 15.4±3.9, sporozoites counts was 17.1±3.4 and 14.9±4.0 compared with control group was 20.5±2.8.As observed significance differences P.0.05 in the mean numbers the oocysts and sporozoites in three replicate plates after 48 h. These results proved that Clarithromycin have effectiveness in inhibition C.parvum in vitro.

Study Effect of Clarithromycin drug on Cryptosporidium Parvum and Efficiency of ELISA Technique in Diagnosis Comparison with Some Traditional Methods in vitro

This research aimed to study effect of Clarithromycin drug on Cryptosporidium Parvum and efficiency of ELISA technique in diagnosis comparison with some traditional methods in vitro. Samples collected from four different sources water were 400 liters:100 liters tap water provider by Al-Kufa District River,100 liters house tank water,100 liters from sea of Al-Najaf and 100 liters stagnant water during period from October 2018 to April 2019.Examined samples by three laboratory methods: Modified Ziehl-Neelson stain, Flotation by saturated sugar solution method and ELISA to detecting from Cryptosporidium oocysts in the water as used Clarithromycin drug effective against C.parvum in vitro. Capability test was performed by impregnation of isolate on cell monolayers and determination the parasite counted after 48 h from the incubation at 37°C. Differ concentrations from Clarithromycin were 4, 8, 16 and 32 mg/L respectively compared with control group.Results of the current study showed that oocysts were found in 182 from 400 liters 45.5% as showed that height oocysts in stagnant water then followed Sea of Al-Najaf were 55% and 45% respectively, height percentage of oocysts were in April and March were 69.09% and 66% while decrease percentage of infection in October was 25%. As showed study results presence significant differences in efficiency of ELISA technique was 83.33% at detection from oocysts compared with Flotation by saturated sugar solution and Modified Ziehl-Neelson stain methods were 66.66% and 61.11% respectively. Shown results this study that Clarithromycin drug in concentrations of 16 and 32 mg/L caused decrease in number oocysts was 17.3±3.5 and 15.4±3.9, sporozoites counts was 17.1±3.4 and 14.9±4.0 compared with control group was 20.5±2.8.As observed significance differences (P?0.05) in the mean numbers of oocysts and sporozoites in three replicate plates after 48 h. These results proved that Clarithromycin have effectiveness in inhibition C.parvum in vitro

Determination of the viability of Toxoplasma gondii oocysts by PCR real-time after treatment with propidium monoazide.

ABSTRACTThis study aimed to investigate a methodology for discriminating viable and non-viable T. gondii oocysts in water. Analyses included two steps: (i) microscopic investigation with vital dyes; (ii) molecular investigation, using a real time PCR (qPCR), after parasite treatment (or not) with propidium monoazide (PMA). The method was called qPCR-PMA. Oocyst aliquots were incubated (15 min) at 25 ºC or 100 ºC and analyzed by microscopy, after trypan blue and neutral red staining. Microscopic investigation determined viable and non-viable oocysts. For the molecular investigation, both aliquots of oocysts were treated with PMA. Non-viable oocysts, after PMA treatment, exhibited an inhibition of DNA amplification by qPCR. Although analyses were carried out with oocysts treated experimentally, these results suggest that qPCR-PMA can be a useful strategy to distinguish viable and non-viable T. gondii oocysts in water safety testing, showing if water is safe to drink.

Patterns of Transmission and Sources of Infection in Outbreaks of Human Toxoplasmosis.

We report on apparent temporal progression of probable sources of infection and transmission routes for global human toxoplasmosis outbreaks as described in published articles. We searched the Scientific Electronic Library Online, Web of Science, PubMed, and Scopus databases for articles on Toxoplasma, toxoplasmosis, and outbreaks. We found that transmission routes for Toxoplasma gondii varied by decade. In the 1960s and 1990s, toxoplasmosis outbreaks mainly occurred through ingestion of cysts in meat and meat derivatives; in the 1980s, through milk contaminated with tachyzoites; in 2000, due to the presence of oocysts in water, sand, and soil; and in 2010, due to oocysts in raw fruits and vegetables. Our study suggests a possible change in the epidemiology of reported toxoplasmosis outbreaks. Because of this change, we suggest that greater attention be paid to the disinfection of vegetables, as well as to the quality of water used for drinking and irrigation.

Toxoplasma gondii Oocyst Infectivity Assessed Using a Sporocyst-Based Cell Culture Assay Combined with Quantitative PCR for Environmental Applications.

Toxoplasma gondii is a ubiquitous foodborne protozoan that can infect humans at low dose and displays different prevalences among countries in the world. Ingestion of food or water contaminated with small amounts of T. gondii oocysts may result in human infection. However, there are no regulations for monitoring oocysts in food, mainly because of a lack of standardized methods to detect them. The objectives of this study were (i) to develop a reliable method, applicable in biomonitoring, for the rapid detection of infectious oocysts by cell culture of their sporocysts combined with quantitative PCR (sporocyst-CC-qPCR) and (ii) to adapt this method to blue and zebra mussels experimentally contaminated by oocysts with the objective to use these organisms as sentinels of aquatic environments. Combining mechanical treatment and bead beating leads to the release of 84% ± 14% of free sporocysts. The sporocyst-CC-qPCR detected fewer than ten infectious oocysts in water within 4 days (1 day of contact and 3 days of cell culture) compared to detection after 4 weeks by mouse bioassay. For both mussel matrices, oocysts were prepurified using a 30% Percoll gradient and treated with sodium hypochlorite before cell culture of their sporocysts. This assay was able to detect as few as ten infective oocysts. This sporocyst-based CC-qPCR appears to be a good alternative to mouse bioassay for monitoring infectious T. gondii oocysts directly in water and also using biological sentinel mussel species. This method offers a new perspective to assess the environmental risk for human health associated with this parasite.IMPORTANCE The ubiquitous protozoan Toxoplasma gondii is the subject of renewed interest due to the spread of oocysts in water and food causing endemic and epidemic outbreaks of toxoplasmosis in humans and animals worldwide. Displaying a sensitivity close to animal models, cell culture represents a real alternative to assess the infectivity of oocysts in water and in biological sentinel mussels. This method opens interesting perspectives for evaluating human exposure to infectious T. gondii oocysts in the environment, where oocyst amounts are considered to be very small.

Cell Culture Infectivity to Assess Chlorine Disinfection of Cryptosporidium Oocysts in Water.

This chapter provides a detailed protocol to assess disinfection efficacy of chlorine against Cryptosporidium oocysts including the core chlorine disinfection assay, the in vitro cell culture infectivity assay, and microscopy analysis and data interpretation.

Quantification and molecular characterization of Cryptosporidium spp. from raw surface water and raw sewage for the monitoring of public water supplies in the metropolitan region of São Paulo, Brazil

Waterborne diseases have emerged as a major public health problem. Cryptosporidium is an important parasite associated with cases of waterborne outbreaks in the world. The complex epidemiology of this protozoan increases the interest for sensitive and rapid methods for the detection of species responsible for infection in humans. In this study, 50 samples were collected from two public water supplies, São Lourenço River (P1A) and Guarapiranga Dam (P2A), and 50 samples of raw sewage were collected from São Lourenço da Serra’s (P1E) and Taboão da Serra’s vertical wells (P2E), between January and December of 2013. The isolation of oocysts in water was carried out by the USEPA Method 1623 (2005) and raw sewage samples were processed by the centrifugation-filtration method. Genotyping occurred by nested PCR, cloning and sequencing based on 18S rRNA gene. Real time PCR assays (qPCR) were carried out for detection and quantification of oocysts in water samples. According to the results, Cryptosporidium was detected in 12% (3/25) of samples at P1A and 16% (4/25) at P2A in the water by nested PCR. In sewage, the parasite was detected in 20% (5/25) of samples at P1E and 24% (6/25) in P2E vertical well. Quantitative PCR detected 0.79 to 1.85 oocysts/L (52%) at P1A and 0.72 to 1.4 oocysts/L (64%) at P2A. Molecular characterization allowed the identification of C. parvum and C. hominis in water, and C. hominis, C. parvumand C. muris in raw sewage by nested PCR. Cryptosporidium species identified herein belong to a group of organisms of significant relevance in waterborne diseases. Therefore, concentration and characterization methodologies applied in our analyses showed to be useful for environmental surveillance studies. The detection of C. muris, which is associated with anthroponotic species, was helpful in the investigation of likely contamination sources in the environment, confirming the need of expansion in effective measures to protect these water supplies.

Evaluation of solar photocatalysis using TiO2 slurry in the inactivation of Cryptosporidium parvum oocysts in water

Cryptosporidium is a genus of enteric protozoan parasites of medical and veterinary importance, whose oocysts have been reported to occur in different types of water worldwide, offering a great resistant to the water treatment processes. Heterogeneous solar photocatalysis using titanium dioxide (TiO2) slurry was evaluated on inactivation of Cryptosporidium parvum oocysts in water. Suspensions of TiO2 (0, 63, 100 and 200mg/L) in distilled water (DW) or simulated municipal wastewater treatment plant (MWTP) effluent spiked with C. parvum oocysts were exposed to simulated solar radiation. The use of TiO2 slurry at concentrations of 100 and 200mg/L in DW yielded a high level of oocyst inactivation after 5h of exposure (4.16±2.35% and 15.03±4.54%, respectively, vs 99.33±0.58%, initial value), representing a good improvement relative to the results obtained in the samples exposed without TiO2 (51.06±9.35%). However, in the assays carried out using simulated MWTP effluent, addition of the photocatalyst did not offer better results. Examination of the samples under bright field and epifluorescence microscopy revealed the existence of aggregates comprising TiO2 particles and parasitic forms, which size increased as the concentration of catalyst and the exposure time increased, while the intensity of fluorescence of the oocyst walls decreased. After photocatalytic disinfection process, the recovery of TiO2 slurry by sedimentation provided a substantial reduction in the parasitic load in treated water samples (57.81±1.10% and 82.10±2.64% for 200mg/L of TiO2 in DW and in simulated MWTP effluent, respectively). Although further studies are need to optimize TiO2 photocatalytic disinfection against Cryptosporidium, the results obtained in the present study show the effectiveness of solar photocatalysis using TiO2 slurry in the inactivation of C. parvum oocysts in distilled water.

Detection of antibodies against Toxoplasma gondii in Mirounga leonina Linnaeus, 1758 (Pinnipedia, Phocidae) from Elephant Island

Objective: To investigate the presence of antibodies against Toxoplasma gondii (T. gondii) in Mirounga leonina (M. leonina) Linnaeus from Elephant Island, Antarctica. Methods: The animals were anesthetized, restrained, measured, weighed and had their blood collected by venipuncture of intervertebral lumbar epidural vein. Blood samples were collected from 102 individuals. Indirect hemagglutination and serum dilution at a proportion of 1:25 was used for specific immunoglobulin G anti-T. gondii detection. Results: Only one (0.98%) specimen, a newly weaned calf, was seropositive. Conclusions: This study is the highest serological survey for antibody detection against T. gondii in M. leonina. The results suggest a low level of exposure to T. gondii, probably due to the absence of felids in the study area. The seropositivity presented by the elephant seal may be related to the presence of oocysts in water or cysts in their preys. Despite being reported the presence of the parasite in fish and molluscs, there are no records of tissue cysts or oocysts in squid or fish of the diet of M. leonina. Thus, further parasitological studies focused on preys of elephant seals are needed for a better understanding of infection of M. leonina by T. gondii.

HUMAN TOXOPLASMOSIS OUTBREAKS AND THE AGENT INFECTING FORM. FINDINGS FROM A SYSTEMATIC REVIEW.

SUMMARYToxoplasmosis, a worldwide highly prevalent zoonotic infection, is transmitted either by the oocysts, from water and soil, or the tissue cysts, in raw or undercooked infected meat, of Toxoplasma gondii. An ongoing debate is whether there are differences between the clinical and epidemiological characteristics of the outbreaks due to one or the other infective form of the agent. We performed a systematic review, recovering 437 reported outbreaks of which 38 were selected. They were complete reports containing ascribedToxoplasma infecting form, and clinical and demographic data. There was no gender or age group selection in the outbreaks, which were described more often in the Americas. A large number of individuals were affected when oocysts, associated with soil and water contaminated with cat feces, were considered the transmission source. Onset of symptoms occurred early when the infection was ascribed to meat tissue cysts (11.4 ± 6.7 days) with sharpened temporal distribution of cases, while a broader and prolonged appearance of new cases was observed when oocysts in water were the source of the infection (20 ± 7 days, p < 0.001). Such information may be useful in the design and implementation of control strategies.

Metabolomic analysis of Cryptosporidium parvum oocysts in water: A proof of concept demonstration

Metabolomic analysis of Cryptosporidium parvum oocysts in water: A proof of concept demonstration

Inactivation of recalcitrant protozoan oocysts and bacterial endospores in drinking water using high-intensity pulsed UV light irradiation

This constitutes the first study to compare the use of high-intensity pulsed UV light (PUV) irradiation for the novel destruction of harmful protozoan (Cryptosporidium parvum Iowa isolate) oocysts and bacterial (Clostridium perfringens ATCC 13124 and Bacillus cereus ATCC 11178) endospores in artificially-spiked water where these organisms are resistant to conventional chlorination. Experimental results revealed that all three test organisms in their dormant recalcitrant state required extended levels of pulsing to achieve significant reductions in numbers compared to other similarly PUV-treated Escherichia coli ATCC 25922 that is a non-spore forming indicator of faecal pollution in water. 120 pulses at 900 V or 16.2 J per pulse (equivalent to a UV dose of 8.39 μJ cm−2) were required to achieve ca. 2 log C. perfringens spore numbers, whereas a similar level of PUV irradiation reduced both C. parvum oocysts and B. cereus endospores by ca. 5 log orders. A comparative ca. 5 log reduction of E. coli cell numbers was achieved after only 25 pulses at 900 V (equivalent to a UV dose of 1.74 μJ cm−2). A clear trend emerged where the order of resistance to PUV-irradiation observed was C. perfringens endospores &amp;gt; C. parvum oocysts, B. cereus endospores &amp;gt; E. coli cells. This study suggests disinfection kinetic data for the more resistant C. perfringens endospores can be used as a measure of estimating disinfection efficacy of PUV treatments for C. parvum oocysts in water, avoiding the need to use complex animal or cell culture infectivity models that are only available in specialised laboratories with highly trained technicians. This study will inform future studies exploring scale-up of PUV at waste-water treatment plants.

Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

Constructed wetlands – Are they safe in reducing protozoan parasites?

Constructed wetlands have been promoted in recent literature for use in rural communities in developed as well as in developing countries as an appropriate technology to be handled with low operational maintenance costs. Within a joint project supported by BMBF (Project No O2WA0107 and No 02WA0108) research was done concerning the sanitation effect of constructed wetlands on wastewater effluents. This article will focus on the detection and the removal of cysts of Cryptosporidium parvum and Giarda lamblia, those being the most frequently identified pathogenic protozoan parasites worldwide with increasing medical and economical consequences. Two plants, one installed in 2000 as a pilot plant at Langenreichenbach near Leipzig (Saxony, Germany), the other one in routine operation since 1993 in a training center at the town of Belzig (Brandenburg, Germany) were tested for three years. Detection methods from the US EPA (ICR Protozoan Method for Detecting Giardia Cysts and Cryptosporidium Oocysts in Water by a Fluorescent Antibody Procedure (EPA/814-B-95-003;US EPA 1995) were employed in order to assess protozoal and bacterial reduction in the wastewater passing through different combinations of filter beds and fillings. Removal of cysts of Cryptosporidium and Giardia spp. turned out to be a 2 log reduction in all plants. The most effective structural element was a two-stage combination of filter beds leading to the highest removal efficiency both for the protozoan and the bacterial indicator organisms. Also, washed sand (0-2mm grain size) in the filter bed proved to be most effective filter material; the planted reed (phragmites spp.) or willow (salix spp.), however, turned out to be of minor importance for the filtering activity.

Detection of Toxoplasma gondii oocysts and surrogate microspheres in water using ultrafiltration and capsule filtration

While reports on waterborne infections with Toxoplasma gondii are emerging worldwide, detection of this zoonotic parasite in water remains challenging. Lack of standardized and quantitative methods for detection of T. gondii oocysts in water also limits research on the transport and fate of this pathogen through aquatic habitats. Here, we compare the ability of hollow-fiber ultrafiltration and capsule filtration to concentrate oocysts in spiked tap water, fresh surface water, and seawater samples. Detection of T. gondii oocysts in concentrated samples was achieved using molecular methods, as well as visually via epifluorescent microscopy. In addition to oocysts, water samples were spiked with T. gondii surrogate microspheres, and detection of microspheres was performed using flow cytometry and epifluorescent microscopy. Results demonstrate that both water concentration methods followed by microscopy allowed for quantitative detection of T. gondii oocysts and surrogate microspheres. For T. gondii oocysts, microscopy was more sensitive than TaqMan and conventional PCR, and allowed for detection of oocysts in all water samples tested. Compared with flow cytometry, microscopy was also a more cost-efficient and precise method for detection of fluorescent surrogate microspheres in tap, fresh and seawater samples. This study describes a novel approach for quantitative detection of T. gondii oocysts in drinking and environmental water samples. The techniques described for concentrating and detecting surrogate microspheres have broad application for evaluating the transport and fate of oocysts, as well as the efficiency of water treatment methods for removal of T. gondii from water supplies.

Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts.

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.