Detection of Cryptosporidium oocysts in water: techniques for generating precise recovery data

When determining the recovery efficiency of a procedure for the detection of Cryptosporidium or the removal efficiency of a treatment process, it is necessary to accurately enumerate a 'seed dose'. Conventional techniques for this are highly variable and consequently, can result in misleading data. In this study, a flow cytometric method was developed for the production of suspensions of Cryptosporidium oocysts in which the number of organisms could be precisely determined. A Becton Dickinson FACScalibur flow cytometer was employed to produce oocyst suspensions containing 100 oocysts. Analysis of these suspensions resulted in a mean dose of 99.5 oocysts (S.D. = 1.1, %cv = 1.1). These results indicate that the use of such suspensions to seed test systems generates far more accurate data than is presently possible using conventional techniques. In addition, the use of immunomagnetic separation (IMS) for the isolation of oocysts from three different water matrices, after seeding with oocysts counted using flow cytometry, was investigated. The recovery efficiency of the IMS procedure was found to be high, with the percentage recovery of oocysts ranging from 82.3 to 86.3%, and the use of precise numbers of oocysts allowed accurate recovery efficiency data to be generated. A laser scanning instrument (ChemScan RDI) was employed for the rapid detection and enumeration of oocysts after capture using membrane filtration. This technique was found to be faster and easier to perform than conventional epifluorescence microscopy. These findings demonstrate that the ChemScan RDI system may be used as alternative procedure for the routine examination of IMS supernatant fluids for the presence of Cryptosporidium.     

Detection of infectious Cryptosporidium parvum oocysts in surface and filter backwash water samples by immunomagnetic separation and integrated cell culture-PCR.

A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected.     

Maximizing recovery and detection of Cryptosporidium parvum oocysts from spiked eastern oyster (Crassostrea virginica) tissue samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Comparison of two methods for detection of Giardia cysts and Cryptosporidium oocysts in water.

The steps of two immunofluorescent-antibody-based detection methods were evaluated for their efficiencies in detecting Giardia cysts and Cryptosporidium oocysts. The two methods evaluated were the American Society for Testing and Materials proposed test method for Giardia cysts and Cryptosporidium oocysts in low-turbidity water and a procedure employing sampling by membrane filtration, Percoll-Percoll step gradient, and immunofluorescent staining. The membrane filter sampling method was characterized by higher recovery rates in all three types of waters tested: raw surface water, partially treated water from a flocculation basin, and filtered water. Cyst and oocyst recovery efficiencies decreased with increasing water turbidity regardless of the method used. Recoveries of seeded Giardia cysts exceeded those of Cryptosporidium oocysts in all types of water sampled. The sampling step in both methods resulted in the highest loss of seeded cysts and oocysts. Furthermore, much higher recovery efficiencies were obtained when the flotation step was avoided. The membrane filter method, using smaller tubes for flotation, was less time-consuming and cheaper. A serious disadvantage of this method was the lack of confirmation of presumptive cysts and oocysts, leaving the potential for false-positive Giardia and Cryptosporidium counts when cross-reacting algae are present in water samples.     

Concentration and detection of cryptosporidium oocysts in surface water samples by method 1622 using ultrafiltration and capsule filtration

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4',6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.     

An evaluation of methods for the simultaneous detection of Cryptosporidium oocysts and Giardia cysts from water.

Methods for the simultaneous detection of Cryptosporidium parvum oocysts and Giardia cysts from water are described and their relative recovery efficiencies are assessed for seeded samples of both tap and river water. Cartridge filtration, membrane filtration, and calcium carbonate flocculation were evaluated, and steps to optimize the concentration procedures were undertaken. Increasing centrifugation to 5,000 x g, coupled with staining in suspension, was found to increase the overall efficiency of recovery of both cysts and oocysts. Cartridge filtration for both cysts and oocysts was examined by use of 100-liter volumes of both tap and river water. Improvements in recovery were observed for Cryptosporidium oocysts after extra washes of the filters. Calcium carbonate flocculation gave the maximum recovery for both Cryptosporidium oocysts and Giardia cysts and for both water types. A variety of 142-mm membranes was examined by use of 10-liter seeded samples of tap and river water. Cellulose acetate with a 1.2-micron pore size provided the best results for Cryptosporidium oocysts, and cellulose nitrate with a 3.0-micron pore size did so for Giardia cysts.     

Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623

The U.S. Environmental Protection Agency has developed method 1623 for simultaneous detection of Cryptosporidium oocysts and Giardia cysts in water. Method 1623 includes four major steps: filtration, immunomagnetic separation (IMS), fluorescent antibody (FA) staining and microscopic examination. It was noted that the recovery levels following IMS-FA and FA staining were high, averaging more than 92.0% and 89.0% for C. parvum oocysts and G. lamblia cysts, respectively. In contrast, when the filtration step was incorporated, the recovery level of C. parvum oocysts declined significantly to 18.1% in seeded tap water, while a relatively high recovery level of 77.2% for G. lamblia cysts could still be achieved. Further study indicated that the recovery level of C. parvum oocysts could be enhanced significantly when an appropriate amount of silica particles was added to a water sample. The recovery level of C. parvum oocysts was affected by particle size and concentration. The optimal silica particle size was determined to be within the range of 5-40 microm, and the corresponding optimal silica concentration was 1.42 g for 10-l tap water. When both G. lamblia cysts and C. parvum oocysts were spiked into the tap water sample containing the optimum amount of silica particles, the average recovery levels of oocysts and cysts were 82.7% and 75.4%, respectively. The results obtained clearly suggested that addition of an appropriate amount of silica particles could improve the recovery level of C. parvum oocysts significantly and yet there was no noticeable deleterious effect on the recovery level of G. lamblia cysts. Further study indicated that the rotation time in the IMS procedure using the Dynal GC-Combo IMS kit (which was recommended in method 1623) was important for G. lamblia cyst detection. In contrast, the recovery level of C. parvum oocysts was not affected by the rotation time. Furthermore, it was found that the recovery levels of C. parvum oocysts using methods 1622 and 1623 were quite close although different IMS kits were used in the two methods.     

Evaluation of an alternative IMS dissociation procedure for use with Method 1622: detection of Cryptosporidium in water

U.S. EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent antibody and confirmation by the demonstration of 1-4 sporozoites or nuclei after staining with 4',6-diamidino-2-phenyl indole dihydrochloride (DAPI). The purpose of this study was to evaluate a new IMS dissociation, a 10-min incubation at 80 degrees C. Heat dissociation improved the average oocyst recovery from 41% to 71% in seeded reagent water, and from 10% to 51% in seeded river samples. The average DAPI confirmation rate improved from 49% to 93% in reagent water, and from 48% to 73% in river samples. This modification improved both oocyst recovery and confirmation.     

Detection of Infectious Cryptosporidium parvum Oocysts in Surface and Filter Backwash Water Samples by Immunomagnetic Separation and Integrated Cell Culture-PCR

A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected.     

Effect of particles on the recovery of cryptosporidium oocysts from source water samples of various turbidities

Cryptosporidium parvum can be found in both source and drinking water and has been reported to cause serious waterborne outbreaks which threaten public health safety. The U.S. Environmental Protection Agency has developed method 1622 for detection of Cryptosporidium oocysts present in water. Method 1622 involves four key processing steps: filtration, immunomagnetic separation (IMS), fluorescent-antibody (FA) staining, and microscopic evaluation. The individual performance of each of these four steps was evaluated in this study. We found that the levels of recovery of C. parvum oocysts at the IMS-FA and FA staining stages were high, averaging more than 95%. In contrast, the level of recovery declined significantly, to 14.4%, when the filtration step was incorporated with tap water as a spiking medium. This observation suggested that a significant fraction of C. parvum oocysts was lost during the filtration step. When C. parvum oocysts were spiked into reclaimed water, tap water, microfiltration filtrate, and reservoir water, the highest mean level of recovery of (85.0% +/- 5.2% [mean +/- standard deviation]) was obtained for the relatively turbid reservoir water. Further studies indicated that it was the suspended particles present in the reservoir water that contributed to the enhanced C. parvum oocyst recovery. The levels of C. parvum oocyst recovery from spiked reservoir water with different turbidities indicated that particle size and concentration could affect oocyst recovery. Similar observations were also made when silica particles of different sizes and masses were added to seeded tap water. The optimal particle size was determined to be in the range from 5 to 40 micro m, and the corresponding optimal concentration of suspended particles was 1.42 g for 10 liters of tap water.     

Maximizing Recovery and Detection of Cryptosporidium parvum Oocysts from Spiked Eastern Oyster (Crassostrea virginica) Tissue Samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Giardia and Cryptosporidium in water: evaluation of two concentration methods and occurrence in wastewater.

Giardia and Cryptosporidium are important agents of water-borne parasitic diseases. In this work we have examined the recovery efficiency of two methods for concentrating Giardia cysts and Cryptosporidium oocysts from water: a membrane filtration method and a crossflow filtration method. Results demonstrated a higher recovery efficiency for crossflow filtration method in comparison to the membrane filtration method. In addition, Giardia cysts and Cryptosporidium oocysts concentration was evaluated in wastewater samples submitted to chemical flocculation or chemical flocculation followed by slow sand filtration. Results showed that slow sand filtration was capable of reducing the number of Giardia cysts, but not of Cryptosporidium oocysts in wastewater.     

Effects of seeding procedures and water quality on recovery of Cryptosporidium oocysts from stream water by using U.S. Environmental Protection Agency Method 1623

U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter. Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters. Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts. The recoveries between the two seeding procedures were highly correlated (r = 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.     

Recovery of Cryptosporidium oocysts and Giardia cysts from source water concentrates using immunomagnetic separation

Immunomagnetic separation (IMS) procedures for the simultaneous isolation of Cryptosporidium oocysts and Giardia cysts have recently become available. We validated Dynal's GC-Combo IMS kit using source water at three turbidity levels (5000, 500 and 50 nephelometric turbidity units [ntu]) obtained from different geographical locations and spiked with approximately 9--11 (oo)cysts per ml. Mean recoveries of Cryptosporidium oocysts and Giardia cysts in deionized water were 62% and 69%, respectively. In turbid water matrices, mean recoveries of Cryptosporidium oocysts were between 55.9% and 83.1% while mean recoveries of cysts were between 61.1% and 89.6%. Marginally higher recoveries of the heat inactivated (oo)cysts were observed (119.4% Cryptosporidium oocysts and 90.9% Giardia cysts) in deionized water when compared with recoveries of viable (oo)cysts (69.7% Cryptosporidium oocysts and 79% Giardia cysts). Age of (oo)cysts on recoveries using the GC-Combo IMS kit demonstrated no effects up to 20 months old. Recovery of Giardia cysts was consistent for isolates aged up to 8 months (81.4%), however, a significant reduction in recoveries was noted at 20 months age. Recoveries of low levels (5 and 10 (oo)cysts) of Cryptosporidium oocysts and Giardia cysts in deionized water using IMS ranged from 51.3% to 78% and from 47.6% to 90.0%, respectively. Results of this study indicate that Dynal's GC-Combo IMS kit is an efficient technique to separate Cryptosporidium/Giardia from turbid matrices and yields consistent, reproducible recoveries. The use of fresh (recently voided and purified) (oo)cysts, aged (oo)cysts, viable and heat-inactivated (oo)cysts indicated that these parameters do not influence IMS performance.     

Recovery of Cryptosporidium oocysts from small and large volume water samples using a compressed foam filter system

A novel filter system comprising open cell reticulated foam rings compressed between retaining plates and fitted into a filtration housing was evaluated for the recovery of oocysts of Cryptosporidium from water. Mean recoveries of 90·2% from seeded small and large volume (100–2000 l) tap water samples, and 88·8% from 10–20 l river water samples, were achieved. Following a simple potassium citrate flotation concentrate clean-up procedure, mean recoveries were 56·7% for the tap water samples and 60·9% for river water samples. This represents a marked improvement in capture and recovery of Cryptosporidium oocysts from water compared with conventional polypropylene wound cartridge filters and membrane filters.     

Effects of pH and magnetic material on immunomagnetic separation of Cryptosporidium oocysts from concentrated water samples

In this study, we examined the effect that magnetic materials and pH have on the recoveries of Cryptosporidium oocysts by immunomagnetic separation (IMS). We determined that particles that were concentrated on a magnet during bead separation have no influence on oocyst recovery; however, removal of these particles did influence pH values. The optimal pH of the IMS was determined to be 7.0. The numbers of oocysts recovered from deionized water at pH 7.0 were 26.3% higher than those recovered from samples that were not at optimal pH. The results indicate that the buffers in the IMS kit did not adequately maintain an optimum pH in some water samples. By adjusting the pH of concentrated environmental water samples to 7.0, recoveries of oocysts increased by 26.4% compared to recoveries from samples where the pH was not adjusted.     

Detection and discrimination of Cryptosporidium parvum and C. hominis in water samples by immunomagnetic separation-PCR

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.     

A new filter-eluting solution that facilitates improved recovery of Cryptosporidium oocysts from water

Cryptosporidium is a zoonotic coccidian parasite associated with diarrhea, and the disinfectant-resistant oocysts are threats to public health even in industrialized countries. In order to make an accurate assessment of the risk to public health, a detection method that has a high recovery rate of oocysts in water is required. In this study, we developed a new filter-eluting solution that facilitates more efficient recovery of Cryptosporidium oocysts from different kinds of water samples. The filter-eluting solution, referred to as PET, consists of sodium pyrophosphate (0.02%), Tween 80 (0.01%) and trisodium EDTA (0.03%). By using PET instead of conventional filter-eluting solutions, the average recovery rate significantly increased from 25.5+/-15.1% to 43.1+/-13.9% (p<0.05). The improved oocyst recovery was likely due to the increased separation of the oocysts from debris trapped on the filter membrane as well as increased capture of the oocysts by immunomagnetic beads. We recommend that PET be used as the filter-eluting solution for detection of Cryptosporidium oocysts in environmental water.     

Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus)

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are therefore useful in monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels (Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) assays, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a 1 log(10) improvement when samples were first processed by IMS. For tank exposed mussels, TaqMan and conventional PCR methods detected C. parvum in <5% of hemolymph samples. No gill washings from these same mussels tested positive by TaqMan PCR or DFA analysis even with IMS concentration. All methods detected the highest prevalence of C. parvum-positive samples in digestive gland tissues of exposed mussels. In conclusion, the most sensitive method for the detection of C. parvum in oocyst-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive. TaqMan PCR was comparable to conventional PCR for detection of C. parvum oocysts in mussels and additionally allowed for automated testing, high throughput, and semi-quantitative results.     

Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.