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.     

Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from water samples

AIMS: Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from tap water. METHODS AND RESULTS: Ferric sulphate, aluminium sulphate and calcium carbonate were compared for their recovery efficiency of C. parvum oocysts from tap water. Lower mean recovery was achieved by calcium carbonate (38.8%) compared with ferric sulphate (61.5%) and aluminium sulphate (58.1%) for the recovery of 2.5 x 10(5) oocysts l(-1); 2.5 oocysts l(-1) and 1 oocyst l(-1) were adequately purified using ferric sulphate flocculation. In vitro excystation experiments showed that ferric sulphate flocculation does not markedly reduce the viability of oocysts. CONCLUSIONS: Ferric sulphate flocculation is a simple and effective tool for the purification of C. parvum oocysts from tap water. SIGNIFICANCE AND IMPACT OF THE STUDY: The high recovery rates and low impact on oocyst viability provided by ferric sulphate flocculation might be useful for the detection of Cryptosporidium oocysts in environmental water samples.     

Modifications to United States Environmental Protection Agency methods 1622 and 1623 for detection of Cryptosporidium oocysts and Giardia cysts in water

Collaborative and in-house laboratory trials were conducted to evaluate Cryptosporidium oocyst and Giardia cyst recoveries from source and finished-water samples by utilizing the Filta-Max system and U.S. Environmental Protection Agency (EPA) methods 1622 and 1623. Collaborative trials with the Filta-Max system were conducted in accordance with manufacturer protocols for sample collection and processing. The mean oocyst recovery from seeded, filtered tap water was 48.4% +/- 11.8%, while the mean cyst recovery was 57.1% +/- 10.9%. Recovery percentages from raw source water samples ranged from 19.5 to 54.5% for oocysts and from 46.7 to 70.0% for cysts. When modifications were made in the elution and concentration steps to streamline the Filta-Max procedure, the mean percentages of recovery from filtered tap water were 40.2% +/- 16.3% for oocysts and 49.4% +/- 12.3% for cysts by the modified procedures, while matrix spike oocyst recovery percentages ranged from 2.1 to 36.5% and cyst recovery percentages ranged from 22.7 to 68.3%. Blinded matrix spike samples were analyzed quarterly as part of voluntary participation in the U.S. EPA protozoan performance evaluation program. A total of 15 blind samples were analyzed by using the Filta-Max system. The mean oocyst recovery percentages was 50.2% +/- 13.8%, while the mean cyst recovery percentages was 41.2% +/- 9.9%. As part of the quality assurance objectives of methods 1622 and 1623, reagent water samples were seeded with a predetermined number of Cryptosporidium oocysts and Giardia cysts. Mean recovery percentages of 45.4% +/- 11.1% and 61.3% +/- 3.8% were obtained for Cryptosporidium oocysts and Giardia cysts, respectively. These studies demonstrated that the Filta-Max system meets the acceptance criteria described in U.S. EPA methods 1622 and 1623.     

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.     

Comparison of methods for the concentration of Cryptosporidium oocysts and Giardia cysts from raw waters

This study compared the recovery of Cryptosporidium oocysts and Giardia cysts ((oo)cysts) from raw waters using 4 different concentration-elution methods: flatbed membranes, FiltaMax foam, Envirochek HV capsules, and Hemoflow ultrafilters. The recovery efficiency of the combined immunomagnetic separation and staining steps was also determined. Analysis of variance of arcsine-transformed data demonstrated that recovery of Cryptosporidium oocysts by 2 of the methods was statistically equivalent (flatbed filtration 26.7% and Hemoflow 28.3%), with FiltaMax and Envirochek HV recoveries significantly lower (18.9% and 18.4%). Recovery of Giardia cysts was significantly higher using flatbed membrane filtration (42.2%) compared with the other 3 methods (Envirochek HV 29.3%, FiltaMax 29.0%, and Hemoflow 20.9%). All methods were generally acceptable and are suitable for laboratory use; 2 of the methods are also suitable for field use (FiltaMax and Envirochek HV). In conclusion, with recoveries generally being statistically equivalent or similar, practical considerations become important in determining which filters to use for particular circumstances. The results indicate that while low-turbidity or "finished" waters can be processed with consistently high recovery efficiencies, recoveries from raw water samples differ significantly with variations in raw water quality. The use of an internal control with each raw water sample is therefore highly recommended.     

Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain

AIMS: To evaluate the prevalence of Cryptosporidium and Giardia in surface water supplies from the province of Alava, northern Spain, and to investigate possible associations among the presence of these pathogenic protozoa with microbiological, physicochemical and atmospheric parameters. METHODS AND RESULTS: A total of 284 samples of drinking and recreational water supplies were analysed. Cryptosporidium oocysts were found in 63.5% of river samples, 33.3% of reservoirs samples, 15.4% and 22.6% of raw water samples from conventional and small water treatment facilities (respectively), 30.8% of treated water from small treatment facilities, and 26.8% of tap water from municipalities with chlorination treatment only. Giardia cysts were found in 92.3% of river samples, 55.5% of reservoirs samples, 26.9% and 45.2% of raw water samples from conventional and small water treatment facilities (respectively), 19.2% of treated water from small treatment facilities, and 26.8% of tap water from municipalities with chlorination treatment only. The presence of Cryptosporidium and Giardia had significant Pearson's correlation coefficients (P < 0.01) with the turbidity levels of the samples, and a number of significant associations were also found with the count levels for total coliforms and Escherichia coli. The samples were positive for Cryptosporidium significantly (P < 0.05) more frequently during the autumn season than during the spring and winter seasons. No significant differences were found in the seasonal pattern of Giardia. A moderate association (r = 0.52) was found between rainfall and the presence of Cryptosporidium oocysts. CONCLUSIONS: Cryptosporidium and Giardia are consistently found at elevated concentrations in surface waters for human consumption from the province of Alava, northern Spain. SIGNIFICANCE AND IMPACT OF THE STUDY: Water treatments based on rapid filtration process and/or chlorination only are often unsatisfactory to provide safe drinking water, a situation that represents an important public health problem for the affected population because of the risk of waterborne outbreaks.     

Portable continuous flow centrifugation and method 1623 for monitoring of waterborne protozoa from large volumes of various water matrices

AIMS: The aims of this study were to validate a portable continuous flow centrifuge (PCFC) as an alternative concentration step of US-EPA Method 1623 and to demonstrate it's efficacy for recovery of low numbers of protozoa from large volumes of various water matrices. METHODS AND RESULTS: Recoveries of Cryptosporidium parvum oocysts, Giardia intestinalis cysts and Encephalitozoon intestinalis spores spiked into 10-1000 l volumes of various water matrices were evaluated during in-house and collaborative trials. Spiked protozoa were either approved standards or diluted stock samples enumerated according to USEPA Method 1623. Cryptosporidium recoveries exceeded method 1623 criteria and substantially high recoveries were observed for Giardia and E. intestinalis. CONCLUSIONS: Portable continuous flow centrifuge methodology exceeded method 1623 acceptance criteria for Cryptosporidium and could be easily adopted for other protozoa. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCFC could be adopted as an alternative user-friendly concentration method for Cryptosporidium and for monitoring of large volumes of source and tap water for accidental or deliberate contamination with protozoa and potentially with other enteric pathogens. It is anticipated that PCFC would also be equal or superior to filtration for protozoa monitoring in wastewater and effluents.     

Occurrence of Cryptosporidium spp. oocysts in raw and treated sewage and river water in north-eastern Spain

AIMS: To determine the occurrence and levels of Cryptosporidium parvum oocysts in wastewater and surface waters in north-eastern Spain. METHODS AND RESULTS: Samples from five sewage treatment plants were taken monthly and quarterly during 2003. In addition, water was collected monthly from the River Llobregat (NE Spain) during the period from 2001 to 2003. All samples were analysed by filtration on cellulose acetate filters or through Envirocheck using EPA method 1623, followed by immunomagnetic separation and examination by laser scanning cytometry. All raw sewage, secondary effluent and river water samples tested were positive for Cryptosporidium oocysts. Of the tertiary sewage effluents tested, 71% were positive for Cryptosporidium oocysts. The proportion of viable oocysts varied according to the sample. CONCLUSIONS: Two clear maxima were observed during spring and autumn in raw sewage, showing a seasonal distribution and a correlation with the number of cryptosporidiosis cases and rainfall events. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first data on the occurrence of Cryptosporidium oocysts in natural waters in north-eastern Spain.     

Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal

Aims:  Waterborne outbreaks of diarrhoeal illness reported worldwide are mostly associated with Cryptosporidium spp. and Giardia spp. Their presence in aquatic systems makes it essential to develop preventive strategies for water and food safety. This study was undertaken to monitor the presence of Cryptosporidium and Giardia in a total of 175 water samples, including raw and treated water from both surface and ground sources in Portugal.
Methods and Results:  The samples were processed according to USEPA Method 1623 for immunomagnetic separation (IMS) of Cryptosporidium oocysts and Giardia cysts, followed by detection of oocysts/cysts by immunofluorecence (IFA) microscopy, PCR-based techniques were done on all water samples collected. Out of 175 samples, 81 (46·3%) were positive for Cryptosporidium and 67 (38·3%) for Giardia by IFA. Cryptosporidium spp. and G. duodenalis genotypes were identified by PCR in 37 (21·7%) and 9 (5·1%) water samples, respectively. C. parvum was the most common species (78·9%), followed by C. hominis (13·2%), C. andersoni (5·3%), and C. muris (2·6%). Subtype IdA15 was identified in all C. hominis -positive water samples. S ubtyping revealed the presence of C. parvum subtypes IIaA15G2R1, IIaA16G2R1 and IIdA17G1. Giardia duodenalis subtype A1 was identified.
Conclusions:  The results of the present study suggest that Cryptosporidium spp. and Giardia spp. were widely distributed in source water and treated water in Portugal. Moreover, the results obtained indicate a high occurrence of human-pathogenic Cryptosporidium genotypes and subtypes in raw and treated water samples.
Significance and Impact of the Study:  Thus, water can be a potential vehicle in the transmission of cryptosporidiosis, and giardiasis of humans and animals in Portugal.     

Direct counting of Cryptosporidium parvum oocysts using fluorescence in situ hybridization on a membrane filter

This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe.     

Hollow-fiber ultrafiltration of Cryptosporidium parvum oocysts from a wide variety of 10-L surface water samples

An optimized hollow-fiber ultrafiltration system (50 000 MWCO) was developed to concentrate Cryptosporidium oocysts from 10-L samples of environmental water. Seeded experiments were conducted using a number of surface-water samples from the southwestern U.S.A. and source water from four water districts with histories of poor oocyst recovery. Ultrafiltration produced a mean recovery of 47.9% from 19 water samples (55.3% from 39 individual tests). We also compared oocyst recoveries using the hollow-fiber ultrafiltration system with those using the Envirochek filter. In limited comparison tests, the hollow-fiber ultrafiltration system produced recoveries similar to those of the Envirochek filter (hollow fiber, 74.1% (SD = 2.8); Envirochek, 71.9% (SD = 5.2)) in low-turbidity (3.9 NTU) samples and performed better than the Envirochek filter in high-turbidity (159.0 NTU) samples (hollow fiber, 27.5%; Envirochek, 0.4%). These results indicate that hollow-fiber ultrafiltration can efficiently recover oocysts from a wide variety of surface waters and may be a cost-effective alternative for concentrating Cryptosporidium from water, given the reusable nature of the filter.     

Evaluation of an internal positive control for Cryptosporidium and Giardia testing in water samples

AIMS: An internal positive control for Cryptosporidium and Giardia monitoring was evaluated for use in routine water monitoring quality control. The control, known as ColorSeed C&G (BTF Pty Ltd, Sydney, Australia), is a suspension containing exactly 100 Cryptosporidium oocysts and 100 Giardia cysts that have been modified by attachment of Texas Red to the cell wall, allowing them to be differentiated from unmodified oocysts and cysts. The control enables recovery efficiencies to be determined for every water sample analysed. METHODS AND RESULTS: A total of 494 water samples were seeded with ColorSeed C&G and with unlabelled Cryptosporidium and Giardia and then analysed. Additionally, the robustness of the ColorSeed labelling was challenged with various chemical treatments. Recoveries were significantly lower for the ColorSeed Texas Red labelled Cryptosporidium and Giardia than recoveries of unlabelled Cryptosporidium and Giardia. However, the differences in recoveries were small. On average ColorSeed Cryptosporidium recoveries were 3.3% lower than unlabelled Cryptosporidium, and ColorSeed Giardia recoveries were 4% lower than unlabelled Giardia. CONCLUSIONS: ColorSeed C&G is suitable for use as an internal positive control for routine monitoring of both treated and raw water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The small differences in recoveries are unlikely to limit the usefulness of ColorSeed C&G as an internal positive control. The ColorSeed labelling was found to be robust after different treatments.     

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.     

A new method for the concentration of Cryptosporidium oocysts from water

A novel method for the concentration of Cryptosporidium oocysts from water has been developed, based upon the precipitation of calcium carbonate. A 10 1 water sample is treated by adding solutions of calcium chloride and sodium bicarbonate and raising the pH value to 10 with sodium hydroxide. Crystals of calcium carbonate form and enmesh particles in the Cryptosporidium oocyst size range. The crystals are allowed to settle, the supernatant fluid is discarded and the calcium carbonate precipitate dissolved in sulphamic acid. The sample can be concentrated further by centrifugation. Recoveries of oocysts from seeded samples of deionized, tap and river water were in excess of 68%.     

Assessment of methods for detection of infectious Cryptosporidium oocysts and Giardia cysts in reclaimed effluents

This study evaluates the occurrence of Cryptosporidium oocysts and Giardia cysts in reclaimed effluents if method 1623 with the Envirochek capsule filters (standard and high-volume [HV] filters) and a modified version of the Information Collection Rule method (ICR) with the polypropylene yarn-wound cartridge filter are used. The recovery efficiency of the analytical methods was evaluated with samples of reagent, tap, and reclaimed water by using flow cytometer-sorted spike suspensions. (Oo)cyst recovery efficiency determined filter performance and method reproducibility in the water matrix tested. Method 1623 with the Envirochek HV capsule filter generated significantly higher recovery rates than did the standard Envirochek filter and the modified ICR method. Notwithstanding, large variations in recovery rates (>80%) occurred with samples of reclaimed water, and none of the water quality parameters analyzed in the reclaimed effluents could explain such variability. The highest concentrations of indigenous oocysts were detected by method 1623 with the HV filter, which provided a sufficient number of oocysts for further confirmation of infectious potential. Confirmation of species and potential infectivity for all positive protozoan samples was made by using a nested PCR restriction fragment polymorphism assay and the focus detection method most-probable-number assay, respectively. The methodology and results described in the present investigation provide useful information for the establishment of pathogen numeric standards for reclaimed effluents used for unrestricted irrigation.     

Optimization of a reusable hollow-fiber ultrafilter for simultaneous concentration of enteric bacteria,protozoa, and viruses from water

The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures. Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration. In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2- and 10-liter water samples. When known quantities (10(5) to 10(6) CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased Escherichia coli strain K-12 recoveries from 70 to 84% and Salmonella enterica serovar Enteritidis recoveries from 36 to 72%. An E. coli antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water. When E. coli XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 x 10(5) and 2.4 x 10(3) per liter), the recovery efficiencies were 89 and 92%, respectively. The simultaneous addition of E. coli XL1-Blue (9 x 10(5) CFU/liter), Cryptosporidium parvum oocysts (10 oocysts/liter), phage T1 (10(5) PFU/liter), and phage PP7 (10(5) PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively. Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of Cryptosporidium oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery). Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment.     

An immunomagnetic separation-real-time PCR method for quantification of Cryptosporidium parvum in water samples

The protozoan parasite Cryptosporidium parvum is known to occur widely in both raw and drinking water and is the cause of waterborne outbreaks of gastroenteritis throughout the world. The routinely used method for the detection of Cryptosporidium oocysts in water is based on an immunofluorescence assay (IFA). It is both time-consuming and nonspecific for the human pathogenic species C. parvum. We have developed a TaqMan polymerase chain reaction (PCR) test that accurately quantifies C. parvum oocysts in treated and untreated water samples. The protocol consisted of the following successive steps: Envirochek capsule filtration, immunomagnetic separation (IMS), thermal lysis followed by DNA purification using Nanosep centrifugal devices and, finally, real-time PCR using fluorescent TaqMan technology. Quantification was accomplished by comparing the fluorescence signals obtained from test samples with those from standard dilutions of C. parvum oocysts. This IMS-real-time PCR assay permits rapid and reliable quantification over six orders of magnitude, with a detection limit of five oocysts for purified oocyst solutions and eight oocysts for spiked water samples. Replicate samples of spiked tap water and Seine River water samples (with approximately 78 and 775 oocysts) were tested. C. parvum oocyst recoveries, which ranged from 47.4% to 99% and from 39.1% to 68.3%, respectively, were significantly higher and less variable than those reported using the traditional US Environmental Protection Agency (USEPA) method 1622. This new molecular method offers a rapid, sensitive and specific alternative for C. parvum oocyst quantification in water.     

Evaluation of a portable differential continuous flow centrifuge for concentration of Cryptosporidium oocysts and Giardia cysts from water

A portable device was developed and assembled from a stationary differential continuous flow centrifuge usually employed for blood cell separation, for the purpose of concentrating Cryptosporidium and Giardia from large volumes of water. Following compaction onto the wall of the disposable plastic centrifuge bowl and aspiration of residual water, the oocysts and cysts were dislodged by injection of a 20 ml solution containing 0.01% Tween-80 and 1% SDS and vigorous shaking. Following aspiration, the oocysts were pelleted, reacted with specific FITC-conjugated monoclonal antibodies, and enumerated via fluorescence microscopy. The entire procedure required about 2 h. Initially, 55% and 87% of Cryptosporidium oocysts and Giardia cysts, respectively, were recovered from 45 litres of tap water, and 27% and 57%, respectively, from river water. Adjustments in centrifuge speed and flow rates improved recovery to about 90% for Cryptosporidium oocysts and hence, this method compared favourably with the recently developed calcium carbonate flocculation method. It was superior in time requirement and volume flexibility, and showed a distinct advantage over the standard cartridge filtration method in all respects. The continuous flow centrifugation equipment is compact, mobile, flexible, and yields reproducibly high recovery rates. The ease of handling, speed of performance and minimal requirements for post-concentration equipment, reagents and labour make the system highly cost-effective. It appears to offer an improved method, well suited for use by water utilities for monitoring the burden of water-borne protozoan pathogens.     

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.     

Occurrence, source, and human infection potential of cryptosporidium and Giardia spp. in source and tap water in shanghai, china

Genotyping studies on the source and human infection potential of Cryptosporidium oocysts in water have been almost exclusively conducted in industrialized nations. In this study, 50 source water samples and 30 tap water samples were collected in Shanghai, China, and analyzed by the U.S. Environmental Protection Agency (EPA) Method 1623. To find a cost-effective method to replace the filtration procedure, the water samples were also concentrated by calcium carbonate flocculation (CCF). Of the 50 source water samples, 32% were positive for Cryptosporidium and 18% for Giardia by Method 1623, whereas 22% were positive for Cryptosporidium and 10% for Giardia by microscopy of CCF concentrates. When CCF was combined with PCR for detection, the occurrence of Cryptosporidium (28%) was similar to that obtained by Method 1623. Genotyping of Cryptosporidium in 17 water samples identified the presence of C. andersoni in 14 water samples, C. suis in 7 water samples, C. baileyi in 2 water samples, C. meleagridis in 1 water sample, and C. hominis in 1 water sample. Therefore, farm animals, especially cattle and pigs, were the major sources of water contamination in Shanghai source water, and most oocysts found in source water in the area were not infectious to humans. Cryptosporidium oocysts were found in 2 of 30 tap water samples. The combined use of CCF for concentration and PCR for detection and genotyping provides a less expensive alternative to filtration and fluorescence microscopy for accurate assessment of Cryptosporidium contamination in water, although the results from this method are semiquantitative.