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.     

Molecular characterization of cryptosporidium oocysts in samples of raw surface water and wastewater

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

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.     

Tracking host sources of Cryptosporidium spp. in raw water for improved health risk assessment

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

Comparison of method 1623 and cell culture-PCR for detection of Cryptosporidium spp. in source waters

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

Performances of the immunomagnetic separation method for Cryptosporidium in water under various operation conditions.

Immunomagnetic separation (IMS) has been specified as a standard method for the measurement of Cryptosporidium in some countries. In this study, the IMS method was evaluated on the basis of the recovery efficiencies of Cryptosporidium oocysts at various IMS operation conditions. The average recovery for different Cryptosporidium concentrations in deionized water was 82.6 +/- 18.2% (n = 52). No significant change in recovery was observed by altering the debris ratio of the water samples. The efficiency was increased by prolonging the reaction time, and by increasing the amount of immunomagnetic beads. The recoveries of oocysts seeded in an Eppendorf with a small reaction volume were similar to those seeded in glass tubes with 10 times the reaction volume. The recovery efficiency of oocysts was reduced significantly when the reaction buffer was replaced by PBS. In conclusion, this method has good reproducibility and high recovery.     

Routine monitoring of Cryptosporidium oocysts in water using flow cytometry

A flow cytometric method for the routine analysis of environmental water samples for the presence of Cryptosporidium oocysts has been developed. It uses a Coulter Epics Elite flow cytometer to examine water samples and to separate oocysts from contaminating debris by cell sorting. The sorted particles are then rapidly screened by microscopy. The method has been evaluated and compared with direct epifluorescence microscopy on 325 river, reservoir and drinking water samples. The technique was found to be more sensitive, faster and easier to perform than conventional epifluorescent microscopy for the routine examination of water samples for Cryptosporidium.     

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.     

A simple method for extracting DNA from Cryptosporidium oocysts using the anionic surfactant LSS

Detection of low amounts of Cryptosporidium oocysts in raw water sources is considered an important component in the management, prevention and control of Cryptosporidium in drinking water supplies as Cryptosporidium causes massive waterborne outbreaks worldwide. As Cryptosporidium has a robust oocyst that is extremely resistant to chlorine and other drinking water disinfectants, both the freeze-thaw method and DNA extraction kits have been commonly used for extracting and purifying DNA from the oocyst. However, the DNA extraction procedures are time consuming and costly. Therefore, a simple and low-cost method to extract and purify DNA from the robust oocyst has been required. In this study, we discussed a simple method for detecting Cryptosporidium DNA with the anionic surfactant, n-lauroylsarcosine sodium salt (LSS) using the loop-mediated isothermal amplification (LAMP) to eliminate the need for the freeze-thaw method and the DNA extraction kits. As a result, Bst DNA polymerase was inhibited by 0.1% LSS but not 0.01% LSS and 5% Triton X-100 or Tween 20. Although DNA was extracted from the oocysts by incubating with 0.1% LSS at 90°C for 15 min, Bst DNA polymerase was inhibited by 0.1% LSS. The inhibition by 0.1% LSS was suppressed by adding 5% of the nonionic surfactants, Triton X-100 or Tween 20. The concentration of LSS in a LAMP tube was 0.01% while that in an incubation tube was 0.1%, because LSS in an incubation tube was diluted by a factor of 10 at the DNA amplification process. Therefore, we found that ten oocysts of Cryptosporidium parvum could be detected by incubation with 0.1% LSS, without removing LSS or adding the nonionic surfactants in the LAMP method.     

Evaluation of analyzing methods for Giardia and Cryptosporidium in a Taiwan water treatment plant

Giardia sp. and Cryptosporidium sp. have emerged as waterborne pathogens of concern in Taiwan. This study examined both parasites in the actual water samples in southern Taiwan. Method 1623 was characterized by a higher recovery rate and lower detection limit compared with the information collection requirement protozoan method. A significant correlation between water turbidity and Cryptosporidium sp. in raw water samples was found in this study.     

Detection of infectious Cryptosporidium oocysts by cell culture immunofluorescence assay: applicability to environmental samples

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

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.     

Detection of Cryptosporidium in water by using polypropylene cartridge filters

Members of the genus Cryptosporidium are protozoan parasites that cause gastroenteritis in humans and animals and appear to be spread largely by the fecal-oral route. A method was developed for the concentration and detection of Cryptosporidium oocysts in water to assess their occurrence in the environment and potential for waterborne disease transmission. This method was developed by using spun polypropylene cartridge filters. Optimal conditions for concentration, filter elution, filter porosity, and detection were determined. Fluoresceinated monoclonal antibodies were used for oocyst detection. Experiments also were conducted to study the effect of flow rate, low oocyst numbers, and the addition of detergents on recovery and retention of oocysts. The method that was developed was sensitive enough to detect oocysts at levels of less than 1 per liter. Using this method, we isolated Cryptosporidium oocysts from secondarily treated sewage.     

Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples.

The development of a reliable method of using PCR for detection of Cryptosporidium oocysts in environmental samples with oligonucleotide primers which amplify a portion of the sequence encoding the small (18S) subunit of rRNA producing a 435-bp product was demonstrated. The PCR assay was found to provide highly genus-specific detection of Cryptosporidium spp. after release of nucleic acids from oocysts by a simple freeze-thaw procedure. The assay routinely detected 1 to 10 oocysts in purified oocyst preparations, as shown by direct microscopic counts and by an immunofluorescence assay. The sensitivity of the PCR assay in some seeded environmental water samples was up to 1,000-fold lower. However, this interference was eliminated by either flow cytometry or magnetic-antibody capture. Sensitivity was also improved 10- to 1,000-fold by probing of the PCR product on dot blots with an oligonucleotide probe detected by chemiluminescence. Confirmation of the presence of Cryptosporidium oocysts in water samples from the outbreak in Milwaukee, Wis., was obtained with this technique, and PCR was found to be as sensitive as immunofluorescence for detection of oocysts in wastewater concentrates.     

Detection of Cryptosporidium in water by using polypropylene cartridge filters.

Members of the genus Cryptosporidium are protozoan parasites that cause gastroenteritis in humans and animals and appear to be spread largely by the fecal-oral route. A method was developed for the concentration and detection of Cryptosporidium oocysts in water to assess their occurrence in the environment and potential for waterborne disease transmission. This method was developed by using spun polypropylene cartridge filters. Optimal conditions for concentration, filter elution, filter porosity, and detection were determined. Fluoresceinated monoclonal antibodies were used for oocyst detection. Experiments also were conducted to study the effect of flow rate, low oocyst numbers, and the addition of detergents on recovery and retention of oocysts. The method that was developed was sensitive enough to detect oocysts at levels of less than 1 per liter. Using this method, we isolated Cryptosporidium oocysts from secondarily treated sewage.     

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.     

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.     

Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater.

Giardiasis and cryptosporidiosis are diseases caused by the protozoan parasites Giardia lamblia and Cryptosporidium parvum. Waterborne transmission of these organisms has become more prevalent in recent years, and regulatory agencies are urging that source and finished water be screened for these organisms. A major problem associated with testing for these organisms is the lack of reliable methodologies and baseline information on the prevalence of these parasites in various water sources. Our study addressed both of these issues. We evaluated the presence and reduction of Giardia cysts and Cryptosporidium oocysts in sewage effluent by a combination of indirect fluorescent antibody (IFA) staining and PCR. Our results indicated a 3-log reduction of Giardia cysts and a 2-log reduction of Cryptosporidium oocysts through the sewage treatment process as determined by IFA. We developed a nested PCR to detect Cryptosporidium oocysts and used a double PCR to detect Giardia cysts. A 100% correlation was noted between IFA and PCR detection of Giardia cysts while correlation for Cryptosporidium oocysts was slightly less. On the basis of these results, PCR may be a useful tool in the environmental analysis of water samples for Giardia and Cryptosporidium organisms.     

Recovery of Cryptosporidium oocysts from water by a membrane filter dissolution method

A membrane filter dissolution method was used to recover Cryptosporidium oocysts from spiked water. The average recovery rate was 70.5%.