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.     

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.     

Evaluation of the immunofluorescence procedure for detection of Giardia cysts and Cryptosporidium oocysts in water.

The accurate determination of the presence of Giardia cysts and Cryptosporidium oocysts in surface waters requires a reliable method for the detection and enumeration of these pathogenic organisms. Published methods have usually reported recovery efficiencies of less than 50% for both cysts and oocysts. Typically, the losses are greater for Cryptosporidium oocysts than they are for Giardia cysts. The purpose of this study was to examine procedures used for sample collection, elution, concentration, and clarification to determine when losses of cysts and oocysts occurred during processing. The results showed that major losses of cysts and oocysts occurred during centrifugation and clarification. Depending on the centrifugation force, oocyst losses of as high as 30% occurred for each centrifugation step. A 1.15-specific-gravity Percoll-sucrose gradient was needed to optimize recovery of oocysts from natural water samples. Minor improvements in the procedure could be accomplished by selecting a filter other than the recommended 1-micron-pore-size (nominal-porosity) polypropylene filter.     

Species-specific, nested PCR-restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.     

Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum

Cryptosporidium parvum is a waterborne pathogen that poses potential risk to drinking water consumers. The detection of Cryptosporidium oocysts, its transmissive stage, is used in the latest U.S. Environmental Protection Agency method 1622, which utilizes organic fluorophores such as fluorescein isothiocyanate (FITC) to label the oocysts by conjugation with anti-Cryptosporidium sp. monoclonal antibody (MAb). However, FITC exhibits low resistance to photodegradation. This property will inevitably limit the detection accuracy after a short period of continuous illumination. In view of this, the use of inorganic fluorophores, such as quantum dot (QD), which has a high photobleaching threshold, in place of the organic fluorophores could potentially enhance oocyst detection. In this study, QD605-streptavidin together with biotinylated MAb was used for C. parvum oocyst detection. The C. parvum oocyst detection sensitivity increased when the QD605-streptavidin concentration was increased from 5 to 15 nM and eventually leveled off at a saturation concentration of 20 nM and above. The minimum QD605-streptavidin saturation concentration for detecting up to 4,495 +/- 501 oocysts (mean +/- standard deviation) was determined to be 20 nM. The difference in the enumeration between 20 nM QD605-streptavidin with biotinylated MAb and FITC-MAb was insignificant (P > 0.126) when various C. parvum oocyst concentrations were used. The QD605 was highly photostable while the FITC intensity decreased to 19.5% +/- 5.6% of its initial intensity after 5 min of continuous illumination. The QD605-based technique was also shown to be sensitive for oocyst detection in reservoir water. This observation showed that the QD method developed in this study was able to provide a sensitive technique for detecting C. parvum oocysts with the advantage of having a high photobleaching threshold.     

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.     

Occurrence of Cryptosporidium oocysts in sewage effluents and selected surface waters

An existing method for the detection of Cryptosporidium oocysts in water was modified to investigate oocyst prevalence in large volumes of water. Surface waters and sewage effluents were filtered, eluted from the filter, and concentrated using centrifugation. The resultant pellet was then homogenized, sonicated, and placed on a sucrose gradient to separate oocysts from the sediment. The uppermost gradient layer was then examined by immunofluorescence using a labeled monoclonal antibody. Using this technique, average numbers of oocysts detected in raw and treated sewage were 5.18 X 10(3) and 1.30 X 10(3)/L, respectively. Filtered sewage effluents had significantly lower numbers of oocysts (10.0/L). These data show that sand filtration may reduce the concentrations of this parasite in waste waters. Highly variable oocyst numbers were encountered in surface waters. Since Cryptosporidium oocysts are frequently present in environmental waters, they could be responsible for waterborne outbreaks of disease.     

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.     

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.     

Identification of Cryptosporidium oocysts in river water.

Water samples were collected from four rivers in Washington State and two rivers in California and examined for the presence of Cryptosporidium oocysts. Oocyst-sized particles were concentrated from 20-liter samples of water by membrane filtration, centrifugation, and differential sedimentation. The particle concentrate was then deposited on a 25-mm-diameter membrane filter for oocyst identification by indirect immunofluorescence assay. The identification procedure had a limit of detection of about five oocysts per liter. Cryptosporidium oocysts were found in each of 11 river water samples examined. Concentrations ranged from 2 to 112 oocysts per liter. The finding of Cryptosporidium oocysts in all samples examined from six western rivers is noteworthy in light of recent reports indicating that Cryptosporidium sp. is a significant agent of human and animal disease. This finding suggests that waterborne oocysts of this parasite are more important than was previously recognized. More detailed studies are needed to define geographical and temporal distribution, to assess the viability of waterborne oocysts, and to determine the importance of water as a means of transmission.     

Identification of Cryptosporidium oocysts in river water

Water samples were collected from four rivers in Washington State and two rivers in California and examined for the presence of Cryptosporidium oocysts. Oocyst-sized particles were concentrated from 20-liter samples of water by membrane filtration, centrifugation, and differential sedimentation. The particle concentrate was then deposited on a 25-mm-diameter membrane filter for oocyst identification by indirect immunofluorescence assay. The identification procedure had a limit of detection of about five oocysts per liter. Cryptosporidium oocysts were found in each of 11 river water samples examined. Concentrations ranged from 2 to 112 oocysts per liter. The finding of Cryptosporidium oocysts in all samples examined from six western rivers is noteworthy in light of recent reports indicating that Cryptosporidium sp. is a significant agent of human and animal disease. This finding suggests that waterborne oocysts of this parasite are more important than was previously recognized. More detailed studies are needed to define geographical and temporal distribution, to assess the viability of waterborne oocysts, and to determine the importance of water as a means of transmission.     

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.     

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.     

Near real-time detection of Cryptosporidium parvum oocyst by IgM-functionalized piezoelectric-excited millimeter-sized cantilever biosensor

Piezoelectric-excited millimeter-sized cantilever (PEMC) biosensors were fabricated and functionalized with immunoglobulin M (IgM) for the detection of Cryptosporidium parvum oocyst in a flow configuration at 1 mL/min. The detection of 100, 1000, and 10,000 oocysts/mL was achieved with a positive sensor response in less than 1 min. Bovine serum albumin (BSA) was used as a blocking agent in each experiment and was shown to eliminate non-specific binding. The sensor's resonance frequency response correlates with C. parvum oocyst concentration logarithmically. The oocyst attachment rate was found to increase by an order of magnitude in increasing concentration from 100 to 10,000 oocysts/mL. The significance of these results is that IgM-functionalized PEMC sensors are highly selective and sensitive to C. parvum oocyst and therefore, have the potential to accurately identify and quantify C. parvum oocyst in drinking water.     

Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.     

Comparison of most probable number-PCR and most probable number-foci detection method for quantifying infectious Cryptosporidium parvum oocysts in environmental samples

Microbial contamination of public water supplies is of significant concern, as numerous outbreaks, including Cryptosporidium, have been reported worldwide. Detection and enumeration of Cryptosporidium parvum oocysts in water supplies is important for the prevention of future cryptosporidiosis outbreaks. In addition to not identifying the oocyst species, the U.S. EPA Method 1622 does not provide information on oocyst viability or infectivity. As such, current detection strategies have been coupled with in vitro culture methods to assess oocyst infectivity. In this study, a most probable number (MPN) method was coupled with PCR (MPN-PCR) to quantify the number of infectious oocysts recovered from seeded raw water concentrates. The frequency of positive MPN-PCR results decreased as the oocyst numbers decreased. Similar results were observed when MPN was coupled to the foci detection method (MPN-FDM), which was done for comparison. For both methods, infectious oocysts were not detected below 10(3) seeded oocysts and the MPN-PCR and MPN-FDM estimates for each seed dose were generally within one-log unit of directly enumerated foci of infection. MPN-PCR estimates were 0.25, 0.54, 0 and 0.66 log(10) units higher than MPN-FDM estimates for the positive control, 10(5), 10(4) and 10(3) seed doses, respectively. The results show the MPN-PCR was the better method for the detection of infectious C. parvum oocysts in environmental water samples.     

Quantitative and qualitative comparison of density-based purification methods for detection of Cryptosporidium oocysts in turbid environmental matrices

Purification methods for Cryptosporidium oocysts are usually selected on the basis of recovery yield, but the amount of particulate debris in environmental matrices could limit efficiency of oocyst detection by microscopic examination or PCR detection. Previous studies have shown that the standard immunomagnetic separation (IMS) procedure would not be the most suitable method for oocyst purification from turbid matrices. We compared the capacity of Percoll-sucrose flotation and six other density-based purification methods to achieve selective separation of Cryptosporidium oocysts from particulate debris. Rate of oocyst recovery and particulate loading in the purified suspensions were chosen as comparison criteria for the different purification methods. In most earlier studies, the chemical treatments employed to obtain a purified oocyst suspension modify the surface properties of oocysts in spiked samples. Assuming this produces unrealistic conditions affecting the evaluation of purification methods, we performed the present study with native oocysts. Flotation and gradient procedures were tested with and without formaldehyde ethyl acetate (FEA) separation. FEA separation was found to be unsuitable. Filtration and Percoll gradient did not allow selective oocyst separation from debris. Among the purification methods suitable for routine microscopic examination, Percoll-sucrose flotation provided the best recovery rates. For automated enumeration systems or PCR detection, potassium bromide and especially Nycodenz gradients appeared to be the most suitable purification methods. Potassium bromide and Nycodenz gradients provided the best balance between oocyst recovery and particulate load.     

Evaluation of Cryptosporidium parvum oocyst recovery efficiencies from various filtration cartridges by electrochemiluminescence assays

AIMS: To evaluate four types of filtration cartridges for their capacities, efficiency for capture and release of Cryptosporidium parvum oocysts for detection. METHODS AND RESULTS: Filtration cartridges included in this evaluation were IDEXX Filta-Max, Gelman Envirochek HV, Corning CrypTest, and Filterite Sigma+. Various dosages of C. parvum oocysts were spiked into water samples with a wide range of turbidity (10-50 NTU). Electrochemiluminescence assays were employed to enumerate viable or total number of C. parvum oocysts in these eluates. Among the cartridges tested, Filta-Max consistently showed higher oocyst recovery efficiency, especially with large volume, highly turbid water samples. CONCLUSIONS: Filta-Max filter is the best performer because of its higher oocyst recovery efficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: The overall sensitivities of various C. parvum oocyst detection assays in water samples can be improved if highly efficient oocyst recovery filtration cartridges such as Filta-Max are incorporated in sample preparation.     

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.