An immunomagnetic separation polymerase chain reaction assay for rapid and ultra-sensitive detection of Cryptosporidium parvum in drinking water

120

Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method.

Section: Discussionmentioning

confidence: 99%

Evaluation of a Strategy for Toxoplasma gondii Oocyst Detection in Water

Villena

Aubert

Gomis

et al. 2004

120

“…This has greatly reduced the sensitivity of PCR detection of oocysts in various water samples. The PCR inhibitors can be removed by IMS (8,9). This practice led to successful detection of Cryptosporidium oocysts in water samples from the 1993 outbreak in Milwaukee, Wis., by a Cryptosporidium genus-specific PCR technique (9) and to genotyping of C. parvum parasites in surface and filter backwash water samples by an integrated cell culture-PCR technique (3).…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of Cryptosporidium Oocysts in Samples of Raw Surface Water and Wastewater

Xiao

Singh

Limor

et al. 2001

293

225

Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate the humanpathogenic 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.

“…4). Although we do not routinely run controls for PCR inhibitors, they should be sufficiently removed during filtration and IMS (13,16,30). Third, low numbers of oocysts in the environment may go undetected by IFA due to sample dilution and competition of sample debris with fluorescent antibodies.…”

Section: Discussionmentioning

confidence: 99%

Sources and Species of Cryptosporidium Oocysts in the Wachusett Reservoir Watershed

Jellison¹,

Hemond²,

Schauer

2002

Understanding the behavior of Cryptosporidium oocysts in the environment is critical to developing improved watershed management practices for protection of the public from waterborne cryptosporidiosis. Analytical methods of improved specificity and sensitivity are essential to this task. We developed a nested PCRrestriction fragment length polymorphism assay that allows detection of a single oocyst in environmental samples and differentiates the human pathogen Cryptosporidium parvum from other Cryptosporidium species. We tested our method on surface water and animal fecal samples from the Wachusett Reservoir watershed in central Massachusetts. We also directly compared results from our method with those from the immunofluorescence microscopy assay recommended in the Information Collection Rule. Our results suggest that immunofluorescence microscopy may not be a reliable indicator of public health risk for waterborne cryptosporidiosis. Molecular and environmental data identify both wildlife and dairy farms as sources of oocysts in the watershed, implicate times of cold water temperatures as high-risk periods for oocyst contamination of surface waters, and suggest that not all oocysts in the environment pose a threat to public health.