Detection of Cryptosporidium parvum oocysts in municipal water samples by the polymerase chain reaction

Abstract

The enteric protozoan, Cryptosporidium parvum, the causative agent for cryptosporidiosis, has been isolated from drinking water, fecal samples from humans and animals, and environmental samples such as sediment and soil. The currently available water sampling methods for detection of this parasite are labor-intensive and the efficiency of oocyst recovery is poor. A recent improved method utilizing membrane filtration and dissolution followed by polymerase chain reaction (PCR) amplification, and confirmatory nested PCR was evaluated for the sensitive and specific detection of C. parvum oocysts. Detection of PCR products by the ELISA-based Digene SHARP Signal™ System Assay was assessed for sensitivity. Seventy-two municipal water samples ranging in volume from 230 to 1,000 l from southwestern Ontario, Canada were spiked with varying concentrations of formalin-killed C. parvum oocysts for use in this study. Oocyst recovery on the filters was determined by the Merifluor immunofluoresence assay for Cryptosporidium/Giardia. Oocyst detection using the PCR assay showed an 84.7% correlation with immunofluoresence assay (IFA) results. During optimization studies, the correlation between PCR and IFA reached 98%. The sensitivity of a primary PCR assay ranged from 1 to 10 oocysts per reaction, which was equivalent to 10 2 to 10 3 oocysts per 100 l municipal water. The PCR assay also showed potential for application to untreated water samples and naturally contaminated municipal water from a recent Cryptosporidium outbreak. Further application of nested PCR may improve overall sensitivity and specificity for detecting C. parvum in municipal water samples since combined primary and nested PCR results showed 97.2% correlation with IFA. The Digene SHARP Signal™ System assay offers a sensitive and specific alternative for detection of C. parvum amplification products.