DETECTION OF CRYPTOSPORIDIUM-PARVUM OOCYSTS IN MUNICIPAL WATER SAMPLES BY THE POLYMERASE-CHAIN-REACTION

The enteric protozoan, Cryptosporidium parvum, the causative agent for cryptosporidiosis, has been isolated from drinking water, fecal sampl es from humans and animals, and environmental samples such as sediment and soil. The currently available water sampling methods for detectio n of this parasite are labor-intensive and the efficiency of oocyst re covery is poor. A recent improved method utilizing membrane filtration and dissolution followed by polymerase chain reaction (PCR) amplifica tion, 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(TM) System Assay was assessed for sensitivity. Seventy-two municipal water samples ranging in volume fro m 230 to 1,000 1 from southwestern Ontario, Canada were spiked with va rying concentrations of formalin-killed C. parvum oocysts for use in t his study. Oocyst recovery on the filters was determined by the Merifl uor immunofluorescence assay for Cryptosporidium/Giardia. Oocyst detec tion using the PCR assay showed an 84.7% correlation with immunofluore scence assay (IFA) results. During optimization studies, the correlati on between PCR and LFA reached 98%. The sensitivity of a primary PCR a ssay ranged from 1 to 10 oocysts per reaction, which was equivalent to 10(2) to 10(3) oocysts per 100 1 municipal water. The PCR assay also showed potential for application to untreated water samples and natura lly contaminated municipal water from a recent Cryptosporidium outbrea k. Further application of nested PCR may improve overall sensitivity a nd specificity for detecting C. pavum in municipal water samples since combined primary and nested PCR results showed 97.2% correlation with IFA. The Digene SHARP Signal(TM) System assay offers a sensitive and specific alternative for detection of %. parvum amplification products . (C) 1998 Elsevier Science B.V.