A PCR method for the quantitation of Cryptosporidium parvum oocysts in municipal drinking water samples was investigated. Quantitative PCR uses an internal standard (IS) template with unknown target numbers to compare to standards of known concentrations in a standard curve. The IS template was amplified using the same primers used to amplify a portion of a 358 bp gene fragment that encodes a repetitive oocyst wall protein in C. parvum. Municipal water samples spiked with known numbers of C. parvum oocysts were tested by quantitative PCR using the IS and the Digene SHARP Signal System Assay for PCR product detection. The absorbance readings for target DNA and IS templates versus the number of molecules of the target DNA were plotted to generate standard curves for estimating oocyst numbers. The method allowed the quantitation of oocysts from log 3 to log 5 spiked into municipal water samples.

Cryptosporidium parvum is a protozoan parasite responsible for an increasing number of outbreaks of gastrointestinal illness worldwide. In this report, we describe development of sample preparation protocols for polymerase chain reaction (PCR)-based detection of C. parvum in fecal material and environmental water samples. Two of these methods were found adequate for isolation of Cryptosporidium DNA from filtered water pellet suspensions. The first involved several filtration steps, immunomagnetic separation and freeze–thaw cycles. The second method involved filtration, addition of EnviroAmp™ lysis reagent, freeze–thaw cycles and precipitation of the DNA with isopropanol. Using nested PCR, we detected 100 oocysts/ml of filtered water pellet suspension, with either of the above sample preparation procedures. Nested PCR increased sensitivity of the assay by two to three orders of magnitude as compared to the primary PCR. The detection limit for seeded fecal samples was 10-fold higher than for filtered environmental water pellet suspension. Nested PCR results showed 62.4 and 91.1% correlation with immunofluorescence assay (IFA) for fecal samples and filtered environmental water pellet suspensions, respectively. This correlation decreased to 47.2% and 44.4%, respectively, when only IFA positive samples were analyzed. However, in fecal samples contaminated with a high number (>105/g) of C. parvum oocysts, this correlation was 100%.

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 102 to 103 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.

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

A simple, rapid, and inexpensive spot test incorporating the substrate pyrrolidonyl naphthylamide was used to examine pyrrolidonyl peptidase activity among 800 bacterial strains belonging to the families Enterobacteriaceae and Vibrionaceae. The pyrrolidonyl naphthylamide test was found to be particularly useful in separating Citrobacter spp. (100% positive) from Salmonella spp. (0.4% positive) and Escherichia coli (0% positive). Furthermore, it would appear to offer a safer alternative to the traditional potassium cyanide test for differentiating citrobacters from salmonellae.