The detection of Cryptosporidium and the resolution of mixtures of species and genotypes from water.

Environmental concentrations of Cryptosporidium require molecular assays with ultra-sensitive detection limits and which provide critical information on genetic diversity within the genus, a feat particularly challenging from a diagnostics point of view. In this study, the performance of repetitive nested PCR-RFLP and limiting template dilution repetitive nested PCR-RFLP were assessed for their ability to detect Cryptosporidium and resolve mixtures of species and genotypes on microscope slides prepared by USEPA Method 1623 from raw water samples. Seventy percent of water samples positive for Cryptosporidium oocysts by immunofluorescent microscopy tested positive by molecular assays and resulted in species/genotype identification.

Molecular and phylogenetic approaches for assessing sources of Cryptosporidium contamination in water.

The high sequence diversity and heterogeneity observed within species or genotypes of Cryptosporidium requires phylogenetic approaches for the identification of novel sequences obtained from the environment. A long-term study on Cryptosporidium in the agriculturally-intensive South Nation River watershed in Ontario, Canada was undertaken, in which 60 sequence types were detected. Of these sequence types 33 were considered novel with no identical matches in GenBank.