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. Multiple species/genotypes were detected in 41% of the samples, including 30 samples from which 3 species/genotypes were detected and 11 samples where 4 species/genotypes were detected. In all, 29 species or genotypes were detected which were represented by the 102 different sequences identified. Of these, 64 were considered novel as no matches were available in GenBank. These results support the use of repetitive and limiting template approaches for the detection and resolution of Cryptosporidium from the environment as well as further supporting the use of DNA sequencing as the most appropriate tool for identifying Cryptosporidium species and genotypes from water.

► 70% of Method 1623 positive samples were also positive by repetitive nested PCR.
► 102 different Cryptosporidium 18S rRNA sequences were detected.
► 29 species and genotypes of Cryptosporidium were detected.
► Repetitive and limiting template approaches successfully resolved mixtures of Cryptosporidium.