Cryptosporidium parvum and Cryptosporidium hominis are two related species of apicomplexan protozoa responsible for the majority of human cases of cryptosporidiosis. In spite of their considerable public health impact, little is known about the population structures of these species. In this study, a battery of C. parvum and C. hominis isolates from seven countries was genotyped using a nine-locus DNA subtyping scheme. To assess the existence of geographical partitions, the multilocus genotype data were mined using a cluster analysis based on the nearest-neighbor principle. Within each country, the population genetic structures were explored by combining diversity statistical tests, linkage disequilibrium, and eBURST analysis. For both parasite species, a quasi-complete phylogenetic segregation was observed among the countries. Cluster analysis accurately identified recently introduced isolates. Rather than conforming to a strict paradigm of either a clonal or a panmictic population structure, data are consistent with a flexible reproductive strategy characterized by the cooccurrence of both propagation patterns. The relative contribution of each pattern appears to vary between the regions, perhaps dependent on the prevailing ecological determinants of transmission.

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum β-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same β-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.