Simple sequence repeat markers have played an important role in elucidating the epidemiology of human and animal cryptosporidiosis. The drawback of sequence length polymorphisms is that nucleotide substitutions remain undetected. As some laboratories have opted for using length polymorphisms, while others have relied on sequencing, there is a need to compare both methods. We used a diversified set of unique length polymorphisms and matching nucleotide sequences to assess the ability of each genotyping protocol to discern clusters of related Cryptosporidium parvum isolates. We found a weak correlation between the two distance measures for individual markers. This analysis was extended to four-locus genotypes based on sequence length data or concatenated sequences from the same loci. We interrogated these data to assess whether one would reach the same conclusions regardless of the genotyping method. Clusters of isolates generated with the concatenated sequences were not observed with amplicon length, indicating that inferences on the structure of a Cryptosporidium population depend on the genotyping method. Moreover, isolate clusters derived from concatenated sequences were dependent on the algorithm used to calculate distances. These results emphasize the need for harmonizing genotyping tools, not only by selecting informative markers, but also by standardizing the entire genotyping method.

Trypanosoma rangeli can infect humans and the same domestic and wild animals and triatomine vectors infected by T. cruzi in Central and South America. This overlapping distribution complicates the epidemiology of Chagas disease because of the cross-reactivity between T. rangeli and T. cruzi antigens. We have studied T. rangeli strains isolated from different geographical regions using the mini-exon gene as a genetic marker. Two pairs of oligonucleotides directed to this gene were designed in order to detect specifically T. rangeli DNA by PCR assays. This assay was highly sensitive, able to amplify the target sequence using the equivalent DNA content of a single parasite as template, and demonstrated no cross-reactivity with T. cruzi DNA. T. rangeli SC-58 strain, isolated in southern Brazil, showed a distinct electrophoretic pattern from the other T. rangeli strains tested. Low stringency single specific primer-PCR (LSSP) assays were able to detect sequence polymorphisms at the mini-exon gene among T. rangeli strains. Sequence comparisons of this gene revealed that the SC-58 strain was genetically distinct from strains isolated in Central America and northern South America. In addition to insertion/deletion events, the presence of microsatellite repeats in the non-transcribed region of the gene contribute to the intra-species variability.