Development of a workflow for identification of nuclear genotyping markers for Cyclospora cayetanensis

Cyclospora cayetanensis is an intestinal parasite responsible for the diarrheal illness, cyclosporiasis. Molecular genotyping, using targeted amplicon sequencing, provides a complementary tool for outbreak investigations, especially when epidemiological data are insufficient for linking cases and identifying clusters. The goal of this study was to identify candidate genotyping markers using a novel workflow for detection of segregating single nucleotide polymorphisms (SNPs) in C. cayetanensis genomes. Four whole C. cayetanensis genomes were compared using this workflow and four candidate markers were selected for evaluation of their genotyping utility by PCR and Sanger sequencing. These four markers covered 13 SNPs and resolved parasites from 57 stool specimens, differentiating C. cayetanensis into 19 new unique genotypes.