Cyclospora cayetanensis is a human parasite transmitted via ingestion of contaminated food or water. Cases of C. cayetanensis infection acquired in the United States often go unexplained, partly because of the difficulties associated with epidemiologic investigations of such cases and the lack of genotyping methods. A Multilocus Sequence Typing (MLST) method for C. cayetanensis based on five microsatellite loci amplified by nested PCR was described in 2016. The MLST loci had high variability, but many specimens could not be assigned a type because of poor DNA sequencing quality at one or more loci. We analyzed Cyclospora-positive stool specimens collected during 1997-2016 from 54 patients, including 51 from the United States. We noted limited inter-specimen variability for one locus (CYC15) and the frequent occurrence of unreadable DNA sequences for two loci (CYC3 and CYC13). Overall, using the remaining two loci (CYC21 and CYC22), we detected 17 different concatenated sequence types. For four of five clusters of epidemiologically linked cases for which we had specimens from >1 case-patient, the specimens associated with the same cluster had the same type. However, we also noted the same type for specimens that were geographically and temporally unrelated, indicating poor discriminatory power. Furthermore, many specimens had what appeared to be a mixture of sequence types at locus CYC22. We conclude that it may be difficult to substantially improve the performance of the MLST method because of the nucleotide repeat features of the markers, along with the frequent occurrence of mixed genotypes in Cyclospora infections.

Cyclosporiasis is an infection caused by Cyclospora cayetanensis, which is acquired by consumption of contaminated fresh food or water. In the United States, cases of cyclosporiasis are often associated with foodborne outbreaks linked to imported fresh produce or travel to disease-endemic countries. Epidemiologic investigation has been the primary method for linking outbreak cases. A molecular typing marker that can identify genetically related samples would be helpful in tracking outbreaks. We evaluated the mitochondrial junction region as a potential genotyping marker. We tested stool samples from 134 laboratory-confirmed cases in the United States by using PCR and Sanger sequencing. All but 2 samples were successfully typed and divided into 14 sequence types. Typing results were identical among samples within each epidemiologically defined case cluster for 7 of 10 clusters. These findings suggest that this marker can distinguish between distinct case clusters and might be helpful during cyclosporiasis outbreak investigations.