Genotyping Canadian Cyclospora cayetanensis Isolates to Supplement Cyclosporiasis Outbreak Investigations

Abstract

Cyclospora cayetanensis is an emerging foodborne parasite that causes cyclosporiasis, an enteric disease of humans. Domestically acquired outbreaks have been reported in Canada every spring or summer since 2013. To date, investigations into the potential sources of infection have relied solely on epidemiological data. To supplement the epidemiological data with genetic information, we genotyped 169 Canadian cyclosporiasis cases from stool specimens collected from 2010 to 2021 using an existing eight-marker targeted amplicon deep (TADS) scheme specific to C. cayetanensis as previously described by the US Centers for Disease Control and Prevention (CDC). This is the first study to genotype Canadian Cyclospora cayetanensis isolates, and it focuses on evaluating the genotyping performance and genetic clustering. Genotyping information was successfully collected with at least part of one of the markers in the TADS assay for 97.9% of specimens, and 81.1% of cyclosporiasis cases met the minimum requirements to genetically cluster into 20 groups. The performance of the scheme suggests that examining cyclosporiasis cases genetically will be a valuable tool for supplementing epidemiological outbreak investigations and to minimize further infections. Further research is required to expand the number of discriminatory markers to improve genetic clustering.

Keywords: Canadian outbreaks; Cyclospora cayetanensis; cyclosporiasis; foodborne pathogen; genotyping; targeted amplicon deep sequencing (TADS).

Figure 1

Yearly and provincial distribution of the 169 Cyclospora cayetanensis-positive unpreserved fecal specimens from cyclosporiasis case patients received at the NML in Guelph from 2010 to 2021 from four provinces: Ontario (ON), Québec (QC), Newfoundland and Labrador (NL), and British Columbia (BC).

Figure 2

Distribution of haplotypes observed for the Nu_CDS1-4 markers of Canadian Cyclospora cayetanensis-positive fecal specimens from 2010 to 2021. The majority of these specimens were homozygous at these four loci, with ≤10% of the specimens being heterozygous at either Part A or Part B of the loci or both. A new haplotype (haplotype 3: Hap2 in Part A and Hap3 in Part B) was recorded at the Nu_CDS2 loci in a 2010 specimen.

Figure 3

Cluster dendrogram of Canadian Cyclospora cayetanensis positive fecal specimens from 2010 to 2021. The dendrogram was generated by applying Ward’s clustering method to the ensemble matrix of pairwise distances that was calculated from 137 Canadian cyclosporiasis case patients (colored) that passed the sequencing requirements and 1077 US specimens from 2018 and 2019 (gray) that were provided with the software as a reference population. Twenty genetic clusters were identified by Module 3, which contained Canadian cyclosporiasis cases (colored), and the number of specimens pertaining to each cluster are as follows: cluster 1 (n = 3), cluster 2 (n = 12), cluster 3 (n = 38), cluster 4 (n = 1), cluster 5 (n = 5), cluster 6 (n = 8), cluster 7 (n = 11), cluster 8 (n = 2), cluster 10 (n = 22), cluster 11 (n = 5), cluster 12 (n = 4), cluster 13 (n = 2), cluster 14 (n = 2), cluster 15 (n = 7), cluster 16 (n = 2), cluster 17 (n = 1), cluster 19 (n = 4), cluster 20 (n = 1), cluster 21 (n = 4), and cluster 22 (n = 3).