Cyclospora cayetanensis is a foodborne parasite that causes cyclosporiasis, an enteric illness in humans. Genotyping methods are used to genetically discriminate between specimens from cyclosporiasis cases and can complement source attribution investigations if the method is sufficiently sensitive for application to food items. A very sensitive targeted amplicon sequencing (TAS) assay for genotyping C. cayetanensis encompassing 52 loci was recently designed. In this study, we analyzed 66 genetically diverse clinical specimens to assess the change in phylogenetic resolution between the TAS assay and a currently employed eight-marker scheme. Of the 52 markers, ≥50 were successfully haplotyped for all specimens, and these results were used to generate a hierarchical cluster dendrogram. Using a previously described statistical approach to dissect hierarchical trees, the 66 specimens resolved into 24 and 27 distinct genetic clusters for the TAS and an 8-loci scheme, respectively. Although the specimen composition of 15 clusters was identical, there were substantial differences between the two dendrograms, highlighting the importance of both inclusion of additional genome coverage and choice of loci to target for genotyping. To evaluate the ability to genetically link contaminated food samples with clinical specimens, C. cayetanensis was genotyped from DNA extracted from raspberries inoculated with fecal specimens. The contaminated raspberry samples were assigned to clusters with the corresponding clinical specimen, demonstrating the utility of the TAS assay for traceback efforts.

中文翻译：

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评估最近开发的环孢子虫基因分型方法的遗传分辨率提高和源追踪效用

卡耶坦环孢子虫是一种食源性寄生虫，可引起环孢子虫病（一种人类肠道疾病）。基因分型方法用于对环孢子虫病病例的标本进行基因区分，如果该方法对于食品的应用足够敏感，则可以补充来源归因调查。最近设计了一种非常灵敏的靶向扩增子测序 (TAS) 测定法，用于对包含 52 个基因座的 C. cayetanensis 进行基因分型。在这项研究中，我们分析了 66 个遗传多样性的临床样本，以评估 TAS 测定法和目前采用的八标记方案之间系统发育分辨率的变化。在 52 个标记中，≥50 个标记已成功对所有样本进行单倍型分析，这些结果用于生成分层聚类树状图。使用先前描述的统计方法来剖析分层树，将 66 个样本分别分解为 TAS 和 8 位点方案的 24 个和 27 个不同的遗传簇。尽管 15 个簇的样本组成相同，但两个树状图之间存在显着差异，凸显了包含额外基因组覆盖范围和选择基因分型目标位点的重要性。为了评估将受污染的食品样本与临床样本进行基因关联的能力，从接种粪便样本的覆盆子中提取的 DNA 对 C. cayetanensis 进行了基因分型。受污染的覆盆子样本被分配到具有相应临床样本的簇中，这证明了 TAS 检测在追溯工作中的实用性。