BACKGROUND Outbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990's. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing. METHODS We developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline. RESULTS This approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study. CONCLUSIONS Our results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.

中文翻译：

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使用完整线粒体基因组的靶向富集和新一代测序对农产品和临床样品中的环孢子虫进行分子分型。

背景技术自20世纪90年代中期以来，环孢子虫病（一种由卡耶坦环孢子虫引起的腹泻疾病）的爆发一直是美国的公共卫生问题。2018 年，美国报告了 2299 例国内获得的环孢子虫病病例，原因是与不同新鲜农产品相关的多起大规模疫情。由于缺乏针对 C. cayetanensis 的标准化分子流行病学工具，疫情调查受到阻碍。对于其他顶端复门球虫寄生虫，线粒体基因组等多拷贝细胞器 DNA 已用于检测和分子分型。方法我们开发了一个工作流程，从香菜样本和临床样本中获取完整的线粒体基因组序列，用于对 C. cayetanensis 分离株进行分型。6.3 kb 长的 C. cayetanensis 线粒体基因组通过 PCR 在四​​个重叠的扩增子中扩增，这些扩增子来自从香菜中提取的基因组 DNA，接种有卵囊，以及通过诊断方法从 C. cayetanensis 呈阳性的粪便样本中提取。制备混合扩增子的 DNA 序列文库，并通过下一代测序 (NGS) 进行测序。使用定制的生物信息学管道组装序列读数。结果这种方法使我们能够对研究样本中完整的线粒体基因组进行测序。2014 年诊断的环孢子虫病患者的 24 份粪便样本的线粒体基因组中的序列改变，例如单核苷酸多态性 (SNP) 谱和插入和缺失 (InDels)，表现出区分能力。基于完整线粒体基因组序列的距离矩阵创建的聚类树状图表明，本研究分析的 2014 年卡耶塔尼西斯爆发分离株之间存在明显的菌株水平多样性。结论我们的结果表明，线粒体基因组序列的基因组分析可能有助于将疫情病例与来源联系起来。