Cyclospora

Solarczyk, Piotr, 2021, Host Range of Cyclospora Species: Zoonotic Implication, Acta Protozoologica 60, pp. 13-20 : 16-17

Drawbacks of Cyclospora molecular diagnostic in animals

As a result of the limitation of the microscopic assay, molecular-based methods have been developed for the detection of Cyclospora in various type of samples to assess infection risk ( Chacín-Bonilla 2008; Kitajima et al. 2014; Lalonde et al. 2016). To establish a reliable zoonotic outcome, microscopic analysis must be supported by molecular results. Because the pathogen is usually present in very low numbers in fecal samples, the detection is a very challenging task. In humans, molecular assays for Cyclospora detection are primarily dependent on the quality and purity of the genetic material, so a priori choice of DNA extraction method to isolate parasite genetic material from animals is a crucial step as well ( da Silva et al. 1999; Qvarnstrom et al. 2015; Paulos et al. 2016; Qvarnstrom et al. 2018). To date, the data of differences in usability of commercial- ly available DNA extraction kits in animals fecal samples is restricted. To overcome current molecular genotyping problems, three genetic loci such a region within the small subunit ribosomal RNA gene ( SSU rRNA), the 70 kilodalton heat shock protein ( HSP 70) gene, and the ribosomal internal transcribed spacer ( ITS) ( Sulaiman et al. 2013; Olivier et al. 2001) were primarily developed to improved detection C. cayetanensis DNA in human fecal samples. According to the literature, SSU-rDNA and ITS gene fragments were used for Cyclospora typing in animals ( Relman et al. 1996; 30 Zhao et al. 2013). However, some caution should be required as the C. cayetanensis populations may be heterogeneous. The pathogen is a sexually reproducing organism and any isolate may have genetically heterogeneous sequences. Through this process, sporozoites in a single sporocyst are thought to be genetically identical, while the sporocysts in a single oocyst can be genetically distinct ( Shirley et al. 1996; Mzilahowa et al. 2007). Therefore, one Cyclospora oocyst is heterozygous, possessing up to two alleles for any given marker and amplicons may vary in their sequences. New genotyping information for C. cayetanensis , derived from mitochondrial genome markers, should be helpful in animal source tracking studies. Next-Generation Se- quencing ( NGS) is the best technique for such studies ( Nascimento et al. 2019; Houghton et al. 2020, Cinar et al. 2020).

NGS shotgun, metabarcoding and commercially available diagnostic test

Progress on the improvement of emerging molecular tools to Cyclospora DNA detection has been observed but it is mostly fronted for humans (Qvarnstrom et al. 2018). Recent advances in modern sequencing technologies and availability of efficient software led to com- plete C. cayetanensis mitochondrial and apicoplast genomes ( Cinar et al. 2015, Cinar et al. 2016, Ogedengbe et al. 2015; Cama and Ortega 2018). The new NGS strategy used on deep sequencing platforms gains from the increasing availability, speed, and decreasing costs. In general, it is based on two approaches. The first is shotgun metagenomics, which profiles the entire micro- bial diversity consisting of both pathogenic and neutral microbiome of the host. This technique demands the knowledge of partial or whole Cyclospora reference genome, which is then compared to the shotgun data following quality processing, curation, and assembly datasets. The shotgun method may be promising to identify and develop novel target loci of C. cayetanensis ( Qvarnstrom et al. 2015) . The whole genome of C. cayetanensis is estimated to be 44 megabase pairs with ~7500 genes ( Liu et al. 2016). Cyclospora mitochondrial genome is ~6200 base pairs (bp) in length, whereas the circular apicoplast genome is ~34,000 bp and encodes complete machinery for protein synthesis. The second NGS approach is based on metabarcoding of the small ribosomal RNA subunit (18S), which targets predefined domains using specific primers. This NGS system seems to be extremely useful in terms of the development of new Cyclospora diagnostic assays ( Qvarnstrom et al. 2015; Nascimento et al. 2016; Liu et al. 2016). Cinar and coworkers described NGS mo- lecular typing of C. cayetanensis identifying potential genomic markers such as single nucleotide polymorphisms ( SNP) and insertion-deletions that could theoretically be used for Cyclospora detection and pathogen subtyping in clinical samples ( Cinar et al. 2020). These promising results were obtained by typing the mitochondrial genome. It was suggested that the diversity of C. cayetanensis and could be used to link outbreaks or even single infection cases to a source. Multicopy and linear mitochondrial genomic sequences observed in C. cayetanensis may also be used for the detection and genotyping of other Cyclospora species ( Cinar et al. 2015; Qvarnstrom et al. 2018).

The development of rapid diagnostic molecular tests has improved the detection of various protozoan pathogens thanks to higher throughput capacity ( Verweij and Verweij 2014). Besides user-friendly software and equipment independence, the ultimate goal of such tests should be better affordability, sensitivity and specificity. Currently, the BioFire FilmArray panel is the only commercially available product capable of detecting C. cayetanensis in addition to 22 enteropathogenic agents (including four protozoan species). Buss and coworkers described the sensitivity and specificity of this test during a cyclosporiasis outbreak in the USA ( Buss et al. 2013). In another study, over one and half thousand clinical stool samples were analyzed, showing that the sensitivity and specificity of this test for C. cayetanensis was 100% ( Buss et al. 2015; Murphy et al. 2019). Up to now, no reports were published on the use of this commercial test to analyze samples from animals.