Comparison of two novel one-tube nested real-time qPCR assays to detect human-infecting Cyclospora spp.

Human-infecting Cyclospora spp. currently include three coccidian parasites that cause the gastrointestinal disease cyclosporiasis in humans. They are often spread through contaminated produce, including leafy greens and berries. The increased availability of sensitive molecular tests for the diagnosis of cyclosporiasis is an important advancement, allowing public health agencies to better understand the scope and source of cyclosporiasis outbreaks. To improve the diagnosis of infected patients, rapidly detect outbreaks, and keep the food supply safe, it is important to continue to develop sensitive, reliable, and inexpensive tests to detect human-infecting Cyclospora spp. In this report, we describe the development and evaluation of two novel one-tube nested qPCR assays for the detection of human-infecting Cyclospora spp. in clinical stool samples, one targeting cytb and the other targeting coxI. Of these, the assay targeting the cytb mitochondrial locus possessed strong performance characteristics compared to a routinely used 18S assay, including a markedly improved (approximately 10-fold lower) relative detection limit of 0.613 oocysts per gram of feces. This is compared to coxI that has a relative detection limit equal to that of the 18S assay. Given the strong performance characteristics of the cytb assay, we propose that it may be useful to diagnostic laboratories wishing to screen clinical fecal specimens suspected of containing human-infecting Cyclospora spp.

Automated summary

Human-infecting Cyclospora spp. are coccidian parasites that cause cyclosporiasis, a gastrointestinal disease, in humans. The development of sensitive molecular tests has greatly improved the diagnosis of cyclosporiasis outbreaks and the understanding of its source. In this study, two novel one-tube nested qPCR assays were developed to detect human-infecting Cyclospora spp. in clinical stool samples. The assay targeting the cytb mitochondrial locus showed better performance compared to the routine 18S assay. This finding suggests that the cytb assay may be useful for diagnostic laboratories screening clinical fecal specimens suspected of containing human-infecting Cyclospora spp.