Molecular Detection for the Apicomplexan Parasites Cyclospora cayetanensis and Cryptosporidium spp.

Abstract

Background: Cyclospora cayetanensis and Cryptosporidium spp. are intestinal Apicomplexan parasites that can cause severe diarrheal disease in children and immunocompromised hosts. Diagnostic challenges using routine diagnostic methods lead to an underreporting of these parasites, particularly in resource-limited settings. Establishing affordable molecular detection techniques will allow for the reliable determination of these parasites’ prevalence in those settings. Objective: The overarching objective was to optimize polymerase chain reaction (PCR) protocols to detect the presence of C. cayetanensis and Cryptosporidium spp. in human stool samples. Once optimized, these protocols will be used to undertake epidemiological research in Honduras. Methods: To optimize the C. cayetanensis PCR assay, we utilized a previously identified sample that contained C. cayetanensis oocysts, as confirmed by epifluorescence microscopy and safranin staining. Two additional samples from a Honduran epidemiological study on soil-transmitted helminths (STH) reported positive by the modified Ziehl-Neelsen staining were also used. The PCR protocol comprised the amplification of the 18S rRNA gene. To optimize the Cryptosporidium spp. PCR assay, a first step was to identify positive samples among donated specimens from a Honduran epidemiological study on STH. Initial screening was done with an enzyme immunoassay (copro-antigen ELISA). The PCR protocol comprised the amplification of the COWP gene and subsequent species identification using RFLP. Results: The C. cayetanensis and Cryptosporidium spp. PCR assays were both optimized. The C. cayetanensis PCR assay revealed one positive sample of the three tested. The positive sample using epifluorescence microscopy and safranin staining showed the corresponding 18S rRNA gene band at ~501 bp. As for the Cryptosporidium PCR assay, only one was PCR-positive out of 4 ELISA-positive samples. RFLP analysis of this sample revealed a possible mixed infection by both C. hominis and C. parvum. Conclusions: Both protocols can now be used to analyze human stool samples in future collaborative epidemiological research with our partners in Honduras.