High-resolution Melting Curve (HRM) analysis in genotypic discrimination of Cryptosporidium isolates from stool of Egyptian children

Cryptosporidiosis is a recognized child infectious killer and the second cause of diarrheal disease and death in infants. Assessing Cryptosporidium spp. genetic diversity is a real goal to elucidate its transmission dynamics and to design preventive measures in absence of effective treatment. Cryptosporidium isolates in stool of Egyptian children were detected using Acid Fast (AF) staining, copro-nPCR/RFLP assay and real time PCR high-resolution melting (HRM) curve analysis assay. Stool samples were collected from 335 children complaining of diarrhea and other GIT symptoms, attending the outpatient clinic of Abu El Reesh hospital, Kasr Al-Ainy School of Medicine, Cairo University. Two genotypes C. hominis and C. parvum were identified in 43 isolates from Egyptian children by copro-nPCR targeting COWP gene and HRM assay. Real time PCR HRM curve analysis, a closed-tube genotyping method, targeting ITS-2 gene confirmed the results of copro-nPCR/RFLP. It is simple, rapid, has more sample throughput, analysis capacities and data storage with less carry-over contamination and cost.