A novel 5-Plex qPCR-HRM assay detecting human diarrheal parasites

Background Intestinal parasitic diseases occur worldwide, and their diagnosis poses considerable challenges.Cryptosporidiumspp.,Entamoeba histolytica, Giardia intestinalis,(and, arguably,Dientamoeba fragilisandBlastocystisspp.) are among the most important and common parasitic protozoans causing diarrhea. Several multiplex real-time PCR assays have been developed for the synchronous detection of these parasites. However, most assays include the use of hydrolysis probes, increasing the cost of stool examination. In this study, we designed and evaluated a real-time PCR protocol, based on high-resolution melting (HRM) curve analysis, to simultaneously detect and differentiate five gastrointestinal parasites. Results Using a blinded panel of 143 clinical samples with laboratory diagnostic data to evaluate the method, we obtained a 95.8% concordance with conventional methods. Moreover, 4.2% of the samples were positive forD. fragilisand 2.8% additionalCryptosporidiuminfections were found with our multiplex assay. Our method is sensitive and specific for the selected parasites with the additional possibility of being run in single-plex as a backup control for mixed infections. Conclusions The assay is a convenient and cost-effective method that could contribute to a quicker and accurate diagnosis as well as to more targeted therapies of parasite-derived diarrhea. Finally, this new multiplex PCR assay could also be instrumental in epidemiology studies on these parasites.