Abstract
BackgroundIntestinal parasitic diseases occur worldwide, and their diagnosis poses considerable challenges. Cryptosporidium spp., Entamoeba histolytica, Giardia intestinalis, (and, arguably, Dientamoeba fragilis and Blastocystis spp.) 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.ResultsUsing 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 for D. fragilis and 2.8% additional Cryptosporidium infections 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.ConclusionsThe 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.
Highlights
Intestinal parasitic diseases occur worldwide, and their diagnosis poses considerable challenges
These fragments were submitted to Basic local alignment search tool (BLAST) and used for the design of shorter fragments for the multiplex Polymerase chain reaction (PCR)
We developed a multiplex real-time PCR (5-plex qPCR) followed by high resolution melting (HRM) curve analysis targeting protists associated with diarrhea in humans
Summary
Intestinal parasitic diseases occur worldwide, and their diagnosis poses considerable challenges. Several multiplex real-time PCR assays have been developed for the synchronous detection of these parasites. Human intestinal protozoan infections can lead to significant morbidity and mortality when not diagnosed timely and treated appropriately. Such infections are typically more associated with low-income countries. Cryptosporidium spp., Entamoeba histolytica, and Giardia intestinalis are the most important parasitic protozoans causing diarrhea [2, 3]. Giardiasis and cryptosporidiosis are major causes of moderate to severe diarrhea in both, developing and developed countries, leading to considerable mortality worldwide [2, 3]. Amoebiasis is the third most frequent cause of death from protozoan parasitic diseases, with high prevalence in developing countries [4, 5]
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