A novel sensitive hexaplex high-resolution melt assay for identification of five human Plasmodium species plus internal control

Background: The diagnosis of malaria infection in humans remains challenging, further complicated by mixed Plasmodium species infections, potentially altering disease severity and morbidity. To facilitate appropriate control measures and treatment, rapid, sensitive, and specific detection assays, including those for the second minor species, would be required. This study aimed to develop a multiplex high-resolution melting (hexaplex PCR-HRM) assay with seven distinct peaks corresponding to five Plasmodium species of the Plasmodium genus, and an internal control to limit false negatives providing quality assurance testing results. Methods: Five species-specific primers for human malaria species were designed targeting on the Plasmodium 18 small subunit ribosomal RNA (18S rRNA) and mitochondrial genes. The hexaplex PCR-HRM was developed for the simultaneous and rapid detection and differentiation of five human Plasmodium spp. The limit of detection (LoD), sensitivity, and specificity of the assay were evaluated. Artificial mixing was used to assess the ability to determine the second minor species. Furthermore, a hexaplex PCR-HRM assay was used to identify 120 Plasmodium-infected clinical isolates from Kanchanaburi, Western Thailand, where malaria is endemic. Results: The hexaplex PCR-HRM assay detected the targeted genome of five Plasmodium species at levels as low as 2.354–3.316 copies/uL with 91.76 % sensitivity and 98.04 % specificity. In artificial mixing, the assay could detect minority parasite species at 0.001 % of the predominant parasite population. Plasmodium vivax infections (99 %) accounted for the majority of malaria cases in Kanchanaburi, Thailand. Conclusions: The developed hexaplex PCR-HRM assay we present in this study is a novel approach for multiplexing the Plasmodium genus and detecting five Plasmodium species with the advantage of detecting second minority parasite species. The developed one-step assay without any nesting protocols would reduce the risks of cross-contamination. Moreover, it also provides a simple, sensitive, specific, and low-cost approach for optional molecular detection of malaria.