A multifaceted molecular approach to surveillance of leishmaniasis: Identification of sand fly species, Leishmania parasites, and blood meal sources using high-resolution melting analysis

Leishmaniasis is a significant public health concern in large parts of the world including Israel, with limited diagnostic tools available for effective surveillance and control. Traditional methods for sand fly species identification, Leishmania detection, and blood meal analysis are time-consuming and prone to errors. To address these challenges, this study aimed to develop PCR-high resolution melt (HRM) assays to accurately determine sand fly species, Leishmania infection and blood meal sources in sand flies. Field-collected sand flies from all regions of Israel were used for the validation of three PCR-HRM assays. These included 254 sand fly males and females identified morphologically for species verification; 1,120 unfed females for Leishmania detection, and 538 engorged females for blood meal identification. PCR products were subjected to HRM curve analysis, and results were compared to nucleotide sequencing and sand fly morphology. Eleven sand fly species, 25 different host species blood meals and four Leishmania species were discerned and each presented a specific HRM pattern. Of the 1,658 analyzed females, 16 (1%) were positive for Leishmania, and the species identified were: Leishmania major, L. tropica, L. infantum and L. donovani. Blood meal source was identified in 520 (96.7%) engorged females. Blood from four animal species (domestic cat, rock hyrax, European hare, cow) accounted for 53% of the sand fly blood meals and the remaining 47% came from 21 other animal species. The sand fly species distribution showed that L. major and L. donovani vectors were mostly prevalent in arid southern Israel while L. tropica and L. infantum vectors were abundant in central and northern Israel. These results present the current knowledge of the different Leishmania species life cycles, vectors, and host species present in Israel and substantiate the utility of the assays developed herein which combine the advantages of PCR and the discriminatory power of HRM.