Abstract
BackgroundThe blood-feeding behaviour of female sand flies may increase their likelihood of acquiring and transmitting Leishmania parasites. Studies on the host usage by these insects may thus improve our understanding of the Leishmania transmission risk in leishmaniasis-endemic areas. Here, we developed a fast multiplex real-time PCR assay for simultaneous detection of dog, human and Leishmania DNA in sand flies.MethodsPrimers and TaqMan probes targeting the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes of dog and human, respectively, were combined in a multiplex assay, which also includes primers and a TaqMan probe targeting the Leishmania minicircle kinetoplast DNA.ResultsThe multiplex assay was 100% specific, with analytical sensitivities of 103 fg/reaction for dog and human and 1 fg for Leishmania. By testing field-collected engorged female sand flies (95 Migonemyia migonei and two Nyssomyia intermedia), 50 M. migonei were positive for one or two targets (positivity rates: 45.4% for human, 4.1% for dog and 12.4% for Leishmania DNA).ConclusionsThis multiplex real-time PCR assay represents a novel fast assay for detecting dog, human and Leishmania DNA in female sand flies and therefore a tool for assessing the risk of Leishmania transmission to these hosts in areas of active transmission.
Highlights
The blood-feeding behaviour of female sand flies may increase their likelihood of acquiring and trans‐ mitting Leishmania parasites
We developed a fast multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of dog and human blood meals and Leishmania parasites in sand flies, with high analytical sensitivity and specificity, as well as relatively low cost
Specificity, sensitivity and linearity of the singleplex and multiplex assays The sets of primers and probes specific for dog cox1, human cytb and Leishmania kinetoplast DNA (kDNA) detected only the expected target
Summary
The blood-feeding behaviour of female sand flies may increase their likelihood of acquiring and trans‐ mitting Leishmania parasites. An important factor influencing the dynamics of Leishmania parasite transmission in endemic areas is the feeding behaviour of female sand flies. Investigations of the blood meals of various species of sand flies are crucial towards a better assessment of the risk of Leishmania transmission in areas where leishmaniasis are endemic. Quantitative real-time PCR [12, 13] and PCR followed by amplicon sequencing [14] demonstrated promising results, with high-level sensitivity All these methods generated important information about the feeding behaviour of sand flies, they may present several drawbacks, such as low sensitivity and specificity (e.g. ELISA and precipitin test) and high cost (e.g. mass spectrometry and PCR followed by amplicon sequencing) [12, 15, 16]
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