Natural infection of Phlebotomus argentipes with Leishmania and other trypanosomatids in a visceral leishmaniasis endemic region of Nepal

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Abstract
Monitoring Leishmania infection in sand flies is important for understanding the eco-epidemiology of kala-azar and assessing the impact of the recently launched kala-azar control programme in the Indian subcontinent. We applied a PCR technique that targets rRNA genes to estimate the natural incidence of Leishmania infection in sand flies sampled in six villages of the Terai region of Nepal. Amplifications were made on 135 pools of sand flies and confirmed by sequencing. Seven pools were found to be PCR positive: in five of them we identified the rDNA signature found in Leishmania spp., whereas two other pools revealed a sequence compatible with other trypanosomatids. Different methodologies were applied to evaluate the infection rate from pools of unequal size and estimated the infection rate to range from 0.468% to 0.578% for the Leishmania group and from 0.185% to 0.279% for the non-Leishmania group. Our results highlight the diversity of flagellate infections likely to be encountered in Phlebotomus argentipes populations. Our methodology allows clear discrimination of Leishmania from other trypanosomatids and should be applied on larger insect samples or in longitudinal studies.