Amplicon-based next-generation sequencing reveals the co-existence of multiple Leishmania species in patients with visceral leishmaniasis

•Visceral leishmaniasis can occur due to co-infection with several Leishmania spp.•Co-infection in visceral leishmaniasis changes our understanding of the disease.•Several trypanosomatids can be identified per sample using this protocol.•Amplicon-based next-generation sequencing is a cost-effective method in molecular epidemiological studies.