Development of an amplicon-based Next Generation Sequencing protocol to identify Leishmania species and other trypanosomatids in Leishmaniasis endemic areas. Development of an amplicon-based Next Generation Sequencing protocol to identify Leishmania species and other trypanosomatids in Leishmaniasis endemic areas

Trypanosomatids infections are an important public health threat affecting many low-income countries across the tropics, particularly in the Americas. Trypanosomatids can infect many vertebrate, invertebrate and plant species, and play an important role as human pathogens. Amongst these clinically relevant pathogens are species from the genus Leishmania and Trypanosoma. Mixed trypanosomatids infections remains a largely unexplored phenomenon. Herein, we describe the application of an amplicon-based next-generation sequencing (NGS) assay to detect and identify trypanosomatids species in mammalian reservoirs, human patients, and sand fly vectors through Leishmania endemic regions. Sixty-five samples from different departments of Colombia, including two samples from Venezuela, were analyzed: 49 samples from Cutaneous Leishmaniasis (CL) patients, eight from sandflies, two from domestic reservoirs (Canis familiaris), and 6 from wild reservoirs (Phyllostomus hastatus). DNA from each sample served to identify the presence of trypanosomatids through conventional PCR using as target the Heat Shock Protein 70 (HSP70) gene. PCR products underwent sequencing by Sanger and Next Generation Sequencing (NGS), and trypanosomatids species were identified using blastn against a reference database built from the trypanosomatids-derived HSP70 sequences. The alpha and beta diversity indexes of amplicon sequence variants were calculated for each group. Results revealed the presence of mixed infections, with more than two Leishmania species, in 34% of CL samples analyzed. Trypanosoma cruzi was identified in samples from wild reservoirs as well as in sand fly vectors. Co-infection events with three different Leishmania species were identified in domestic reservoirs. These findings depose the traditional paradigm of Leishmaniasis being a single-species driven infection and redraw the choreography of host-pathogen interaction in context of multiparasitism. Further research is needed to decipher how co-infections may influence disease progression. This knowledge is key to developing an integrated approach for diagnosis and treatment.