We used Sanger and amplicon-based sequencing (NGS) in samples from different mammals to identify trypanosomatid infections in many departments of Colombia. A total of 174 DNA samples were analyzed by conventional PCR of a fragment from the heat shock protein 70 (Hsp70) gene: 18 humans, 83 dogs, and 73 wild mammals. The positive samples were sequenced by Sanger. From these, 27 samples were sent to amplicon-based sequencing of the same gene fragment. Data obtained was used to make diversity analyses.. An overview of the trypanosomatid (Kinetoplastida: Trypanosomatidae) parasites infecting several mammal species in Colombia

Background: Trypanosomatids are one of the most important parasites for public health due to their impact on human, animal, and plant health. Diseases associated with these pathogens manifest mainly in poor and vulnerable populations, where social, environmental, and biological factors modulate the case’s incidence and geographical distribution. Methods: We used Sanger and amplicon-based sequencing (NGS) in samples from different mammals to identify trypanosomatid infections in many departments of Colombia. A total of 174 DNA samples were analyzed by conventional PCR of a fragment from the heat shock protein 70 (Hsp70) gene: 18 humans, 83 dogs, and 73 wild mammals. The positive samples were sequenced by Sanger. From these, 27 samples were sent to amplicon-based sequencing of the same gene fragment. Data obtained was used to make diversity analyses. Results: A total of 112 samples were positive for PCR by Hsp70 fragment, these corresponded to: 22.3% Leishmania spp., 18.8% L. amazonensis, 9.8% L. braziliensis, 13.4% L. infantum, 8% L. panamensis, and 27.8% Trypanosoma cruzi. Comparison of the identified species by the two sequencing technologies used resulted in 97% of concordance. Alpha and beta diversity indices were significant, mainly for dogs as well as an interesting index of coinfection events in the analyzed samples: different Leishmania species, the simultaneous presence of T. cruzi, and even T. rangeli in one of the samples analyzed. Moreover, a low presence of L. braziliensis was observed in samples from wild mammals. Interestingly, for our knowledge, this is the first report of Leishmania detection in Hydrochaeris hydrochaeris (capybara) in Colombia. Conclusions: The Hsp70 fragment used in this study is an optimal molecular marker for trypanosomatid identification in many hosts and allows the identification of different species in the same sample when amplicon-based sequencing is used. However, the use of this fragment for molecular diagnosis through conventional PCR should be carefully interpreted due to this same capacity to identify several parasites. This point is of pivotal importance in highly endemic countries like South American countries, owing to the co-circulation of different genera from the Trypanosomatidae family. The findings show an interesting starting point for One Health approaches, in which coevolution and vector-host interactions can be studied