Molecular diagnosis of trypanosomatids in Didelphis marsupialis reveals risk areas for Trypanosoma cruzi transmission and sympatric circulation with T. rangeli in the metropolitan area of Bucaramanga, Santander, Colombia

The adaptation of wild animals to urban environments can lead to increased contact with humans and a higher risk of exposure to zoonotic agents. <i>Didelphis marsupialis</i> (common opossum) is an important reservoir of <i>Trypanosoma cruzi</i> and <i>Leishmania</i> spp., which commonly affect human populations in Latin America. Therefore, this study aimed to assess the frequency of trypanosomatid infections and characterize <i>T. cruzi</i> DTUs in common opossums from the Metropolitan Area of Bucaramanga (MAB), Santander, Colombia. A total of 70 individuals from four municipalities (Bucaramanga, Floridablanca, Girón, and Piedecuesta) were analyzed by PCR using blood samples, of which 14.3 % (95 % CI: 7.95-24.3 %) tested positive for trypanosomatids. Next-generation sequencing of 18S and Hsp70 genes in positive samples identified <i>T. cruzi</i> DTU TcI and <i>T. rangeli</i> in nine (12.9 %, 95 % CI: 6.91-22.66 %) and two (2.86 %, 95 % CI: 0.79-9.83 %) samples, respectively, including one case of co-infection (1.43 %, 95 % CI: 0.04-7.7 %). A heatmap revealed a high concentration of <i>T. cruzi</i>-positive cases in peripheral neighborhoods of Bucaramanga adjacent to forested areas. This study confirms the presence of an enzootic transmission cycle of <i>T. cruzi</i> in the MAB, highlighting the role of <i>D. marsupialis</i> as an important reservoir, particularly in peripheral neighborhoods of Bucaramanga. The sympatric circulation of <i>T. cruzi</i> and <i>T. rangeli</i> in opossums from the MAB introduces new epidemiological challenges for Chagas disease control in these areas, emphasizing the need for improved diagnostic strategies to differentiate both parasites in patients and epidemiological studies including vectors and reservoirs.