Genetic diversity of natural populations of Trypanosoma cruzi in clinical samples from patients with oral Chagas disease in Venezuela: Follow-up after treatment with trypanocidal drugs

Abstract

Oral transmission of Chagas disease (OChD) is an increasingly important aspect in the epidemiology. Venezuela has reported the two largest outbreaks described so far, affecting a total of 192 people mostly children. The long-term impact after treatment on the dynamics of infection in natural populations of T. cruzi is still unclear. In this sense, we proposed a genetic characterization of T. cruzi populations present in peripheral blood in order to differentiate responder or non-responder patients and predict the response to treatment (Tmt). We performed quantification of the parasitic load by qPCR, genetic typing of T. cruzi populations by multiplex qPCR of nuclear genome markers and RFLP-PCR for the hypervariable region of T. cruzi kDNA to demonstrate changes in Minicircle signatures (Ms) of the parasite populations present in 41 clinical samples from 15 patients. To reflect the genetic diversity found, Jaccard distances (Jd) values were compared. This clinical monitoring confirmed the presence of T. cruzi DNA in 26 post-treatment samples up to 9 years after Tmt. These results reveal 100 % therapeutic failure for both outbreaks of OChD, classifying these patients as non-responders to Tmt. All samples showed homogeneity at the DTU level, being typified as TcI. The Ms showed a high degree of polymorphism, with 73 % of total post-Tmt samples with Jd values close to 1. Analyzing the dynamics of each patient`s population separately, in all post-Tmt samples the change in Ms variability respect to pre-Tmt sample was not statistically significant. This variability does not reflect a natural or induced clonal selection process driven by the etiological Tmt; on the contrary, it could be associated with the clonal histotropism process evidenced in natural T. cruzi infections. In conclusion, these strategies of molecular characterization of parasite DNA were useful to detect Tmt failure and find out the lack of parasite population selection with Tmt in these OChD settings.