Chagas disease, caused by Trypanosoma cruzi, remains a significant global health challenge, particularly in the molecular diagnostics of low parasitemia during the chronic phase. This highlights the critical need for enhanced diagnostic methodologies. In response, this study evaluates the effectiveness of an automated magnetic beads-based DNA extraction method in improving the molecular diagnosis of Chagas disease compared to the traditional silica column-based extraction. Accordingly, this research seeks to enhance the DNA yield, purity, and sensitivity of real-time PCR (qPCR) assays for detecting T. cruzi satDNA. Blood samples spiked with guanidine–EDTA solution and varying concentrations of T. cruzi were used to compare the two extraction methods. The results indicated that the magnetic bead-based method outperformed the silica column in terms of DNA concentration, purity, and earlier detection of T. cruzi satDNA. Although both methods had similar limits of detection at a 95% confidence interval, the magnetic bead-based approach demonstrated higher sensitivity and reproducibility, particularly in low-parasitemia samples. The findings suggest that the magnetic beads-based DNA extraction method offers a more reliable, faster, and more sensitive alternative for diagnosing chronic Chagas disease, potentially improving clinical outcomes by enabling more accurate and earlier parasite detection.

Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of T. cruzi DNA in açaí, the evaluation of parasites’ viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various T. cruzi DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for T. cruzi DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable T. cruzi. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of T. cruzi in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption.

Chagas disease (CD) is a neglected tropical disease caused by the flagellate protozoan Trypanosoma cruzi, representing a major socioeconomic challenge. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression, and several pathogens, including T. cruzi, can modulate host miRNA networks. In this context, we hypothesized that host-derived miRNAs could serve as biomarkers in chronic CD. Given the intrinsic lability of RNA, we evaluated the efficacy of a 6 M guanidine-HCl/0.2 M EDTA solution, widely used in the molecular detection of T. cruzi DNA, in preserving mRNAs and miRNAs when mixed in a 1:1 ratio with human blood. Samples with or without guanidine were enriched with exogenous miRNAs (cel-miR-39 and cel-miR-54) and stored at 4 °C. RNase P expression was also evaluated in blood samples stored for up to 120 days and in samples from patients with CD, allowing direct comparison of mRNA stability over time. Samples preserved with guanidine-EDTA showed Ct values that were 4 to 5 cycles lower for all targets analyzed and demonstrated greater RNA stability over time. Taken together, these findings demonstrate that guanidine-EDTA robustly preserves mRNA and miRNAs in human blood, expanding the feasibility of molecular analyses in retrospective samples and corroborating its potential application in the studies of biomarkers of therapeutic response and prognosis in CD.