The Development of a One-Step RT-qPCR for the Detection and Quantification of Viable Forms of Trypanosoma cruzi in Açai Samples from Areas at Risk of Chagas Disease through Oral Transmission

Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as a & ccedil;a & iacute;, 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 a & ccedil;a & iacute; 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 a & ccedil;a & iacute;, the evaluation of parasites' viability using molecular methods in this type of sample and differentiating between live and dead parasites in a & ccedil;a & iacute; pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in a & ccedil;a & iacute; pulp samples. This protocol enables the stabilization and preservation of nucleic acids in a & ccedil;a & iacute;, 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 a & ccedil;a & iacute; samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in a & ccedil;a & iacute; samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using a & ccedil;a & iacute; 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 a & ccedil;a & iacute; pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from a & ccedil;a & iacute; consumption.