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 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.

The selective obtaining of amyrins from Amazonian Protium oleoresins / La obtención selectiva de amirinas a partir de oleorresinas de Protium amazónicas

SUMMARY The oleoresin produced by species of genus Protium sp. is rich in alpha and betaamyrins, two triterpenes with many pharmacogical activities. Considering the need to make the improved obtainment of these products feasible, this study sought to optimize techniques for the extraction and isolation of amyrins from resin. Two methods of extraction (maceration and sonication) with different solvents were compared to direct isolation from crude resin. The isolation of triterpenes was performed by chromatographic columns and the yields of extracts and fractions were analyzed by analysis of variance. The best extraction solvent for amyrins was hexane for both maceration and sonication methods (38.16±2.06% and 37.67±8.21%, respectively). There was no statistical difference between these methods and the direct method (32.05±2.40%). Additionally, the direct method is cheaper and more environmentally friendly. Thus, this study showed that it is possible to obtain a large quantity of amyrins by means of cheap, fast and ecological methods.

RESUMEN La oleorresina producida por especies del género Protium sp. es rica en amirinas alfa y beta, dos triterpenos con muchas actividades farmacológicas. Esta investigación buscó optimizar las técnicas de extracción y aislamiento de amirinas de la resina para hacer factible la obtención mejorada de esos productos. Se compararon dos métodos de extracción (maceración y sonicación) con diferentes solventes con aislamiento directo de la resina cruda. El aislamento de los triterpenos se realizó mediante columnas cromatográficas y los rendimientos de extractos y fracciones fueron hechos mediante análisis de varianza. El mejor solvente para la extracción de amirinas fue el hexano para ambos métodos de maceración y sonicación (38,16±2,06% y 37,67±8,21%, respectivamente). No hubo diferencia estadística entre estos métodos y el método directo (32,05±2,40%). Además, el método directo es más barato y ecológico. De este modo, esta investigación demostró que es posible obtener una gran cantidad de amirinas a través de métodos rápidos, baratos y ecológicos.