Validation of a novel multiplex real-time PCR assay for Trypanosoma cruzi detection and quantification in a?ai pulp

Abstract In Brazil, orally acquired T. cruzi infection has become the most relevant transmission mechanisms from public health perspective. Around 70% of new Chagas disease cases have been associated with consumption of contaminated food or beverages. A?ai (Euterpe oleracea and Euterpe precatoria) is currently one of the most commercialized Amazonian fruits in the Brazilian and international markets. Therefore, it has become important to incorporate in the production process some procedures to measure out effective hygiene and product quality control required by global market. Molecular methods have been developed for rapid detection and quantification of T. cruzi DNA in several biological samples, including food matrices, for epidemiological investigation of Chagas disease and food quality control. However, a high-performance molecular methodology since DNA extraction until detection and quantification of T. cruzi DNA in a?ai berry pulp is still needed. Herein, a simple DNA extraction methodology was standardized from the supernatant of a?ai berry pulp stabilized in a Lysis buffer. In addition, a multiplex real time qPCR assay, targeting T. cruzi DNA and an Exogenous Internal Positive Control was developed and validated, using reference from all T. cruzi DTUs and commercial samples of a?ai pulp, from an endemic municipality with previous history of oral Chagas disease outbreak. Thus, a high-sensitivity qPCR assay, that could detect up to 0.01 parasite equivalents/mL in a?ai, was reached. As of the 45 commercial samples analyzed, 9 (20%) were positive for T. cruzi. This high-sensitive, fast and easy-to-use molecular assay is compatible with most of the laboratories involved in the investigations of oral Chagas disease outbreaks, representing an important tool to the epidemiology, control and surveillance of Chagas disease. Author SummaryOral transmission of Chagas disease has acquired an increasingly importance on the disease epidemiology. Most of the orally acquired Chagas Disease cases are related to the consumption of fresh foods or drinks, as sugar cane juice, a?ai berry pulp and bacaba wine, contaminated with triatomines or its feces. In Brazil, it has recently caused numerous outbreaks and has been linked to unusually severe acute infections. So far, the evaluation of the potential for oral transmission of Chagas disease through the consumption of a?ai-based products is mostly determined by clinical or parasitological methods. Despite the recent advances, a highly sensitive, reproductible and properly validated real time PCR assay for the molecular diagnostic of T. cruzi in a?ai pulp samples is still missing. Herein, a simple and reproducible multiplex real-time PCR assay was developed to the detection and quantification of T. cruzi DNA in a?ai pulp samples. This methodology, that includes a simple step for sample stabilization and DNA extraction based on silica-membrane spin columns, can be useful for analyzing orally transmitted acute Chagas disease outbreaks.