Validation of a novel multiplex real-time PCR assay for Trypanosoma cruzi detection and quantification in açai 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. Açai (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 açai berry pulp is still needed. Herein, a simple DNA extraction methodology was standardized from the supernatant of açai berry pulp stabilized in a 6M Guanidine-HCl/0.2M EDTA 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 açai 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 açai, 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.

Oral transmission of Chagas disease from a One Health approach: A systematic review

“…These berries have become popular globally and are currently the most commercialised Amazonian fruit in the Brazilian and international markets. A study of the presence of DNA from T. cruzi showed that it could be detected in a 20% of acai samples [13]. Hence it is essential to implement hygienic measures and food quality controls in the commercial chain.…”

Comparison of the Influence of Different Nucleic Acid Extraction Assays on the Sensitivity of Trypanosoma cruzi-Specific Real-Time PCR

“…Poverty-related Chagas disease, which is, e.g., endemic in resource-limited tropical settings in South America [1], has been of increasing interest for the development of molecular diagnostic test assays in the recent two decades [2][3][4][5][6][7][8][9][10][11][12][13][14][15]. However, different from other systemic parasitic diseases such as malaria, for which excellent diagnostic accuracy of molecular diagnostic approaches has been repeatedly shown [16][17][18], the reliability of molecular techniques for the diagnosis of Chagas disease has often been less convincing in previous studies [8,[10][11][12][13][14].…”

Açai (Euterpe precatoria Mart. Arecaceae) from the Northern Region of Bolivia is not contaminated with Trypanosoma cruzi

“…Oral transmitted T. cruzi infection through consumption of açaí juice has been detected in several locations of Brazil but has not been reported in Bolivia yet. There are reports of oral transmitted infections in the Brazilian municipalities of Coari (Finamore-Araujo et al 2021), Manaos (Santana et al 2019), Amazonas State, Pará State (de Mattos et al 2017) and Amapá State (Rodrigues 2015). However, most of these municipalities are still far from the Bolivian border, with few reports (less than 10 cases) in the neighbor states of Acre and Rondonia (Shikanai-Yasuda and Carvalho 2012).…”