Extraction of Trypanosoma cruzi DNA from food: a contribution to the elucidation of acute Chagas disease outbreaks

Ferreira, Renata Trotta Barroso; Melandre, Aline Martins; Cabral, Maria Luiza; Branquinho, Maria Regina; Cardarelli-Leite, Paola

doi:10.1590/0037-8682-0414-2015

Cited by 18 publications

(13 citation statements)

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“…Namely, the characteristic dark color of the fruit, which is associated with high anthocyanin concentrations and a large amount of organic matter [34,35], greatly limit the possibility of parasites detection during microscopic visualization. In this context, molecular methods can overcome these limitations regarding analysis directly from food, and provide specific diagnosis [26,41,50,52,63] and T. cruzi genotyping [51]. The qPCR developed in the present study showed improved sensitivity, in which was possible to detect the DNA corresponding to 0.01 T. cruzi equivalents/mL in the sample.…”

Section: Discussionmentioning

confidence: 87%

“…T. cruzi transmission via the oral route is not a recent event, since it represents the main route of contamination between vectors and animals and also is one of the main mechanisms of parasite dispersion among mammals [11,18]. Despite that, only after 2004 foodborne outbreaks of Chagas disease became an event most frequently discussed and investigated [63]. Originally sporadic reported, orally acquired Chagas disease seem to be increasing among populations in several Brazilian states [17][18][19] and in other endemic Latin American countries as well [19,24,62,64].…”

Section: Discussionmentioning

confidence: 99%

“…However, in the Northern region of Brazil, the oral transmission poses significant importance, mainly due to the daily diet based on the consumption of food in natura, which means unpasteurized homemade or artisan fresh food [19,22]. Regarding the food ingested, açai, which is macerated in order to produce a paste or drink, has been identified as the most frequent food involved in cases of orally acquired acute Chagas disease in Brazil [22,63,65].…”

Section: Discussionmentioning

confidence: 99%

“…Real Time qPCR, infection rates in complex food matrices may be underestimated since the chemical characteristics of açai may lead to a reduction of the PCR reaction due to the presence of contaminants and inhibitors[63]. The use of negative and positive controls is essential to ensure the reliability of the reaction, avoiding false-positive or false-negative results.…”

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confidence: 99%

“…Presently, there are several methods available for DNA extraction from different sorts of samples and they can be based on commercial kits or can be in-house methods. Ferreira et al[63] compared the quantity and quality of T. cruzi DNA isolated from artificially contaminated açai samples, whereby different DNA extraction methods were applied. In the study, the selected commercial kit proved not to be adequate for T. cruzi detection in food samples while one same performance and, consequently, PCR was always negative even though acai samples were contaminated.As a first attempt to increase the sensitivity of the methodology in this study,…”

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confidence: 99%

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Validation of a novel multiplex real-time PCR assay forTrypanosoma cruzidetection and quantification in açai pulp

Finamore-Araujo

Faier-Pereira

Brito

et al. 2020

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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 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 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.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, açai 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 açai-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 açai 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 açai 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.

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