"Bunch of acai berries sign": a new radiological sign in patients with CNS involvement in Chagas disease.

Chagas disease is an infection caused by Trypanosoma cruzi, a parasite endemic in Latin America. Acute involvement of the CNS by Chagas has been considered rare, but presumed reactivation of chronic disease in immunosuppressed patients has been the subject of recent reports. Our objective is to describe the clinical and imaging characteristics of four patients with Chagas disease and CNS involvement, and the patients had to have available MRI and a diagnosis confirmed by biopsy. The imaging findings were similar, highlighting the presence of focal cerebral lesions with hypointensity on T2-WI, and these lesions assume a "bunch of acai berries appearance", a fruit involved in the transmission of T. cruzi. The post Gd T1-WI shows punctate enhancement. Knowledge of this pattern may be crucial to recognize this disease in immunocompromised patients from endemic areas.

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

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.

SYBR Green qPCR Technique for the Detection of Trypanosoma cruzi in Açaí Pulp.

Chagas disease, which is found widely in Latin America and has a great impact on public health, is caused by the parasite Trypanosoma cruzi. It is a neglected parasitic disease that urgently requires rapid diagnostic methods. The objective of this study was to develop a SYBR Green real-time quantitative polymerase chain reaction (qPCR) technique for the direct identification and quantification of T. cruzi from experimentally contaminated açai fruit samples. We used discrete typing units, TcI, containing 3.5 × 104 cells/mL, to infect the pulp of the açai fruit. This was followed by DNA extraction using a standardized procedure. The DNA samples were quantified and amplified at specific time and temperature intervals. The specificity of the oligoinitiators used in the qPCR assays was estimated by calculating the primer dissociation curve (melting curve) along with a detection threshold using different concentrations of DNA. The method used here demonstrated good efficiency and precision for the detection and quantification of T. cruzi DNA, with a detection limit of 2.65 × 10-14 g/µL DNA. The qPCR technique presented here could serve as an important tool for the diagnosis of T. cruzi parasites in açai.

Virulence of Trypanosoma cruzi from vector and reservoir in in natura açaí pulp resulting in food-borne acute Chagas disease at Pará State, Brazil.

BACKGROUND: In recent decades some outbreaks of food-borne acute Chagas disease (ACD) in humans were identified by clinical and epidemiological characterization after association through the ingestion of açaí pulp probably contaminated with Trypanosoma cruzi. Whereas Belém and Abaetetuba stood out as important risk regions for disease transmission, the importance of Rhodnius pictipes, and Philander opossum for the biological cycle of T. cruzi, and data from agribusiness market of açaí, to study T. cruzi from vector and reservoir of the Brazilian Amazon region is critical for this context. Thus, the purpose of this study was to verify the infective capacity and the virulence of T. cruzi in açaí pulp from vector and reservoir at Pará State experimentally. METHODS: 105T. cruzi I in in natura açaí pulp from Belém at Pará State, at room temperature, after forced sieving, by intraperitoneal, gavage or oral route of inoculation in B6.129S7Rag1-/-tmMom/J Unib allowed food-borne ACD analysis using common light microscopy. PRINCIPAL FINDINGS: T. cruzi in in natura açaí pulp from R. pictipes (Val-De-Cans Forest, Belém, and Ajuaí River, Abaetetuba, Pará), and P. opossum (Combu Island, Belém, Pará) caused ACD and death between 17 and 52 days after experimental infections in murine immunodeficient hosts. CONCLUSIONS: T. cruzi from different sources and locations at Pará State in in natura açaí pulp retained its infective capacity and virulence, and can cause new outbreaks of ACD by oral transmission. Additionally, quality basic education will facilitate efficient hygiene practices throughout the açaí productive chain can eradicate food-borne ACD in the coming decades.

Molecular detection of Trypanosoma cruzi in acai pulp and sugarcane juice.

Chagas disease, caused by Trypanosoma cruzi affects about 6-8 million people worldwide. Although transmission by triatomine insects has been controlled, other means of transmission maintain the infection. These forms of transmission are responsible for introducing Chagas disease in other non-endemic countries of the world. Thus, Chagas disease, nowadays is a worldwide health problem. In Brazil, acai pulp and sugarcane juice have been associated with Chagas disease outbreaks. The difficulties in isolation of the parasite from foods are hampering source tracking which could allow the confirmation of an implicated food commodity in these outbreak investigations. To address this scientific gap, we evaluated the performance of real-time PCR (qPCR) for detecting T. cruzi in acai pulp and sugarcane juice. All experiments were performed with acai pulp and sugarcane juice samples contaminated with different concentrations of T. cruzi. In assays with qPCR, the results showed that the ideal procedure for T. cruzi identification in acai pulp and sugarcane juice consisted of: i. centrifugation; ii. DNA extraction with a commercial kit for stool matrix; and iii. qPCR using a specific molecular marker for T. cruzi. The seeding in LIT medium of experimentally contaminated foods was effective in detecting the parasitic load by qPCR. The efficacy of qPCR was also verified testing food samples crushed with infected Triatomines. In conclusion, this methodology can be used to perform rapid diagnosis in outbreaks, facilitating measures in disease control.

qPCR for the detection of foodborne Trypanosoma cruzi.

Here we presented a potential real-time PCR (qPCR) method with public health importance and relevance for detection of Trypanosoma cruzi in açai pulp. There is not a current process to identify T. cruzi in açai, that ensures innocuity of this food concerning oral transmission. First, six new primers were designed using the DNA sequences of T. cruzi y152 and Emerald strains obtained from GenBank. For primers evaluation and titration they were validated regarding the amplification and not with the fluorophore chosen 1ngµL-1 of the T. cruzi DNA as target. For determination of the ideal concentration the titration of the primers drawn in this study showed T. cruzi DNA amplification in five primer pairs at concentrations 100, 200 and 300nM and DNA fixed concentrations at 1ngµL-1. For standardization all reactions were performed in triplicate with 5.0µL and positives and negatives controls were included in every run. As positive control DNA from two genotypes TcI and TcII were used. As negative control the reaction product without DNA of the parasite was used. The best primer concentration, for the expected fragments, was 300nM. From six primers improved the Ep1F/Ep1R primer detected 1×10-4ngµL-1 for both genotype of the parasite. The Bp1F/Bp1R showed amplification for 1.70.10-7ngµL-1 for TcI and 4.31.10-8ngµL-1 for TcII, based on the standard curve. The last step we tested the selected primers in qPCR for monitoring T. cruzi in açai pulp experimentally contaminated. The recovery rate for the TcII was 71%, whereas in açai samples contaminated with TcI it was 76%.

Condições higienicossanitárias da cadeia produtiva do açaí na região do Maracanã, São Luís, MA / Hygienic and sanitary conditions of the açaí productive chain in the region of Maracana, Sao Luis, MA

Objetivou-se, neste estudo, avaliar as condições higienicossanitárias da cadeia produtiva do açaí desde a colheita até o processamento da polpa realizada na região do Maracanã, São Luís-MA enfatizando os processos de colheita do fruto, debulha, armazenamento, transporte e extração da polpa para comercialização. Concomitantemente, buscou-se avaliar se os produtores envolvidos tinham conhecimento sobre os agentes etiológicos de doenças alimentares, em especial da Doença de Chagas, cujo agente é transmitido por via oral pelo consumo do açaí. Para esta avaliação foi elaborado um questionário com 30 perguntas, baseado no Manual de Práticas de Colheita e Manuseio do Açaí proposto pela Embrapa (2006) e a aplicação se deu por meio de entrevistas pessoais. Os resultados com relação às medidas de higiene que devem ser adotadas durante a preparação da polpa de açaí, foram classificados como satisfatórios na sua maioria, conforme o manual, entretanto, os conhecimentos sobre a transmissão da doença de Chagas pelo açaí, foram escassos. Deste modo, é fundamental que atividades de conscientização devem ser desenvolvidas na comunidade para corroborar na prevenção desta e de outras doenças.

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

INTRODUCTION: Before 2004, the occurrence of acute Chagas disease (ACD) by oral transmission associated with food was scarcely known or investigated. Originally sporadic and circumstantial, ACD occurrences have now become frequent in the Amazon region, with recently related outbreaks spreading to several Brazilian states. These cases are associated with the consumption of açai juice by waste reservoir animals or insect vectors infected with Trypanosoma cruzi in endemic areas. Although guidelines for processing the fruit to minimize contamination through microorganisms and parasites exist, açai-based products must be assessed for quality, for which the demand for appropriate methodologies must be met. METHODS: Dilutions ranging from 5 to 1,000 T. cruzi CL Brener cells were mixed with 2mL of acai juice. Four Extraction of T. cruzi DNA methods were used on the fruit, and the cetyltrimethyl ammonium bromide (CTAB) method was selected according to JRC, 2005. RESULTS: DNA extraction by the CTAB method yielded satisfactory results with regard to purity and concentration for use in PCR. Overall, the methods employed proved that not only extraction efficiency but also high sensitivity in amplification was important. CONCLUSIONS: The method for T. cruzi detection in food is a powerful tool in the epidemiological investigation of outbreaks as it turns epidemiological evidence into supporting data that serve to confirm T. cruzi infection in the foods. It also facilitates food quality control and assessment of good manufacturing practices involving acai-based products.

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

Abstract: INTRODUCTION: Before 2004, the occurrence of acute Chagas disease (ACD) by oral transmission associated with food was scarcely known or investigated. Originally sporadic and circumstantial, ACD occurrences have now become frequent in the Amazon region, with recently related outbreaks spreading to several Brazilian states. These cases are associated with the consumption of açai juice by waste reservoir animals or insect vectors infected with Trypanosoma cruzi in endemic areas. Although guidelines for processing the fruit to minimize contamination through microorganisms and parasites exist, açai-based products must be assessed for quality, for which the demand for appropriate methodologies must be met. METHODS: Dilutions ranging from 5 to 1,000 T. cruzi CL Brener cells were mixed with 2mL of acai juice. Four Extraction of T. cruzi DNA methods were used on the fruit, and the cetyltrimethyl ammonium bromide (CTAB) method was selected according to JRC, 2005. RESULTS: DNA extraction by the CTAB method yielded satisfactory results with regard to purity and concentration for use in PCR. Overall, the methods employed proved that not only extraction efficiency but also high sensitivity in amplification was important. CONCLUSIONS: The method for T. cruzi detection in food is a powerful tool in the epidemiological investigation of outbreaks as it turns epidemiological evidence into supporting data that serve to confirm T. cruzi infection in the foods. It also facilitates food quality control and assessment of good manufacturing practices involving acai-based products.

Distantiae transmission of Trypanosoma cruzi: a new epidemiological feature of acute Chagas disease in Brazil.

BACKGROUND: The new epidemiological scenario of orally transmitted Chagas disease that has emerged in Brazil, and mainly in the Amazon region, needs to be addressed with a new and systematic focus. Belém, the capital of Pará state, reports the highest number of acute Chagas disease (ACD) cases associated with the consumption of açaí juice. METHODOLOGY/PRINCIPAL FINDINGS: The wild and domestic enzootic transmission cycles of Trypanosoma cruzi were evaluated in the two locations (Jurunas and Val-de Cães) that report the majority of the autochthonous cases of ACD in Belém city. Moreover, we evaluated the enzootic cycle on the three islands that provide most of the açaí fruit that is consumed in these localities. We employed parasitological and serological tests throughout to evaluate infectivity competence and exposure to T. cruzi. In Val-de-Cães, no wild mammal presented positive parasitological tests, and 56% seroprevalence was observed, with low serological titers. Three of 14 triatomines were found to be infected (TcI). This unexpected epidemiological picture does not explain the high number of autochthonous ACD cases. In Jurunas, the cases of ACD could not be autochthonous because of the absence of any enzootic cycle of T. cruzi. In contrast, in the 3 island areas from which the açaí fruit originates, 66.7% of wild mammals and two dogs displayed positive hemocultures, and 15.6% of triatomines were found to be infected by T. cruzi. Genotyping by mini-exon gene and PCR-RFLP (1f8/Akw21I) targeting revealed that the mammals and triatomines from the islands harbored TcI and Trypanosoma rangeli in single and mixed infections. CONCLUSION/SIGNIFICANCE: These findings show that cases of Chagas disease in the urban area of Belém may be derived from infected triatomines coming together with the açaí fruits from distant islands. We term this new epidemiological feature of Chagas disease as "Distantiae transmission".