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Early diagnosis, efficient clinical support, and proper antifungal therapy are essential to reduce death and sequels caused by cryptococcosis. The emergence of resistance to the antifungal drugs commonly used for cryptococcosis treatment is an important issue of concern. Thus, the in vitro antifungal susceptibility of clinical strains from northern Brazil, including C. neoformans VNI (n = 62) and C. gattii VGII (n = 37), to amphotericin B (AMB), 5-flucytosine, fluconazole, voriconazole, and itraconazole was evaluated using the Etest and Vitek 2 systems and the standardized broth microdilution (CLSI-BMD) methodology. According to the CLSI-BMD, the most active in vitro azole was voriconazole (C. neoformans VNI modal MIC of 0.06 μg/ml and C. gattii VGII modal MIC of 0.25 μg/ml), and fluconazole was the least active (modal MIC of 4 μg/ml for both fungi). Modal MICs for amphotericin B were 1 μg/ml for both fungi. In general, good essential agreement (EA) values were observed between the methods. However, AMB presented the lowest EA between CLSI-BMD and Etest for C. neoformans VNI and C. gattii VGII (1.6% and 2.56%, respectively, P < .05 for both). Considering the proposed Cryptococcus spp. epidemiological cutoff values, more than 97% of the studied isolates were categorized as wild-type for the azoles. However, the high frequency of C. neoformans VNI isolates in the population described here that displayed non-wild-type susceptibility to AMB is noteworthy. Epidemiological surveillance of the antifungal resistance of cryptococcal strains is relevant due to the potential burden and the high lethality of cryptococcal meningitis in the Amazon region.

Background Several cases of food-borne acute Chagas disease (ACD) have been reported in the Brazilian Amazon so far. Up to 2004, the occurrence of ACD by oral transmission, associated with food consumption, was rare. Recent cases of ACD in Brazil have been attributed to the consumption of juice from the açai palm containing reservoir animals or insect vectors waste, infected with Trypanosoma cruzi. This study aimed to determine the T. cruzi contamination rate and to genotype the parasite in food samples prepared from açai, which are commercialized in Rio de Janeiro and the Pará States in Brazil. Methods The amplificability of DNA extracted from açai samples, and T. cruzi and Triatominae detection were performed by conventional PCR. Molecular characterization was done by multilocus PCR analysis, to determine the parasite discrete type units (DTUs) based on the size of PCR products in agarose gels, using the intergenic region of the spliced leader (SL), 24 Sα rDNA and nuclear fragment A10 as targets. Results From the 140 samples of açai-based products analyzed, T. cruzi DNA was detected in 14 samples (10%); triatomine DNA was detected in one of these 14 samples. The parasite genotyping demonstrated that food samples containing açai showed a mixture of T. cruzi DTUs with TcIII, TcV and TcI prevailing. Conclusions In this study, the molecular detection and identification of T. cruzi from açai-based manufactured food samples, was performed for the first time. Although parasite DNA is a marker of possible contamination during food manufacturing, our findings do not indicate that açai is a source of Chagas disease via oral transmission per se, as live parasites were not investigated. Nevertheless, a molecular approach could be a powerful tool in the epidemiological investigation of outbreaks, supporting previous evidence that açai-based food can be contaminated with T. cruzi. Furthermore, both food quality control and assessment of good manufacturing practices involving açai-based products can be improved, assuring the safety of açai products.

O desenvolvimento de metodologia altamente discriminatória para a identificação e caracterização de genótipos das espécies de plantas medicinais regulamentadas pelo sistema público de saúde brasileiro (SUS) é de suma importância para o controle de qualidade destas espécies como matérias-primas na produção de medicamentos fitoterápicos, consequentemente, minimizar o risco sanitário associado à ineficácia terapêutica devido ao uso de matéria prima de identidade duvidosa. Por isto, foi utilizado o método RAPD-PCR para a elaboração de um perfil genético de três espécies de plantas medicinais regulamentadas pelo SUS do Brasil: Mikania glomerata, Maytenus ilicifolia e Schinus terebinthifolius, a partir de exemplares destas plantas, que foram cedidas pela Coleção Temática de Plantas Medicinais do Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro. Os 60 iniciadores utilizados no RAPD-PCR com o DNA das três espécies geraram 1284 produtos amplificados que variaram de 100-1500 pb. Foram selecionados cinco iniciadores que geraram no total 76 fragmentos entre 200-1100 pb com astrês espécies, sendo os iniciadores OPG18, OPA7 e OPG17 para a Mikania glomerata, os iniciadores OPG20, OPC13 e OPA11 para a Maytenus ilicifolia e OPA4, OPA18 e OPG14 para a Schinus terebinthifolius e os iniciadores OPA17 e OPC6 para as três espécies. Os perfis resultantes permitiram a identificação eficiente das espécies. Foram identificados iniciadores que geraram um único fragmento que poderão servir para desenhar um iniciador específico, que poderá ser usado na identificação da planta em produtos como monofarmacos e associações. ----------------------------------------------------------------------------------------------- Development of a highly discriminatory method for the identification of genotypes and species of medicinal plants regulated by the Brazilian public health system (SUS) is of paramount importance for the quality control of these species as raw material in the production of herbal medicines in order to minimize the health risks associated with ineffective therapy using raw materials of uncertain identity. We used RAPD-PCR to generate genetic profiles of three medicinal plant species regulated by SUS in Brazil, Mikania glomerata, Maytenus ilicifolia, and Schinus terebinthifolius, from the specimens of these plants donated by the Theme Collection of Medicinal Plants of the Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro. The 60 arbitrary primers used for RAPD-PCR with DNA from the three species generated 1284 amplified products ranging from 100 to 1500 bp. Five primers generating a total of 76 fragments between 200 and 1100 bp from the three species were selected: OPG18, OPA7, and OPG17 for Mikania glomerata; OPG20, OPC13, and OPA11 for Maytenus ilicifolia; OPA4, OPA18, and OPG14 for Schinus terebinthifolius; and OPA17 and OPC6 for the three species. The profiles generated allowed the effective discrimination of the species. Primers were identified that generated a single fragment that can serve to determine a specific marker for use in the identification of plant products either in isolation or in association.

The Brazilian government has approved many transgenic maize lines for commercialization and has established a threshold of 1% for food labeling, which underscores need for monitoring programs. Thirty four samples including flours and different types of nacho chips were analyzed by conventional and real-time PCR in 2011 and 2012. The events MON810, Bt11, and TC1507 were detected in most of the samples, and NK603 was present only in the samples analyzed in 2012. The authorized lines GA21, T25, and the unauthorized Bt176 were not detected. All positive samples in the qualitative tests collected in 2011 showed a transgenic content higher than 1%, and none of them was correctly labeled. Regarding the samples collected in 2012, all positive samples were quantified higher than the threshold, and 47.0% were not correctly labeled. The overall results indicated that the major genetically modified organisms detected were MON810, TC1507, Bt11, and NK603 events. Some industries that had failed to label their products in 2011 started labeling them in 2012, demonstrating compliance with the current legislation observing the consumer rights. Although these results are encouraging, it has been clearly demonstrated the need for continuous monitoring programs to ensure consumers that food products are labeled properly.

It is widely accepted that quantitative reference materials (RM), are indispensable tools for verification of the precision and accuracy of analytical measurements. The RM can be used by food microbiology laboratories, as part of their quality assurance programmes, to achieve their quality control. In Brazil, Anvisa RDC No. 12/01 specifies the enumeration of coliforms as one of the parameters for evaluating cheese quality. The aim of this study was to produce a quantitative RM for proficiency testing (PT) for use in the testing of enumeration of coliforms in cheese matrixes. A sample of an ultra-filtered cheese with a coliforms count of

The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R²) demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct) values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target.A extração de DNA é uma etapa crítica na análise de organismos geneticamente modificados em alimentos por PCR em tempo real. Neste trabalho, os métodos CTAB e DNeasy forneceram preparações de DNA em quantidade e com qualidade a partir de amostras de proteína texturizada de soja, fórmula infantil e extrato de soja. Em relação ao Material de Referência Certificado contendo 5% de soja RoundupReady® , nenhum dos métodos forneceu DNA de boa qualidade. Entretanto, o teste de diluição realizado nos extratos de CTAB não demonstrou interferência de substâncias inibidoras. As eficiências das amplificações do alvo lectin não foram estatisticamente diferentes e os coeficientes de correlação (R²) demonstraram alto grau de correlação entre o número de cópias e os valores de Ct. A ANOVA demonstrou ajuste das curvas de regressão e ausência de desvios significativos. As eficiências das amplificações do alvo p35S, utilizando extratos DNeasy, não foram estatisticamente diferentes e os valores de R² ficaram acima de 0,98, sem desvios de linearidade. Dois valores de R², utilizando extratos CTAB, foram menores que 0,98, correspondendo a menor grau de correlação edesvio de linearidade em uma corrida. A análise comparativa dos Cts dos alvos p35S e lectin não demonstrou diferenças significativas entre as curvas analíticas da amplificação de cada alvo.

The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R²) demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct) values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target. A extração de DNA é uma etapa crítica na análise de organismos geneticamente modificados em alimentos por PCR em tempo real. Neste trabalho, os métodos CTAB e DNeasy forneceram preparações de DNA em quantidade e com qualidade a partir de amostras de proteína texturizada de soja, fórmula infantil e extrato de soja. Em relação ao Material de Referência Certificado contendo 5% de soja RoundupReady® , nenhum dos métodos forneceu DNA de boa qualidade. Entretanto, o teste de diluição realizado nos extratos de CTAB não demonstrou interferência de substâncias inibidoras. As eficiências das amplificações do alvo lectin não foram estatisticamente diferentes e os coeficientes de correlação (R²) demonstraram alto grau de correlação entre o número de cópias e os valores de Ct. A ANOVA demonstrou ajuste das curvas de regressão e ausência de desvios significativos. As eficiências das amplificações do alvo p35S, utilizando extratos DNeasy, não foram estatisticamente diferentes e os valores de R² ficaram acima de 0,98, sem desvios de linearidade. Dois valores de R², utilizando extratos CTAB, foram menores que 0,98, correspondendo a menor grau de correlação edesvio de linearidade em uma corrida. A análise comparativa dos Cts dos alvos p35S e lectin não demonstrou diferenças significativas entre as curvas analíticas da amplificação de cada alvo.

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.

Analysis of food products consisting of, or produced from, genetically modified organisms (GMOs) is required to verify compliance with labeling legislation and to detect any possible unauthorized transgenic crops. With these goals, 240 samples of soy-derived foodstuffs and 25 samples of maize-derived foodstuffs were analyzed from 2004 to 2007. All samples positive for Roundup Ready® soybean were quantitatively examined using the TaqMan® GMO 35S Soy kit. In food containing soy, 68 (28.3%) were shown to contain GM soy, whereas in food containing maize, neither Bt 176 nor MON 810 maize were found. Quantitative analysis revealed GMO contents ranging from 0.05 to 1% in 43 (63.2%) samples, and more than 1% in 25 (36.8%) samples. The absolute and relative limits of detection (LODs) were approximately 10 copies and 0.0125%, respectively, and the absolute and relative limits of quantification (LOQs) were approximately 40 copies and 0.05%, respectively, suggesting sufficient sensitivity to quantify genetically modified (GM) materials below and above the legal threshold of 1%. The presence of GM material in these samples was not indicated on their labels, indicating that none of these food products had been appropriately labeled. These results clearly demonstrate the need for a monitoring program of food products by the Brazilian regulatory authorities.

Wheat flour is regarded as one of the main ingredients used in food preparation and is therefore present in virtually all Brazilian meals. In 2000, the per capita consumption of wheat flour corresponded to 53 kg and was distributed as follows on the Brazilian market: 47% bakeries, 14% pasta industries, 20% domestic consumption, 8% biscuit industries, 5% bread industries and 6% in other segments. The objective of this study was to evaluate the presence of soybeans and Roundup Ready® soybeans in wheat flour samples and wheat flour-based preparations for human consumption, and to verify the adequacy of the labels with respect to the legislation using Real Time PCR quantitative analysis. INCQS/FIOCRUZ analyzed 16 wheat flour samples of 10 different brands. Fifteen (15) samples showed positive results for the le1 lectin gene, showing the presence of soybeans in these products, and twelve (12) samples showed positive results for the CP4 EPSPS gene, specific for the RR® soybean, demonstrating the use of genetically modified soybean in these products. All the analyzed samples classified as wheat flour-based food preparations for human consumption, known commercially as cake, quiche, pancake and cookie mixes, tested positive for the RR® soybean bean, and should therefore include the symbol established by Regulation No 2658 of 22ndDecember, 2003 on the label.

The occurrence of acute Chagas disease (ACD) by oral transmission related to food consumption, until 2004, was a little known or investigated event. Currently becomes frequent in the Amazon region and is related to the occurrence of recent outbreaks in several states. Recent cases in Brazil of ACD are related to the consumption of acai juice, considered essencial food in the diet of the population in the Northern region. the acai berry has been appreciated both for its nutritional value, as by its characteristic flavor, ceasing to be consumed in the producing regions, extending throughout the brazilian territory and even exported to other countries. The aim of this study was to highlight the new challenges faced by public health agencies in preventing the transmission of Chagas disease by consumption of acai. Although there are important strategies being implemented by Brazil to combat Chagas disease transmitted via food, there is still a need for incentives to search for that knowledge generated assist in understanding the oral transmission of this parasite and its better interpretation of epidemiological, prevention and control. Besides the implementation of good Hygiene Practices, good Manufacturing Practices and the rapprochement between science institutions and producers of acai to contribute in solving this problem.

A NBR ISO/IEC 17025:2005, uma das normas mais utilizadas em laboratórios de ensaio, descreve como um de seus critérios para a garantia da qualidade dos resultados analíticos a participação periódica dos laboratórios em ensaios de proficiência (EP). Os analitos, utilizados nos EP são materiais de referência (MR) provenientes de um mesmo lote, e devem apresentar características de homogeneidade e estabilidade. O objetivo deste estudo foi produzir um MR qualitativo destinado ao ensaio de pesquisa de Listeria monocytogenes em matriz queijo pela técnica de liofilização. Para a produção do MR, foi utilizado como matriz o queijo Minas frescal (QMF) ultrafiltrado. A matriz foi distribuída em frascos, contaminada com a bactéria alvo e submetida à liofilização, tendo a sacarose como crioprotetor. O MR produzido foi considerado homogêneo e estável na temperatura de ≤-70°C durante todo o período estudado (10 meses). O material apresentou estabilidade a 4, 25, 30 e 35°C por quatro dias e a -20°C por 48 dias, e os resultados estatísticos indicam tendência à estabilidade. Conclui-se que o material apresentou todos os requisitos necessários de um MR de qualidade e poderia ser transportado aos laboratórios participantes de um EP à temperatura máxima de 35°C por até quatro dias, uma vez que os resultados indicaram a manutenção da concentração celular nesse período. Esse foi o primeiro trabalho a descrever uma metodologia de produção de MR contendo L. monocytogenes em matriz queijo. The standard most used in testing laboratories, ISO/IEC 17025:2005, describes the participation of laboratories in periodic proficiency testing (PT), as a criteria for quality assurance of analytical results. The analyses used in PT are reference materials (MR) from the same lot, and must have characteristics of homogeneity and stability. This study aimed to produce a qualitative RM for detection of Listeria monocytogenes assays in cheese matrix. For the production of RM, Minas Frescal cheese (MFQ) was used as matrix. The matrix was distributed in flasks, contaminated with the target bacteria and submitted to freeze-drying. Sucrose was used as cryoprotector. The RM produced was considered homogeneous and stable at -70°C during the entire period of study (10 months). The material showed stability at 4, 25, 30 and 35°C for 4 days and at -20°C the RM showed stability for 48 days, and the statistical results indicate a tendency to maintain stability. It was concluded that the material showed all the requirements of an RM quality and could be transported to the laboratory participants of a PT at 35°C up to 4 days, since the results indicate the maintenance of cell concentration during this period. This is the first study to describe a methodology for producing MR containing L. monocytogenes in cheese matrix.

O uso de materiais de referência (MR) é uma das principais ferramentas utilizadas para garantia e controle da qualidade de laboratórios de microbiologia de alimentos. No Brasil, a RDC n.º 12/01 da Anvisa prevê como um dos parâmetros para a avaliação da qualidade de queijos a enumeração de estafilococos coagulase positiva (ECP). O grande desafio na produção de MR destinados a ensaios microbiológicos é a instabilidade natural dos micro-organismos, o que dificulta o desenvolvimento e a manutenção desses MR. O objetivo deste estudo foi produzir um MR quantitativo destinado ao ensaio de enumeração de ECP em matriz queijo. Uma amostra de queijo ultrafiltrado com contagem de ECP

The enterococci are important nosocomial pathogens with a remarkable capacity of expressing resistance to several antimicrobial agents. Their ubiquitous nature and resistance to adverse environmental conditions take account for their ability to colonize different habitats and for their potential for easy spreading through the food chain. In the present study we evaluated the distribution of species and antimicrobial susceptibility among enterococcal isolates recovered from food obtained in retail stores in Rio de Janeiro, Brazil. The following species were identified among 167 isolates obtained from poultry meat and 127 from pasteurized milk: Enterococcus faecalis (62.6%), E. casseliflavus (17.3%), E. durans (6.5%), E. gallinarum (3.0%), E. gilvus (2.4%), E. faecium (2.0%), E. hirae (1.4%), and E. sulfureus (1.0%). The overall percentages of antimicrobial resistant isolates were: 31.2 % to tetracycline, 23.8% to erythromycin, 11.3% to streptomycin, 4.3% to chloramphenicol, 3.9% to gentamicin, 1.4% to norfloxacin, 1.1% to imipenem, 0.7% to ciprofloxacin, nitrofurantoin, and penicillin, and 0.4% to ampicillin. Intermediate resistance was detected in frequencies varying from 0.5% for linezolid to 58.2% for erythromycin. None of the isolates showed resistance to glycopeptides. High-level resistance to aminoglycosides was observed in 13.1% of the isolates. Multiresistance was observed in E. faecalis, E. casseliflavus, E. faecium, E. gallinarum, E. durans and E. gilvus.