Comparative antifungal susceptibility analyses of Cryptococcus neoformans VNI and Cryptococcus gattii VGII from the Brazilian Amazon Region by the Etest, Vitek 2, and the Clinical and Laboratory Standards Institute broth microdilution methods.

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.

Detection and genotyping of Trypanosoma cruzi from açai products commercialized in Rio de Janeiro and Pará, Brazil.

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.

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.

Avaliação de um Sistema de Banco de Dados para Coleção de Fungos de Referência / Evaluation of a Database System for Reference Fungi Collection

De acordo com o Guia para Operação de Centros de Recursos Biológicos da Organização para Cooperação e Desenvolvimento Econômico (OCDE), os Centros de Recursos Biológicos devem dispor os dados descritivos do e sua origem ao Global Biological Resource Center Network. Essa deliberação ratifica a preocupação da validação de ferramentas utilizadas no tratamento de dados pelas coleções e laboratórios prestadores de serviços no escopo indireto de suas rotinas, visando à qualidade. A avaliação do Sistema de Banco de Dados da Coleção de Culturas de Fungos de Referência (INFOGER_FUNGOS), do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), de acordo com a norma ISO/IEC nº 17.025:2005, estabelece um marco na qualidade e integridade das informações, economizando tempo na elaboração das estruturas do sistema permitindo ao profissional diretamente envolvido com a coleção e suas particularidades seja seu administrador. Este trabalho teve como objetivo demonstrar um método de avaliação de um sistema de gerenciamento de dados no cumprimento de sua finalidade atingindo níveis de qualidade satisfatórios. Ele servirá de modelo para avaliações de sistemas utilizados em coleções de micro-organismos dentro das normas da Qualidade e Acreditação de seus serviços e produtos, e colaborar no estabelecimento de padrão de dados baseado em experiências brasileiras, adaptado e estendido a modelos existentes.

Desenvolvimento de material de referência para ensaio de proficiência em microbiologia de alimentos / Development of reference materials for proficiency tests in food microbiology

Ensaios de proficiência (EP) são considerados importantes ferramentas para a condução de programas controle de qualidade, que possibilitam efetuar a avaliação da habilidade de laboratórios em obter resultados precisos. No Brasil é restrita a oferta de EP na área de microbiologia de alimentos. Além disso, os altos custos diminuem a participação regular de laboratórios nacionais nesses ensaios. O presente estudo teve como objetivo realizar a avaliação da técnica de liofilização no preparo de materiais de referência (MR) para EP destinados ao ensaio de detecção de Salmonella spp. em leite. Foi determinada a concentração do inóculo utilizado no preparo de dois lotes e, também, foi padronizado o procedimento de contaminação da matriz. Para monitorar a qualidade dos MR produzidos foram estabelecidos ensaios de controle, dentre eles o teste da homogeneidade e da estabilidade em longo e curto prazo. A técnica de liofilização mostrou ser adequada para a produção de MR de qualidade e aplicável para EP. Os MR apresentaram estabilidade à temperatura de estoque (-20ºC) durante os três primeiros meses, porém foi observada perda de viabilidade após doze meses de armazenamento. Na estabilidade em curto prazo, os MR foram estáveis a 4°C, contudo apresentaram redução significativa no número de células quando mantidos a temperaturas de 25ºC e 37ºC durante sete dias.

Proficiency tests are considered relevant tools of quality control programs used to monitor the laboratory performance and to assess the reliability of tests results. In Brazil, the provision of proficiency testing (PT) is restricted to food microbiology area. Furthermore, high costs reduce the regular participationof national laboratories in these tests. This study evaluated the freeze-drying technique employed forpreparing reference materials (RM) to be used in the PT schemes for detecting Salmonella spp. in milk.The concentration of inoculums used for preparing two batches of RM was determined, and the proceduresfor matrix contamination were standardized. The control trials were established in order to monitor thequality of the produced RM, and among them, there are long and short-term homogeneity and stability assays. The freeze-drying technique proved to be a suitable procedure for producing RM for PT. The RM were stable for the first three months when stored at -20ºC, nevertheless, viability loss could be observed after 12-month storage. The RM were stable at 4°C at short-term stability, although when the products were kept at 25°C and 37°C for seven days, a significant reduction in the number of cells could be observed.