Molecular typing of Mycobacterium bovis isolated in the south of Brazil

Bovine tuberculosis is a major infectious disease of the cattle. In this study, 85 M. bovis isolates from 162 lymph nodes, obtained from a herd of cattle on a farm in southern Brazil, were evaluated using spoligotyping and VNTR. The strains were grouped into five clusters and five orphans, showing a heterogenic genetic profile, what could represent diverse geographic origins of the introduced cows and/or the frequent movement of cattle between different properties.

Mycobacterium tuberculosis belonging to family LAM and sublineage RD Rio: common strains in Southern Brazil for over 10 years

A sublineage of Mycobacterium tuberculosis called RD Rio was described in 2007. Although only recently described, this strain may have been present previously in the population, and its identification in clinical isolates will elucidate bacterial transmission dynamics and host-pathogen interactions. This study evaluated the clonal diversity of the RD Rio sublineage in clinical isolates from Rio Grande-RS obtained between 1998 and 2001. Among the 45 samples analyzed by the MIRU-VNTR method, there were six clusters with two samples each and 33 orphan strains with unique pattern. The strains were distributed across several different lineages including LAM (34.04%), X (14.89%), Haarlem (12.77%), UgandaI (10.64%), S (4.26%), NEW-1 (2.13%) and Cameroon (2.13%); 14.89% of the strains matched to multiple lineages. RD Rio strains were present in 28.9% of the samples and 81.25% of the identified strains belonged to the LAM family. The high clonal diversity observed in this study is a constant feature in this region. The RD Rio sublineage has been in Rio Grande-RS since 1998. The continued monitoring of RD Rio in clinical isolates will enhance the understanding of its epidemiological significance.

Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

Growth kinetics of Mycobacterium tuberculosis measured by quantitative resazurin reduction assay: a tool for fitness studies

We standardized a method to evaluate the growth kinetics of Mycobacterium tuberculosis by measuring quantitatively the reduction of resazurin by spectrophotometry. Growth curves and the rate of growth of twenty-one M. tuberculosis clinical isolates were determined. The method showed technical simplicity and is inexpensive to assess the fitness of each isolate.

Comparative evaluation of the Nitrate Reductase Assay and the Resazurin Microtitre Assay for drug susceptibility testing of Mycobacterium tuberculosis against first line anti-tuberculosis drugs

Tuberculosis remains as a serious infection disease of worldwide distribution, with high morbidity and mortality, mainly in low socio-economic condition countries. The state of emergency of tuberculosis caused by the resistant and multidrug-resistant (MDR) strains, became the main threat to the tuberculosis treatment and control programs. A fast detection method for the resistant strains will allow the implementation of an adequate treatment and contribute for controlling the dissemination of these resistant strains. This study evaluated the performance of the nitrate reductase assay in solid (NRA-LJ) and liquid (NRA-7H9) media, to determine the susceptibility to first line anti-tuberculosis drugs: isoniazid (INH), rifampicin (RMP), ethambutol (EMB) and streptomycin (SMR). Both methods NRA-LJ and NRA-7H9 were evaluated among 18 strains with a known susceptibility profile. The resazurin microtiter assay (REMA) was performed as a reference method. One hundred percent of accordance was observed between NRA-7H9 and REMA for the four tested drugs. When the NRA-LJ method was compared to REMA, the sensitivity and the specificity to INH, RMP, EMB and SMR were 100%, 100 %, 85.7%, 76.9% and 80%, 100%, 75% and 80%, respectively. From the 57 clinical isolates of M. tuberculosis evaluated by NRA-7H9 and REMA, 56 (98.2%) were sensitive to all antibiotics tested (INH, RMP, EMB and SMR) by the NRA-7H9 method, while three of these strains were resistant to INH by REMA. One strain showed resistance to INH and RMP for both methods, and MIC of 1.0 µg/ml to INH for both methods, while MIC of 1.0 and 2.0 µg/ml to RMP for REMA and NRA-7H9, respectively. The three assays showed a high level of agreement for rapid detection of rifampicin and isoniazid resistance. Regarding rapidness, the detection of color change in the NRA method is within instants as compared to the overnight incubation required for the REMA test. NRA might represent an inexpensive and alternative assay for rapid detection of resistance in low-income countries.

A tuberculose permanece como uma séria doença infecciosa, com distribuição mundial, alta morbidade e mortalidade, ocorrendo principalmente em paises com baixa condição econômica. O estado de emergência da tuberculose causada por cepas resistentes e multirresistentes tornou-se uma importante ameaça para o tratamento e programas de controle da tuberculose. Uma rápida detecção de cepas resistentes permitirá a implantação de um tratamento adequado e contribuirá para controlar a disseminação destas cepas. Este estudo avaliou a performace do ensaio nitrato redutase em meio sólido (NRA-LJ) e meio líquido (NRA-7H9), para determinar a sensibilidade frente aos fármacos antituberculosos de primeira linha: isoniazida (INH), rifampicina (RMP), etambutol (EMB) and estreptomicina (SMR). Ambos os métodos, NRA-LJ e NRA-7H9, foram avaliados com 18 cepas com conhecido perfil de sensibilidade. O ensaio de microplaca com resazurina (REMA) foi utilizado como método de referência. A concordância observada entre NRA-7H9 and REMA foi de 100% para os quatro fármacos testados. Quando o método NRA-LJ foi comparado com o REMA, a sensibilidade e especificidade para INH RMP e SMR foram de 100%, 100%, 85,7%, 76,9% e 80%, 100%, 75% and 80%, respectivamente. Dos 57 isolados clinicos de M. tuberculosis avaliados por NRA-7H9 e REMA, 56 (98.2%) foram sensíveis a todos antibióticos testados (INH, RMP, EMB e SMR) pelo método NRA-7H9, enquanto três destas cepas foram resistentes para INH pelo REMA. Uma cepa mostrou resistência para INH e RMP por ambos os métodos, e CMI de 1,0 µg/ml para INH para ambos os métodos, enquanto CMI de 1,0 e 2,0 µg/ml para RMP pelo REMA e NRA-7H9, respectivamente. Os três ensaios mostraram um alto nível ded concordância para uma rápida detecção de resistência a rifampicina e isoniazida. Com relação à rapidez na obtenção dos resultados, a detecção na mudança de cor nos métodos NRA é imediata enquanto para o método REMA é necessário incubar overnight. Os métodos NRA podem representar uma alternativa, de baixo custo e rápida detecção de resistência, em países com poucos recursos.

Microcolony detection in thin layer culture as an alternative method for rapid detection of Mycobacterium tuberculosis in clinical samples

Tuberculosis remains one of the major causes of mortality worldwide. The early detection of new cases is an important goal in the program of tuberculosis control. Several methodologies for rapid and accurate laboratorial diagnosis have been developed, however, some of these techniques are expensive or cumbersome, making their implementation in low-income regions unfeasible. In this study, the thin layer culture method was compared with conventional culture method and it was observed that it provides earlier results and a presumptive species identification, being adequate alternative method for rapid laboratory diagnosis.

A tuberculose permanece como uma das principais causas de mortalidade em todo o mundo. A detecção precoce dos casos novos é um importante ponto nos programas de controle da tuberculose. Diversas metodologias para um rápido e acurado diagnóstico laboratorial tem sido desenvolvidas, entretanto, algumas destas técnicas são de alto custo ou de difícil execução, tornando inexeqüível sua implementação em locais com poucos recursos. Neste trabalho o método de cultivo em camada delgada foi comparado ao método de cultivo convencional verificando-se apresenta resultados positivos em menor tempo com uma identificação presuntiva da espécie de micobactéria, sendo assim um método alternativo adequado para o diagnóstico laboratorial rápido.