Performance of a molecular assay to detect Mycobacterium tuberculosis complex DNA in clinical specimens: multicenter study in Brazil.

BACKGROUND: In high tuberculosis (TB) burden countries, there are few data on the performance of new molecular commercialised assays developed locally. OBJECTIVE: To evaluate the performance of a new molecular commercialised assay for TB diagnosis (Detect-TB) in three laboratories. METHODS: A total of 302 sputum samples from an equal number of patients with presumptive diagnosis of pulmonary tuberculosis (PTB) were submitted for routine smear microscopy, culture, and Detect-TB assay at three different sites in Brazil (the cities of Caxias do Sul, São Paulo and Canoas). FINDINGS: Seventy four (24.7%) TB cases were diagnosed (65 bacteriologically confirmed). When compared to smear microscopy/culture results, the overall sensitivity and specificity of Detect-TB assay was 84.6% (CI 95%; 73.7-91.6) and 93.1% (CI 95%; 89.1-95.8), respectively. When compared to bacteriological and clinical diagnostic criteria, the sensitivity and specificity of Detect-TB assay was 74.3% (CI 95%; 63.3-82.9) and 92.9% (CI 95%; 88.7-95.6), respectively. Among the three sites - Caxias do Sul, São Paulo and Canoas - the sensitivity and specificity were respectively 94.7% and 97.8%; 71.4% and 93.9%, 82.1% and 88.9%. MAIN CONCLUSIONS: These findings suggest that the Detect-TB assay could be applied routinely in reference laboratories across different regions in Brazil.

Performance of a molecular assay to detect Mycobacterium tuberculosis complex DNA in clinical specimens: multicenter study in Brazil

BACKGROUND In high tuberculosis (TB) burden countries, there are few data on the performance of new molecular commercialised assays developed locally. OBJECTIVE To evaluate the performance of a new molecular commercialised assay for TB diagnosis (Detect-TB) in three laboratories. METHODS A total of 302 sputum samples from an equal number of patients with presumptive diagnosis of pulmonary tuberculosis (PTB) were submitted for routine smear microscopy, culture, and Detect-TB assay at three different sites in Brazil (the cities of Caxias do Sul, São Paulo and Canoas). FINDINGS Seventy four (24.7%) TB cases were diagnosed (65 bacteriologically confirmed). When compared to smear microscopy/culture results, the overall sensitivity and specificity of Detect-TB assay was 84.6% (CI 95%; 73.7-91.6) and 93.1% (CI 95%; 89.1-95.8), respectively. When compared to bacteriological and clinical diagnostic criteria, the sensitivity and specificity of Detect-TB assay was 74.3% (CI 95%; 63.3-82.9) and 92.9% (CI 95%; 88.7-95.6), respectively. Among the three sites - Caxias do Sul, São Paulo and Canoas - the sensitivity and specificity were respectively 94.7% and 97.8%; 71.4% and 93.9%, 82.1% and 88.9%. MAIN CONCLUSIONS These findings suggest that the Detect-TB assay could be applied routinely in reference laboratories across different regions in Brazil.

Novas tecnologias para estudo da tuberculose: uma análise da detecção e transmissão de M. tuberculosis circulante / New technologies for the study of tuber culosis: an analysis of the detection and transmission of circulating M. tuberculosis

Além de identificar os doentes com tuberculose (TB), é importante monitorar os genótipos de M. tuberculosis circulantes. Mesmo após a implantação do Xpert® MTB/RIF, o diagnóstico é ainda realizado apenas pela baciloscopia, que apresenta baixa sensibilidade, na maioria dos laboratórios. OBJETIVO: Utilizar análises de DNA para o diagnóstico e identificação dos genótipos circulantes em uma população do Rio Grande do Sul, Brasil. METODOLOGIA: Amostras clínicas foram analisadas pelo Detect-TB (Labtest,MG), por PCR em tempo real (Xpert® MTB/RIF) e comparados a baciloscopia. A genotipagem foi realizada por spoligotyping. RESULTADOS: A acurácia do Detect-TB para a identificação da TB foi similarao Xpert® MTB/RIF, sendo que o Detect-TB foi mais custo-efetivo quando utilizado em conjunto com a baciloscopia. Os genótipos LAM5, RDRio e like-Pinni2, relacionados a resistência ao tratamento, estavam sendo transmitidos neste grupo, e a maioria dos resistentes a isoniazida (78,5%)e dos resistentes a rifampicina (92,1%) apresentavam as mutações mais conhecidas. CONCLUSÃO: A aplicação de tecnologias de DNA pode auxiliar no controle de TB, tanto no diagnóstico rápido quanto na identificação de perfis resistentes, viabilizando tratamento adequado aos pacientes.

In addition to detecting patients with tuberculosis (TB), it is important tomonitor circulating M. tuberculosis genotypes. Even after the implantation of Xpert® MTB/RIF, the diagnosis is still based only by bacilloscopy, which have a low sensitivity, in most laboratories. OBJECTIVE: To use DNA analysis for diagnosis and identification of circulating genotypes in a population of Rio Grande do Sul, Brazil. METHODOLOGY: Clinical samples were analyzed by Detect-TB (Labtest,MG), real-time PCR (Xpert® MTB/RIF) and compared to bacilloscopy. Genotyping was performed by spoligotyping. RESULTS: The accuracy of Detect-TB was similar to Xpert® MTB / RIF, butDetect-TB was more cost-effective when used with bacilloscopy. The genotypesLAM5, RDRio and like-Pinni2, related to treatment resistance, were being transmitted among this group, and the majority of the resistant to isoniazid (78.5%) and the resistant to rifampicin (92.1%) presented themost known mutations. CONCLUSION: The application of DNA technologies can help in the controlof TB, both in rapid diagnosis and in the identification of resistant profiles, allowing adequate treatment to the patients.

Sequence and structural characterization of tbnat gene in isoniazid-resistant Mycobacterium tuberculosis: identification of new mutations.

The present study was carried out to investigate the presence of polymorphism in the N-acetyltransferase gene of 41 clinical isolates of Mycobacterium tuberculosis, that were resistant to isoniazid (INH) with no mutations in the hot spots of the genes previously described to be involved in INH resistance (katG, inhA and ahpC). We observed single nucleotide polymorphisms (SNPs) in ten of these, including the G619A SNP in five isolates and an additional four so far un-described mutations in another five isolates. Among the latter SNPs, two were synonymous (C276T, n=1 and C375G, n=3), while two more non-synonymous SNPs were composed of C373A (Leu→Met) and T503G (Met→Arg) were observed in respectively one and two isolates. Molecular modeling and structural analysis based in a constructed full length 3D models of wild type TBNAT (TBNAT_H37Rv) and the isoforms (TBNAT_L125M and TBNAT_M168R) were also performed. The refined models show that, just as observed in human NATs, the carboxyl terminus extends deep within the folded enzyme, into close proximity to the buried catalytic triad. Analysis of tbnat that present non-synonymous mutations indicates that both substitutions are plausible to affect enzyme specificity or acetyl-CoA binding capacity. The results contribute to a better understanding of structure-function relationships of NATs. However, further investigation including INH-sensitive strains as a control group is needed to get better understanding of the possible role of these new mutations on tuberculosis control.