A descriptive study on isoniazid resistance-associated mutations, clustering and treatment outcomes of drug-resistant tuberculosis in a high burden country.

PURPOSE: To describe katG and inhA mutations, clinical characteristics, treatment outcomes and clustering of drug-resistant tuberculosis (TB) in the State of São Paulo, southeast Brazil. METHODS: Mycobacterium tuberculosis isolates from patients diagnosed with drug-resistant TB were screened for mutations in katG and inhA genes by line probe assay and Sanger sequencing, and typed by IS6110-restriction fragment-length polymorphism for clustering assessment. Clinical, epidemiological and demographic data were obtained from surveillance information systems for TB. RESULTS: Among the 298 isolates studied, 127 (42.6%) were isoniazid-monoresistant, 36 (12.1%) polydrug-resistant, 93 (31.2%) MDR, 16 (5.4%) pre-extensively drug-resistant (pre-XDR), 9 (3%) extensively drug-resistant (XDR) and 17 (5.7%) susceptible after isoniazid retesting. The frequency of katG 315 mutations alone was higher in MDR isolates, while inhA promoter mutations alone were more common in isoniazid-monoresistant isolates. Twenty-six isolates phenotypically resistant to isoniazid had no mutations either in katG or inhA genes. The isolates with inhA mutations were found more frequently in clusters (75%) when compared to the isolates with katG 315 mutations (59.8%, p = 0.04). In our population, being 35-64 years old, presenting MDR-, pre-XDR- or XDR-TB and being a retreatment case were associated with unfavourable TB treatment outcomes. CONCLUSION: We found that katG and inhA mutations were not equally distributed between isoniazid-monoresistant and MDR isolates. In our population, clustering was higher for isolates with inhA mutations. Finally, unfavourable TB outcomes were associated with specific factors.

Prospecção de novos candidatos a fármacos antituberculose: uma abordagem in vitro / Prospecting new candidates for antituberculosis drugs: an in vitro approach

A tuberculose (TB) é uma doença infecciosa causada pelo Mycobacterium tuberculosis que apresenta alta morbidade e mortalidade. Atualmente, a tuberculose afeta 9,9 milhões de pessoas em todo o mundo e é responsável por cerca de 1,3 milhões de mortes, sendo um sério problema de Saúde Pública global. O tratamento da TB é composto por uma associação de fármacos e dura seis meses. A frequência de doentes infectados com isolados resistentes aos fármacos de primeira linha, principalmente os isolados multirresistentes, é uma ameaça mundial que caracteriza um importante problema de saúde pública no controle da TB em vários países. Existem poucos medicamentos para o tratamento da TB muti-resistente e com base nesta problemática o presente estudo visou a avaliação do potencial farmacológico in vitro de fármacos aprovados, utilizando a abordagem de reposicionamento de fármacos. A partir de uma triagem in vitro de 89 fármacos, foram selecionados os que apresentaram atividade antituberculose para a avaliação da concentração inibitória mínima, os fármacos foram avaliados em exposição a 23 isolados do complexo Mycobacterium tuberculosis e a cepa ATCC H37 RV 27294 in vitro utilizando o ensaio de resazurina. Além disso, foram realizados ensaios de citotoxicidade contra células de mamíferos NCTC L929 em cultura, determinando-se os Índices de Seletividade in vitro. Dentre os 89 fármacos testados, quatro (amiodarona, flunarizina, ivermectina e pentamidina) apresentaram atividade antituberculose a 10 µM. A Concentração Inibitória Mínima (CIM) 90% da amiodarona resultou em 5-10 µM, flunarizina 10 µM, ivermectina 0,15 ­ 10 µM e pentamidina 1,25-10 µM. Os índices de seletividade observados para a amiodarona foram de 4,21 ­ 8,43, flunarizina >20, ivermectina 1,17 ­ 78,47 e pentamidina 14,40 ­ 115,2. Com base na atividade e citotoxicidade da amiodarona, foi realizado um ensaio fluorimétrico utilizando Sytox Green para avaliar a permeabilidade de membrana plasmática do complexo M. tuberculosis na presença deste fármaco. Foi observado que a amiodarona não alterou a permeabilidade da membrana plasmática do Complexo M. tuberculosis, tendo seu mecanismo de ação letal por outra via.

Correlating genetic mutations with isoniazid phenotypic levels of resistance in Mycobacterium tuberculosis isolates from patients with drug-resistant tuberculosis in a high burden setting.

We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis isolates from drug-resistant TB patients from São Paulo state, Brazil. Isolates resistant to the critical concentration of isoniazid in MGIT (0.1 µg/mL) were screened for mutations in katG 315 codon, inhA promoter region and rpoB RRDR by MTBDRplus assay and subjected to determination of isoniazid resistance levels by MGIT 960. Discordances were resolved by Sanger sequencing. Among the 203 isolates studied, 109 (54%) were isoniazid-monoresistant, 47 (23%) MDR, 29 (14%) polydrug-resistant, 12 (6%) pre-XDR and 6 (3%) XDR. MTBDRplus detected isoniazid mutations in 75% (153/203) of the isolates. Sequencing of the entire katG and inhA genes revealed mutations in 18/50 wild-type isolates by MTBDRplus (10 with novel mutations), resulting in a total of 32/203 (16%) isolates with no mutations detected. 81/83 (98%) isolates with katG 315 mutations alone had intermediate resistance. Of the 66 isolates with inhA C-15T mutation alone, 51 (77%) showed low-level, 14 (21%) intermediate and 1 (2%) high-level resistance. 5/6 (83%) isolates with mutations in both katG and inhA had high-level resistance. Inferred mutations corresponded to 22% (16/73) of all mutations found in rpoB. Mutations detected in katG regions other than codon 315 in this study might be potential new isoniazid resistance markers and could explain phenotypic resistance in some isolates without katG and inhA classic mutations. In our setting, 16% of isoniazid-resistant isolates, some with high-level resistance, presented no mutations either in katG or inhA.