RESUMO
Introducción. Una parte de los aislamientos de Mycobacterium tuberculosis multirresistente también presenta resistencia a la etionamida. Es importante determinar si la resistencia a la isoniacida es independiente o se cruza con la resistencia a la etionamida, ya que si sucede lo segundo habría que reevaluar el tratamiento antituberculoso de segunda línea. La prueba molecular GenoType MTBDR plus ® detecta las mutaciones asociadas con la resistencia a isoniacida y podría detectar la resistencia cruzada a la etionamida. Objetivo. Evaluar la prueba GenoType MTBDR plus ® y comparar su desempeño con el de la secuenciación, en la detección de mutaciones en el gen katG y en el promotor inhA en aislamientos clínicos de M. tuberculosis multirresistente. Materiales y métodos. Se utilizaron el estuche comercial GenoType MTBDR plus 1.0 ® y la secuenciación para evaluar mutaciones en el gen katG y en el promotor inhA en 30 aislamientos de M. tuberculosis multirresistente con resistencia a la etionamida. La cepa de laboratorio H37Rv y tres aislamientos sensibles a los medicamentos de primera línea, sirvieron de control. Resultados. Al comparar los resultados de la secuenciación y de la prueba GenoType MTBDR plus ® , el índice kappa fue de 1. Todos los aislamientos resistentes a la isoniacida y la etionamida tenían las mutaciones detectadas con la prueba GenoTypeMTBDR plus ® en el gen katG, y 40 % de ellos, las detectadas en el promotor inhA. Mediante secuenciación se encontraron, además, mutaciones en katG en posiciones diferentes a las detectadas por la prueba GenoType MTBDR plus ® . Conclusión. La prueba GenoTypeMTBDR plus ® tiene la capacidad de detectar rápidamente la resistencia a isoniacida. Además, los resultados del estudio sugieren que también podría utilizarse como prueba de tamización para detectar la resistencia cruzada a etionamida.
Introduction: A variable proportion of isolates of multidrug-resistant Mycobacterium tuberculosis also presents resistance to ethionamide. It is important to determine whether resistance to isoniazid is independent or crossed with resistance to ethionamide, given that this could lead to the re-evaluation of second-line anti-tuberculosis treatment. The GenoType MTBDR plus ® molecular test is used for the detection of MDR-MTB, as it identifies mutations associated with resistance to isoniazide and could detect cross-resistance with ethionamide. Objective: To evaluate the performance of GenoType MTBDR plus ® in comparison with sequencing in the detection of mutations in gene katG and promotor inhA in clinical isolates of multidrug-resistant M. tuberculosis . Materials and methods: The GenoType MTBDR plus 1.0 ® commercial kit and sequencing were used to evaluate mutations in gene katG and promotor inhA in 30 multidrug-resistant M. tuberculosis isolates that were resistant to ethionamide. The laboratory strain H37Rv and three pan-sensitive isolates acted as controls. Results: The kappa index for the comparison between the results of sequencing and GenoType MTBDR plus ® was 1. All the isolates resistant to isoniazid and ethionamide had the mutations detected by GenoTypeMTBDR plus ® in the katG gene and 40% of them in promotor inhA. Sequencing also revealed katG mutations in positions different to those detected by GenoType MTBDR plus ® . Conclusion: GenoType MTBDR plus ® is able to detect resistance to isoniazid rapidly. Our results suggest that it could also be used to screen for cross-resistance with ethionamide.
Assuntos
Humanos , Oxirredutases/genética , Proteínas de Bactérias/genética , Catalase/genética , Técnicas de Tipagem Bacteriana/métodos , Análise de Sequência de DNA/métodos , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Etionamida/farmacologia , Técnicas de Genotipagem , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , DNA Bacteriano/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas/genética , Etionamida/metabolismo , Genótipo , Isoniazida/metabolismo , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Antituberculosos/metabolismoRESUMO
Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, still causes higher mortality than any other bacterial pathogen until now. With the emergence and spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR-TB) strains, it becomes more important to search for alternative targets to develop new antimycobacterial drugs. Lupulone is a compound extracted from Hops (Hurnulus lupulus), which exhibits a good antimicrobial activity against M. tuberculosis with minimal inhibitory concentration (MIC) value of 10 µg/mL, but the response mechanisms of lupulone against M. tuberculosis are still poorly understood. In this study, we used a commercial oligonucleotide microarray to determine the overall transcriptional response of M. tuberculosis H37Rv triggered by exposure to MIC of lupulone. A total of 540 genes were found to be differentially regulated by lupulone. Of these, 254 genes were upregulated, and 286 genes were downregulated. A number of important genes were significantly regulated which are involved in various pathways, such as surface-exposed lipids, cytochrome P450 enzymes, PE/PPE multigene families, ABC transporters, and protein synthesis. Real-time quantitative RT-PCR was performed for choosed genes to verified the microarray results. To our knowledge, this genome-wide transcriptomics approach has produced the first insights into the response of M. tuberculosis to a lupulone challenge.
Assuntos
Antituberculosos/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Terpenos/metabolismo , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The acetylator status of 200 pulmonary tuberculosis patients was determined by sulphadimidine (SDM) acetylation in (1) urine and (2) serum. In both estimations, distribution of the patients with regard to the acetylation rates was found to be bimodal. Based on the percent frequency distribution of acetylated SDM, the antimode for urine and serum was 65% for the former and 45% for the latter. Patients acetylating above 65% or 45% of administered SDM were taken as rapid acetylators, while those acetylating less, were slow acetylators. The ratio of slow to rapid acetylators in urine and serum was 58 : 42 and 66 : 34 respectively. The renal clearance of acetylated fraction of SDM was considerably greater as compared to that of the unacetylated fraction. Agreement between the two methods as evaluated by the kappa statistic was 0.71. The determination of the acetylator status by the SDM acetylation test in the urine is simple to perform and has acceptable accuracy. It may be used as an acceptable substitute to serum for estimating the isoniazid inactivation status which is of considerable importance when patients are treated with daily/ intermittent/once-weekly drug regimens for tuberculosis.
Assuntos
Acetilação , Adolescente , Adulto , Idoso , Anti-Infecciosos/metabolismo , Antituberculosos/metabolismo , Humanos , Isoniazida/metabolismo , Pessoa de Meia-Idade , Fenótipo , Sulfametazina/metabolismo , Tuberculose Pulmonar/genéticaAssuntos
Adolescente , Adulto , Pessoa de Meia-Idade , Humanos , Masculino , Feminino , Acetiltransferases , Antituberculosos/efeitos adversos , Fenótipo , Acetilação , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Chile , Estudos Prospectivos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Isoniazida/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Antituberculosos/metabolismoRESUMO
A atividade metabólica do M. tuberculosis sob diversas condiçöes experimentais foi estudada utilizando um sistema radiométrico automático, capaz de quantificar o 14CO2 produzido pela oxidaçäo de substâncias marcadas com Carbono-14. As experiências realizadas incluíram: a) vias metabólicas; b) determinaçäo dos tempos de detecçäo para inoculaçöes de diversas magnitudes; c) efeito da filtraçäo sobre a reprodutibilidade dos resultados; d) influência de meio hostil; e) determinaçäo das concentraçöes inibitórias mínimas para hidrazida, estreptomicina, etambutol e rifampicina; f) tempo de duplicaçäo para o M. tuberculosis e M. bovis. Estes microorganismos metabolizaram até 14CO2 o 14C-formato, (U-14C) acetato, (u-14C) glicerol, (1-14C) ácido palmítico, (1-14C) ácido láurico, (u-14C) L-ácido málico, (U-14C) D-glicose e (1-14C) D-glicose, mas näo (1-14C) L-glicose, (u-14C) glicina ou (U-14C) piruvato. Usando 14C-formato, (1-14C) ácido palmítico ou (1-14C) ácido láurico foi possível detectar 10 bacilos/frasco em 24-48 horas e até 10 bacilos/frasco em 16-20 dias. Resultados reprodutíveis foram obtidos sem filtrar a suspensäo de bactérias, desde que cultivadas em meio 7H9 líquido com 0,05% de polissorbato 80 e homogenizadas antes da experiência. Drogas que bloqueiam a síntese protêica apresentaram concentraçäo inibitória mínima menor com o método radiométrico do que com o convencional. O tempo médio de duplicaçäo para o M. bovis e várias cepas de M. tuberculosis com diversas substâncias marcadas foi 9 + ou - 1 horas