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1.
Mem Inst Oswaldo Cruz ; 109(3): 307-14, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24821057

RESUMO

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.


Assuntos
Proteínas de Bactérias/genética , Catalase/genética , Farmacorresistência Bacteriana Múltipla/genética , Mutação/genética , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Colorimetria , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA , Técnicas de Genotipagem , Isoniazida/farmacologia , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/efeitos dos fármacos , Hibridização de Ácido Nucleico , Rifampina/farmacologia
2.
J Clin Microbiol ; 51(5): 1606-8, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23467605
3.
Mem Inst Oswaldo Cruz ; 104(5): 710-4, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19820830

RESUMO

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC-->ACC (Ser-->Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100% of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Catalase/genética , Farmacorresistência Bacteriana/genética , Isoniazida/farmacologia , Mutação/genética , Mycobacterium tuberculosis , Colorimetria/métodos , DNA Bacteriano/análise , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase
4.
Front Microbiol ; 8: 711, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28496433

RESUMO

Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis. A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55, and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality that is often disregarded by the tuberculosis specialists in favor of the almost undisputed importance of antibiotic target-gene mutations for the resistance in M. tuberculosis.

5.
Mem. Inst. Oswaldo Cruz ; 109(3): 307-314, 06/2014. tab
Artigo em Inglês | LILACS | ID: lil-711730

RESUMO

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.


Assuntos
Proteínas de Bactérias/genética , Catalase/genética , Farmacorresistência Bacteriana Múltipla/genética , Mutação/genética , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Colorimetria , DNA Bacteriano/genética , Técnicas de Genotipagem , Isoniazida/farmacologia , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/efeitos dos fármacos , Hibridização de Ácido Nucleico , Rifampina/farmacologia
6.
Mem. Inst. Oswaldo Cruz ; 104(5): 710-714, Aug. 2009. ilus
Artigo em Inglês | LILACS | ID: lil-528078

RESUMO

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100 percent of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Catalase/genética , Farmacorresistência Bacteriana/genética , Isoniazida/farmacologia , Mycobacterium tuberculosis , Mutação/genética , Colorimetria/métodos , DNA Bacteriano/análise , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase
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