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1.
Mem Inst Oswaldo Cruz ; 115: e190407, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32321155

RESUMEN

BACKGROUND: Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES: To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS: Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS: The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS: qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.


Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana/genética , Marcadores Genéticos/genética , Isoniazida/farmacología , Mutación/genética , Mycobacterium tuberculosis/genética , ADN Bacteriano/genética , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad
2.
Mem. Inst. Oswaldo Cruz ; 115: e190407, 2020. tab
Artículo en Inglés | LILACS-Express | LILACS | ID: biblio-1101275

RESUMEN

BACKGROUND Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.

3.
Braz. j. microbiol ; 49(3): 641-646, July-Sept. 2018. tab, graf
Artículo en Inglés | LILACS | ID: biblio-951817

RESUMEN

Abstract DNA genotyping of Mycobacterium tuberculosis has been widely applied in the understanding of disease transmission in many countries. The purpose of this study was to genotype the strains of M. tuberculosis isolated in patients with new tuberculosis (TB) cases in Minas Gerais, as well as to compare the similarity, discriminatory power, and agreement of the clusters between the IS6110 Restriction Fragment Length Polymorfism (RFLP) and 12 loci Variable Number Tandem Repeat - Mycobacterial Interspersed Repetitive Units (MIRU-VNTR) techniques. It was observed that 32% (66/204) of the isolated strains in the RFLP-IS6110 and 50.9% (104/204) of the isolated strains in the MIRU-VNTR presented a similarity of equal to or above 85%. The RFLP-IS6110 and MIRU-VNTR proved to contain a high discriminatory power. The similarity index resulting from the RFLP showed no recent transmission. Good agreement was observed between the techniques when clusters were detected; however, the best epidemiological relationship was found when using the RFLP-IS6110.


Asunto(s)
Humanos , Tuberculosis/microbiología , Polimorfismo de Longitud del Fragmento de Restricción , Técnicas de Tipificación Bacteriana/métodos , Repeticiones de Minisatélite , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados/métodos , Mycobacterium tuberculosis/aislamiento & purificación , Brasil , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética
4.
Braz J Microbiol ; 49(3): 641-646, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29249565

RESUMEN

DNA genotyping of Mycobacterium tuberculosis has been widely applied in the understanding of disease transmission in many countries. The purpose of this study was to genotype the strains of M. tuberculosis isolated in patients with new tuberculosis (TB) cases in Minas Gerais, as well as to compare the similarity, discriminatory power, and agreement of the clusters between the IS6110 Restriction Fragment Length Polymorfism (RFLP) and 12 loci Variable Number Tandem Repeat - Mycobacterial Interspersed Repetitive Units (MIRU-VNTR) techniques. It was observed that 32% (66/204) of the isolated strains in the RFLP-IS6110 and 50.9% (104/204) of the isolated strains in the MIRU-VNTR presented a similarity of equal to or above 85%. The RFLP-IS6110 and MIRU-VNTR proved to contain a high discriminatory power. The similarity index resulting from the RFLP showed no recent transmission. Good agreement was observed between the techniques when clusters were detected; however, the best epidemiological relationship was found when using the RFLP-IS6110.


Asunto(s)
Análisis del Polimorfismo de Longitud de Fragmentos Amplificados/métodos , Técnicas de Tipificación Bacteriana/métodos , Repeticiones de Minisatélite , Mycobacterium tuberculosis/aislamiento & purificación , Polimorfismo de Longitud del Fragmento de Restricción , Tuberculosis/microbiología , Brasil , Humanos , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética
5.
Mem Inst Oswaldo Cruz ; 112(11): 769-774, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29091137

RESUMEN

BACKGROUND: The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB. METHOD: We studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible. FINDINGS: Among the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. MAIN CONCLUSIONS: Both methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil.


Asunto(s)
Técnicas Bacteriológicas/métodos , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Brasil , Genotipo , Humanos , Técnicas de Diagnóstico Molecular , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tuberculosis Resistente a Múltiples Medicamentos/microbiología
6.
Mem. Inst. Oswaldo Cruz ; 112(11): 769-774, Nov. 2017. tab
Artículo en Inglés | LILACS | ID: biblio-894852

RESUMEN

BACKGROUND The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB. METHOD We studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible. FINDINGS Among the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. MAIN CONCLUSIONS Both methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil.


Asunto(s)
Humanos , Técnicas Bacteriológicas/métodos , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Mycobacterium tuberculosis/genética , Brasil , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Patología Molecular , Genotipo
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