Pyrosequencing for rapid detection of Mycobacterium tuberculosis resistance to rifampin, isoniazid, and fluoroquinolones.

Bravo, Lulette Tricia C; Tuohy, Marion J; Ang, Concepcion; Destura, Raul V; Mendoza, Myrna; Procop, Gary W; Gordon, Steven M; Hall, Geraldine S; Shrestha, Nabin K

Bravo, Lulette Tricia C; Tuohy, Marion J; Ang, Concepcion; Destura, Raul V; Mendoza, Myrna; Procop, Gary W; Gordon, Steven M; Hall, Geraldine S; Shrestha, Nabin K.

Afiliación

Bravo LT; Cleveland Clinic, Cleveland, Ohio 44195, USA.

J Clin Microbiol ; 47(12): 3985-90, 2009 Dec.

Article en En | MEDLINE | ID: mdl-19846642

RESUMEN

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.

Asunto(s)

Antituberculosos/farmacología; Farmacorresistencia Bacteriana; Isoniazida/farmacología; Mycobacterium tuberculosis/efectos de los fármacos; Ofloxacino/farmacología; Rifampin/farmacología; Análisis de Secuencia de ADN/métodos; Proteínas Bacterianas/química; Proteínas Bacterianas/genética; Farmacorresistencia Bacteriana/genética; Fluoroquinolonas/farmacología; Genotipo; Humanos; Pruebas de Sensibilidad Microbiana; Mutación; Mycobacterium tuberculosis/genética; Fenotipo; Factores de Tiempo

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Rifampin / Ofloxacino / Análisis de Secuencia de ADN / Farmacorresistencia Bacteriana / Isoniazida / Mycobacterium tuberculosis / Antituberculosos Tipo de estudio: Diagnostic_studies / Evaluation_studies Límite: Humans Idioma: En Revista: J Clin Microbiol Año: 2009 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos

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