RESUMEN
Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC TM MGIT TM 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.
Asunto(s)
Antituberculosos/farmacología , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Proteínas Bacterianas/genética , Catalasa/genética , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN , Farmacorresistencia Bacteriana/genética , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación , Mycobacterium tuberculosis/genética , Oxidorreductasas/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC TM MGIT TM 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.
Asunto(s)
Humanos , Antituberculosos/farmacología , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Proteínas Bacterianas/genética , Catalasa/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Mutación , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/genética , Oxidorreductasas/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Real-time (RT)-PCR increases diagnostic yield for bacterial meningitis and is ideal for incorporation into routine surveillance in a developing country. We validated a multiplex RT-PCR assay for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in Brazil. Risk factors for being culture-negative, RT-PCR positive were determined. The sensitivity of RT-PCR in cerebrospinal fluid (CSF) was 100% (95% confidence limits, 96.0%-100%) for N. meningitidis, 97.8% (85.5%-99.9%) for S. pneumoniae, and 66.7% (9.4%-99.2%) for H. influenzae. Specificity ranged from 98.9% to 100%. Addition of RT-PCR to routine microbiologic methods increased the yield for detection of S. pneumoniae, N. meningitidis, and H. influenzae cases by 52%, 85%, and 20%, respectively. The main risk factor for being culture negative and RT-PCR positive was presence of antibiotic in CSF (odds ratio 12.2, 95% CI 5.9-25.0). RT-PCR using CSF was highly sensitive and specific and substantially added to measures of meningitis disease burden when incorporated into routine public health surveillance in Brazil.
Asunto(s)
Meningitis Bacterianas/microbiología , Vigilancia de la Población/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Técnicas de Tipificación Bacteriana , Brasil , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Recuento de Leucocitos , Masculino , Meningitis Bacterianas/sangre , Meningitis Bacterianas/líquido cefalorraquídeo , Persona de Mediana Edad , Análisis Multivariante , Neisseria meningitidis/clasificación , Neisseria meningitidis/genética , Reproducibilidad de los Resultados , Factores de Riesgo , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Real-time (RT)-PCR increases diagnostic yield for bacterial meningitis and is ideal for incorporation into routine surveillance in a developing country. We validated a multiplex RT-PCR assay for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in Brazil. Risk factors for being culture-negative, RT-PCR positive were determined. The sensitivity of RT-PCR in cerebrospinal fluid (CSF) was 100% (95% confidence limits, 96.0%100%) for N. meningitidis, 97.8% (85.5%99.9%) for S. pneumoniae, and 66.7% (9.4%99.2%) for H. influenzae. Specificity ranged from 98.9% to 100%. Addition of RT-PCR to routine microbiologic methods increased the yield for detection of S. pneumoniae, N. meningitidis, and H. influenzae cases by52%, 85%, and 20%, respectively. The main risk factor for being culture negative and RT-PCR positive was presence of antibiotic in CSF (odds ratio 12.2, 95% CI 5.9-25.0). RT-PCR using CSF was highly sensitive and specific and substantially added to measures of meningitis disease burden when incorporated into routine public health surveillance in Brazil