Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify Mycobacterium tuberculosis bacilli and to detect
rifampin and
isoniazid resistance in
DNA extracts from sputum samples. We studied three genes: rpoB, which is associated with
rifampin resistance, and katG and inhA, which are associated with
isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with
pulmonary tuberculosis with positive
auramine-
rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for rpoB and inhA genes and 96.5% (198 of 205) for the katG gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for
rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for
isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for
rifampin. The phenotypic method for
isoniazid reported 13 resistant strains (> or = 0.1 microg/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that
rifampin-resistant M.
tuberculosis could be detected in
DNA extracted from
auramine-
rhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of
auramine-
rhodamine-positive specimens obtained from patients with culture-documented
pulmonary tuberculosis. Similarly, this occurs in half of the
isoniazid-resistant M.
tuberculosis DNA extracted from
auramine-
rhodamine-positive specimens.