Mutations in an 81-bp region of the rpoB gene associated with rifampin resistance were studied in 41 rifampin-resistant clinical strains of Mycobacterium tuberculosis isolated in Turkey. Fourteen different rpoB alleles, three of which had not been reported before, were found. A reverse hybridization-based line probe assay (the Inno-LiPA Rif.TB test) for rapid detection of the mutations was evaluated with these isolates. Rifampin resistance was correctly identified in 23 of 41 isolates (56.1%) with the kit's R probes specific for these mutations. Seventeen of 41 isolates (41.5%) yielded hybridization patterns, with at least one negative signal obtained with the S probes for the wild type. One isolate was identified as rifampin sensitive by the line probe assay. The rate of concordance of the results of the line probe assay with the results of the in vitro susceptibility test was high (97.6%). These results demonstrate that the line probe assay kit may be useful for the rapid diagnosis of rifampin-resistant tuberculosis.Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent, annually leading to 2 million deaths worldwide (4). As the incidence of tuberculosis has increased, there has been a corresponding rise in the incidence of drug-resistant strains of M. tuberculosis. Early diagnosis of the disease and rapid identification of resistance to primary antituberculosis agents are essential for efficient treatment and control of multidrug-resistant (MDR) strains. Rifampin (RIF) is one of the most potent antituberculosis drugs; therefore, resistance to RIF often results in high clinical relapse rates, particularly if RIF resistance is associated with resistance to other antituberculosis drugs (5, 14). Moreover, more than 90% of RIF-resistant isolates are also resistant to isoniazid; therefore, detection of RIF resistance could also identify MDR strains (3, 5, 17). The incidence of pulmonary tuberculosis in Turkey was 35.5 per 100,000 population in 2000. In the Aegean region, 8.2% of M. tuberculosis strains isolated between 1999 and 2001 were found to be resistant to RIF. During the same period, the incidence of resistance to both RIF and isoniazid was 6.8% (6).Collectively, DNA sequencing studies demonstrate that more than 95% of RIF-resistant M. tuberculosis strains have a mutation within the 81-bp hot-spot region (codons 507 to 533) of the RNA polymerase B subunit (rpoB) gene (9,10,12,13,15,16,18,20,21,23,24). In addition, other studies reveal that mutations associated with RIF resistance can also occur in other regions of the rpoB gene, although these occur less frequently (7,8). Several molecular methods have been developed in recent years to evaluate the rpoB gene for RIF resistance mutations, including DNA sequencing, line probe assay, and analysis with DNA microarrays (19). These molecular tests can also serve as a means of detecting molecular epidemiological markers, since the relative frequencies of the alleles associated with resistance can vary geographically ...

Although DNA sequencing method has not been used routinely for detecting resistance of Mycobacterium tuberculosis to antituberculosis drugs, it is suggested for the investigation of gene mutations causing resistance. Using DNA sequencing (Automated Applied Biosystems), we attempted to determine mutations in the 81-bp cor region (rifampin resistance determining region) of the rpoB gene in 48 Mycobacterium tuberculosis strains found to be rifampin resistant by classical phenotypic methods. Of the 48 strains, 46 (95.8%) were found to have rpo gene mutations with 13 different types while in two (4.2%) of the 48 strains, no mutations were detected. None of the strains had mutations at more than one codon. Point mutations at the 531st (52.1%) and 526th (18.9%) codons were frequent. The most frequent point mutation was Ser531Leu, and it was found in 21 (43,8 %) of 48 strains. This is the first study from Turkey, reporting Ser522Leu point mutation in one isolate and deletion of 515th codon (ATG - Met) in two isolates.