Characterization of rpoB Mutations in Rifampin-Resistant Clinical Isolates of Mycobacterium tuberculosis from Turkey by DNA Sequencing and Line Probe Assay

Abstract

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 ...

Improved rapid molecular diagnosis of multidrug-resistant tuberculosis using a new reverse hybridization assay, REBA MTB-MDR

“…The mechanism of resistance to RIF involves missense mutations, small deletions or insertions in the rpoB gene encoding the b-subunit of RNA polymerase. Studies from diverse countries have shown that 95-96 % of all RIFresistant isolates have mutations within an 81 bp 'core region' of rpoB (Bártfai et al, 2001;Cavusoglu et al, 2002;Herrera et al, 2003;Mani et al, 2001;Telenti et al, 1993;Yue et al, 2003). In contrast, but perhaps not unexpectedly given its highly complex mechanism of action, the mutations causing INH resistance are located in more than three genes and regions (Barry et al, 1998;Slayden & Barry, 2000).…”

Frequency of mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis clinical isolates from Sudan

“…Knowledge on the geographical distribution of the rpoB resistant alleles is essential for the development of diagnostic tools. Although sequence analysis of the rpoB gene has been shown to be effective for detecting RIF-resistance alleles in over 90% of RIF-resistant isolates from different geographical regions [6,7], there is only limited information from Sudan [8]. In order to find Sudanspecific mutations, that could potentially be used for the optimization of the current detection tools of RIF resistance alleles, we determined in this study the mutation profile occurring within an 81-bp fragment of the rpoB gene, in a collection of MDR clinical isolates of Mycobacterium tuberculosis isolated from Khartoum, Sudan.…”

“…Mutations within the 81-bp hot-spot region (codons 507 to 533) account for more than 90% of RIF-resistant M. tuberculosis strains [6,7], but still there was a proportion of strains phenotypically RIFresistant but lacking mutation within the hot-spot region. Yue et al, 2003 [14] have reported that 10% of phenotypically RIF-resistant M. tuberculosis isolates did not show any mutations at the 81-bp region.…”

Evaluación de la técnica Xpert® MTB/RIF para la detección de Mycobacterium tuberculosis complex en muestras extra-pulmonares

“…La detección de MTBc por técni-cas moleculares es de gran utilidad porque ha permitido un diagnóstico más rápido respecto del cultivo y más sensible con respecto a la baciloscopia, desarrollándose en los últimos años, varias pruebas diagnósticas [7][8][9][10][11][12] . Una de estas es el ensayo Xpert ® MTB/RIF, que se realiza en el sistema GeneXpert ® (Cepheid, Sunnyvale, CA).…”