Molecular Characterization of Rifampin-Resistant Isolates of Mycobacterium tuberculosis from Hungary by DNA Sequencing and the Line Probe Assay

Abstract

Two regions of rpoB associated with rifampin resistance were sequenced in 29 rifampin-resistant (determined by the proportion method) isolates of Mycobacterium tuberculosis obtained from patients from three counties in Hungary. Of the 29 resistant strains, 27 had a mutation in either the 81-bp region (26 strains) or the N-terminal region (1 strain), while the other 2 strains had no mutations in either region. The locations and frequencies of the mutations differed from those previously reported. The most common mutation in this study, D516V, was found in 38% of the Hungarian strains, a frequency 2 to 10 times higher than that found in studies from other countries. These same 29 isolates were also evaluated with the Inno-LiPA Rif. TB test (LiPA), a reverse hybridization assay for the rapid detection of rifampin resistance. Although LiPA detected the presence of an rpoB mutation in 26 of the resistant isolates, the type of mutation could not be determined in 4 isolates because the mutations present were not among those included on the LiPA strip. In addition, a silent mutation in one of the rifampin-susceptible control strains was interpreted as rifampin resistant by LiPA. These findings demonstrate the importance of validating this rapid molecular test by comparison with DNA sequence results in each geographic location before incorporating the test into routine diagnostic work.The recent worldwide increase in the incidence of drugresistant strains of Mycobacterium tuberculosis has highlighted the need for faster and more accurate detection of resistance to rifampin (RMP), one of the most important antituberculosis drugs (17). RMP is most effective in killing actively metabolizing M. tuberculosis, and resistance to RMP often results in high clinical relapse rates (5, 15). Because of the prolonged turnaround time for conventional susceptibility testing, patients infected with drug-resistant tuberculosis may be inadequately treated and thus remain infectious for longer periods than those infected with susceptible strains.Based on collective observations that mutations resulting in an amino acid change within the 81-bp core region of the RNA polymerase ␤-subunit (rpoB) gene are found in more than 96% of RMP-resistant M. tuberculosis strains, several molecular methods have been developed for the rapid (24-to 48-h) detection of mutations in this region (3,10,16,18,24,25,(28)(29)(30). In addition, other studies revealed that mutations associated with RMP resistance can also occur in other regions of the rpoB gene, although less frequently (6,7,23). It has also been shown elsewhere that the information provided by these molecular tests can serve as a molecular epidemiological marker since the relative frequency of the alleles associated with resistance can vary geographically (10,20).Therefore, the aim of the present study was to determine the drug resistance profile of 29 RMP-resistant M. tuberculosis isolates obtained in East Hungary and to detect and identify mutations present in the rpoB gene. Two molecular assays we...

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