Detection of Rifampin Resistance among Isolates ofMycobacterium tuberculosisfrom Mozambique

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Abstract
Rifampin resistance in respiratory isolates of Mycobacterium tuberculosis from Mozambique was detected by screening for point mutations using polymerase chain reaction (PCR) and DNA sequence analysis. The target template was a 350-bp fragment of rpoB encoding the β-subunit of the RNA polymerase. Of the 66 strains studied, 38 were rifampin resistant by susceptibility testing with the radiometric method, 3 were intermediately resistant, and 25 were susceptible to rifampin. In 39 of the 41 rifampin-resistant strains, base-substitutions in the rpoB fragment were detected, and a total of 13 distinct mutations affecting 6 amino acids were observed. One of these mutations (His → Thr in amino acid 526) was not previously described. The isolates were also investigated by restriction fragment length polymorphism (RFLP) analysis using the insertion element IS6110 as a hybridization probe. A total of 47 RFLP patterns were identified, with up to 9 isolates having the same RFLP pattern. Strains with the same RFLP pattern harbored different mutations in rpoB, suggesting that acquisition of rifampin resistance followed the spread of a rifampin-susceptible clone. The data showed that rifampin resistance can be detected with a high sensitivity by DNA sequence analysis of this fragment of rpoB. However, a few strains with rifampin resistance due to factors other than base substitutions in rpoB could be missed.