LAUSR

OBJECTIVE To evaluate the utility of a sequencing procedure for the entire rpoB gene of Mycobacterium tuberculosis to identify mutations pre, within and post rifampicin resistance determinant region (RRDR) in isolates circulating in a region affected by tuberculosis. METHODS Five primers were designed, with which five DNA fragments of rpoB were obtained, sequenced by Sanger and analyzed in silico in order to identify mutations over the entire rpoB gene in rifampicin sensitive and resistant tuberculosis. RESULTS It was possible to analyze the entire rpoB gene in 5 rifampicin sensitive and 15 rifampicin resistant isolates. A total of 36 mutations were identified. Two mutations were found pre RRDR, nine within RRDR and 25 post-RRDR. The most frequent mutations within RRDR were S531 L (53%), followed by S512 T (20%), all of which were found in rifampicin resistant isolates. Of the 25 mutations found post-RRDR, 14 were only in resistant isolates, and the most frequent was D853 N present in 85% of isolates. Mutations E818 K, D836 N and T882 P were observed in 80% of the rifampicin resistant and sensitive isolates. CONCLUSIONS The proposed sequencing method allowed identification of mutations in the entire rpoB gene. This procedure represents a useful tool for the diagnosis of rifampicin resistance. The number of mutations found raises new questions about the diversity of mutations in the rpoB gene and their role in rifampicin resistance in regions were tuberculosis is endemic.