Confirmation of silent mutations in the rpoB gene locus of <i>M. tuberculosis </i>isolates using pyrosequencing and phenotypic DST

More than 95% cases of rifampicin resistance in M.tuberculosis strains can be attributed to mutations in 81-bp hotspot region of rpoB gene locus called Rifampicin Resistance Determining Region. These mutations can be detected by rapid molecular diagnostic techniques such as Line Probe Assay (LPA), but detection of silent or noncanonical mutations on LPA may not necessarily confer rifampicin resistance and result in false rifampicin resistance reporting. These silent mutations can be confirmed by phenotypic Drug Sensitivity Testing (DST) and/or by DNA sequencing. This study was undertaken to confirm the silent or noncanonical mutations using phenotypic DST (Gold standard) and pyrosequencing, a rapid real time method for sequencing small DNA segments by synthesis. A total of 300 DNA extracts comprising of 101 silent/noncanonical mutation strains, 101 pan-sensitive, 96 MDR-TB and 02 rifampicin mono-resistant strains were processed for pyrosequencing following detection by LPA. Pyrosequencing was performed with sequence analysis mode of  PyroMark Q96 ID system (Qiagen, Valencia, CA). All 300 sputum concentrates were also processed for phenotypic DST, using solid and liquid culture methods. Pyrosequencing detected mutations in all 101 silent/noncanonical mutation strains, but 02 strains did not produce amino acid change and hence, no alteration in protein structure (true silent mutations). Maximum mutations were seen in 526 codon (35 strains) followed by 511 codon region (30 strains). Two novel mutations were reported: substitution in 529 codon CGA – CAA and 517-518 codon deletion. However, only 53/101 rifampicin resistant strains were rifampicin resistant by MGIT 960 and 59/101 by solid DST. This proves that low-level rifampicin resistance linked to specific rpoB mutations could be missed by phenotypic DST and this could be attributed to the critical concentration of rifampicin used. 101 pan-sensitive, 96 MDR-TB and 02 rifampicin mono-resistant strains used as controls were confirmed by pyrosequencing and showed 100% concordance with phenotypic DST. Hence, to conclude, pyrosequencing is a confirmatory tool for detection of rifampicin resistance in silent or noncanonical mutation cases in M. tuberculosis and reiterates the fact that liquid DST may miss some rifampicin resistance conferring mutations compared to solid DST, further suggesting that the gold standard for rifampicin resistance be reconsidered.