Correlation of mutations detected by INNO-LiPA with levels of rifampicin resistance in Mycobacterium tuberculosis

Abstract

Background & objectives: Due to emergence of drug resistance in Mycobacterium tuberculosis, there is a need to have accurate and rapid methods for detection of drug resistance to important drugs like rifampicin. The present study was aimed at evaluation of a commercially available INNO-LiPA assay, for the detection of mutation in rpoB gene region of M. tuberculosis and correlate these mutations with levels of rifampicin resistance for assessing their clinical relevance.

Methods: Fifty five well-characterized isolates of M. tuberculosis deposited from various regions of India in Mycobacterial Repository Centre at the CJILOMD, Agra were subjected to susceptibility testing for rifampicin at various concentrations of drug viz., 10, 40, 64, 128 microg/ml on Lowenstein- Jensen (LJ) medium. rpoB gene fragment (260 bp) was amplified using Rif-TB amplification kit and after hybridization, detection was done by using INNO-LiPA Rif TB kit.

Results: The rpoB gene could be amplified from DNA extracted from all the 55 culture isolates and showed clear hybridization pattern with M. tuberculosis complex specific probes on LiPA strips. Mutations detected were correlated with degree of rifampicin resistance. All the sensitive isolates (identified by MIC) were identified as rifampicin sensitive (100%) by INNO-LiPA as they exhibit positive for wild type 'S' probes and negative for 'R' probes. Two of the 5 isolates, resistant at 10 microg/ml and 40 microg/ml had either D516V, H526Y mutations or unknown mutations. Thirty (85.71%) isolates resistant at clinically relevant levels (64,128microg/ml) exhibited double, triple or more 'R' type mutations (R(2(D516V)), R(4a(H526Y)), R(4b(H526D)), R(5(S531L))) as well as unknown mutations present at 'S' probes region whereas remaining isolates did not show any mutation by this method. This method could identify with definitiveness 60 per cent ( 21/35) isolates as rifampicin resistant as mutations observed in others were also present in isolates with low levels of resistance.

Interpretation & conclusion: The results indicate that INNO-LiPA Rif TB test is a rapid and easy to use method for detection of mutations associated with rifampicin resistance in M. tuberculosis. However, as some of these mutations are also present in isolates with low degree of resistance which are still microbiologically sensitive to rifampicin, there is a need to improve this assay by exclusion of some of the current probes and inclusion of more probes.