Discordance in Genotypic and Phenotypic anti-tuberculosis drug susceptibility results: time to reconsider critical concentration.

Abstract

ObjectiveTo correlate rpoB mutations found on the sanger sequencing in Mycobacterium tuberculosis (MTB) isolates with Minimum Inhibitory Concentrations (MICs) to the rifampicin. MethodsWe assessed the minimum inhibitory concentrations (MICs) for 151 archived clinical MTB isolates that were determined phenotypically susceptible to RIF (101;66.89%) and remaining fifty (50;33.11%) were resistant to RIF by BACTEC MGIT SIRE DST. MIC values were determined using colorimetric redox indicator (Resazurin/REMA) method and results were correlated with rpoB gene mutations associate with rifampicin resistance found. ResultsComparing the MIC and critical concentration, we found that 15 of these 101 (14.85%) isolates were misclassified by MGIT-960 as sensitive at standard critical concentration (1.0{micro}g/mL) though these were found to have low-level RIF resistance by CRI assay (MIC 0.50{micro}g/mL to 1.0{micro}g/mL) and sanger sequencing. We found that all of 15 isolates contained non-synonymous mutations, the commonest being the Ile572Phe (7, 46.66%), followed by Leu533Pro (3, 20.0%), His526Leu (2, 13.33%), His526Asn+Ile572Phe (1), Asp516Tyr (1), and Leu533Pro+Pro564Arg (1). These mutations are reported to confer low-level RIF resistance. But we did not find any mutation at MIC <0.25g/mL. ConclusionWe found that a significant number of MTB isolates have phenotypic and genotypic discordance. Taking 1.0{micro}g/mL of rifampicin as critical concentration, isolates from approximately 15% patients are misidentified as susceptible to rifampicin, even when these strains carry low level drug resistance conferring mutations and have potential to develop clinical MDR-TB.