Discordance in genotypic and phenotypic drug susceptibility results: time to reconsider critical concentration of rifampicin.

: The objective of this study is to correlate rpoB mutations found on the new-generation sequencing (NGS) in Mycobacterium tuberculosis (MTB) isolates with minimum inhibitory concentrations (MICs) to the rifampicin (RIF). We assessed the minimum inhibitory concentrations for 151 archived clinical MTB isolates that were determined phenotypically susceptible to RIF (101; 66.89%), and the remaining 50 (50; 33.11%) were resistant to RIF by BACTEC MGIT SIRE DST. MIC values were determined using the colorimetric redox indicator (Resazurin/REMA) method, and results were correlated with rpoB gene mutations associated with rifampicin resistance found. Comparing 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 µg/mL) though these were found to have low-level RIF resistance by CRI assay (MIC 0.50 to 1.0µg/mL) and NGS. We found that all 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+Ile572 Phe (1), Asp516Tyr (1), and Leu533Pro+Pro564 Arg (1). These mutations are reported to confer low-level RIF resistance. But we did not find any mutation at MIC ≤0.25 µg/mL. We found that a significant number of MTB isolates have phenotypic and genotypic discordance. Taking 1.0 µg/mL of rifampicin as a critical concentration, isolates from approximately 15% of patients are misidentified as susceptible to rifampicin, even when these strains carry low-level drug resistance-conferring mutations and have the potential to develop clinical multidrug-resistant tuberculosis.

Objective: Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide, killing millions every year. The emergence of multidrug-resistant and extensively drug-resistant TB forms poses a challenge to the TB control programs. In the past few decades, several molecular tests for rapid detection and drug resistance determination have been developed. But these can miss the genetic mutations that confer low-level resistance to rifampicin (RIF), a critical constituent for treating drug-susceptible TB. On the other hand, for the phenotypic methods, a cutoff value is fixed, known as critical concentration (CC). The current WHO-endorsed CC for rifampicin is 1.0 μg/mL in liquid culture for confirmation of drug resistance; because of that in this system too, low-level RIF resistance may not be correctly identified. Therefore, it is important that either the CC for phenotypic methods is lowered or the specific mutations are included in the molecular tests. This study provides important insights in that direction.