Reevaluating Rifampicin Breakpoint Concentrations for Mycobacterium tuberculosis Isolates with Disputed rpoB Mutations and Discordant Susceptibility Phenotypes

In this study, rifampicin resistance breakpoints based on MICs of disrupted rpoB mutants of Mycobacterium tuberculosis (MTB) were explored using the Mycobacteria Growth Indicator Tube (MGIT) system and microplate alamarBlue assay (MABA). Sixty-one MTB isolates with disputed low-level rifampicin resistance-associated rpoB mutations and 40 RIF-susceptible wild-type isolates were included. Among the 61 resistant isolates, 25 (41.0%) had MICs >= 2.0 mg/L via MABA, while 16 (262%) were identified as RIF resistant via MGIT. Epidemiological cut-off (ECOFF) values obtained using MABA and MGIT were 025 and 0.125 mg/L, respectively. Based on 0.125 mg/L as a tentative critical concentration (CC), MABA RIF resistance-detection sensitivity was 93.4%, prompting the reduction of the MGT CC to 0.125 mg/L, given that only a single isolate (1.6%) with the borderline mutation would be misclassified as susceptible to RIF based on this CC. Based on DNA sequencing of RRDR as the gold standard, the diagnostic accuracy of MGIT (99.0%) was significantly higher than that of MABA (91.1%). MICs of Leus"Pro mutant isolates were negatively correlated with time to liquid culture positivity (TTP) in our analysis (R = 0.957, P < 0.01). In conclusion, our results demonstrated missed detection of a high proportion of rifampicin-resistant isolates based on the WHO-endorsed CC. Such missed detections would be avoided by reducing the optimal MGIT RIF CC to 0.125 mg/L In addition, MGIT based on reduced CC outperformed MABA in detecting borderline RIF resistance, with MABA MIC results obtained for isolates with the same mutation correlating with MTB growth rate. IMPORTANCE Tuberculosis (TB) is still one of the world's leading infectious disease killers. The early and accurate diagnosis of RIF resistance is necessary to deliver timely and appropriate treatment for TB patients and improve their clinical outcome. Actually, a proportion of MTB isolates with disputed rpoB mutations present a diagnostic dilemma between Xpert and phenotypical drug susceptibility testing (pDST). Recently, WHO reported a pragmatic approach by lowering critical concentration (CC) to boost sensitivity of resistance detection of pDST. Therefore, a detailed analysis of the association between RIF susceptibility and disrupted mutations within rpoB gene would lay a foundation to assess the diagnostic accuracy of pDST with lowering RIF CC. In this study, we aim to determine the MICs of MTB isolates with disrupted mutations by MGIT and microplate alamarBlue assay (MABA). We also aimed to determine the optimal breakpoints for MTh isolates with these mutations.