Refined understanding of the impact of the Mycobacterium tuberculosis complex diversity on the intrinsic susceptibility to pretomanid.

Previous work reported unprecedented differences in the intrinsic in vitro susceptibility of the Mycobacterium tuberculosis complex (MTBC) to pretomanid (Pa) using the Mycobacteria Growth Indicator Tube (MGIT) system. We tested 125 phylogenetically diverse strains from all known MTBC lineages (1-9) without known Pa resistance mutations and four strains with known resistance mutations as controls. This confirmed that MTBC, unlike most bacteria-antimicrobial combinations, displayed substantial differences in the intrinsic susceptibility relative to the technical variation of Pa MIC testing. This was also the case for the Middlebrook 7H11 (7H11) medium, demonstrating that these differences were not specific to MGIT. Notably, lineage 1 was confirmed to have intrinsically elevated MICs compared with lineages 2, 3, 4, and 7 (L2-4/7), underlining the urgent need for WHO to publish its decision of whether lineage 1 should be deemed treatable by BPaL(M), the now preferred all-oral regimen for treating rifampin-resistant tuberculosis. Lineages 5 and 6, which are most frequent in West Africa, responded differently to Pa, with lineage 5 being more similar to L2-4/7 and lineage 6 being more susceptible. More data are needed to determine whether 7H11 MICs are systematically lower than those in MGIT. IMPORTANCE: This study confirmed that the Mycobacterium tuberculosis complex lineage 1, responsible for 28% of global tuberculosis cases, is less susceptible to pretomanid (Pa). It also refined the understanding of the intrinsic susceptibilities of lineages 5 and 6, most frequent in West Africa, and lineages 8 and 9. Regulators must review whether these in vitro differences affect the clinical efficacy of the WHO-recommended BPaL(M) regimen and set breakpoints for antimicrobial susceptibility testing accordingly. Notably, regulators should provide detailed justifications for their decisions to facilitate public scrutiny.

Re-evaluation of critical concentrations of antituberculosis fluoroquinolones in the Mycobacteria Growth Indicator Tube 960 system.

Background: Fluoroquinolones (FQs) have substantial activity against the Mycobacterium tuberculosis complex (MTBc) by preventing bacterial DNA synthesis through DNA gyrase inhibition. The reference standard for FQ-resistance testing is phenotypic drug-susceptibility testing (pDST) based on growth inhibition of MTBc in drug-containing Mycobacteria Growth Indicator Tube system (MGIT) media at a critical concentration (CC) that differentiates phenotypically wild-type from nonwild-type MTBc and at a clinical breakpoint that identifies strains that will likely still respond to treatment at higher doses. Despite the recent introduction of powerful new TB drugs, highly sensitive detection of clinically defined FQ resistance remains key. Method: In this study, we re-evaluated the current WHO-recommended CCs of Lfx (1.0 mg/ml), Mfx (0.25 mg/ml), Gfx (0.25 µg/ml), and the nowadays, obsolete CC of Ofx (2.0 mg/ml) for MGIT, using 147 MTBc isolates with known gyrA and gyrB sequences including both high-and low-level FQ resistance-conferring mutants. We tested a wide range of drug concentrations covering the current and former/obsolete WHO-recommended CCs for FQs and some intermediate concentrations to challenge the current WHO-recommended CCs. Results: The specificity of all four CCs was 100%. The sensitivities varied: 92.4% for Ofx and Lfx, 85.7% for Mfx, and 83.2% for Gfx. Lowering the CC of Mfx to 0.125 mg/ml would allow to correctly classify all wild-type and mutant isolates while lowering the CC of Gfx to 0.125 mg/ml would still misclassify some gyrA/gyrB mutants as susceptible. Conclusion: Based on our findings, a minimal inhibitory concentration of 0.125 mg/ml on MGIT medium is a more appropriate CC for Mfx and probably also as a surrogate for overall FQ resistance in the MTBc.

Middlebrook 7h11 reduces invalid results and turnaround time of phenotypic drug-susceptibility testing of M. tuberculosis.

Background: Phenotypic drug-susceptibility testing (pDST), which relies on growth inhibition in the drug-containing media, remains a challenge for fastidious Mycobacterium tuberculosis complex (MTBc) isolates due to insufficient growth on the growth controls (GC). Middlebrook 7H11 (M7H11) medium contains casein hydrolysate, which may favor the growth of such strains. Method: In this study, we tested whether M7H11 reduces invalid results due to insufficient growth on the GCs and the turnaround time (TAT) of pDST for MTBc compared to Middlebrook 7H10 (M7H10) without affecting the accuracy of the pDST results and how it differs between rifampicin- and isoniazid-susceptible non multi-drug resistant (non-MDR), MDR and MDR with additional resistance to fluoroquinolones (Pre-XDR) MTBc isolates. We compared the proportions of invalid pDST results due to lack of growth on the GCs, TATs of valid parallel drug-susceptibility testings as an indicator of speed of MTBc growth, and colony-forming unit (CFU) count on the most diluted GC of the parallel pDSTs after equal incubation periods as an indicator of growth abundance on M7H11 and M7H10. We also analyzed the agreement between the pDST results of the same drug or drugs in the same drug class, tested in parallel on both media. Results: For MDR and pre-XDR isolates, relative to M7H10, M7H11 significantly reduced the occurrence of invalid pDST results due to insufficient growth on the GCs (odds ratio [OR] = ∞ [95% confidence interval (CI) 1.9-∞], P = 0.004 for MDR, OR = ∞ [95% CI 3.3-∞], P = 0.0001 for pre-XDR) and the TAT of pDSTs (OR = 17 [95% CI 2.6-710.4], P = 0.0001 for MDR, OR = 9.3 [95% CI 4.0-26.5], P < 0.0001 for pre-XDR). The growth abundance of MTBc on M7H11 was significantly higher compared to M7H10 (17 CFU on M7H10 vs. 28 on M7H11), irrespective of drug-resistance profiles. The agreement between the pDST results between the two media was high (Cohen's k > 0.98). Conclusion: Our study findings suggest that M7H11 is preferred over M7H10 for pDSTs of MTBc isolates.