Epidemiological cut-offs for Sensititre susceptibility testing of Mycobacterium tuberculosis: interpretive criteria cross validated with whole genome sequencing.

Universal drug susceptibility testing (DST) is an important requirement of the End TB Strategy. The Sensititre broth micro-dilution assay (BMD) tests multiple drugs quantitatively. We defined interpretive criteria for this assay and analysed genotypic-phenotypic relationships. 385 Mycobacterium tuberculosis clinical isolates were processed for BMD and whole genome sequencing. The epidemiological cut-off value 99% (ECV99) amongst genotypically wild type (gWT) strains defined susceptibility. Minimum inhibitory concentration distributions of the resistance-associated variants (RAVs) for each drug were analysed. Susceptibility (µg/mL) criteria were determined as follows: rifampicin (≤0.125), isoniazid (≤0.25), ethambutol (≤2.0), moxifloxacin (≤0.5), levofloxacin (≤1.0), amikacin (≤2.0), kanamycin (≤8.0), capreomycin (≤4.0), clofazimine (≤0.25) and linezolid (≤2.0). Most drugs showed clear separation between gWT and RAV. Isoniazid showed a tri-modal pattern with 14/17 strains at ECV99 harbouring a fabG1 c. -15C > T RAV. Ethambutol RAVs at embB codons 306, 405 and 497 were responsible for resistance and showed differential distributions. Moxifloxacin RAVs (gyrA codon 90) were a dilution or two higher than the ECV99 while gyrB RAVs were uncommon and showed drug specific resistance propensity. Interpretive criteria established were robust facilitating progress towards universal DST and individualised precision medicine. This study demonstrates the value of quantitative DST to accurately interpret mutation data.

Defining Bedaquiline Susceptibility, Resistance, Cross-Resistance and Associated Genetic Determinants: A Retrospective Cohort Study.

BACKGROUND: Bedaquiline (BDQ) is a novel agent approved for use in combination treatment of multi-drug resistant tuberculosis (MDR-TB). We sought to determine BDQ epidemiological cut-off values (ECVs), define and assess interpretive criteria against putative resistance associated variants (RAVs), microbiological outcomes and cross resistance with clofazimine (CFZ). METHODS: A retrospective cohort study was conducted. Minimal inhibitory concentrations (MIC) to BDQ were determined using 7H9 broth microdilution (BMD) and MGIT960. RAVs were genetically characterised using whole genome sequencing. BDQ ECVs were determined using ECOFFinder and compared with 6-month culture conversion status and CFZ MICs. FINDINGS: A total of 391 isolates were analysed. Susceptible and intermediate categories were determined to have MICs of ≤0.125µg/ml and 0.25µg/ml using BMD and ≤1µg/ml and 2µg/ml using MGIT960 respectively. Microbiological failures occurred among BDQ exposed patients with a non-susceptible BDQ MIC, an Rv0678 mutation and ≤2 active drug classes. The Rv0678 RAVs were not the dominant mechanism of CFZ resistance and cross resistance was limited to isolates with an Rv0678 mutation. INTERPRETATION: Criteria for BDQ susceptibility are defined and will facilitate improved early detection of resistance. Cross- resistance between BDQ and CFZ is an emerging concern but in this study was primarily among those with an Rv0678 mutation.