Intra-Host Evolution Among Drug-Resistant Tuberculosis Patients Failing Treatment.

Background: Understanding () intra-host evolution of drug resistance is important for successful drug-resistant tuberculosis (DR-TB) treatment and control strategies. This study aimed to characterise the acquisition of genetic mutations and low-frequency variants associated with treatment-emergent drug resistance in longitudinally profiled clinical isolates from patients who experienced DR-TB treatment failure.

Patients And Methods: We performed deep Whole Genome Sequencing on 23 clinical isolates obtained longitudinally across nine timepoints from five patients who experienced DR-TB treatment failure enrolled in the CAPRISA 020 InDEX study. The minimum inhibitory concentrations (MICs) were established on the BACTEC™ MGIT 960™ instrument on 15/23 longitudinal clinical isolates for eight anti-TB drugs (rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, linezolid, clofazimine, bedaquiline).

Results: In total, 22 resistance associated mutations/variants were detected. We observed four treatment-emergent mutations in two out of the five patients. Emerging resistance to the fluoroquinolones was associated with 16- and 64-fold elevated levofloxacin (2-8 mg/L) and moxifloxacin (1-2 mg/L) MICs, respectively, resulting from the D94G/N and A90V variants in the gene. We identified two novel mutations associated with elevated bedaquiline MICs (>66-fold): an emerging frameshift variant (D165) on the gene and R409Q variant on the gene present from baseline.

Conclusion: Genotypic and phenotypic resistance to the fluoroquinolones and bedaquiline was acquired in two out of five patients who experienced DR-TB treatment failure. Deep sequencing of multiple longitudinal clinical isolates for resistance-associated mutations coupled with phenotypic MIC testing confirmed intra-host evolution.