Characterisation of Bedaquiline Resistance-Associated Mutations in Drug-Resistant Tuberculosis Phenotypes

Technical Summary: Characterisation of Bedaquiline Resistance-Associated Mutations in Drug-Resistant Tuberculosis Phenotypes in Eswatini

Problem Statement
Bedaquiline (BDQ) has become a cornerstone of treatment for drug-resistant tuberculosis (DR-TB), yet resistance to this drug is emerging. In Eswatini, a high TB-burden country, the prevalence of BDQ resistance is reported at an alarming 36%. Despite this, there is a critical lack of data profiling the specific genetic mutations associated with BDQ resistance within the Eswatini population. Standard diagnostic methods, such as culture-based drug susceptibility testing and molecular assays (e.g., GeneXpert, Line Probe Assay), often fail to detect certain clinically relevant resistance mutations or cannot fully characterize complex resistance mechanisms. This gap hinders the timely selection of optimal therapies and effective public health responses.

Methodology
This prospective study enrolled 60 participants with various DR-TB phenotypes (HR-TB, RR-TB, MDR-TB, and pre-XDR-TB) from four major health facilities across three regions of Eswatini (Manzini, Shiselweni, and Hhohho) between June 2022 and November 2023.

• Sample Processing: Mycobacterium tuberculosis isolates were cultured using the BACTEC MGIT™ 960 system. DNA was extracted from subcultures using the cetyltrimethylammonium bromide (CTAB) method.
• Sequencing: Culture-based Whole-Genome Sequencing (WGS) was performed on the Illumina NextSeq platform, generating 150-base-pair paired-end reads with a minimum depth of 100X.
• Bioinformatics: Raw sequencing data were analyzed using TB-Profiler (version 6.6.6) to identify single-nucleotide polymorphisms (SNPs) and resistance-associated variants based on the WHO mutation catalogue (2nd edition).
• Statistical Analysis: Associations between specific mutations and DR-TB phenotypes, as well as phenotypic BDQ resistance, were evaluated using SPSS version 25, employing Chi-square tests to determine statistical significance.

Key Results
The study identified a complex landscape of genetic variability regarding BDQ resistance:

• Prevalence of Mutations: 35% (21/60) of isolates harbored at least one mutation associated with confirmed BDQ resistance. However, the majority of isolates (63%) carried mutations classified as "interim" (lacking sufficient clinical data for permanent resistance classification) or of "uncertain significance." Specifically, the paper notes that n=34 (57%) contained mutations classified as interim, while n=1 (2%) were of uncertain significance.
• Predominant Variants: The most frequent mutations observed were Rv0676c_p.Ile948Val (92%) and Rv1979c_c.-129A>G (83%). According to WHO classifications, these are interim resistance-associated mutations, often linked to Lineage 4 strains common in Eswatini, rather than primary drivers of phenotypic resistance.
• Confirmed Resistance: The Rv0678_p.Met146Thr mutation was the predominant confirmed resistance-associated variant, found in 16 isolates. Notably, this mutation was significantly associated with phenotypic BDQ resistance (p < 0.001).
• Phenotype-Genotype Correlation:
  • Rv0678_p.Met146Thr was significantly associated with MDR-TB (39%) and pre-XDR-TB (75%) phenotypes (p = 0.011).
  • Rv1979c_c.129A>G, Rv0676c_p.Ile948Val, Rv3245c_p.Met517Leu, and Rv0676c_c.1065G_T showed significant associations with various DR-TB phenotypes (p < 0.05).
  • Crucially, while several mutations were associated with DR-TB phenotypes, they did not show a statistically significant association with phenotypic BDQ resistance.
• Pre-existing Resistance: A significant finding was that 25% of the study population (15/60), specifically those identified as HR-TB cases with no prior BDQ exposure, possessed pre-existing Rv0678/mmpR5 mutations.

Significance and Claims
The authors claim this study provides the first WGS characterization of BDQ resistance-associated mutations in Eswatini. The primary significance of the findings lies in the distinction between mutations that are merely lineage-associated polymorphisms and those that confer actual phenotypic resistance.

• Diagnostic Implications: The study suggests that relying solely on treatment history to predict BDQ resistance is inadequate, as resistance-associated variants (specifically Rv0678/mmpR5) are circulating in the population independent of direct drug pressure.
• Surveillance Value: The detection of Rv0678_p.Met146Thr in HR-TB cases without prior exposure underscores the necessity for proactive, population-level genomic surveillance.
• Clinical Guidance: The authors conclude that while many isolates carry genetic alterations in BDQ-related genes, only Rv0678_p.Met146Thr demonstrated a strong genotype-phenotype concordance for BDQ resistance in this cohort. Therefore, integrating WGS into routine DR-TB surveillance is essential to distinguish between lineage-specific variants and true resistance determinants, thereby guiding personalized treatment strategies and preventing the initiation of suboptimal regimens.

The paper modestly notes limitations, including a relatively small sample size and the cross-sectional nature of the study, which prevents determining if pre-existing mutations contribute to the progression from HR-TB to MDR-TB. Future longitudinal studies are recommended to investigate these dynamics.