Resistance-gene minority variation and delayed microbiological response in rifampicin-resistant tuberculosis

Routine whole-genome sequencing (WGS) interpretation of Mycobacterium tuberculosis resistance focuses mainly on consensus-level mutations. Minority variants, quantified by allele frequency (AF), may indicate resistant subpopulations under drug pressure, but their association with treatment response in rifampicin-resistant or multidrug-resistant tuberculosis (RR/MDR-TB) remains unclear.

We retrospectively analysed RR/MDR-TB patients treated in Ningbo, China, from 2021 to 2025, integrating clinical, phenotypic, treatment, microbiological, and WGS data. Minority resistance-associated variants (RAVs) were defined as low-frequency variants matching WHO V2 mutation catalogue entries at AF ≥ 3%, with AF ≥ 5% defining the primary higher-frequency subset. Per-drug analyses excluded patients whose target genes had median depth <200×. Associations with microbiological response were assessed using covariate-adjusted logistic regression, with higher-frequency and treatment-matched variants examined secondarily.

We analysed 229 RR/MDR-TB patients with linked WGS, treatment, and microbiological follow-up data. Fixed RAVs (WHO V2 catalogue-annotated) were carried by 220/229 patients (median 3, IQR 2-5). Minority RAVs at AF ≥ 3% were detected in 13 patients. Overall minority-RAV burden was not clearly associated with microbiological response after adjustment. The clearest signal involved treatment-matched AF ≥ 5% minority RAVs, whose carriers had a higher 6-month culture-positivity rate than non-carriers (4/7 [57.1%] vs 40/198 [20.2%]; adjusted odds ratio [aOR] 7.21, 95% CI 1.38-43.68; p = 0.020; q = 0.034), with a modest AUC increase from 0.675 to 0.695. Six-month positivity was highest among patients with both high fixed RAV burden and treatment-matched AF ≥ 5% minority RAVs (4/5 [80.0%] vs 15/106 [14.2%] in the low-burden non-carrier reference; Fisher's exact p = 0.003).

Minority variants showed limited overall prognostic value, but higher-frequency treatment-matched variants provided a coherent signal for 6-month culture positivity when considered with fixed RAV burden. These exploratory findings support a cautious, treatment-aware review of selected minority variants and require external validation before clinical use.