Genomic markers of drug resistance in populations with minority variants.

Minority variants of harboring mutations conferring resistance can become dominant populations during tuberculosis (TB) treatment, leading to treatment failure. Our understanding of drug-resistant within-host subpopulations and the frequency of resistance-conferring mutations in minority variants remains limited. sequences recovered from liquid cultures of culture-confirmed TB cases notified between January 2017 and December 2021 in New South Wales, Australia were examined. Potential drug resistance-conferring minority variants were identified using LoFreq, and mixed populations of different strains (≥100 SNPs apart) were examined using QuantTB. A total of 1831 routinely sequenced strains were included in the analysis. Drug resistance-conferring minority variants were detected in 3.5% (65/1831) of sequenced cultures; 84.6% (55/65) had majority strains that were drug susceptible and 15.4% (10/65) had majority strains that were drug resistant. Minority variants with high-confidence drug resistance-conferring mutations were 1.5 times more common when the majority strains were drug resistant. Mixed strain populations were documented in 10.0% (183/1831) of specimens. Minority variants with high-confidence drug resistance-conferring mutations were more frequently detected in mixed strain populations (2.7%, 5/183) than in single strain populations (0.6%, 10/1648; = 0.01). Drug-resistant minority variants require monitoring in settings that implement routine sequencing. The frequency with which drug-resistant minority variants are detected is likely influenced by pre-culture requirement. Culture-independent sequencing methods should provide a more accurate reflection of drug-resistant subpopulations.