Next-generation sequencing reveals major and minority HIV-1 drug resistance mutations in treatment-experienced individuals with virological failure.

We analyzed major and minority HIV-1 drug resistance mutations in treatment-experienced individuals with virological failure using next-generation sequencing. Conventional population-based sequencing has limited sensitivity for variants present at low intra-host frequencies, which may obscure resistant subpopulations in heavily treated patients. A total of 188 plasma samples collected between 2013 and 2015 were evaluated, of which 147 were successfully amplified and sequenced on the Illumina MiSeq platform. Minority variants were defined as resistance-associated mutations detected at frequencies from 1% to <20% of high-quality mapped reads. Major and minority resistance-associated mutations were identified across the analyzed regions, with low-frequency variants detected mainly in protease and reverse transcriptase. These variants included clinically relevant substitutions associated with reduced susceptibility to protease inhibitors, NNRTIs, and NRTIs that would likely remain undetected by conventional sequencing. Integrase inhibitor-associated mutations were uncommon but were detected in patients with no reported prior exposure to integrase inhibitors, suggesting undocumented exposure, transmitted resistance, or other unexplained resistance patterns. Overall, NGS provided additional resolution for HIV-1 resistance profiling in individuals with extensive antiretroviral exposure by revealing low-frequency resistant subpopulations in protease and reverse transcriptase. These findings support the use of NGS as a complementary approach for HIV-1 resistance surveillance and interpretation of virological failure when considered alongside treatment history, viral load, sequencing depth, mutation frequency, and validated analytical thresholds.