Comparison of Different HIV-1 Resistance Interpretation Tools for Next-Generation Sequencing in Italy

background: next-generation sequencing (NGS) is gradually replacing Sanger sequencing for HIV genotypic drug resistance testing (GRT). this work evaluated the concordance among different NGS-GRT interpretation tools in a real-life setting. methods: routine NGS-GRT data were generated from viral RNA at 11 italian laboratories with the AD4SEQ HIV-1 solution v2 commercial kit. NGS results were interpreted by the smartVir system provided by the kit and by two online tools (HyDRA web and stanford HIVdb). NGS-GRT was considered valid when the coverage was >100 reads (100×) at each PR/RT/IN resistance-associated position listed in the HIVdb 9.5.1 algorithm. results: among 629 NGS-GRT, 75.2%, 74.2%, and 70.9% were valid according to smartVir, HyDRA Web, and HIVdb. considering at least two interpretation tools, 463 (73.6%) NGS-GRT had a valid coverage for resistance analyses. the proportion of valid samples was affected by viremia <10,000-1000 copies/mL and non-b subtypes. mutations at an NGS frequency >10% showed fair concordance among different interpretation tools. conclusion: this Italian survey on NGS resistance testing suggests that viremia levels and HIV subtype affect NGS-GRT coverage. within the current routine method for NGS-GRT, only mutations with frequency >10% seem reliably detected across different interpretation tools.