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We sought to define the prevalence of pretreatment integrase strand transfer inhibitor (INSTI) resistance and assess the transmission networks of those with pretreatment INSTI resistance.A retrospective cohort study of HIV-positive patients with genotypic resistance testing sent to a single referral laboratory in North Carolina between 2010 and 2016.We linked genotype and public health data for in-care HIV-positive individuals to determine the prevalence of INSTI resistance among treatment-naive (defined as those with a first genotype ≤3 months after diagnosis) and treatment-experienced (defined as those with a first genotype >3 months after diagnosis) patients. We performed molecular and phylogenetic analyses to assess whether pretreatment INSTI resistance mutations represented clustered HIV transmission.Of 8825 individuals who contributed sequences for protease, reverse transcriptase, or INSTI genotypic resistance testing during the study period, 2784 (31%) contributed at least one sequence for INSTI resistance testing. Of these, 840 were treatment-naive individuals and 20 [2.4%, 95% confidence interval (CI): 1.5, 3.6%] had INSTI mutations; only two (0.2%, 95% CI: 0.02, 0.9%) had major mutations. Of 1944 treatment-experienced individuals, 9.6% (95% CI: 8.3, 11.0%) had any INSTI mutation and 7.0% (95% CI: 5.9, 8.3%) had major mutations; the prevalence of INSTI mutations among treatment-experienced patients decreased overtime (P < 0.001). In total 12 of 20 individuals with pretreatment INSTI mutations were part of 10 molecular transmission clusters; only one cluster shared identical minor mutations.The prevalence of major pretreatment INSTI resistance is very low. Pretreatment INSTI mutations do not appear to represent clustered HIV transmission.