Objective: 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. Design: 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. Methods: 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. Results: 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. Conclusion: The prevalence of major pretreatment INSTI resistance is very low. Pretreatment INSTI mutations do not appear to represent clustered HIV transmission
