PROJECT SUMMARY HIV drug resistance may compromise the UNAIDS 90-90-90 treatment goals and is a major hurdle to sustainable antiretroviral therapy success. Though resistance testing is recommended by guidelines for patient care in developed countries, there are existing research gaps in its understanding; genotypic-phenotypic correlations in diverse HIV-1 subtypes, the relationship between minority drug resistance variants and treatment outcomes, and reasons for discordances between genotype and treatment success or failure remain unclear. These research gaps are of particular concern in resource-limited settings, where limited medications and sub-optimal monitoring are common; and in children and adolescents, a vulnerable population with the need for life-long therapy, who have higher levels of HIV-1 RNA, a wider treatment gap with fewer on therapy, lower rates of suppression, limited formulations and more non-adherence. The long-term goal of our research team is to provide new evidence to improve the clinical disease management children and adolescents living with HIV in resource-limited settings. The purpose of this proposal is to address existing drug resistance research gaps in a previously established, carefully characterized cohort of 499 children and adolescents living with HIV in Kenya. To do this, we will use our successful collaboration with the Academic Model Providing Access to Healthcare (AMPATH) in Kenya, one of the largest HIV programs in sub-Saharan Africa, and uniquely leverage existing resources from our ongoing R01 AI120792 on perinatally-infected children and adolescents at AMPATH (MPI Kantor and Vreeman). We hypothesize that comprehensive investigations of genotypic-phenotypic and resistance-treatment outcome discordances in diverse non-B subtypes will resolve some of these existing research gaps and optimize patient care in settings where it is most needed. We will address this hypothesis with the following Aims: (1) Investigate genotype-phenotype correlations; (2) Evaluate etiologies for treatment failure in the presence of a ?susceptible genotype?; and (3) Evaluate etiologies for treatment success in the presence of a ?resistant genotype?. To achieve these aims, we will use already available samples (collected between 2016-2018) from the existing cohort of Kenyan children and adolescents living with non-B subtypes (Aim 1); longitudinally follow the cohort for four years (Aims 2 and 3); collect blood samples bi-annually and assess adherence; identify participants that are eligible for proposed additional investigations; conduct in vitro phenotyping to examine genotypic-phenotypic correlations; and perform near full-length next generation sequencing on RNA and DNA to investigate minority resistance variants and alternative mechanisms of resistance. The proposal is innovative in the uniqueness of the cohort studied, the comprehensive proposed scientific investigations, and study design and evaluation of discordant genotype- treatment outcomes scenarios. These investigations and directly addressing the existing knowledge gaps in HIV-1 drug resistance in a particularly-vulnerable population with diverse non-B subtypes, will have a high impact on improving treatment outcomes in children, adolescents, and adults living with HIV.