PROJECT SUMMARY Unhealthy alcohol use, common among persons with (PWH) and at risk for HIV, is associated with multiple deleterious effects that increase the risk of HIV transmission. However, identifying individuals with unhealthy alcohol use in practice remains challenging because self-report consistently underrepresents true alcohol consumption. An objective measure of alcohol use, with clearly defined associations with clinical outcomes relevant for HIV prevention and transmission, would eliminate the time and resources dedicated to trying to subjectively quantify alcohol use, and would instead direct efforts towards swift and effective clinical interventions. Phosphatidylethanol (PEth) holds tremendous untapped potential as an objective, quantitative alcohol biomarker that could fill this unmet need. However, PEth has primarily been used in a qualitative and inconsistent manner which complicates interpretation and hinders actionable interventions. PEth's application to HIV prevention and treatment is also limited, especially with contemporary HIV pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART) with injectable long-acting agents such as cabotegravir (CAB-LA) and cabotegravir/rilpivirine (CAB/RPV-LA). Delays in PEth concentration results in practice also impedes real-time interventions. This proposal will address these significant knowledge gaps and is directly responsive to RFA- AA-21-016. The long-term objective of this work is to advance PEth as an objective and actionable alcohol biomarker for HIV prevention and treatment. This will be accomplished as follows: Aim 1. Establish the relationship between PEth concentrations, PrEP adherence, and HIV acquisition among persons at risk for HIV. Using a seminal PrEP trial, HPTN-083, which compared CAB-LA vs. emtricitabine/tenofovir disoproxil fumarate (F/TDF) for HIV prevention in men and transgender women who have sex with men, the relationship between PEth and (1) time to drop-out, (2) time between cabotegravir/placebo injections, (3) intracellular tenofovir-diphosphate concentrations (an objective PrEP adherence biomarker) and (4) the risk of acquiring HIV will be established. Aim 2. Determine the ability of PEth to predict the undetectable=untransmissible (U=U) threshold among PWH with adherence barriers. To prevent HIV transmission and end the epidemic, suppression of viral replication to <200 copies/mL among PWH is essential. ACTG A5359 is a trial comparing CAB/RPV-LA vs. oral ART in PWH with a history of non-adherence to ART. A5359 includes a 12-24 week oral ART induction period with economic incentives (Step 1), followed by randomization to continued oral ART vs. monthly CAB/RPV-LA injections for 52 weeks (Step 2). The primary objective is to determine the ability of PEth in Step 1 to predict failure to achieve the U=U threshold in Steps 1 and 2. Aim 3. Develop a point-of-care test for PEth in whole blood. Using a commercially available miniature mass spectrometer, a point...