Project Summary Over 20 million people living with HIV (PLHIV) are receiving antiretroviral therapy and require HIV viral load testing to identify cases of virological failure and provide actionable information to guide alternative clinical treatment. Current methods for HIV viral load measurement rely on quantitative PCR (qPCR) or digital PCR (dPCR), which are commonly restricted to highly resourced central laboratories and there is a need to decentralize HIV viral load monitoring to enable rapid, clinic-based or home self-testing viral load measurements. We have identified a novel method for implementing digital isothermal amplification that leverages the characteristic viscous reaction buffer of recombinase polymerase amplification (RPA) isothermal amplification chemistry and commercially available porous membranes. We propose to apply amplification nucleation site analysis (ANSA) for HIV-1 viral load monitoring to accurately quantify HIV-1 RNA over clinically relevant HIV-1 subtypes and viral loads. We propose two exploratory aims to demonstrate that ANSA can achieve the required viral load dynamic range and quantitative precision across HIV-1 subtypes.