Abstract: Current self-tests for HIV rely on detection of antibodies to the virus, and thus are not useful during the early stages of infection. These assays are also not appropriate for patients on antiretroviral therapy who may be at risk for viral rebound, as pre-existing anti-HIV antibodies mask changes in viral load. Conversely, current HIV diagnostics based on detection of viral RNA are sensitive, but too complex and expensive to be appropriate for self-testing. To address this critical need, we will develop the first enzyme-free sample-to-answer HIV viral load self-test, in which a fingerstick volume of blood is passively processed leading to amplification of the isolated HIV genomic RNA. The RNA amplification strategy will rely on hairpin cascade reactions (HCR), a DNA nanotechnology approach with proven single-copy sensitivity in other contexts, in a sessile droplet implementation. The entire assay will be implemented in an inexpensive, disposable, paper-based consumable. In the R61 phase, we propose to: (1) building on methods we have previously validated, optimize passive processing of whole blood coupled with paper-based viral lysis and RNA isolation; (2) complete development of HCR-based amplification of HIV genomic RNA; (3) integrate sample processing and RNA amplification into a single consumable; and (4) obtain feedback on assay design and usability from patients at risk for HIV infection, and develop a pilot manufacturing plan. In the R33 phase, we will (1) scale production of the assay with contract manufacturers in quantities suitable for pilot testing, and (2) complete testing of the assay with a cohort of 1000 patients at-risk for HIV infection, recruited from patients visiting the Emergency Department at the University of Rochester Medical Center.