Parent Grant: 1DP2DA051910 Parent Grant Title: Point-of-use Acute HIV Infection Diagnostic for Substance Using Populations Supplement Title: Expansion of Point-of-use Acute HIV Infection platform for Hepatitis C Virus An estimated 2.3 million individuals with HIV infection are co-infected with hepatitis C (HCV). In the US, people who use drugs (PWUD) are at high risk for both infections. HIV accelerates liver cirrhosis caused by HCV and contributes to the 350,000 deaths that occur due to HCV-related liver disease each year. Unfortunately, existing widely used rapid diagnostic tests (RDTs) for HCV do not detect the virus itself but instead detect only the anti-HCV antibodies produced by the infected individual. This antibody-based detection presents two challenges. First, host antibodies develop long after initial exposure to HCV which can allow for infections to remain undetected and potentially spread to other individuals. Second, anti-HCV antibodies remain even after HCV clearance and can result in false positive results that do not differentiate a current infection from a past infection. In contrast, test targeting the virus itself, such as nucleic acid amplification tests (e.g. RT-PCR), have the potential to detect both acute and chronic HCV infections. However, these assays typically require cold-chain storage of reagents, significant sample preparation, and extensive laboratory infrastructure to run. While PWUD are willing to get tested for HIV and HCV at non-traditional sites such as rural pharmacies (Duong et al 2019), current laboratory-based RT-PCR tests are unable to reach these individuals who are already underserved by traditional healthcare. Thus, there is an urgent need for a radically new approach to diagnose HCV among PWUD in order to engage infected individuals in the care continuum at the earliest stages of infection and prevent progression and transmission of HCV. In this supplement, we propose to adapt our current platform technology for acute HIV or order to to detect acute HCV thresholds above the clinically relevant 3000 HCV IU/mL (Reipold et al 2017) at point-of-use settings. Our ongoing DP2 is based on our handheld sample-to-answer technology, an automated microfluidic rapid autonomous analytical device (microRAAD). We have demonstrated that microRAAD is capable of detecting HIV virons directly from a whole blood samples. We incorporated dried amplification reagents and wax valves in paper-based substrates with resistive heating elements and low-power circuitry. By combining feedback-controlled heating of the RT-LAMP assay and wax valves with paper’s capillary flow, our assembled device automatically isolates viral particles from human blood, lysis the virus, amplifies HIV-1 RNA, and transports products to a detection zone with familiar visual lateral flow assay readout. This entire process requires only 3 simple steps for the user: first, a drop of blood is added to the sample pad, next, the user dispenses a buffer solution, and after a brief waiting period, the user reads the visual yes/no result. We have demonstrated that the fully integrated microRAAD detects as few as 10^5 HIV-1 vp/reaction directly from whole blood in under 90 minutes. In the ongoing DP2, we are working with local populations of PWUD and HIV service providers to evaluate feasibility, acceptability, and usability of the technology and to further reduce this detection to below 10^3 HIV vp/mL by combining p24 protein-based targeting and RT-LAMP together as well as improving clinical relevance via integration of an internal amplification control. Our next steps to adapt MicroRAAD to acute HCV detection will include: 1) Develop and optimize a novel assay for HCV detection targeting the conserved regions of the genome. For HCV assay design we will compare the 5’non-coding region (5’-NCR) and the 3’untranslated region (3’UTR) which have both been used in clinical HCV viral load assays. While the 5’-NCR is used in western assays, it’s known that the variability in this region is somewhat divergent from the sequences circulating worldwide. Therefore, we will also consider the 3’UTR for RT-LAMP design. We will generate at least two primers to each region and will compare the time to amplification (TTA), limit of detection (spiking in 0-10^7 IU/mL of synthetic RNA purchased from ATCC), and resistance to whole blood and plasma spiked into each sample at concentrations ranging from 0-20%. 2) Expand our community-based participatory research with PWUD and their service providers to evaluate HCV-focused detection at the point-of-use in concert with HIV detection. This will include performing feasibility and acceptability studies to evaluate the operational, clinical, and market needs specific for HCV detection by and for PWUD at the POU, evaluate how socio-cultural factors and systemic barriers to care among PWUD impact their HCV testing and integration into the current HIV care continuum and assess usability of device prototypes to determine the best operational protocols among stakeholders. This platform for point-of-use HCV/HIV detection could transform both testing and treatment of HIV and HCV among PWUD by bringing acute infection detection to substance abuse treatment facilities, needle exchanges, and injection drug clinics and will elucidate the targeted interventions needed to remove barriers to care. References: Reipold IE, Easterbrook P, Trianni A, Panneer N, Krakower D, Ongarello S, Roberts T, Miller V, Denkinger C. Optimising diagnosis of viraemic hepatitis C infection: the development of a target product profile. BMC Infect Dis. 2017 Nov 1;17(Suppl 1):707. doi: 10.1186/s12879-017-2770-5. PMID: 29143620; PMCID: PMC5688443. Duong M, Delcher C, Freeman PR, Young AM, Cooper HLF. Attitudes toward pharmacy-based HCV/HIV testing among people who use drugs in rural Kentucky. J Rural Health. 2022 Jan;38(1):93-99. doi: 10.1111/jrh.12564. Epub 2021 Mar 5. PMID: 33666274; PMCID: PMC8418619.

PARENT GRANT NARRATIVE People who use drugs, are 22 times more likely to acquire HIV than the average population and are at higher risk of transmitting HIV to others, yet are less likely to be diagnosed due to limited healthcare access. The goals of this proposal are to understand how systemic barriers to care limit HIV testing and treatment among people who use drugs, and to develop and evaluate a highly sensitive handheld diagnostic for acute HIV infection that can be used to diagnose HIV among people who use drugs in order to prevent transmission of HIV during the earliest, and most communicable, stage of infection.