PROJECT ABSTRACT In the United States alone, over 2 million people live with active hepatitis C virus (HCV) infections, and up to 140,000 new infections occur each year. Since 2013, highly effective direct-acting antiviral therapy has been available, allowing HCV to be cured with short courses of well-tolerated medications. However, HCV diagnostics now present the greatest barrier to elimination. The current diagnostic workflow involves two- stage testing with initial screening for HCV antibodies followed by molecular testing for viral RNA. Therefore, an accurate, simple, rapid, and affordable molecular test for viral RNA is needed to increase detection of HCV and important coinfections (such as HIV), eliminate cumbersome two-stage testing, and allow rapid planning of subsequent care after a single visit. The development of such an economical, rapid HCV molecular test is the expressed purpose of the NIH Notice of Special Interest (NOT-AI-23-001) to which this proposal is a response. However, two key barriers exist to low-cost implementation of molecular methods for RNA viruses: 1) lack of automated stand-alone low-cost RNA extraction and stabilization technologies, and 2) complicated designs and/or expensive proprietary detection technologies for isothermal methods. Research in this proposal overcomes these barriers with novel chemistries and bespoke microfluidics to enable minimally trained healthcare workers to perform on-demand HCV/HIV duplex molecular testing in the clinic. To do so, Aim 1 will adapt and automate RNA Sample Extraction and Stabilization (RNAES) technology for HCV/HIV RNA extraction from capillary whole blood. Building on newly obtained preliminary results, the current protocol will be streamlined to extract and store HCV/HIV RNA from plasma, RNAES technology will be adapted for whole blood, and 3D-printed programmed stand-alone microfluidics will be created to automate RNAES and create a sample- in, RNA-out device. Along with this, Aim 2 will apply chemical innovations and microfluidics to Transcription- mediated amplification (TMA) for standalone colorimetric detection of HCV/HIV RNA. Preliminary data shows that detection can be achieved with a colorimetric readout suitable for low-complexity settings and a simple hand- mixed microfluidic will create a self-contained readout and avoid potential contamination from adding reagents post-amplification. In Aim 3, external user testing and a clinical evaluation of the HCV/HIV detection system will be performed in partnership with an external testing site at Stanford University. Separately, based on the final RNAES whole blood device and workflow, a prospective study to test capillary blood samples from individuals with HCV and/or HIV will be carried out. This proposal has been carefully designed to enable rapid scaling and translatability to the market. Achieving either device from Aim 1 or Aim 2 will represent a significant advance that can be leveraged to improve HCV testing. Combined, these technologies will have an important positive impact on RNA virus detection through development of an economical, agile molecular detection system.

PROJECT NARRATIVE Current hepatitis C virus diagnostics present the greatest barrier to viral cure and elimination, and an accurate, simple, rapid, and affordable molecular test for viral RNA is needed to increase detection of hepatitis C virus along with important coinfections such as human immunodeficiency virus. This proposal seeks to eliminate key barriers to low-cost molecular testing for RNA viruses using novel chemistries and bespoke microfluidics that enable minimally trained healthcare workers to perform on-demand duplex molecular testing in the clinic. Achieving device development proposed in Aim 1 or Aim 2 will represent a significant advance that can be leveraged to improve hepatitis C testing, and combined, these technologies will have an important positive impact on RNA virus detection with an economical, agile molecular detection system.