Nucleic Acid Amplification Testing for Point-of-Care Diagnostics of Bloodborne Viruses

Abstract

Access to effective diagnostics remains a critical barrier to healthcare equity in low-resource settings, where bloodborne diseases like HIV, hepatitis C, and Zika continue to significantly contribute to global morbidity and mortality rates. Early diagnosis is essential for timely treatment initiation, disease monitoring, and transmission prevention, yet access to centralized laboratory testing remains limited in many regions due to infrastructure, equipment, and personnel requirements. Point-of-care (POC) molecular diagnostics have the potential to overcome these barriers by providing access to rapid, sensitive, and low-cost testing at the point of patient care. The widespread implementation of point-of-care nucleic acid amplification tests (POC NAATs) remains constrained due to the complexity of sample preparation, amplification, detection, and overall user requirements. This dissertation addresses several key challenges in the development of simplified POC NAATs by improving paper-based isothermal NAAT reactions, simplifying sample preparation for blood, testing with clinical samples, and reducing user steps through workflow integration. To enhance amplification performance in paper-based recombinase polymerase amplification (RPA) reactions, I developed a vibration-based platform to improve reagent redistribution and reaction kinetics within porous membranes. This approach significantly enhanced reaction performance, resulting in improved overall amplification, lower limits of detection, and reduced time-to-threshold when compared to typical unmixed paper-based reactions. To address challenges associated with sample preparation from blood-derived samples, I developed and integrated an extraction-free workflow that enables the direct addition of thermally incubated samples into scalable RT-RPA reactions. In parallel, I formulated a concentrated custom RPA rehydration buffer that improved assay repeatability, robustness, and increased allowable sample volume. The extraction-free amplification workflow was evaluated using both cultured HIV in plasma and HCV clinical samples. Using a scalable reaction format, the assay achieved a sensitivity of 800 copies/mL for HIV plasma. Successful amplification from HCV clinical samples further demonstrated the feasibility of the approach and provided early validation that this workflow could successfully process and detect viral RNA from clinical serum specimens. Finally, I developed a sample-to-answer cartridge and reader NAAT platform designed to further reduce user steps, minimize instrumentation requirements, and maintain an overall assay time under 30 minutes. By integrating sample preparation and amplification components into a more streamlined format, this platform moves toward a practical implementation of molecular testing in decentralized settings. Overall, this work advances the development of simplified, low-cost, and portable molecular diagnostic workflows that are better suited for POC use in resource-limited environments. Through improvements in amplification performance, sample preparation simplification, and device integration, these contributions help address critical barriers to the broader development of POC molecular diagnostics.