Paper-based Nucleic Acid Amplification Testing at the Point-of-Care for HIV Viral Load Monitoring

Abstract

Major advancements have been made in the past forty years to combat the HIV pandemic. Viral load testing, a type of nucleic acid amplification test (NAAT), is a crucial tool in early detection, monitoring antiretroviral therapy efficacy, and maintaining viral suppression. However, there is a significant lack in access to routine viral load testing, particularly in low- and middle-income countries where the burden of disease is often the highest. Current platforms are prohibitively expensive and require trained technicians, stable electricity, and established cold-chain logistics. These requirements limit traditional NAAT platforms to centralized laboratories, reducing access and delaying linkage to appropriate care. There is an urgent need to develop point-of-care (POC) NAAT devices for disseminated HIV viral load testing. In this dissertation, I describe novel technologies and advancements in various areas of the NAAT workflow for point-of-care implementation: sample preparation, amplification, and detection. While this work is focused primarily on HIV, many of the same processes can be applied to other bloodborne viruses, such as Hepatitis B, Hepatitis C, Zika virus, Ebola virus, or malaria.I first describe a paper-and-plastic device that leverages isotachophoresis (ITP) for the electrokinetic extraction and purification of target DNA from a whole blood sample with minimal user steps. The device integrates on-chip high efficiency blood fractionation, proteolytic digestion of plasma proteins, and isotachophoretic extraction of nucleic acids with off-chip recombinase polymerase amplification (RPA). I detail steps towards modifying this device for extraction of viral HIV RNA. This requires the development and integration of lyophilized RNase inactivation chemistries onto paper membranes. I then discuss a novel method for the quantification of RPA reactions on paper membranes for point-of-care settings using amplification nucleation site analysis and show that this method can more accurately quantify extracted viral HIV RNA across subtypes compared to traditional tube-based reactions. The work presented in this dissertation describes advancements towards paper-based point-of-care platforms for low-cost disseminated viral load testing (and NAAT testing as a whole) with minimal user steps and cost.