Development towards Sensitive Point-of-Care Tests for HIV and COVID-19

Abstract

Human immunodeficiency virus (HIV), with approximately 39 million people living with HIV globally, persists as one of the foremost challenges to global health. Sensitive HIV testing is needed for both early diagnosis and routine viral load monitoring in patients receiving antiretroviral therapy (ART). However, the most sensitive testing approach, rt-qPCR, is limited to centralized laboratories. A sensitive point-of-care HIV test using fingerstick samples could expand testing coverage, especially in low-resource settings. In the thesis, I explored two independent approaches aiming towards a simple yet sensitive HIV test. The first approach targets the reverse transcription activity of the reverse transcriptase (RT) enzyme encapsulated inside HIV. Specifically, engineered nucleic acid substrates are added for HIV RT to generate complementary DNA (cDNA), followed by amplification and detection mediated by LAMP or CRISPR-Cas systems. In addition to HIV detection, the phenotypic HIV RT activity assay holds potential for HIV drug resistance screening and could be adapted for other pathogen-derived enzyme targets. The second approach directly adds fingerstick plasma into large-volume RT-LAMP for extraction-free HIV RNA detection. The results highlight the feasibility of scaling up LAMP reaction volumes, providing a fresh perspective on isothermal amplification.In response to the COVID-19 pandemic started in 2019, we have additionally targeted improving the sensitivity of rapid antigen tests that are widely adopted for point-of-care COVID-19 diagnosis. While saliva offers a sample volume of up to 5mL, most antigen tests can only accommodate 50-100μL samples, leading to an inevitable sensitivity loss. We have developed a simple streptavidin-biotin-based agglutination system that could enrich analytes from larger sample volumes to improve assay sensitivity. Besides COVID-19, we anticipate the simple agglutination system could be employed for other targets to facilitate processing larger sample volumes with enhanced sensitivity.