Overcoming barriers to point-of-care nucleic acid amplification testing

Event start: 2026-07-01T18:00:00Z Event end: 2026-07-01T19:00:00Z Location: BioScience Research Collaborative In this thesis I address aspects of three key bottlenecks limiting clinical translation of isothermal nucleic acid amplification tests (isothermal NAATs) – (1) sample preparation, (2) assay integration and clinical validation, and (3) implementation analysis in real-world use environments. Addressing these bottlenecks is important to increase clinical access to highly sensitive and specific nucleic acid diagnostics where laboratory testing is unavailable. Currently, most people globally cannot access NAAT tests when they are needed. Isothermal NAATs, which operate at a single temperature reducing instrumentation requirements and processing time, have been developed to address this need. Despite substantial research and development on isothermal NAATs for many indications, very few have achieved widespread clinical adoption. To address the need for simple sample preparation suitable to the point-of-care, I focused on extraction-free sample preparation and developed a framework to guide the design and validation of extraction-free sample preparation methods based on gaps identified in the literature (Chapter 2 and 3). This framework focuses on identifying potential amplification inhibitors, determining the lytic efficiency of different methods, and creating appropriate model samples. This framework can be used by test developers to co-design an amplification assay and suitable extraction-free sample preparation workflow. When I then applied this framework in Chapter 3 to design an extraction-free sample preparation workflow for an assay detecting β-globin from buccal swabs and blood, I was able to design extraction-free sample preparation that consistently detected target from both buccal swabs and blood. I then applied the framework I developed in Chapters 4 and 5 to optimize extraction-free sample preparation for assays for two indications. In Chapter 4, to address the need for assay integration and clinical validation, I optimized SCD-RPA, an isothermal NAAT to diagnose sickle cell anemia and sickle cell trait. I integrated the assay with a modified version of the previously developed sample preparation workflow and validated it in a multi-country study with 161 patient samples collected in both a high- and a low-resource setting. SCD-RPA demonstrated robust performance with an overall genotype prediction accuracy of 96.9%. In Chapter 5, I applied the framework for extraction-free sample preparation to HPV-LAMP, a test being developed for cervical cancer screening, and achieved good analytical performance (30.4 HPV16 positive cells, 7.6 HPV18 positive cells, and

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