UC San Diego

Viral, fungal, and bacterial infections often share similar or overlapping symptoms that complicate diagnosis and impede appropriate and timely treatment. Infections can spread to the bloodstream and cause sepsis, a severe and quickly progressing condition that necessitates rapid identification and treatment of the underlying pathogen. However, most emerging pathogen detection technologies for sepsis are follow up tests after a culture yields a positive result or have narrow adoption due to limited coverage of pathogen targets, low sensitivity, and high costs. This dissertation advances universal digital PCR and high resolution melting (U-dHRM) by developing novel methods to integrate multiple cross-kingdom universal primers to accurately identify known and novel fungal, viral, and bacterial pathogens with high sensitivity and low cost directly from low volume (1mL) samples in a single test.In Chapter 1, to expand U-dHRM’s capabilities by establishing a universal fungal assay, we targeted bronchoalveolar lavage fluid (BAL) as an ideal sample type suited for clinical validation which commonly encounters additional clinically relevant genera in patients suspected of invasive mold infection (IMI). By demonstrating U-dHRM’s speed, ability to simultaneously identify and quantify clinically relevant mold pathogens in polymicrobial samples, and detect emerging opportunistic pathogens to possibly aid treatment decisions and improve patient outcomes we show promise as a separate or combination diagnostic approach to standard mycological tests. In Chapter 2, we expand the bacterial database and address additional strategies for U-dHRM to discriminate species with multiple strains that present unique clinically relevant diseases. A 2-step rapid assay for syphilis detection and typing by eubacterial species distinction and hypervariable gene targeting is described using qPCR and HRM. In Chapter 3, we advance U-dHRM with a cross-kingdom multiplex assay for neonatal infections, the most critical patient population U-dHRM is ideally suited for with the potential to overcome low starting blood and low expected pathogen loads. We detect and differentiate a panel of the most common neonatal pathogens in a single fast assay that has the potential to advance the field of diagnostics to yield more timely and accurate treatment.