Adaptive molecular diagnostics for respiratory pathogens: Applying COVID lessons to broader clinical challenges

Project Summary Nearly five years have passed since the emergence of SARS-CoV-2, during which time this virus has become the most tested-for pathogen in the history of laboratory medicine. After initial shortages, SARS-CoV-2 diagnostics were ultimately launched at a massive scale, including gold-standard nucleic acid amplification tests and rapid antigen assays. This unprecedented period clearly illustrated how mass-produced commercial products are necessary to meet routine diagnostic needs at a population level. At the same time, the pandemic also exposed key weaknesses in this market-driven pipeline, including an inability to address rapidly evolving testing scenarios and more specialized patient care. The COVID-19 emergency has ended, although similar challenges remain for other pathogens that cause acute respiratory infections. Fortunately, the diagnostic field also stands in a position to reflect on the pandemic and apply its lessons to remaining deficits. In this R01 renewal, therefore, we will continue delivering novel molecular techniques for identifying/characterizing acute respiratory infections, but as strategically informed by the SARS-CoV-2 experience. A recurrent diagnostic gap throughout the pandemic involved adaptability: that is, the responsiveness of the diagnostic pipeline to rapidly evolving and specialized clinical needs. Accordingly, we propose to develop adaptive - yet imminently practical - molecular methodologies that build upon traditional PCR. However, more than just validating discrete products, these efforts will champion novel technologies that can be customized by individual laboratories across testing settings (whether clinical, public health, or research), addressing unmet needs for both established respiratory pathogens and unknown future threats. Critically, these tools will be made possible through a continued collaboration between an engineering-focused assay development group, together with the clinical laboratory enterprise of an academic health system. Our design goals will address three interconnected challenges facing respiratory testing: (i) rapid pathogen genotyping, without the need for frank sequencing; (ii) high-order pathogen multiplexing, without the need for inflexible (and expensive) commercial microfluidic-based platforms; and (iii) practical epidemiologic surveillance, achievable at local levels. Specifically, Aim 1 will create a pipeline for the design of ligation-coupled PCR, with combinatoric mediator-probe readout, for comprehensive variant detection and strain typing. Aim 2 will consolidate syndromic respiratory testing into a single-vessel format, in which the fluorescent readouts of numerous pathogens are binned according to their clinical `actionability'. Finally, pivoting to epidemiologic testing, Aim 3 will combine specimen-pooling with concentration techniques from wastewater analysis, creating new opportunities for cost-effective surveillance. Together, these Aims will enable respiratory assays that can be adapted to local institutional needs - whether motivated by research, epidemiology, or patient-level clinical care (and its stringent regulatory requirements). In this way, the proposed research program will apply lessons from the pandemic well beyond just SARS-CoV-2.