B-240 Respiratory Scout: fast and accurate home molecular testing for the detection and discrimination of influenza A/B, and SARS-CoV-2

Background Influenza and SARS-CoV-2 infections impose a massive burden on patients, clinics, and laboratories. Rapid, accurate, and affordable molecular diagnostic tools to diagnose early infections at home would save time and costs, and bolster the efficacy of test-to-treat programs. Thus far, at-home options have lacked early detection capabilities or are too expensive for widespread adoption into American households. Respiratory Scout is a low-cost (under $20/test), PCR-accurate molecular test for Flu A/B and SARS-CoV-2. The test runs on a low cost tech-enabled platform that facilitates access to truly convenient follow up care. Methods The Respiratory Scout assay utilizes 3 spectrally distinct channels to detect four molecular targets. Scout’s patented Loop-de-Loop (LdL) reverse transcription loop-mediated amplification (RT-LAMP) technology allows for programmable fluorescence gain for individual pathogen targets without impacting reaction time. This allows for higher-order multiplexing beyond a “single dye, single channel” paradigm. This technology is leveraged to include an internal human control and differentiation between influenza types A and B, and SARS-CoV-2 in a single tube on a 3-channel reader, the Scout Hub. The one-pot reaction simplifies test kit consumables and manufacturing, therefore dramatically lowering cost of goods. Pilot lyophilized assay beads were produced in-house. Beads were rehydrated with volunteer-collected nasal matrix (NM), spiked with whole influenza A, influenza B , or SARS-CoV-2 viruses, and run on Scout Hubs with the Scout Connect mobile app. Results The Flu A LoD was approximately 1000 copies/rxn, and the Flu B LoD was approximately 10,000 copies/rxn; all reactions amplified in under 30 minutes. Adjusting the relative brightness of Flu A/B Loop-de-Loop probes enabled clear discrimination between Flu A and B over the entire range of titrated whole virus input. Simulated co-infections resulted in accurate detections as well. An algorithm was developed to detect and differentiate between each target. Conclusion Respiratory Scout has similar sensitivity to leading molecular FDA-authorized or cleared testing systems, offering PCR-accurate results at rapid antigen test pricing. Sensitivity can be further improved with additional assay optimization. LdL technology allows for higher-order multiplexing via differential brightness in a single channel, saving instrumentation costs and complexity. Low cost molecular testing for common respiratory viruses would greatly reduce patient load in clinics and improve the American respiratory patient experience.