Laboratory-based RT-PCR, the current gold standard for COVID-19 testing, can require a turnaround time of 24 to 48 h from sample collection to result. The delayed time to result limits the effectiveness of centralized RT-PCR testing to reduce transmission and stem potential outbreaks.

The lateral-flow immunoassay (LFA) is a well-established diagnostic technology that has recently seen significant advancements due in part to the rapidly expanding fields of paper diagnostics and paper-fluidics. As LFA-based diagnostics become more complex, it becomes increasingly important to quantitatively determine important parameters during the design and evaluation process. However, current experimental methods for determining these parameters have certain limitations when applied to LFA systems. In this work, we describe our novel methods of combining paper and radioactive measurements to determine nanoprobe molarity, the number of antibodies per nanoprobe, and the forward and reverse rate constants for nanoprobe binding to immobilized target on the LFA test line. Using a model LFA system that detects for the presence of the protein transferrin (Tf), we demonstrate the application of our methods, which involve quantitative experimentation and mathematical modeling. We also compare the results of our rate constant experiments with traditional experiments to demonstrate how our methods more appropriately capture the influence of the LFA environment on the binding interaction. Our novel experimental approaches can therefore more efficiently guide the research process for LFA design, leading to more rapid advancement of the field of paper-based diagnostics.

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SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

Background As the COVID-19 pandemic continues to cause substantial morbidity and mortality, there is an increased need for rapid, accessible assays for SARS-CoV-2 detection. Methods Here we present a clinical evaluation and real-world implementation of the INDICAID COVID-19 Rapid Antigen Test (INDICAID Rapid Test). A multi-site clinical evaluation of the INDICAID Rapid Test using prospectively collected samples from symptomatic subjects was performed. The INDICAID Rapid Test was then implemented at COVID-19 outbreak screening centers in Hong Kong to screen individuals for COVID-19 to prioritize confirmatory RT-PCR testing among asymptomatic populations. Results The clinical evaluation in symptomatic patient populations demonstrated a positive percent agreement and negative percent agreement of 85.3% (95% confidence interval [95% CI]: 75.6% - 91.6%) and 94.9% (95% CI: 91.6% - 96.9%), respectively, when compared to laboratory-based RT-PCR testing. When used during outbreak testing of 22,994 asymptomatic patients, the INDICAID Rapid Test demonstrated a positive percent agreement of 84.2% (95% CI: 69.6% - 92.6%) and a negative percent agreement of 99.9% (95% CI: 99.9% - 100%) compared to laboratory-based RT-PCR testing. When incorporated in a testing algorithm, the INDICAID Rapid Test reduced time to confirmatory positive result from an average 10.85 hours (standard RT-PCR only) to 0.84 hours, depending on the algorithm. Conclusion The INDICAID Rapid Test has excellent performance when compared to laboratory-based RT-PCR testing, and when used in tandem with RT-PCR, reduces the time to confirmatory positive result.