Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout

P. Wang
University of Pennsylvania,
United States

Keywords: SARS-CoV-2, antigen detection, COVID-19, digital assay


High sensitivity SARS-CoV-2 antigen assays are desirable to mitigate false negative results. Limited data is available to quantitate and track SARS-CoV-2 antigen burden in respiratory samples from different populations. Methods: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection (LOD) of 0.5 pg/mL (10.6 fM) nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. A computer vision and machine learning-based automatic microbubble image classifier was developed to accurately identify positives and negatives. Antigen dynamics was tracked in 38 ICU COVID inpatients and 13 immunocompromised COVID patients. Results: Compared to rRT-PCR methods, the Microbubbling Antigen Assay demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. Conclusions: The MSAA enables sensitive and specific detection of acute infections, quantitation and tracking of antigen dynamics, and may serve as a screen method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.