Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking

Chen, Hui; Li, Zhao; Feng, Sheng; Richard-Greenblatt, Melissa; Hutson, Emily; Andrianus, Stefen; Glaser, Laurel J; Rodino, Kyle G; Qian, Jianing; Jayaraman, Dinesh; Collman, Ronald G; Glascock, Abigail; Bushman, Frederic D; Lee, Jae Seung; Cherry, Sara; Fausto, Alejandra; Weiss, Susan R; Koo, Hyun; Corby, Patricia M; Oceguera, Alfonso; O’Doherty, Una; Garfall, Alfred L; Vogl, Dan T; Stadtmauer, Edward A; Wang, Ping

BACKGROUND: High sensitivity SARS-CoV-2 antigen assays are desirable to mitigate false negative results. Limited data are available to quantify 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 fmol/L) nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives, and quantified and tracked antigen dynamics in ICU COVID inpatients and immunocompromised COVID patients. RESULTS: Compared to qualitative RT-PCR methods, the MSAA 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, the 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, quantification and tracking of antigen burden, and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.