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

Area of Science:

• Virology
• Immunology
• Medical Diagnostics

Background:

• High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are crucial for minimizing false negative results in coronavirus disease 2019 (COVID-19) diagnosis.
• Limited data exist on quantifying and tracking SARS-CoV-2 antigen levels in respiratory samples across diverse patient populations.

Purpose of the Study:

• To develop and validate a novel, high-sensitivity SARS-CoV-2 antigen assay with smartphone readout for accurate detection and quantification.
• To track SARS-CoV-2 antigen dynamics in intensive care unit (ICU) and immunocompromised COVID-19 patients.

Main Methods:

• Development of the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with a limit of detection of 0.5 pg/mL nucleocapsid antigen.
• Utilization of a computer vision and machine learning-based classifier for automatic microbubble image analysis.
• Quantification and tracking of antigen dynamics in nasopharyngeal (NP) swabs from ICU and immunocompromised COVID-19 patients.

Main Results:

• The MSAA demonstrated high agreement with RT-PCR (97% positive, 97% negative agreement) in a clinical validation study of 372 NP swabs.
• Antigen positivity decreased with days-after-symptom-onset in immunocompetent individuals, contrasting with prolonged detection in immunocompromised patients.
• Total microbubble volume correlated inversely with cycle threshold values and days-after-symptom-onset, indicating antigen burden.

Conclusions:

• The MSAA provides sensitive and specific detection of acute SARS-CoV-2 infections.
• This assay enables quantification and tracking of viral antigen burden, aiding in longitudinal studies.
• The MSAA can identify patients with active viral replication, warranting close monitoring and potential viral sequencing.