Taqman PACMAN: a simple molecular approach for positive rapid antigen test confirmation during periods of low prevalence

• Published in: Microbiology spectrum 2024-05, Vol.12 (5), p.e0407323-e0407323
• Main Authors: McCracken, Gregory R, Patriquin, Glenn, Hatchette, Todd F, Davidson, Ross J, Goodall, Barbara, Barrett, Lisa, MacDonald, James, Heinstein, Charles, Pettipas, Janice, Ross, John, LeBlanc, Jason J
• Format: Article
• Language: English
• Subjects: Antigens, Viral; Clinical Microbiology; COVID-19; COVID-19 - diagnosis; COVID-19 - epidemiology; COVID-19 Nucleic Acid Testing - methods; COVID-19 Serological Testing - methods; extraction; False Positive Reactions; Humans; molecular; Nucleic Acid Amplification Techniques - methods; PCR; Prevalence; rapid antigen; Real-Time Polymerase Chain Reaction - methods; Research Article; SARS-CoV-2; SARS-CoV-2 - genetics; SARS-CoV-2 - immunology; SARS-CoV-2 - isolation & purification; Sensitivity and Specificity
• ISSN: 2165-0497; 2165-0497
• DOI: 10.1128/spectrum.04073-23

Antigen-based rapid diagnostic tests (Ag-RDTs) were widely deployed to enhance SARS-CoV-2 testing capacity during the COVID-19 pandemic. Consistent with national guidance for low prevalence settings, positive Ag-RDTs were confirmed using nucleic acid amplification tests (NAATs) to avoid false positive results. However, increasing demands for positive Ag-RDT confirmation competed with other testing priorities in clinical laboratories. This work hypothesized that real-time RT-PCR without nucleic acid extraction (NAE) would be sufficiently sensitive to support positive Ag-RDT confirmation. Ag-RDT and NAAT results from community-based asymptomatic testing sites prior to the omicron variant wave were compared to calculate the weekly false positive rate (FPR) and false detection rate (FDR). Real-time RT-PCR was compared with and without NAE using 752 specimens previously tested positive for SARS-CoV-2 using commercial NAATs and 344 specimens from Ag-RDT-positive individuals. The impact of SARS-CoV-2 prevalence on laboratory resources required to sustain Ag-RDT confirmation was modeled for the RT-PCR with and without NAE. Overall, FPR was low [0.07% (222/330,763)] in asymptomatic testing sites, but FDR was high [30.7% (222/724)]. When RT-PCR was compared with and without NAE, 100% concordance was obtained with NAAT-positive specimens, including those from Ag-RDT-positive individuals. NAE-free RT-PCR significantly reduced time to results, human resources, and overall costs. A 30.7% FDR reaffirms the need for NAAT-based confirmation of positive Ag-RDT results during low SARS-CoV-2 prevalence. NAE-free RT-PCR was shown to be a simple and cost-sparing NAAT-based solution for positive Ag-RDT confirmation, and its implementation supported data-driven broader Ag-RDT deployment into communities, workplaces, and households. Rapid antigen testing for SARS-CoV-2 was widely deployed during the COVID-19 pandemic. In settings of low prevalence, national guidance recommends that positive antigen test results be confirmed with molecular testing. Given the high testing burden on clinical laboratories during the COVID-19 pandemic, the high volume of positive antigen tests submitted for confirmatory testing posed challenges for laboratory workflow. This study demonstrated that a simple PCR method without prior nucleic acid purification is an accurate and cost-effective solution for positive rapid antigen test confirmation. Implementing this method allowed molecular confirmatory tes