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

Abstract

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.

Importance: 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 testing for positive antigen tests to be sustained as antigen testing was expanded into large populations such as workplaces, schools, and households.

Keywords: COVID-19; PCR; SARS-CoV-2; extraction; false positive; molecular; prevalence; rapid antigen.

Fig 1

Testing strategies used in Nova Scotia for low and high SARS-CoV-2 prevalence. The large gray and blue arrows represent the overall specimen throughput supported by commercial and laboratory-developed NAATs, respectively. (A) During low SARS-CoV-2 prevalence, nucleic acid extraction (NAE)-free RT-PCR (solid green arrow) supported positive Ag-RDTs confirmation without impacting routine specimen workflows using pooling on commercial NAATs. If positive Ag-RDT samples had been introduced into routine testing (red dashed arrows), it would have impacted testing strategies such as specimen pooling, where additional work would have been triggered to resolve positive pools (blue dashed arrows).

Fig 2

Anticipated impact of prevalence on the Abbott Panbio Ag-RDT false discovery rate (FDR). FDRs were modeled in relation to prevalence based on sensitivity and specificity values described by (i) the manufacturer “best case scenario” at 91.4% and 99.8% (dotted black line), (ii) symptomatic SARS-CoV-2 cases in systematic review (18) at 75.1% and 99.5% (dotted blue lines), (iii) asymptomatic cases in the same review (18) at 48.9% and 98.1% (solid blue lines), (iv) symptomatic cases in the COVAG study (19) (dotted orange lines) at 74.32% and 99.73%, (v) asymptomatic cases in the COVAG study (19) at 23.28% and 99.96% (solid orange lines), and (vi) “worst case scenario” represented as the World Health Organization’s (WHO) minimal criteria for Ag-RDTs test performance at 80% and 97% (dashed black line).

Fig 3

Justifying positive Ag-RDTs confirmation with NAAT during periods of low SARS-CoV-2 prevalence. (A) SARS-CoV-2 Ag-RDT positivity and number of Ag-RDTs performed in asymptomatic community testing sites from 21 November 2020 to 21 December 2021. (B) Number of positive Ag-RDTs detected and proportion confirmed by NAAT following specimen recollection.

Fig 4

Comparison of real-time RT-PCR with and without NAE. For analytical sensitivity comparisons, 10-fold serial dilutions of a quantified SARS-CoV-2 specimen were used to compare RT-PCR with (blue) and without NAE (orange). The average threshold cycle (Ct) values obtained for each dilution are displayed for both Rt-PCR target: (A) E gene and (B) RdRp. In (C) and (D) are the distribution of Ct values for RT-PCR with (blue) or without NAE (orange) obtained from 257 positive NAAT specimens recollected from individuals that tested positive with Ag-RDTs. C represents the results of the E gene target, whereas D represents those of RdRp.

Fig 5

Impact of SARS-CoV-2 prevalence on resources required to support Ag-RDT confirmation testing with RT-PCR with or without an NAE. Daily Ag-RDTs administered are depicted for a population of 100,000. The impact of SARS-CoV-2 prevalence is depicted: (A) for the number of RT-PCR runs and instruments required, (B) for the human resources and work shifts required, and (C) for RT-PCR and total costs.