Comparison of the analytical and clinical sensitivities of 34 rapid antigen tests with prevalent SARS-CoV-2 variants of concern during the COVID-19 pandemic in the UK

Abstract

Antigen-detection rapid diagnostic tests (Ag-RDTs) have become a central pillar for the management of coronavirus disease worldwide due to their speed and ease of use and are now being developed for use in other emerging outbreaks. Like other viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is subject to rapid mutation as it spreads, and new variants of concern (VOCs) emerge frequently, posing a significant challenge for the detection of newer, highly mutated variants. It is, therefore, important that the performance of Ag-RDTs is regularly evaluated, particularly in outbreak scenarios where rapid diagnostics are key to limiting disease spread. Here, we present a comprehensive evaluation of the analytical and clinical sensitivities of 34 commercially available Ag-RDTs with five SARS-CoV-2 VOCs, all of which were highly prevalent in the UK at various times between 2019 and 2023. This study highlights the importance of regular evaluation of the Ag-RDT performance, with several Ag-RDTs demonstrating a reduced performance with some VOCs. We conclude that a regular performance evaluation through our proposed pipeline, combined with a broad consensus approval threshold across global organizations, is essential to maintaining the effectiveness of Ag-RDTs as a disease management tool during outbreaks.IMPORTANCEAntigen-detection rapid diagnostic tests (Ag-RDTs) came to global prominence during the coronavirus disease pandemic, where they offered a quick and simple at-home diagnostic, which could be used to manage disease spread. A major ongoing challenge for the broad use of Ag-RDTs is the speed at which new SARS-CoV-2 variants emerge, each of which has the potential to reduce the performance of available Ag-RDTs. As Ag-RDTs are explored for use in other viral disease outbreaks, pipelines for the regular evaluation of test performance are essential for ensuring that Ag-RDTs can be employed effectively. Here, we have developed a robust pipeline for the large-scale evaluation of commercially available Ag-RDTs against several major SARS-CoV-2 variants, which can be adapted and applied to other emerging outbreaks to ensure that test performance is maintained as a virus evolves.

Keywords: B.1.1.529; COVID-19; Delta; Omicron; SARS-CoV-2 variant of concern (VOC), B.1.617.2; antigen detection; limit of detection (LOD); rapid diagnostic test (RDT).

Fig 1

Heatmap comparing the LODs of 34 Ag-RDTs using the Ancestral (WT), Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) Omicron (BA.1), and Omicron (BA.5) variants on PFU/mL (left) and RNA copies/mL (right). Ag-RDT brands are given on the y axis, while the SARS-CoV-2 strain is given on the x axis. The blue colors indicate LODs fulfilling the DHSC (for PFU/mL) and WHO criteria (for RNA copies/mL), while the red colors indicate LODs that fail to meet these criteria * = P ≤ .05 between VOCs. Data of the Ancestral, Alpha, and Gamma have been taken from our previously published work (21).

Fig 2

Boxplot of the SARS-CoV-2 viral load (RNA copies/mL) as quantified by RT-qPCR on the NP swabs collected from participants recruited to FALCON between January 2021 and March 2022. Whiskers indicate 95% confidence intervals, and the horizontal line indicates the median. Asterisks indicate the statistical significance between different VOCs as determined by the Kruskal-Wallis test (ns = non-significant, * = P ≤ .05, ** = P ≤ .01)

Fig 3

Limit of detection analyses of the upper-respiratory samples positive by RT-qPCR for five SARS-CoV-2 Ag-RDT tests (Covios, Hotgen, Onsite, Flowflex, and SureStatus) using NP swabs. The log10 RNA copies on the x axis were plotted against a positive (1.0) or negative (0.0) Ag-RDT result on the y axis. Fill curves show the logistic regressions of the viral load on the Ag-RDT result; the vertical dashed lines indicate the log10 RNA copies subjected to the test at which 50 and 95% LOD of the samples are expected to be positive based on the regression results. No significant differences were observed.

Fig 4

Positive and negative Ag-RDT results according to the RT-qPCR cycle threshold (Ct) values. The P-values indicate the results of the Bonferroni-adjusted post-hoc tests performed following three-way factorial ANOVA to compare Ag-RDT+ and Ag-RDT− within each variant and test brand. Asterisks indicate comparisons where Cohen’s d ≥ 0.5 and P < 0.05, indicating a significant difference between the genomic Ct values of RT-qPCR+/Ag-RDT+ and RT-qPCR+/Ag-RDT− samples.

Fig 5

Flowchart comparing the sample collection and Ag-RDT assessment methods in the retrospective and prospective studies. Retrospective study details are on the left-hand side of the chart, while prospective study details are on the right.