Analytical Sensitivity of Lateral Flow Devices against SARS-CoV-2 Omicron Subvariants BA.4, BA.5, and BA.2.75

Rapid antigen testing has played a pivotal role in the COVID-19 pandemic, enabling quick identification of individuals with SARS-CoV-2 infection, and rapid institution of clinical and public health measures. In many settings, including Australia, rapid antigen testing is now the main modality of testing, with only one-third of SARS-CoV-2 infections now diagnosed using PCR assays (1). The ongoing emergence of new SARS-CoV-2 variants requires constant vigilance in diagnostic assay performance, including assessments of the analytical and clinical sensitivity of rapid antigen tests. Although amino acid changes in the SARS-CoV-2 spike protein are the characteristic feature of SARS-CoV-2 variants, mutations in the nucleocapsid protein (the diagnostic target of most antigen tests) can occur (1). To date, however, the performance of most antigen tests has been assessed against previously circulating variants, including the Delta and Omicron B.1. 1.529 lineages (2, 3). Accordingly, we undertook a rapid assessment of the analytical sensitivity of six antigen tests, commonly used in our setting, against the Omicron sublineages BA.4, BA.5, and BA.2. 75. Representative Delta, BA.4, BA.5, and BA.2. 75 isolates were obtained from clinical samples referred to the Victorian Infectious Diseases Reference Laboratory (VIDRL) in Melbourne, Australia. We included a Delta isolate to enable comparison with our previous analytical assessments of antigen kit performance (3, 4). SARS-CoV-2 genomic sequencing of virus isolates was performed following cell culture using the Oxford Nanopore Rapid Barcoding (SQK-RBK110.96) and Midnight RT PCR Expansion (MRT001; both Nanopore technologies) kits. SARS-CoV-2 consensus sequences were generated using the ARTIC Sequencing report generated by the inbuilt Nextflow wf-artic supplied by Oxford Nanopore on their GridION instrument. Lineage designation was assigned using both Nextclade and Pangolin v3.1. 16. Isolates were grown as previously described (3) and harvested when cytopathic effect (CPE) was observed. For each variant, we constructed a 10-fold dilution range of quantified virus spanning;2 108 to 2 105 copies/mL, corresponding to N gene cycle threshold (Ct) values of;19 to ;29 on an in-house real-time RT-PCR assay (5), and using droplet digital PCR (ddPCR) (3). Testing was performed in quadruplicate using live virus at Biosafety Level 3, and limit of detection (LOD) was defined as the last dilution where all four replicates were positive. Additional testing was performed on three of the kits most commonly used for testing in our setting. Twenty BA.5 and five BA.4 clinical isolates were selected with Ct values 20.1 to 29.0. BA.5 was selected as this is the current dominant variant in Victoria. Sample volumes applied to the test cassette and incubation time were followed as per manufacturer’s instructions. Results interpretation was performed by two readers as previously described (3). A third reader was used to resolve discordant results. The analytical sensitivity of all kits was similar for Delta, BA.4, BA.5, and BA.2. 75 (Fig. 1). All kits detected Delta, BA.4, BA.5, and BA.2. 75 at 6 log10 copies/mL (Ct 25), and only three kits Editor Yi-Wei Tang, Cepheid Copyright © 2022 Mackenzie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Deborah A. Williamson, deborah.williamson@unimelb.edu. au. The authors declare no conflict of interest.