Analytical sensitivity of seven SARS-CoV-2 antigen-detecting rapid tests for Omicron 1 variant 2

22 The emergence of novel SARS-CoV-2 variants of concern (VOCs) requires investigation of a 23 potential impact on diagnostic performance, such as Antigen-detecting rapid diagnostic tests 24 (Ag-RDT). Although anecdotal reports have been circulating that Omicron is in principle 25 detectable by Ag-RDTs, no published data are a yet available for the newly emerged Omicron 26 variant. Here, we have performed an analytical sensitivity testing with cultured virus in seven 27 Ag-RDTs for their sensitivity to Omicron compared to data earlier obtained on VOCs Alpha, 28 Beta, Gamma and Delta and a pre-VOC isolate of SARS-CoV-2. Overall, we have found a 29 tendency towards lower sensitivity for Omicron compared to pre-VOC SARS-CoV-2 and the 30 other VOCs across tests. Importantly, while analytical testing with cultured virus may be a 31 proxy for clinical sensitivity, is not a replacement for clinical evaluations which are urgently 32 needed for Ag-RDT performance in Omicron-infected individuals. 33

The emergence of novel SARS-CoV-2 variants of concern (VOCs) requires investigation of a potential impact on diagnostic performance.SARS-CoV-2 antigen-detecting rapid diagnostic tests (Ag-RDT) offer quick, cheap and laboratory-independent results at the point of care. 1 Although sensitivity is lower compared to RT-PCR, they enable reliable detection of high viral loads associated with infectious virus presence, making them important public health tools. 2,3ver, the majority of Ag-RDT validation studies were performed prior to the emergence of SARS-CoV-2 variants of concern (VOC). 4VOC Omicron was first reported at the end of November from South Africa and is characterized by a high number of mutations compared to earlier circulating SARS-CoV-2. 5 The majority of mutations are located in the Spike protein, that are, according to preliminary data, associated with considerable escape from neutralization by both disease-and vaccine derived antibodies, and probably also associated lower vaccine effectiveness. 6,7 ,8 ,9 ,10Current epidemiological data show that Omicron circulation is associated with a steep increase in case numbers as well as an increased risk of reinfections. 11nd the Spike mutations, Omicron has also mutations in the nucleocapsid, which is the target of almost all Ag-RDTs.3][14] In addition, a deletion (Del31-33) is found in the nucleocapsid of Omicron, as well as another mutation P13L, which is present in some, but not all Omicron sequences.No information on a potential impact of these mutations on Ag-RDTs performance is available so far.Anecdotal reports were circulating on positive detection of Omicron-confirmed patient samples by Ag-RDTs but no published data on Ag-RDT sensitivity for Omicron is available so far.
. Here, we have evaluated test analytical sensitivity using cultured SARS-CoV-2 Omicron variant, in comparison with earlier data on isolates of the other VOCs (Alpha, Beta, Gamma and Delta) and an early-pandemic (pre-VOC) SARS-CoV-2 isolate (B.1.610)6][17] All viruses were isolated from clinical samples.
Isolates were grown in Vero-E6 cells as described previously. 16The Omicron variant which was initially isolated on Vero-TMPRSS cells, then further passaged with a stock passage (p2) prepared on VeroE6.Vero TMPRSS were kindly received from National Institute for All Ag-RDT assays were performed according to the manufacturers' instructions with the exception that 5 μL of virus dilution was directly added to the proprietary buffer, and then applied to the Ag-RDT in duplicates under BSL3 conditions. 17Ag-RDT buffer without virus was used as a negative control.Any visible test band in the presence of a visible control band was considered as positive.

When assessing by infectious virus titers (PFU/mL) (Fig 1), analytical sensitivity to detect
Omicron was lower than for the other VOCs in most of the tests evaluated.One test, Flowflex (ACON biotech) showed the highest overall sensitivity for all SARS-CoV-2 isolates used compared to the others, and here, Omicron was detected with even slightly higher sensitivity than Delta but still lower than Alpha, Beta, Gamma and pre-VOC SARS-CoV-2.
Of note, while in this analysis the previous VOCs Alpha, Beta, Gamma and Delta were mainly detected with comparable or even higher sensitivity compared to pre-VOC SARS-CoV-2, here Omicron is the first VOC which showed a tendency towards lower analytical sensitivity across assays.However, we have also observed considerable heterogeneity in sensitivity patterns across variants and between individual assays in this analytical testing using cultured virus.
Differences in analytical sensitivity between Ag-RDTs might be explained by the different epitopes used in each test, potentially affected by the mutations in the nucleocapsid.If the lower sensitivity towards Omicron that we observed here is confirmed by findings from clinical validations, the use of Ag-RDTs in the early symptomatic period of an Omicron infection or in asymptomatic patients could be less reliable, with important implications for public health measures.
Importantly, while analytical testing with cultured virus may be a proxy for clinical sensitivity, is not a replacement for clinical evaluations and has several limitations, e.g.ratio between infectious virus, viral protein and RNA copies might differ between patient specimens and cultured virus isolates.In addition, other factors, such as in vivo shedding of infectious virus and overall viral loads could further influence clinical test performance.Therefore, further studies on diagnostic accuracy of Ag-RDTs for the newly emerged VOC Omicron are urgently needed to guide public health responses.Analytical sensitivity for early-pandemic SARS-CoV-2 B.1.610,Alpha, Beta, Gamma and Delta have already been published before but were added here for consistency reasons and better interpretability of the data on Omicron. 15,16

Biological
Standards and Controls (NIBSC, Cat.Nr. 100978).The following mutations and deletion in the nucleocapsid were present in the original patients' sequence as well as in the virus isolate of the passage used in this study: R203K, G204R, P13L, Del31-33.The starting dilution of infectious titers for all viruses used in this study was 4.24 log10 PFU/mL.Seven Ag-RDTs were used: I) Panbio COVID-19 Ag Rapid test device (Abbott); II) Standard Q COVID-19 Ag (SD Biosensor/Roche); III) Sure Status (Premier Medical Corporation), the three latter being WHO-EUL approved and thus of high global public health relevance, 18 IV) 2019-nCoV Antigen test (Wondfo); V) Beijng Tigsun Diagnostics Co. Ltd (Tigsun); VI) Onsite COVID-19 Ag Rapid Test (CTK Biotech); VII) ACON biotech (Flowflex), several of them being on the waiting list for WHO-EUL approval.