Association of Results of Four Lateral Flow Antibody Tests with Subsequent SARS-CoV-2 Infection

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine coverage remains incomplete, being only 15% in low-income countries. Rapid point-of-care tests predicting SARS-CoV-2 infection susceptibility in the unvaccinated may assist in risk management and vaccine prioritization. We conducted a prospective cohort study in 2,826 participants working in hospitals and Fire and Police services in England, UK, during the pandemic (ISRCTN5660922). Plasma taken at recruitment in June 2020 was tested using four lateral flow immunoassay (LFIA) devices and two laboratory immunoassays detecting antibodies against SARS-CoV-2 (UK Rapid Test Consortium’s AbC-19 rapid test, OrientGene COVID IgG/IgM rapid test cassette, SureScreen COVID-19 rapid test cassette, and Biomerica COVID-19 IgG/IgM rapid test; Roche N and Euroimmun S laboratory assays). We monitored participants for microbiologically confirmed SARS-CoV-2 infection for 200 days. We estimated associations between test results at baseline and subsequent infection, using Poisson regression models adjusted for baseline demographic risk factors for SARS-CoV-2 exposure. Positive IgG results on each of the four LFIAs were associated with lower rates of subsequent infection with adjusted incidence rate ratios (aIRRs) of 0.00 (95% confidence interval, 0.00 to 0.01), 0.03 (0.02 to 0.05), 0.07 (0.05 to 0.10), and 0.09 (0.07 to 0.12), respectively. The protective association was strongest for AbC-19 and SureScreen. The aIRR for the laboratory Roche N antibody assay at the manufacturer-recommended threshold was similar to those of the two best performing LFIAs at 0.03 (0.01 to 0.10). Lateral flow devices measuring SARS-CoV-2 IgG predicted disease risk in unvaccinated individuals over a 200-day follow-up. The association of some LFIAs with subsequent infection was similar to laboratory immunoassays. IMPORTANCE Previous research has demonstrated an association between the detection of antibodies to SARS-CoV-2 following natural infection and protection from subsequent symptomatic SARS-CoV-2 infection. Lateral flow immunoassays (LFIAs) detecting anti-SARS-CoV-2 IgG are a cheap, readily deployed technology that has been used on a large scale in population screening programs, yet no studies have investigated whether LFIA results are associated with subsequent SARS-CoV-2 infection. In a prospective cohort study of 2,826 United Kingdom key workers, we found positivity in lateral flow test results had a strong negative association with subsequent SARS-CoV-2 infection within 200 days in an unvaccinated population. Positivity on more-specific but less-sensitive tests was associated with a markedly decreased rate of disease; protection associated with testing positive using more sensitive devices detecting lower levels of anti-SARS-CoV-2 IgG was more modest. Lateral flow tests with high specificity may have a role in estimation of SARS-CoV-2 disease risk in unvaccinated populations.


discussion/interpretation
The KM graphs are helpful. What would be the authors explanation for these plateauing? Could this reflect access to vaccination? Worth discussion?
Could KMs be generated for the lab assays (at recommended threshold, and potentially alternative thresholds) Minor: Line 231 should read "NOT"? Line 242, missing? Refs -need some formatting e.g. refs 1,7 The authors may be interested in this recent paper:10.1093/cid/ciac629 Reviewer #2 (Comments for the Author): The authors evaluated a large (N=2826) cohort of plasma taken from individuals recruited between June 2020 and January 2021 of the SARS-CoV-2 pandemic to determine if the use of rapid immunoassays could provide data indicating immune protection (or diseases susceptibility) from SARS-CoV-2. Their results indicate that positive IgG results were associated with lower rates of subsequent infection. The sample size, methodology and statistical methods used in this study are appropriate.
Major Comment: Given that this study was conducted in the first year of the pandemic before vaccines were widely available, and also prior to the emergence of the Delta, Omicron and BA.5 variants, the authors need to provide additional commentary regarding the applicability of this data in late 2022. As the authors mention, ~65% of the world's population have received at least one vaccine dose, so this data may no longer apply to that population, and while it's true that vaccination rates are substantially lower in low income countries, those individuals are now going to be exposed to BA.5, or possibly another emerging variant. Do the authors have data indicating that the LFIAs employed in this study perform similarly well for BA.5? What scenarios do the authors envision for applying this methodology to the broader population now that vaccination rates are higher overall? Additionally, what was the rationale behind the LFIAs chosen for this study, was it a matter of convivence? Did the authors perform a comparison study with other LFIAs and chose the most sensitive and specific assays? What is the performance data , ie, sensitivity, specificity, ppv, npv of these tests?
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Response to Reviewers
Reviewer 1 Overall, this paper sets out to address an important, unresolved, question and reads very clearly. I only have a few minor suggestions for modification which might help strengthen the paper • Thank you very much for your review. We have made the following changes below.
Rationale: the potential for an LFIA based approach is probably stronger to inform vaccination policy (e.g., by identifying groups with lower response) than individual management. The intro/discussion could be strengthened on this as well as relevance of these findings to vaccination induce immune responses • We have added a sentence to the first paragraph of the introduction: "In vaccinated populations, understanding individual risk could also help to monitor immune responses to vaccination and inform prioritisation of booster delivery". • We have also added a sentence to the last paragraph of the discussion: "In the context of higher vaccination rates, LFIAs could also play a role in assessing protection after vaccination and prioritising the delivery of booster vaccines to groups with lower antibody levels".
It would be helpful to provide the performance characteristics of the tests within the paper to help follow the discussion/interpretation  (Figure 4)(21). SureScreen was estimated to have the highest positive predictive value, and OrientGene and Biomerica the highest negative predictive values, when detecting antibody at the manufacturer's cut-off value and relative to an ELISA-based gold standard (21)." The KM graphs are helpful. What would be the authors explanation for these plateauing? Could this reflect access to vaccination? Worth discussion?
• Thank you for your comment. With regard to the plateauing observed in the KM graphs, this could possibly reflect decline in incidence of SARS-CoV-2, population protection being acquired, or increased vaccination. However, we have declined to comment in the paper.
Could KMs be generated for the lab assays (at recommended threshold, and potentially alternative thresholds) • We have generated KMs for the Roche and EuroImmun lab assays at the recommended threshold and added them to Figure 2, alongside the KMS for the LFIAs.
Line 231 should read "NOT"?
• We have corrected this typo and it now reads "than individuals who did not have detectable antibodies".
• We have corrected this typo and it now reads "This concern has been addressed for the SureScreen device, for which data has now been published showing very similar accuracy on finger-prick samples taken from individuals and serum samples analysed in a laboratory." Refs -need some formatting e.g. refs 1,7 • We have improved the formatting of the references such that the organisation is correctly displayed.
The authors may be interested in this recent paper:10.1093/cid/ciac629 • Thank you for highlighting this very relevant paper. We have now cited it in the introduction and the discussion.

Reviewer 2
The authors evaluated a large (N=2826) cohort of plasma taken from individuals recruited between June 2020 and January 2021 of the SARS-CoV-2 pandemic to determine if the use of rapid immunoassays could provide data indicating immune protection (or diseases susceptibility) from SARS-CoV-2. Their results indicate that positive IgG results were associated with lower rates of subsequent infection. The sample size, methodology and statistical methods used in this study are appropriate.
• Thank you very much for your review. We have made the following changes below.
Major Comment: Given that this study was conducted in the first year of the pandemic before vaccines were widely available, and also prior to the emergence of the Delta, Omicron and BA.5 variants, the authors need to provide additional commentary regarding the applicability of this data in late 2022. As the authors mention, ~65% of the world's population have received at least one vaccine dose, so this data may no longer apply to that population, and while it's true that vaccination rates are substantially lower in low income countries, those individuals are now going to be exposed to BA.5, or possibly another emerging variant. Do the authors have data indicating that the LFIAs employed in this study perform similarly well for BA.5?
• We agree that this is an important point to discuss and have added more detail in the second paragraph of the discussion to highlight this as an important caveat: "Firstly, it applies to a historical cohort of unvaccinated individuals and prior to the emergence of variants such as Delta, Omicron, and BA.5 (33). The performance of LFIAs might be different in the context of immunity gained from vaccination (in addition to or instead of natural infection) and to estimate protection against different circulating SARS-CoV-2 variants with differential antibody neutralisation; this is an important area for future research (8)." • We have also emphasised the need for further research to explore if LFIAs perform similarly well for other variants in the final paragraph of the discussion: "An ongoing programme of field studies would be required to show whether the LFIA-associated protection seen in this study extends to self-read and healthcare worker-read tests; to currently prevalent SARS-CoV-2 strains which may differ from the strains circulating during this study (36,37); and (if surveillance of vaccinated individuals were contemplated) to vaccinated individuals." What scenarios do the authors envision for applying this methodology to the broader population now that vaccination rates are higher overall?
• We have added a sentence to the first paragraph of the introduction highlighting that LFIAs could still play a role in the context of higher vaccination rates: "In vaccinated populations, understanding individual risk could also help to monitor immune responses to vaccination and inform prioritisation of booster delivery (5-8)". • We have also emphasised this in the final paragraph of the discussion: "In the context of higher vaccination rates, LFIAs could also play a role in assessing protection after vaccination and prioritising the delivery of booster vaccines to groups with lower antibody levels." Additionally, what was the rationale behind the LFIAs chosen for this study, was it a matter of convivence? Did the authors perform a comparison study with other LFIAs and chose the most sensitive and specific assays?
• We have added the rationale behind choosing these LFIAs in the 'Lateral flow immunoassays' paragraph of the Methods section: "These devices had been selected by the UK Department of Health and Social Care's New Tests Advisory Group on the basis of test and performance data available, and our previous research has described their sensitivity and specificity (21)" What is the performance data, ie, sensitivity, specificity, ppv, npv of these tests?
• We have added a section in the Discussion describing the performance data and citing our previous paper which contains more detailed information on test performance: "Of the four LFIAs included in our study, comparative testing on a large, well-characterised sample set showed specificity was highest in SureScreen ( , which could detect lower levels of SARS-CoV-2 antibodies, including levels at which disease risk is elevated (Figure 4)(21). SureScreen was estimated to have the highest positive predictive value, and OrientGene and Biomerica the highest negative predictive values, when detecting antibody at the manufacturer's cut-off value and relative to an ELISA-based gold standard (21)."