Risk factors for SARS-CoV-2 seroprevalence following the first pandemic wave in UK healthcare workers in a large NHS Foundation Trust

Abstract

Background: We aimed to measure SARS-CoV-2 seroprevalence in a cohort of healthcare workers (HCWs) during the first UK wave of the COVID-19 pandemic, explore risk factors associated with infection, and investigate the impact of antibody titres on assay sensitivity. Methods: HCWs at Sheffield Teaching Hospitals NHS Foundation Trust were prospectively enrolled and sampled at two time points. We developed an in-house ELISA for testing participant serum for SARS-CoV-2 IgG and IgA reactivity against Spike and Nucleoprotein. Data were analysed using three statistical models: a seroprevalence model, an antibody kinetics model, and a heterogeneous sensitivity model. Results: Our in-house assay had a sensitivity of 99·47% and specificity of 99·56%. We found that 24·4% (n=311/1275) of HCWs were seropositive as of 12th June 2020. Of these, 39·2% (n=122/311) were asymptomatic. The highest adjusted seroprevalence was measured in HCWs on the Acute Medical Unit (41·1%, 95% CrI 30·0–52·9) and in Physiotherapists and Occupational Therapists (39·2%, 95% CrI 24·4–56·5). Older age groups showed overall higher median antibody titres. Further modelling suggests that, for a serological assay with an overall sensitivity of 80%, antibody titres may be markedly affected by differences in age, with sensitivity estimates of 89% in those over 60 years but 61% in those ≤30 years. Conclusions: HCWs in acute medical units and those working closely with COVID-19 patients were at highest risk of infection, though whether these are infections acquired from patients or other staff is unknown. Current serological assays may underestimate seroprevalence in younger age groups if validated using sera from older and/or more severe COVID-19 cases.

Establishing SARS-CoV-2 membrane protein-specific antibodies as a valuable serological target via high-content microscopy

“…Using HCM, we observed a statistically significant difference in signal intensity for N and S antibodies between pre-pandemic and SARS-CoV-2-positive samples ( Figures 2 A and 2C). As previously reported, 27 we found that IgG levels were significantly elevated for N and S in patients hospitalized after SARS-CoV-2 infection (inpatient) compared to non-hospitalized individuals (outpatient), with these trends also seen in ELISA absorbances for the sample sets ( Figures S4 A, S4B, S4E, and S4F). ELISA-based detection of N or S IgG in SARS-CoV-2-positive samples had marginally higher sensitivity than HCM when specificity was fixed at 100% ( Figure 2 B, N ELISA (185/196, 94.4%) vs. N HCM (182/196, 92.9%), Figure 2 D, S ELISA (195/196, 99.5%) vs. S HCM (192/196, 98.0%)), Tables S1 and S2 ) with a greater number of samples from COVID-19 inpatients containing detectable N or S IgG ( Figures S4 C, S4D, S4G, and S4H, Tables S1 and S2 ).…”

“…Plasma samples used were from healthcare workers (HCWs) recruited at Sheffield Teaching Hospitals NHS Foundation Trust (STH) as part of the COVID-19 Humoral Immune Responses in front-line healthcare workers (HCWs) study (HERO), sampled in May and June 2020. 27 Further longitudinal plasma samples from HCWs were used from a prospective, observational, cohort study (PITCH), where in Sheffield, participants were recruited under the Sheffield Teaching Hospitals (STH) Observational Study of Patients with Pulmonary Hypertension, Cardiovascular Disease and other Respiratory Disease (STH-Obs). 44 , 45 Regulatory approval was provided by HRA and Health and Care Research Wales (HERO - 20/HRA/2180), and the Yorkshire and Humber – Sheffield Research Ethics Committee (STHObs - 18/YH/0441).…”

“…ELISA for S and N proteins were performed as previously described. 27 High binding microtiter plates (either Immulon 4HBX; Thermo Scientific, 6405, or Nunc MaxiSorp; Thermo Scientific, 442404) were coated overnight at 4°C with 50 μl/well SARS-CoV-2 protein diluted in PBS (pH 7.4). Either full-length spike produced in mammalian cells, 46 or nucleocapsid produced in E.coli were used as antigens.…”

Omicron BA.1/BA.2 infections in triple-vaccinated individuals enhance a diverse repertoire of mucosal and blood immune responses

“…Nucleocapsid IgG was assessed using an ELISA as previously described (36). High-binding 96-well ELISA plates (Immulon 4HBX; Thermo Scientific, 6405) were coated overnight at 4°C with 50 µL/well full-length untagged nucleocapsid protein produced in E. coli (Uniprot ID P0DTC9 (NCAP_SARS2), diluted to 2 µg/mL in 7.4 pH phosphate buffered saline (PBS).…”

“…To allow quantification of antibody concentration, we included a 12-step standard curve consisting of sera pooled from convalescent SARS-CoV-2-confirmed patients, calibrated to the WHO International Standard for anti-SARS-CoV-2 immunoglobulin (NIBSC, 20/136), with results reported in binding antibody units/mL (BAU/mL). Samples that were considered negative based on previously determined thresholds (36) were assigned a value of 1.04 BAU/mL, which was half the value of the lowest point on the standard curve.…”