Patients
Patients with immune-mediated inflammatory diseases (IMID; N=534) were recruited in centers in Erlangen, Nuremberg and Bamberg (FAU Erlangen-Nuremberg, IMID practices in Erlangen and Bamberg). These centers are specialized in the treatment of IMID patients in the field of rheumatology, gastroenterology and dermatology. Patients were offered to participate if they were on stable (> 3 months) treatment with cytokine inhibitors, comprising either therapeutic antibodies and receptors (such as those neutralizing tumor necrosis factor alpha or interleukins- 6, -17 or -23) or chemical agents (such as Janus Kinase inhibitors). In addition, a cohort of IMID patients (N= 259) receiving no cytokine inhibitors within the last three months was recruited in the same centers. Center distribution of IMID patients with and without cytokine inhibitors was identical.
Controls
In addition, two control groups were analyzed. The first (N=285) included health care (HC) professionals (doctors, nurses and technicians), who work in the aforementioned institutions and are involved in the treatment, diagnostics and research on IMID patients. Subjects were contacted personally as well as by e-mail and invited to participate in the study. None of HC professionals denied participating in this study as their interest in receiving coronavirus diagnostics was very high. In addition and since the exposure of the HC control to infectious agents may be higher than in the general population, an additional control group with healthy subjects not involved in health care was analyzed (“non-health care control”, NHC; N=971). This group was composed of two populations: A cohort of healthy subjects (N=329) from the district of Erlangen-Höchstadt and the city of Erlangen, that has been established via field campaigns to assesses healthy ageing. These subjects were not allowed to have a diagnosis of any IMID. Part of this cohort has been described previously (Berlin A, et al. Osteoarthritis Cartilage. 2019;27:1043-1047) and numbers have been expanded since then. The second part was a cohort of firefighters (N=651) from the same region (district of Erlangen-Höchstadt and the city of Erlangen), which was recruited by an organized field campaign in March and April 2020. Recruitment was centrally organized by the volunteer fire brigade officers, who invited all firefighters of their sub-units to participate (via the corresponding fire brigade mailing lists). All firefighters agreed to participate in this study. As the prevalence of anti-SARS-Cov2 antibodies was very similar in the non-firefighter cohort (2.12%) and the firefighter cohort (2.36%.), the two NHC control cohorts were pooled.
Ethical approval
Ethical approval (#157_20 B) to conduct this analysis was granted by the institutional review board (IRB) of the University Clinic of Erlangen. Written informed consent was obtained from the study participants.
Demographic characteristics
Age, sex and body mass index were documented in all patients and controls. In addition, history of smoking, arterial hypertension, diabetes mellitus and chronic lung diseases, which put patients at risk for severe COVID-19 infection were documented.
Assessment of symptoms and social contacts
The study groups were exposed to questions on clinical symptoms (cough, rhinitis, throat pain, fever, headache, fatigue, musculoskeletal pain, anosmia, shortness of breath and diarrhea) and social contacts (contact with infected individuals, travel to at risk areas, working outside home) provided by the German federal authority for infectious diseases (Robert Koch Institute) and disseminated through routes of public communication, as well as social distancing rules imposed by the government at the beginning of the study period. Data about symptoms and social contacts were self-documented (present/absent, complied/not-complied) using a standardized form during the study period (February 1st to April 15th, 2020). These data were self-documented and retrieved when serum analysis was done after 8 weeks.
Anti-SARS-CoV-2 antibody testing
Serum samples were taken between March 18th and April 30th for anti-SARS-CoV-2 IgG tests. Immunoglobulin G (IgG) antibodies against the S1 domain of the spike protein of SARS-CoV-2 where tested by the recent CE version (April 2020) of the commercial enzyme–linked immunosorbent assay from Euroimmun (Lübeck, Germany) using the EUROIMMUN Analyzer I platform and according to the manufacturers protocol. Optical density was determined at 450 nm with reference wavelength at 630nm. A cut-off of ≥0.8 (OD450nm) was considered as positive. This assay has very high (>99%) specificity (see also https://www.ukbonn.de/ C12582D3002FD21D/vwLookupDownloads/Streeck_et_al_Infection_fatality_rate_of_SARS_CoV_2_infection2.pdf/%24FILE/Streeck_et_al_Infection_fatality_rate_of_SARS_CoV_2_infection2.pdf) and has been approved for diagnostic use in the United States (see https://www.centerforhealthsecurity.org/resources/COVID-19/serology/Serology-based-tests-for-COVID-19.html#sec2. Assays were performed in line with the guidelines of the German Medical Association (RiliBAK) with stipulated internal and external quality controls.
Tests used for validation of positive results
Two reference tests for IgG antibodies against the SARS-CoV-2 were used, one of them a chemo-luminescent assay (CLIA) for detecting antibodies against the spike and nucleocapsid proteins (Shenzhen Yhlo Biotech, iFlash-SARS-CoV-2, Cat #C86095G, Shenzhen, China). This assay is based on magnetic beads coated with SARS-CoV-2 spike and nucleocapsid antigen for the detection of specific IgG using a fully automated iFlash Immunoassay Analyzer (Shenzhen Yhlo Biotech). The assays were performed according to the manufacturer’s protocols. The IgG titre was automatically calculated as arbitrary units (AU/ml) and the cut-off value for a positive test was 10 AU/ml. In addition, an enzyme–linked immunosorbent assay detecting antibodies against the nucleocapsid protein (Immundiagnostik, Bensheim, Germany). Optical density was determined at 450 nm with reference wavelength at 630nm. A cut-off of ≥0.5 (OD 450nm) was considered as positive.
In-house SARS-CoV-2 ELISA
An in-house ELISA was prepared according to published coating protocols (ref. 1). In detail, 100 µl of the coating solution, consisting of 1.5µg/ml of one of the above indicated SARS-CoV-2 antigens in 1xPBS, were applied to wells of a ninety-six well plates (Costar), incubated overnight at 4°C. The following antigens were used: recombinant SARS-CoV-2 Spike Protein, S1 Subunit (1-Us-Tag); SARS-CoV-2 Spike S1 receptor binding domain (RBD) (His-Tag) (both Sino Biological, Beijing, China); SARS-CoV-2 (201 9-nCoV) Meridian Biosciences (Memphis, TX); Spike Protein (S2 ECD, His tag); SARS-CoV-2 (COVID-19) nucleocapsid protein(Sino Biological, Beijing, China). After removing the coating solution Liquid plate sealer animal free (#163 050, Candor, Germany) was added to the plates at room temperature (RT) for 1 hour as blocking solution. Serum samples diluted 1:100 in 1% bovine serum albumin in 1xTBS/0,01% Tween were added to the plates for 30 min at room temperature. The following samples were used: (1) Controls with anti-SARS-CoV-2 IgG negative IgG in the Euroimmun ELISA (N=6), (2) Controls with anti-SARS-CoV-2 IgG positive IgG in the Euroimmun ELISA (N=6), (3) patients with COVID-19 infection and positive viral RNA test (N=6), patients with endemic human coronavirus infection during the pre-SARS-CoV-2 era in spring 2019 (N=5). Plates were washed three times with 200ul per well of 1xTBS/0.01 % Tween. Next, a 1:15.000 dilution of goat anti-human IgG-horseradish peroxidase (HRP) conjugated secondary antibody (Dianova) was prepared in HRP-Protector solution (#222 050, Candor, Germany) and added to the wells for 15 min. Plates were again washed thrice with 1xTBS/0.01 % Tween. Once completely dry, 100 ul of 3, 3′,5 ,5′-Tetramethylbenzidine Liquid (TMB, Sigma Aldrich) Substrate solution was added to each well of the plates for 15 minutes and then the reaction was stopped by addition of 100 μL per well of 0.16 M sulfuric acid. Absorbance was measured at 450nm. The positive cut off was equal to the mean of the OD values of the negative control wells on the respective plate plus three times the standard deviation of the OD value distribution from the negative control samples.
Statistics
We summarized participant characteristics using means, standard deviations and percentages as appropriate. For anti-SARS-CoV2 IgG positivity (≥0.8 OD450nm) exact 95% confidence intervals were constructed based on the Poisson approximation to the binomial distribution. We estimated relative risks of seropositivity in study groups using the NHC group as the reference and adjusting for age, sex and sampling-date using a Poisson regression model with robust sandwich standard errors (ref. 2). Adjustment for sampling date was achieved using the cumulative confirmed COVID-19 case-counts reported by the Robert Koch Institute for Erlangen and Erlangen-Höchstadt on the date of serum sampling (https://experience.arcgis.com/experience/478220a4c454480e823b17327b2bf1d4; accessed on 07.05.2020 at 16.24). We reasoned that these case counts would approximate the overall risk of exposure to SARS-CoV-2 from the onset of the pandemic to the date of sampling. We constructed 4x2 contingency tables for binary categories of (i) patient-reported history of contact with persons having a febrile respiratory tract infection, (ii) absence from workplace, such as working from home, being unemployed or retired, and (iii) travel to high-risk regions. Using these contingency tables we calculated and plotted standardized Pearson’s residuals showing the deviation of the observed frequencies in each study group from expected frequencies of the relevant category. Results for chemo-luminescent detection of anti-S1 spike protein antibodies (Yhlo Biotech) and anti-nucleocapsid antibodies (Immunodiagnostik) were compared between S1-positive and S1-negative subjects using Mann Whitney U test. A p value of less than 0.05 was considered as significant. All analyses were done using R v.3.5.3 software (R Foundation for Statistical Computing, Vienna, Austria) and GraphPad Prism v8.1 (GraphPad Software, San Diego, USA).
Data availability
Anonymized raw data and R syntax to reproduce the analyses, figures, tables in the published article are available.
Methods References
- Amanat F, Stadlbauer D, Strohmeier S, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv 2020:2020.03.17.20037713.
- Barros, A.J. & Hirakata, V.N. Alternatives for logistic regression in cross-sectional studies: an empirical comparison of models that directly estimate the prevalence ratio. BMC Medical Research Methodology 2003;3, 21. doi: 10.1186/1471-2288-3-21.