Methods

Study design and setting

The IA-COVID study presented here is a prospective cross-sectional study conducted in a real-life clinical setting at a Swiss University Hospital (Figure 1). First ad interim results have been published previously [21]. Inselspital is the largest tertiary hospital in Switzerland, covering a catchment area of more than 1 million inhabitants (German and French-speaking areas). The study protocol was approved by the appropriate ethical committee (Kantonale Ethikkommission Bern #2020-00683) and the institutional authorities. All participants signed informed consent. The study was conducted in accordance with the declaration of Helsinki.

Population

Two groups of individuals representing distinct target populations were included between April and November 2020 (Figure 1): (a) consecutive inpatients with suspected SARS-CoV-2 infection (ill patients at risk for complications), and (b) healthcare workers at Inselspital (healthy individuals tested for surveillance measures). The inclusion criteria for inpatients were: (1) hospitalization in Inselspital, (2) tested at least once for SARS-CoV-2 by real-time polymerase chain reaction (RT-PCR; nasopharyngeal swab), (3) aged 18 or older, and (4) signed informed consent. Only inpatients with more than 4 days of residual sample material were considered. The inclusion criteria for healthcare workers were: (1) medical staff at Inselspital since February 2020, (2) aged 18 or older, and (3) signed informed consent. Healthcare workers were contacted using e-mail lists. A representative subset of patient samples was analyzed with the live SARS-CoV-2 neutralization assay (see below). Additionally, 102 anonymized biobank samples collected from inpatients between December 2018 and February 2019 were analyzed (Liquid Biobank Bern; www.biobankbern.ch). Healthcare workers were contacted by e-mail lists.

Definition of diagnoses

We defined a positive RT-PCR from a nasopharyngeal swab as the primary reference standard test representing a “confirmed SARS-CoV-2” infection (COVID-19). We defined “SARS-CoV-2” as negative if (a) RT-PCR was negative in all nasopharyngeal swabs conducted or (b) RT-PCR not performed. Following applicable regulations, all inpatients and medical staff were supposed to be tested in case of symptoms. Biobank samples were classified as negative as they had been collected before the pandemic (i.e., winter 2018/2019).

Handling of samples and collection of data

To ensure adequate pre-analytical conditions, blood was taken following an established in-house protocol. Samples were collected using plastic syringes containing serum or lithium heparin, respectively (S-Monovette®, Sarstedt, Germany). Residual sample material was used in the case of inpatients; serum and heparin tubes were used in medical staff. Samples were immediately transported to the laboratory and centrifuged within 30 minutes using an established protocol [22].
Pseudonymized demographical, clinical, and laboratory data were extracted from the patient documentation and transferred by the Insel Data Science Center (IDSC) [23]. A REDCap database survey was constructed to collect data of medical personnel (demographic, symptoms, comorbidities, and risk factors).

Selection of immunoassays

To study all major serological testing strategies, we selected seven immunoassays with different analytical techniques (enzyme-linked immunosorbent assay, ELISA; electrochemiluminescence immunoassay, ECLIA; chemiluminescence immunoassay; CLIA; lateral flow immunoassay, LFI) and epitopes of the SARS-CoV-2 (receptor binding domain of the spike glycoprotein, RBD; S1/S2 domain of the spike glycoprotein; nucleocapsid, N). Key characteristics of the tests are given in Table 2. Considering the results of our own interim analysis and other publications [21], we decided to include assays capturing IgG and pan-Ig.
An in-house anti-RBD ELISA was developed and validated as previously described [21]. An ELISA capturing antibodies against the S1 domain was employed (Euroimmune AG, Lübeck, Germany). Besides, an ELISA determining antibodies against N was used (Epitope Diagnostics Inc, San Diego, CA, USA). Three high-throughput tests were selected: a pan-Ig ECLIA capturing antibodies against N (Roche diagnostics, Rotkreuz, Switzerland), an IgG CLIA detecting anti-S1/S2 antibodies (DiaSorin S.p.A., Saluggia, Italy), and an IgG CLIA measuring anti-N antibodies (Abbott Laboratories, Sligo, Ireland). Aiming to use a sensitive and precise LFI, we employed a pan-Ig LFI capturing antibodies against an extended RBD protein, which is suggested to be a superior antigen compared to canonical RBD constructs (BÜHLMANN Laboratories, Schönenbuch, Switzerland) [24].

Detection of SARS-CoV-2 specific antibodies

Laboratory technicians and researchers were blinded to the RT-PCR results and clinical details. All ELISA measurements were done on a DSX automated ELISA system device (DYNEX Technologies, Chantilly, VA, USA) as previously described; instructions of the manufacturers were followed [21]. For the in-house ELISA, 96-well plates were coated, blocked, washed repeatedly, and secondary polyclonal anti-human IgG was added. Results were measured at OD450-620, and samples with an OD > 0.5 were considered positive. Concerning the anti-S1 ELISA measured at OD450-620, antibody values were expressed as a ratio (ODsample/ODcalibrator), and ratios above 0.8 were assigned positive. With regard to the anti-N ELISA, measurements were conducted at OD450-620 and the cut-off was calculated by the following formula: 1.1*(mean ODnegative control + 0.10).
The anti-N ECLIA was conducted on a Cobas 8000 analyzer (Roche diagnostics, Rotkreuz, Switzerland). The cut-off was calculated based on the calibrator measurements and a cut-off index s/c ≥ 1.0 was considered positive [25]. Anti-N CLIA was determined on an Architect i2000SR (Abbott AG, Baar, Switzerland). The cut-off was determined by dividing the measurement of the samples by the mean calibrators result; an index ≥ 1.4 was considered positive [26]. The anti-S1/S2 CLIA was determined on a Liaison XL (DiaSorin S.p.A., Saluggia, Italy); arbitrary units ≥ 12.0 were classified positive [27].
The anti-RBD+ LFI was conducted as previously described [24]. 10µL serum was transferred to the application pad and designated chase buffer was added. Results were analyzed after 15 minutes using the Quantum Blue® Reader (3rd generation; BÜHLMANN Laboratories, Schönenbuch, Switzerland) that measures the test line intensity of the LFI in Grey Values [GVs]. Based on the limit of blank plus four times the standard deviation, samples showing GVs of at least 1.5 are considered positive for SARS-CoV-2 antibodies. For this study, GVs were also measured and evaluated quantitatively.

Detection of SARS-CoV-2 by real-time PCR

Nasopharyngeal swabs (Copan FLOQSwabs and Copan UTM Viral Transport medium; Copan, Brescia, Italy) were transported at room temperature and stored at 4° C until processing. Three different methodologies for nucleic acid testing (NAT) were employed in the study period. A laboratory-developed test (LDT) PCR workflow was utilized [28] and two commercial, fully automated assays (Seegene Allplex 2019-nCoV Assay, Seegene, Seoul, Korea; Roche cobas® SARS-CoV-2 Assay, Roche Diagnostics, Rotkreuz, Switzerland). Details have been reported previously [21]. RT-PCR was done in clinical practice, and laboratory technicians were not aware of the index test results.

Live SARS-CoV-2 neutralization assay

A representative subset of patients was selected for analysis using a live SARS-CoV-2 neutralization assay (n=201). To identify potential differences between immunoassays, we included all complex cases (RT-PCR+/anti-S1-; RT-PCR-/anti-S1+; RT-PCR+/asymptomatic patients; anti-S1+/anti-N-; anti-S1-/anti-N+) and other antibody-positive and negative patients. Technicians and researchers conducting and interpreting the neutralization assay were blinded regarding RT-PCR results, serological test details, and clinical details.
Neutralizing antibodies were determined by the inhibition of virus-induced cytopathic effect (CPE). Methodological details have been described previously [21]. Briefly, sera were incubated at 56°C for 30 minutes, centrifuged (13’000 rpm for 10 minutes), and diluted 1:8 in cell culture medium. Duplicates of five-fold serial dilutions were prepared in 96-well plates and mixed with 100 TCID50 of infectious SARS-CoV-2 in equal volume (BetaCoV/France/IDF0372/2020; National Reference Centre for Respiratory Viruses (Institut Pasteur, Paris, France). After incubation at 37°C for one hour, dilutions were transferred to confluent Vero E6 cells (provided by Prof. Dr. Volker Thiel, University of Bern, Bern, Switzerland) and incubated at 37°C, 5%CO2 and >85% relative humidity for three days. CPE was assessed by crystal violet staining. Wells with an undisturbed cell layer were rated as (-), and signs of CPE were rated as (+). Full neutralization titer was defined as the serum dilution without signs of CPE (-) in both duplicates with a limit of detection (LOD) of 1:16. [21].

Statistical analysis

Descriptive statistics were used to describe the distribution of serological test results in individuals with and without COVID-19, overall and in subgroups. Diagnostic accuracy measures were calculated using pre-defined cut-offs of the serological index tests in relation to the reference standard as defined above (RT-PCR). Two-by-two tables were generated, sensitivities and specificities calculated, receiver-operating-characteristics (ROC) curves created, and c-statistics calculated. Serological test results were standardized using z-scores for test comparison in the context of the neutralization assay. [21]. Antibody response between subgroups was compared using unpaired t-test; P-values were reported without adjustment for multiple testing. As a power analysis, we used a method proposed by Bujang et al. [29]. We considered a prevalence of 6% (COVID-19) and a power of 0.8 to detect differences in specificity of 0.05 between assays. Analyses were carried out using the Stata 14.2 statistical software (StataCorp. 2014. Stata statistical software: Release 14. College Station, Tx: StataCorp LP). Figures were created using Prism 6 (GraphPad Software, Inc., LaJolla, California, United States).

Patient and public involvement

The study protocol, the questionnaire, and the first results have been presented on different occasions at Inselspital, and participant feedback was requested and incorporated.