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.