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SARS-CoV-2 serological tests can generate false positive results for samples from patients with chronic inflammatory diseases

13 Nov 2020-medRxiv (Cold Spring Harbor Laboratory Press)-

TL;DR: The majority of the verified serological assays were sensitive to interfering antibodies in samples from patients with chronic inflammatory diseases and therefore may have poor specificity in this context.

AbstractObjectives Patients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless the tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays with samples from patients with chronic inflammatory diseases collected before April 2019, thus defined as negative. Methods Samples from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and RF +/- systemic lupus erythematosus (SLE, n=10), were tested with 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed multiplex bead-based assay. Results Six LFA and the in-house IgG assay gave the correct negative results for all samples. However, the majority of assays (n=13), gave false positive signal with samples from patients with RA and SLE. This was most notable in RF positive RA samples. MS samples did not give any false positive in any of the assays. Conclusion The majority of the verified serological assays were sensitive to interfering antibodies in samples from patients with chronic inflammatory diseases and therefore may have poor specificity in this context. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays.

Topics: Rheumatoid factor (54%), Population (51%)

Summary (3 min read)

INTRODUCTION

  • Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID19), which emerged as a pandemic late 2019 (1).
  • As a result, there has been substantial concern during the pandemic as to the potential increased risk COVID-19 disease severity and mortality among these patient groups (4).
  • These autoantibodies are also present in a variety of other diseases as well as in the general population and may increase with age, smoking and chronic infection (10, 11).
  • Clinical manifestation of SLE is heterogeneous and can affect multiple organs.
  • Due to the substantial global demand, SARS-CoV-2 serological testing has been rapidly developed and released to the market.

Rheumatoid Factor Detection Methods

  • Analysis IgA, IgG and IgM isotypes of RA samles from the EIRA cohort and SLE samples was performed using the EliA immunoassay on the Phadia 2500 instrument and the cutoff values as stated in the manufacturer’s instructions (Phadia GmbH, Frontiers in Immunology | www.frontiersin.org 3 Uppsala, Sweden) (24).
  • Serum samples of RA patients treated with IFX were analyzed for IgM RF using laser nephelometry technique.

SARS-CoV-2 Serological Detection Methods

  • Two Enzyme-Linked Immunosorbent Assays and 17 rapid diagnostic lateral flow assays (LFA) were included.
  • The brand name, antigen, manufacturer determined specificity and sensitivity, are outlined in Table 2.
  • All tests were performed according to manufacturer instructions and using serum.

Commercial Lateral Flow SARS-CoV-2 Assays

  • LFAs are designed to enable point of care analyses and can generate immediate results with read-outs as bands in small cartridges.
  • The results were compared to an in-house multiplex bead-based and validated SARS-CoV-2 serological assay developed at SciLifeLab and KTH Royal Institute of Technology as previously described (27).
  • The antigens were immobilized on magnetic color coded beads (MagPlex, Luminex Corp) and plasma/serum IgG that bound to the antigens were detected by an R-phycoerythrin conjugated goat anti-hIgG (Invitrogen, H10104).
  • The cut-off for seropositivity was defined as signals above the mean +6 SD of the 12 negative controls included in each assay.

Commercially Available SARS-CoV-2 ELISA Kits

  • The two included ELISAs were performed according to the manufacturers’ instructions.
  • The first ELISA used to detect IgG against SARS-CoV-2 (test A, Table 2) was the recomWell SARSCoV-2 IgGElisa kit (MikrogenDiagnostik GmbH,Germany).
  • In addition, Frontiers in Immunology | www.frontiersin.org 4 they also tested specificity using samples from people with conditions that present with atypical immune system activity including EBV infection, pregnancy, ANA and RF-positive subjects.
  • The cut-off for positivity was calculated according to the manufacturer’s instructions.
  • Not specified Not specified R Wondfo, Guangzhou, China Wondfo Biotech SARS-CoV-2 Antibody Test Not specified W195 Combined: IgM +IgG:99.57% S Innovita Biological Technology Co Ltd China* 2019-nCoV Ab Test (Colloidal Gold) Spike and Nucleocapsid protein^ Not specified IgM: 100% IgG: 100% Ma y 2021 | Volu ^Emergency Use Authorization (EUA) Serology Test Performance | Food and Drug Administration (FDA).

Statistical Analyses

  • Rate of false positive signals were determined as the number of positive samples divided by the total number of samples tested for each assay.
  • Statistical analyses and figures were generated using GraphPad Prism (version 8.2.1).
  • The statistical difference between RF positive and RF negative RA subsets were calculated with Fishers exact test.
  • The other groups were too small to make any meaningful statistical evaluations and thus these results are only presented as descriptive analyses.

Commercial LFA and ELISA Assays

  • Serum samples from 47 RA patients (with two samples from one of the patients), 10 SLE and 10 MS patients were evaluated using 19 SARS-CoV-2 commercial serological assays and compared to an in-house developed multiplex bead-based assay (27).
  • For the 17 LFAs evaluated for specificity using 25 RA samples (from 24 patients of which 20 were treatment naïve and 4 were treated with infliximab) that were positive for RF, 10 assays had unspecific signal detected for at least one immunoglobulin isotype .
  • None of the two ELISAs (test A and B) gave any false positive signals with these samples.
  • No associations were identified between RF isotypes and false positive anti-SARS-CoV-2 IgM or IgG signal in the SLE samples.

SciLifeLab and KTH In-House Validated SARS-CoV-2 Serological Assay

  • Due to insufficient sample volume only 66 of the 68 samples were analyzed using the in-house developed multiplex bead-based assay for IgG detection as described above (27).
  • All samples analyzed using this method were classified as negative.
  • The only two samples not included were the two infliximab treated samples from the same patient (Table S1).

DISCUSSION

  • Serological assays are necessary tools in a pandemic, both for determining the proportion of the population already subjected to the infection and for the individual to confirm past infection and present immunity.
  • In the case of SARS-CoV-2, it seems that a small proportion of the individuals who have been infected do Frontiers in Immunology | www.frontiersin.org 7 not develop antibodies, at least not as determined by currently available serological assays (29).
  • Therefore, an independent verification of sensitivity and specificity of such assays is often required.
  • The authors see two false positive signals in the IgG test for RF positive RA and SLE samples.
  • In conclusion, serological assays could be sensitive to interfering antibodies, as shown in sera from persons with autoimmune diseases.

ETHICS STATEMENT

  • Samples and data were collected with informed consent in compliance with the Helsinki Declaration.
  • The ethics committee waived the requirement of written informed consent for participation.

AUTHOR CONTRIBUTIONS

  • All authors contributed to conceptualization, execution, writing, review, and editing of the manuscript.
  • All authors approved the final version of the manuscript.

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False Positive Results in SARS-CoV-2
Serological Tests for Samples
From Patients With Chronic
Inammatory Diseases
Nastya Kharlamova
1,2
, Nicky Dunn
1,2
, Sahl K. Bedri
1,2
, Svante Jerling
1,2
,
Malin Almgren
1,2
, Francesca Faustini
3
, Iva Gunnarsson
3
, Johan Rönnelid
4
,
Rille Pullerits
5,6
, Inger Gjertsson
5
, Karin Lundberg
2,3
, Anna Månberg
7
, Elisa Pin
7
,
Peter Nilsson
7
, Sophia Hober
8
, Katharina Fink
1,9,10
and Anna Fogdell-Hahn
1,2
*
1
Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden,
2
Center for Molecular Medicine, Karolinska
Institutet, Stockholm, Sweden,
3
Department of Medicine Solna, Division of Rheumatology, Karolinska Institutet and
Rheumatology, Karolinska University Hospital, Stockholm, Sweden,
4
Department of Immunology, Genetics and Pathology,
Uppsala University, Uppsala, Sweden,
5
Department of Rheumatology and Inammation Research, Institution of Medicine,
Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden,
6
Department of Clinical Immunology and
Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden,
7
Division of Afnity Proteomics, Department of
Protein Science, KTH Royal Institute of Technology, SciLifeLab, Stockholm, Sweden,
8
Department of Protein Science, KTH
Royal Institute of Technology, Stockholm, Sweden,
9
Department of Neurology, Karolinska University Hospital,
Stockholm, Sweden,
10
Centrum for Neurology, Academical Specialist Centrum, Stockholm, Sweden
Patients with chronic inammatory diseases are often treated with immunosuppressants
and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological
tests will improve our understanding of the infection and immunity in this population,
unless they tests give false positive results. The aim of this study was to evaluate the
specicity of SARS-Cov-2 serological assays using samples from patients with chronic
inammatory diseases collected prior to April 2019, thus dened as negative. Samples
from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or
without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-
CCP2) and systemic lupus erythematosus (SLE, n=10) with or without RF, were analyzed
for SARS-CoV-2 antibodies using 17 commercially available lateral ow assays (LFA), two
ELISA kits and one in-house developed IgG multiplex bead-based assay. Six LFA and the
in-house validated IgG assay correctly produced negative results for all samples.
However, the majority of assays (n=13), gave false positive signal for samples from
patients with RA and SLE. This was most notable in samples from RF positive RA patients.
No false positive samples were detected in any assay using samples from patients with
MS. Poor specicity of commercial serological assays could possibly be, at least partly,
due to interfering antibodies in samples from patients with chronic inammatory diseases.
For these patients, the risk of false positivity should be considered when interpreting
results of the SARS-CoV-2 serological assays.
Keywords: SARS-CoV-2, autoimmunity, autoantibod ies, diagnost ics, rheu matoid arthritis, systemic lupus
erythematosus, multiple sclerosis, rheumatoid factor
Frontiers in Immunology | www.frontiersin.org May 2021 | Volume 12 | Article 6661141
Edited by:
Dimitrios Petrou Bogdanos,
University of Thessaly, Greece
Reviewed by:
Ana Barrera-Vargas,
Instituto Nacional de Ciencias Me
dicas
y Nutricio
n Salvador Zubira
n
(INCMNSZ), Mexico
Tao Li,
National Center of Biomedical Analysis
(NCBA), China
*Correspondence:
Anna Fogdell-Hahn
Anna.Fogdell-Hahn@ki.se
These authors have contributed
equally to this work and
share rst authorship
Specialty section:
This article was submitted to
Autoimmune and
Autoinammatory Disorders,
a section of the journal
Frontiers in Immunology
Received: 09 February 2021
Accepted: 06 April 2021
Published: 03 May 2021
Citation:
Kharlamova N, Dunn N, Bedri SK,
Jerling S, Almgren M, Faustini F,
Gunnarsson I, Rönnelid J, Pullerits R,
Gjertsson I, Lundberg K, Månberg A,
Pin E, Nilsson P, Hober S, Fink K and
Fogdell-Hahn A (2021) False Positive
Results in SARS-CoV-2 Serological
Tests for Samples From Patients With
Chronic In ammatory Diseases.
Front. Immunol. 12:666114.
doi: 10.3389/fimmu.2021.666114
ORIGINAL RESEARCH
published: 03 May 2021
doi: 10.3389/fimmu.2021.666114

INTRODUCTION
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)
is the causative agent of the coronavirus disease 2019 (COVID-
19), which emerged as a pandemic late 2019 (1). The cumulative
number of infected and fatal cases can be followed at the Johns
Hopkins University COVID-19 Dashboard (2). Patients with
chronic inammatory disease are often treated with
immunomodulatory treatments and therefore potentially more
susceptible to infec tions (3) . As a result, there has been
substantial concern during the pandemic as to the potential
increased risk COVID-19 disease severity and mortality among
these patient groups (4). There is limited evidence about their
risk of severe COVID-19, or knowledge of how their disease or
immunomodulatory treatment may affect either their pre-
existing immunity or ability to develop protective immunity
following inf ection (5, 6). Approximately 6% of th e worlds
population are affected by chronic inammatory diseases
which i ncludes conditions such as multiple sclerosis (MS),
rheumatoid arthritis (RA) and systemic lupus erythematosus
(SLE) (7). These are generally progressive diseases and although
for the majority there are no cures, treatment is centered around
slowing disease progression with immunomodulatory
treatments. The hallmarks of autoimmune diseases are
inammation, loss of self-tolerance and the presenc e of
autoantibodies. MS is a chronic inammatory disorder
restricted to the central nervous system, characterized by
demyelination, axonal loss and the formation of sclerotic
plaques. The worldwide prevalence is estimated to be 2.2
million cases, but with large geographical variation (8). RA is a
heterogeneous chronic inammatory disease, which affected
close to 5 million people globally by 2010 and with prevalence
increasing due to the increased aging of the human population
(9). The disease is characterized by synovial inammation and
the formation of the pannus, which causes cartilage and bone
destruction, joint dysfunction, pain and disability. Rheumatoid
factor (RF) and anti-citrullinated protein antibodies (ACPA),
often detected as a nti-cyclic citrullinated peptide (CCP)
antibodies, are the most frequent and the most studied RA-
related autoantibodies. RF is an antibody reactive with the Fc
portion of IgG, mainly consisting of IgM in Caucasian RA
populations, but also IgG and IgA RF are present. Although
RF is detected in approximately 70% of RA patients, the presence
of RF is not specic for RA. These autoantibodies are also present
in a variety of other diseases as well as in the general population
and may increase with age, smoking and chronic infection ( 10,
11). SLE is a systemic inammatory disease of the connective
tissue, characterized by a loss of self-tolerance and leading to
production and deposition of a large panel of autoantibodies and
immune complexes formation (12). Clinical manifestation of
SLE is heterogeneous and can affect multiple organs.
Approximately 25% of SLE patients have RF (13), but these
patients can also have anti-nuclear antibodies (ANA) and anti-
double-stranded (ds) DNA antibodies.
Serological tests are useful for determining past infection and
present immunity. The presence of IgM antibodies indicates a
recent infection, whereas presence of IgG antibodies indicates
possible long-lasting immunity (14). Important information can
be achieved by having access to reliable serological methods
during a pandemic; to identify seropositive people for
convalescent plasma donations; guide policies and ease
restrictions on human mobility based on sero-epidemiological
evidence; ensure immunity to allow key workers to return to
work after exposure; and evaluate vaccine development studies
and vaccine strategies.
Due to the substantial global demand, SARS-CoV-2
serological testing has been rapidly developed and released to
the market. The assays are validated before release and also often
independently veried before being approved (15, 16). However,
the panel of samples used to d etermine specicity is often
focused on r uling out cross-reactivity with othe r viral
infections an d might not include serum from patien ts with
chronic inammatory diseases, even though it is recommended
(
16). Based on experience from development and validation of
serology assays for measuring anti-drug antibodies (ADA) in
persons with chronic inammatory disease, it is recommended
to show specicity against drug naïve patient serum, as
antibodies present in patients with autoimmune diseases are
known to interact with reagents in serological assays and give
unspecic signal (17, 18). Given the signicant role serological
tests may have as useful wide-spread screening tools for
immunity, it is important to verify the specicity of SARS-
CoV-2serologicaltestsinasimilarwayforspecicity i n
patient groups with autoimmune diseases, using samples that
were collected before the pandemic.
The aim of this pilot study was to verify the specicity in a
number of the commercially available SARS-CoV-2 serological
tests, using a panel of samples from patients with different
chronic inammatory diseases collected before the SARS-CoV-
2 outbreak as negative controls, to get an indication of the extent
of the issue for further developments, validatio ns and
verications of serology assays.
MATERIALS AND METHODS
Patient Serum Samples
To evaluate specicity of SARS-C oV-2 s erolo gical assays in
patients wit h chron ic inammatory diseases, a selection of
negative control samples was retrieved from the biobank
(n=68). To exclude individuals with risk of previous exposure
to SARS-CoV-2 infection, only samples collected before April
2019, (1-22 years since time of c ollection, Table 1), were
included in the study. Serum samples were se lected from
patients with MS (n=10), RA (n=47), of which 2 samples were
from the same patient), or SLE (n=10) (Table 1).
TheMSpatientswerediagnosedaccordingtothe2017
updated McDonald criteria (19). The RA diagnoses were
determined according to the 1987 revised American College of
Rheumatology disease classication criteria by rheumatologists,
within 12 months after the rst symptoms of joint disease (20).
For the SLE patients, the diagnoses were determined according to
the American College of Rheumatism (21) and/or The Systemic
Lupus Erythematosus Collaborating Clinics (22).
Kharlamova et al. False Positive SARS-CoV-2 Serological Tests
Frontiers in Immunology | www.frontiersin.org May 2021 | Volume 12 | Article 6661142

MS patient samples were collected in a research laboratory
providing routine testing for anti-drug antibodies (ADAs) at the
Centre for Molecular Medicine, Karolinska Institutet in
Stockholm and had been treated with interferon beta (IFNb).
Three MS samples were ADA positive. Of the RA samples, 40
were from the Swedish population-based case control study
Epidemiological Investigation of RA (EIRA) and had not been
treated with any disease modifying anti-rheumatic drug
(DMARD) (23). Of these patients, 20 were RF and anti-CCP2
positive (50%); six were RF negative but anti-CCP2 positive
(15%), and 14 were both RF and anti-CCP2 negative (35%) (24).
The additional seven RA patient samples were retrieved from a
prospective study cohort (Sahlgrenska University Hospital,
Gothenburg) and were iniximab (IFX) treated. Of these seven
patient samples, three were RF and anti-CCP2 positive; two were
RF negative but anti-CCP2 positive; one was RF positive but
anti-CCP2 negative, and one sample was both RF and anti-CCP2
negative. T he SLE samples were obtained from a study
investigating the development of ADA against rituximab
(RTX), and therefore all patients were RTX treated. Five of ten
samples was anti-rituximab ADA positive (Table S1). All of the
SLE patients had ANA and four of them anti-dsDNA.
This retrospective cohort study was approved by the Ethics
Review Authority in Stockholm and Gothenburg (202023/04,
dnr 2020-01649, 2012/1550-31/3, dnr 96-174). Samples and data
were collected with informed consent in compliance with the
Helsinki Declaration.
Assays
Rheumatoid Factor Detection Methods
Analysis IgA, IgG and IgM isotypes of RA samles from the EIRA
cohort and SLE samples was performed using the EliA
immunoassay on the Phadia 2500 instrument and the cutoff
values as stated in the manufacturers instructions (Phadia GmbH,
Uppsala, Sweden) (24). Serum samples of RA patients treated with
IFX were analyzed for IgM RF using laser nephelometry technique.
Anti-Cyclic Citrullinated Peptide Assay
Anti-CCP2 IgG in EIRA was previously determined using the
Immunoscan CCPlus
®
ELISA (Euro-Diagnostica , Malmö,
Sweden), in accordance with the manufacturer´s instructions.
SARS-CoV-2 Serological Detection Methods
A total of 19 commercially available serological assays were
evaluated in this study and compared to an in-house assay.
Two Enzyme-Linked Immunosorbent Assays (ELISA) and 17
rapid diagnostic lateral ow assays (LFA) were included. These
tests were assigned a letter from A S(Table 2) and referred to
as such in text and gures in this study. The brand name, antigen,
manufacturer determined specicity and sensitivity, are outlined
in Table 2. All tests were performed according to manufacturer
instructions and using serum.
Commercial Lateral Flow SARS-CoV-2 Assays
LFAs are designed to enable point of care analyses and can
generate immediate results with read-outs as bands in small
cartridges. These rapid lateral ow tests are developed for whole
blood, serum and plasma. At time of testing, the appropriate
volume of serum was applied to the designated well and then the
buffer was added. After the recommended incubation period, the
presence and intensity of the bands were investigated and graded
from negative to four levels of positivity by the same operator.
An In-House Validated SARS-CoV-2
Serological Assay
The results were compared to an in-house multiplex bead-based
and validated SARS-CoV-2 serological assay developed at
SciLifeLab and KTH Royal Institute of Technology as previously
described (27). In brief, IgG r eactivity was analyzed in a
high-throughput and multiplex bead-based format utilizing
384-well plates and Fle xMap3D in strumentations (Luminex
Corp) for read-out (27). Reactivity against three different
in-house produced viral protein variants was used to differentiate
between positive and negative samples: Spike trimers comprising
the prefusion-stabilized spike glycoprotein ectodomain (28)
(expressed in HEK and puried using a C-terminal Strep II tag),
Spike S1 subunit (expressed in CHO and puried with HPC4 tag),
and the Nucleocapsid protein (expressed in E. coli and puried
using an N-terminal His-tag). The antigens were immobilized on
magnetic color coded beads (MagPlex, Luminex Corp) and
plasma/serum IgG that bound to the antigens were detected by
an R-phycoerythrin conjugated goat anti-hIgG (Invitrogen,
H10104). Reactivity against at least two out of the three viral
antigens included in the panel was required for positive read out.
The cut-offfor seropositivity was dened as signals above the mean
+6 SD of the 12 negative controls included in each assay. The
method utilizing the combination of the three antigens has been
found to have 99.2% sensitivity (99.6%, 99.2%, 96.7%, respectively,
for the three antigens individually) and 99.8% specicity (98.9%,
99.1%, 98.4%, respectively, for the three antigens individually)
based on 243 positive controls (dened as >16 days after onset or
TABLE 1 | Patients characteristics.
Rheumatoid
Arthritis
Multiple
Sclerosis
Systemic
Lupus
Erythematosus
Patients (n) 47 10 10
Age (years median,
min - max)
53 (18-71) 46 (39-70) 35.5 (30-60)
Female (n, %) 33 (70) 7 (70) 9 (90)
IgM RF positive (n, %) 24 (51) n/a 0
Anti-CCP2 positive
(n, %)
31 (66) n/a n/a
Treated with IFX
(n, %)
7 (15) 0 0
Treated with RTX
(n, %)
0 0 10 (100)
Treated with IFN
beta-1a (n, %)
0 10 (100) 0
ADA positive (n, %) n/a 3 (30) 5 (50)
Time period of
sampling
1998 - 2006* 2003 - March
2019
2003 - 2018
n, number; RF, rheumatoid factor; anti-CCP, anti-cyclic citrullinated p eptide; IFX,
iniximab; RTX, rituximab; IFN interferon; ADA, anti-drug antibodies; n/a, not applicable;
RA, rheumatoid arthritis.
*Time period of sampling for RA patients treated with IFX: 2018 - March 2019.
Kharlamova et al. False Positive SARS-CoV-2 Serological Tests
Frontiers in Immunology | www.frontiersin.org May 2021 | Volume 12 | Article 6661143

positive PCR) and 442 negative controls (dened as collected 2019
and earlier) samples.
Commercially Available SARS-CoV-2 ELISA Kits
The two included ELISAs were pe rformed ac cording to the
manufacturers instructions. The rst ELISA used to detect IgG
against SARS-CoV-2 (test A, Table 2) was the recomWell SARS-
CoV-2 IgG Elisa kit (Mikrogen Diagnostik GmbH, Germany). This
assay is an indirect ELISA which uses highly puried recombinant
nucleocapsid protein from SARS-CoV-2 as an antigen. The
manufacturer had determined the pote ntial interference of
antibodies against other pathogens that might induce clinical
symptoms similar to those of a SARS-CoV-2 infection (including
for example seasonal coronaviruses, inue nza A virus, RSV,
Mycoplasma pneumoniae, Chlamydia pneumoniae). In addition,
they also tested specicity using samples from people with
conditions that present with atypical immune system activity
including EBV infection, pregnancy, ANA and RF-positive
subjects. The cut-off for positivity was calculated according to the
manufacturers instructions.
The second ELISA test was the EDI Novel Coronavirus COVID-
19 IgG Elisa Kit (Epitope Diagnostics, Inc., San Diego, USA) to
detect IgG (test B, Table 2). This is an in vitro diagnostic and CE
marked indirect ELISA with plates coated with peptides from the
SARS-CoV-2 nucleocapsid antigen. Specicity of this assay was
determined by the manufacturer using anti-inuenza A, anti-
inuen za B, Hepatitis C virus (HCV), ANA and respiratory
syncytial virus (RSV). The cut-off for positivity was determined
according to the manufacturers instructions. The manufacturer
states that a positive result may be due to past or present infection
TABLE 2 | Description of the SARS-CoV-2 serological assays and the test codes used in this study.
Test
Code
Manufacturer Kit Name Antigen/Target Catalogue
number
Company reported
assay speci city
A Mikrogen Diagnostik GmbH,
Germany*
recomWell SARS-CoV-2 IgG ELISA kit Nucleocapsid protein 7304 IgG: 98.7%
B Epitope Diagnostics, Inc., San
Diego, USA
EDI Novel Coronavirus COVID-19 IgG ELISA Kit Nucleocapsid protein KT-1032 IgG: 100%
C Jiangsu Medomics medical
technology Co., Ltd, China
Rapid IgM-IgG combined Antibody Test Kit for SARS-CoV-
2 (ICA)
Spike protein
(RBD MK201027)
201030 Not specied
D Salafa Oy, Salo, Finland Salacor (Biohit) SARS-CoV-2 IgG/IgM rapid test kit Nucleocapsid protein COV-01-S IgM: 99.2%
IgG: 99.9%
E Salafa Oy, Salo, Finland Sienna SARS-CoV-2 IgG/IgM rapid test kit Spike protein (RBD) 102222 IgM: 100%
IgG: 98.8%
F Liming Bio-Products Co., Ltd.
Jiangsu, China
StrongStep_SARS-CoV-2 IgM/IgG_REF502090_ Antibody
Rapid Test
Nucleocapsid and
Spike protein
502090 IgM: 100%
IgG: 98,7%
G Zhejiang Orient Gene Biotech
Co., Ltd. (China)
COVID-19 IgG/IgM Rapid Test Cassette alt.
HEALGEN_ COVID-19 IgG/IgM Rapid Test Cassette (Whole
Blood/Serum/Plasma_REF GCCOV-402a
Nucleocapsid and
Spike protein (25)^
GCCOV-
402a
IgM: 98.46%
IgG: 98.46%
H InTec Products inc., Haicang
Xiamen, China
INTEC_ Colloidal Gold (whole blood/Serum/Plasma) Rapid
SARS-CoV-2 Antibody (IgM/IgG)
Nucleocapsid protein
(25)
ITP16001-
TC25
Combined IgM+IgG:
98%
I Sugentech Inc., South Korea* SGTi-ex COVID-19 IgM/IgG Nucleocapsid and
Spike protein^
COVT025E IgM: 98.3% (90% FDA
August 2020)
IgG:100%
J Xiamen Biotime Biotechnology
Co., Ltd. China
SARS-CoV2 IgG/IgM Rapid Qualitative Test Spike protein^ BT1301 Not specied
K Zhuhai Livzon Diagnostics Inc.
(China)
COVID-19 IgG/IgM Lateral ow Rapid Test Cassette Nucleocapsid protein Not
specied
IgM: 99.7%
IgG: 99.4%
L Abbott Point of Care Inc. USA* Panbio COVID-19 lgG/lgM Rapid Test Device Nucleocapsid protein
(26)
ICO -T402 IgM: 92.8%
IgG: 92.8%
M SureScreen Diagnostics Ltd, UK SureScreen Diagnostics COVID-19 IgG/IgM Rapid Test
Cassette (Whole blood/serum/Plasma)
Spike protein/RBD COVID19C IgM: 99.2%
IgG: 99.2%
N Wuhan Easy Diagnosis
Biomedicine Co., Ltd (China)*
COVID-19 (SARS-CoV-2) IgM/IgG Antibody Test kit Not specied SA-2-D IgM: 100%
IgG: 100%
O Zhuhai Encode Medical
Engineering Co., Ltd., China*
SARS-CoV-2 IgG/IgM Rapid test S1-RBD and
nucleocapsid protein
RCD-422 IgM: 100%
IgG: 100%
P Jiangsu SuperbioBiomedical Co.,
Ltd, China
SARS-CoV-2 (COVID-19) IgM/IgG Antibody Fast Detection
Kit (Colloidal Gold)
Spike and
nucleocapsid protein^
B00502 IgG: 95.8%
IgM: 95.8%
Q Lumigenex (Suzhou) Co., Ltd.
China
Lumigenex SARS-CoV-2 IgG/IgM Antibody Rapid Test Kit Spike and
nucleocapsid protein
Not
specied
Not specied
R Wondfo, Guangzhou, China Wondfo Biotech SARS-CoV-2 Antibody Test Not specied W195 Combined: IgM
+IgG:99.57%
S Innovita (Tangshan) Biological
Technology Co Ltd China*
2019-nCoV Ab Test (Colloidal Gold) Spike and
Nucleocapsid
protein^
Not
specied
IgM: 100%
IgG: 100%
^Emergency Use Authorization (EUA) Serology Test Performance | Food and Drug Administration (FDA).
*Stated in the instructions to have tested interference with RA and/or RF or other autoantibodies.
As of January 2021 tests C, G, I, K, N, O, P are not FDA approved.
For updated status of assays visit https://www.nddx.org/covid-19/pipeline.
Kharlamova et al. False Positive SARS-CoV-2 Serological Tests
Frontiers in Immunology | www.frontiersin.org May 2021 | Volume 12 | Article 6661144

with SARS-CoV-2 but not due to other coronavirus strains, such as
coronavirus HKU1, NL63, OC43, or 229E.
Statistical Analyses
Rate of false positive signals were determined as the number of
positive samples divided by the total number of samples tested
for each assay. Statistical analyses and gures were generated
using GraphPad Prism (version 8.2.1). The statistical difference
between RF positive and RF negative RA subsets were calculated
with Fishers exact test. The other groups were too small to make
any meaningful statistical evaluations and thus these results are
only presented as descriptive analyses.
RESULTS
Commercial LFA and ELISA Assays
Serum samples from 47 RA patients (with two samples from one
of the patients), 10 SLE and 10 MS patients were evaluated using
19 SARS-CoV-2 commercial serological assays and compared to
an in-house developed multiplex bead-based assay (27).
The overall results of all 68 samples are illustrated in Figure
1. A total of six commercial LFAs (test G, H, J, K, R and S)
reached 100% specicity for both IgG and IgM including all
chronic inammatory disease cohorts patients (n=67). Notably,
all samples from MS patients (n=10) were negative for both IgM
and IgG in all 20 assays.
For the 17 LFAs evaluated for specicity using 25 RA samples
(from 24 patients of which 20 were treatment naïve and 4 were
treated with iniximab) that were positive for RF, 10 assays had
unspecic signal detected for at least one immunoglobulin
isotype (Figures 2 and 3). Five assays had unspecic signal for
both IgM and IgG in a few up to a majority of the RA samples
(test C: IgM 19/20, IgG 8/20; test D: IgM 19/20, IgG 2/20; test M:
IgM 4/20, IgG 3/20; test N: IgM 6/20, IgG 1/20; and test P: IgM 1/
20, IgG 1/20). Unspecic IgM signal, without unspecic IgG
signal, was detected in four LFAs (test E: 5/20; test F: 16/20; test
O: 20/20; and test Q: 19/20). In one LFA, only the IgG test gave
unspecic signal (test L: 1/20). In contrast, only ve assays
detected unspecic signal in RA samples that were RF negative
(n= 23), with ve detecting IgM and one detecting IgG (test D:
IgM 1/23; test F: IgM 1/23; test M: IgM 2/23, IgG 2/23; for test N:
FIGURE 1 | Overview of false positive results in all samples for 19 different serological tests. Six LFA tests (G, H, J, K, R & S) gave no false positive results. The false
positivity rate of the remaining tests ranged between 2 - 45%. The test code keys are described in Table 2. The two ELISA assays (test A and B) were only tested
for IgG.
Kharlamova et al. False Positive SARS-CoV-2 Serological Tests
Frontiers in Immunology | www.frontiersin.org May 2021 | Volume 12 | Article 6661145

Citations
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02 Mar 2021
TL;DR: Potential use of glucocorticoids, biologics, JAK inhibitors, etc., blocking the effects of pro-inflammatory cytokines for treatment of COVID-19 is discussed.
Abstract: The coronavirus 2019 pandemic (coronavirus disease, COVID-19), etiologically related to the SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus-2), has once again reawakened healthcare professionals’ interest towards new clinical and conceptual issues of human immunology and immunopathology. An unprecedented number of clinical trials and fundamental studies of epidemiology, virology, immunology and molecular biology, of the COVID-19 clinical course polymorphism and pharmacotherapy have been conducted within one year since the outbreak of 2019 pandemic, bringing together scientists of almost all biological and physicians of almost all medical specialties. Their joint efforts have resulted in elaboration of several types of vaccines against SARS-CoV-2 infection and, in general, fashioning of more rational approaches to patient management. Also important for COVID-19 management were all clinical trials of biologics and “targeted” anti-inflammatory drugs modulating intracellular cytokine signaling, which have been specifically developed for treatment immune-mediated inflammatory rheumatic disease (IMIRDs) over the past 20 years. It became obvious after a comprehensive analysis of the entire spectrum of clinical manifestations and immunopathological disorders in COVID-19 is accompanied by a wide range of extrapulmonary clinical and laboratory disorders, some of which are characteristic of IMIRDs and other autoimmune and auto-in-flammatory human diseases. All these phenomena substantiated the practice of anti-inflammatory drugs repurposing with off-label use of specific antirheumatic agents for treatment of COVID-19. This paper discusses potential use of glucocorticoids, biologics, JAK inhibitors, etc., blocking the effects of pro-inflammatory cytokines for treatment of COVID-19.

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TL;DR: The outbreak of the 2019 novel coronavirus disease (COVID-19) has induced a considerable degree of fear, emotional stress and anxiety among individuals around the world.
Abstract: The outbreak of the 2019 novel coronavirus disease (COVID-19) has induced a considerable degree of fear, emotional stress and anxiety among individuals around t

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"SARS-CoV-2 serological tests can ge..." refers background in this paper

  • ...The cumulative number of infected and fatal cases can be followed at the Johns Hopkins University COVID-19 Dashboard.(2) Patients with chronic inflammatory disease are often treated with immunomodulatory treatments and therefore potentially more susceptible to infections....

    [...]

  • ...Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an enveloped positive sense single stranded RNA virus and the causative agent of the coronavirus disease 2019 (COVID-19) which emerged as a pandemic in the human population late 2019.1 The cumulative number of infected and fatal cases can be followed at the Johns Hopkins University COVID-19 Dashboard.2 Patients with chronic inflammatory disease are often treated with immunomodulatory treatments and therefore potentially more susceptible to infections.3 As a result, there has been substantial concern during the pandemic as to the potential increased risk COVID-19 disease severity and mortality among these patient groups....

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16 Mar 2020-Science
TL;DR: It is estimated that 86% of all infections were undocumented before the 23 January 2020 travel restrictions, which explains the rapid geographic spread of SARS-CoV-2 and indicates that containment of this virus will be particularly challenging.
Abstract: Estimation of the prevalence and contagiousness of undocumented novel coronavirus [severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2)] infections is critical for understanding the overall prevalence and pandemic potential of this disease. Here, we use observations of reported infection within China, in conjunction with mobility data, a networked dynamic metapopulation model, and Bayesian inference, to infer critical epidemiological characteristics associated with SARS-CoV-2, including the fraction of undocumented infections and their contagiousness. We estimate that 86% of all infections were undocumented [95% credible interval (CI): 82–90%] before the 23 January 2020 travel restrictions. The transmission rate of undocumented infections per person was 55% the transmission rate of documented infections (95% CI: 46–62%), yet, because of their greater numbers, undocumented infections were the source of 79% of the documented cases. These findings explain the rapid geographic spread of SARS-CoV-2 and indicate that containment of this virus will be particularly challenging.

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Additional excerpts

  • ...There also seem to be some pre-existing immunity present in the population, as determined by memory T cell reactivity(23) 24 and the estimated prevalence of infected individuals in comparisons to the proportion that succumb in severe disease.(25)...

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Journal ArticleDOI
25 Jun 2020-Cell
TL;DR: Using HLA class I and II predicted peptide ‘megapools’, circulating SARS-CoV-2−specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively, suggesting cross-reactive T cell recognition between circulating ‘common cold’ coronaviruses and SARS.
Abstract: Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide "megapools," circulating SARS-CoV-2-specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%-27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4+ T cells in ∼40%-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating "common cold" coronaviruses and SARS-CoV-2.

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Journal ArticleDOI
10 Mar 1995-Cell
Abstract: Structural similarity between viral T cell epitopes and self-peptides could lead to the induction of an autoaggressive T cell response Based on the structural requirements for both MHC class 11 binding and TCR recognition of an immunodominant myelin basic protein (MBP) peptide, criteria for a data base search were developed in which the degeneracy of amino acid side chains required for MHC class 11 binding and the conservation of those required for T cell activation were considered A panel of 129 peptides that matched the molecular mimicry motif was tested on seven MBP-specific T cell clones from multiple sclerosis patients Seven viral and one bacterial peptide efficiently activated three of these clones Only one peptide could have been identified as a molecular mimic by sequence alignment The observation that a single T cell receptor can recognize quite distinct but structurally related peptides from multiple pathogens has important implications for understanding the pathogenesis of autoimmunity

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01 Oct 2020-Cell
TL;DR: It is shown that SARS-CoV-2 elicits robust, broad and highly functional memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19.
Abstract: SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19.

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