SARS-CoV-2 serological tests can generate false positive results for samples from patients with chronic inflammatory diseases
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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References
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"SARS-CoV-2 serological tests can ge..." refers background in this paper
...IgM-RF is the isotype commonly measured in most clinical laboratories, and detected in 60-80% of RA patients,(29) 32 but might appear also in other diseases.(29) 32 33 In the current study, we were not able to detect any specific associations between occurrence of RF IgM or RF IgG and false positivity for IgM/ IgG antiSARS-Cov-2 in RA patients, since the RF positive RA sera were specifically selected to be highly positive for all RF isotypes simultaneously....
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...The presence of IgM antibodies indicates a recent infection, whereas presence of IgG antibodies indicates possible longlasting immunity.(13) 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 evidences; ensure immunity to allow key workers to return to work after exposure; and evaluate vaccine development studies and vaccine strategies....
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...4 variety of other diseases as well as in the general population and may increase with age, smoking and chronic infection.(9) 10 SLE is a systemic inflammatory 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....
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