Correctly Interpreting SARS CoV 2 Serologic Assays
The authors of Tang et al. (1) have incorrectly concluded that the Abbott SARS CoV 2 assay demonstrated higher sensitivity and specificity than the EUROIMMUN Anti SARS CoV 2 IgG assay, despite also stating that overlapping of confidence intervals showed differences were statistically insignificant. Therefore, the authors contradict their own conclusions as they found no significant difference between the EUROIMMUN and the Abbott assays for detection of IgG antibodies to SARS CoV 2. Of concern, for the specificity controls, the authors used 153 samples. Of these, 50 were from uncharacterized donors sampled in 2015 and the other 103 samples used were without description of the sampling date. We can only assume that the 80 samples from symptomatic patients with negative RT PCR for SARS CoV 2 noted in the study were sampled during the pandemic. The sampling date for the remaining 23 specimens is unclear: 5 samples from patients with positive RT PCR for other human coronaviruses without sampling date, 4 samples from patients with Influenza A or B without characterization of the type or conformation for the disease and 14 random samples with known outcomes of unrelated antibodies against CMV or EBV and rheumatoid factor. We regard the use of PCR negative samples taken during the pandemic for negative controls in the calculation as problematic and believe that most in the field would agree with that conclusion. RT PCR cannot be used as a "gold standard" in this setting, given that the "false" negative rate of RT PCR is approximately 20% depending on the study (2) Negative results in individuals that have already been infected may arise because of analytical failure, but may also be attributable to timing of sampling, low nasal carriage of virus, or low viral load. It therefore remains plausible that positive results in this group are true positives. Overall, 5 patients compared with one patient were positive out of the 80 symptomatic RT PCR negative individuals using EUROIMMUN and Abbott assays, respectively. Additional serial sampling of these patients would be required to accurately determine their immune response against SARS CoV 2. In a study of 1756 preand postpandemic normal individuals, specificity of the EUROIMMUN Anti SARS CoV 2 IgG assay was 99.0% (3) We further believe that the condensed report of the sensitivity based on all 103 samples from only 48 patients taken at different time points postonset of symptoms (PSO) and/or positive results in RT PCR also portrays a biased picture. In general, IgG antibodies can be detected at a median of 14 days' PSO and IgG peaks at 21 25 days PSO. Long et al. 2020 showed an increase in virus specific IgG was observed during the first 3 weeks PSO, and the positivity rate reached approximately 100% at 17 19 days PSO (4) Segregation of time points for determining sensitivity of IgG assays is imprecise as published in this study, and mostly before IgG response maturation. CDC recommendations call for medical use of IgG serology to determine past infection, not an acute infection, and use of acute rather than convalescent sera to determine "sensitivity" of an IgG serology is incorrect. Beavis et al. demonstrated a EUROIMMUN Anti SARS CoV2 IgG sensitivity of 100% in convalescent sera of previously documented RT PCR positive individuals (5) The study cites and was performed according to an earlier CE marked assay package insert. While the kit and assay instructions remained unchanged, the currently available kit was FDA EUA approved based on additional data (3) We recognize that the literature on serology is rapidly evolving, but FDA websites are constantly updated (3) In total, 597 COVID19 patient samples were included in our EUA submission. Additional studies have been published or are ongoing. The authors state it is unclear whether neutralizing antibodies target the spike protein or the nucleocapsid protein. On the contrary, experience with SARS CoV 1 and recent studies on SARS CoV 2 suggest that neutralizing antibodies target the external S spike protein, particularly the S1 subunit (the antigen in the EUROIMMUN assay) and the receptor binding domain contained within the S1 domain, preventing viral entry into the host cell, rather than the internal N nucleocapsid protein (the antigen in the Abbott assay) We and others continue to study neutralization. In conclusion, we believe strongly the authors' study does not accurately represent true sensitivity and specificity of the EUROIMMUN IgG ELISA.
Clinical chemistry