Case-Control Study of Individuals with Discrepant Nucleocapsid and Spike Protein SARS-CoV-2 IgG Results

Abstract

Background: Laboratory-based methods for SARS-CoV-2 antibody detection vary widely in performance. However, there are limited prospectively-collected data on assay performance, and minimal clinical information to guide interpretation of discrepant results.

Methods: Over a 2-week period, 1080 consecutive plasma samples submitted for clinical SARS-CoV-2 IgG testing were tested in parallel for anti-nucleocapsid IgG (anti-N, Abbott) and anti-spike IgG (anti-S1, EUROIMMUN). Chart review was conducted for samples testing positive or borderline on either assay, and for an age/sex-matched cohort of samples negative by both assays. CDC surveillance case definitions were used to determine clinical sensitivity/specificity and conduct receiver operating characteristics curve analysis.

Results: There were 52 samples positive by both methods, 2 positive for anti-N only, 34 positive for anti-S1 only, and 27 borderline for anti-S1. Of the 34 individuals positive for anti-S1 alone, 8 (24%) had confirmed COVID-19. No anti-S1 borderline cases were positive for anti-N or had confirmed/probable COVID-19. The anti-N assay was less sensitive (84.2% [95% CI 72.1-92.5%] vs 94.7% [95% CI 85.4-98.9%]) but more specific (99.2% [95% CI 95.5-100%] vs 86.9% [95% CI 79.6-92.3%]) than anti-S1. Abbott anti-N sensitivity could be improved to 96.5% with minimal effect on specificity if the index threshold was lowered from 1.4 to 0.6.

Conclusion: Real-world concordance between different serologic assays may be lower than previously described in retrospective studies. These findings have implications for the interpretation of SARS-CoV-2 IgG results, especially with the advent of spike antigen-targeted vaccination, as a subset of patients with true infection are anti-N negative and anti-S1 positive.

Keywords: COVID-19; SARS-CoV-2; serology.

Fig. 1.

Flow chart of consecutive plasma samples tested for SARS-CoV-2 IgG. The 115 samples positive/borderline for either or both Abbott anti-nucleocapsid IgG (anti-N) and EUROIMMUN anti-spike protein IgG (anti-S1) were all included as cases. Out of the 965 samples negative by both assays, 115 controls were selected for chart review based on one-to-one matching for availability of demographic data, age, and sex (see Supplemental Table 2).

Fig. 2.

Log-transformed Abbott anti-nucleocapsid IgG (anti-N) index value vs EUROIMMUN anti-spike protein IgG (anti-S1) optical density ratio for the age and sex-matched cases/controls (n = 230). The horizontal dashed line represents the anti-N threshold of positivity (1.4); the 2 vertical dashed lines represent the anti-S1 threshold for borderline (0.8) and positive (1.1), respectively. The top right corner contains dual-positive samples, the bottom left corner contains dual-negative samples, and the remaining points represent samples with discordant results. The different colors/shapes represent A) IgG results, B) exposure history, C) respiratory nucleic acid amplification test (NAAT) results, D) symptom history according to Centers for Disease Control (CDC) clinical case criteria, E) inpatient and/or intensive care unit (ICU) admission history, F) case classification based on CDC surveillance case definitions.

Fig. 3.

Receiver operating curve characteristics for A-B) Abbott anti-nucleocapsid IgG (anti-N) and C-D) EUROIMMUN anti-spike protein IgG (anti-S1). While the 2 assays have similar areas under the curve (AUCs) with overlapping 95% confidence intervals (95% CI), the anti-N assay has a more favorable sensitivity (solid red line) vs specificity (dashed blue line) profile as compared to the anti-S1 assay. The Abbott anti-N index threshold for positivity could be lowered from 1.4 to 0.6 to improve sensitivity without a substantial decrement to specificity.