Diagnostic performances and thresholds: The key to harmonization in serological SARS-CoV-2 assays?

Abstract

Background: The evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibody (Ab) assay performances is of the utmost importance in establishing and monitoring virus spread in the community. In this study focusing on IgG antibodies, we compare reliability of three chemiluminescent (CLIA) and two enzyme linked immunosorbent (ELISA) assays.

Methods: Sera from a total of 271 subjects, including 64 reverse transcription-polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 patients were tested for specific Ab using Maglumi (Snibe), Liaison (Diasorin), iFlash (Yhlo), Euroimmun (Medizinische Labordiagnostika AG) and Wantai (Wantai Biological Pharmacy) assays. Diagnostic sensitivity and specificity, positive and negative likelihood ratios were evaluated using manufacturers' and optimized thresholds.

Results: Optimized thresholds (Maglumi 2 kAU/L, Liaison 6.2 kAU/L and iFlash 15.0 kAU/L) allowed us to achieve a negative likelihood ratio and an accuracy of: 0.06 and 93.5% for Maglumi; 0.03 and 93.1% for Liaison; 0.03 and 91% for iFlash. Diagnostic sensitivities and specificities were above 93.8% and 85.9%, respectively for all CLIA assays. Overall agreement was 90.3% (Cohen's kappa = 0.805 and SE = 0.041) for CLIA, and 98.4% (Cohen's kappa = 0.962 and SE = 0.126) for ELISA.

Conclusions: The results obtained indicate that, for CLIA assays, it might be possible to define thresholds that improve the negative likelihood ratio. Thus, a negative test result enables the identification of subjects at risk of being infected, who should then be closely monitored over time with a view to preventing further viral spread. Redefined thresholds, in addition, improved the overall inter-assay agreement, paving the way to a better harmonization of serologic tests.

Keywords: Antibodies; COVID-19; Clinical performances; SARS-CoV-2.

Fig. 1

IgG antibody levels for Maglumi, iFlash, Liaison, Euroimmun and total antibody levels of Wantai are reported after Log10 scale transformation. Patients/Subjects categories: blood donors (Pre-COV), negative autoimmune patients (AI), negative healthcare workers (NegHW), SARS-CoV-2 positive patients, subdivided into Mild (Mild), Moderate (Mod) and Severe (Sev) symptoms. Statistically significant differences: for Maglumi and iFlash, Pre-COV, AI and NegHW vs SARS-CoV-2 positive patients (p < 0.01 for all); for Liaison, Pre-COV, AI and NegHW vs SARS-CoV-2 positive patients (p < 0.01 for all) and Mild vs Sev (p = 0.017); for Euroimmun, AI and NegHW vs SARS-CoV-2 positive patients (p < 0.01 for all) and Mild vs Sev (p = 0.006); for Wantai, p = not significant for all.

Fig. 2

IgM antibody levels are reported for Maglumi, iFlash and Wantai. IgA antibody levels are reported for Euroimmun. Log10 scale transformation was used. Blood donors (Pre-COV), negative autoimmune patients (AI), negative healthcare workers (NegHW), SARS-CoV-2 positive patients, subdivided into Mild (Mild), Moderate (Mod), and Severe (Sev) symptoms. Statistically significant differences: for Maglumi, Pre-COV and AI vs NegHW and SARS-CoV-2 positive patients (p < 0.01 for all) and Mild vs Mod and Sev (p < 0.01 and p = 0.022, respectively); iFlash, Pre-COV and AI vs NegHW and SARS-CoV-2 positive patients (p < 0.01 for all) and Mild vs Sev (p = 0.041, respectively); for Euroimmun, NegHW vs Sev (p = 0.009) and Mild vs Sev (p = 0.044); for Wantai, NegHW vs SARS-CoV-2 positive patients (p = 0.03 for Mild, p < 0.01 for Mod and Sev).

Supplementary Fig. 1

Supplementary Fig. 2