Clinical Performance of the Elecsys Electrochemiluminescent Immunoassay for the Detection of SARS-CoV-2 Total Antibodies

In the context of COVID-19, a wide range of serology immunoassays with different SARS-CoV-2 antigen recognition and antibody specificity have been developed to complement reverse transcription PCR (RT-PCR) assays (1,). Serological testing is useful for diagnosis and characterization of the course of the disease, identification of convalescent plasma donors, epidemiology studies, lockdown exit programs, and COVID-19 vaccine development (2, 3,). Given the widespread dissemination of these new methods and the limited experience with these assays, it is crucial for laboratories to rigorously validate these methods before broad introduction into routine clinical practice. Independent validations are also needed to ensure that the assays are in line with expected analytical and clinical performance specifications (1–4). This study is the first to report the external validation of a new electrochemiluminescent immunoassay, the Elecsys anti-SARS-CoV-2 from Roche Diagnostics. This test allows the detection of total antibodies (including IgG) specifically directed against the SARS-CoV-2 nucleocapsid and is performed on the cobas® e801 module. The test result is given as a cutoff index. According to the manufacturer, a result <1.0 is considered negative, and a result ≥1.0 is considered positive (5). The within- and between-run imprecision (CV) on 5 patient pools (cutoff index means of 0.081, 1.0, 8.7, 24, and 54) varied from 0.8% to 3.3% and from 1.2% to 3.6%, respectively. Sample storage complied with the conditions listed in the package insert. This retrospective study was conducted from May 6 to 12, 2020, at the clinical biology laboratory of the Clinique Saint-Luc Bouge (SLBO). Serum samples (n = 140) obtained from 97 patients with a confirmed RT-PCR SARS-CoV-2 diagnosis were used to determine the clinical sensitivity of the assay. RT-PCR on respiratory samples (nasopharyngeal swab samples) was performed on the LightCycler® 480 Instrument II using the LightMix® Modular SARS-CoV-2 E-gene set (Roche Diagnostics). Serum samples were subdivided into different categories based on the number of days after a positive RT-PCR test, as follows: 0–6 days, 45 sera; 7–13 days, 35 sera; 14–20 days, 24 sera; 21–27 days, 15 sera; ≥28 days, 21 sera. Among the 60 samples collected, ≥14 days after positive detection by RT-PCR and using the manufacturer’s cutoff, the Elecsys anti-SARS-CoV-2 immunoassay identified 55 true-positive and 5 false-negative samples. The diagnostic sensitivity was 91.7% (95% CI, 81.6%–97.2%). Using the optimal cutoff provided by ROC curve analyses (i.e., >0.165) improved the performance of the test to give a sensitivity of 100% (95% CI, 94.0%–100%; Fig. 1). Clinical performance of the Elecsys anti-SARS-CoV-2 assay subdivided by time since reverse transcription PCR (RT-PCR) positivity or the onset of symptoms. Cross-reactivity refers to the cross-reactivity test group. *Unaffected by the cutoff used (≥1.0 or >0.165). The dotted lines indicate the manufacturer’s cutoff (in black) and the optimized cutoff (in gray). COI, cutoff index. A sensitivity analysis was also performed considering the date of symptom onset. Among the 97 patients, data about time of symptom onset were available for 92 patients. The collected samples (n = 129) were subdivided into different categories according to the number of days after the onset of symptoms, as follows: 0–6 days, 22 sera; 7–13 days, 28 sera; 14–20 days, 26 sera; 21–27 days, 23 sera; ≥28 days, 30 sera. Among the 79 samples evaluated ≥14 days after the onset of symptoms and using the manufacturer’s cutoff, the Elecsys anti-SARS-CoV-2 assay identified 72 true-positive and 7 false-negative samples. The diagnostic sensitivity was 91.1% (95% CI, 82.6%–96.4%). Using the ROC curve cutoff (i.e., >0.165) improved the performance of the tests, with sensitivity of 95.1% (95% CI, 88.0%–98.7%). Analyses of serum samples obtained ≥28 days after symptom onset provided sensitivity of 96.7% (95% CI, 82.8%–99.9%) and 100% (95% CI, 88.9%–100%) with the manufacturer and the optimized cutoff, respectively (Fig. 1). Considering samples obtained before 14 days (from RT-PCR positivity or symptom onset), sensitivity was not sufficient to be reliable in clinical practice (Fig. 1). Non-SARS-CoV-2 sera (n = 79) collected before the COVID-19 pandemic (between January 2019 and December 2019) with potential cross-reactions (cross-reactivity test group) were also analyzed. Samples in this group included positive antinuclear antibodies (n = 5), antithyroglobulin antibody (n = 1), anti-Treponema pallidum antibodies (n = 2), antistreptolysin O (n = 1), antithyroid peroxidase antibodies (n = 4), chikungunya antibody (n = 1), direct Coombs (n = 1), hepatitis B antigen (n = 4), hepatitis C antibodies (n = 7), hepatitis E antibodies (n = 4), HIV antibodies (n = 2), IgA Chlamydia pneumoniae (n = 1), IgG Chlamydia trachomatis (n = 1), IgG Coxiella burneti (n = 2), IgM Borrelia (n = 1), IgM Coxiella burnetii (n = 1), IgM cytomegalovirus (n = 5), IgM Epstein-Barr virus viral capsid (n = 5), IgM Mycoplasma pneumoniae (n = 6), IgM parvovirus B19 (n = 7), IgM Toxoplasma gondii (n = 5), influenza antibodies (n = 6), irregular agglutinins ( n = 2), and rheumatoid factor (n = 5). The calculated specificity was 100% (95% CI, 95.44%–100.0%). Using the ROC curve cutoff (i.e., >0.165) had no effect on the measured diagnostic specificity (Fig. 1). The optimal ROC cutoff showed excellent clinical performance ≥14 days after RT-PCR positivity or the onset of COVID-19 symptoms. Additional studies are needed to confirm the best cutoff. Expert societies are urged to provide guidance on the best time after RT-PCR positivity or symptom onset to perform serological investigations, which are important determinants of the true positivity rate. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. J. Favresse, statistical analysis, administrative support, provision of study material or patients; C. Eucher, administrative support, provision of study material or patients; M. Elsen, administrative support, provision of study material or patients; J. Douxfils, statistical analysis. Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: J. Douxfils, chief executive officer and founder of Qualiblood sa. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: Roche Diagnostics provided the kits for the validation. Expert Testimony: None declared. Patents: None declared. Other Remuneration: J. Douxfils, personal fees from Diagnostica Stago, Roche, Roche Diagnostics, Daiichi-Sankyo, and Portola, outside the submitted work. Acknowledgment: We thank the personnel of the Saint-Luc Bouge laboratory for technical assistance.