Goals
Molecular assays on nasopharyngeal swabs remain the cornerstone of COVID-19 diagnosis. The high technicalities of nasopharyngeal sampling and molecular assays, as well as scarce reagent resources, limit our testing capabilities. Several strategies have failed, to date, to completely alleviate this testing process (eg, saliva sampling or antigen testing of nasopharyngeal swabs). We evaluated the clinical performance of ELISA detection of SARS-CoV-2 nucleocapsid antigen (N-antigen) in serum or plasma using the COVID-19 Quantigene® assay (AAZ, France).
Methods
Yields were determined on 63 serum samples from 63 non-COVID patients and 227 serum samples (165 patients) from the French COVID and CoV-CONTACT cohorts with RT-PCR confirmed SARS-CoV-2 infection, including 142 samples. serum (114 patients) obtained within 14 days of symptom onset.
- Patients and ethics
Negative samples comprised 50 pre-pandemic samples (collected between December 2, 2019, and January 13, 2020) and 13 pandemic samples from patients not infected with SARS-CoV-2 that tested positive for other microbial antigens (i.e., antigen NS1, HBs antigen, HIV-1 p24 antigen, HKU1 coronavirus or malaria antigens). Positive samples were collected between January 25, 2020, and September 2, 2020, from study participants, included in the French COVID (clinicaltrials.gov NCT04262921) and CoV-CONTACT (clinicaltrials.gov NCT04259892) cohorts.
The following serum samples from those patients, when collected at the physician’s discretion, were also included. They gave their informed written consent for the use of their samples for research. Ethics approval was granted by the French Ethics Committee CPP-Ile-de-France 6 (RCB ID: 2020-A00256-33 and RCB ID: 2020-A00280-39) and the French National Data Protection Commission ( approval #920102).
For COVID-19 patients, available serum samples were classified into different categories based on delay from symptom onset: serum collected ≤14 days after symptom onset (142 serum samples from 114 patients), samples serum samples collected >14 days after symptom onset (81 serum samples from 72 patients), serum samples collected from asymptomatic patients (three serum samples from three patients), and patient with no symptom onset date (one sample from patient serum).
- Assessment of N antigen level
Before analysis, serum samples were stored at –80°C. N-antigenemia levels were determined with an ELISA CE-IVD microplate assay, COVID-Quantigene® (AAZ), according to the manufacturer’s recommendations. Briefly, in each well of 96-well microplates previously coated with anti-SARS-CoV-2 N antibodies, 50 μL of a solution containing biotinylated anti-SARS-CoV-2 N antibodies and 50 μL of serum were added. After incubation at 37°C for 60 min, plates were washed five times with phosphate buffer.
Then, 100 μL of a solution containing horseradish peroxidase-conjugated streptavidin was added, followed by incubation for 30 min at 37 °C. The plates were washed five times with the phosphate buffer solution, then 50 μL of a solution containing the substrate (3,3′,5,5′-tetramethylbenzidine (TMB)) and 50 μL of a second solution containing urea. After 15 min at 37°C, the colourimetric reaction was stopped by adding 50 µL of H2SO4.
Absorbance values were measured at 450nm, with the reference set at 630nm. Standards made with recombinant N antigens were assayed on each plate to quantify N antigenemia levels for each patient’s sample. Since the purpose of this study was to evaluate the sensitivity of this new assay, samples with titers greater than 180 pg/mL were not diluted for accurate quantification.
- RT-PCR assays
For all patients included in this study, the diagnosis of SARS-CoV-2 infection was made in the virology department of the Bichat-Claude Bernard University Hospital using RT-PCR on nasopharyngeal swabs, as recommended.
Different techniques were performed during the study period for nasopharyngeal samples, due to frequent shortage issues and fast turnaround time requirements: RealStar® SARS-CoV-2 (Altona, Hamburg, Germany), Cobas® SARS-CoV-2 ( Roche Diagnostics, Branchburg, NJ, USA), Simplexa® COVID-19 Direct (DiaSorin, Gerenzano, Italy), BioFire® SARS-CoV-2 (BioMerieux, Salt Lake City, UT, USA), QIAstat -Dx® Respiratory SARS-CoV-2 (Qiagen, Hilden, Germany) and NeumoDX® (Qiagen, Hilden, Germany) using IP2 Institute Pasteur and WHO E gene primers. Gene cycle threshold (Ct) values, available for all techniques except Simplexa® COVID-19 Direct and BioFire® SARS-COV-2, were used as an indicator of viral load for 104 samples from 91 patients with swabs. and matched nasopharyngeal sera (ie collected in the same 24 hr).
For a subset of 146 samples, corresponding to 89 patients included in the French COVID-19 cohort, paired serum and plasma samples were available, allowing the presence of viral RNA in plasma to be determined. Briefly, viral nucleic acids were extracted from 200 μL of plasma with the MagNA Pure LC Total Nucleic Acid Isolation Kit – Large Volume (Roche Diagnostics, Branchburg, NJ, USA) and eluted at 50 μL. RT-PCR was performed on 10 μL of eluate using the RealStar® SARS-CoV-2 assay (Altona, Germany), according to the manufacturer’s recommendations. Samples with RT-PCR cycle threshold values greater than 40 were considered negative.
- Detection of anti-SARS-CoV-2 nucleocapsid IgG
For a subset of 85 serum samples, corresponding to 80 patients (ICU patients: n = 21, ward patients: n = 36, and outpatients: n = 23), we performed a chemiluminescent microparticle immunoassay that detects anti-IgG. N (Architect SARS-CoV-2 IG assay, Abbott). The results were reported as a signal for the cut-off value (S/Co). The positivity threshold was set at 1.4, as recommended by the manufacturer.
Results
The specificity was 98.4% (95% CI 95.3-100). Sensitivity was 79.3% overall (180/227, 95% CI, 74.0–84.6) and 93.0% (132/142, 95% CI, 88.7–97.2) within 14 days of symptom onset. Ninety-one of the included patients had serum samples and nasopharyngeal swabs collected in the same 24 hours. Among those with high nasopharyngeal viral loads, ie, Ct values below 30 and 33, only 1/50 and 4/67 were negative for N-antigenemia, respectively. Among those with a negative nasopharyngeal RT-PCR, 8/12 had positive N-antigenemia; The lower respiratory tract was explored in six of these eight patients, showing positive RT-PCR in five cases.
Discussion
This is the first evaluation of a commercially available serum N antigen detection assay. It exhibits robust specificity and sensitivity within the first 14 days after symptom onset. This approach provides a valuable new option for COVID-19 diagnosis, requiring only one blood draw and is easily scalable across clinical laboratories.
Keywords
Antigen, antigenemia, Blood, COVID-19, Diagnosis, Plasma, SARS-CoV-2, Serum