Serum SARS-CoV-2 Antigens for the Determination of COVID-19 Severity

Abstract

The diagnostic of SARS-CoV-2 infection relies on reverse transcriptase polymerase chain reactions (RT-PCRs) performed on nasopharyngeal (NP) swabs. Nevertheless, false-negative results can be obtained with inadequate sampling procedures, making the use of other biological matrices worthy of investigation. This study aims to evaluate the kinetics of serum N antigens in severe and non-severe patients and compare the clinical performance of serum antigenic assays with NP RT-PCR. Ninety patients were included in the study and monitored for several days. Disease severity was determined according to the WHO clinical progression scale. Serum N antigen levels were measured with a chemiluminescent assay (CLIA) and the Single Molecular Array (Simoa) assay. Viremia thresholds for severity were determined and proposed. In severe patients, the peak antigen response was observed 7 days after the onset of symptoms, followed by a decline. No real peak response was observed in non-severe patients. Severity thresholds for the Simoa and the CLIA provided positive likelihood ratios of 30.0 and 10.9 for the timeframe between day 2 and day 14, respectively. Sensitive detection of N antigens in serum may thus provide a valuable new marker for COVID-19 diagnosis and evaluation of disease severity. When assessed during the first 2 weeks since the onset of symptoms, it may help in identifying patients at risk of developing severe COVID-19 to optimize better intensive care utilization.

Keywords: COVID-19; RT-PCR; SARS-CoV-2; antigenic assay; prognosis test.

Figure 1

Study flow diagram. ^# In some kinetic models, asymptomatic patients and patients with positive SARS-CoV-2 spike immunoglobulin G were excluded. * Multiple sample inclusion criteria were applied to investigate different possible cut-offs for severity: (a) earliest antigenemia value since symptom onset within the day 2 to day 14 window for a particular patient; (b) mean of all antigenemia values of samples collected within the day 2 to day 14 window for a particular patient; and (c) the maximal antigenemia value obtained within the day 2 to day 14 window. All these inclusion strategies reported one value per patient (see Supplemental Figure S2).

Figure 2

Kinetics of antigenemia since the onset of symptoms in non-severe and severe patients. The grey dotted lines correspond to the positivity cut-off of each antigen assay, as found by ROC curve analyses. The black dotted line corresponds to the positivity cut-off of the iFlash assay, as declared by the manufacturer. The red dotted lines correspond to the severity cut-off of each antigen assay, as found by the ROC curve analyses for the day 2–day 14 window. Only patients with symptoms and negative for SARS-CoV-2 Spike IgG directed against the spike protein were included in this kinetics representation.

Figure 3

Levels of serum antigen according to the delay since the onset of symptoms (<3, 4–10, 11–20 and >20 days) and severity. Blue dots correspond to non-severe patients and red dots to severe patients. The grey dotted lines correspond to the positivity cut-off of each antigen assay, as found by ROC curve analyses. The black dotted line corresponds to the positivity cut-off of the iFlash assay, as declared by the manufacturer. Medians are represented on top of each whisker box. Only p-values < 0.05 are represented.

Figure 4

(A) Positive and negative SARS-CoV-2 Spike IgG results in serum according to antigenemia. The grey dotted lines on the Y-axis correspond to the positivity cut-off of each antigen assay, as found by ROC curve analyses. The black dotted line on the Y-axis of the iFlash panel represents the cut-off specified by the manufacturer. (B) Linear regression of antigenemia obtained with the two antigen assays versus the amount of SARS-CoV-2 Spike IgG. The grey dotted line on the X-axis represents the positivity cut-off for IgG directed against the spike protein.