Nucleocapsid Antigenemia Is a Marker of Acute SARS-CoV-2 Infection

Abstract

Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is essential for diagnosis, treatment, and infection control. Polymerase chain reaction (PCR) fails to distinguish acute from resolved infections, as RNA is frequently detected after infectiousness. We hypothesized that nucleocapsid in blood marks acute infection with the potential to enhance isolation and treatment strategies. In a retrospective serosurvey of inpatient and outpatient encounters, we categorized samples along an infection timeline using timing of SARS-CoV-2 testing and symptomatology. Among 1860 specimens from 1607 patients, the highest levels and frequency of antigenemia were observed in samples from acute SARS-CoV-2 infection. Antigenemia was higher in seronegative individuals and in those with severe disease. In our analysis, antigenemia exhibited 85.8% sensitivity and 98.6% specificity as a biomarker for acute coronavirus disease 2019 (COVID-19). Thus, antigenemia sensitively and specifically marks acute SARS-CoV-2 infection. Further study is warranted to determine whether antigenemia may aid individualized assessment of active COVID-19.

Keywords: COVID-19; SARS-CoV-2; antigenemia; nucleocapsid.

Figure 1.

A, Schematic of the process for COVID-19 status assignment. Samples from patients with no record of positive SARS-CoV-2 respiratory testing were only considered negative if corresponding negative respiratory testing occurred on the same day. Due to the lack of a gold standard for active SARS-CoV-2 infection, samples from individuals with history of positive SARS-CoV-2 testing were labeled based on earliest known positive SARS-CoV-2 respiratory test and time since symptom onset. *Samples with postwindow-positive SARS-CoV-2 testing were labeled negative if a negative SARA-CoV-2 test was available following the sample but before the positive test. Otherwise the sample was labeled unknown. B, Flow chart of categorization and labeling process indicating number of samples assigned to each group. Abbreviations: COVID-19, coronavirus disease 2019; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 2.

A, Prevalence of antigenemia and serum or plasma nucleocapsid levels for blood samples by category. Unexpected results (presence of nucleocapsid in the convalescent and same-day negative groups, absence of nucleocapsid in the acute group) are examined in Supplementary Tables 2–5. B, ROC curve for diagnostic performance of detectable antigenemia with reference to a −14/+3 day window for acute infection. The additional curves progressively exclude ambiguous categories. C, Impact on sensitivity and specificity of varying the window period, which defines the reference standard for acute COVID-19. D, AUC for the same varied window periods. E, Antigenemia compared to RT-PCR Ct value for those specimens with a Ct value available from the clinical laboratory on the same day. Symbols correspond to assay and gene target with horizontal line linking Ct values for different targets detected in the same sample. This includes data from 4 assays on 3 thermocycler platforms described in further detail in the Supplementary material. Abbreviations: AUC, area under the curve; CDC, Centers for Disease Control and Prevention; COVID-19, coronavirus disease 2019; Ct, cycle threshold; N, nucleocapsid; ORF, open reading frame; ROC, receiver operating characteristic; RT-PCR, reverse transcription polymerase chain reaction; S, spike; Se, sensitivity; Sp, specificity.

Figure 3.

A and B, Serum or plasma nucleocapsid plotted against time since diagnosis (A) and symptom onset (B). Samples without antigen detected are shown stacked on the horizontal axis. Four samples with antigenemia beyond 41 days are listed in the box and 93 samples without antigenemia between 41 and 351 days after earliest diagnosis are not shown. C, Serum or plasma nucleocapsid in patients whose COVID-19 course was described as asymptomatic in clinical records. The x-axis reflects time between first known positive respiratory test and the day the blood sample used in our analysis was collected. Shape and shading of each symbol classify serological status of asymptomatic patients. D, Serum or plasma nucleocapsid for individuals with positive nasopharyngeal RT-PCR on the same day as blood sample collection. Shape and shading of each symbol classify clinical status of patients. Abbreviations: COVID-19, coronavirus disease 2019; N, nucleocapsid; RBD, receptor-binding domain; RT-PCR, reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 4.

A, Comparison of serum or plasma nucleocapsid levels in individuals with and without SARS-CoV-2–specific antibodies. Samples were tested by in-house developed serological tests for nucleocapsid- and receptor binding domain-specific IgG as well as receptor binding domain-specific IgA and IgM. Levels of nucleocapsid are plotted and compared in samples stratified by seropositivity for each antibody type. B, Comparison of nucleocapsid levels by serostatus pattern. RBD positive/N negative is most consistent with prior vaccination. RBD positive /N positive is most consistent with natural infection with or without prior vaccination. Abbreviations: Ig, immunoglobulin; N, nucleocapsid; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 5.

Comparison of serum or plasma nucleocapsid levels by (A–C) severity and (D and E) inflammatory biomarkers. B and C, Intubation includes intubation within 30 days before or after the blood sample was collected. D and E, Individuals with severe COVID-19 as defined by the composite of 30-day intubation or mortality are highlighted. Abbreviations: COVID-19, coronavirus disease 2019; CRP, C-reactive protein; FEU, fibrinogen equivalent unit.