Viral Antigen and Inflammatory Biomarkers in Cerebrospinal Fluid in Patients With COVID-19 Infection and Neurologic Symptoms Compared With Control Participants Without Infection or Neurologic Symptoms

Key Points Question Are cerebrospinal fluid (CSF) SARS-CoV-2 antigens associated with central nervous system inflammation in patients with COVID-19? Findings Of 44 patients with COVID-19 (23 neurosymptomatic) included in this hospital-based cross-sectional study, CSF nucleocapsid antigen was detectable in 89% of patients with available data and was significantly correlated with immune activation markers (neopterin and interferon γ). Moreover, neurosymptomatic patients had a more pronounced inflammatory CSF profile compared with neuroasymptomatic patients that could not be attributed to differences in COVID-19 severity. Meaning These results suggest that viral components may contribute to central nervous system immune responses without direct viral invasion and highlight the clinical importance of neurologic symptoms.


Viral diagnostic methods
Nucleic acid from 200 μL nasal swab medium, plasma or CSF was extracted from the clinical samples in a MagNA Pure 96 instrument using the Total Nucleic Acid isolation kit (Roche, Basel, Switzerland). RT-PCR targeting the RNA-dependent RNA polymerase (RdRP) region was performed by a QuantStudio 6 instrument (Applied Biosystems, Foster City, CA, USA) using a probe and the primers RdRP_Fi, GTCATGTGTGGCGGTTCACT and RdRP_Ri, CAACACTATTAGCATAAGCAGTTGT 1 . Viral load was estimated using the cycle threshold (Ct) values using formula (47 − Ct)/3.4 = log10 copies/sample. Ct values ≤38 were considered as positive.

MSD S-PLEX CoV-2 N, MSD S-PLEX CoV-2 S, and MSD S-PLEX Proinflammatory Panel I assay design
The MSD S-PLEX assay kits (Meso Scale Discovery, Rockville, MD) employ a sandwich immunoassay format and electrochemiluminescence (ECL) detection. The assays are carried out in specially designed 96-well plate consumables having integrated screen-printed carbon ink electrodes on the bottom of each well that are used as solid-phase supports for binding reactions, and as the source of electrical energy for inducing ECL from ECL labels in binding complexes on their surfaces. The kits use MSD's ultra-sensitive S-PLEX ECL format, which provides additional signal enhancement and sensitivity relative to conventional ECL formats. Sample quantitation was achieved using a calibration curve generated with a recombinant antigen standard, and fit to a four parameter logistic (4PL) model. The LOD for all S-PLEX assays was determined as the concentration (based on a 4PL fit to a calibration curve) that provides a signal 2.5 standard deviations above the blank signal. For graphing and analysis, any concentrations below the limit of detection (LOD) were assigned the LOD value, and any concentrations above the highest calibration standard were assigned the concentration of the highest calibration standard.
The N antigen assay 2-4 utilizes recombinant full-length N-protein as a standard, and monoclonal capture/detection antibodies generated by MSD against the full-length recombinant N protein. The S antigen assay 3 utilizes recombinant receptor binding domain (RBD) of the S1 subunit of the spike protein as a standard, and monoclonal capture/detection antibodies generated by MSD against recombinant RBD. The assay cut-offs for classifying samples as N or S antigen negative or positive were established in previous clinical studies of respiratory or plasma samples 2,3 .
All S-PLEX cytokine assays used recombinant cytokines as calibrator material and monoclonal antibodies as capture and detection antibodies. Anchoring of the calibrators to WHO or NIBSC standards is described in the package insert. LLOQ of the assays was defined as the lowest concentration that gave consistently CVs of less than 25% (<30% for IL-1b) and analyte recovery between 80% and 120% (75%-125% for IL-1b).

Neuroimaging
Neuroimaging was not included in the study protocol, and availability of imaging was limited in the early part of the pandemic due to infection prevention and control measures. However, brain computed tomography (CT) scan was performed in 19/23, and MRI in 4/23 patients in the NS-group. No evidence of encephalitis, acute hemorrhage, microhemorrhage or ischemia was found in imaging studies, while signs of global cortical atrophy were found in one, and small vessel disease in 4 patients.

Serum to CSF antigen ratios
The median (IQR) ratio of serum:CSF N-Ag was 42 (18-89). Overall, concentrations of serum N-Ag were markedly higher compared with S-Ag concentrations ( Table 2). Although the borderline concentrations and small number of detectable samples of S-Ag in CSF prohibited an accurate calculation of the corresponding ratio for serum:CSF S-Ag ratio, the number of CSF samples with positive S-Ag detection was within an expected range considering the measured serum concentrations of S-Ag given that S-Ag had a comparable serum:CSF ratio to N-Ag.