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 | Infectious Diseases | JAMA Network Open | JAMA Network
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Figure 1.  SARS-CoV-2 Cerebrospinal Fluid (CSF) Antigen Detection
SARS-CoV-2 Cerebrospinal Fluid (CSF) Antigen Detection

Detection of nucleocapsid antigen and spike antigen in CSF in patient groups and controls. A, Nucleocapsid antigen was positive in 31 of 35 (14 of 16 neuroasymptomatic and 17 of 19 neurosymptomatic) patients and 0 of 41 controls. B, Spike antigen was positive in 1 patient (0 of 41 controls). Positive assay cutoffs (dashed lines) were 0.32 pg/mL for nucleocapsid antigen and 0.41 pg/mL for spike antigen. Points represent individual values; lines and whiskers represent medians and IQRs.

Figure 2.  Neuroinflammatory Cerebrospinal Fluid (CSF) Biomarker Profiles in Groups of Patients With COVID-19 and Controls
Neuroinflammatory Cerebrospinal Fluid (CSF) Biomarker Profiles in Groups of Patients With COVID-19 and Controls

Profiles of CSF biomarkers of inflammation/immune activation (A-H) and neuroaxonal injury (I) in the 21 neuroasymptomatic and 23 neurosymptomatic patient groups, patient control groups (n = 41), and healthy control groups (n = 10). Boxes indicate the median and IQR, whiskers indicate 10th and 90th percentiles, small white squares indicate means, and values above or below are drawn as individual points. Concentrations are shown as unadjusted values. Dashed lines indicate assay levels of detection (LODs). Dotted lines indicate upper normal reference values. β2M indicates β2-microglobulin; IFN-γ, interferon γ; IL, interleukin; TNF-α, tumor necrosis factor α.

Figure 3.  SARS-CoV-2 Nucleocapsid Antigen Correlations
SARS-CoV-2 Nucleocapsid Antigen Correlations

A, A close correlation was found between cerebrospinal fluid (CSF) and serum SARS-CoV-2 nucleocapsid antigen. Individual points are shown using unadjusted nucleocapsid antigen concentrations in patients with COVID-19. The median (IQR) ratio of serum-CSF nucleocapsid antigen was 42 (range, 18-89). B and C, CSF neopterin and interferon (IFN) γ concentrations are shown as unadjusted individual values in relation to CSF nucleocapsid antigen. B, Dotted line represents upper normal reference for CSF neopterin. D and E, CSF SARS-CoV-2 nucleocapsid antigen was significantly correlated with CSF neopterin (D) and CSF IFN-γ (E) adjusted for time of CSF sampling. Individual values are adjusted for time of sampling and are reported as the day 10 equivalent concentration. All correlation coefficients (r) and statistical significances were estimated using partial correlation adjusted for time from symptom onset to CSF sampling day.

Table 1.  Baseline Characteristics for the Patients With COVID-19 and Control Groups
Baseline Characteristics for the Patients With COVID-19 and Control Groups
Table 2.  Biomarker Concentrationsa
Biomarker Concentrationsa
1.
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Paterson  RW, Benjamin  LA, Mehta  PR,  et al; UCLH Queen Square COVID-19 Biomarker Study Group.  Serum and cerebrospinal fluid biomarker profiles in acute SARS-CoV-2-associated neurological syndromes.   Brain Commun. 2021;3(3):fcab099. doi:10.1093/braincomms/fcab099 PubMedGoogle ScholarCrossref
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Yilmaz  A, Marklund  E, Andersson  M,  et al.  Upper respiratory tract levels of severe acute respiratory syndrome coronavirus 2 RNA and duration of viral RNA shedding do not differ between patients with mild and severe/critical coronavirus disease 2019.   J Infect Dis. 2021;223(1):15-18. doi:10.1093/infdis/jiaa632 PubMedGoogle ScholarCrossref
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WHO Working Group on the Clinical Characterisation and Management of COVID-19 infection.  A minimal common outcome measure set for COVID-19 clinical research.   Lancet Infect Dis. 2020;20(8):e192-e197. doi:10.1016/S1473-3099(20)30483-7 PubMedGoogle ScholarCrossref
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Yilmaz  A, Mellgren  Å, Fuchs  D,  et al.  Switching from a regimen containing abacavir/lamivudine or emtricitabine/tenofovir disoproxil fumarate to emtricitabine/tenofovir alafenamide fumarate does not affect central nervous system HIV-1 infection.   Infect Dis (Lond). 2019;51(11-12):838-846. doi:10.1080/23744235.2019.1670352 PubMedGoogle ScholarCrossref
20.
Hagberg  L, Cinque  P, Gisslen  M,  et al.  Cerebrospinal fluid neopterin: an informative biomarker of central nervous system immune activation in HIV-1 infection.   AIDS Res Ther. 2010;7:15. doi:10.1186/1742-6405-7-15 PubMedGoogle ScholarCrossref
21.
Gaetani  L, Höglund  K, Parnetti  L,  et al.  A new enzyme-linked immunosorbent assay for neurofilament light in cerebrospinal fluid: analytical validation and clinical evaluation.   Alzheimers Res Ther. 2018;10(1):8. doi:10.1186/s13195-018-0339-1 PubMedGoogle ScholarCrossref
22.
Yilmaz  A, Blennow  K, Hagberg  L,  et al.  Neurofilament light chain protein as a marker of neuronal injury: review of its use in HIV-1 infection and reference values for HIV-negative controls.   Expert Rev Mol Diagn. 2017;17(8):761-770. doi:10.1080/14737159.2017.1341313 PubMedGoogle ScholarCrossref
23.
Rosengren  LE, Ahlsén  G, Belfrage  M, Gillberg  C, Haglid  KG, Hamberger  A.  A sensitive ELISA for glial fibrillary acidic protein: application in CSF of children.   J Neurosci Methods. 1992;44(2-3):113-119. doi:10.1016/0165-0270(92)90004-W PubMedGoogle ScholarCrossref
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Lee  MH, Perl  DP, Nair  G,  et al.  Microvascular injury in the brains of patients with Covid-19.   N Engl J Med. 2021;384(5):481-483. doi:10.1056/NEJMc2033369PubMedGoogle ScholarCrossref
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Remsik  J, Wilcox  JA, Babady  NE,  et al.  Inflammatory leptomeningeal cytokines mediate COVID-19 neurologic symptoms in cancer patients.   Cancer Cell. 2021;39(2):276-283.e3. doi:10.1016/j.ccell.2021.01.007 PubMedGoogle ScholarCrossref
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    Original Investigation
    Infectious Diseases
    May 23, 2022

    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

    Author Affiliations
    • 1Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
    • 2Region Västra Götaland, Department of Infectious Diseases, Sahlgrenska University Hospital, Gothenburg, Sweden
    • 3Meso Scale Diagnostics LLC, Rockville, Maryland
    • 4Institute of Biological Chemistry, Medical University of Innsbruck, Biocenter, Austria
    • 5Institute of Medical Biochemistry, Medical University of Innsbruck, Biocenter, Austria
    • 6Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
    • 7Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden
    • 8Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden
    • 9Department of Neurodegenerative Disease, UCL Institute of Neurology, London, England
    • 10Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden
    • 11UK Dementia Research Institute at UCL, London, England
    • 12Hong Kong Center for Neurodegenerative Diseases, Clear Water Bay, Hong Kong, China
    JAMA Netw Open. 2022;5(5):e2213253. doi:10.1001/jamanetworkopen.2022.13253
    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.

    Abstract

    Importance  Neurologic symptoms are common in COVID-19, but the central nervous system (CNS) pathogenesis is unclear, and viral RNA is rarely detected in cerebrospinal fluid (CSF).

    Objective  To measure viral antigen and inflammatory biomarkers in CSF in relation to neurologic symptoms and disease severity.

    Design, Setting, and Participants  This cross-sectional study was performed from March 1, 2020, to June 30, 2021, in patients 18 years or older who were admitted to Sahlgrenska University Hospital, Gothenburg, Sweden, with COVID-19. All patients had CSF samples taken because of neurologic symptoms or within a study protocol. Healthy volunteer and prepandemic control groups were included.

    Exposure  SARS-CoV-2 infection.

    Main Outcomes and Measures  Outcomes included CSF SARS-CoV-2 nucleocapsid antigen (N-Ag) using an ultrasensitive antigen capture immunoassay platform and CSF biomarkers of immune activation (neopterin, β2-microglobulin, and cytokines) and neuronal injury (neurofilament light protein [NfL]).

    Results  Forty-four patients (median [IQR] age, 57 [48-69] years; 30 [68%] male; 26 with moderate COVID-19 and 18 with severe COVID-19 based on the World Health Organization Clinical Progression Scale), 10 healthy controls (median [IQR] age, 58 [54-60] years; 5 [50%] male), and 41 patient controls (COVID negative without evidence of CNS infection) (median [IQR] age, 59 [49-70] years; 19 [46%] male) were included in the study. Twenty-one patients were neuroasymptomatic and 23 were neurosymptomatic (21 with encephalopathy). In 31 of 35 patients for whom data were available (89%), CSF N-Ag was detected; viral RNA test results were negative in all. Nucleocapsid antigen was significantly correlated with CSF neopterin (r = 0.38; P = .03) and interferon γ (r = 0.42; P = .01). No differences in CSF N-Ag concentrations were found between patient groups. Patients had markedly increased CSF neopterin, β2-microglobulin, interleukin (IL) 2, IL-6, IL-10, and tumor necrosis factor α compared with controls. Neurosymptomatic patients had significantly higher median (IQR) CSF interferon γ (86 [47-172] vs 21 [17-81] fg/mL; P = .03) and had a significantly higher inflammatory biomarker profile using principal component analysis compared with neuroasymptomatic patients (0.54; 95% CI, 0.03-1.05; P = .04). Age-adjusted median (IQR) CSF NfL concentrations were higher in patients compared with controls (960 [673-1307] vs 618 [489-786] ng/L; P = .002). No differences were seen in any CSF biomarkers in moderate compared with severe disease.

    Conclusions and Relevance  In this study of Swedish adults with COVID-19 infection and neurologic symptoms, compared with control participants, viral antigen was detectable in CSF and correlated with CNS immune activation. Patients with COVID-19 had signs of neuroaxonal injury, and neurosymptomatic patients had a more marked inflammatory profile that could not be attributed to differences in COVID-19 severity. These results highlight the clinical relevance of neurologic symptoms and suggest that viral components can contribute to CNS immune responses without direct viral invasion.

    Introduction

    Neurologic symptoms are common in patients hospitalized with COVID-19 and include anosmia, encephalopathy, encephalitis, and cerebrovascular manifestations, including stroke.1-3 Several mechanisms have been proposed to contribute to the central nervous system (CNS) pathogenesis, including direct damage caused by viral CNS invasion, direct or indirect consequences of the systemic inflammatory response, viral interaction with the neurovascular unit or microvascular injuries, and thromboembolic events.4,5

    The neuroinvasive capacity of SARS-CoV-2 is questionable. Although virus has been suggested to enter the CNS via olfactory sensory neurons, neuropathologic studies6,7 have shown conflicting results. Furthermore, viral detection in brain tissue is often sporadic, of low levels, and not clearly linked to neuropathologic signs of infection.8 In cerebrospinal fluid (CSF), viral RNA can only be detected at very low levels in a few cases, and characteristic signs of neurotropic viral infections (CSF pleocytosis and blood-brain barrier injury) are often mild or absent.4,9 Although viral RNA is rarely detected, intrathecal immune activation has been a distinguishing feature in patients with neurologic symptoms.10,11 Biomarker signs of neuroaxonal injury have also been observed, most evident in patients with encephalitis or stroke.10,12-14

    The cause of the CSF immune response in COVID-19 in the absence of detectable viral RNA is not clear. SARS-CoV-2 viral nucleocapsid antigen (N-Ag) can be detected in plasma during the acute phase of infection and is potentially useful as a diagnostic as well as prognostic marker in COVID-19.15 However, little is known about the presence of viral antigens in CSF. The aims of this study were to investigate whether viral antigens could be detected in CSF in patients with COVID-19 and to compare CSF biomarker profiles of patients with or without neurologic symptoms as well as in patients with moderate compared with severe COVID-19.

    Methods
    Study Population, Procedures, and Viral Diagnostics

    This prospective cross-sectional study included adult (≥18 years of age) patients with confirmed SARS-CoV-2 infection admitted to Sahlgrenska University Hospital in Gothenburg, Sweden, who had undergone a diagnostic lumbar puncture because of neurologic symptoms or as part of a research protocol. This study was performed from March 1, 2020, to June 30, 2021. All participants (or next of kin if the patient was not able) provided written informed consent according to the Declaration of Helsinki.16 The study protocol was approved by the Swedish Ethical Review Authority. The report follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cross-sectional studies.

    SARS-CoV-2 infection was confirmed by viral RNA detection in real-time polymerase chain reaction assays of nasopharyngeal swab or plasma (n = 1) specimens or in-hospital seroconversion (n = 1). SARS-CoV-2 RNA was analyzed by real-time polymerase chain reaction in swab specimens and cell-free CSF and plasma samples as previously described (eMethods in the Supplement).10,17 Serum SARS-CoV-2 antibodies were used as complementary diagnostic tests and were analyzed using commercial assays (Architect, Abbott Laboratories, and iFlash 1800, YHLO). SARS-CoV-2–specific IgG was not analyzed in CSF.

    Lumbar puncture and venipuncture for CSF and blood sampling were performed in direct sequence. Clinical and neurologic status was recorded by the attending physician (infectious disease specialist or neurologist) in an electronic medical records database. Participant ethnicity was investigator observed using electronic medical records. The patient cohort was divided into groups according to neurologic status: neuroasymptomatic or neurosymptomatic. Disease severity was evaluated according to the World Health Organization Clinical Progression Scale.18

    Two separate control groups were included in the study. At the time of antigen and cytokine analysis, prospective control samples were unavailable. Consequently, we used archived (−80 °C) CSF and serum samples from age-matched COVID-19–negative patient controls who had undergone a diagnostic lumbar puncture because of initial suspicion of CNS infection, with CSF analyses indicating no sign of ongoing CNS disease. For the other included CSF analyses, additional archived samples were unavailable. Instead, a second control group of healthy COVID-19–negative volunteers recruited prospectively during the pandemic was used.

    SARS-CoV-2 Antigen Detection

    Detection of SARS-CoV-2 nucleocapsid and spike proteins was performed using MSD SPLEX CoV-2 N and MSD S-PLEX CoV-2 S assay kits (Meso Scale Discovery). The assays were run according to protocols in the kit package inserts (eMethods in the Supplement). The CSF and plasma samples were run undiluted (25 μL per well). Sample quantitation was achieved using a calibration curve generated using a recombinant antigen standard. 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 its value. The LOD values were 0.16 pg/mL for the nucleocapsid assay and 0.28 pg/mL for the spike assay, and the assay cutoffs (concentrations used for classifying samples as antigen positive) were 0.32 pg/mL for the nucleocapsid assay and 0.41 pg/mL for the spike assay (eMethods in the Supplement).

    Biomarker Analyses

    IgG and albumin concentrations were measured by immunoturbidimetry on a Cobas instrument (Roche Diagnostics). The IgG index and albumin ratio were calculated as previously described.19 Cerebrospinal fluid neopterin was measured using a commercially available immunoassay (Brahms).20 Cerebrospinal fluid β2-microglobulin (β2M) was measured using the N latex β2M kit on the Atellica NEPH 630 System (Siemens Healthcare GmbH). The CSF NfL was measured using a previously described in-house sandwich enzyme-linked immunosorbent assay.21 The NfL concentrations were age adjusted and reported as the equivalent for 65 years of age as previously described.22 The CSF glial fibrillary acidic protein concentration was measured using an in-house enzyme-linked immunosorbent assay as previously described.23 Cytokine concentrations were measured using the novel S-PLEX Proinflammatory Panel I. The S-PLEX format provides signal enhancement that achieves sensitivity in femtograms per milliliters (eMethods in the Supplement). The CSF and plasma samples were tested undiluted.

    Statistical Analysis

    All statistical analyses were performed using Prism software, version 9.0.0 (GraphPad Software) or SPSS, version 26 (IBM Inc). Numerical variables were log10 transformed where appropriate. Differences between patients with COVID and controls and differences in CSF NfL concentrations between the groups of patients with COVID-19 were estimated by Welch 2-sided t tests. Comparisons of other CSF biomarkers, as well as the first principal component of the cytokines neopterin and β2M, between the groups of patients with COVID-19 were performed using analysis of covariance, adjusting for time from symptom onset to CSF sampling. Because CSF NfL is known to increase gradually during the weeks after a CNS injury, this analysis was not adjusted for sampling time to avoid overadjustment. Correlations among CSF biomarkers were analyzed using partial correlation, adjusting for time of CSF sampling. Partial correlation coefficients are reported as r unless otherwise stated. Differences in baseline characteristics between patient groups were analyzed using t tests for continuous variables and Fisher exact test for categorical variables. All tests were 2-sided, and P < .05 was considered statistically significant.

    Results
    Study Participants

    Forty-four patients (median [IQR] age, 57 [48-69] years; 30 male [68%] and 14 female [32%]), 10 healthy controls (median [IQR] age, 58 [54-60] years; 5 male [50%] and 5 female [50%]), and 41 patient controls (COVID negative without evidence of CNS infection) (median [IQR] age, 59 [49-70] years; 19 male [46%] and 22 female [54%]) were included in the study. All patients were admitted to the hospital with moderate (n = 26) or severe (n = 18) COVID-19 according to the World Health Organization Clinical Progression Scale.18 Twenty-one patients with COVID-19 were neuroasymptomatic, and 23 were neurosymptomatic. The neurosymptomatic group included patients with encephalopathy (n = 21), encephalitis (n = 1), and Guillain-Barré syndrome (n = 1). Encephalitis was defined as altered mental status and CSF pleocytosis (white blood cell count, ≥5/μL [to convert to ×109/L, multiply by 0.001]), viral detection in CSF, or supporting evidence of CNS infection (neuroimaging or electroencephalography). Baseline characteristics in the different groups are shown in Table 1. The median (IQR) ages were 57 (48-69) years for the patients with COVID-19, 58 (54-60) years for the healthy controls, and 59 (49-70) years for the patient controls.

    Neurosymptomatic patients tended to be older, had a lower proportion of severe COVID-19, and were consequently less likely to be treated with betamethasone (22% vs 48%; P = .02). The time from any COVID-19 symptom onset to CSF sampling was significantly shorter compared with the neuroasymptomatic group (median [range], 9 [3-16] vs 13 [3-21] days; P = .008). The CSF samples were acellular (white blood cell count, ≤4/μL) in all patients except in 2 neuroasymptomatic patients (white blood cell count, 6 and 7/μL) and 1 patient with encephalitis (white blood cell count, 261/μL). Three patients in the neurosymptomatic group died during hospitalization. Neuroimaging was performed in a subset of participants (eResults in the Supplement).

    Viral Detection

    All CSF samples tested negative for SARS-CoV-2 RNA, whereas 14 patients (11 of 20 neuroasymptomatic and 3 of 15 neurosymptomatic) had detectable viral RNA in plasma. The median cycle threshold (Ct) value for detectable plasma samples was 36.0 (range, 31.3-38.0). Concentrations of serum and CSF SARS-CoV-2 nucleocapsid and spike antigens are given in Table 2. The SARS-CoV-2 N-Ag was detectable in CSF in 31 of 35 patients (89%) (0 of 41 controls). The measured CSF N-Ag concentrations were not significantly different between the groups of patients with COVID-19 according to neurosymptoms (Figure 1) or disease severity (eTable 1 in the Supplement) after adjustment for CSF sampling day. Cerebrospinal fluid SARS-CoV2 spike antigen (S-Ag) was positive in 1 patient. Serum-CSF ratios are given in the eResults in the Supplement.

    CSF Biomarkers

    Cerebrospinal fluid biomarker concentrations are given in Table 2. The albumin ratio was above the upper normal reference (10.2) in 5 of 43 patients (12%), but no significant difference was seen in the albumin ratio between the groups of patients with COVID-19 and the control groups. All patients with COVID-19 and controls had a normal IgG index. Patients with COVID-19 had significantly higher CSF concentrations of neopterin, β2M, interleukin (IL) 2, IL-6, IL-10, and tumor necrosis factor α (TNF-α) compared with controls (Table 2 and Figure 2).

    Comparing the groups of patients with COVID-19, significantly higher concentrations of several biomarkers were seen in neurosymptomatic compared with neuroasymptomatic patients (Figure 2). However, because the CSF biomarker concentrations had similar trajectories depending on when the CSF sample was drawn, we adjusted the analysis for time from COVID-19 symptom onset to CSF sampling to avoid overestimation of the effect size because time of sampling was a significant covariate for CSF biomarker concentrations. Details of CSF biomarkers on the basis of sampling time are shown in eFigure 1 in the Supplement. Because median time from symptom onset to CSF sampling was significantly shorter in neurosymptomatic patients, we included sampling day in the model. After adjusting for CSF sampling day, the higher observed concentrations of IFN-γ, neopterin, IL-6, and TNF-α in the neurosymptomatic group (Figure 2) remained statistically significant for IFN-γ (neurosymptomatic-neuroasymptomatic ratio, 2.0; 95% CI, 1.1-3.7; P = .03), whereas CSF neopterin (ratio, 1.4; 95% CI, 0.99-1.9; P = .06), IL-6 (ratio, 1.8; 95% CI, 0.97-3.5; P = .06), and TNF-α (ratio, 1.4; 95% CI, 0.99-1.8; P = .06) fell just above the threshold for significance (Table 2). We compared the first principal component of the cytokines, β2M, and neopterin between the groups of patients with COVID-19 as a single test of difference in immunomarkers and found a significant difference (0.98; 95% CI, 0.36-1,61; P = .003), which remained significant after adjustment for time of sampling (0.54; 95% CI, 0.03-1.05; P = .04).

    Age-adjusted median (IQR) CSF NfL concentrations were significantly higher in patients with COVID-19 compared with controls (960 [673-1307] ng/L vs 618 [489-786] ng/L; P = .002), whereas no significant difference was found between the groups of patients with COVID-19 (Table 2 and Figure 2). Notably, 7 of 20 neurosymptomatic patients (35%) had concentrations of CSF NfL equivalent for 65 years of age above the upper normal reference (1577 ng/L), whereas CSF NfL equivalent for 65 years of age concentrations were within normal limits in all individuals in the other groups. We found no significant differences in any of the CSF biomarkers in patients with moderate compared with severe disease after adjusting for CSF sampling day.

    A close correlation was found between serum and CSF concentrations of SARS-CoV-2 N-Ag (r = 0.84; P < .001). Initial analysis also indicated a significant association between CSF N-Ag and the immune activation markers CSF neopterin, β2M, and IFN-γ, but after adjusting for CSF sampling day, correlations remained significant only for CSF neopterin (r = 0.38, P = .03) and IFN-γ (r = 0.42; P = .01) (Figure 3).

    Serum Biomarkers

    Serum biomarker concentrations followed a similar pattern of immune activation compared with CSF. Additional statistical differences were seen for several biomarkers between the groups of patients with COVID-19 in serum compared with CSF, as well as between patients with COVID-19 and controls. Time-adjusted differences in serum neopterin, β2M, TNF-α, IFN-γ, IL-4, and IL-2 were seen between the groups of patients with COVID-19, with higher concentrations found in neurosymptomatic patients. Compared with controls, patients with COVID-19 had higher serum concentrations of all biomarkers except IL-12p70 and IL-1β (eTable 2 and eFigure 2 in the Supplement).

    We used an albumin ratio–adjusted biomarker index ([CSF concentration/serum concentration]/albumin ratio) to explore differences in intrathecal biomarker proportions between the patient groups. Of all studied biomarkers, only IL-4 showed a significant difference (neurosymptomatic-neuroasymptomatic ratio, 2.7; 95% CI, 1.1-6.7; P = .03), suggesting that CNS symptoms were not associated with selective intrathecal production of the inflammatory biomarkers.

    Discussion

    In this study of patients hospitalized with acute COVID-19, we found that despite the absence of viral RNA, SARS-CoV-2 N-Ag was readily detectable in CSF in most individuals using a novel ultrasensitive assay. Furthermore, CSF N-Ag was correlated with the CSF immune activation biomarkers neopterin and IFN-γ. Our findings indicate that SARS-CoV-2 viral antigens are associated with a CNS immune response without apparent viral invasion of the CNS. Although CSF N-Ag concentrations were not significantly different between patient groups, patients with COVID-19 had higher concentrations of CSF NfL compared with controls, and neurosymptomatic patients had a more marked immune activation biomarker profile, suggesting that the magnitude of the CNS immune response, possibly triggered by viral components, contributes to the neuropathogenesis of COVID-19.

    Although the neuroinvasive capacity of SARS-CoV-2 is questionable,6-9,24-28 intrathecal immune activation is a prominent feature in several CSF studies.4,10,11,29 The presence of SARS-CoV-2 N-Ag may partially explain the apparent discrepancy between the lack of viral RNA detection and the CNS immune response in COVID-19, but antigen detection has rarely been included in CSF studies. Nucleocapsid or spike antigen was not detected in any CSF samples of 13 patients with cancer and severe COVID-19 with neurologic symptoms using a commercial enzyme-linked immunosorbent assay, presumably related to assay sensitivity, although the time of sampling may have contributed to the lack of antigen detection.29 In plasma, as confirmed in the current study, N-Ag was detected in most patients during early SARS-CoV-2 infection.15 We found a high degree of correlation between serum and CSF N-Ag concentrations (r = 0.84; P < .001) and a median serum-CSF ratio of 42 (range, 18-89), suggesting that CSF N-Ag originates from systemic sources, either by passive diffusion across the blood-brain barrier or as a consequence of viral interaction with the neurovascular unit, possibly including infection of neurovascular pericytes.30-32 The markedly lower serum concentrations in combination with the potential loss of S-Ag by binding to angiotensin-converting enzyme 2 on cells lining the ventricular system may have influenced the low detection rate of S-Ag in CSF.

    Patients with COVID-19 had significantly increased CSF concentrations of neopterin, β2M, IL-2, IL-6, IL-10, and TNF-α compared with controls, indicating CNS-specific inflammation and confirming previous findings of increased neopterin and β2M in patients with COVID-19 with neurologic symptoms.10,11 Although increased CSF concentrations of IL-6 and TNF-α have also been described previously,11,12,33 results are less consistent across studies.13,34 Although other CNS infections and inflammatory conditions can generate significantly higher inflammatory CSF biomarker concentrations,13,35 the CSF inflammatory biomarker profile seen in patients with COVID-19 in our study remains a consistent finding and adds to previous reports11,29,36,37 indicating the importance of CNS immune activation in the neuropathogenesis of COVID-19.

    The underlying mechanism of neurologic symptoms (particularly encephalopathy) during COVID-19 is not clear. Of interest, we found that patients with neurologic symptoms had significantly higher time-adjusted CSF concentrations of IFN-γ and a more marked inflammatory biomarker profile compared with neuroasymptomatic patients. Age-adjusted CSF NfL, representing neuroaxonal injury, was also significantly higher in patients with COVID-19 compared with controls. In addition, all patients with CSF NfL concentrations above the upper normal reference were found in the neurosymptomatic group. Overall, we found a notable correlation between CSF NfL and CSF TNF-α, IL-1β, and IL-17A, suggesting that immune cell activation and the CNS inflammatory response may result in neuroaxonal injury. Altogether, these findings indicate that the CNS immune response may be an important driver of CNS pathogenesis, similar to other CNS inflammatory conditions or infections.11,13,29,38 The principal difference between patient groups appeared to be in the magnitude of the immune response, suggesting that neuropathogenesis in COVID-19 with neurologic symptoms represents a continuum of inflammation, where the intensity of the inflammatory response (likely in combination with individual premorbid vulnerability) may be a determining factor in the development of CNS symptoms. In turn, CNS symptoms may reflect harmful effects on the brain measurable as neuroaxonal injury (CSF NfL). Notably, CSF biomarkers followed a similar pattern compared with serum, where differences in biomarkers between groups of patients with COVID-19 as well as controls were more evident, indicating that the CNS inflammatory response likely constitutes a continuous part of the systemic infection. However, qualitative differences in the immune response in neurosymptomatic compared with neuroasymptomatic patients remains a possibility that needs to be investigated further. As described by others,14 no significant differences were found in CSF glial fibrillary acidic protein concentrations between any of the patient groups and controls, indicating that astrocyte activation may not be a major contributor to CNS pathogenesis in COVID-19.

    Few previous reports have included CSF analyses from patients with COVID-19 without neurologic symptoms. In a small study33 of patients in the intensive care unit, neurologic syndromes were associated with higher CSF concentrations of several cytokines and chemokines. However, observations made exclusively in patients in the intensive care unit may be subject to bias that can be attributed to critical illness and organ failure. We did not find any significant differences in CSF biomarkers in patients with moderate compared with severe COVID-19. Moreover, the lower proportion of patients with severe COVID-19 in those with neurologic symptoms compared with the neuroasymptomatic group (30% vs 52%) further suggests that neurologic manifestations represent a specific pathological process within the CNS that cannot be attributed solely to systemic infection.

    The time from symptom onset to CSF sampling was an important covariate for CSF biomarker concentrations. The timing of CSF sampling has varied considerably in previous studies.11-14,39 Given the inflammatory character of acute COVID-19, sampling at a later time may not reflect the processes that initiate CNS pathology. Moreover, few studies have adjusted for CSF sampling day, which may have influenced results.13 In the current study, all CSF samples were collected during the acute phase of COVID-19 (median of 12 days from symptom onset). Although we believe this timing is important for evaluating viral antigen detection and initial CNS immune responses, the CSF NfL is known to have a slower initial kinetic and longer half-life, where additional CSF sampling later in the disease course would be of interest for further evaluating the association of CNS inflammation with markers of neuronal injury.40,41

    Strengths and Limitations

    Strengths of this study include the prospective design, the inclusion of neuroasymptomatic as well as neurosymptomatic patients, and the inclusion of control groups. However, the study also has several limitations. The sample size, although large in comparison with other published reports,11,12,42 is still relatively small, and neuroimaging was not included consistently. Encephalopathy was the dominating neurologic manifestation, although the definition of encephalitis differed slightly from those used in some previous studies43 of COVID-19 with neurologic symptoms. Furthermore, the study did not include nonhospitalized patients with mild COVID-19. The small study size and exploratory approach prohibited evaluation of the potential prognostic value of the studied biomarkers. Of necessity, the study included 2 different control groups. Notably, the patient controls used for antigen and cytokine analyses had CSF samples drawn for clinical suspicion of CNS infection. Although no infection was subsequently diagnosed, some individuals may have had other undiagnosed CNS conditions that potentially affected CSF biomarker concentrations. It is highly unlikely that this would exaggerate differences compared with the COVID-19 samples, although we cannot rule out an opposite effect. In addition, we cannot exclude that other residual confounding factors may have influenced results.

    Conclusions

    In this cross-sectional study of Swedish adults with COVID-19 infection and neurologic symptoms, compared with control participants, SARS-CoV-2 N-Ag was detectable in CSF in most patients with COVID-19, despite the absence of detectable CSF viral RNA, and was correlated with CNS immune activation. Furthermore, neurosymptomatic patients had a more pronounced immune activation profile compared with neuroasymptomatic patients, and patients with COVID-19 had signs of neuroaxonal injury compared with controls. These observations could not be attributed to differences in COVID-19 severity, where no differences in CSF biomarkers were seen in patients with moderate compared with severe disease.

    Although confirmatory studies evaluating the significance of viral antigen detection in CSF are needed, these results suggest that viral components may contribute to CNS immune responses without direct viral invasion into the CSF compartment, which could partly explain the apparent discrepancy between the scarcity of CSF viral RNA detection and the marked CNS inflammatory response seen in patients with COVID-19 with neurologic symptoms. The results of this study also highlight the importance of neurologic symptoms, including encephalopathy. These findings have important potential implications for clinical treatment of patients with COVID-19, including the use of antiviral therapies, as well as for the continued importance of including CSF analyses in future studies of CNS pathogenesis and treatment strategies.

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    Article Information

    Accepted for Publication: April 4, 2022.

    Published: May 23, 2022. doi:10.1001/jamanetworkopen.2022.13253

    Correction: This article was corrected on June 22, 2022, to fix some incorrect labels in Figures 1 and 2; the label “Neurosymptomatic patients” had been incorrectly written as “Asymptomatic patients.”

    Open Access: This is an open access article distributed under the terms of the CC-BY License. © 2022 Edén A et al. JAMA Network Open.

    Corresponding Author: Arvid Edén, MD, PhD, Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital, Gothenburg SE-416 50, Sweden (arvid.eden@gu.se).

    Author Contributions: Dr Edén had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

    Concept and design: Edén, Schöll, Sigal, Stengelin, Gisslén.

    Acquisition, analysis, or interpretation of data: Edén, Grahn, Bremell, Aghvanyan, Bathala, Fuchs, Gostner, Hagberg, Kanberg, Kanjananimmanont, Lindh, Misaghian, Nilsson, Sigal, Stentoft, Studahl, Yilmaz, Wang, Stengelin, Zetterberg, Gisslén.

    Drafting of the manuscript: Edén, Gisslén.

    Critical revision of the manuscript for important intellectual content: All authors.

    Statistical analysis: Edén, Bathala, Misaghian, Nilsson, Wang, Gisslén.

    Obtained funding: Edén, Schöll, Gisslén.

    Administrative, technical, or material support: Bathala, Fuchs, Gostner, Kanberg, Kanjananimmanont, Lindh, Misaghian, Schöll, Sigal, Stentoft, Yilmaz, Wang, Stengelin, Gisslén.

    Supervision: Edén, Grahn, Bremell, Sigal, Studahl, Gisslén.

    Conflict of Interest Disclosures: Dr Schöll reported receiving grants from the Wallenberg Foundation, Swedish Research Council, and Swedish Brain Foundation during the conduct of the study. Dr Zetterberg reported receiving advisory board fees from AbbVie, Alector, Annexon, Artery Therapeutics, AZTherapies, CogRx, Denali, Eisai, Nervgen, Pinteon Therapeutics, Red Abbey Labs, Passage Bio, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, lecture fees from Cellectricon, Roche, Fujirebio, Alzecure, and Biogen, and cofounding and owing stock in Brain Biomarker Solutions outside the submitted work. Dr Gisslén reported receiving grants from the Swedish state under an agreement between the Swedish government and the county councils, SciLifeLab from the Knut and Alice Wallenberg Foundation, and the Swedish Research Council during the conduct of the study. No other disclosures were reported.

    Funding/Support: Dr Gisslén is supported by grants KAW 2020.0182 and KAW 2020.0241 from the SciLifeLab National COVID-19 Research Program, financed by the Knut and Alice Wallenberg Foundation, the Swedish state, under agreement ALFGBG-717531 between the Swedish government and the county councils and agreement 2021-06545 of the Swedish Research Council. Dr Zetterberg is a Wallenberg Scholar supported by grant 2018-02532 from the Swedish Research Council, grant 681712 from the European Research Council, grant ALFGBG-720931 from the Swedish State Support for Clinical Research, grant 201809-2016862 from the Alzheimer Drug Discovery Foundation, grants ADSF-21-831376-C, ADSF-21-831381-C, and ADSF-21-831377-C from the AD Strategic Fund, the Alzheimer's Association, the Olav Thon Foundation, the Erling-Persson Family Foundation, grant FO2019-0228 from the Stiftelsen för Gamla Tjänarinnor, Hjärnfonden, Sweden, the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement 860197, grant JPND2021-00694 from the European Union Joint Program for Neurodegenerative Disorders, and the UK Dementia Research Institute at UCL. Dr Schöll is supported by grant KAW2014.036 from the Knut and Alice Wallenberg Foundation (Wallenberg Centre for Molecular and Translational Medicine), grants 2017-02869, 2021-02678, and 2021-06545 from the Swedish Research Council, the Swedish state under the agreement between the Swedish government and the county councils, ALF agreements ALFGBG-813971 and ALFGBG-965326, and grants FO2021-0311 from the Swedish Brain Foundation.

    Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

    Meeting Presentation: Presented at the Conference on Retroviruses and Opportunistic Infections (CROI) 2022 (virtual); February 16, 2022.

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