key: cord-0897644-kebrwb6t authors: Virhammar, Johan; Nääs, Anja; Fällmar, David; Cunningham, Janet L.; Klang, Andrea; Ashton, Nicholas J.; Jackmann, Sven; Westman, Gabriel; Frithiof, Robert; Blennow, Kaj; Zetterberg, Henrik; Kumlien, Eva; Rostami, Elham title: Biomarkers for central nervous system injury in cerebrospinal fluid are elevated in COVID‐19 and associated with neurological symptoms and disease severity date: 2021-01-19 journal: Eur J Neurol DOI: 10.1111/ene.14703 sha: 71015d1272f8abf2bf665f9b1b00b6f4b3693056 doc_id: 897644 cord_uid: kebrwb6t BACKGROUND AND PURPOSE: Neurological symptoms have been frequently reported in hospitalized patients with coronavirus disease 2019 (COVID‐19), and biomarkers of central nervous system (CNS) injury are reported to be increased in plasma but not extensively studied in cerebrospinal fluid (CSF). This study examined CSF for biomarkers of CNS injury and other pathology in relation to neurological symptoms and disease severity in patients with neurological manifestations of COVID‐19. METHODS: Nineteen patients with neurological symptoms and mild to critical COVID‐19 were prospectively included. Extensive analysis of CSF, including measurement of biomarkers of CNS injury (neurofilament light chain [NfL] protein, glial fibrillary acidic protein [GFAp], and total tau), was performed and compared to neurological features and disease severity. RESULTS: Neurological symptoms included altered mental status (42%), headache (42%), and central (21%) and peripheral weakness (32%). Two patients demonstrated minor pleocytosis, and four patients had increased immunoglobulin G levels in CSF. Neuronal autoantibody testing using commercial tests was negative in all patients. Increased CSF levels of NfL protein, total tau, and GFAp were seen in 63%, 37%, and 16% of patients, respectively. Increased NfL protein correlated with disease severity, time in intensive care, and level of consciousness. NfL protein in CSF was higher in patients with central neurological symptoms. CONCLUSIONS: Although limited by the small sample size, our data suggest that levels of NfL protein, GFAp, and total tau in CSF are commonly elevated in patients with COVID‐19 with neurological symptoms. This is in contrast to the standard CSF workup where pathological findings are scarce. NfL protein, in particular, is associated with central neurological symptoms and disease severity. Coronavirus disease 2019 (COVID- 19) , is a pandemic caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). A significant number of case reports and case series have described different types of neurological complications in COVID-19 [1] [2] [3] . The neurological manifestations are broad and may be caused by a direct effect of the virus on the nervous system or by a parainfectious or postinfectious immune-mediated inflammation [4] . However, neurological complications may also be secondary to critical illness and a long stay in an intensive care unit (ICU). Recently, neurochemical evidence of acute central nervous system (CNS) injury in patients with COVID-19 was shown in the form of increased plasma levels of neurofilament light chain (NfL) protein, a marker of axonal injury, and of glial fibrillary acidic protein (GFAp), a marker of astrocytic injury [5, 6] . In this regard, few studies have investigated the cerebrospinal fluid (CSF) in patients with COVID-19 [7, 8] , which is less sensitive than plasma to confounding release of neuromarkers (e.g., from peripheral nerves). Lumbar puncture (LP) is an important tool to evaluate critically ill patients with neurological symptoms, as it can reveal both the underlying pathology and the severity of injury to the nervous system. There are few reports on results from CSF analysis in patients with , and prospective studies with comprehensive CSF and neurological investigations are rare. The aim of this study was to describe clinical characteristics in relation to findings in CSF analyses among patients with COVID-19 and neurological symptoms. This was a prospective single-center study. Patients with confirmed COVID-19 and at least one new-onset neurological symptom were included from April 2020 until July 2020. Patients had either a positive polymerase chain reaction (PCR) test for SARS-CoV-2 in upper and/or lower airway samples [9, 10] Included patients were investigated with LP if this was required as part of their routine evaluation. In patients without strong indication for LP, the procedure was optional. The National Institutes of Health (NIH) criteria for COVID-19 severity grading were used to classify patients as mild, moderate, severe, or critical [11] . As a measure of respiratory status, the lowest PaO 2 /FiO 2 ratios at any time before the LP were documented for patients treated in the ICU. Microbiology, Uppsala University Hospital, as previously described [12] . All routine plasma and CSF analyses including interleukin-6 (IL- CSF NfL protein and GFAp concentrations were measured using in-house enzyme-linked immunosorbent assays, as previously described in detail [13, 14] . CSF T-tau concentration was measured using Lumipulse technology in accordance with the kit insert from the manufacturer (Fujirebio, Ghent, Belgium). Plasma NfL protein, GFAp, and T-tau measurements were performed using single molecule array assays on an HD-X Analyzer (Human Neurology 4-Plex A assay, N4PA advantage kit, 102153), as previously described [5] . A single batch of reagents was used; intraassay coefficients of variation were < 8% for all analytes. Autoantibodies in CSF and serum (NMDAR, LGI1, CASPR2, GABA B1 R, GABA B2 R, AMPA1, AMPA2, Ri, Yo, Ma2, CV2, Hu, and amphiphysin) were analyzed using a commercial assay (Euroimmun, Lübeck, Germany). Data are presented as median (interquartile range [IQR]) or number (%). The Mann-Whitney U test was used for comparing continuous parameters between groups. Correlations between clinical parameters and CSF findings were tested using Spearman rank correlation. In the figures, the biomarker data have been log-transformed to achieve near-normal distribution. A p value of <0.05 was considered significant. The statistical analysis was performed using SPSS version 27 (IBM, Armonk, NY). COVID-19 was confirmed in 32 patients through positive PCR for SARS-CoV-2 in upper and/or lower airway samples and in one patient with IgG for SARS-CoV-2 in serum. Twelve patients had contraindications for LP (all related to high doses of low molecular weight heparin or oral anticoagulants), and one patient declined the investigation. Therefore, LPs could be performed in 20 out of 33 patients. One patient suffered from a small traumatic subarachnoid hemorrhage and a skull fracture after a head trauma 2 days before the LP and was excluded. The remaining 19 patients were the main focus of this study. The median time between onset of symptoms and LP was 23 days (IQR = 6-43). Descriptive data are presented in Table 1 , and detailed characteristics of each case are given in Table 2 . The most common neurological symptoms at the time of LP were altered mental status (n = 8, 42%) and headache (n = 8, 42%), followed by peripheral weakness (n = 6, 32%) and anosmia (n = 5, 26%). All neurological symptoms and respiratory support are presented in Table 3 . PCR for SARS-CoV-2 was positive in one patient (5%), and there was a pleocytosis in two patients (11%). CSF albumin level was increased in one patient (5%), denoting disruption of the blood-CSF barrier. Four patients (21%) had elevated intrathecal IgG Tables 2 and 4 ). There was a correlation between number of days in the ICU and NfL protein (r = 0.72, p < 0.001) but not for T-tau and GFAp In 11 patients, plasma samples were analyzed for NfL protein, T-tau, and GFAp in plasma. There was a strong correlation between CSF and plasma levels for both NfL protein (r = 0.98, p < 0.001) and GFAp (r = 0.97, p < 0.001). For T-tau, no correlation could be demonstrated. A head computed tomography scan or magnetic resonance imaging was performed in 17 patients as part of the clinical workup. No pathological findings could be detected in eight (47%) patients. A more detailed description of the pathological findings is presented in Table 2 . In this prospective study, we present data on biochemical, inflammatory, and neuronal injury biomarkers in the CSF and plasma of patients with COVID-19 and neurological symptoms. The main finding is that a majority of patients had a negative standard CSF workup, whereas markers of neuronal injury were increased. Even though the neurological symptoms were severe in some of the patients, the standard CSF workup tended to be negative in a majority of patients, and no specific pattern for COVID-19 could be identified. Only a few cases had mild pleocytosis and increased IgG in CSF, and one patient had oligoclonal bands, which is in line with recent reports [15] [16] [17] [18] . Animal models in mice of coronaviruses suggest that viral entry into the CNS can occur [19, 20] . SARS-CoV-2 is known to have a neuroinvasive propensity, and there are case reports with RNA detection in CSF that indicate a direct invasion of the virus into the CNS [21, 22] . In our study, we could detect viral RNA in only one patient, which is in parity with recent reports [8, 22] . In the absence of direct findings of viral meningitis or encephalitis in the vast majority of patients with COVID-19, the mechanism of the brain injury implied by increased NfL protein and T-tau remains to be elucidated. Increased plasma and CSF levels of NfL protein have previously been shown in patients with sepsis-associated encephalopathy [23] . Surgery and anesthesia may cause increased plasma levels of both NfL protein and T-tau [24] . No patient in the study underwent surgery the weeks before inclusion and only three patients were anesthetized and treated with invasive ventilation at the time of LP, but 10 patients did at some point before the LP. However, respiratory dysfunction as measured by the lowest PaO 2 / FiO 2 ratio during invasive ventilation before the LP did not correlate with any of the markers of neuronal injury. Other confounders such as comorbidity may also be an issue. One patient had epilepsy and autism, but continuous electroencephalography did not reveal seizure activity at the time of the study. Another patient suffered from a short cardiac arrest in the ICU (33 days before LP) but was resuscitated within 60 s. Exclusion of these two patients from statistical analysis did not alter any of the main results or conclusions. Previous studies on herpes encephalitis have shown that NfL protein levels are often far more elevated than what was seen in this study [25] . Furthermore, the time-series data recently published by Westman et al. [26] illustrate that the kinetics of NfL protein after acute infectious encephalitis is relatively slow, with a peak approximately 1 month after onset of disease. This means that timing of the CSF sampling in relation to onset of disease, as well as in relation to ICU care and other confounders, is an important covariate when assessing NfL protein levels. We found a strong correlation between plasma and CSF levels of NfL protein and GFAp, suggesting that plasma levels of these biomarkers parallel CSF levels in patients with COVID-19. The strong correlation indicates a steady-state passage across the blood-CSF barrier irrespective of disease severity, and is consistent with a negligible barrier injury. This is further supported by the findings that only one patient had increased albumin in CSF. This is in contrast to viral meningitis or encephalitis, where differences in disease severity cause the level of barrier injury to vary among patients [27] . The raised levels of T-tau in CSF and plasma have only rarely been explored in relation to COVID-19 [8] . Thirteen of the included patients did not undergo LP, thereby making the cohort less representative for the COVID-19 disease. Importantly, the study was not designed to assess incidence or prevalence of neurological symptoms related to COVID-19, but rather to select patients with whom we had optimal access to information on the clinical course and were able to perform LP. Still, not all patients had the exact same set of investigations performed due to the clinical situation. Furthermore, the effective half-life is reported to be 12 to 24 h for GFAp, about 10 h for T-tau, but several weeks for NfL protein, and patients were included at different time points along the disease trajectory, which may have affected the results [29, 30] . The study is cross-sectional without longitudinal follow-up data from CSF, final neurological diagnosis, and outcome. In conclusion, our results show that the standard CSF workup is normal in a majority of patients with COVID-19 and new onset neurological symptoms. CSF biomarkers related to CNS injury are increased, indicating COVID-19-related brain damage. NfL protein, in particular, is indicative of disease severity and may be a valuable tool for monitoring neuroprotective effects of new therapies. Future studies in larger samples are needed to explore and understand the genesis of neurological injury in COVID-19 patients. The authors report no disclosures relevant to this study. 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