key: cord-0741015-6os6ovoe
authors: Schweitzer, Finja; Goereci, Yasemin; Franke, Christiana; Silling, Steffi; Bösl, Fabian; Maier, Franziska; Heger, Eva; Deiman, Birgit; Prüss, Harald; Onur, Oezguer A.; Klein, Florian; Fink, Gereon R.; Di Cristanziano, Veronica; Warnke, Clemens
title: Cerebrospinal Fluid Analysis Post–COVID‐19 Is Not Suggestive of Persistent Central Nervous System Infection
date: 2021-11-22
journal: Ann Neurol
DOI: 10.1002/ana.26262
sha: fd43d3da142eb0a47a289ae2877e444adce9c01b
doc_id: 741015
cord_uid: 6os6ovoe

This study was undertaken to assess whether SARS‐CoV‐2 causes a persistent central nervous system infection. SARS‐CoV‐2–specific antibody index and SARS‐CoV‐2 RNA were studied in cerebrospinal fluid following COVID‐19. Cerebrospinal fluid was assessed between days 1 and 30 (n = 12), between days 31 and 90 (n = 8), or later than 90 days (post–COVID‐19, n = 20) after COVID‐19 diagnosis. SARS‐CoV‐2 RNA was absent in all patients, and in none of the 20 patients with post–COVID‐19 syndrome were intrathecally produced anti–SARS‐CoV‐2 antibodies detected. The absence of evidence of SARS‐CoV‐2 in cerebrospinal fluid argues against a persistent central nervous system infection as a cause of neurological or neuropsychiatric post–COVID‐19 syndrome. ANN NEUROL 2021

S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection primarily targets the upper and lower respiratory tract, causing dry cough and fever. Neurological and neuropsychiatric manifestations have been associated with coronavirus disease , ranging from mild to fatal at all disease stages irrespective of disease severity. 1, 2 Interestingly, immunofluorescence and polymerase chain reaction (PCR) analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of COVID-19 revealed persistent detection of SARS-CoV-2 RNA and specific immunoreactivity in the small bowel in 50% of individuals. 3 It appears reasonable to assume that SARS-CoV-2 may also reach the central nervous system (CNS) via several routes, including the transcribial, hematogenous, and lymphatic routes, or via axonal transport or transsynaptic transfer. 4 Histopathological data revealing viral RNA transcripts and particles by transmission electron microscopy in brain tissue may suggest CNS infection. 5 Therefore, symptoms such as cognitive impairment or fatigue persisting for >90 days (post-COVID-19) following acute respiratory COVID-19 might be caused by SARS-CoV-2 persistence in the CNS.

Systematic studies of SARS-CoV-2 RNA detection in cerebrospinal fluid (CSF) from patients with neurological symptoms early during COVID-19 and in patients with post-COVID-19 may help address the question of an acute and/or persistent CNS infection with SARS-CoV-2. SARS-CoV-2 RNA was infrequently detected in the CSF in single cases and case series, [6] [7] [8] [9] [10] with all these cases reported within the first 90 days of the respiratory infection.

In addition to molecular assays, the SARS-CoV-2specific CSF antibody index (AI SARS-CoV-2 ) allows calculation of an intrathecally produced antibody fraction and might provide indirect evidence of CNS infection.

The AI is in clinical use for chronic CNS infections such as herpes virus encephalitis, subacute sclerosing panencephalitis, and neuroborreliosis, 11 and is under investigation for progressive multifocal leukoencephalopathy. 12 This study aimed to clarify whether SARS-CoV-2 persistently infects the CNS, with SARS-CoV-2 RNA from CSF and the AI SARS-CoV-2 as outcome measures.

The data and biomaterial were derived from a prospective cohort study at baseline, collected at 2 tertiary university hospitals in Germany (Cologne/Berlin) between April 2020 and April 2021 from patients hospitalized or presenting at the specialized post-COVID-19 outpatient clinic. The study was approved by the institutional review board of the University of Cologne (20-1501) and Berlin (EA2/066/20) and registered in the German Clinical Trials Register (DRKS00024434). Patients between 18 and 99 years of age and with neurological or neuropsychiatric symptoms during or after PCR-confirmed COVID-19 were eligible for the study following written informed consent.

Detection of SARS-CoV-2 RNA in CSF Viral nucleic acids were extracted from CSF and serum samples (200μl) using the innuPREP Virus DNA/RNA Kit-IPC16 and the automated platform InnoPure C16 touch (20μl eluate volume; Analytik Jena, Jena, Germany). To assess SARS-CoV-2 (N and E gene) RNA reverse transcriptase (RT)-PCR cycle threshold (Ct) levels, samples were analyzed using the LightMix SarbeccoV E gene plus EAV control (TIB Molbiol, Berlin, Germany) and N gene (inhouse primer sets in multiplex PCR) as previously described. 13 Assays were carried out on LightCycler 480 (Roche Diagnostics, Mannheim, Germany). Samples with a weak signal in the RT-PCR assay were reanalyzed using a 1-step RT droplet digital (dd) PCR multiplex assay targeting SARS-CoV-2 E, RdRp, and N with a limit of detection of 5 viral RNA copies per reaction as previously described, 14 and 2 additional commercial tests. The Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA) with a limit of detection of 0.005 PFU/ml for N gene and 0.02 PFU/ml for E gene (PFU is defined as plaqueforming unit), and the Cobas SARS-CoV-2 assay on the automated Cobas 6800 (Roche Diagnostics) with a limit of detection of 0.0063 50% tissue culture infectious dose (TCID50)/ml for SARS-CoV-2 ORF1a/b and 0.0082 TCID50/ml for E gene were used.

To determine the AI SARS-CoV-2 , SARS-CoV-2 immunoglobulin class G (IgG) was quantified in diluted CSF and serum samples using the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) targeting the S1 domain of the spike protein (Euroimmun Diagnostik, Lübeck, Germany). Results were expressed semiquantitatively as the ratio of extinction probe and extinction calibrator. CSF samples were generally diluted at 1:2; if antibody concentration exceeded the standards provided, additional 1:20, 1:40, or 1:80 dilutions were required. Serum samples were diluted at 1:101, 1:404, and 1:1010; a few samples required further 1:2020 and 1:4040 dilutions. AI SARS-CoV-2 was calculated based on SARS-CoV-2 IgG in serum and CSF, and albumin and total IgG to estimate specific intrathecal antibody synthesis as previously described. 11 According to the manufacturer's recommendations, serum SARS-CoV-2-specific IgG values were chosen for calculations for which the optical density (OD) was closest to 1 and closest to the OD detected for the corresponding CSF sample.

We analyzed 40 patients after PCR-confirmed SARS-CoV-2 infection treated for neuropsychiatric manifestations of COVID-19, and an available matching CSF-serum pair (Fig 1) . CSF was assessed between days 1 and 30 (acute COVID-19, n = 12), between days 31 and 90 (ongoing COVID-19, n = 8), or later than 90 days (post-COVID-19, n = 20) after the COVID-19 diagnosis. Patients in the acute COVID-19 group were older (p < 0.001), and the frequency with a severe or critical COVID-19 disease course was higher as compared to during ongoing and post-COVID-19 (10 of 12, 83.3% vs 7 of 28, 25.0%). A majority of the patients in the post-COVID-19 group complained of cognitive deficits (17 of 20, 85.0%), verified using a screening test in 4 of 15 tested patients (26.7%), (Fig 2) . Regarding SARS-CoV-2-specific antibodies in CSF, 11 patients within the first 90 days of infection and with detectable antibodies had higher levels than 13 patients with post-COVID-19 (median RU = 84.7 vs 7.4, p < 0.001). Anti-SARS-CoV-2 antibody levels in CSF inversely correlated with time since the detection of SARS-CoV-2 RNA in the respiratory tract (see Fig 2) . In 1 patient, an intrathecally produced anti-SARS-CoV-2 antibody fraction was determined as assessed by AI SARS-CoV-2 . This was noted 39 days after the detection of SARS-CoV-2 RNA in the respiratory tract (Tables 1A  and 2A ). In this patient, CSF was taken to further evaluate delirium and ocular motility dysfunction. At the time of sampling, the patient suffered from acute respiratory distress syndrome due to ongoing COVID-19, complicated by multiple organ dysfunction and septicemia. The same patient showed borderline CSF AIs to measles and rubella (1.42 and 1.37, respectively, negative for varicella-zoster virus).

As the key finding of our study, neither fundamental CSF findings, nor various PCR protocols, nor IgG-based SARS-CoV-2-directed antibody measures were suggestive of replicative CNS infection as the cause of neuropsychiatric symptoms in post-COVID-19. These post-COVID-19 patients had suffered from a mild course of the acute infection, and cognitive deficits were among the leading complaints. The median age of 50 years was within the range of published post-COVID-19 cohorts. [15] [16] [17] We noted an elevated AI SARS-CoV-2 in 1 patient with severe ongoing COVID-19 infection, possibly explained by polyspecific immune activation, matching the absence of SARS-CoV-2 RNA from CSF, and borderline AI indexes toward other viruses.

The current evidence for direct viral brain invasion in COVID-19 is conflicting; the frequent detection of SARS-CoV-2 in brain reported by one group 5 was not confirmed by others. 18, 19 These autopsy studies included older individuals that deceased from COVID-19, demographics that substantially differed from our post-COVID-19 patients. The same is true for published CSF studies assessing only the acute or ongoing phases of COVID-19, 20 and lacking systematic antibody analyses. Owing the limitations to our study, we cannot definitely preclude CNS infection; the sample size is small, a CSF PCR may fail to detect virus latently infecting brain tissue, and an IgG-based AI SARS-CoV-2 directed against the spike protein may miss other immune responses.

Nevertheless, despite these limitations, CSF studies such as ours are needed to further explore the still elusive pathogenesis of post-COVID-19. Whereas neuropsychiatric symptoms during acute COVID-19 could be explained by hyperinflammation, hypoxemia, hypoperfusion, dehydration, glucose dysregulation, and sedation, 1 they remain unexplained in post-COVID-19. [15] [16] [17] Latent infection, viral persistence, virus-induced autoimmunity, persistent structural, functional, or metabolic changes following infection, and psychosocial stress are among the alternative nonexclusive explanations. 1 Currently, post-COVID-19 is defined as "signs and symptoms that develop during or after an infection consistent with COVID-19, continue for more than 12 weeks and are not explained by an alternative diagnosis" (www. nice.org.uk/guidance). Such a definition based on a temporal association with preceding COVID-19 illustrates the need for biomarker studies to more precisely differentiate post-COVID-19 from pre-or coexisting other conditions, given the relatively young patient population with complaints of cognitive deficits several months after SARS-CoV-2 infection. We thank V. Worm, D. Wilken, and Dr E. Brüsehaber of Euroimmun for technical and material support and assistance with interpreting AI data. Open Access funding enabled and organized by Projekt DEAL.

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Long COVID in a prospective cohort of home-isolated patients

Characterizing long COVID in an international cohort: 7 months of symptoms and their impact

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Microvascular injury in the brains of patients with Covid-19

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This study was supported by the German Research Foundation (PR 1274/8-1, FR 4479/1-1, WA4101/2-1).

F.S., Y.G., C.F., S.S., V.D.C., and C.W. contributed to the conception and design of the study; all authors contributed to the acquisition and analysis of data. F.S., Y.G., and C.W. contributed to drafting the text or preparing the figures; all authors critically revised the manuscript for important intellectual content.

CW received personal compensation from BioNTech for participating in an educational discussion. The other authors have nothing to report.