key: cord-0940334-blc3mnbj
authors: Bernard-Valnet, R.; Perriot, S.; Canales, M.; Pizzarotti, B.; Caranzano, L.; Castro-Jimenez, M.; Epiney, J.-B.; Vijiala, S.; Salvioni Chiabotti, P.; Anichini, A.; Salerno, A.; Jaton, K.; Vaucher, J.; Perreau, M.; Greub, G.; Pantaleo, G.; Du Pasquier, R.
title: CSF of SARS-CoV-2 patients with neurological syndromes reveals hints to understand pathophysiology
date: 2020-11-04
journal: nan
DOI: 10.1101/2020.11.01.20217497
sha: 1b0d5d2c36a3f10d72e69d32f4155952bf220d45
doc_id: 940334
cord_uid: blc3mnbj

Objective: Coronavirus disease (COVID-19) has been associated with a large variety of neurological disorders. However the mechanisms underlying these neurological complications remain elusive. In this study we aimed at determining whether neurological symptoms were caused by SARS-CoV-2 direct infection of by pro-inflammatory mediators. Methods: We checked for SARS-CoV-2 RNA by RT-qPCR, SARS-CoV-2-specific antibodies and for 48 cytokines/chemokines/growth factors (by Luminex) in the cerebrospinal fluids (CSF) +/- sera of a cohort of 17 COVID-19 patients with neurological presentation and 55 neurological control patients (inflammatory [IND], non inflammatory [NIND], multiple sclerosis [MS]). Results: We found SARS-CoV-2 RNA and antibodies specific for this virus in the CSF of 0/17 and 8/16 COVID-19 patients, respectively. The presence of SARS-CoV-2 antibodies was explained by a rupture of the blood brain barrier (passive transfer) in 6/16 (38%). An intrathecal synthesis of SARS-CoV2-specific antibodies was present in 2/16 patients. Of the four categories of tested patients, the CSF of IND exhibited the highest level of chemokines (CCL4, CCL5, CXCL8, CXCL10, CXCL12, and CXCL13), followed by the CSF of MS patients (CXCL12, and CXCL13). There was no significant difference between COVID-19 and NIND patients, even if some chemokines (CCL4, CCL5, CXCL8, andCXCL10) tended to be higher in the former. Interestingly, among COVD-19 patients, the CSF of those with a severe disease (encephalitis/encephalopathy) contained higher levels CXCL8 and CXCL10 than those with other neurological presentations. Interpretation: Our results do not show obvious SARS-CoV-2 infection of the central nervous system, but point to a mild inflammatory reaction reflecting an astrocytic reaction. Methods: We checked for SARS-CoV-2 mRNA by qPCR, SARS-CoV-2-specific antibodies and for 49 cytokines/chemokines/growth factors (by Luminex) in the cerebrospinal fluid (CSF) +/- serum of a cohort of 17 COVID-19 patients with neurological presentation and 55 neurological controls (inflammatory, non inflammatory, multiple sclerosis). Results: We found SARS-CoV-2 mRNA and antibodies specific for this virus in the CSF of 0/17 and 8/16 COVID-19 patients, respectively. The presence of SARS-CoV-2 antibodies was explained by a rupture of the blood brain barrier (passive transfer) in 6/16 (37,5%), but an intrathecal synthesis of SARS-CoV2-specific antibodies was present in 2/17.As compared to SARS-CoV-2-negative NIND patients, the CSF of IND patients exhibited the highest level of chemokines (CCL4, CCL5, CXCL8, CXCL10, CXCL12, and CXCL13), followed the CSF of MS patients (CXCL12, and CXCL13). There was no difference between COVID-19 patients with neurological diseases compared to NIND even if some chemokines (CCL4, CCL5, CXCL8, andCXCL10) tended to be higher than NIND. Interestingly, among COVD-19 patients, the CSF of those with a severe disease (encephalitis/encephalopathy) contained higher levels CXCL8 and CXCL10 than those with other neurological presentations. Interpretation: Our results confirm the absence of obvious SARS-CoV-2 infection of the central nervous system and point to a mild inflammatory reaction reflecting an astrocytic reaction.

Corona viruses' outbreaks have been repeatedly associated with neurological disorders.

Indeed, human tropic coronaviruses seem able to reach the central nervous system and are found in brain necropsies and in cerebrospinal fluid (CSF) of severe acute respiratory syndrome coronavirus (SARS-CoV) patients 1,2 . In mouse model, it has been shown that a strain of human tropic coronavirus is able to reach the olfactory bulb trough the cribriform plate and then to spread through a neuron-to-neuron transmission 3 .

In line with this neurological tropism, Coronavirus Disease 2019 (COVID-19) has shown a large range of neurological complications 4 that may be classified as followed: critical care-related neurological syndromes either central (sub-cortical deficit characterized by attention and executive dysfunction) or peripheral (critical care-associated polyneuropathies or myopathies ) 5 ; anosmia/dysgueusia 6 ; myelo-meningo-encephalitis 7, 8 ; Guillain-Barré Syndrome (GBS), and its variant affecting cranial nerves (Miller-Fischer Syndrome) 9 ; and cerebrovascular disease (strokes) 10 .

If data from previous outbreaks point to a neurotropism of the coronaviruses, the pathophysiology underlying SARS-CoV-2-related neurological deficits remains elusive.

Indeed, to date, SARS-CoV-2 has only been rarely found in the CSF, suggesting that direct brain infection is not obvious 11, 12 . Thus, the main hypotheses to explain neurological complications in COVID patients point at mechanisms either related to low grade presence of the virus in the CNS, to cytokine storm or to the presence of an auto-immune response, such as anti-neuronal antibodies by analogy to what occurs in autoimmune encephalitis. However, data firmly establishing one or the other hypothesis are still missing. Yet, the fact that encephalitis/encephalopathies caused by SARS-CoV-2 may respond to corticosteroids 7, 13 suggest involvement of immune mechanisms.

In an attempt to decipher mechanisms underlying neurological symptoms, we looked at SARS-CoV-2-encoding RNA, SARS-CoV-2-specific antibodies and at a panel of 48 cytokines/chemokines/growth factors in the CSF of 72 study patients. Seventeen of them were infected with SARS-CoV-2, and 55 were control SARS-CoV-2-negative patients suffering from inflammatory, including MS or non-inflammatory neurological disorders. We found that SARS-CoV-2 patients tend to have signs of blood brain barrier opening and possible astrocytes activation, but no strong immune response in the CSF or obvious CNS infection by the virus.

Study population. All consecutive patients seen at Lausanne University Hospital (CHUV) during the first wave of COVID-19 (March to end of May 2020) with any neurological All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 4, 2020. ;  manifestations and for whom a lumbar puncture including SARS-CoV-2 PCR was performed, were included in this study. The control cohort consisted of patients who were diagnosed with inflammatory neurological disorder (IND), non-inflammatory neurological disorder (NIND) or multiple sclerosis (MS) prior to the COVID-19 wave, thus who were SARS-CoV-2-negative by definition, and had been enrolled in the COOLIN'BRAIN cohort between 2005 and 2020.

Samples (serum and CSF) for MS were all harvested during a clinical relapse of the disease before any treatment with corticosteroids.

Ethics. This study was approved by Canton de Vaud Ethical Committee (CER-VD) in the frame of CORO-NEURO study (authorization n° 2020-01123) and COOLIN-BRAIN study (authorization n°2018-01622). All patients included in this study signed specific informed consent.

SARS-CoV-2 PCR. SARS-CoV-2 tests in CSF specimens were performed using our automated platform with an in-house RT-qPCR targeting the E-gene with the primers and probe described by Corman and colleagues 14 . SARS-CoV-2 tests in respiratory specimens were performed either using our automated platform (at the beginning of the pandemic) or using the cobas SARS-CoV-2 test on the cobas 6800 instrument (Roche, Basel, Switzerland), since 24th March 2020. Both methods were compared and exhibited 99.2% of concordant results 15 .

Anti-SARS-CoV2 IgG specific to the native trimeric Spike (S) protein were quantified using a multiplex bead assay as previously described 16 Graphical representation and statistical analysis. Graphical representation and statistics were generated using PRISM software (version 8.1.2, GraphPad software, La Jolla, Ca, USA). All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.01.20217497 doi: medRxiv preprint Multiple group analysis were made using Kruskal-Wallis test with Dunn's multiple comparison test. For analysis with 2 group, Mann-Whitney test was used to determine statistical significance. Heatmaps and k clustering were made using ClustVis web-based tool 18 .

From March to May 2020, 30 patients benefited from a research of SARS-CoV-2 by RT-qPCR and had a concomitant lumbar puncture. Among them, the diagnosis of COVID-19 was established in 17 based either on positive nasopharyngeal swab RT-qPCR (88%) or serology (12%). Clinical description of SARS-CoV-2 patients is summarized in Table 1 . These 17 patients presented with various neurological presentation including encephalopathy (7), encephalitis (2), myelitis (1), optic neuritis (1), Guillain-Barré Syndrome (1), mononeuritis multiplex (1) and headache (3) ( Table 2 .) Half of them were admitted to Intensive Care Unit (ICU) and 6 (35%) required mechanical ventilation. No prominent MRI abnormalities were found, especially neither leptomeningeal enhancement nor diffusion restriction, in contrast with a previous report 19 . All electroencephalographic (EEG) recording showed some abnormalities, consisting mainly in encephalopathic slowing but also irritative activity in one patient with encephalitis. Several patients also exhibited alterations in nerve conduction, including mononeuritis multiplex, polyradiculopathy and critical myopathy/polyneuropathy. (Table 2. ).

COVID-19 patients showed abnormal lumbar puncture characterised mainly by elevation of protein level and elevated albumin index. Furthermore, oligoclonal bands were found in a minority of patients and consisted mostly in identical bands in the serum and the CSF (type 4; Table 2 ). These features point toward an opening of the blood brain barrier. Pleocytosis was encountered only in few patients with encephalitic presentation. As expected, the CSF profile was characterized by high protein level and pleocytosis in most IND patients and oligoclonal bands restricted to the CSF (type 2) in all MS patients ( Table 2 ). All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.01.20217497 doi: medRxiv preprint

All 17 COVID-19 patients enrolled in this study undergone a RT-qPCR for detection of SARS-CoV-2 RNA in the CSF. It came back negative in all, despite positivity for most (88%) in the nasal swab (Fig. 1C ).

Most tested patients (8/9) had positive serology for SARS-CoV-2 in the blood with high titers (positivity threshold >6). Two patients with IND had SARS-CoV-2-specific antibodies above the positive threshold in the blood, however the overall titer was low (Fig. 1C ). Of note, both patients presented with pathologies regularly associated with cross-reactive antibodies: sarcoidosis and paraneoplastic syndrome.

Antibodies against SARS-CoV-2 were detected in the CSF of half of COVID patients. Of interest, there was evidence of an intrathecal synthesis of SARS-CoV-2-specific antibodies in 2/17 patients (Reiber index > 2; data not shown). Conversely, SARS-CoV-2 antibodies were not detected in the CSF of any control study patients, except for the patient suffering from neurosarcoidosis, where it was also positive in the serum (Fig. 1C) .

First, the analysis of the cytokine panel in the serum of the 9 SARS-CoV-2-infected patients for whom it was available showed an increased cytokine production compared to the serum of SARS-CoV-2-negative patients with other neurological disorders, inflammatory (IND) or not (NIND) (Fig. 1D ). As previously demonstrated IL-6, CXCL-10 and IL-1-RA were significantly elevated in SARS-CoV-2-infected patients (Fig. 1E ). Contrary to previous works, increase of CXCL8 and TNFa were not observed in the serum of our patients ( Fig. 1E and data not shown) 20, 21 .

While the cytokine profile of SARS-CoV-2-infected patients with neurological conditions was inflammatory for some in the serum (especially for ICU patients), this was not the case in the CSF. Indeed, the analyses performed in the CSF revealed that SARS-CoV-2-infected patients were mainly clustered with NIND and MS ones, while the CSF of IND patients exhibited a stronger immune signature, characterized by elevated levels of several cytokines, chemokines and growth factors ( Fig. 2A) . This unbiased clustering was confirmed by individual cytokine/chemokine analysis with significant elevation of IL-6, CCL4, CCL5, CXCL8, CXCL10, CXCL12, CXCL13, G-CSF and VEGF-A in the CSF of IND patients (Fig. 2B and data not shown). Of note, we observed an increased level of several chemokines, including CXCL8 and CXCL10, and to a lesser extent CCL4 and CCL5, in SARS-CoV-2-infected patients compared All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.01.20217497 doi: medRxiv preprint to NIND controls, but which did not reach significance (Fig. 2B) . Interestingly, this increase was more pronounced in SARS-CoV-2-infected patients with a more severe involvement of the CNS (eg. encephalopathy, encephalitis, myelitis), as compared to the ones with milder COVID-19 associated neurological disorders (headaches without meningitis) or with predominant peripheral nerve involvement (Fig. 2C) .

Even if they did not display strong immune signature, MS patients showed higher levels of CXCL12, CXCL13 and G-CSF in the CSF (Fig. 2B and data not shown) .

In this study, we attempted to understand the pathogenesis of neurological impairments in the context of COVID-19 disease.

First, we did not detect SARS-CoV-2 RNA in the CSF of any of our 17 patients (Fig 1.B ). Only few authors reported that, in severe encephalitis with neuronal and astroglial destruction, SARS-CoV-2 RNA could be found in the CSF but this remains an exception 12 . Interestingly, in vitro experiments using human induced pluripotent stem cells (hiPSC)-derived brain organoids demonstrated the capacity of SARS-CoV-2 to infect neurons and astrocytes but at a very low yield 22, 23 . Thus the lack of SARS-CoV-2 detection in the CSF does not rule out the presence of viral RNA in the brain parenchyma, but does suggest that, if present, its concentration is too low to be detected.

Second, we found that half of our SARS-CoV-2-infected patients exhibited virus-specific antibodies in their CSF (Fig 1.C) . Two of them showed evidence of an intrathecal synthesis of antibodies against SARS-CoV-2. These findings suggest that a humoral immune response against the virus may take place in the CNS, but this mechanism may concern only a subset of patients. Indeed, the fact that we found an increased permeability of the blood-brain barrier (increased albumin in the CSF, type 4 oligoclonal bands) in almost all SARS-CoV-2-infected patients suggests that a large proportion of the virus-specific antibodies found in the CSF may come from the periphery. Nevertheless, wherever these antibodies come from, they may be instrumental in the antiviral response in the brain. Supporting these findings, authors recently showed that the CSF from a COVID-19 patient displayed neutralizing antibodies able to prevent neuronal infection in an hiPSC-derived brain organoid model 22 .

Finally, our results bring an unprecedented insight on the inflammatory mechanisms involved in neurological complications of COVID-19. Indeed, we first compared to inflammatory and non inflammatory neurological controls and have been able to show that, contrary to patient with inflammatory disorders, SARS-CoV-2-infected patients do not display specific immune signature in the CSF. Interestingly, the sera of these SARS-CoV-2-infected patients exhibited All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Our observation of blood brain barrier alterations in COVID patients also favours the hypothesis of astrocytes activation. Indeed, astrogliosis can lead to the disruption of the blood brain barrier via reduction of astrocytic gap junctions, among other mechanisms 30 . In addition, astrocytes dysfunction, even in absence of a strong inflammatory milieu, can affect directly neuronal functioning and integrity, such as suggested by high neurofilament levels in the serum of COVID-19 patients 27 .

To conclude, our results suggest that moderately severe neurological complications in SARS-CoV-2-infected patients are not due to a major viral infection of the brain nor to a massive inflammatory response in this organ. However, the absence of such a massive response does not mean that the brain of COVID-19 patients with neurological features is unharmed by inflammation. Indeed, it is possible that the CSF only imperfectly reflects the inflammatory mechanisms taking place in brain tissue. Supporting this hypothesis, we can refer to our cohort of patients with active MS, whom we know exhibit a solid inflammation in the brain that needs corticosteroids to be tamed, but in the CSF of whom only a slight elevation of few chemokines can be detected. Precisely, others have reported that corticosteroids improve the neurological symptoms in COVID-19 patients with neurological complications 7,13 .

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 4, 2020. ; (20) 15 0 (0) 9 (60) 6 (40) 0 (0) 0 (0) preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted November 4, 2020. ; All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.01.20217497 doi: medRxiv preprint Expression is represented in log10 scale. K-means clustering was used to determine patients clusters (Cluster 1, n=1; Cluster 2, n=11; Cluster 3, n=59). Cytokines/chemokines with no variations across all patients are not displayed. Period refer if patient were sampled during COVID-19 pandemy or before it (prior) (B) Bar plot representation (mean ± SEM) with log10 scale of CCL4, CCL5, CXCL8, CXCL10, CXCL12 and CXCL13 expression in the CSF of patient with SARS-CoV-2 infection (red circles, n=16), IND (green circles, n=21) or NIND (blue circles, n=19). Statistical significance comparing to NIND group calculated using Kruskal-Wallis with correction for multiple comparisons (adjusted p:* ≤ 0.05 *** ≤ 0.001, **** ≤ 0.001). (C) CCL4, CCL5, CXCL8 and CXCL10 expression (mean ± SEM) in SARS-CoV-2 infected patient according to neurological presentation either Headache/peripheral nerve/else (white bar, n=5) or encephalopathies/encephalitis/myelitis (grey bar, n=11). Representation corrected by detection limit. Statistical significance was calculated using Mann-Whitney test (p:* ≤ 0.05).

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.01.20217497 doi: medRxiv preprint

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We are grateful to Mrs Géraldine Le Goff for her help in collecting study subjects samples. This work was made possible by grants to RDP from the Swiss National Foundation 320030-179531 and from the Swiss Multiple Sclerosis Foundation.

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