key: cord-0733615-8l7mdk9l
authors: Li, Yan‐Chao; Zhang, Yan; Tan, Bai‐Hong
title: What can cerebrospinal fluid testing and brain autopsies tell us about viral neuroinvasion of SARS‐CoV‐2
date: 2021-03-25
journal: J Med Virol
DOI: 10.1002/jmv.26943
sha: 447c1b1616a45f1d0881d27bd0fc4c6c545b4839
doc_id: 733615
cord_uid: 8l7mdk9l

To provide instructive clues for clinical practice and further research of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, we analyzed the existing literature on viral neuroinvasion of SARS‐CoV‐2 in coronavirus disease 2019 (COVID‐19) patients. To date, SARS‐CoV‐2 has been detected in the cerebrospinal fluid (CSF) or brain parenchyma in quite a few patients, which provide undeniable evidence for the neuroinvasive potential of this novel coronavirus. In contrast with the cerebrum and cerebellum, the detection rate of SARS‐CoV‐2 was higher in the olfactory system and the brainstem, both of which also showed severe microgliosis and lymphocytic infiltrations. As compared with the number of patients who underwent viral testing in the central nervous system (CNS), the number of patients showing positive results seems very small. However, it seems too early to conclude that the neuroinvasion of SARS‐CoV‐2 is rare in COVID‐19 patients because the detection methods or sampling procedures in some studies may not be suitable or sufficient to reveal the CNS infection induced by neurotropic viruses. Moreover, the primary symptoms and/or causes of death were distinctly different among examined patients, which probably caused more conspicuous pathological changes than those due to the direct infection that usually localized to specific brain areas. Unfortunately, most autopsy studies did not provide sufficient details about neurological symptoms or suspected diagnoses of the examined patients, and the documentation of neuropathological changes was often incomplete. Given the complex pathophysiology of COVID‐19 and the characteristics of neurotropic viruses, it is understandable that any study of the CNS infection may inevitably have limitations.

It is possible that the potential neuroinvasion of SARS-CoV-2 plays a role in the peculiar respiratory manifestations. 8, 9 The first-hand clinical report on neurological involvement associated with SARS-CoV-2 infection became available online shortly after the appearance of this novel coronavirus. 10 Thereafter, Moriguchi et al. 11 and Xiang et al. 12 provided the first evidence of SARS-CoV-2 in the cerebrospinal fluid (CSF) of COVID-19 patients.

Since then, the neuroinvasive potential of SARS-CoV-2 has attracted more and more attention. 13 However, to date, only a few patients with COVID-19 were tested positive for SARS-CoV-2 in the CSF. 13 Similarly, many decreased patents with COVID-19 who underwent brain autopsies were reported to show negative results for SARS-CoV-2 detection in the brain parenchyma. 7, 13 Given these observations, the direct invasion of SARS-CoV-2 in the central nervous system (CNS) is considered to be rare, and therefore may not be responsible for the neurological manifestations of patients with COVID-19.

However, it should be noted that the examined patients in many studies exhibited different neurological features, and therefore probably had different underlying diseases. 13 In many cases, the neuropathological changes caused by hypoxia-ischemia, strokes, toxic metabolic changes, multiorgan failure, or cytokine storming appeared so striking that it was difficult to conclude whether the neuroinvasion of SARS-CoV-2 played a pathogenic role or not. 14 Since the beginning of 2020, SARS-CoV-2 has spread rapidly all over the world and caused a profound impact on human health, lifestyles, economy, politics, and even the world pattern. As the pandemic of COVID-19 has aroused great public concerns, related scientific papers are increasing in number in an explosive manner. 7 Therefore, it is necessary to make a systematic analysis of the existing literature so as to reveal instructive clues for clinical practice and further research of SARS-CoV-2 infection.

An exhaustive search of case reports, cohort studies, series of cases, postmortem studies, and clinical trials related to the possible neuroinvasion of SARS-CoV-2 was performed through PubMed/MED-LINE and COVID-19-related preprints from medRxiv and bioRxiv from December 1, 2019, to October 31, 2020. In addition, the references of relevant articles were also scanned for additional studies related to SARS-CoV-2 and CNS infection.

The papers on COVID-19 were retrieved by using "novel coronavirus disease 2019 or COVID 19 or severe acute respiratory syndrome coronavirus 2 or SARS CoV 2 or 2019 novel coronavirus or 2019 nCoV" in Title/Abstract (Strategy 1). To reveal the involvement of the nervous system in COVID-19, the following keywords in title/abstract were combined with Strategy 1: "neurological or nervous system or CNS or PNS or brain or cerebrum or cerebral or cerebellum or cerebellar or thalamus or thalamic or hippocampus or hippocampal or pons or pontes or pontine or brainstem or oblongata or medulla oblongata or spinal cord or cerebrospinal or neural or neuron or nerve." Reviews, meta-analyses, opinion, correspondence, perspective, and letters to the editor containing no original data of interest were excluded from quantitative analysis. Only case reports or series studies that reported patients diagnosed with COVID-19 based on positive SARS-CoV-2 polymerase chain reaction (PCR) or serologic testing were included in this study. The titles and abstracts were first screened, and the full texts and supplementary files were then obtained from the library of Jilin University. The papers were selected based on their relevance as to whether the CSF or the brain was tested for SARS-CoV-2. Due to our limited capacity, the cut-off time of this review was set as October 31, 2020.

The purpose of this study was to search for evidence of the neuroinvasion of SARS-CoV-2. Therefore, our analysis was focused on the relationship between the primary symptoms of examined patients with COVID-19 and their CSF testing results for SARS-CoV-2, white blood cells (WBCs), and intrathecal antibodies against SARS-CoV-2. By contrast, the results for the measurement of CSF biomarkers of inflammation and neuronal injury were excluded from this study.

In total, we identified 97 relevant papers which reported 468 COVID-19 patients who underwent CSF PCR testing for SARS-CoV-2 (Tables 1-3 , S1 and S2). Among these patients, only 30 patients (30/468, 6.4%) from 25 papers were reported to show positive results (Tables 1 and S1).

Among the 30 patients with positive CSF testing, the primary symptoms or possible diagnoses were provided for 24 

In total, we identified 28 autopsy studies that reported structural abnormalities in 134 (66.4%) of 202 patients who died from COVID-19 (Tables 4 and S3) . Among 202 patients, 108 (108/202, 53.5%)

were further tested for SARS-CoV-2 in the neural tissues (Tables 5   and S3 ). Among 134 patients with brain abnormalities, SARS-CoV-2

RNA and viral proteins were detected in 31 ( (Table 5) .

T A B L E 4 Structural abnormalities in different brain areas and the detection of SARS-CoV-2 in the abnormal regions (x/number) CSF testing is currently the only clinically acceptable invasive method to evaluate CNS responses to infection. However, the detection rate of SARS-CoV-2 in the CSF is highly dependent on the types of diseases and the time of sample collection. 45 The titers of viruses in the CSF may change over the course of a patient's illness due to possible CSF clearance. Therefore, CSF testing may fail to give positive results due to delayed sampling. 44 Although some patients showed negative results for SARS-CoV-2 in the CSF, the possibility of CNS infection cannot be completely excluded in these patients, as demonstrated in some autopsy studies. 46, 47 On the other hand, viral detection via PCR testing is not 100% sensitive due to genetic variability in the virus itself or technical factors. 44 It has been pointed out that CSF testing will give falsepositive results if a sample has been contaminated by blood. Moreover, a positive SARS-CoV-2 PCR does not definitively indicate that neuroinvasion is responsible for a given constellation of symptoms. 44 Taken together, from the data of CSF testing, the presence of SARS-CoV-2 or anti-SARS-CoV-2 antibodies with intrathecal antibody synthesis has been found in the CSF in a total of 37 of 468 (7.9%). Although these findings are not conclusive evidence, they strongly indicate the possible invasion of SARS-CoV-2 in the CNS in some COVID-19 patients.

Postmortem examination is known as the most definitive mean to assess viral neuroinvasion in patients with COVID-19. 14 Since Paniz-Mondolfi et al. 46 reported the first autopsy evidence of SARS-CoV-2 viral particles in the brain parenchyma of a COVID-19 patient by electron microscopy and PCR assays on April 21, 2020, increasing autopsy studies on patients with COVID-19 have been published. 39, Among these studies, structural abnormalities are widely observed in the olfactory bulb/tract, brainstem, cerebellum, and cerebrum. The most common findings are hypoxic injury and vascular accidents, which is well consistent with the known hypoxemia and hypercoagulable state of blood in most decreased COVID-19 patients. However, this is not contradictory to the neuroinvasion of SARS-CoV-2, as SARS-CoV-2 RNA and/or viral proteins have been detected in the brain in some patients with hypoxic brain injury and/ or brain vascular accidents. 48, 54, 59, 63, 66, 71 Microglial activation is a common pathological feature during neuronal injury induced by a variety of insults. Therefore, it may not be surprising to find microglial activation in the compromised brain regions in more than half of the examined cases. Interestingly, the detection rate of SARS-CoV-2 is much higher in the brain regions with microgliosis and/or lymphocytic infiltrations, relative to those with hypoxic brain injury or vascular accidents. Moreover, severe microglial proliferation is most commonly observed in the medulla oblongata. 48, 54, 56, 59, 67, 72 These findings indicate that the inflammatory responses in some specific brain areas cannot be LI ET AL.

| 4253 attributed to only the hypoxemia or vascular accidents in critical patients with COVID-19.

Trans-neuronal transfer is known as a unique way for neurotropic viruses to infect the nervous system. 73 The high incidence of olfactory disorder in COVID-19 patients supports the hypothesis that olfactory mucosa/nerve may be one of the portals for SARS-CoV-2 to enter the CNS. 8, 9, 73, 74 Consistently, SARS-CoV-2 RNA and viral proteins have been detected in the olfactory system in 14 (58.3%) of 24 and 5 (83.3%) of 6 tested patients, respectively.

Moreover, 50 (37.3%) of 134 tested patients showed severe microgliosis and/or lymphocytic infiltrations in the olfactory nerve, olfactory bulb, or olfactory cortex. 59, 67 The brainstem is comprised of many important structures, and the wide anatomical connections make it an easily accessible CNS target for SARS-CoV-2 from peripheral infection sites. 7,75 Consistently, the brainstem was found to show a high detection rate of SARS-CoV-2, 59,60,62 as well as the most severe microgliosis and/or lymphocytic infiltrations. 48, 54, 56, 58, 67, 72 These findings support the speculation that the brainstem may be a major target in the CNS for SARS-CoV-2.

To date, PCR or quantitative real-time polymerase chain reaction (qRT-PCR) techniques revealed positive results for SARS-CoV-2 in the brain in 56 (51.9%) of 108 tested cases, while immunohistochemistry, using antibodies against viral nucleocapsid and/ or spike proteins, revealed positive results in the brain in 25 (29.4%) of 85 tested cases. The detection rate by PCR assays was much higher than that by immunohistochemistry in almost all tested brain regions (Table 5 ).

It is reported that some COVID-19 patients had a detectable level of SARS-CoV-2 in the blood. 76 patients who were tested negative on qRT-PCR analysis of SARS-CoV-2 RNA in the brain tissues, viral proteins were detectable by immunohistochemistry in the medulla oblongata. 59 Noteworthily, Matschke et al. 57 found that the presence of SARS-CoV-2 in the brain was not associated with the severity of neuroimmune activation. At first sight, this finding seems surprising, but it may be consistent with the characteristics of neurotropic viruses as they can hide in neurons from the surveillance of the immune system. 78 Therefore, the immune response will not be effectively activated in the infected areas unless the initially infected neurons have been significantly damaged. 7 Whether technically or ethically, it is a great challenge to carry out brain autopsies on human beings, especially for patients with infectious diseases. It is a pity that most autopsy studies did not provide sufficient details about neurological symptoms or suspected diagnoses of the examined patients. Moreover, documentation of neuropathological changes in COVID-19 patients was often incomplete in many reports. These are not conducive for further analysis of the relationship between the autopsy findings and clinical manifestations. Due to technical or ethical factors, complete brain removal was difficult or even was not permitted in some studies.

However, incomplete or random sampling of brain tissue is not sui- 

At present, SARS-CoV-2 has been detected in the CSF or brain parenchyma in quite a few patients, which provide undeniable evidence for the neuroinvasive potential of this virus. The detection of SARS-CoV-2 in the olfactory mucosa/nerve/bulb coincides with the olfactory dysfunction reported in most COVID-19 patients and therefore supports the use of the olfactory pathway by SARS-CoV-2

to enter the CNS. The discovery of brainstem abnormalities and the presence of SARS-CoV-2 in some medullary neurons support the brainstem as a major target of SARS-CoV-2 in the CNS. The possible damage to the brainstem respiratory center is worthy of further study, as it may be responsible for the high incidence of severe respiratory distress syndrome in COVID-19 patients.

As compared with the number of patients who underwent viral testing in the CNS, the number of patients with positive results seems very small. However, it should not be simply concluded that the neuroinvasion of SARS-CoV-2 is rare in COVID-19 patients because the detection methods or sampling procedures in some studies may not be suitable or sufficient to reveal the CNS infection induced by neurotropic viruses. Moreover, the primary symptoms and/or causes of death were significantly different among COVID-19 patients, and probably caused more conspicuous pathological changes than those due to direct infection, which usually localized to specific brain areas. Given the complex pathophysiology of COVID-19 and the characteristics of neurotropic viruses, it is understandable that any study of the CNS infection may inevitably have limitations.

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The authors declare that there are no conflict of interests.

Yan-Chao Li conceived and designed the study. Yan Zhang and Bai-Hong Tan helped to search and interpret the literature. All authors critically reviewed and approved the final version of the paper.

The data in this study can be obtained upon request from the corresponding author.