Speaker: ‘More work’ needed to identify cause of neurological symptoms in COVID-19
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Viral RNA load was low and did not appear associated with other pathological features among a small cohort of fatal COVID-19 cases, according to study results presented during the American Academy of Neurology annual meeting.
The findings, which were simultaneously published in The New England Journal of Medicine, did show ischemic hypoxia among all 18 cases.
“This work was conducted early on during the COVID-19 pandemic, during which time we knew about the respiratory-related illness of the disease as well as the neurological symptoms associated with it, including headache, stroke, nausea and others,” Erica Normandin, PhD candidate at the Broad Institute and Harvard Medical School, said during her presentation. “There are many ways that COVID-19 could cause neurological symptoms, but we wanted to focus on whether the virus was invading the central nervous system, establishing infection in the brain tissue and whether that is what caused these symptoms. At this early stage, we did not know what to expect, but we envisioned a study that would provide a comprehensive early portrait of whether there is virus in the brain and how much of it was where it was, as well as how and why it got there.”
Normandin and colleagues sought to examine the neuropathological findings and quantified SARS-CoV-2 viral burden among 18 consecutive fatal COVID-19 cases (median age, 62 years; 78% men) at Brigham and Women’s Hospital. They performed SARS-CoV-2 immunohistochemistry and reverse transcription quantitative polymerase chain reaction on the medulla and frontal lobe with olfactory nerve among 16 cases and 10 brain sections among two cases.
“We chose the frontal olfactory and medulla because they were the regions most suspected that might have virus present given the information that we had at the time,” Normandin said.
Common presenting neurologic symptoms included myalgia in three patients, headache in two and decreased taste in one patient. Of note, 11 patients were placed on mechanical ventilation.
Researchers detected acute hypoxic injury in the cerebrum, hippocampus and cerebellum among all patients, rare foci of perivascular lymphocytes in two patients and focal leptomeningeal inflammation in one patient.
Viral RNA load was low and did not appear associated with other pathological features, Normandin said.
Results of 10 specimens from two cases were equivocal (viral load < 5 copies/mm³) in four and five sections. Among the 16 other cases, three medulla sections and three frontal lobe and olfactory sections were positive (between 5 copies/mm³ and 59.4 copies/mm³). All other sections were equivocal or negative.
“When we sequenced these samples to a depth of more than one million paired reads, we identified tons of SARS-CoV-2 reads in lung samples,” Normandin said. “Conversely, among the 10 brain samples from subject 5, we did not identify a single SARS-CoV-2 read.”
However, for the two brain samples with the highest viral loads detected, researchers did identify a few SARS-CoV-2 reads.
“This was not enough to proceed with viral genome analyses, but it was expected and consistent with the quantitative PCR results,” Normandin said. “In fact, when comparing results from the samples, there is excellent correlation between the two samples. This gives us a lot of confidence in our methods and findings.”
Researchers additionally found that immunohistochemical staining for SARS-CoV-2 nucleocapsid protein was negative in neurons, glia, endothelium and immune cells.
“We did, however, consistently identify hypoxic ischemia among all cases,” Normandin said. “This seems to indicate that non-central nervous system organ damage is the most likely cause of neurological symptoms in fatal cases. We did not find strong evidence for virus causing pathology directly and many reports since have been consistent with this finding. As far as what is causing these neurological symptoms, there have been hypotheses born out of this study and others, but there is still more work to be done to get to the bottom of this.”
References:
- Normandin E, et al. Abstract S31.003. Presented at: 2021 Annual Meeting (virtual meeting); April 17-22, 2021.
- Soloman IH, et al. N Eng J Med. 2020;doi:10.1056/NEJMc2019373.
Perspective
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Jennifer A. Frontera, MD, FNCS
Neurological events have been widely reported in the context of both acute and post-acute COVID-19 infection. While many disorders, such as toxic metabolic encephalopathy or ischemic stroke, appear to be related to secondary effects of COVID-19, including hypoxia, hypercoagulability or multisystem organ failure, the pathophysiology underlying other apparent COVID-19-related complications remains unclear.
Much attention has been given to the possible neurotropic capabilities of COVID-19 as an explanation for symptoms ranging from anosmia/dysgeusia to dysautonomia to cognitive impairment. Indeed, a handful of neuropathological studies of COVID-19 decedents have identified SARS-CoV-2 RNA by PCR in the central nervous system and some have additionally identified SARS-CoV-2 spike and/or nucleocapsid protein in or around brain tissue by immunohistochemistry and confocal microscopy studies. However, the double-edged sword of PCR is its ability to detect and amplify small amounts of genetic material, and studies that have reported PCR positivity have either identified low RNA copies or not reported viral levels. Such low levels of RNA could represent blood contamination or regional contamination from non-central nervous system tissue (such as olfactory epithelium or mucosal cells). Data revealing detection of SARS-CoV-2 RNA in single cell analysis or via in situ hybridization in nervous system tissue is lacking. Additionally, long post-mortem intervals prior to neuropathological evaluation can lead to autolysis, which can confound morphological and molecular analyses. Furthermore, neuropathological studies that have detected SARS-CoV-2 RNA have either not found widespread neuronal or glial involvement or not found co-localization of viral RNA with other neuropathological injury. It is conceivable that initial neurological injury may be caused by a mechanism other than neuro-invasiveness (such as hyperinflammation), which could lead to a permissive blood brain barrier and passive movement of viral particles into brain tissue. In this scenario, SARS-CoV-2 virions may merely represent innocent bystanders.
In the abstract presented by Normandin and colleagues, data is presented from 18 COVID-19 autopsies. The authors report findings similar to their original New England Journal of Medicine paper, which identified acute hypoxic injury as a pervasive neuropathological feature in all subjects. Additionally, they found that while there was limited evidence of viral RNA by RT-qPCR, some sections from in the medulla, frontal lobe and olfactory region yielded higher RNA copies; however, viral load did not correlate with other histopathological findings and was inconsistent with the interval from onset of COVID-19 symptoms to death. Immunohistochemical studies did not identify SARS-CoV-2 nucleocapsid protein in any nervous system cell type. Notably, only six patients had symptoms that might be construed as neurological, and five of these had myalgias or headache, which are non-specific. It is possible that neurological symptoms were present but undetected in some patients, particularly those that were mechanically ventilated and likely sedated prior to death.
Understanding underlying mechanisms of neurological injury following COVID-19 infection is critical to advancing therapeutics targeting debilitating acute and post-acute neurological symptoms. The socioeconomic impact of COVID-19 related neurological sequalae could be profound if lingering symptoms interfere with an individual’s ability to retain employment, engage in household responsibilities or even participate in leisure activities.
Further evaluation of the contribution of neuroinflammation, blood-brain barrier disruption, autoantibodies or other possible pathophysiology underpinning SARS-CoV-2 related neurological injury is urgently needed.
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Normandin E, et al. Abstract S31.003. Presented at: 2021 Annual Meeting (virtual meeting); April 17-22, 2021.
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