Skip to main content

Transforming the understanding
and treatment of mental illnesses.

Neurologic and Psychiatric Effects of SARS-CoV-2 Meeting: Day 1

Watch on YouTube.

Transcript

Neurologic and Psychiatric Effects of SARS-CoV-2 Meeting: Day 1

Transcript

DR. JOSEPH: Welcome to the Neurologic and Psychiatric Effects of SARS-CoV-2 Infection meeting organized by the National Institute of Mental Health, National Institute of Neurological Disorders and Stroke, and National Institute on Aging.  Thank you very much for joining.  My name is Jeymohan Joseph, and I'm a branch chief at the Division of AIDS Research at NIMH. 

I'd like to spend a few minutes talking about the background, goals, and structure of the meeting.  Emerging data suggests that as high as 30 percent of COVID patients experience neurologic and psychiatric symptoms, ranging from ischemic strokes, encephalitis, and mental health issues, such as anxiety, depression; psychosis and delirium and PTSD are also seen in some patients; loss of smell, fatigue and malaise are quite common.  You'll hear a lot more in-depth discussion of these topics during the meeting.

For a proportion of patients, symptoms last for weeks or months after acute infection has passed.  New symptoms may appear after acute infection has passed, whether or not they had symptoms during the acute infections.  Some of these symptoms include fatigue, cognitive difficulties, brain fog, sleep problems, fever, anxiety, and depression.  These health effects of the virus are termed post-acute sequelae of SARS-CoV-2 infection, and you'll hear a lot more about this on the second day of the meeting.

So the goals of the meeting are really to examine emerging data related to epidemiology and pathophysiology of neurologic and psychiatric complications of SARS-CoV-2 infection in acute setting as well as in relation to post-acute sequelae of SARS-CoV-2 infection.  Interactions with other CNS infections, such as HIV, will also be examined.  A panel will be convened to discuss future gaps and priorities in relation to post-acute sequelae of SARS-CoV-2.

A few words about the structure of the meeting.  Day one will be focused on acute COVID-19.  Clinical neurologic and psychiatric manifestations and associated markers of inflammation, neuroimaging, and brain autopsy studies will be discussed.  Neuro-COVID in the setting of HIV and autoimmune disease, such as MS, will also be addressed.

Day two will be focused on post-acute sequelae of SARS-CoV-2.  We'll hear patient perspectives, patient-led research, and journalistic perspectives.  Special issues such as racial disparities in neuro-COVID susceptibility, access to care will be addressed.  Finally we will hear a global perspective with special emphasis on recent second wave infection in India.  We'll also have a unique session that will highlight resources for neuro-COVID research.  These include model systems, cohort, biobanks, and WHO global forum.

Finally, we'll have a roundtable with leaders from 12 NIH institutes who will discuss IC interests and resources for addressing neurologic and psychiatric effects of SARS-CoV-2 infection.  Dr. Josh Gordon from NIMH and Dr. Walter Koroshetz from NINDS have kindly agreed to lead this discussion.

I want to acknowledge the organizing committee who worked really hard to pull this meeting together, Dr. Serena Spudich from Yale who'll be moderating the first day of the meeting, Dr. Avi Nath from NINDS, who will moderate the second day of the meeting, and Dr. Eliezer Masliah from the National Institute on Aging.

It's now my great honor -- oh, sorry, I have to do some housekeeping notes.  Participants will be muted in listen-only mode and cameras will be turned off.  Please submit your questions via Q&A box any time during the presentations.  If you have technical difficulties hearing or viewing the webinar, please note these in the Q&A box and our technicians will work to fix this problem.  You can also send an email to the address listed there.

Now it is my great honor to introduce Dr. Joshua Gordon, director of the U.S. National Institute of Mental Health to provide opening remarks for this meeting.  Dr. Gordon, I'll turn it over to you.

DR. GORDON: Thank you, Dr. Jeymohan, and I want to thank the organizing committee for putting together a really tremendous agenda.  The issues of neurologic and psychiatric effects of SARS-CoV-2 infection for me, like probably for some of you, is actually intensely personal.  I have a very good friend who was unfortunate enough to contract COVID, and although she had a relatively I would say moderate case, not requiring hospitalization, she has had persistent cognitive symptoms ever since, and one of the first questions that she asked me was is this because of the virus?  These were early days.  This was last spring, and we really didn't know. 

But I think one thing that is becoming clearer as Jeymohan noted and as you'll hear more about throughout the course of the next couple of days is that neuropsychiatric illness following SARS-CoV-2 infection is certainly common, and it seems more common than with other serious respiratory illnesses.

We need to understand this.  We need to understand it not just because of the hundreds of thousands of Americans and millions of people around the world who have been infected with SARS-CoV-2 already, but also because we know that this pandemic is not over.  It's not over around the world, and it is not over in the United States.  So anything we can learn about it to prevent it and, importantly, to reduce the symptomatology is crucial.

NIMH is one of several brain institutes at NIH, and we are collaboratively working together as is evidenced by this workshop, but also many other efforts to understand and study treatment of the neurologic and psychiatric impacts of SARS-CoV-2 infection.

With regard specifically to the psychiatric symptomatologies, there's really three different populations to consider.  First, of course, is the indirect effects of the pandemic, and I say this first because that's, despite the many, many again hundreds of thousands of Americans who've been infected with it, we've all been affected by the pandemic and we know that there are serious psychiatric symptoms in the setting of the pandemic, even for those who are not infected, and although that's not the next two days, it is important to remember and to take into account as we study COVID-19, if for no other reason than we have to ensure that when we're studying individuals who've been infected, that we understand how they compare to the rest of us who have been affected but not infected.

Second is those who've been directly infected by SARS-CoV-2 who do not have a history or psychiatric illnesses.  We have to know if the mental illness symptoms that we're seeing are part of the COVID syndrome directly caused by the virus or if they're caused by some other pathophysiologic response to that virus.  We have some basic questions about that.  How does this new onset illness differ from that seen with other serious respiratory illnesses?  We know that at least in hospitalized patients, it seems that neuropsychiatric illness is more common in response to SARS-CoV-2 than, say, influenza, but we need to know more about how it differs and how it is similar.

And importantly, and this goes back to my friend's story, we know that neuropsychiatric symptomatology is associated with the severity of the disease, but what is the incidence of neuropsychiatric symptoms in the context of mild or moderate illness?  We're hearing more and more anecdotal reports that you can have neuropsychiatric symptoms despite a relatively mild course, and we need to understand that.

So in addition to all of us, in addition to those who have been infected with SARS-CoV-2, we at NIMH also care deeply about individuals with mental illness who've been affected directly or indirectly by the SARS-CoV-2 pandemic.  In terms of direct effect, we know, thanks to work by some of my colleagues, including my fellow institute director Nora Volkow of the National Institute of Drug Abuse and her team's work, that people who have mental illnesses and substance use disorders before infection with SARS-CoV-2 are more likely to be infected and if infected, particularly with serious mental illness, are more likely to suffer more severe cases and more likely to die.  In fact, individuals for example with schizophrenia are ten times more likely than the general population to contract SARS-CoV-2 and if they do contract SARS-CoV-2, they are three times more likely than individuals without schizophrenia to die from it.

So these are serious numbers and serious effects.  So we need to think about the general population, and we need to think about individuals who have been infected with SARS-CoV-2, and we need to think about individuals with preexisting neurologic and psychiatric complications as we consider these effects of this virus.

I'm really looking forward to learning more from you all over the course of the next two days, and I'm looking forward to the results of the studies that we've already funded and to funding the next generation of research.  I think one piece of good news is here is we have lots and lots of tools at our disposal as you'll see over the next couple of days to study this issue, and with all the energy and effort that's being directed, I think we can look forward to some real discovery some that you will hear about today and some that will be exposed over the next couple of years.

Thank you very much again, and I'm looking forward to hearing from all of you.

DR. RAUSCH: Thank you, Dr. Gordon, and welcome, everyone.  I am Dianne Rausch, and I'm the director of the Division of AIDS Research at the National Institute of Mental Health.  We're very pleased to be participating in this important meeting on the neurological and psychiatric effects of SARS-CoV-2 infection.

The Division of AIDS Research supports a broad research agenda that encompasses basic and clinical neuroscience of HIV infection, as well as basic and applied behavioral science to prevent new infections and limit morbidity and mortality among those infected.  From the beginning of the HIV epidemic, we have supported an integrated research program to understand the viral, molecular, and cellular mechanisms involved in the pathophysiology of HIV-induced CNS dysfunction and the mechanisms of establishment and persistence of HIV viral reservoirs in the CNS.

Neuro HIV investigators have been studying the impact of HIV and inflammation on the CNS for the past 30 years.  They're currently poised to play a strong role in the study of the impact of SARS-CoV-2 on the CNS.  There are similarities between the CNS consequences of HIV and SARS-CoV-2 in symptomology, symptomatology, and outcomes, such as cognition, mood and anxiety disorders, depression, et cetera, and there are many common pathways that may be impacting these comorbidities.  These include inflammation and immune dysfunction where systemic and neuroinflammation are present both with SARS-CoV-2 and HIV, leading to impact on the blood/brain barrier and the disruption of cytokine/chemokine homeostasis.

That dysregulation is documented both with SARS-CoV-2 and HIV, leading to a disruption of the microbiota brain/gut axis that can disrupt CNS function.  Both infections show breakdown of blood/brain barrier and widespread glial cell activation, neuronal injury despite very few infected cells in the CNS suggesting indirect mechanisms of neuroglial injury, and many of the model systems and concepts that have been developed to study neuropathogenesis of HIV infection could also be applied to SARS-CoV-2.  This meeting will examine many of the SARS-CoV-2 induced pathophysiologic mechanisms that result in some of the neurological and psychiatric effects seen, and this can be very informative for both diseases.

I'd now like to introduce Dr. Serena Spudich, the professor of neurology at Yale University, who will moderate the first day of the meeting.  So take it over, Serena.  Thank you.

DR. SPUDICH: Thanks so much, Dr. Rausch.  Welcome, everyone, and thanks so much for joining the meeting today.

Before I introduce the first speaker, I wanted to just mention a couple of logistic issues about how sessions for the two days are designed.  So both days, we will be starting with a plenary talk by an invited speaker, and then following that, we have divided the topics for the two days of these meetings into thematic sessions, and we'll have a number of talks by different speakers within a certain theme, and at the end of each session, we will have a group Q&A session that will be live with the speakers from those sessions.  We'll be taking your questions from the Q&A entered in the Zoom and moderating those questions between a group of speakers.

We will also have -- we have a planned 30-minute break today, planned between 12:45 and 1:15 p.m. U.S. Eastern time, and we'll also be ending both days with panel discussions.  Today we have one panel discussion, kind of to review some of the topics that were discussed in the first day.  Tomorrow we actually have two different panel discussions, both between some of the speakers and investigators, as well as among the NIH leaders.

So the other thing I wanted to mention is that detailed speaker bios are available on the website, and you can read about each of the different speakers and many of the panelists in those bios.  For the introductions over the next two days, we're going to be extremely brief, just to introduce people so they can get into the content of their presentations.

So it's my great honor to introduce Dr. Stanley Perlman from the University of Iowa, in the United States, who is going to be discussing neurologic complications of coronaviruses.  Thanks so much, Dr. Perlman.

Agenda Item: Plenary Talk: Overview of Neurologic Complications for Coronaviruses

DR. PERLMAN: I am Stanley Perlman, from the University of Iowa.  I'm going to be talking about providing an overview of neurologic complications for coronaviruses in general, with a little bit of discussion of COVID-19, but I know there are many other speakers who are going to discuss this at greater length than I'm going to.

I want to thank the organizers for inviting me.  I want to start off by showing this picture of a coronavirus.  This was done by a tattoo artist who was put out of business by the pandemic.  He gave me this last summer, and I think it's a good representation of the way we view COVID-19. 

I've been interested in brain infections caused by coronavirus for a long time.  This is a slide from one of our early studies from many years ago, probably over 20 years ago now, where we were interested in different kind of CNS disease caused by coronavirus.  This is a murine coronavirus, and what it does is it infects primarily, this particular strain, the white matter of the spinal cord, it infects no other organs in the body.  And what one can see as one looks at the middle panel here, one looks at the middle panel, is that if one doesn't have T cells or B cells -- these are RAG1 knockout mice -- then one sees virus infection of many cells in the white matter.  That's shown here.  But the white matter itself looks very good, and there's no macrophages and the axons underlying the white matter look to be healthy as well.

If one adds back T cells, within seven days one sees destruction of the myelin, elimination of the virus, and macrophages infiltrating into the white matter of the spinal cord.  So this has really been a central interest of mine for many, many years.  Why is it that this coronavirus causes an infection of the spinal cord that in itself doesn't seem to be too harmful, but as soon as the host tries to eliminate it, now there's marked immunopathology, and I think this is consistent with what we see in COVID-19 as well, this disparity between the infection and the host response to it.

Going over some of the coronavirus infections of the CNS before SARS-CoV-2, one of the common cold coronaviruses, OC43, has been postulated to be a factor in the initiation or exacerbation of multiple sclerosis, much like what I showed in the previous slide.  But this has not really been supported by data.  Another common cold coronavirus, 229E, has also been found in some patients with MS, but again, without any real evidence of role in pathology.

Then there's one case of lethal OC43-induced encephalitis in an 11-month-old SCID patient.  This was first reported in the New England Journal, and this shows some of the data.  This is probably the only report that I think is really believable showing overwhelming infection.  This child had severe combined immunodeficiency syndrome.  What one can see here is rarefaction of neurons as well as extensive labeling of antigen here, shown in panel D.  So this is considered the best illustration I think of a OC43-caused encephalitis.

In SARS, there were many cases, much like with SARS-CoV-2, there's many patients who developed some sort of neurological or psychiatric sequelae after the acute infection.  Some patients developed psychosis during the acute infection.  This was attributed to steroid use, personal vulnerability, or psychosocial stressors.  And then after the infection resolved in those who survived the infection, there was reports of neurologic and psychiatric complaints.  And we were particularly interested in this because of some of our mouse work, but I think that evidence for this being directly virological, as opposed to the widespread use of steroids and the widespread use of ventilators, it was very hard to prove one way or the other.

Then finally, there's occasional reports of detection of viral RNA protein in the CNS on autopsy.  This is going, I think this is very similar to what we're seeing now with SARS-CoV-2.  There's a little bit of evidence for viral infection, but the vast majority of evidence so far shows that in fact direct infection of the CNS doesn't occur to a great extent.

This is one of the two papers that describes this.  This is from Journal of Experimental Medicine, and just looking here in the lower panel, these are the only brain figures in the manuscript.  What the authors say here is that by in situ hybridization, they're seeing evidence of virus infection.  And this is really the vast majority of what they're claiming to be viral infection, so whether there is or not is I think still an open question.  Some of these others -- this is a negative control for the in situ hybridization, and this is I think someone who died from a traffic accident.  So there's staining here, but whether it's real, one would like to see more fields from even from this individual patient and from other samples. 

And then this is a second patient of somebody who died from SARS.  This is a 39-year-old physician, and these figures show here we can see encephalomalacia, with a larger version of it shown here.  And vasocongestion.  And some hemorrhage in the brain of this person.

And here, this is a lovely picture of a virus, but it's actually of a Vero cell that was infected with lysates from the brain of this person who was infected.  So this is, I think, a decent confirmation that virus was there.  We don't really know how much, though.  The next slide shows some information about that. 

Here, by immunohistochemistry, we can see some evidence of virus in infected neurons.  But again, it's one case, so it would suggest that the brain was rarely involved directly by virus infection, similar to what we're seeing now.  Whether this was a consistent finding across this whole brain is really hard to tell from the manuscript.

In terms of MERS, we have much less information.  Not that we had so much about SARS, but we have even less from MERS.  MERS is really a camel virus that occasionally spills over to humans, and in the nine years since we've had the outbreak began, there's been 2,500 cases, so not very many cases.  There's now about one or two cases a week in Saudi Arabia.  There's still a very high mortality, about 35 percent.  And this is because patients usually have comorbidity.  Neurological disease is very rare.  I know of a report describing three patients with acute neurological disease and there's scattered others as well, and some survivors, just like with SARS, have neurological and psychiatric disease, but as with SARS, many of these patients were on ventilator support or received steroids.  And we know nothing about the brain really, because only two autopsies were performed. 

So there's no information on the virus in the brain at the time of death.  These two autopsies were also not perfect, either.  One was somebody who was preserved for 10 days before the autopsy was actually performed.  The reason for this is because for cultural reasons there's no autopsies in Saudi Arabia, and then there was a sub-outbreak in Korea, and at that time people were so worried about the spread of the infection that all the brains were burned.  So there was no samples left for doing autopsies.

I know I was asked to talk primarily about the prior coronavirus infections in terms of the brain infection, but there really isn't much, so I'm just going to over a few points here about COVID-19, and then of course the rest of this two-day meeting will discuss these in much greater detail.  So what we know from so far in a very broad view, and this, as I said, will be talked about much more after me, there's several neurologic, psychiatric sequelae recognized in COVID-19 survivors, and these include brain fog and cognitive defects, strokes, seizures, encephalopathy.  There's a worry that there's increased risk of neurodegenerative disease, but I don't think we know this yet.  Guillain Barre syndrome and anosmia and ageusia.

The thing that that it's really been remarkable is that the sequelae may last for several months, they may be disabling, but we really have not detected virus in the brain.  Occasionally there have been reports of it but they're not necessarily the best reports, and some of the data are hard to believe, so we don't really know what the role of the virus is in this infection. 

It seems that CNS damage may result from poorly understood effects of proinflammatory mediators or thrombosis.  How this virus is working is really unclear, because there's so many viruses that cause severe respiratory disease that don't have the same multiorgan effects that SARS-CoV-2 does.  So this is an area, of course, of importance, not just for the brain, but also for the heart and other organs that are affected indirectly by the virus.

One possibility is that the virus products do something, so whether the virus has a hit-and-run effect on endothelial cells in different organs or in the organs themselves is not known, whether the surface glycoprotein, which may or may not be released, we don't really know what levels are in the blood, whether this is having an effect is not known.  This was not widely seen with SARS, so even though the infections are similar, the consequences do seem to be different. 

What I'm going to talk for the rest of the time about is just some work that we've done with anosmia, because this is such an important complication of COVID-19, it's well-established.  It generally resolves within a short period of time.  However, it may persist for longer periods of time with variable resolution, and it can also be associated with parosmia and phantosmia, particularly phantosmia, which suggests that there's also central involvement -- just not involvement of the olfactory epithelia.  Some of the aspects of this can be investigated by using mice.

This is the one phenomenon where I think actual direct viral infection actually has a role.  And that's shown here in the next slide.  This is -- we're not the only ones to show this, but this is one example.  This is a 46-year-old who succumbed to COVID-19, and this is four days post-diagnosis.  We obtained nasal tissue and we performed immunostaining on this tissue, and what one can see here is that the N-protein is readily detectable in these cells.  From the position of these cells, we don't have labels for sustentacular cells, but from the position of these cells we believe they are sustentacular cells.  And around these sustentacular cells are myeloid cells.  That's what's shown in green here.  And this will be something I'll show you also seems to occur in mice infected with SARS-CoV-2.

We became interested in this initially because of the 60-year-old woman who first came to our attention in March 2020.  She was someone who was absolutely fine but lost her sense of smell and taste.  She couldn't smell perfume or food or anything else.  So she was otherwise perfectly fine.  She mostly recovered her senses of smell and taste within 14 days, but at eight months these were not fully recovered, and I think this is a common story, that people are mostly back to normal, but some people are not completely back to normal.

And at the same time that we saw her, there were reports of anosmia and ageusia appearing, and so she figured that she had COVID-19, she had been at a wedding, but we didn't really know.  And then eight months later in November 2020, she became interested in knowing whether her anosmia was caused by COVID-19.  So what we did is we obtained blood for antibody and T cell analysis, and we put this patient at as an antibody titer of 1:48, which was not very high, but she also had T cell responses, which are shown by these dots here.  This is cells that are responding to stimulation by either the N-protein or the S-protein of SARS-CoV-2.  And without stimulation there's none of these cells expressing interferon-gamma TNF, but with stimulation we can see these cells are clearly present. 

So we decided to investigate these in animals, and I'm not going to spend much time on some of these animals, but just to say that we know that in terms of the common cold coronaviruses, OC43-infected mice develop acute encephalitis.  They're not useful for respiratory disease, but they develop brain disease, which, as I said before, is not really typical for humans to develop.  We developed these K18-hACE2 mice, which were extremely susceptible to brain infection, so that one only needed three virus plaque-forming units for a lethal brain disease. 

And on the other hand, when we had a mouse-adapted virus for SARS, which is different than these transgenic mice, mice developed no brain disease, and very similarly for MERS, we developed transgenic mice which developed encephalitis, but when we used normal mice that could, with a mouse-adapted virus, basically we saw no brain infection.  These were not really normal mice, but they expressed a human receptor in place of the mouse receptor, so different from the transgenic mice.  Nonhuman primates infectable by either SARS or MERS, there was no evidence for CNS disease.

To use mice to study COVID-19, we developed several models.  I already mentioned the ACE2 transgenic mice.  And importantly, for what I'm going to say here, other groups prior to us showed that you could infect mice with SARS-CoV-2 if you modified the virus slightly.  There's only one to two amino acid incompatibilities between mouse ACE2 and the virus.  So we did this as well.  this is just showing -- I'm going to show data from both the K18 and mice infected with human virus, and mice infected with our mouse-adapted virus. 

And in both cases what we see is widespread infection of sustentacular cells here, and we also see infection of the olfactory and respiratory epithelium and the sinus epithelium, but this is really the most important -- widespread sustentacular cell infection much like what we see in humans. 

This again shows that sustentacular cells are infected and not olfactory neurons, that the green cells are neurons and the red cells are the sustentacular cells that are infected.  There's no overlap.  Then we wanted to see if these mice developed anosmia, so we used several tests.  I'll just show one here.  This is female mice distinguishing novel from familiar scents, and these experiments were done by a postdoctoral fellow in my lab, and what one can see is an uninfected mouse spends most of her time, in this case, around the novel scent, that's shown in blue, but by two or three days after infection, she can no longer distinguish the familiar from the novel scents.

Then when one looks at the recovery from this process, one can see here, just looking at the scent discrimination test, that initially the mouse spends most of their time on the novel scent, and then this goes away with time.  There's little difference.  But by 21 days after infection the mice are again spending more time with the novel scents.  We did the same things with varied food tests, which again show the mice are basically back to normal by 21 days.  This shows how long it takes to find buried food.

So what's causing anosmia in these mice?  We don't have all the answers yet.  But this is one thing we see; we see that this destruction of cilia, this is pretty widespread even though the sustentacular cell infection is not that widespread and doesn't cover most of the epithelium, but still there's widespread cilia loss, and at the same time we see loss of olfactory receptors.  This is ten different receptors, and just looking at this one here, one can see expression levels that are quite variable but high.  By two and six days after infection, reception levels are very low.  Twelve days they've more or less recovered to normal.

And the only thing that we can see, we don't see much changes in cytokines.  We see changes in just CXCL10, and I'm not going to show that.  And also we don't see much differences in cell infiltrates.  But what we do see is that myeloid cells, which are usually concentrated in the basal layer here, have all moved into the epithelium, or many of them have moved into the epithelium, around sites of virus infection.  And we think this is similar to what we saw in the human samples and may contribute to anosmia.

To summarize what I've said about anosmia, mice infected with SARS-CoV-2 we think are useful for studying anosmia.  I didn't really show this, but as in patients, female mice develop more severe anosmia than males, even though COVID-19 is more severe in males in general.  Olfactory sensory neurons, like in humans, are not infected, but sustentacular cells are.  There are important for olfactory sensory neuron homeostasis, and they may produce inflammatory mediators that indirectly affect OSN function.

With that I'd like to stop, and I thank the audience.

DR. SPUDICH: Thank you very much, Dr. Perlman.

Agenda Item: SESSION I: Clinical Neurologic and Psychiatric Manifestations of Acute COVID-19

DR. SOLOMON: Hi, my name is Professor Tom Solomon, at the University of Liverpool in the UK.  I want to thank you for inviting me to this meeting.  I've been asked to talk about central nervous system manifestations of SARS-CoV-2. 

I head a large research group in Liverpool here.  You can see we have various team leaders managing various aspects of our work, and I'm also the director of Brain Infections UK and Brain Infections Global and also the UK's emerging infections research unit.  I'm going to tell you a little bit about our work in each of these areas, CNS COVID disease.

First of all, the HPRU.  We've worked in the past on Ebola and Zika, and because we are set up as a rapid reaction unit to study emerging infection problems, we were actually in position to study the very first hospitalized cases in the UK, as you can see here, and in fact, our network of hospitals have now recruited 200,000 hospitalized patients into this study called the ISARIC 4C study, which has also underpinned many of the other national initiatives, including our viral genetics intuitive, human genetics, et cetera.

The first paper we produced was on 200,000 hospitalized patients here, and I thought I'd just present a bit of data from this, because it was clear from the start that things like fatigue and confusion and headache were prominent in hospitalized patients.  Also that dementia and chronic neurological disorders were common comorbidities, which it subsequently turns out do indeed increase the risk of a poor outcome.

So the ISARIC study showed that about 70 percent of patients have a neurological symptom, which might just be a headache.  About 10 percent have a syndrome, and then about 1 percent end up with a neurological diagnosis.  But what was clear from the start when these neurological manifestations or associations were being reported was that there seemed to be a wide array of presentations, so we pulled these together in this review we published in Lancet in July 2020.

This showed that about 10 percent of patients had encephalopathy, about 1 percent a true encephalitis.  But also of course, other things were being reported, like Guillain-Barre syndrome.  We pointed out in our paper that one has to be clear that trying to determine whether this presentation is actually caused by the disease, or is it just a coincidence?  Obviously, if you have millions of people infected with a virus, then there are going to be by chance other presentations that are coincidental.

We also encouraged people to use standardized case definitions, which we produced here in the journal, because people were using a wide array of definitions in different centers.  So then we used definitions like these in our first UK study, which was run in collaboration with a series of professional bodies across the UK, and the first reports of 150 or so patients was in Lancet Psychiatry. 

What you can see here is that, in red on the left, the time to presentation for our patients in our CoroNerve study, as it was called, was approximately the same as those with COVID in general.  That's the blue line.  And then also the age distribution shown on the right there, was similar.

The main presentations were patients with cerebrovascular disease, altered mental status, and then other problems.  Interestingly, the patients with neuropsychiatric presentations, including altered mental status, tended to be a bit younger than those with cerebrovascular disease.  We've written up now in more detail the first 260 patients in the study, and I'll just draw your attention to box B here, which shows quite a lot of overlap in the primary diagnoses of these patients, particularly cerebrovascular disease and delirium.  And then even if we look within stroke types or stroke subtypes, there's quite a range of overlapping presentations.   

With our Brain Infections Global network, we have looked in more detail at some of these presentations, so we produced these case record forms linked to the standardized case definitions, and made them freely available via the Brain Infections Global website, and then we said to people who downloaded them, would they be interested in contributing data to an individual patient data meta-analysis, which is currently with the journals. 

And what you can see on the left, we had nearly 2,000 patients for whom we had IPD data, and they came mostly from North America and Europe, but also, importantly, we did have some patients from other settings, including lower- and middle-income countries.  What we found is that actually encephalopathy was the most commonly reported presentation at about 50 percent, with cerebrovascular events about 25 percent, and then other neurological syndromes about 25 percent.

Importantly, for 93 percent of patients, the general COVID symptoms preceded the neurological features, but that meant for 7 percent, the actual presentation was with neurological disease in patients who had no other obvious COVID disease.  Also, importantly, a third of patients developed their neurological problem after hospital admission, suggesting that there might be scope for intervention.  A poor outcome was more common in those with cerebrovascular events, and also the risk factors for poor outcome were different.  So, in the cerebrovascular patients, it was basically marks of severe disease -- breathlessness, elevated D-Dimer -- whereas those with encephalopathy didn't have the same risk factors.

Overall mortality was 30 percent, and the hazard of death was less for patients in the WHO European region, but increased for those from lower- and lower-middle-income countries. 

I'll just come back to this interest in those who deteriorate.  Our most recent publication from the ISARIC study I mentioned earlier, this now is 74,000 patients, nearly half of whom deteriorated after hospital admission, including need for ventilator support, critical care, or death, and we've identified these predictors of deterioration, which do include a low Glasgow coma scale score.

So, the critical thing is we're seeing all these encephalopathy patients with altered mental status, but in terms of viral invasion of the central nervous system, the things we normally look for, like virus in the CSF, this is very rare.  People have begun looking at autopsies.  Initially the suggestion was it was pretty rare to detect virus.  More recently we've seen data suggesting that it's more commonly detected.  I know some of these papers will be discussed later, so I'm not going to go into great detail.  But I think the interesting thing is that the areas where the virus has been detected is not the same as the areas where there's a little bit of inflammation, inflammatory change.  So it suggests there's something different going on.

Now, neurologists traditionally say that the retina is the window on the brain, because we can actually see nervous tissue.  But if that's the case, perhaps for SARS-CoV-2 the nose has been the front door, because this has really given us a clue as to what might be going on.  Here's a single patient who had anosmia, and this is an MRI scan.  You can see in image C there that the olfactory bulbs are swollen, and by the time the sense of smell has returned to normal, this swelling has gone down in image D there. 

This rather elegant study suggest what might be going on, and effectively it is not the olfactory neurons but it's the supporting cells, the sustentacular cells, which have the critical ACE2 receptor to allow viral entry.  The idea is that perhaps it's inflammation in these cells which is disturbing the olfactory neurons and impeding smell. 

But on the other hand, there are more recent data -- these are autopsy data on patients who didn't particularly have neurological complications, there were a range of problems, and these data suggest that the virus is perhaps getting in through the olfactory route and then spreading to other neuronal centers, which are connected to the olfactory pathways.  And there seems to be colocalization of virus in neurons, as we can see here.  But again, there does not appear to be the kind of destructive process of neurons and associated perivascular inflammatory change in the same parts of the brain that we see with viruses like Japanese encephalitis virus, Herpes simplex virus, which do cause a rip-roaring encephalitis.

Supporting this idea that spread might be via the olfactory route are these data from an unpublished study in the UK.  This is a fabulous study of the UK imaging biobank, 40,000 subjects in this COVID sub-study, and there are 394 who had a scan done and then they developed COVID, and then they had another scan some months later.  These have been compared with some controls who had interval scans but did not develop COVID.  What the data show is a loss of gray matter in the limbic cortical areas, those which are directly linked to the primary olfactory and gustatory systems, suggesting that perhaps this is a route of viral spread, but when virus gets to these areas it's not causing a frank encephalitis, but is causing changes which result in neuronal loss. 

So how do we put all this together?  There seem to be three disease mechanisms, broadly speaking.  Firstly, there is this clear inflammatory systemic inflammatory response, which can cause vascular leak, and inflammation around vessels, but not necessarily those parts of the brain where the virus has entered.  Secondly, there's this prothrombotic state, which causes frank strokes, but also microvascular thrombosis and hemorrhage.  And then finally, there are data showing the virus invades the vascular epithelium and can cause perturbation here, and as we've seen, sometimes it does get across into the nervous tissue, including microglia and astrocytes, perhaps also sometimes neurons themselves. 

And maybe it's the balance of these three different disease mechanisms that determines whether this particular patient is going to present with a vascular problem or this encephalopathy delirium syndrome, or just anosmia.  We're looking at this in more detail now in our national COVID CNS study, and also through a case control study which we're running globally.  We're looking for risk factors for development of neurological disease. 

Finally, I'll just end with some more data from our ISARIC study, and this is one beginning to look at some of the inflammatory markers and biomarkers, and broadly speaking, there are some antiviral markers which you can see don't vary in severity from 1 to 6, but the markers of coagulation do increase with more severe disease, as do the markers of inflammation.  And interestingly, the principal component and network analysis shows a couple of markers, IL-6 and GM-CSF, seem to be critical in the disease mechanism. 

We also had samples from the flu pandemic back in 2009 and were able to compare with them, and you'll see that whereas IL-6 is elevated both in flu and COVID, GM-CSF is only elevated in fatal COVID disease.  So we think this may be a critical mediator of the disease progression.

We're working closely through the Global COVID-19 Neuro Research Coalition to support the WHO, and I know Tarun Dua is going to be talking about some of this later on. 

Finally, just to let you know about our COVID neuro webinars which happen every month.  Many of you have attended these already.  And in December, at the encephalitis conference in London, there will be quite a strong focus on neurological COVID disease, and I hope some of you will make it in person to this hybrid meeting.

Finally, just to mention the people who have been funding our work, and I’ll stop there.  Thank you very much for your interest.

DR. PARDO-VILLAMIZAR: Thank you to NIH and the organizers of the symposium for the invitation.  I'd like to discuss today aspects related with what is assumed to be immune-mediated neurological disorders associated with COVID-19.  This is an interesting topic that is important for clinicians and healthcare providers. 

I don't have any disclosures.  My funding is coming from the NIH and the Bart McLean Fund for Neuroimmunology Research.

Every time I discuss aspects related with neurological problems triggered by infectious disorders, I emphasize on the different aspects that determine what are the major factors associated with neurological problems.  Mostly the pathogen, in this case the virus, on the other side the host, that actually will determine the magnitude of immunological reactions due to genetic susceptibility and the presence of factors in that host.  But the most important is obviously the pathogen, particularly the ability of that virus to produce neurotropism or the ability to invade central nervous system tissues. 

So we learned in the past several months about SARS-CoV-2, and the most important aspect of this pathogenesis is that we have been discussing in past several months about neurotropism, neurovirulence, and neuroinvasiveness.  But the reality is that we are still learning about that and in my view, particularly from the perspective of neuropathology and neurovirology, at this moment there is not a full demonstration that SARS coronavirus-2 is neurotropic.  And obviously produce neurological complications, but most of those complications are not necessarily related with a virus invasion of neurons or glial cells.

This is a difficult topic, and obviously controversial topic.  But the most important aspect of the discussion is COVID-19 definitely triggers neurological problems.  COVID-19 triggers neurological problems because there are associated metabolic disturbances, vascular and coagulation disorders, and in minor degrees, some degree of immunological reactivity that may affect central nervous system function and eventually produce neurological manifestation.  It is still unknown what is the magnitude of autoimmunity triggered by coronavirus in the brain or central nervous system.  Again, it's very unclear if the virus has neurotropism, and the evidence at this moment actually do not support that view.

In addition to SARS and COVID and triggering neurological disorders, COVID unmasks neurological disorders, and this is one important aspect for the clinician, because many of those metabolic disturbance, coagulation, vascular disorders, are going to unmask neurological problems that eventually may be confused as being produced primarily by COVID-19.  But the other thing that is really important is COVID-19 coexists with other neurological problems and the mechanism of disease in those neurological problems may worsen in the setting of COVID-19.

An important aspect of COVID-19 is the setting in which we have observed these complications, and most of the descriptions are coming from stages like the critical and severe stage in which there are obviously several complications, most of them are associated with vascular abnormalities, encephalopathies, due to metabolic disturbances.  But for purposes of our discussion, the main issues is what is going on with acute or post-viral immunological syndromes?  We are talking about disorders that are very frequent in viral and infectious disorders that trigger a neurological complication like Guillain-Barre or in cases of encephalitis, myelitis of ADEM.

Other speakers are going to focus on post-COVID-19 conditions, and I'd like to basically focus a little bit on what we believe are immune-mediated neurological complications.  Obviously, there is a long list of those neurological complications that may emerge in the setting of infectious disorders, particularly the viral disorders, and that is very well-known with other viruses, particularly flaviviruses or other type of pathological conditions induced by infections.  That they are basically associated with presence of encephalitis, myelitis, or polyradiculoneuritis in the form of Guillain-Barre or granular layer involvement.

In the case of Guillain-Barre is the classical autoimmune manifestation triggered by infectious disorder, and this is very well established and very well known that many viral disorders produce acute demyelinating forms or acute neuronal-axonal form of Guillain-Barre.  We know that very well in the setting of previous epidemics of viral disease like happened in the case of Zika in 2016, in the Americas, that produced a very clear evidence of an association of a viral disease with emergence of Guillain-Barre.  And we know that also with other microbial infections, like campylobacter jejuni, similar to what we have observed recently in two outbreaks of Guillain-Barre in Peru in 2018 and 2019.

However, what is going on with Guillain-Barre in COVID-19, it has been very well described.  Our colleagues in Europe, in Italy, and United Kingdom, and other countries, have described clearly that cases of Guillain-Barre emerge in the setting of COVID-19.  The major question is, is COVID-19 trigger a major outbreak of those disorders, similar to Guillain-Barre, and the answer is coming from epidemiological studies, and perhaps the very well-recognized study from the United Kingdom showed that Guillain-Barre basically didn't increase, or the incidence of Guillain-Barre didn't increase, in the setting of COVID-19 epidemic or pandemic, and that has been very well documented by these epidemiological analyses. 

Neurologists have describe COVID-19 and Guillain-Barre and many of our colleagues in Italy, Spain, Europe, have described clusters of Guillain-Barre in the setting of COVID-19, but the reality is that the majority of us who are working on Guillain-Barre haven't seen any evidence at all that COVID-19 is a major culprit in triggering Guillain-Barre.  One of the recent studies by Dr. Umapathi in Singapore showed a similar finding to what has been demonstrated by our colleagues in the United Kingdom.

Now, in the case of central nervous system complications, there is very good evidence that some cases emerge resembling encephalitis and encephalomyelitis or even myelitis.  However, epidemiological cases or epidemiological studies have shown that those cases are not necessarily overwhelmingly high as compared with other cases like cerebrovascular disease or metabolic complications in the setting of COVID-19. 

What is every interesting is, and what is lacking, is a very thorough documentation that many of those cases of, quote, encephalitis or, in quotes, encephalomyelitis are really associated with primary inflammatory disorders, primary immune-related disorders, and it is clearly evident from neuropathological studies that there is demonstration of microvascular ischemic and metabolic disease in some of these cases that underwent pathological studies and it is very clear that many of the cases actually lack a very good demonstration by gold standard of neuropathology that there is an active process of adaptive immune reaction in the brain of these cases.

We have an interesting observation.  I'd like to outline two cases that actually expose the challenge that we are dealing with COVID-19 and these autoimmune complications.  This is a patient that was admitted to our service.  It's a 40-year-old woman who was admitted with a diagnosis of extensive white matter and gray matter changes, very suggestive of ADEM.  The studies actually were initiated because she show up with seizures, and in the setting of seizures she was evaluated thoroughly, including a spinal fluid analysis that was normal, MRIs that showed evidence of this abnormality. 

Interestingly, this woman actually evolved from focal seizures to generalized seizures and to status epilepticus, to the point that her status epilepticus was refractory and was difficult to control.  That actually was very difficult situation and we were forced to actually to do extensive testing not only in the spinal fluid, blood, but finally in the brain, and the brain interestingly showed that there was not necessarily overwhelming evidence of inflammation, but rather extensive microvascular disease and vasculopathic, microangiopathic changes, and some of those, actually, yes, were associated with some sort of inflammation, but that inflammation was restricted to the perivascular compartment of the perivascular unit.  Obviously, there was an overwhelming amount of activation of glial cells, including astroglia and microglial cells.

Interestingly, in this patient the only way to control the seizure activity was providing treatment with an IL-6 receptor inhibitor like tocilizumab that was able to control these things.  So the question is, is this really a classical form of ADEM?  Now, this patient, fortunately, got better, and the disease basically slowed down, and she improved the magnitude of clinical problems as well as MRI abnormalities.

The second case illustrates clearly a challenge in the diagnosis of ADEM.  This is another woman in her early 20s that experienced COVID-19 with clear respiratory symptoms that didn't produce too much of neurological problems at the beginning, but almost 45 days later, she presented to a neurologist service in another institution with a progressive somnolence, ataxia, cognitive decline, and speech abnormalities.  The MRI clearly showed evidence of abnormalities in the white matter and, more interestingly, there was evidence of this extensive spinal cord disease characterized by these extensive lesions, mostly located in the posterior compartment and some of them actually has enhanced with gadolinium.  The assessment of the retina with optic coherence tomography clearly show areas of damage of the retinal nerve ganglion cell layer and a very marked decrease in the retinal ganglion cell layer. 

Now, this patient eventually was treated in another institution with IV methylprednisolone, plasma exchange, IVIG, and later with rituximab, and she came to our institution for a second opinion about her encephalomyelitis, and actually it was very interesting because during the assessment of all her previous workup, it was very evident that this patient had experienced a quite significant metabolic disturbance in presence of normal vitamin B12, but had a very elevated level of methylmalonic acids. 

So this is a patient that actually COVID-19 unmasked a cyanocobalamin deficiency, and this patient was treated for cyanocobalamin deficiency and is getting much better.  But this is an example of COVID-19 unmasking a neurological problem and metabolic disturbance and a problem that actually is not necessarily directly associated with COVID-19 inflammatory reaction, but rather COVID-19 unmasking a neurological problem.

So I'd like to emphasize that in addition to COVID-19 producing neurological problems, COVID-19 unmasks many neurological problems, including some demyelinating disorders, some autoimmune disorders, and some other disorders in which the clinician need to be aware about such conditions. 

Thank you so much, and I hope that we can clarify some aspects of these issues in the question-and-answer session.  Thank you.

DR. FRONTERA: Hi, my name is Jennifer Frontera.  I am a neurointensivist and a stroke doctor, and I'm going to talk today about neurovascular events in COVID-19.  These are my disclosures.  I have a couple of grants from NIH that deal with COVID, and I sit on the WHO Brain Health taskforce for COVID.

In my very quick ten minutes, I'm going to cover ischemic stroke, hemorrhagic stroke, cerebral venous thrombosis, and I took the liberty to add in global hypoxia/hypoxic ischemic brain injury, which is like a global stroke to the brain, effectively, because I think it has a lot of implications for post-COVID manifestations.

This is just to give a sense of the prevalence of each of these types of complications in patients with COVID.  This is a prospective study we did in New York City in the spring of 2020, where we looked at about 4,500 consecutive RT-positive, SARS-CoV-2 positive patients, and we prospectively screened them for new neurological deficits.  Of those with neurologic deficits, which was about 14 percent of the entire cohort, you can see the breakout here. 

Over half of them had toxic/metabolic encephalopathy; that was by far the most common sequela.  Ten percent had ischemic stroke, 4 percent had hemorrhagic stroke, and 10 percent had anoxic or hypoxic brain injury, among those that had a new neurological event.

Other large studies have shown, and this is from the UK, that over the six-month time since the pandemic started, the rates of intracranial hemorrhage and ischemic stroke seemed to be higher in patients that have COVID-19 as compared to other forms of respiratory illness such as influenza.  So this red line is looking at hemorrhagic stroke prevalence over this timeframe compared to those with other types of viral illnesses, and here is ischemic stroke over here.  So there is something unique about SARS-CoV-2 as compared to other types of viral illnesses that seems to predispose patients for neurovascular events.

So I'll start off by going into ischemic stroke.  Ischemic stroke amongst SARS-CoV-2 patients is still relatively uncommon, in about 0.5 to 1.8 percent of patients with SARS-CoV-2 has been described worldwide.  However, compared to other forms of respiratory infection or influenza, stroke seems to occur at a higher rate. Compared to influenza, for example, 1.6 percent of patients with COVID had an ischemic stroke, compared to 0.2 percent of patients hospitalized with influenzas, and this was almost an eightfold higher risk.  These patients also have a higher risk of death if they have SARS-CoV-2 with their ischemic stroke than other types of pneumonia. 

Not surprisingly, stroke incidence and prevalence increased with increase in COVID severity, and higher stroke rates were seen inpatients that were more sick or critically ill.  These patients also have higher mortality rates, up to five-and-a-half times higher, compared to non-COVID historical controls or contemporary SARS-CoV-2 negative controls.

I'm just going to show you one example of many studies, and this is one that we did in the spring of 2020 at NYU, where we looked at patients who had COVID, those who were COVID-negative and contemporaneous, and those who were historical controls from the years prior 2019, and we just looked at stroke subtypes as an example.  And you can see cryptogenic stroke seemed to happen much more frequently as a mechanism of stroke than it did amongst COVID-negative patients or historical controls. 

Same thing for ESUS, or embolic stroke of undetermined source, was much more common, and that goes along with the increase in rates of cryptogenic stroke.  And death, as you might imagine, was much higher in patients who also had COVID with ischemic stroke.  And these types of results in terms of mechanisms of stroke seemed to bear out in other large studies where cryptogenic stroke seems to be more common, which the begs the question over how much hypercoagulability may be contributing to stroke in these patients. 

This is a meta-analysis of almost 68,000 patients with ischemic stroke across a variety of different studies, and this is the risk of stroke amongst patients with COVID.  The risk is almost fourfold higher compared to contemporary SARS-CoV-2 negative controls and historical controls.  Similarly, if you look at the risk of cryptogenic stroke compared to controls, it's fourfold higher.  So this does bear out in large meta-analyses.

I like this schematic because it gives other possible mechanisms of stroke that could be unique to SARS-CoV-2, so for example, myocardial injury, myocarditis, might put you at risk for an embolic stroke.  There's also the hypercoagulable state that we discussed, which could even put you at risk for a paradoxical stroke if you have a PFO and a venous sinus thrombosis or venous thrombosis.  Other factors, including leakiness of the blood/brain barrier, which might affect your ability to autoregulate and put you at risk for hemorrhagic stroke, as an example. 

There's also traditional risk factors at play that always apply, seem to continue to apply in SARS-CoV-2 -- Afib, elevated cholesterol, diabetes, hypertension.

Hemorrhagic stroke, fortunately this is less frequent, because it tends to be more devastating, and we documented this in about 0.4 percent of patients who are hospitalized with COVID.  And this is just examples from different patients to show you that they're rather unusual, the types of bleeds that we were observing. 

This patient here, for example, has diffuse hypoxic brain injury and then hemorrhaged into what was already an infarcted brain when fully anticoagulated.  Here's a patient that was not fully anticoagulated, but proceeded to have over a period of days new, multicompartmental hemorrhages at the gray-white junction, including subarachnoid hemorrhage, very unusual patterns that sometimes you might see in patients with traumatic brain injury or sometimes patients that have amyloid angiopathy and a lot of small vessel injury.

Here is a SWI gradient echo sequence in a COVID patient, and you can just see it is riddled at the gray-white junction with all of these microhemorrhages, and possibly micro-thromboses with microhemorrhages.  So there's quite unusual patterns of hemorrhage that we were seeing in these patients. 

When we looked at our own patients, we found that anticoagulation was very highly correlated with the risk of intraparenchymal hemorrhage.  This is, again, in the spring of 2020, so a lot of these patients were prophylactically anticoagulated based on their D-Dimer levels.  What we did find, there was over fivefold risk of intraparenchymal hemorrhage with anticoagulation, even after adjusting for other traditional risk factors.

So what's going on with these patients?  Well, here's that same MRI I showed you with a lot of these microbleeds.  You can see pathologically, this is on brain sectioning, this kind of microhemorrhage at the gray-white junction, this is postmortem MRI, you see the sort of same thing.  Here, on HNU light microscopy, you can see actually thrombus in these small vessels, and some of them have some inflammation in the vessel wall, but the micro-thromboses seem to be prominent across multiple different neuropathological studies.

Here's a study from New England Journal looking at the blood/brain barrier, to some extent.  So this is olfactory bulb and this is pons, and there's some fibrinogen staining in the parenchyma.  So fibrinogen should not be in brain parenchyma.  That means that it must be coming out of blood vessels.  Here is an example of a blood vessel with some disrupted collagen vascular basal lamina, suggesting possible breakdown of blood/brain barrier.

Other neuropathologic studies have not shown blood/brain barrier disruption.  This is a study of 41 patients from Columbia that recently came out, in fact, look like the tight junctions were intact.  However, what their paper did notice is 100 percent of the patients had hypoxic brain injury, and when you look across different neuropath studies, this seems to be one of the most common findings.  A lot of them had acute infarcts in various locations, almost 20 percent had hemorrhages, and interestingly, they noted that ischemic stroke seems to be adjacent to a lot of these hemorrhages, and they hypothesized that perhaps many of these hemorrhages are hemorrhagic conversion to small ischemic strokes.  Indeed, it's possible that you have micro-thromboses in these small vessels that subsequently hemorrhage.

In regards to micro-thromboses, macro-thrombosis, cerebral venous thrombosis, or cerebral sinus thrombosis, the major point I want to say about this is that the risk of this occurring is much higher in the context of COVID than it is after vaccine.  And even in the context of COVID it's relatively rare.  So less than 0.1 percent of patients with SARS-CoV-2 will have this cerebral sinus thrombosis.  So that's eight cases per 10,000 patients.  It frequently is associated with intraparenchymal hemorrhage, but it's a very uncommon cause of stroke in our SARS-CoV-2 patient overall.

Post-vaccine, this has gotten a lot of press.  The risk of sinus thrombosis is higher with adenovirus vectors, such as J&J, AstraZeneca, Sinovax, et cetera, and less common with mRNA type of vaccines like Pfizer and Moderna.  This is a little bit older data, but from the CDC, the risk of cerebral sinus thrombosis from J&J vaccine was about one per million.  For Pfizer at the time, zero per 100 million, and for Moderna, about 3.5 per 100 million.  So orders of magnitude more common in adenovirus vaccines, but again, far, far less common than if you were to have COVID, where it's 8 per 10,000 cases.  The take-home message is that it's still much safer to be vaccinated than it is to have COVID.

Lastly, global hypoxia.  These are just images of patients with hypoxic brain injury.  In our cohort it happened in about 1.4 percent of hospitalized patients, so more common actually than ischemic stroke, and here's some of the imaging that we saw at various timepoints from the hypoxic insult.  You can see perfusion restriction in vulnerable territories like the cortex layers 3, 5, and 6.  Here at the striatal neurons of the basal ganglia, Purkinje cells in the cerebellum, and then white matter changes that happen typically in a delayed fashion.  Here you can also see these punched-out areas on this MRI that suggest possible medullary vein thrombosis that may have contributed to this picture here or been exacerbated in the context of hypoxia. 

The issue with, I think, these hypoxic patients, and the reason I bring this up, is again because of the post-acute sequelae that could occur in these folks.  If you look at autopsy series, about 47 percent of all COVID neuropathologic studies identified some components of hypoxic brain injury, and on the patient level, 28 to 100 percent of patients neuropathologically had evidence of hypoxic injury, depending on which study that you're looking at.  So it's a relatively common finding across these studies.

If we look mechanistically, the concern is that potentially there's downstream effects from hypoxia, and hypoxia inducible factors, HIF1A for example, that are turned on in the context of low brain oxygen and have a series of pathways that could promote even a neurodegenerative type of picture, pathologically. 

So here's a schematic, for example.  We know that Alzheimer's disease, for example, there is an infectious type of pathology or hypothesis for the development of Alzheimer's type pathology that could occur, that is related to elevated levels of cytokines, blood/brain barrier breakdown, and even hypoxia can contribute to this cascade, again, via upregulation of amyloid production, decreased amyloid breakdown, and increased neurofilament tangle production.  So what happens long-term in these patients is certainly a concern in terms of what could be cognitive consequences. 

I will just show you very quickly, we looked at serum biomarkers of cognition in these patients while they were hospitalized and compared them to non-COVID patients that were either normal, had mild cognitive impairment, or Alzheimer's disease, and this is just some markers.  We used SiMoA technology so it’s extremely sensitive to small levels of these biomarkers, for tau, NfL, UCHL1 and GFAP. And you can see that in some of the COVID patients the levels of these neurodegenerative biomarkers was substantially higher than even amongst Alzheimer's patients.  Now, what's unclear is if this is just a one-time phenomenon and these are going to go back to normal or if they continue to rise over time, or if this is a static insult or progressive insult.

We also did look at these same biomarkers in patients that had toxic-metabolic encephalopathy during hospitalization.  Significantly higher in those patients versus those who did not have any neurological manifestations, higher in patients who died, and lower in patients that were discharged home, and this was found across almost all of the markers that we looked at.  So certainly neurodegeneration markers are playing some role.  We just have to figure what exactly the mechanism is and the pathology.

To summarize, we actually found global hypoxia in about 1.5 percent of patients.  So more common than ischemic stroke, in 1 percent; intraparenchymal hemorrhage in about half a percent; and cerebral vein thrombosis in less than 0.1 percent.  Cryptogenic stroke seems to be more common; maybe hypercoagulability is playing a role.  Hemorrhagic strokes are interestingly happening in unusual locations, and there might be a suggestion of small-vessel micro-thromboses followed by subsequent hemorrhage.  Blood/brain barrier disruption may play a role.

Cerebral sinus thrombosis is not common.  It's even less common postvaccine, so get vaccinated.  Hypoxic brain injury may be the largest effect that we're dealing with, in terms of long-term neurodegenerative consequences.  This is an area of active study, and I certainly think deserves attention as we move forward looking at post-acute sequelae of COVID.

Thank you very much for your attention, and thanks for joining us today.  And I'd like to thank the organizers for inviting me.

DR. WHITTAKER: Hi.  This is Liz Whittaker.  Today I'm going to be talking about CNS implications of multisystem inflammatory syndrome in children, or MIS-C.  Or as we call it in the UK, PIMS.  So apologies if I use both words interchangeably.  I have no disclosures today.

I guess as a pediatrician, we've had a really different pandemic, compared to those who look after adult patients.  Children have made up a very tiny proportion of those who've been hospitalized or who've died with COVID-19, and if we look at the spectrum of disease, they've largely had asymptomatic or mild disease, with probably less than 1 percent experiencing severe disease like adults do, and a very tiny proportion experiencing MIS-C or PIMS, and this post-infectious inflammatory syndrome, which is kind of highlighted in this infographic here.  And then the other group of patients we don't know so much about are those who have persistent symptoms after COVID, which appear to be a group of children and young people that we probably should have more interest in.

Children have had a really tough time.  I'm just showing you here some newspaper headlines from the UK, just showing how there's lots of chatter about them being in school, and are they transmitting the virus, and should we be vaccinating them, is it all their fault that everyone gets sick.  And it's been a really stressful time, and I think that this is a really important thing that impacts on children's experience of the pandemic as well.

I'm just going to run through this case very quickly, and apologize if it's too fast, but this is a classic example of someone who presented with MIS-C or PIMS.  So it's an 11-year-old girl who had flulike symptoms with tummy pain and a sore neck at home for a few days, came to hospital, was found to have possible abdominal sepsis and seen by the surgeons and started on antibiotics, before having a really significant deterioration requiring lots of cardiac support and very significant inflammation on her blood picture, including abnormal cardiac enzymes. 

And actually, she was so unwell, her heart was working so poorly, that she had to be transferred to our local ECMO center, extracorporeal membrane oxygenation center, and she was treated with 16 days of high-dose steroids before she was extubated, with thankfully really good cardiac recovery and normal cognition.  And unsurprisingly, she had some common complications of being in critical care, but actually made it home within 10 days.  She has made a more or less full recovery. 

This is one of the kind of really severe end-of-spectrum cases of these children who've presented with this post-inflammatory syndrome, and this figure just illustrates that if we look at adult hospitalizations in France, in orange, they occurred at this time, and on a different scale, these cases of PIMS or MIS-C were occurring four to five weeks later.  Which suggests that it's a post-infectious phenomenon.

We've seen, in the UK, about 750 of these children, and in the United States, about 4,000, with this very classic picture, but it also, which I didn't mention for her, a proportion of these young people have headaches and confusions, and the most severe group have cardiac involvement and end up in critical care, on inotropes, et cetera.  And they have a very distinct blood picture which is similar to adults with severe COVID including coagulopathy, significant inflammation, and cardiac enzyme involvement. 

These children all have very significant changes in their skin and mucocutaneous areas, as you can see in these images.  And from the very beginning, it has been identified that a number of these children and young people have neurological abnormalities which were associated with changes on MRI imaging, as can be seen on the left of this slide, and so in this first group of 27 children, four of them had significant changes, and two of whom did not make a full recovery but, interestingly, had normal CSFs and no cells or no raised protein.  And at that time there was lots of hypotheses about what the mechanism was.

This much bigger cohort from the United States of nearly 1,700 patients included 22 percent who had neurological involvement, and of those, about 12 percent, had life-threatening illness.  What we can see in the under-5s is that they presented with fits, and more commonly than the older children, but that like our patient that headaches, confusion, and fatigue and weakness, were quite commonly seen in the older cohort, along with anosmia which we've seen in adults and children with COVID.  And in addition, we can see that there's this association with higher inflammation in these children and young people.  The images here really demonstrate how significant this is.

We know that being in intensive care and being unwell, in its own right, can have detrimental effects on children's psychiatric and cognitive outcomes, and this one study from our center here has shown that there is an association between outcomes and inflammation during the admission of the patient.  Just looking at the long-term follow-up of the PIMS cohort from Great Ormond Street of 46 children, what the picture shows is the spectrum of symptoms in the beginning and how they waned, but by six months the most predominant symptoms which were still experienced were neurological.  And just to say that this was a very severe end-of-the spectrum group of children, because they were all transferred to Great Ormond Street, so there were none of the milder end, and just demonstrating, as I said, 52 percent of them had neurological involvement at presentation, which is higher than the usual cohorts. 

They required quite a lot of intensive care input, and interestingly, there was abnormal neurology identifiable on examination at six months from a variety of things, including dysmetria, abnormal eye movements, and differences in gait, as well as fatigue and concerning quality-of-life questionnaire changes, and a huge amount of anxiety both from the children and young people and their parents, although reassuringly, the vast majority were back in fulltime education. 

So what is the pathogenesis of this?  Is it a direct viral effect on the brain tissue?  Is there a parainfectious phenomenon whereby there are abnormal systemic inflammation that affects the blood/brain barrier because it's leaky at that time?  Is it a vasculitis or endothelial injury pathogenesis?  Or is it a more commonly seen postinfectious inflammation?  And I think at this point we don't really know, but we're really keen to find out, and so we're doing a study which is exploring imaging changes, blood markers, but also outcomes of these children and young people after admission with PIMS.

So we hypothesize: our null hypothesis is that there's no impact on mental health and cognition, and that the treatments that we use which are not insignificant are also not associated with the impact.  So we are comparing the clinical spectrum and outcome of these children and young people and their neurodevelopmental outcomes.  We want to know if all children are at risk, or just those who have neurological presentations at the beginning, and if those early biomarkers of inflammation and MRI findings are associated with long-term poor outcomes.

So what are we measuring?  We're doing some complex neuroimaging, including pathology detection, looking for atrophy, and changes in the axonal areas.  We're doing a variety of blood pictures looking at serum neurofilaments, along with immunology, and looking at whether the serology is persisting or waning.  And then finally we're doing some both physician and patient assessments of cognitive and psychiatric outcomes, including quality of life outcomes. 

This is all under way.  We've recruited a large number of children already, but yet don't have any data.  And just to finish up, to think about how this might link in with some of the other concerning features that we see in children, we know that post-infectious conditions exist for many other infections.  What we don't know about post-COVID syndrome in children is how many children are affected, how much it affects their function, what causes it, what their treatments should be, and what their outcomes are like. 

We know that in the UK that about 8 percent of children experience symptoms at about five weeks, and in other cohorts it's much lower, around 2 percent.  These are all kind of biased observed questions, and so what we really need is some better data, and so in the UK we're doing the CLoCK study, which is a very large prevalence study of persistent symptoms in children and young people who have had confirmed infection, but also have not had infection, to try and explore some of these epidemiological questions. 

In addition, using both the DIAMONDS cohort, which is a diagnostic study that's running across Europe, as well as linking in with colleagues in San Diego for an NIH-funded PreVAIL study, we're hoping to be able to come up with a diagnostic signature.  And excitingly, we already have some suggestion that there is a distinctive signature for MIS-C from children with COVID-19, Kawasaki disease, and sepsis.  This may lead us to a better point-of-care test, but we clearly need to do further phenotyping.  Can we identify those who have got abnormal neurology, and will this be useful from a prognostic perspective? 

In addition, we're recruiting children with persistent symptoms post-COVID to see if we can assist in their diagnosis as well.

So, in summary, this is a rare but serious inflammatory condition, and at least 22 percent present with brain involvement, but at the moment we don't know yet if they have worse outcomes or what the implications are for their long-term prognosis, and in addition, it isn't clear what the relationship is with post-COVID syndrome in children.

Thank you very much for listening, and here are my acknowledgements.

DR. SPUDICH: Fantastic talks from this group of speakers.  I really appreciate that whirlwind amount of data that we've heard.  We have about six minutes for a Q&A session, so I'm going to ask Dr. Solomon, Dr. Pardo, Dr. Frontera, and Dr. Whittaker to please turn on your cameras, and I'm going to be selecting some questions from the Q&A box throughout the meeting.  This is how the Q&A is going to run.

I'd like to start with a question for Dr. Solomon, which was posed by Dr. Howard Gimmelman(?), asking is GM-CSF and IL-6 a cause or a reaction to the disease?  And I suspect what he means is a cause or reaction to the neurologic aspects of disease.

DR. SOLOMON: Thanks very much.  Those data I presented were on the whole ISARIC Consortium, so they weren't selectively patients with neurological disease, but is it cause or effect?  I think we think it is a response to the virus.  It's part of this strong immune response that we've been hearing about from all the speakers today, and the question then is why do some people get this strong response, and is that because they are getting more virus onboard, which there's some suggestion that more virus load is associated with more disease, more severe disease?  Or is it actually that their body is responding perhaps because of some polymorphisms in critical genes? 

As people will know, there are some data suggesting critical polymorphisms, though I'm not sure of any specifically for those genes.  But I'd be interested to hear what others have to say on that.

DR. SPUDICH: Maybe Dr. Pardo, I don't know if he has any comment on that related to the immunologic responses.

DR. PARDO-VILLAMIZAR: Our own experience evaluating cytokines, particularly in CSF, and also in the serum compartment, points out that some of the cytokine responses actually are very likely coming from the autoglia responses.  Again, in a paper that was published a couple of months ago by our group, we pointed out that the signature of IL-6 and other markers of cytokine in the CSF actually was very similar in patients with COVID-19 as compared with patients with vascular disease and stroke.  And if you take out the COVID-19, basically any patient with a stroke will have increases in IL-6 and increases in TNF-alpha.  So that's not specific for COVID-19; it's basically specific for the vascular injury and ischemic injury.  So we didn't see any evidence at all in the CSF or specific increases that are related with COVID-19.

DR. FRONTERA: Serena, I was wondering if I could ask you about the autoantibody paper in Nature that came out from the Yale group, looking at autoantibodies to certain things like IL-1 and cytokines and chemokines that may exacerbate the inflammatory response in these patients?  I don't know if you're familiar with that study or if you're able to comment on how that may play a role.

DR. SPUDICH: Jennifer, I would love to have the opportunity to have Dr. Farhadian, who's going to be presenting that data in the next session, talk about that in the Q&A, so maybe we can hold that question to let Dr. Farhadian reply. 

Actually, I have a question for you that was put into the chat from the audience, and it would just be nice to be able to get to that.  So there's a question directed to you asking in terms of encephalopathy that we see in, I think, hospitalized COVID patients probably, which do you think is playing the largest role -- microvascular and endothelial damage that you talked about, or the neurodegenerative inflammation that you indicated with the blood markers that you showed?

DR. FRONTERA: When we looked at it, we broke down etiologies of encephalopathy, and the most common were septic encephalopathy, which we know has a relationship with cytokine responses.  Uremic encephalopathy, because of the coincidence of acute renal failure in a substantial percentage of critically ill COVID patients.  And then hypoxic, and I think the hypoxia is certainly a major player, and a lot of the neurodegenerative markers that we're seeing, though they are specific to neuronal tissues or glial tissue, it's not specific necessarily to COVID.  And these things might be also observed in patients that have profound hypoxia or are critically ill. 

So we don't know yet.  Ideally, we would like to measure these in a controlled population as well, that have ARDS or some other thing like that, to try to get a handle on if it's really COVID-specific, or it's just due to profound hypoxia and related metabolic disorders. 

DR. SPUDICH: Thank you.  And I think some of the subsequent talks will talk a little bit more about some of these same markers of the pathological findings and might help to shed light on some of those questions.

Maybe one question for Dr. Whittaker, the question is about as a pediatrician, are you seeing increased incidence of tic disorders?  And that was actually something we've seen a little bit here at Yale and had that question raised as well, in children who have had coronavirus-19 or in post-MIS-C children.

DR. WHITTAKER: Not necessarily post-MIS-C children, but I know that there has been an increased recognition of Tourette's and tics and OCD, but also most of the other mental health disorders that children and young people have been experiencing.  So we've seen a real spike in eating disorders, as well as, obviously, anxiety and depression.  And it's really hard to pick apart how much of that is directly viral mediated and how much of that is due to the mitigations of the pandemic and the environment they find themselves in. 

So I know there are colleagues that Tom might know in Liverpool who are working on this and we're collaborating with them, both in the context of follow-up of the MIS-C patients, but also the post-COVID persistent symptom, long-COVID cohort, to try and understand exactly how much of this is virologically driven.  But I don't think we have an answer, but there is definitely an increase in children presenting with these conditions, but what's caused it, I think, is less clear at this time. 

DR. SPUDICH: Fantastic.  Thanks everyone  I know that was very brief.  The questions actually will be recorded, and will be passed back to the NIH, and hopefully can also be shared with the speakers for further discussion and comment.  We will be closing this session, and so thanks again to all the speakers in this session for joining, and we will be introducing the next session.

So the next session is entitled Human Translational Studies of Neurologic Disorders in Acute COVID-19: Blood and CSF Studies, and this is actually going to include markers of inflammation, neuroimaging, and brain autopsy studies.  So I'm going to introduce the speakers from this session.  They include Dr. Magnus Gisslen from University of Gothenburg in Sweden, Dr. Shelli Farhadian from Yale University in the USA, Dr. Dima Hammoud who is at the NIH in Bethesda, Maryland; Dr. Govind Nair, who is also at the NIH in Bethesda, Maryland; Markus Glatzel, who is at the University of Hamburg in Germany; and Dr. Rebecca Folkerth at New York University in New York.

So again, we're going to be having sort of a speed date with all of these speakers giving their presentations, and then we'll have some time for a Q&A session at the end.  Thanks very much.

Agenda Item: SESSION II: Human Translational Studies of Neurologic Disorders in Acute COVID-19: Blood and CSF Study

DR. GISSLEN: My name is Magnus Gisslen.  I am an infectious disease physician and a professor of infectious diseases at the University of Gothenburg in Sweden.  I'm happy to have this opportunity to give a short presentation about viral, immune, and neuronal injury biomarkers in acute COVID-19.

As we have heard, neurological manifestation is common.  They are common in acute COVID-19.  I would not discuss symptoms, but the corresponding CSF and blood biomarker characteristics.  There are proposed mechanisms of neuropathogenesis.  First, it could be a direct CNS damage caused by viral neuroinvasion, could be a CNS cytokine-release syndrome, it could be indirect consequences of the systemic inflammatory response, or it can also be microvascular injuries and thromboembolic events are the cause of the CNS symptoms.

So what can CSF and blood biomarkers tell us?  First, when looking at the detection of SARS-CoV-2 RNA in the cerebrospinal fluid and detection with PCR, in this review, 27 published studies and case reports of in total 449 CSF samples, with available PCR from the CSF, showed that SARS-CoV-2 RNA was detected in only 2.7 percent, and also when the viral RNA was detected, in those cases, the levels were either very low or not reported in the studies.

A literature review published in February by Lewis et al showed that 7 percent of the 238 CSF samples from different studies were positive.  In our own data, you can see a comparison of SARS-CoV-2 levels in the upper respiratory tract in blue, in blood in red, and in CSF in green.  You can see in CSF, the levels are very low, mostly undetectable, in a few cases indeterminate levels in the CSF, in blood slightly higher, but much, much higher levels in the upper respiratory tract.

What about intrathecal immune activation?  First, when it comes to cerebrospinal fluid, white blood cell count, and in this same review from Lewis, they had CSF white blood cell count from 409 patients all with COVID-19 associated neurological symptoms, and as you can see in this graph, symptoms are relatively severe with a lot of encephalopathy and coma and other severe symptoms.  Despite that, CSF pleocytosis with a limit of 5 cells per microliter was seen only in 15 percent.

Similarly, in our cohort, where it was sort of an even lower frequency of pleocytosis, that was probably as a consequence of different types of patient cohort, in the one patient here with high CSF white blood cell count was a typical meningoencephalitis, a condition that exists in COVID-19, but it seems to be relatively rare.

Then markers of active cellular immune system.  First, several studies have analyzed different cytokines in the CSF and compared to blood, and I will not talk about that at all.  It will be covered by Shelli Farhadian who is the next speaker.  But we're looking at neopterin and beta 2 microglobulin, there is marker of activated cellular immune system, and neopterin is mainly produced of activated macrophages and microglia.  Very high levels are found in blood but also with particularly high levels in the CSF.  Those levels are comparable to other types of encephalitis like herpes encephalitis, varicella zoster,  and other types of encephalitis.

Next, we're looking at markers of neuronal injury, and neurofilament light protein.  This is a marker that we have looked a lot at in, for example, in HIV, but it's also useful, very useful, in other types of CNS injury.  It's a marker of neural injury.  It's a sensitive and specific to ongoing axonal injury.  NfL is increased, well, here you can see the age-adjusted levels.  It increased with age, and therefore you need to take age into account, but here is sort of age-adjusted levels.  Eight out of 31 patients had increased NfL in the acute phase.  They were hospitalized moderate to severe disease, moderate there is hospitalized with oxygen aid and severe they are intensive care patients.  Majority of those were neuro-symptomatic, most encephalopathy.

In another study led by Garcia and colleagues, they looked at NfL and also various immune activation markers.  They were analyzed in CSF, and different levels compared to different CNS severity were compared with other conditions.

If you look at NfL, you can see here that patients in yellow would have those with COVID-19, and those with headache as only CNS-associated symptom at levels comparable to controls, while those with encephalopathy, although you can see it's very few cases, but the levels were comparable with other CNS infections and other CNS conditions.  Also COVID-associated stroke at very high levels, as have other patients with stroke.

This is another study comparing NfL with severity, and you can see an inverse correlation with the Glasgow Coma Scale in panel b, and an association between NfL levels and days on ICU at panel a, and this raises an important question that I do not have any good answer to.  That is, do NfL levels in ICU-treated COVID-19 differentiate from other severe infections requiring ICU.  There are actually very little data on that, and you can assume the patient in ICU, whatever condition, can have neuronal injury.  So that is something that is needed to look more at.

Plasma and CSF NfL are normally highly correlated, and plasma levels normally affect CSF levels, and thus CNS neuronal injury.  Plasma levels are normally 50 to 100 times lower than CSF levels.  However, a caveat this that in patient with peripheral neuropathy, plasma NfL can be increased without CNS engagement.  Here is a study where 47 patients with mild to severe disease, with majority were neuro-asymptomatic, but what you could find and see, the plasma NfL were increased in severe disease, severe disease were mainly patients in ICU.  Plasma GFAp, marker of astroglial injury or activation, were increased also in moderate disease and in severe disease.  But there were no difference in NfL in patients with mild or moderate COVID-19 compared to healthy controls in acute phase.

Another very recent publication from Prudencio and colleagues found an association between blood NfL levels and outcome, and both for the risk of intubation of ICU admission, length of hospital stay, and et cetera.

So what happens with these markers when they are followed longitudinally?  Well, this is a study looking at the longitudinal follow-up of 100 patients and about 50 controls with varying disease severity.  I'm showing the severe cases here.  And in total, 51 of 97, 53 percent, self-reported persistent neurological symptoms they had the symptoms at 6 months post-acute infection.

Increased NfL and GFAp were seen in severe disease in the acute phase, and as you can see in NfL, the highest plasma NfL were seen about 30 to 40 days after symptom onset.  And that is sort of similar that is seen, for example, after a stroke where you have increasing NfL that is highest in about three or four or five weeks after the injury.

Then all of those biomarkers, both NfL and also GFAp normalized in, I wouldn't say, all patients, but in all groups, regardless of self-reported CNS symptoms.  So there were no indication of ongoing CNS injury in patients with post-acute neurological sequelae.

In conclusion, the neurological impact of SARS-CoV-2 during acute infection is significant and closely linked to the severity of COVID-19.  Although the degree of neuro-invasiveness is still under investigation, SARS-CoV-2 infections clearly generate significant immunological response in the CNS.  Biomarker evidence of astrocytic and neuronal injury can be seen primarily in patients with severe disease, and there is still very little available data in patients with post-acute COVID-19 CNS symptoms.

Thank you very much.

DR. FARHADIAN: Good morning, everyone.  Thank you, Dr. Spudich and Dr. Nath, for including me in today's session.  Today I'll be talking about my group's recent work assessing the immune cell landscape in the CSF to gain insights into neuroimmune responses during SARS-CoV-2 infection.

As you've already heard today from Dr. Gisslen and others, although neurological and psychiatric symptoms are frequent during acute COVID-19, viral neuroinvasion of the CNS appears to be a rare or short-lived phenomenon, at least based on viral PCR of autopsy brain tissue and CSF.  In contrast, several brain autopsy studies during COVID-19 show evidence for CNS immune activation in affected patients.  Here I'm showing you just a few examples from studies published over the last several months highlighting what are some of the common immune findings, including nodules of activating microglia and T cell infiltration in the brain.

But of course autopsy studies are limited and do not reflect the CNS immune state in living humans.  We asked whether we could assess for neuroimmune responses in patients with acute COVID-19 by studying CSF immune cells as we had done previously in adults with HIV infection.

To do this, we utilized the IMPACT study at Yale, working with many investigators and led by the group that I've shown here, back in March of 2020 we formed a biorepository study to enroll hospitalized patients with acute COVID-19.  Research participants which have now numbered over 350 donated tissue samples and in some cases, this included CSF.  Today I'll be focusing on our findings from our initial cohort of patients who donated CSF.

Our study was designed to compare blood and CSF immune parameters in patients with COVID-19 and matched uninfected controls.  Using these tissue, we measured transcriptional changes and antibody responses in the CSF compared to the peripheral blood to identify CNS-specific immune responses.  Prior work from other groups suggested that cytokine storm or exuberant cytokine responses in the peripheral blood associates with severe COVID-19 disease.  We wondered whether the inflammatory profile in the CSF would reflect that of the blood.

To do this, we measured the levels of 71 inflammatory cytokines in the CSF and blood of our COVID patients and controls.  In this small sample, we found that IL-1 and IL-12 were elevated in the CSF, but not in the blood of COVID-19 patients compared to controls.  More importantly, we found that cytokines that are elevated in the CSF during COVID-19 are not elevated in the plasma and vice versa.

Using a slightly different approach, Remsik and colleagues at Memorial Sloan Kettering likewise examined CSF and blood cytokine levels within the same COVID patients and similarly to us also found that the CSF demonstrates a divergent cytokine profile when compared to the plasma of COVID-19 patients.  Overall, these studies demonstrate a compartmentalized immune response to COVID-19 in the central nervous system.

We then turned toward assessing transcriptional changes in CSF immune cells to understand immune pathways that are affected during COVID-19 infection.  Here I'm showing you results from single cell RNA sequencing of blood and CSF immune cells in five COVID-19 patients and 11 healthy controls.  We looked at gene expression within the different immune cell types and asked whether there were gene expression signatures that were found in the CSF of COVID-19 patients.  We found that both CD4 and CD8 T cells in the CSF of COVID patients demonstrated upregulation of inflammatory pathways, including IL-1 and IL-12 as I've highlighted here, which nicely validates our earlier cytokine results.

I also want to highlight that at the same time a group led by Heming performed a similar study and likewise found evidence for T cell activation in the CSF.  Taken together, the single cell RNA seq data from our two groups, which I've highlighted in the box, shows that compared to the blood, CSF T cells in COVID-19 demonstrate upregulation of T cell activation pathways, increased IL-1 and IL-12 signaling, increased CD4 exhaustion, and clonal expansion of CD4 T cells in the CSF unique to those found in the blood.

So what I've shown you so far has primarily focused on cellular immunity in the CNS.  But what about humoral responses?  To better understand antiviral antibodies in the CSF, we collaborated with Dr. Michael Wilson's group who had developed a custom multiplex ELISA that assesses for antibodies against numerous SARS-CoV-2 epitopes.  What I'm showing you here at the top is data for antibody reactivity in healthy controls and in COVID-19 patients.  You can see that not surprisingly, none of the healthy controls shown here at the top had antibodies against SARS-CoV-2 epitopes in either plasma or in the CSF.  In contrast, all of our COVID-19 cases had antibodies against SARS-CoV-2 epitopes in both the plasma and in CSF.

Moreover, we found that the antibody profile was different in the CSF when compared to the plasma.  So for example, focusing here on the first patient, COVID-1, this patient, as you can see, in the plasma has antibodies against total spike protein, the receptor binding domain, but none against nucleocapsid protein, whereas in the CSF this patient had antibodies against spike, RBD, as well as the nucleocapsid.

When we look at the other patients in the cohort as well, we see different antibody profiles in the plasma and in the CSF for the same patient.  These results suggest that antiviral antibodies in the CSF do not simply reflect passive transfer of antibodies from the periphery, but rather, that antibodies in the CSF may be being produced locally in response to a local CNS present viral antigen.

But how does this square away with the finding that CSF rarely contains viral nucleic acid, which Dr. Gisslen has shown you already?  To get at this question, we worked with Eric Song, a wonderful MD/PhD student in Dr. Akiko Iwasaki's lab, who developed a mouse model of COVID-19.  In this model, the human H2 receptor is expressed in either the lung alone, the brain alone, or the lung and the brain, and thus a targeted infection can be delivered to either organ alone or to both of them, and you can see here the mouse that has infection only in the lung, a mouse that's infected in both the lung and the brain, and a mouse that's been infected only in the brain.

When we focus on the mouse that has infection of both the brain and the lung and measure viral RNA by PCR, we find detectable virus in the brain on day 3 and day 7 post-infection and in the lung, as expected.  Surprisingly, though, despite robust infection of the brain, we cannot detect virus by PCR in the CSF.  This suggests that CSF viremia may be a poor indicator of brain infection.

Moreover, when we measure anti-spike antibodies in the various compartments, we find that only mice with productive brain infection have detectable antiviral antibodies in the CSF.  Mice who are infected only in the lungs do not have CSF antibodies.

One of our most striking findings from the single cell RNA seq was that CSF from COVID-19 patients contained a higher frequency of B cells when compared to the CSF from controls, which typically contains very few B cells.  We wondered whether these expanded B cell populations were producing antibodies targeting the virus or whether they were producing autoreactive antibodies.  To directly address this question, we cloned individual monoclonal antibodies from clonally expanded B cell receptor sequences in the CSF and the blood.

Shown here are the frequencies of the top five B cell receptor clones in the blood and in the CSF of our COVID-19 participant who had the highest frequency of CSF B cells.  Of note, there was no overlap between the top sequences found in the blood and in the CSF.  We used these sequences from these B cell receptors to make monoclonal antibodies.

So to start, using the same multiplex ELISA panel I showed you before, we found that one of the CSF-derived monoclonal antibodies and two of the peripheral blood derived monoclonal antibodies targeted SARS-CoV-2 spike protein.  None of the other monoclonal antibodies recognized other SARS-CoV-2 antigens on the Luminex panel.

But what were these B cell clones targeting?  Clearly these were not antiviral.  Given reports of new onset humoral autoimmunity in COVID-19, we wondered whether some of the CSF-derived monoclonal antibodies might be autoreactive against neural tissue.  We worked with Chris Bartley and Sam Pleasure to test all the monoclonal antibodies we had created for anti-neural immunoreactivity by anatomic mouse brain tissue staining.

In this approach, the monoclonal antibody is incubated with rodent brain section and counterstained with the fluorescent antibody.  We found that four out of the five CSF-derived monoclonal antibodies showed some degree of anti-neural immunoreactivity, and I'm showing you some of the examples of the immunostaining here.

This includes monoclonal antibody C2, which as you'll recall is the CSF-derived monoclonal antibody that we had previously demonstrated was anti-spike.  This suggests cross-reactivity between an antibody that is both antiviral and also appears to be targeting brain tissue.  Given these findings of monoclonal antibodies derived from the CSF that seem to be exhibiting anti-neural activity, we went back and tested whole CSF from our COVID-19 cases to see if the other COVID-19 cases might also harbor autoantibodies in the CSF.

We found that five out of seven cases we tested were immunoreactive to mouse brain tissue when compared to controls, in which two out of six were reactive.  Moreover, the COVID-19 CSF was immunoreactive at higher titers than controls.

So to conclude, CSF studies suggest that immune-mediated processes rather than or in addition to simple neuroinvasion may potentially contribute to neurological sequelae of COVID-19.  We found that cytokine levels and T cell transcriptomes show a compartmentalized cellular immune response in the CNS.  We found that all patients we tested had antiviral antibodies in the CSF, and that most of our patients also had autoimmune anti-neural antibodies in the CSF.

With that, I want to thank the many people who helped contribute to this work and especially those who I've shown you their pictures and highlighted their names here.  This was really a collaborative effort, and I'm happy to take any questions.  Thank you so much.

DR. HAMMOUD: Good morning.  My name is Dima Hammoud.  I'm a senior investigator at the Center for Infectious Disease Imaging at the NIH.  Today I'll be talking about neuroimaging and COVID-19.

I'll start by talking a little bit about the acute and subacute imaging findings which were mostly described in the early days of the pandemic, mostly in association with severely infected COVID-19 patients.  Then I'll talk a little bit more in details about the subacute and chronic imaging findings that we are seeing now, and that seem to reflect abnormalities in survivors and long haulers.

So as I said, in the early days of the pandemic, we started seeing lots of reports about vascular complications in COVID-19 patients.  The most common ones were strokes.  They were more commonly to be arterial, more likely than venous, and more commonly seen in the posterior circulation like in this patient of ours who presented with right PCA infarct.  Most of those, however, were seen in patients who were already predisposed to stroke by other cardiovascular disease risk factors, and they were very sick, and obviously the addition of coagulopathy and inflammatory pathways in the setting of DIC or cytokine storm did not improve their chances.

Hemorrhagic changes were also reported a lot.  They were either extra-axial or sometimes massive intraparenchymal hematomas, but again, they were relatively nonspecific, seen mostly in severely affected patients, and commonly associated with DIC and coagulopathy and sometimes predisposing factors like trauma or being on anticoagulants.

A slightly more unusual pattern of hemorrhage was described by a few groups.  Mostly these microhemorrhagic changes seen at the white gray matter junction, internal capsules, and middle cerebellar peduncles and corpus collosum, and they were associated with severe, mostly in patients in the ICU.

But again, if you go back and check the literature, you find that similar findings have been already described in other patients with severe illness, they were referred to as critical-illness microbleeds, mostly in patients who were in the ICU for sepsis or high-altitude exposure.  The etiology is unknown, but it is assumed to be a combination of hypoxemia, micro-angiopathy, and potentially DIC and thrombosed vessels.

Another manifestation is encephalitis and encephalopathy.  This is the first case of encephalopathy described in a patient from Michigan, a case of acute hemorrhagic necrotizing encephalopathy, mostly involving the thalami and medial temporal lobes.  But again, nonspecific, as had been already described, especially in children with influenza and other viral infections, and this is likely to be due to the cytokine storm associated with COVID-19 rather than the virus itself.

Another entity is leukoencephalopathy with reduced diffusivity.  These are mostly areas of restricted diffusion, confluent involving mostly the white matter of the cerebral hemispheres, as well as the middle cerebellar peduncles, but also similar to previously described reports of delayed post-hypoxic encephalopathy, for example.  Again, a manifestation of severe disease and ICU patients.

There were a few reports of leptomeningeal enhancement in a series of 58 patients, approximately 40 percent had focal leptomeningeal enhancement, mostly seen in post-contrast FLAIR imaging.  These have been described in other entities like in HIV, for example, MS, and other neuroinflammatory conditions, and recently a paper by Lewis and colleagues found out that even though those patients are more likely to have the virus in the CSF, most of them do not.  So it's more likely to be a manifestation of disease rather than actual involvement of the virus.

So now about the more relevant or the long-term effects of COVID-19 and what this imaging is telling us about those.  There are quite a few papers.  This one was published in early 2021.  They evaluated 51 patients, 32 of whom had had severe disease.  Although none of the patients had neurologic manifestations in the acute stage and none of them had any evidence of structural abnormalities on MRI, when they looked carefully at volumetric measures, perfusion measures, and diffusion and DTI measures, they found that there were significant differences especially in patients who had severe disease.  For example, one of the findings is decreased cortical thickness in different locations in the brain, mainly the left insula, as you can see here, but also left hippocampus and superior temporal gyrus, suggesting a chronic effect on the brain, and also supported by the fact that most of these changes correlated significantly with inflammatory markers like CRP, procalcitonin, and interleukin 6.

So it's possible that all of these residual findings we are seeing in survivors and long haulers are due to the acute exposure to inflammatory changes in the periphery.

A more recent paper which is not peer reviewed yet, but it's a very promising report, included a -- it's from the UK biobank, and it was a large multimodal brain imaging study.  The interesting thing about it is that it had a longitudinal setting.  So participants had already been scanned before they contracted the virus, and then they were invited again to be imaged after they had contracted the virus and recovered.

They had large numbers, approximately 400 patients and 400 controls, all of whom were scanned twice and all of whom were matched for age, sex, ethnicity, and time between the two scans.

In this study, the investigators used two approaches.  The first one was hypothesis-driven analysis.  They concentrated on regions which they believed would be involved including the primary or secondary cortical gustatory and olfactory areas, and they found that on the left side of the brain, there was actually an effect in COVID-19 patients.  There was a longitudinal loss of volume and thickness in various locations like the parahippocampal gyrus and orbitofrontal cortex, and these findings were further supported by a more exploratory approach where the left parahippocampal gyrus and lateral orbitofrontal cortex, both of those locations, the thickness of the gray matter was significantly decreased in patients who had COVID compared to those who didn't.

An interesting finding in this paper, however, is this ratio of brain volume to estimated total intercranial volume.  So in other words, it's how much brain there is inside the skull.  There seems to be a loss of volume in COVID-19 patients which was not seen in the controls, and that's interesting, because it could reflect a generalized neurodegenerative process and neuronal loss that goes beyond the limbic system and olfactory system.  So we have to wait for the final report to better understand what that means.

There were quite a few FDG studies as well, and this one, 15 subjects were evaluated, almost 30 days after they were infected.  Those were sick patients.  They had at least one neurologic symptom each, and the imaging showed predominant frontoparietal hypometabolism, when they compared it to 45 controls, which correlated highly with cognitive assessment.

Interestingly, more recently, the same group showed updated data on their cohort.  This was presented at the Society of Nuclear Medicine meeting in June, where they reevaluated eight subjects six months later, and they found that they did improve.  So these areas here show hypermetabolism compared to their own baselines.  But then when they compared them again to controls, there were still foci of hypometabolism, suggesting that residual deficits were still measurable.

A similar study was performed by another group where patients were evaluated acutely, one month later, and then six months later, there was a definite improvement of the hypometabolism, which was described at the subacute stage.  So all of this supports a theory of improved functional outcome or improved hypometabolic abnormalities in those patients, whether they were reversed completely or not, it's unclear. 

But not all of the studies had consistent results.  This one was -- they included 35 patients with persistent functional complaints and they were all imaged approximately a month to maybe five months after infection, and there was also FDG hypometabolism involving bilateral rectus/orbital gyri bilaterally, including olfactory gyrus, so that's interesting.

But there was also involvement of the right temporal lobe, including the amygdala and hippocampus, the right thalamus, and in this specific study, there were abnormalities seen in the midbrain and in the cerebellum.  And all of these correlated significantly with numerous functional complaints, as well as hyposmia, anosmia, and a variety of other symptomatology.

So maybe not super consistent, but all pointing towards a certain amount of damage that might or might not be reversible.  So what's the future of neuroimaging in COVID-19?  We will probably be seeing largescale studies, mostly MR imaging studies, but also probably PET imaging studies that will better demonstrate whether there's permanent damage and neurodegeneration or not.  We will likely get more information from diffusion, perfusion, and functional MRI images as well.

Another potential thing is the evaluation of animal models of COVID-19.  We recently looked at an animal model of Ebola virus, and we found that there was a disruption of the blood/brain barrier and increased fluid and contrast accumulation in the brains of those animals after inoculation, which was consistent with immunofluorescence showing albumin leakage in the brains of the euthanized animals.

So we believe this reflects blood/brain barrier disruption likely to due to the infection as well as peripheral inflammation.  So considering a cytokine storm occurs also in COVID-19, the question is there a similar process happening in COVID-19 patients which could account for blood/brain barrier disruption and neuronal damage?  We won't be able to find out unless we have a validated COVID-19 model of the infection.

With that, I would like to conclude and thank you for your attention.

DR. NAIR: Hello, my name is Govind Nair.  I am a staff scientist and direct the Quantitative Imaging Core Facility at the NINDS.

I want to thank the organizers, especially Jeymohan Joseph and Avindra Nath for inviting me here to talk on postmortem MRI in COVID-19.  I also want to thank Avi for getting me involved in this research in the first place.  I want to point out that some of the results shown in this presentation have been reported earlier this year in the NEJM paper.

I have no financial interests or relationships to disclose regarding this. 

Before I show you the data from COVID-19 brains, I want to briefly describe the need for postmortem imaging.  Traditionally, brain is sectioned into smaller slabs and blocks at the time of autopsy to look for gross abnormalities.  Histological studies is performed on these regions and also on standard blocks.  However, when there's little a priori information on the site of pathology, such as in the case of brain metastasis or in COVID-19, then this process becomes increasingly inefficient.

This is especially true if one is looking for tiny foci of pathological changes.  Postmortem imaging using MRI offers a window into the pathological changes in the brain before the brain is sectioned.  In this slide, you can see the whole brain, which has been fixed in formalin for over two weeks, being set up in a container for MRI.  The container fits inside MRI and the brain is typically imaged for about 48 hours.  Images with various contrast can be acquired at about 150-to-300-micron isotropic resolution using this method.

Of course the postmortem imaging can also be performed on brain sections that has already been cut, and these slabs are imaged at 100-micron isotropic resolution or better.  Here you can see a movie of postmortem images from the brain of a patient who was clinically diagnosed with multiple sclerosis.  As the movie plays, you may be able to identify MS lesions in the white matter and maybe even a few in the gray matter.  The point here is of course that one knows the pathology and its location before the brain is cut and of course the smaller the lesion, the more useful this technique becomes.

Another advantage of doing MRI is that it is more sensitive to picking up pathological changes than visual observation.  In this study, it was reported that more than 40 percent of the MS lesions visible on the MRI were neither visible on gross examination, nor palpable on the surface of the slab.  Recently we published postmortem MRI performed at cellular resolution.  These images are from a piece of normal-appearing cortex.  MRI is on the left, shown next to DAB Turnbull, and LFB-PAS staining on the middle and right.  While the best resolution from MRI remains at least an order of magnitude lower than that from basic microscopy, we are still able to identify individual iron-laden oligodendrocytes and myelin sheaths around axons.

But the idea that postmortem scans can be helpful for understanding pathology is almost as old as MRI itself.  Here is a letter to the editor of Lancet written in 1984 stating that the best way to correlate clinical changes with MRI was to be able to perform MRI immediately before and after death and after fixation and correlate with histology.  However, in the middle of the COVID-19 pandemic last year, all this was not possible.  We performed our experiments on fixed tissue that was shipped to us by our collaborators and sometimes we did not even have complete clinical information.

This is a partial list of patients who donated their brain for the study postmortem.  As you can see, majority of the donors were male and elderly.  Most of the donors had past medical history of diabetes and hypertension.  Post-COVID, most patients had respiratory symptoms including four who had ARDS.  These are images from a patient who had reperfusion injury, which is clearly visible on the brain slab.  We extracted a small piece of tissue surrounding the bleed and MRI performed at 100-micron isotropic resolution is shown in the movie.

One can clearly appreciate penetrating vessels that run to and from the PL surface.  A closer look at the image on the extreme right shows two kinds of signal from these results.  One highlighted with yellow shows that dark or hypointense vessels, indicating presence of iron, probably from hemoglobin.  But there are vessels such as the one highlighted in red which shows brighter hyperintense signal within it.

The bright signal seems to be more prevalent near the site of the bleed, and make or respond with thrombosis within the vessel.  Bright and dark vessels were also seen in other brains that we imaged, including the ones that did not have any evidence of reperfusion injury.  Here is an example from the basal ganglia.  We were able to closely match the MRI in the center with IHC for fibrinogen on the right, bright vessels seems to correspond with vascular condition.

Fibrinogen stain also shows leakage in the vicinity of the congested vessels, with some evidence of thrombosis as seen in this CD61 IHC.

Here is another example of vascular changes seen in COVID-19.  In this case, fibrinogen leakage seems to correspond to regions of signal intensity changes in the MRI, albeit the presence of iron in substantia nigra dominates the MRI signal changes.  Vascular thrombosis is also more extensively appreciated in this section, and as well as on the MRI. 

Our initial hypothesis was to look for signal changes in the parenchyma of the olfactory bulb and in the respiratory centers located in the brainstem.  We know that clinically these are the functions that are most affected in COVID-19.

This is a coronal section through the olfactory bulb on its side.  MRI was performed at 25-micron isotropic resolution.  One can appreciate signal changes in the MRI which mostly correlated with fibrinogen leakage as well as collagen deposition in the blood vessels, indicating the thinning of basal lamina of the endothelial cells.  There's also evidence of increased microglial activation in this region, as indicated by the IBA1 staining.

In the brainstem, we were able to appreciate several regions of punctate hypointensities possibly indicating microbleeds.  You can see this in the 3D multiplanar reformat images on the left and it's highlighted by arrows in images from three different donors.  The signal is different from the linear hypointensities described earlier, as they are punctate.  But they often lie adjacent to the linear signal, making them difficult to spot.  Nevertheless, they can be visualized using multiplanar reformatting.  You can see a few of them clustered around in the brainstem in the bottom right image.

Finally, we were also able to see signal intensity changes in the parenchyma of the brainstem, separate from those seen within blood vessels.  The top series of images you can see signal changes in the brainstem corresponded well with regions of collagen deposition, as well as fibrinogen leakage.  It is possible that these are early demyelinative changes in these regions.

Further studies are being performed to better understand all the MRI signal changes in this tissue from the COVID patients.  Some of these congested vessels as well as microbleeds are visible in the cerebellum, although the microbleeds in this case need to be more carefully studied.

In the interests of time, I will only show one comparative study.  This one is from UBL in Brussels.  They performed in situ imaging within a day of death in 19 subjects and found that much of the same pathological changes as we saw in the ex vivo study.  This includes diffuse hyperintensities, increased visualization of blood vessels, microhemorrhages, and even subcortical hemorrhagic lesions.  They also found some significant changes in and around olfactory bulb in the subject.

So in summary, we were able to identify several features on postmortem MRI from COVID donors.  There were hyperintensities in the blood vessels indicating a vascular congestion, and also we were able to identify thrombosis in several of these vessels.  They were punctate hypointensities, which were indicative of microbleeds.  There are some indications of MRI signal changes in regions of increased blood/brain barrier permeability as indicated by the fibrinogen IHC.  This was seen in olfactory bulb as well as in the brainstem regions.

It is also to be noted that MRI contrast is sensitive to far fewer items and include only things like iron and myelin.  So depending upon the downstream effects of this fibrinogen leakage, its effect on MRI signal might be different.

In addition, most of these cases were acute COVID infections, where this is duration of merely a few days to a couple of weeks.  This timeframe may not be sufficient to induce demyelinating changes in the brain.  However, these observations here can be applied to design pulse sequences for in vivo studies.  However, there are also several limitations to the studies.  The major one being that there were no appropriate control tissue.  Even pre-COVID imaging in some of these subjects would have provided a lot of information on the specificity of these changes to SARS-CoV-2 infection.

I want to stop here and thank my collaborators, especially Myoung Hwa Lee and Steve Dodd, who helped with all the MRIs.  I also want to thank Alan Koretsky and Avi Nath, all the imaging was performed on 11.7 Tesla magnet in the LFMI, but we also used the other scanners in the other core facilities for correcting preliminary data.

I want to thank Marco Hefti and our extramural collaborators for helping out with acquiring the tissue in the middle of the pandemic, and of course I want to thank all the organ and tissue donors and their next of kin for making this research possible.  Thank you for your attention, and I'm happy to take questions on the online session.

DR. GLATZEL: So, my name is Markus Glatzel.  I am from Hamburg, Germany, and I will present you some data on neuropathology of COVID-19 and hoping that this helps to clear this disease.

Okay.  So the whole meeting is about psychiatric and neurological features of COVID-19, which I will call neuro-COVID-19 now.  I think that neuropathology can make a big contribution to solving the riddle of COVID-19, and we are asking ourselves if there is a neuropathological correlate to neuro-COVID-19.  We also are investigating if we can find the virus in the CNS and, if yes, how the virus gains access to the CNS, and of course, ultimately we would like to know how the virus leads to neuro-COVID-19.

So the first question: is there a neuropathological correlate for neuro-COVID-19, and what we did here in Germany actually in Hamburg, we were autopsying COVID-19 patients very early on, actually in the first wave of the pandemic.  We autopsied every single individual at least in our state.  This put us in a situation where pretty early on we had enough material to look in this in detail, and now we've looked at over more patient brains from patients dying from COVID-19 in a standardized fashion where we look at least six different brain regions using markers of glial response, but also markers for neuroinflammation and markers giving us an idea about the immune reaction in the brain. 

This has put us into a situation where I think we can now talk about the neuropathology of COVID-19, and if you ask me the nutshell what is the neuropathology of COVID-19, I will tell you it's a perivascular glial neuroinflammatory response that leads to activation of microglia and infiltration of T lymphocytes, and it's most pronounced in the brainstem and in the cerebellum.

We don't see obvious neuronal damage in these brains, but we see indirect evidence for axonal damage.  For example, if we look at APP accumulation in axons, we can see that we have much more accumulation in axons in COVID-19 brains than we see in the control patients.

Of course, we are asking ourselves is what we're seeing, is it above what we're seeing in controls?  Is it something specific?  For this, we look at COVID-19 here in blue, but also at age and sex matched control patients and, importantly, also at patients that are under invasive ventilation, these ECMO patients here.

If we compare this to a classical paradigm disease of neuroinflammation, multiple sclerosis, and what we see is that both in the presence of activated microglia cells but also presence of macrophages, presence of diverse T cells, we can see more of these in COVID-19 than in the controls, also in the invasive treatment controls, but of course, it's less than we see in this paradigm disease of neuroinflammation multiple sclerosis.

So I think we can answer the question if there's a correlate in neuropathology for neuro-COVID-19 for COVID-19 with a definite yes.  There is a correlate for this.  It's something we see.  It seems to be specific.

Next question is, of course, can we find the virus in the brain?  We checked this also pretty early on, and we and also other groups researchers have found that, yes, in the brain you can find virus both using QPCR but also using immune staining and antibodies to viral proteins, but it's a very low level.  It's much, much lower than we see in the lungs or pharynx and it's even lower than what we see in kidneys, for example.  So it's about 50 percent of patients and it's very, very low level.

Where can we see it?  Well, if we look at the viral proteins, and if we see it, you can see it for example around vessels and around endothelia, and here this brown signal here, this is actually viral proteins stained in a vessel in the brain.  Very rarely we can also see virus in the parenchyma, and here actually what we see is in green viral proteins, in I believe it's an activated microglia cell, in the brain parenchyma actually, this was in the brainstem.

So I think, yes, we can also find the virus in the brain, and the question how virus gets there would be a lecture on its own, so I will not tell you anything about this.  We've looked in this also in some detail.  I'll just give you my take on this.  So I think the blood/brain barrier crossing is probably the most important route the virus takes to get into the brain.

But the real big question is how does the virus lead to symptoms of neurology but also psychiatry, and of course there can be directly virus-induced, or there could also be indirectly an immune-mediated.  In order to answer this question, let me just go back a little bit in history, also go back a little bit in how we looked at these patients, to patient autopsies that we performed very early on.  It's a 29-year-old previously healthy male that died with a fulminant course of COVID-19.  So this individual was completely healthy before he contracted the virus, and he had a pretty rapid disease onset, only two or three days of symptoms and then he died.  He did not undergo any treatment.  He died in his bed.  So he didn't die in the hospital.

At autopsy, of course, we saw these typical lung changes, but you see here when I say neurovascular pronounced glial activation and neurovascular pronounced microglial activation, and this is what prompted us to look in this in more detail.  We did this -- actually it's done by Susanne Krasemann and the (indiscernible) Institute -- by spatial transcriptomics where we looked specifically at the neurovascular niche.  So we only looked at around 20 to 30 cells, which make up the neurovascular niche.  So this is blood/brain barrier cells, but also some immune cells which are around the niche, and what we saw is we saw that compared to controls again we saw that there's induction of interferon-gamma-mediated signaling pathways, but also an induction of antigen processing and presentation pathways.  So this gives us the first hint onto what is going on at least in the perivascular niche.

We also saw early on is that in COVID-19, we can see string vessels, and string vessels are certain type of capillary remnants.  So when capillaries die, the inner band dies out first and the connective tissue strand stays longer, and this actually what string vessels are.  We know them from Alzheimer disease, and this is how they look like.  They are this kind of shrunken endothelia.  We saw these also in COVID-19, and we wondered what is going on?  How can this be?

For this, Markus Schwaninger is the person that is a researcher in Lubeck here in Germany.  He is a big expert in string vessels, and he checked this in COVID-19 patients and in controls, and if you quantify actually, really you can see much more of these string vessels occurring in patients dying from COVID-19.  He also in another collaboration checked this also in mouse and hamster models of the disease, and yes, also in these models in mid-phase of the disease, you can see much more of these string vessels occurring.

Of course, the question is how can this be?  What is underlying mechanism here?  This is something that Markus Schwaninger was lucky, or he is a big expert in NEMO, and he has shown that if you delete NEMO in the brain, in brain endothelia, you get string vessel formation, and independent studies looking at targets of the main protease of SARS-CoV-2, this NSP5, have found that NEMO as in other coronaviruses is a target of this main protease. 

So this put together, we looked at this specifically.  So we looked does NEMO, does the main SARS-CoV-2 protease cut NEMO?  And yes, it does.  It does so in endothelial cells from mice, endothelial cells from humans, but also in a recombinant approach you see ascending doses of this main protease lead to like cutting of NEMO.

So in the end, I think we have a first hint on what could be going on in the brain.  So we think that there are direct virus-induced effects really at the endothelia via the SARS-CoV-2 main protease and via its target NEMO, and then there are also indirect effects, which are immune mediated, maybe caused by the vascular damage, but also maybe caused by other mechanisms which I showed you before in the spatial transcriptomics become active.

So this is now the end of my presentation.  I thank you for listening to me, and of course, this has only been possible by a big collaboration and in Germany, we are very lucky because we have fused forces in this national autopsy network where now all autopsies are looked at together, we can act fast, and we can also get big enough cohorts for high enough powered studies.

Thank you again for your attention, and I will take questions now.

DR. FOLKERTH: Hi, my name is Rebecca Folkerth.  I'm a neuropathologist at the Office of the Chief Medical Examiner in New York City.

Coronavirus has been with us for a long time, and the current pandemic is similar in some ways to the previous epidemics of SARS and MERS, which not only had respiratory illness, but also neurologic symptoms as listed here.  This has been thought to be related to the neurotropism of the virus, directly infiltrating the olfactory neuroepithelium as well as the ability to cross the blood/brain barrier.

But I would like to bring up the possibility that sudden death may represent a neurologic symptom, and that is because from our point of view at the New York Office of the Chief Medical Examiner, we saw a huge rise in the volume of calls to our office related to sudden unexpected deaths in patient homes or in the streets, and this was much higher than expected for our normal rate of deaths in 2019 figure below.  If we look at exactly one year after the initiation of the crisis, we certified more than 30,000 deaths due to COVID just in New York City, and you can see again the peaks here in the pandemic and then the more recent hump that we've just been through.

So this meant for us quite a huge task to collect decedents from either hospitals where they died or out in the field in their homes, and for this we had to use force multipliers, contract vendors, and funeral homes and refrigerated trailers, both at our facilities and at hospitals for their overflowing morgues.  We had to construct a whole unit in Brooklyn, in the Navy yard, and this is still in use.  We had to staff it with our personnel to track and store all of the decedents.

So of course this is why sudden death to me is part of something I would like to put forward as a symptom of COVID-19.  We did do 27 autopsies on individuals with COVID.  Eight had influenza-like illnesses, 15 did not, 4 had influenza-like illness symptoms but recovered, and then had sudden collapse.  I've highlighted in blue here all of the individuals who were either found dead, had a witnessed collapse or were found down, and two of them had agitated delirium suggesting again possible neurologic syndromes that we couldn't otherwise be aware of.

The presence of lung injury was noted in 67 percent of our cases.  We had subtle brain inflammation in 65 percent and acute neuronal ischemia in half of our cases.  Now you'll notice that this is a different number.  We're still analyzing with specialized imaging and so on some of these other cases.  I'll show you the neurovascular injury that we were able to detect.  Notably, we did not have many of the other medical illnesses that were described in the hospitalized cohorts.

This is an example of the perivascular leptomeningeal inflammation.  Leptomeningeal here, perivascular here.  These were mostly T cells.

We did have encephalitis with neuronophagia and microglial nodules seen here, occasional foci with neuronal loss here shown in the dorsal motor nucleus of the vagus.  I'd like to point out to you involvement of the insula which, as you know, is a central autonomic control site, sometimes referred to as the cardunculus, again suggesting that this may play a role in sudden death.

We did have focal vascular pathology, including fibrinoid necrosis of small vessels.  Almost demyelinative but not quite, and then a basal arteritis here in a cerebral artery without any leptomeningeal inflammation around it.

These are the cellular reactions, representative reactions that we saw.  These were published in our letter to the New England Journal at the end of last year.  This was work in part done in the laboratory of Dr. Nath.

What we found and that I'd like to focus for a minute on is in 7T scans of tissue sections, fixed tissue sections from 16 New York cases and three University of Iowa cases from Marco Hefti, is the finding of abnormal signal and fibrinogen outside of vascular profiles in the tissue pretty convincing for vascular leakage in a good number of our cases.  Similar changes were noted in an in vivo case reported by the Mass General group in Boston, and these corresponded in an individual who went on to die with petechiae and micronecroses.

Other reports showed sparse neuroinflammation.  This is a report by Isaac Solomon and colleagues from Brigham and Women's Hospital again showing similar to what we saw, a few microglial nodules, inflammation, negative immunohistochemistry for SARS-CoV-2, sparse copy numbers on quantitative PCR in a couple of sites, and I put arrows on the medulla again, because I find that interesting.  The German group also showed very similar findings in six cases, all of whom were hospitalized, but I do want to highlight that they found changes in some of these same brainstem nuclei, and they did not see infarcts in the cases.

This is a nice single case by the Mayo group of a hospitalized individual who developed petechiae and ADEM-like lesions with demyelination without axonal injury and then another area of gray matter, they found a microinfarct. 

The largest series by far is that of the Mt. Sinai group in which 58 hospitalized patients came to autopsy and they found in a third infarcts as you see here with microthrombi and ischemia and also ADEM-like lesions.  They also demonstrated ACE2 vascular expression by immunohistochemistry. 

So these findings have all led to what is, I think, probably the most current model suggesting that COVID induces vascular injury with changes in the endothelium, increases in permeability, prothrombotic and proinflammatory consequences.

Now I quickly want to show you some changes in a small cohort of children that we looked at, and I won't go through all of the details here.  These have been published.  There hasn't been much published in the way of pathology.  This is showing the one case with hypersensitivity myocarditis.

In our children, we had four cases, and some of them at least had markedly elevated proinflammatory markers.  One of them had a coinfection with adenovirus.  Two of these cases died quickly and were not resuscitated, and two underwent hospital treatment with ECMO, one of them had an infarct related to ECMO, the others showed inflammation similar to what we saw in the adults.

This is one of our pediatric cases who underwent postmortem specimen scanning of the brain.  Here you see cloudy leptomeninges, a similar hyperintense vascular lesion here, which corresponded to this very vessel with inflammation and vacuolation around it, microglial nodule in the brainstem, and microinfarct here.

Also seen in two of our four children was a very interesting finding of suggesting autonomic nervous system involvement.  This is a myenteric ganglion with necrotic neurons and inflammation, a periadrenal sympathetic ganglion, also with similar changes, and occasionally we saw fibrinoid vessel and endothelial prominence in peripheral autonomic nerves.

So I'd like to summarize and leave you with some food for thought.  The extent and nature of the neuropathology is variable and subtle.  Neuroinflammation is sparse, but it's seen in key autonomic sites, central and peripheral.  Could be a substrate for sudden death.  The neurovascular effects are disproportionate to the degree of inflammation, and infrequently associated with virus, at least in the studies that I've shown you so far.  Arteritis is rare, but given that these are relatively small cohorts, they could be significant.

Secondary effects on the nervous system are a consequence particularly in hospitalized patients, and we need to be alert to the possible long-term consequences, the so-called PASC, and that these may represent a continued risk for sudden death even after recovery.

I'd like to acknowledge the individuals at my agency, my collaborators in these various sites, and I'd like to thank you for your attention.

DR. SPUDICH: So thank you to the speakers for a series of just absolutely fabulous talks, and I think that actually although there was some diversity in what was presented, there's a lot of intersecting topics in this group of talks.  So I'd like to ask Drs. Gisslen, Farhadian, Hammoud, Nair, Glatzel, and Folkerth to please turn on your cameras and thank you.  I think you've all done that.

We are going to launch into a 15-minute Q&A session.  This is going to take us 5 minutes into the planned break, but I think the Q&A will be really important and interesting for this group of talks.  So we really appreciate everyone's time.

I'm going to start out actually with a question to Dr. Farhadian which is a follow-up from what Dr. Frontera asked in the previous session, and she's articulated her question.  So I'll read it.  In regards to autoantibodies -- and she's referring to a specific paper in which you were involved that came out of Yale -- is whether the group has looked at genetic polymorphism for some of the inflammatory receptors, and I think maybe Dr. Farhadian, could you comment on what that paper showed in terms of autoantibodies, cytokines, and immune markers?

DR. FARHADIAN: Sure.  So thank you so much, Dr. Frontera, for the question.  The paper that you're referring to, which was a study that was led by Dr. Ring and Dr. Iwasaki at Yale, screened hundreds of hospitalized patients with COVID-19 for a diverse set of autoantibodies.  So it wasn't specific to neuronal targets, and compared them to uninfected healthcare workers and found a high proportion of hospitalized patients contained autoantibodies in their serum by a new methodology that was developed for this exact purpose.

So I think your question is really interesting, because you're asking about the hosts themselves and are there host factors and genetic polymorphisms, say, as you mentioned, inflammatory receptors or otherwise, it's a really interesting angle to COVID research.  Obviously the COVID human genome studies have been focusing on that, and the groups at Rockefeller and elsewhere.

We haven't specifically looked at that with regards to neurological outcomes, into the presence of antibodies to neuronal tissue, I'm not aware of any group that has, but I think it's an important question because it really focuses on underlying host susceptibility, which kind of -- sorry, Serena, I don't want to jump the gun, but I think it is maybe a little bit connected to one of the other questions that came up in the chat box.  Someone was asking about the diverse, the heterogeneity and clinical and pathological manifestations, any thoughts on what could be contributing or driving to this.  I don't know the answers.  Certainly there are many contributors, but one of course might be host susceptibility perhaps having to do with genetic polymorphism.

DR. SPUDICH: Wonderful.  Thanks so much, Dr. Farhadian, and I think I actually was going to also ask a couple of the other speakers to weigh in on this particular question.  So the question, again, is there's considerable heterogeneity in clinical and pathological manifestations of neuro-COVID.  I think COVID in general, but particularly for neuro-COVID, are there any thoughts on what could be contributing to or driving this heterogeneity?  I was wondering if Dr. Gisslen might want to comment on whether there were -- since he's looked at some large groups of patients and specimens, were there markers that seemed more related to certain kinds of clinical outcomes and is there any clue as to what could be potentially underlying some of this heterogeneity?

DR. GISSLEN: Thank you.  These are great questions that I don't really have no clear answers to.  One thing that we have found is that when it comes to acute phase, looking at the immune activation, it looks like a lot of -- it doesn't look like there is a clear correlation with clinical symptoms, neuro symptoms.  We see a huge immune activation also in this without -- in the CNS, without -- or in the CSF, without CNS symptoms.  Though we're looking at neuro markers, for example, for astroglial markers or for neuronal markers, there is probably at least a correlation to acute phase.  But we haven't looked at sort of host factors that I think is probably very important and not only for COVID, that might also be the case in other viral infection, other infection.

For example, how does it look like in influenza, with influenza encephalitis, for example?  So it's a lot of work to be done there, I think.

DR. SPUDICH: I am wondering if actually any of the pathologists have any insight into this question, as well.  It seems that we've seen a variety of different kinds of pathology described, including using MRI on the autopsy tissue, and I am not sure whether there's any knowledge of how any of the pathological findings have been associated with any of the clinical manifestations.  Many of these obviously were severely ill COVID patients, which is why we're looking at autopsy specimens, but I wonder if either Dr. Folkerth or Dr. Glatzel have any comment on that association.  It looks like Dr. Glatzel is raising his hand.  So do you want to try to answer that?

DR. GLATZEL: I think the thing is that the people that die are usually older individuals, and they have all disease histories, no?  We see a lot of patients -- I have looked at more than 100 brains now, and medium age is 75 or something.  Of course, one third of them have Alzheimer disease changes, and this of course also influences -- they also have an ongoing inflammation in the brain.  Then another 50 percent have Parkinson's disease, and all of these things which preexist influence how COVID presents in the brain, I think.

So I think in the future, we probably have to do, have to look at subgroups, you know, in larger cohorts.  This is at least my take.

DR. SPUDICH: It seems with the autopsy studies, that's a real challenge, because to get many, many autopsy specimens is something that is --

DR. GLATZEL: Then you have to pool.  You have to collaborate.  Then it's possible.

DR. SPUDICH: Right, and you made the point that in Germany that's actually sort of intrinsic in the system there, which is fantastic.

DR. GLATZEL: We just got like a -- it's a grant that brings together all autopsy centers and they pool the samples.  That's it.  It's a small grant only, but yeah.

DR. SPUDICH: Well, that is an important thing that hopefully we can be talking about some more tomorrow when we're talking to the institutes here at the NIH.

Maybe I'll move on to another postmortem type of question which was I think directed specifically at Dr. Folkerth, which was you mentioned that there were some of the brain specimens that you were looking at were individuals in a sudden death kind of cohort, and the question is, I think, the interest was it in previously hospitalized patients, and here the question said, or in long COVID patients with a more mild form of acute COVID?  I kind of remember that it's neither, but can you describe exactly what those patients were like?

DR. FOLKERTH: Thank you.  That is a very good question and I think key to what I was hoping to present in my talk, which is that the cohort of hospitalized patients and their neuropathology is quite different from those who die suddenly.  Our cohort for the most part was individuals who died at home or in other unwitnessed settings, including in the subway and on the street.  So what's interesting about that is we don't have complications of therapy overlaid on what we see.  The bad thing is we don't have very good understanding of what their neurologic complications or symptoms were.  We only know in some of them that they had a couple days of cough and fever and so on, and that is what caused us to actually do the swabs on them.

Unfortunately, because of the political situation at the time in New York state and the difficulty in getting swabs from the federal government, we were not even swabbing individuals who were coming into our office, and I told you we had thousands of people, decedents coming in.  So we don't know what the real denominator is, and we weren't able to do autopsies on every case like Dr. Glatzel and I totally agree, the best way to do that is to try to pool resources.

We, instead, had to only look at cases for which we had to do an autopsy to certify the death, and that included violent deaths, suicides, homicides, things like that.  So it's a unique population.  Or ones in whom we didn't have prior medical illness that would allow us to certify the death, and those were the ones that we had to do the autopsy on.  So it's a bit of a unique cohort, but I think really important and I want to again raise the question of whether -- and this also dovetails with some of the other comments, whether the parts of the brain that are involved are those that mediate autonomic cardiorespiratory responses, and I think we are obliged to look into that a bit more.  Thank you.

DR. SPUDICH: Thanks so much.  I would like to ask a question to Dr. Hammoud, and it's a little bit of a tweak of a question that was just asked sort of to the group in general.  But many of the neuroimaging reports that we've seen in the literature throughout the pandemic have been of hospitalized patients, and we all know that's because even getting a hospitalized patient into the MRI scanner in the time of the peak concerns of COVID was very difficult.  But do you know of any data that routinely looked at individuals who had more mild forms of the disease during the time they had COVID and conducted any kind of neuroimaging?

DR. HAMMOUD: Thank you very much, Serena, for the question.  I kind of agree with the other speakers that there has been a lot of heterogeneity in our imaging literature, especially as far as the results and the types of imaging.  I think it's mostly due to the fact that people have been looking at small samples of patients and the populations that have been looked at are very different.  So lots of differences like gender, severity of disease, when they were imaged after the infection, and other kind of like associated neurologic presentations.

As far as the mild disease, I think what we really need at this point are follow-up studies of those, and there hasn't been a large sample that I know of where only mild disease patients have been looked at.  The largest I think is the one from the UK biobank.  The good thing about that one is it includes asymptomatic as well as very mild disease, and the beauty of it is that it's longitudinal.

The changes that they have described in their preliminary reports were mostly volumetric and gray matter thickness, but I know from corresponding with the first author that they're now looking at other findings like DTI findings and susceptibility weighted imaging findings as well, and she said that they're analyzing this data and hopefully will get a better explanation eventually.

But as far as like smaller reports here and there, there hasn't been any consistent that I know of.  For example, there was one report which showed increased gray matter volume rather than decreased gray matter volume, which goes against all the other studies and could be due to the acuity of the disease in those patients.

So heterogeneity of the populations is our main problem in imaging.  Maybe if we can pool the imaging or somebody can undergo a comprehensive meta-analysis of all the studies that have been published, it would be of use.  But at this point, we're as confused as pathologists are.

DR. SPUDICH: Thank you for, again, pointing out the paper and actually the first author put a link to the exact medRxiv paper in the link, which I think is really valuable and many people have mentioned it, and more people have mentioned it tomorrow.  So I think that is a useful finding.

I am going to then direct a question to Dr. Nair, and one of the things that we've talked about and we talked about in the clinical session is that there may be vascular injury and we heard about overt strokes, and it seems to me that some of your 7-Tesla data looking at brain tissue suggests that there may be microvascular injury that may not be obvious on clinical imaging.  It's a little bit of a hypothesis, but could you talk about the possibility that there may be some more microvascular injury at more subtle levels in people who have more mild disease?  Is that something that's hypothetical or that we could be testing for in live patients?

DR. NAIR: So I think that's a real possibility.  We have seen microhemorrhages and even thrombotic changes that, you know, in patients who are non-COVID.  So it is possible to pick it up with MRI.  And that is one of our techniques that we are using, Dima, to image post-COVID subjects, yes.  That's a possibility for sure.

And there is very specific techniques, like T2* imaging that can really pick it up in a very sensitive manner.

DR. SPUDICH: I think that is an interesting tool, thinking about going forward, and hopefully we have fewer and fewer acute COVID cases here, but obviously worldwide there are still many cases and there may be places that those kinds of studies can be used, longitudinally looking at people during milder forms of acute COVID and going on.

There are a couple of very specific questions.  So this is to Dr. Farhadian.  Have autoantibodies against ACE2 been detected?

DR. FARHADIAN: We didn't find that in our work, and I don't believe that it was part of the panel, although I'm not sure.  But I think there have been a couple of preprints on this issue, but it's not something that I'm expert in.

DR. SPUDICH: Okay.  I think that we can wrap up this terrific discussion.  So thank you so much to all of the speakers for being available for this live Q&A.  It's really important, and I think the talks included a lot of sort of complementary data and some things that I think can help us all think in new ways about these problems.

There are a couple of clinical questions in the chat, and I think some of these will be discussed probably in either the panel discussion or tomorrow.  So really thank you all for joining, for your fantastic talks, and for the discussion here, and we will wrap up right now for a 30-minute break.  So returning at 1:20 p.m. U.S. Eastern time, and we'll look forward to session III that will follow after the break.  Thanks so much, everyone.

(Break)

Agenda Item: SESSION III: Co-Morbidities and NeuroCOVID

DR. SIGAL: Hi, my name is Alex Sigal from the Africa Health Research Institute in Durban South Africa. I’ll be telling about SARS-CoV-2 as a moving target in the interactions of HIV co-infection and SARS-CoV-2 variants of concern. I’ll be making a couple points, first COVID19 disease severity in people with HIV is influenced by the infecting variant. And second, advanced HIV disease may delay SARS-CoV-2 clearance and lead to intra-host evolution of apparent adaptations found in variants of concern.

This is becoming very familiar, variants emerge either because they’re more transmissible or because they escape previous immunity, or both. And here is how the variant, the beta variant first found in South Africa looked like in November 2020 when it kind of exploded.

At that point Sandile Cele in my lab started to outgrow this variant, which was difficult, but he managed it. And once he had it, so this is a life virus neutralization assay, and the point of it is to test the effect of the variant on neutralization capacity of convalescent plasma or plasma from vaccinated people, to see if it’s the same or different relative to the actual virus which produced the immunity.

So basically the assay is you add virus to the convalescent plasma, or to vaccinated plasma, or to antibodies, you dump that on the cells, and you put a layer of gel over it so that the infection stays local, and you’re counting these foci of infection.

And when we did that what kind of struck us very strongly is that if you use the matched virus in plasma, that means in South Africa there are now three infection waves, the first one was with non-variants of concern, the second one was with the beta, and now the third one is the delta.

So if you use this virus from the first infection wave, not a variant of concern, added to the matched plasma from that same wave, you find that it gets neutralized pretty effectively by many of the plasmas we tested. Not so the beta variant, which is here called 501Y.V2, the earlier name. It is affected, but not much.

And this was what we found for the majority of cases. So we had about tenfold decrease in neutralization when you added the beta variant to convalescent plasma which was elicited by non-variants of concern. This was also done by another group here in South Africa using a serum neutralization assay. If you use the match plasma to the beta variant, what was elicited by the beta variant, that cross-neutralizes pretty well the earlier variant.

Now, these variants are not only to escape from neutralizations, but in fact they could do other things. In this example they can mediate escape from the innate immune response or the interferon response. So they can do kind of a bunch of stuff.

And we have an ongoing cohort to monitor what kind of happens with people that are infected, and one of the things we monitor is symptoms. And what we found in the first wave was that you don’t really have a difference in symptoms between people living with HIV, which are about 40 percent of our cohort, relative to HIV negative. 40 percent is about right for the population where we are.

But in the second wave, which is the beta variant wave, in fact there was a significant difference in one of these disease severity symptoms or outcomes, and that’s people living with HIV require supplemental oxygen, which is much more common than HIV negative participants, and we can see it here. Interestingly, this effect, we do not see more severe disease necessarily in the HIV negative participants with the beta variant.

And I’ll just go through this very quickly, but this associated basically with the low CD4 count. So individuals with low CD4 count were more prone to higher disease severity, here on a scale from one to three where three is highest, not so much if you cut it by HIV status it was not as clear. And if you look at who has the low CD4 counts, basically people living with HIV generally, that’s the overwhelming majority. The second point is that advanced HIV disease may delay SARS-CoV-2 clearance and lead to intra-host evolution.

And here we track the participant, mostly retroactively, that had advanced HIV with persistent SARS-CoV infection. You can see the CT value, which is the inverse of the viral load for SARS-CoV-2 stays consistent for about half a year in the 20 range. This participant also had a CD4 count in single digits, and viral load that was not suppressed.

And what we found once we got onto this case is that this participant was failing antiretroviral therapy, this participant was switched to a dolutegravir regimen on day 190, and very soon after that HIV was controlled, and after that he also had control of SARS-CoV-2, so it looks like this. So the control of SARS-CoV-2 is important to control the advanced HIV infection.

And when we looked at what’s going on at these different timepoints, going from bottom to top, from day zero to in this case 106, you see a quasi-species of mutations that we’re picking up. Some of them we know are associated with escape from antibodies, like the E484K, and some that we know less. And these mutations are actually changing through time.

So even the E484K, which arose fairly early, it actually got lost after a while. And the outgrowths on these viruses, the outgrown viruses were not exactly the same as the most common circulating genotype, but they exist within these genotypes. And we tested the three of them, a couple of early ones and one late one, day 190, and we found certainly this looks like escape.

So this early virus when challenged with plasma from convalescent participants from the same wave was neutralized pretty well. Not so much the later, what we call visit seven virus, which was from a swab on day 190. And when you look at slightly later virus, the visit three virus at day 20 at post symptom onset, that was a little more comparable to the visit seven virus. So certainly these look like variant.

So to conclude, there’s more severe disease in people living with HIV in the beta variant infection wave. SARS CoV-2 evolves similar mutations to variants of concern in a participant with advanced HIV. Evolved mutations lead to escape from neutralization. Control of HIV with antiretroviral therapy may be key to preventing evolution of SARS-CoV-2 in people with advanced HIV.

And maybe lastly, and these two bits of data really brought it home to me, is that SARS-CoV-2 biology may change between variants and within one person if infection persists. And these are, it was a big team of people, these are just a partial list, but I want to especially thank Farina, Sandile, Tulio, Richard, and our clinical collaborators in the Bill and Melinda Gates Foundation. Thank you.

DR. SINGER: Hello, I am Dr. Elyse Singer, and I’m here to talk about HIV positive SARS-CoV-2 autopsy cases from the National NeuroAIDS Tissue Consortium. I am professor of neurology at UCLA and the PI of one of the consortium members, the National Neurological AIDS Bank. Our consortium has been around since 1998, funded by NIH. We have four collection sites at UCLA, UCSD, Galveston, and Mt Sinai, and a data coordinating center run out of University of Nebraska and the EMIS Corporation.

We have been following HIV positive adults at high risk of death who consent to participate in data and sample collection of their tissues after death. Eligibility criteria for our cohort include advanced HIV/AIDS, older age, and high risk of mortality.

Subjects undergo serial standardized neuropsychologic and Neuromedical examinations and fluid collections during life and rapid autopsy in the event of their death. We make biobank samples available to researchers through a standardized request process. And we occasionally accept some non-enrolled immediate postmortem donations, which are characterized by chart review as available.

There’s a tremendous advantage of having a longitudinal study during the pandemic like this, because our cohort is very high risk, sort of a sentinel cohort, being that we’re located in all these large cities across the country. We have a pre-COVID19 baseline on most of the subjects, we have overlap of the epicenters of the epidemic, which was not something we expected or planned for, but this is what happened, and we have the research infrastructure and expertise in collection and storage of infectious samples, which many places do not have. We’re not funded to specifically perform SARS-CoV research, but I think we may be a good model for future research such as research on post-acute COVID sequelae.

Our UT Galveston site harvested five cases of which one is an NNTC case with preexisting data, and that one case will be made available to the public in the future for research. The remainder are autopsies done by the PI, Dr. Gailman, on non-NNTC subjects, and therefore will not get permission to share those with the public.

As you can see, the past medical history of the individuals showed that they have very severe medical problems. Most of them were older. And if you look through the neuropathology you will see a tremendous amount of vascular disease, and occasional other types of pathology, such as Alzheimer’s disease, not surprisingly because many of these patients were probably in a nursing home.

The next group were seven HIV positive SARS-CoV-2 cases harvested at the Mt. Sinai site. One of these had been in the NNTC premortem. The remainder were collected by the PI, DR. Susan Morgello and some of them will be made available for future use to scientific public. Again, you’re going to see in these cases a tremendous amount of vascular disease, including some sickle cell disease, which was likely preexisting, a lot of stroke related changes and microthrombi, and a fair amount of hypoxic ischemic changes.

There’s also one case with an aspergillus superinfection, which is not unusual given that many of the patients had been treated with steroids as part of their therapy. Again, you see one person with an underlying tauopathy, again probably consistent with age, and at the high vulnerability of the older population for COVID.

Our third group comes from the NNAB, my site. These were all prospectively characterized and collected patients who had signed up for the autopsy program and had up to 18 years of premortem follow-up. All of them were older, the youngest was 51, the oldest was 79. We also saw some hypoxic ischemic changes and a good deal of vascular disease.

At least one patient had a history of vascular dementia, which I had diagnosed and worked up prior to his death. Another one, our relatively youngest patient, had fairly severe medical problems, was on dialysis for end stage renal disease. And we also discovered at autopsy that she was developing cancer of the lung, which we had not even been aware of during her physical exams.

We took a deeper dive into these three NNTC cases and took some postmortem tissue samples from the olfactory nerve or bulb as available, frontal lobe, medulla, and lung. These were sent to Dr. Tom Lane at UCI who kindly performed RNA scope with a probe specific for CoV-2 spike transcripts. Negative controls were also compared for the probe of interest, and the SARS CoV-2 RNA appears as red on the slides. The summary was that the RNA scope was negative in all the brain samples and positive in one of the three lung samples.

And this was the last case in 2102, a 54-year-old right-handed female, who had been a longstanding HIV survivor, a low nadir CB4, but under excellent control at the time that she was harvested. She had a history of neurocognitive impairment but was best described mainly to non-HIV disorders as well as her HIV.

She had numerous comorbidities, the highlights of which were severe obesity, end stage renal disease and hypo-hemodialysis, adrenal adenoma, major depression and hyperglycemia. Interestingly, this case, unlike the other two, had died within four days of becoming symptomatic. She hadn’t been intubated, she did require some supplemental oxygen, and she had COVID pneumonia and volume overload, and the final cause of death was probably an acute MI.

As I stated the general pathology was remarkable for this unexpected adenocarcinoma in her lungs, and the neuropathology was significant for hypoxic ischemic changes, and vascular disease. SARS CoV-2 was detected in her lungs but not in her brain. As you can see, no spike probe showed up in either of the brain samples or the olfactory nerve, but was positive in lung. The remainder of the cases were negative.

In summary, most of our HIV positive COVID cases seem to have a similar profile. They were older, they had multiple comorbidities, they had a low nadir CD4 count, and a neurologic or psychiatric history. This is pretty consistent with the national profile I think.

All of ours contracted, all the ones that we study prospectively at NNAV contracted SARS-CoV-2 while they were in ART with excellent HIV control. We’re questioning whether they have some type of permanent immune damage as all of them had very low nadir CD4s in the past prior to beginning ART We did not detect SARS-CoV-2 in our CNS autopsy tissue, it’s unclear why.

It could have been in the CNS early and cleared prior to death, maybe we need to do a different type of test probe. Notably the case with the positive findings in lung died very quickly, as opposed to the other cases who died after more prolonged course.

And alternatively maybe the CNS neuropathology was due to hypoxia or inflammation, or autoantibodies. Nothing gets done in science without a village, and I’d like to acknowledge all of these wonderful people who contributed, including Dr. Lane and his team at UCI, our neuropathologists, Drs. Yong, Magaki, Said, and the wonderful NNAB staff, as well as the NIH for funding our consortium. Thank you.

DR. BAR-OR: HI, I am Amit Bar-Or from the University of Pennsylvania. I have been charged with this fairly broad topic on COVID outcomes in MS, autoimmune disease, or the immunosuppressed. These are my disclosures. And I thought under this broad umbrella to talk a little bit about SARS-CoV-2/COVID outcomes in MS patients, and outcomes particularly in MS patients as an autoimmune disease on immune therapies. And then have been given special permission to tell you about some hot off the press data related to COVID vaccine outcomes in MS patients on B cell targeting anti-CD20 therapy.

So to start with the outcomes in MS patients, there is good reason for us to have been concerned as the pandemic rolled out that individuals with CNS inflammation may be particularly susceptible to issues with SARS-CoV-2 and COVID infection. And this depicts the cascades that have been invoked for SARS-CoV-2 infection. If you look on the right and you’re an MS professional you might think of this as the MS pathophysiologic cascade. So obviously concerned about interactions.

And I’ll just show one example, not to reiterate what you’ve heard in great detail about COVID related changes in the CNS, but this is an example of mass cytometry spatial profiling that actually used multiple sclerosis as control brains, and they provided this nice picture of all the changes that you’ve heard of, and while focusing on features that would distinguish COVID from controls, including MS, they observed multiple changes in innate and adaptive immune activation, all of which are also seen in MS.

So we’ve been braced to see what would happen with people with CNS inflammation as part of their background autoimmune disease, and SARS-CoV-2, and in fact little is known still to date about interactions of CNS COVID and CNS inflammation in MS. People with MS appear to experience all of the acute as well as long COVID related complications that can be seen in the background population.

There’s no obvious increase or for that matter decrease in the risks of COVID outcome. There’s no obvious increase or for that matter decrease in the risks of COVID outcomes severity just by virtue of having MS. And conversely there’s no evidence of increased MS activity, although every once in a while a case report will pop out, but larger sample sets have not reiterated any concerns of increased disease activity with the infection.

And in people living with MS, predictors of severe outcome are essentially as per the general population, age, black race and comorbidities, increasing the risk of severe outcomes, as well as the level of disability, the neurological disability, and if they were treated recently with steroids for relapse. Which then brings in the issue of concomitant immune directed therapies and COVID outcomes.

So we’ll talk about outcomes in MS patients as an example of an autoimmune disease on immune therapies, although of course across autoimmune diseases they differ, they differ in terms of the background risks, the combinations of treatments that they’re on, and so on. So just focusing on MS to illustrate some principles.

And I’ll use one of the several published large registry studies looking at immune therapy impact on COVID19 severity in MS, the Italian registry. And in this busy slide what you see on the left-hand side are the multiple disease modifying therapies or DMTs that these MS patients might have been on.

And the graphic shows you with unity no difference between treatment versus no treatment versus where the point estimate and the confidence intervals are for each one of these different treatments. And what you’ll appreciate is that for the most part, most therapies, ranging from anti-VLA4, fumarate, cladribine, teriflunomide, S1P receptor modulation and so on are not implicating any increase or decrease in severe COVID outcomes with these treatments.

Interferon interestingly, and this has now been replicated in other studies, may confer protection from severe COVID outcomes, and there are some mechanistic reasons or rationale behind that. And ocrelizumab, an anti-CD20 is the one that has been associated in several but not all registry studies with some increased risk in severe outcomes, hospitalization, ICU stays and even death.

And a follow-up to the same Italian registry shows you here at the top relapsing-remitting MS, at the bottom progressive MS, and you’re looking here at anti-CD20 on the left, no therapy in the middle, and other DMTs, with the greys being pneumonia or hospitalization, and the darks being more severe outcomes with ICU and death.

First, the proportions are higher in the progressive MS at the bottom versus the relapsing remitting. And again you see here that anti-CD20 appears to be associated with worse outcomes. And the other DMTs may in fact be associated with some improved outcomes relative to no therapy, and this may of course be in keeping with suspected mechanisms underlying some of the COVID complications, which include an immune response to the target organs of the host.

So a summary of the outcomes. Most therapies in MS with a broad range of mechanisms of action do not appear associated with increased risk of severe COVID. Some, interferon, perhaps teriflunomide, may even be protective. Recent steroid use and the use of anti-CD20 appears to be associated with a small increased risk of severe outcomes.

So now transitioning in the second part to this hot off the press dataset, COVID vaccine outcomes in patients with autoimmune disease, and particularly on immune depleting therapies, have represented a very important knowledge gap, important implications for patients and those around them.

And the early indication is that there are substantially attenuated antibody responses in patients on anti-CD20, not entirely surprising given that you’re depleting the cells that are precursors to the antibody producing cells. It’s a major knowledge gap, particularly for the cell-based responses which have been generally harder to study.

So we’ve recently completed a vaccine study shown here five timepoints of blood draws prior to the first dose, 10 to 12 days after the first, just prior to the second injection. These are the RNA vaccines. Then 10-12 days after the second. And four weeks or so after the second injection. And we are looking at antibody responses to spike in RBD, as well as cell-based responses, both antigen specific memory B cells as well as activated T cells, both CD4 and CD8, and spike-antigen specific T cell antigen specific responses.

So the spike antibodies shown for the healthy controls and the MS patients, you can see the nice trajectory replicating recently published data on healthy controls, and you see a range of responses with attenuation and delay in the MS patients treated with the anti-CD20 summarized on the right. So replicating some recent emergence of data describing an attenuated response, although if you wait longer more and more patients with MS will mount these responses.

There’s a pretty good correlation of the serologies with B cell reconstitution and longer anti-CD20 inter-dose interval. The grey dots are the healthy controls, high numbers of B-cells, or proportions, and high serologic responses. And the MS patients showed a range where based on the lymphocyte repletion after the dose interval event ICD20 you may get more of the antibodies produced.

When we look using tetramer and flow cytometry at memory B cells specific to the SARS-CoV-2 spike protein, again on the left the trajectories in healthy control, and much attenuated with some patients not mounting as summarized on the right, to the spike protein. And below, also to the RBD, a similar panel with substantial attenuation in the anti-CD20 treated patients of SARS-CoV-2 specific memory B cells to both spike in RBD.

On the T cell side we were able to do at different time points a multiparametric flow cytometry analysis, then looking at cluster analyses based on a series of markers of capturing different profiles of CD4 T cells, any you can see here several clusters or metaclusters as we refer to at the different time points, the grey are the healthy controls, the orange are the MS.

And for example, metacluster seven, which happens to be TH1 T cell responses, you can see the initial expected bump with the first vaccine injection, the second vaccine injection bump, and you do get somewhat attenuated but pretty decent CD4 T cell activation in the MS patients as compared to the controls.

And now looking at antigen specific AIM assay spike tetramer reactive, you can see here that you’re getting the normal healthy control, and quite a decent response with some attenuation and delay in the MS context.

And a similar analysis, going through this very quickly for the CD8 T cells, where again you see as per the expected results in the healthy controls a decent increase to the initial vaccine injection, and interestingly a suggestion of a heightened response in the population after the second injection, which is very much reiterated at the level of the spike antigen specific AIM assay responses, where you can see here actually accentuated CD8 T-cell responses to the spike protein in the anti-CD20 treated MS patients, especially after the second vaccine. This is a second surprise and very interesting we think in terms of what it teaches us.

So we identify substantially attenuated serological responses, mildly attenuated CD4 T cell responses I didn’t show you, but that’s primarily an attenuation of T-folic helper T cells, and an augmented CD8 T cell response. And of course, correlations with protection need to be confirmed, but we also are getting some interesting implications to the cooperativity between B cells and T cells in the context of SARS-COV-2 vaccine responses, and likely reiterating some of the observations with natural infection. And I will stop here and thank you for your attention.

DR. TROYER: Hello, and thank you to the National Institute of Mental Health and the other supporting institutes at NIH for inviting me here today. I’m Dr. Emily Troyer, and I’m a general and child and adolescent psychiatric, and also a postdoctoral fellow in the Department of Psychiatry at University of California San Diego, and my position is supported by an NIMH T32 training grant.

So in April of 2020 my colleagues and I published a review in Brain, Behavior, and Immunity, where we explored potential neuropsychiatric symptoms in the context of the novel pandemic. And I’d like to acknowledge my colleagues here, Dr. Suzy Hong and Dr. Jordan Cone.

At the time, which seems like ages ago at this point, what we tried to do was synthesize data related to psychiatric symptoms following past viral outbreaks, and integrate that with knowledge gained from modern psychoneuroimmunology studies in order to hypothesize about potential neuropsychiatric effects of SARS-CoV-2 infection.

And our position was that diverse symptoms could potentially accompany or follow infection in some individuals over variable periods of time. And that the host antiviral response might play a role in eliciting those symptoms.

So theoretical mechanisms could include theoretical cytokine network dysregulation leading to neuroinflammatory responses like microglial activation, along with compromised blood-brain barrier integrity, leading to peripheral immune cell transmigration into the CNS, and ultimately disruption of neurotransmission.

Also, in early 2020 a meta-analysis was published of neuropsychiatric symptoms in survivors of severe infection during past coronavirus outbreaks, namely SARS-CoV-1 and MERS-CoV. And they demonstrated that cognitive, mood, anxiety, and trauma related symptoms were common, psychosis was also noted both in the acute illness and at long-term follow-up, which was an average of about three years after illness, and strikingly at that long-term follow-up almost a quarter of survivors had not returned to work.

And if we shift to early in the SARS-CoV-2 pandemic, we start to appreciate that there is a bidirectional relationship, in that COVID19 infection is associated with neuropsychiatric symptoms, both acutely and in the first three months following infection, and also preexisting neuropsychiatric disorders might influence not only risk for COVID infection but related outcomes like need for hospitalization and even mortality.

So now looking forward, if we try to understand longer term post COVID psychiatric symptoms, we see that in the several months following infection, about up to six months, sleep disturbances, depressed mood, anxiety and trauma related symptoms are among the most common reported.

It’s interesting to note that in the case of depression and anxiety studies don’t seem to suggest a relationship between severity of COVID infection and risk for symptoms, whereas there does seem to be a relationship between severity of infection and risk for psychosis. In fact some studies even suggest milder COVID infection is associated with increased risk for depression and anxiety.

It’s also important to note that the literature on psychiatric symptoms so far is rapidly evolving, and still has some important limitations. So much of the literature for example is based on studies of adult cohorts following hospital discharge, and so the samples are maybe enriched for more severe infection.

And most studies are observational and lacking control groups, and it’s frequently unclear if the psychiatric symptoms reported in survivors represent an exacerbation of a preexisting illness or onset of novel psychiatric disorders.

So in April of this year a large EMR based study was published which doesn’t suffer from some of the limitations I just discussed, in that their sample isn’t enriched for a particular severity of COVID infection, and they’re able to compare exposure to COVID versus exposure to other medical conditions.

And they examined several neurologic and psychiatric outcomes and showed that almost a quarter of survivors had a mood, anxiety, or psychotic disorder diagnosis within the first six months after COVID infection. And for almost nine percent of survivors this is a new onset psychiatric disorder not present prior to COVID. And they went on to demonstrate that risk for several psychiatric disorders is significantly greater following COVID compared to following influenza or other respiratory tract infections.

So far we’ve talked mostly about psychiatric disorders collectively, but now I’d like to take a moment to look at COVID and trauma related symptoms specifically. And follow-up studies in COVID survivors so far have reported prevalence of PTSD or post-traumatic stress symptoms or PTSS ranging from around five to 45 percent.

And of course, post-ICU PTSD is recognized outside the pandemic. And prevalence of PTSD or PTSS reported in the general public is also significantly increased since the beginning of the pandemic. So it’s difficult to disentangle the degree to which COVID infection specifically influences risk for trauma related symptoms.

And PTSD actually wasn’t originally an outcome of interest in the six-month EMR study we just discussed, but the authors have subsequently published these results and showed that COVID is associated with greater risk of PTSD compared to influenza infection. And similar to psychosis, but unlike depression and anxiety, PTSD does appear to be related to severity of COVID infection.

So now I’ll just take a minute and shift to also discuss PTSD symptoms in people living through the pandemic who haven’t been infected with SARS-CoV-2. And this is also an area of interest in the Hong Laboratory at UCSD. So outside the pandemic, studies show that like several psychiatric disorders people with PTSD have evidence of increased inflammatory cytokines compared to healthy controls, and prospective studies have shown increased concentrations of some acute phase and vascular inflammatory markers at baseline is associated with risk for developing PTSD after trauma exposure.

And of course, in COVID infection we think the host cytokine responses could possibly influence neuropsychiatric symptoms. But it’s unknown if individuals with preexisting conditions that are associated with chronic low-grade inflammation like cardiovascular disease for example are at increased risk of developing trauma related symptoms in the context of the pandemic.

So here we have work in progress from our group where during the pandemic we administered online questionnaires to aging adults with hypertension who are psychiatrically healthy who previously completed a clinical trial of Tai Chi versus health education classes for hypertension. And we screened for trauma related symptoms using the PC-PTSD, and over 40 percent of our respondents endorsed at least one trauma related symptom, while almost nine percent reported three or more trauma related symptoms since onset of the pandemic.

And when we looked at pre-pandemic baseline predictors of trauma symptoms, only female biologic sex and baseline anxiety scores significantly predicted pandemic related trauma symptoms. Now the group endorsing three or more trauma symptoms did have a greater median PRC at baseline, but this did not reach statistical significance, and none of the baseline acute phase or vascular inflammatory markers predicted trauma symptoms during the pandemic in our group.

So the role of baseline chronic low-grade inflammation in risk for pandemic related PTSS in our participants is still unclear, but in future analyses we plan to integrate a larger set of biomarkers along with repeated longitudinal assessments throughout the pandemic.

So just to summarize I’ve provided a rapid overview of psychiatric symptoms in COVID survivors up to about six months following infection, and sleep disturbance, depression, anxiety, and trauma related symptoms seem to be most commonly reported.

It’s important to note that most studies to date have been in adult COVID survivors, and epidemiologic studies of past pandemics suggest post-viral psychiatric sequelae may have some developmental specificity. So longitudinal cohorts of COVID survivors at all stages of development, including in utero, through advanced aging, will be needed going forward.

And of course, we also need longer term follow-up to appreciate trajectories of psychiatric symptoms over time. We need to understand pathogenic mechanisms underlying psychiatric sequelae in order to tailor preventive and treatment strategies. And unraveling the effects of SARS-CoV-2 infection versus pandemic related stressors on risks for some psychiatric symptoms will probably be an ongoing challenge, but both might have targetable and partially overlapping neuroimmune mechanisms.

And finally, we’re almost certainly going to be facing psychiatric symptoms in the population at a scale unprecedented at least in recent history, and we need to continue to build capacity to be able to mitigate the effects of this in the population. And as scientists and clinicians I hope we’ll take this opportunity to continue to break down the delineation between physical and mental health so that we can understand where those shared pathogenic mechanisms and treatment targets lie.

So with that I’d just like to acknowledge our funding institutes at NIH and all of my colleagues in the Hong Laboratory at UCSD, and thank you for your time.

DR. ELY: I want to welcome everybody to the next presentation. My name is Dr. Wes Ely, I am an intensivist at Vanderbilt University, and I am one of the co-directors of the CIBS Center, which is Critical Illness, Brain Dysfunction and Survivorship Center. And I will be co-presenting with David Bennett. David, would you like to introduce yourself?

DR. BENNETT: I am David Bennett, and I’m the Director of the Rush Alzheimer’s Disease Center in Chicago, and I’ve been working with Wes on a study of brains from ICUs.

DR. ELY: David and I are principal investigators on an NIA R01 which is designed to collect and establish a brain repository for critically ill patients who develop acquired dementia, and we’re trying to understand how this relates with COVID and non-COVID patients.

The study I’m going to present to you right now, and then David will merge into the study I was just speaking of, is a study we did in COVID called the COVID D Study, which was published in Lancet respiratory, and in this investigation, I have no disclosures by the way, there were only NIH funding for this, no industry involvement, and this was a study in which we enrolled, and David if you could switch slides please, in which we enrolled 2100 patients.

We have been studying, in COVID you realize that there has been a dramatic amount of overuse of sedation in the ICU. And one of the ongoing R01s we had through NHLBI was this study we just published in the New England Journal of Dexmedetomidine and propofol for severe sepsis.

And we learned in this study that both were basically equivalent if not overused. In COVID patients we have found that propofol and benzodiazepines have been dramatically rampantly used, and that it has even created a shortage in propofol and benzos during the COVID pandemic.

Historically, from 2000-2015, delirium rates in critically ill mechanically ventilated patients were around 60 to 80 percent, a completely crazy high number of acute brain dysfunction in these patients. And we know through many New England Journal, Lancet, and JAMA papers that this brain dysfunction of delirium is a dose related risk factor for severe long-term cognitive impairment for higher death rates, high cost of care, and longer lengths of stay.

In the last five years prior to COVID we were able to drop those delirium rates into the 45-50 percent range using an evidence-based safety bundle called the ABCDEF bundle, or the A-F bundle I like to abbreviate it. COVID rates went way back up into the 80-percentile range and took us back-to-back in the 2000-2015 era for brain dysfunction in ICU patients, and benzo us skyrocketed as well.

So we began to think about all the different types of organ dysfunction in our COVID patients. And you can see on this fairly famous slide lungs, heart, liver, GI tract, you name it, it’s involved, and on the top right you see the brain. All of us have been struggling with how to think of COVID’s relationship with the brain.

I and some residents came up with this mnemonic called the F-COVID, we hate COVID, so F-COVID, and the causes of this delirium really relate to these six factors: the tremendous amount of isolation and loneliness that patients experience, that’s family. Clotting, tremendous amount of clotting.

I’ve got a patient right now with arterial clots in all of her arms and legs, and she’s going to lose her hands and feet. Oxygenation has been profound because of the pneumonia. The virus itself has some degree of neural invasion, I’ll let David tackle that when he gets to his presentation. Patients are immobilized, and they get drugs like benzos.

The solution we use clinically is called the Dr. DRE. We go through each day and we think about diseases that could be causing delirium, sepsis, clotting, CHF et cetera. Drug removal, that is getting rid of potentially deleriogenic drugs like benzos. And environmental issues like eyeglasses, hearing aides, et cetera. So these are just some tips clinically, and now I’d like to present the study.

This is an international, multi-center, retrospective cohort study of over 2000 patients. We prospectively collected the data, but we didn’t know that we were going to do this until the COVID pandemic got so out of control and I started partnering with people around the world to get these data. We performed multi-variable logistic regression, multi-variable regression analysis, and identified the delirium risk factors.

In this investigation, the patients were a median of 64 years old, they were extremely critical ill, the sat scores of 40 show that, 88 percent of them were on the ventilator, and 81 percent had coma for 10 days and 55 percent delirium for three. So you can see that basically we had two weeks of profound cognitive impairment.

In this graph you just see that there was a gargantuan amount of coma early on, that’s the dark blue line, and then as it receded you see the delirium coming on up.

Benzo use in the cohort of COVID patients we studied was about 64 percent for a week, propofol use 71 percent for a week, dexmedetomidine use 44 percent for four days.

A disaster that we discovered from this cohort study was that only 24 percent or 23 percent of people got what we refer to as an SAT or an SBT. That stands for spontaneous awakening trial, then spontaneous breathing trial. And what this refers to is stopping the ventilator and stopping sedation every day to see if the patient can breathe and be awake and alert on their own.

In every ICU population around the world that number should be above 80 percent. This shows a complete noncompliance with a basic safety approach. It would be as if you were getting on an airplane and trying to go from LA to New York and the pilot just said I’m not going to do the safety checklist, and that’s what happened in our COVID ICUs.

Perhaps even more disastrous, look at the family visitation rates down there. Eight percent in person, nine percent virtually. So that means only 17 percent got any interaction with family, and 83 percent nothing at all. That’s very sad.

This shows the risk factors that we analyzed, and David if you’ll hit one more time it will highlight two bars. I just want to focus on two things: sedation overuse, and family underuse. You can see that sedation shows the point estimate far to the right, increasing the risk, and the use of family brings your point estimate to the left, so reducing delirium rates.

If you show the next slide I’ll bring it to a conclusion here. This is a very famous tweet during COVID of this nurse before and after COVID. So before COVID you see her all fresh and well groomed, and then during COVID you see her basically fighting for her life, barely getting by. And this Maslow’s triangle shows where people are in their element of survivorship. And where we want people to be is the top of this pyramid, Maslow’s pyramid. But where we and patients were living is at the very bottom, struggling to get by.

What we learned from this COVID-D study was that delirium and coma have become an epidemic within the pandemic. There has been a doubling of the duration of the amount of acute brain dysfunction in these patients to two weeks or more, which really portends a very dangerous prognosis for the long-term brain function. Since we already know that three to five days of delirium actually increases the risk of dementia and death and many other untoward outcomes.

So the two strongest predictors of this delirium were benzodiazepine overuse and family underuse. We’ve been trying very aggressively in the past six months to counteract these things and get back to the basics of the ABCDEF bundle, which we know decreases length of stay, duration of coma, delirium, et cetera.

And so we’re trying to build a way forward so that we don’t have as much acquisition of brain dysfunction. But I’d like to now turn it over to Dr. David Bennett and let him tell you some of the data we’ve been accruing in our NIH funded investigation. David, thank you so much.

DR. BENNETT: So I realize, I apologize, I thought I was supposed to talk about our brain ICU study, and I had a death in the family, and by the time I got around to this I had already put this talk together, so that’s what I’m going to do.

We got a supplement to our brain ICU study to look at brains from people with and without COVID, and in and outside the ICU. I’ll start by talking a little bit about people who died in the ICU with COVID, and these brains came from Wes and Vanderbilt to Chicago, early in the pandemic when they were fixed for quite a long time.

And so we had 20 adults, 16 male, mostly white, and then like you heard from the other talks earlier these are a lot of old people, and so they get all the other comorbidities that you see with age. Alzheimer’s, Parkinson’s, neocortical Lewy bodies, late CAA, and supervascular disease. And then a number of them had recent infarcs.

And then we’re going to show just a few different things that could in theory be related to COVID. First, we see some microthrombi, and many of these things that I’m going to shown were shown earlier before the break. And microglial nodules. Neuron aphasia, vasculitis, and ring vasculitis, and ring hemorrhages. And these could be related to COVID, but they’re certainly not so sure related to COVID. And these are sick people from an ICU.

And so one of the things we’re hoping to do with this study as we accrue brains from the ICU from people who don’t have COVID is to try and sort out what is due to the ICU experience, because many of these delirium and dementia type findings we see pre-COVID in people without COVID, and just a variety of other very nonspecific factors such as inflammation, lymphocytic coupling, huffings, meningeal lymphocytes and perineal lymphocytes.

And then we also do ex vivo MRI and we identified a number of microbleeds, and like one of the earlier talks this is a nice approach to getting a good survey of the brain in order to help identify sites that are worthy of being investigated microscopically.

In the second half I’m going to switch to non-ICU people. So ICU negative, with and without COVID. Now these are a completely different group of participants. These are actually participants that come from a few ongoing cohort studies that we do in the community in and around the Chicago area and across the country.

And here we’ve been freezing material in the more recent cases, and we started to do a study of single nucleus RNA seq. And so the first thing I’ll show you is the COVID data on the single nucleus RNA seq had quite a few fewer unique genes, and fewer reads per gene, and a higher mitochondrial percentage.

So I should note that the people with and without COVID came from the same cohort that we’ve been following now for many years. But they weren’t done at the same time, the two batches were done differently, because the COVID brains had to be done in the DSL3 laboratory, and the controls from the same cohort done by the same lab, this happens to be at MIT with Li-Huei Tsai, were done prior to COVID. But the cases are matched in terms of demographics.

Here we see people with and without all the cells and then COVID positive. So we’re able to recapitulate fairly well the cellular structure of the single nucleus RNAseq. And here you see all of the cells clustering and the different cell types that we’re able to identify.

And then what we did was we looked for a relative increase in different cell types. And what we see is a relative increase in COVID of astrocytic cells. And then if we do differential expression we can see a number of genes that are differentially expressed related to RNA splicing, related to lysosomes, related to interferon alpha, interferon gamma, prednisones, and then related to COVID. We did not see COVID in these cases, we did do RNA scope, we could not find anything that we were convinced was COVID, either in these cases or in the prior set of cases.

And then here is a sample of a couple particular differences in BA1 pro-inflammation that’s up in COVID, homeostatic, and then NHC proinflammatory genes that are up in COVID. And then I’m going to stop there. And there’s a lot of people to think both at Vanderbilt and Rosh and at MIT that generated these data. And I want to thank you very much.

DR. SPUDICH: Thank you so much and thank you particularly to our live speakers for jumping in and being able to present and share your slides on this platform. So we had a number of outstanding talks, and I think they covered a variety of different aspects which we can consider on comorbidities of COVID, ranging from co-infection with HIV, immunosuppressant states, the condition of having multiple sclerosis or being on multiple sclerosis treatment, and then really focusing on mental health and delirium in COVID.

So I’d like to ask the speakers in this session, Drs. Sigal, Singer, Bar-Or, Troyer, Ely and Bennett to please turn on their cameras, and we have about 15 minutes for a Q&A. We have some good questions already in the chat, and remember the audience members if you have other questions to post it to the Q&A if you want to direct it to the speakers, they’re welcome.

I will start with a question for Dr. Sigal. And let me just confirm that Dr. Sigal is able to still be connected. I’m not actually sure. He may have dropped off. So instead, I’ll start with a question for Dr. Troyer. There are a couple questions actually relevant to Dr. Troyer.

So one says have any studies focused on the social isolation of COVID and its effects on the mental health of the population. I think you talked about that being an issue, but can you go back to the complexity of dealing with that issue specifically?

DR. TROYER: With the issue of loneliness and isolation, that’s certainly something that all of us have faced over the course of the pandemic. There’s a broad literature related to that that I haven’t talked about very much here, because I wanted to focus really on people who had contracted SARS-CoV-2.

But in our cohort we have where we’ve looked at PTSS, we’ve also assessed loneliness and not surprisingly loneliness is highly correlated with depression, anxiety, and trauma related symptoms. We don’t have baseline loneliness data though, to be able to say whether or not that’s a predictor of trauma related symptoms in the pandemic, but in general this is going to be something that’s very difficult to tease apart. We’ve never going to have a clean control group, because all of us have been affected by the psychosocial impact of the pandemic.

DR. SPUDICH: I think the issue of isolation and loneliness has been brought up both in your talk as well as in the talk by Dr. Ely. There’s actually a question by one of the other speakers that’s being posted, saying it’s interesting and sad to see what a strong effect family isolation had on COVID patients in the ICU. So I’m wondering if Dr. Ely can comment on the isolation effect on ICU patients who were hospitalized for non-COVID reasons, and has that been examined.

DR. ELY: I first would like to comment back on the previous question, that we at the CIBS Center, our Critical Illness Brain Dysfunction Survivorship Center are also studying the mental health effects, and we have a bank of 15 people calling hundreds of COVID survivors, long haulers, and we actually have ongoing, I just want people to be aware this is happening, we have multiple support groups every week.

So we have COVID support groups, we have COVID spouse support groups, we have healthcare professional support groups, and they’re also part of ongoing studies. We have a study called Isolate which is actually studying the effect of COVID on hospitalized non-COVID patients. It has been absolutely fascinating.

In our experience the effect of isolation and the loneliness has been a big-time predictor of the acquisition of delirium, but also post-traumatic stress disorder and long-term depression. And so if you think about what an ICU patient experiences after they leave the intensive care unit, we’ve had for 15-20 years the concept of PICS, Post-Intensive Care Syndrome.

And in many ways you can think that long haulers have PICS. If a COVID patient got admitted to the hospital and they are six months later experiencing PTSD, depression, cognitive dysfunction, they’ve got PICS. And that’s their manifestation as a long COVID patient, as a long hauler.

Now, there are, and I’ll make this distinction, there are people who get COVID and never come into the hospital, and they can also have long COVID. I would equate that to MECFS, Myalgic Encephalitis Chronic Fatigue Syndrome. And there’s a lot of people equating the MECFS to long COVID, but if you stick to the area that I study and David studies, we study hospitalized patients, and they have PICS and long COVID, and it’s PTSD, depression, cognitive dysfunction in spades, and there’s no question that the effect, and we’re studying the effect of isolation on it, and it’s not just about those people who contracted COVID, we are also actively studying through NIH supplements the effect on non-COVID patients. And if I can end with an anecdote, the first patient that ever had a visitor during the pandemic at Vanderbilt health hospital was my young lupus patient who has lupus cerebritis, and I just could not take it anymore and I said her parents are coming in.

And she was suffering so much by her parents not being with her, and she didn’t even have COVID. So absolutely this was a travesty, and we have to open the hospitals back up, because it’s part of the treatment plan to have the family there. It’s not a luxury, it’s got to be part of the treatment plan for the patient’s dignity and their self-worth.

DR. BENNETT: If I could make a comment on social isolation which we’ve been studying for a number of years in our COHORT, we did do a burst design during COVID, and went back and did an interview on over 1000 people on who we had premorbid perceived social isolation data.

These are people not in the ICU and not necessarily with COVID, most of them don’t have COVID, probably 99 percent didn’t have COVID. And surprisingly there was very little uptick in the amount of social isolation. And we expected a lot more, compared to premorbid ability.

So I think we need to also consider, and I think one of the other speakers brought this up, is people bring their loneliness with them to COVID, from before. And the ICU is kind of a weird, bizarre, really isolating people as opposed to self-perceived social isolation, which is loneliness.

DR. SPUDICH: Can I follow up with another identified factor in your study, Dr. Ely, which is you talked about the fact that patients who received higher doses of benzodiazepines were also at higher risk of delirium and the sort of prolonged waking up and those kinds of issues. And I wondered did you correct for reasons that those people had higher doses of benzos?

Sometimes more benzos are given for more agitation for example. This is outside of my expertise, but I’m a clinical neurologist and sometimes we see that. So were there reasons that particular patients received higher benzodiazepines that might be actually associated with underlying neurological disease independent of the benzo doses.

DR. ELY: I love that question, and we did. We had the RAS levels, Richmond Agitation Sedation Scale, so we were able to look at the patients who were hyperactively delirious versus hypoactively delirious. And what we found was pretty striking, that it was almost all hypoactive delirium, it wasn’t for agitation. Really what was happening was that patients were experiencing hypoxemic respiratory failure and a need for mechanical ventilator, and so people were giving high dose of benzos purely to deeply sedate people into a coma, and then when they came back out through the coma they landed in delirium for a long time. It was not due to agitation as much as it was they’re on the ventilator, they’re with COVID, and we need them deep.

What we’ve been learning, the take home for me as an intensivist in learning how to take care of COVID patients is I’ve got COVID patients right outside this call room here that I’ve been in with earlier today, and I do not have them deeply sedated, they’re very hypoxemic, I’ve got them on high flow nasal canula, 100 percent, 60 liters, or BIPAP, and I don’t need to sedate them anymore.

So at the beginning of the pandemic we kind of lost our footing, and we were panicky and we were afraid and we treated people in a way that wasn’t in keeping with what we had learned about intensive care medicine over the past 15 years. We’ve now got back to our basics, and we treat them now the original way we used to with the A-F bundle, and we’re not seeing those high doses of benzos and overt propofol shortages anymore, it just took us a while to get our footing.

DR. SPUDICH: I think that is probably true worldwide in terms of how you described the initial ICU care unfortunately. I have a couple of other questions for some of the other speakers. So Dr. Singer, there are sort of two questions for you. Number one, you talked about the fact that you did not detect any SARS-CoV-2 in the brains of these coinfected COVID and HIV autopsy brains.

But as an HIV neurologist, I think about co-infections or inflammation triggering HIV replication, and I don’t know if I missed it, but did you look to see whether there was detectable HIV in the brains, I know most of these were suppressed on therapy, but did you find any HIV detected in the brains?

DR. SINGER: We haven’t looked yet, we’re going to do that. We expected there will be very little however, because these particular individuals not only were very closely studied by us and had multiple undetectable viral loads, but at least two of them were in care facilities where their medicines were administered by other people or supervised.

I’m sure there is some detectable in brain, because we’ve certainly found it in other members of our cohort who did not have SARS-CoV-2, but were long-term survivors with undetectable viral loads in blood and CSF, and we still found something in the brain. But in general, I’m going to suspect that we won’t find very much.

DR. SPUDICH: I think the question would be does the inflammation associated with SARS-CoV-2 or the immune response somehow trigger a release in each application. So I think that’s an interesting question.

DR. SINGER: I suspect that is correct. These patients are very susceptible to delirium in any case, and I would suspect that these patients when they became hypoxic or had their heart attacks were very susceptible, not to mention the fact that at least two out of the three were cognitively abnormal prior to going into the SARS-CoV-2 situation, one had vascular dementia that we had worked up very carefully before he even developed SARS-CoV-2, and the other had multifactor cognitive impairment due probably to being on dialysis and having poorly controlled renal failure.

DR. SPUDICH: That was a little bit my follow-up question, and I think you made such an elegant point that so many of these brains that you were able to examine were from people with multiple medical comorbidities. And so how do we as a field, and I think we talked about this a little bit in the last session, how do we examine these rare autopsy brains for example in people who are coinfected or had HIV and then also have SARS-CoV-2, how do we make sense of what’s specifically due to COVID, HIV, which factors. And you’ve had a long experiencing in gleaning this information from autopsy studies, so how do you do that?

DR. SINGER: I think one of the things that is helpful is that we do have a baseline on that, and in some cases 18 years of baseline. So we have a very good idea of what the patients were like before they got SARS-CoV-2, and none of these people were in terrifically good shape.

We have other people in the study who acquired SARS-CoV-2 who survived, and we’ll get follow-up neuropsych on them. Now it would be even better if we had brain scans as well, but I think that the advantage of having an existing longitudinal cohort is that you have a baseline, and this is I think a tremendously helpful thing.

DR. SPUDICH: Another question I modified a little bit for Dr. Bar-Or, which is a question from the audience about are there similarities between issues seen in solid organ transplantation patients. And the question was whether that’s seen also in HIV patients, but the observation that some individuals have had solid organ transplants, and then we heard from Dr. Sigal some individuals who have chronic HIV seem to have more of a long duration of SARS-CoV-2 shedding, potentially persistent infection. Is that something that you have seen or has been described in the setting of some of the immunotherapies that are used to treat something like multiple sclerosis?

DR. BAR-OR: I think in the context of MS, which is a situation where you’re using immune therapies in otherwise rather healthy, young individuals, probably not particularly representative of some of the other context, transplant context, multiple other medications, older and sicker patients with other comorbidities. So it may inform less, but it also provides a particular window that may help isolate some of the many factors that you had pointed to before.

And what we have not seen at least in the MS population other than in the context of the anti-CD20, where there is a bit of a signal of more severe and more prolonged, including a little bit of an increased frequency of death in the context of SARS-CoV-2 infection and its complications, still the numbers are really too small to be able to say is it more of the infection or more the sequelae of the infection that are producing the issues in this population because of the B cell or with the super imposed B cell defeating therapy. But I think as you get into areas of transplant and other comorbidities with age, this becomes very complex as you were alluding to in asking about before.

DR. SPUDICH: And a bit of tangential question, but also related specifically to MS, I think there’s emerging data that some people with longstanding MS have some potentially more cognitive effects than simply the focal lesions that we all learned about when I was a resident at least. Are you familiar with some of those patients who may already have some mild cognitive injury from MS seeming to have more problems cognitively, either during COVID or after COVID?

DR. BAR-OR: I think we stand to learn about that important question in the studies that are ongoing. There’s the MS Recog which is a multicenter study that’s registry based and already has 1000 MS patients and controls to look at. At the moment the study just reported on physical disability and mental health related anxiety and depression, but not yet on cognition, which is not that easy to formally capture in a quantitative or semi-quantitative way. But the issue is a big one because cognition in fact is something that impairs many people with MS, and not just the relatively few where it’s quite evident early on.

DR. SPUDICH: Maybe we can wrap up by one final question to Dr. Troyer related to this issue of cognition. I think one of the challenges that many of us have had caring for people who have had COVID or are having some lingering symptoms is some seem to have mental health concerns or problems, some seem to have some primary cognitive concerns, and some of these are overlapping.

So is that something that do you have insight into how best to, some of them could be independent, some could be causal between one and the other, and then some of them could be from the same common pathway physiologically. So do you have thoughts about how to untangle those things?

DR. TROYER: That’s a really good question that I don’t know that I have a concise answer for unfortunately. When we think about some of the immune mediated processes though that we’ve talked about happening in the context of COVID infection and in some of these other processes like neurodegenerative or demyelinating processes, outside of the pandemic we see inflammation being associated with psychiatric disorders for sure, and with cognitive disorders, and they can overlap, sometimes they do, sometimes they don’t, but I think it’s going to take us quite some time to really disentangle that. But I think the hope is that we will identify shared mechanisms.

And one of the main questions for me as a psychiatrist going forward is will our current treatments be effective for post-COVID psychiatric symptoms or do we need to think about things like immune modulating treatments or other strategies.

DR. SPUDICH: So I am hoping that some of those things we’ll have an opportunity to talk about tomorrow. And I think all of these types of questions, and thinking then about how do we design the right research studies, many of you already have really relevant studies ongoing, but hopefully this can be more of a discussion as the day and a half continue. Thank you to all of the speakers, and thanks particularly for the effort to be in on the life Q&A, which I think is really fabulous. These were really fantastic talks and discussion.

We’re going to move now into the final portion of today’s session in today’s agenda, which is a panel discussion. And I’m’ absolutely delighted to introduce Dr. Avindra Nath, close colleague, friend, organizer of this meeting who will be leading the panel discussion. So Dr. Nath is clinical director of clinical research at NINDS.

Agenda Item: Panel Discussion

DR. NATH: Thank you Dr. Spudich for that kind introduction. I am delighted to host this panel session, and I’m going to take the opportunity to introduce our three panelists first.

Dr. Masliah is in the National Institutes of Aging, and he is the Director of the Division of Neuroscience. He’s a neuropathologist by training, and he has done extensive research on CNS infections, particularly HIV infection and neurodegenerative diseases.

Dr Susan Morgello is a professor in the Department of Pathology and is also the Director of the Manhattan HIV Brain Bank at Mt. Sinai Medical Center. And she has extensive medical experience against CNS infections, particularly HIV and now recently has done extensive amount of work with COVID as well.

Professor Tom Solomon has been introduced already, but he is the Chair on Neurological Sciences at University of Liverpool, and also the Director of the National Institutes of Health Research Health Protection Research Unit on Emerging Infections. And he has extensive experience in international emerging infections. And you heard his talk today, so I’d like to welcome all three of our panelists.

So what I’m going to do is pick up some questions from the Q&A or from the chat box, and present it to our panelists. But any of the speakers, I know the speakers cannot type in the Q&A, so you’re welcome to just unmute yourself or raise your hand and ask a question.

So to get ourselves started I’ll take one question from the Q&A, and maybe I’ll present that to Dr. Solomon. This is a question with regards to MECFS, and they want to know whether the study of COVID is going to shed light on MECFS, and where the overlap between the two might be.

DR. SOLOMON: Just to clarify, I think you’re saying MECFS as in chronic fatigue syndrome, just to be clear on the terms because we use them slightly differently. I do think, I mean this pandemic has continued to surprise us, every time we think we’re getting somewhere there’s some new twist in the tail, and clearly the whole long COVID story, which again you call it something slightly differently in America at the moment, but we all seem to change the terms every few weeks.

Anyway, you know what I mean. Patients with long-term problems after their COVID infection wasn’t expected, and I think does give us a real opportunity to think about some of the problems that people have after other viral infections.

So we all as neurologists, we see patients with chronic fatigue, and typically we suspect that they’ve been infected with something, we don’t know when and we don’t know what it was but we do know they clearly have ongoing problems, and there may be a mixture of biological, psychological, and social reasons that contribute to the ongoing problems. I think with these patients with long COVID we have a real opportunity to understand this whole area a bit better.

Firstly, though I think we have to distinguish, we’re calling it long COVID in the UK, but there are several different things going on. There are patients who have lung problems after for example severe respiratory disease, so that’s not really a big surprise. A patient who has an ongoing hemiparesis after a COVID related stroke, that’s not a surprise. And I think we need to disentangle it.

And the patients that I think really are of interest are where the patient groups themselves have raised the issues, and those are the people who had relatively mild disease and then have ongoing tardness, brain fog, poor memory, et cetera. Many of those symptoms are very similar to the chronic fatigue symptoms, and I think on this occasion we do know what the virus was, and we do know exactly when it happened, and we have fantastic opportunities to study it better.

So I hope that if we approach it in the right way we will really understand these post-infectious syndromes better, but I would point out that I know some people in the chronic fatigue community have reacted rather against this, and they don’t like the idea that some of these long COVID symptoms have anything to do with chronic fatigue. So we’re clearly going to have a challenge there in terms of how we relate to patient groups, some of whom I think are on the call as well. So let’s see what the thoughts are. But I’d be interested to hear the thoughts of others in the panel.

DR. NATH: Thank you very much. The next question I’m going to direct to Dr. Morgello. We heard a lot about the neuropathology of COVID, but do you think that there are specific findings or specific characteristics that one could look at and say well this is definitely a COVID related brain, or COVID related pathology, or do you think it overlaps with too many other diseases?

DR. MORGELLO: As I am taking a step back, I say okay, and the challenge was Dr. Spudich said what can neuropathologists do to help us to figure out what’s COVID exacerbating as a pre-extant condition, what is COVID triggering as a COVID specific phenomenon, and what is COVID triggering that’s a generic phenomenon that you see with many infections.

So one of the most exciting talks I heard today was Dr. Glatzel who was actually looking at a condition that actually I thought to myself gee, nobody has ever looked at this in infectious diseases, which is the presence of string vessels in the brains of individuals in the neurovascular niche.

And so I did a quick online search, I said okay, I know it’s in neurodegenerative diseases. Has anybody looked to see what is specifically going on with the microvasculature, and have people observed string vessels in other infectious disorders.

A lot of what we are seeing in COVID falls into a generic category. You’ve got the hypoxia, you have hypoxic brain damage, you have a cytokine storm, you have enhanced reactivity in brain microglia. But the idea of specifically destroying a component in the neurovascular unit is something that I think may point a finger to a unique COVID associated phenomena.

And I said this months ago when we had our December conference. I said a lot of the things that I’m thinking about in terms of how we approach COVID are similar to what we see after the effects of chemotherapy and chemo brain. There’s a disruption somehow in the endothelial compartment that results in not only acute phenomena but persistent phenomena beyond the range of when we’re administering either the drug or the infectious agent.

And I really think that we need to be focusing in depth as neuropathologists on the specific things. We know the receptors for this virus are in the vascular tree. We know for a fact that they’re not expressed deep in brain parenchyma, while patients may have in rare occasions indications of a deeper-seated parenchymal persistence, and I don’t want to say infection, because there’s no evidence of brain replication. I think we really need to be focusing on that neurovascular unit to help us get to the truly COVID specific and dissect it out from what may be generic.

DR. NATH: That is actually very helpful. It is quite striking as to how the neurovascular unit turns out to be the most important thing. A number of other infections do the same thing, like dengue, Ebola, others. They attack the endothelial cells predominantly.

DR. MORGELLA: They’re a different mechanism though. What was fascinating to me were a series of patients we saw early in the pandemic that we reported and were reported also by our colleagues in Beth Israel, young individuals with no pre or disposing conditions who develop large vessel occlusions, transient clots in the internal carotid, large artery territorial strokes, no pre-extant conditions, clots resolved, and they’re left with a stroke. And what causes that transient change? It’s varicose triad, it’s got to be one of the three, and this virus we know is binding there. So I think that’s the tell for me.

DR. NATH: Either the virus or just the viral protein alone.

DR. MORGELLA: Either the viral protein or the antibody response, I have these memories of anti-idiotypes and what are we doing with our immunity to the immunity. There’s a lot of depth here we could really go into that’s COVID specific.

DR. MASLIAH: I think we should also add that it all depends on where are we looking, and I think we always think what is happening in the central nervous system specifically and in deep grey matter and white matter areas and so on. We heard this morning also there appears to be also some specific pathology occurring in the olfactory epithelium and the olfactory bulb.

At least to me the pathology that it was showing in the olfactory epithelium and the sustaining cells and so on appeared to be fairly COVID related, and at least the virus could be identified in that area. To me that’s what it looks so far the most neuropath virus specific.

But of course, as Sue mentioned I think if we had focused on the nervous system the vasculopathy seems to be a very interesting place to look in more detail. I think we could learn a lot more about vasculopathy looking at these brains.

DR. SOLOMON: What do you think about looking, like you say, the nose  provides a fantastic opportunity, as neurologists we tend to think of the retina as the window on the brain because we can actually see nervous tissue, but here the nose is acting like the front door in many ways, it’s a route in for the virus, and we have an opportunity to study the relationship between neurological tissue and the accessory tissues in people who are alive potentially because it’s so accessible. Should we be doing more of that, should we be doing more biopsies of people’s noses, would that be an approach?

DR. NATH: We’ve looked into that ourselves. It is not an easy thing to do. So I talked to the otolaryngologist, and what they said was that in order to biopsy the olfactory nerves there you have to have total loss of smell, because the complication rate is significant. If you just want to get nasal mucosa that’s not a problem, but if you need to go deep in there to get something then the complication rate is high enough whereby you cannot do it in asymptomatic individuals.

DR. SOLOMON: I guess even in people who have totally lost their smell you can’t be sure that they wouldn’t get it back over time.

DR. MORGELLO: And the risk/benefit with regard to whether this is truly SARS-CoV-2 specific. If you think about post influenza anosmia and nausea, we know that certain percentages of people who develop flu have the same phenomena, and in fact there have even been neuroimaging studies that have shown atrophy in primary and secondary regions in the brain after influenza.

So the studies beautifully displayed today showing that we’re not infecting the neurons, we’re infecting the sustentacular cells, which is a milieu of it’s creating a cytokine storm which then theoretically can be predisposing to neuronal atrophy or dysfunction or what have you. So I’m not sure, I agree that the nose is the portal, but it’s not the same kind of nose as the mouse nose.

So we know that when we put it in the mouse nose in the trans gene it hops on the neurons and it goes into the primary and secondary order phenomena, and in fact that was really interesting about Dr. Farhadian’s presentation today, their work on these trans genes and what’s actually going on in the mouse models.

And I would caution that group because these mouse transgenic models are really queer birds. Number one, the trans genes aren’t, Dr. Pearlman would probably be a better commentator on this, you don’t see any inflammatory response in these mouse models. It’s totally unlike the MERS DPP4 trans genes, where there’s an abundant immune response, you get vast influx of cells and multifocal distribution. The mouse ACE2 trans genes, you put the virus in the nose, it hops in the neuron, and it sequentially affects first and second order regions of brain. We’re not seeing that in people.

DR. NATH: That is very interesting. Let me go on to a couple of other questions that are popping up. One is for Dr. Masliah, talking about the loss of smell, that’s also a common symptom in Alzheimer’s and maybe in Parkinson’s patients and an early symptom. So what do you think is relationship between COVID and these diseases, and where are the gaps, where should we be looking to understand these relationships? The fear is maybe we’re going to have a large population where we have accelerated dementia, is that a reasonable concern?

DR. MASLIAH: That’s a very interesting question Avi, thank you. I think the key there would be from a technological point of view, the NIH have invested a tremendous amount of funds now on these RADx initiative that includes one for developing rapid test to not only detect but also to distinguish the loss of smell due to COVID versus other neurological disorders, including Parkinson’s, Alzheimer’s disease, and so on, which is really not easy. It might be possible with tests that now have much greater fidelity and ability to discern.

But what we know in terms of neuropathology and the olfactory bulb and nasal cavity in Alzheimer’s and Parkinson’s is that there is mostly considerable accumulation of misfolded proteins, including Tau in Alzheimer’s and alpha-synuclein in Parkinson’s disease, accompanied by microglia cell activation.

As you have shown in COVID we’ve seen olfactory bulb significant microglial cell activation, and there are some reports, not in human, there is one report in the nonhuman primate model of accumulation of alpha-synuclein in olfactory bulb and other regions, maybe that’s a possibility.

There is also the possibility that there is increased assimilation of amyloid, but it’s really hard to tell, I do believe that there is an overlap between some of the pathogenic mechanisms involving COVID-19 and some of the neurodegenerative diseases that might collapse or synergize the olfactory system and olfactory bulb. We really won’t know until all these more sophisticated tests develop, and we have more neuropath studies.

DR. SOLOMON: I just want to, I’d be interested in your opinion, or Eliza’s, or Susan’s, on these data. So clearly with this disease there’s not the kind of frank neuropathology that we see with a virus like herpes simplex virus or Japanese encephalitis virus.

But what do colleagues make of the data, and I know some of this has been discussed, showing the idea that virus gets in through the nose, through the olfactory neurons, and then apparently seems to spread to connected neuronal pathways. And yet is not causing a great amount of damage in these nerves, and certainly is not triggering masses of inflammation. So what do we think, what’s it doing there, why is it doing that, and are there other viruses that behave in similar ways.

DR. NATH: Let me take that question. I am not sure that I’ve seen any convincing data that the virus is getting into the olfactory nerves. The receptor is present on the sustentacular cells, not on the olfactory nerves. Now it’s true if you use some of the other coronaviruses, the OC42 or whatever, those are neurotropic, they will go through the olfactory bulb all the way to the brain stem. But this one doesn’t.

Now, it’s true you have pathology in the olfactory bulb, how do you explain that? One possibility is it’s not the virus, it’s the proteins that are going in and doing the stuff. And we’ve shown that with HIV, there is extensive literature, and most viruses when they replicate they produce a lot of protein, very little gets incorporated into the virus.

All the other proteins get thrown around. I mean DP120, CAD, all these things are toxic, why wouldn’t the other proteins produced by the virus do. So I think it may be an indirectly mediated mechanism, the evidence that you have profound virus traveling through the brain I’m not too sure about that, people may feel differently.

DR. SOLOMON: The paper by Dr. Mineheart and colleagues in Nature and Neuroscience, which again apologies I’ve had to dip in and out of the meeting a little bit today, so I haven’t been able to attend every bit of every session, but showing virus particularly in pathways that are connected. Do you, I think there was some PCR data and some immunohistochemical data. Do you think the jury is still out a little bit on that?

DR. NATH: I think there was a presentation, and they showed that yes, you can find small amounts of virus in small numbers of individuals. We’ve looked very extensively ourselves, we did not just PCR, we did in situ hybridization, we did RNA seq, and now we did hybridization, you pull down and then sequence it, we still can’t convince ourselves. The small amounts of virus you can find, the same thing with for example HIV, you can find small amounts of virus in the brain, it doesn’t explain the pathology, because the pathology is extensive.

DR. MORGELLO: I tend to agree with Avi, but I’m trying my hardest to maintain an open mind. Number one, there are other viruses that do this. A classic one is rabies. And what you see is you see profound neuronal function and death. There’s no inflammatory response, and yet you see trans-synaptic presentations of this virus traveling along. We know that the mouse transgenic models show this, we know that there is a sparse literature in SARS that theoretically demonstrated multimodal, in situ, electron microscopy, the same phenomena.

And we know that there are some case reports of individuals, typically in the fingers of one hand they have seizures, they get a lumbar puncture, it’s acellular, but they have virus present in the CFS in the absence of cells. And for me this sort of pricks up my ears.

I say okay, so maybe there is a tiny fraction of individuals, and what the predisposing conditions are are unclear, but the fact that there are some human case reports with seizure activity, which might induce a neuronal vulnerability state, or something that there’s a first hit and maybe there’s a second hit, I don’t want to totally get rid of it, I don’t believe that’s what’s happening in 99.9 percent of people, but I think we have to be open minded to that small percentage, only because we have a mouse model that says something, and we have these odd case reports where you’re seeing virus in an acellular CSF.

And in fact when I saw that I said oh my god, when you think about the international encephalitis consortium guidelines for what we call encephalitis versus what we might jump into encephalopathy, one of the minor criteria are cells.

DR. NATH: I’d be a little bit careful of making too much from a single case report. The vast majority of people looked at the CSF of people with all kinds of encephalopathic symptoms and haven’t found anything in the CSF.

DR. MORGELLO: I agree, but Dr. Gisslen today there were three cases, I saw those three – I’m trying to be open minded. I’m with you Avi. I said to myself –

DR. NATH: You can find an exception to everything.

DR. MORGELLO: Exactly. And sometimes the exceptions are helpful and sometimes they’re not. I think they’re probably not in the context of the masses of people who are suffering from this disease, but I think that we do have to allow them --

DR. NATH: If you look at rabies, it does not matter if you look at one or 100 of them, you find 100 percent of them have virus in the brain. You won’t find a rabies case without the virus in the brain.

DR. MORGELLO: Again it is not going through the nose.

DR. NATH: However it’s getting there, if you have viral encephalitis, it’s getting there, it’s loaded into the brain.

DR. MORGELLO: I agree with you there 100 percent.

DR. MASLIAH: Just keep in mind that I think a big topic of today’s discussion was heterogeneity of the acute COVID, and probably also the post-acute COVID, very heterogeneous disorder. It’s not like case of rabies or all the neuroinvasive disorders where there is a clear pattern. Here I think we have many different patterns.

Probably these differences in results between labs in terms of finding or not COVID in the brain might also be related not only to methodological, technical, et cetera issues, but maybe also to heterogeneity of disease and talking about different conditions. I think the jury is still out, we need to look into it. But I agree with you Avi, I’m more in the camp that it is not a neuroinvasive virus.

DR. NATH: There are two people who asked the same question here, so maybe we should move onto a slightly different topic, and that relates to dysautonomia. And as being a major post COVID complication of the illness. Would anybody like to comment on that as to what kinds of dysautonomic symptoms are we seeing and what might be the underlying pathophysiology? Maybe Tom you want to start with it.

DR. SOLOMON: This is one I was happy not to start with, I’ll just tell you why. We mostly focused on acute disease I guess because we all started early with acute disease, and there are other colleagues in the UK doing some of these large studies looking at the long, at some of the range of problems.

DR. NATH: Maybe I’ll take that. We have done a fair bit of work on it ourselves. So we are putting together a cohort of individuals where we primarily want to study dysautonomia, and the few that we’ve studied were quite struck by the fact that really they do have all the criteria for POTS So you stand them up, their heart rate goes up, the blood pressure will fall, and it can be erratic after a period of time.

Now one has to be a little bit careful because you can also get myocarditis and you can get AV blocks, primary and secondary AV blocks and first degree and second-degree AV blocks in these patients. So one has to make sure that you’re not seeing an overlap in cardiac syndrome. And then Dr. Folkerth even showed that there was inflammatory infiltrates in some of the sympathetic ganglia. So there could be reasons for that.

We looked at some of the carotid bulbs and we found inflammatory infiltrates there too. So there could be multiple factors as to what causes dysautonomia in these patients. And besides blood pressure changes they can manifest with constipation, loose stools, there can be GI symptoms, there can be low grade favor, or they can form a Reynolds type phenomenon, the patients complain of tingling in their hands and feet, it’s not a neuropathy but actually it is a vasoconstriction.

DR. SOLOMON: I’ll ask a question Avi. Obviously we are getting interested in the complications of vaccines. As you know in the UK, I guess because we have been using a number of the adenovirus vector vaccines, seen a lot of the thrombosis, thrombocytopenia patients, but what about patients with dysautonomia after vaccines? I’ve seen one or two people who may have had that.

DR. NATH: We’ve seen a number of them, have a collection of 30 patients with dysautonomia. They usually have complaints suggestive of a small fiber neuropathy in addition to that, they go hand in hand. We’ve done skin biopsy on at least four of them. Interestingly the skin biopsies were normal. But maybe it’s too early, it will take a while before we see the pathological changes in the nerves themselves.

Anecdotally we’ve treated a couple with IVIG, they did show improvement for whatever it’s worth, it’s an open label treatment, so I don’t know what to make of it. Some patients on the outside have been treated with cortical steroids, and they’ve reported variable amounts of improvement. So my guess is it’s likely an immune mediated phenomenon, secondary to the vaccine for some reason. But it is responsive to immunotherapy.

I think we’re over the hour, so I want to thank each one of you and all the speakers today that did a phenomenal job, and especially for those overseas who have been here late into the night, I look forward to seeing you again tomorrow. Thanks again. And thanks Dr. Spudich for carrying the entire session, you did a phenomenal job, not easy.

DR. SPUDICH: Thanks, Avi. I think we’re closing out the meeting for today, and we’ll be inviting everyone to join tomorrow morning at 10:00 AM US Eastern Time for another action-packed agenda. But thanks everyone for joining, and thanks for the terrific speakers and panelists. That last panel was really fabulous. Thank you.