Learn about the evolving Ebola epidemic and its various aspects including disease prevention, management and treatment, response to the epidemic, ethical considerations, and the post-Ebola global health landscape.
Laboratory Testing, Pathogenesis, and Pathology, Part 2
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From the course by Emory University
Ebola Virus Disease: An Evolving Epidemic
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From the lesson
Laboratory Issues
Upon completion of this module, learners will be able to: Discuss clinical laboratory considerations for Ebola Virus Disease.Differentiate between persons under investigation and persons with confirmed EVD. Identify where and how EVD laboratory testing is performed. Recognize the safety concerns surrounding the disposal of specimens and human remains. Explore the immune response to EVD and discuss the implications for future therapeutics and vaccines.
Meet the Instructors
Dabney P. Evans, PhD, MPHAssistant Professor
Hubert Department of Global Health. Rollins School of Public Health. Director, Center for Humanitarian Emergencies.
Carlos del Rio, MDChair
Department of Global Health at the Rollins School of Public Health
So lets talk a little bit about parthenogenesis and pathology,
that means how the virus really causes disease in patients.
So the virus contains multiple proteins, it has the nucleic acid in the center,
and then it contains a variety of proteins that basically cover
the nucleic acid material.
And then, it has proteins that allow the nucleic acid to be
replicated or create more virus.
And it has a covering of the virus.
So, some important proteins to remember are the VP35,
which is a polymerase cofactor that the virus has.
Another protein, which is a matrix, a VP40 protein.
These two proteins are very interesting because as soon as the virus gets into
the cell, they basically tell the cell to stop producing
any other things that they need to be producing.
And it basically stops the production of one protein that's very
important that fights the virus, which is called interferon.
So both the BP35, and the BP40, basically tells
the cell that has been infected by the Ebola virus not to produce interferon.
The interferon is the substance that
basically signals other cells to come fight the virus.
So the virus is basically saying, I want you to reproduce me,
but don't let other cells come and get rid of new cells that I already infected.
The other protein that the virus has that also helps
the virus to continue to reproduce, and to continue to produce more viruses,
and to escape the immune mechanisms of the cell,
is a protein that's called soluble glycoprotein, or SGP.
This particular protein, basically, the virus produces more than it needs.
This protein is usually lining the virus covering.
And by producing more of this protein,
more of this SGP, the antibodies that
the human body is gonna be producing are gonna continue to be produced.
But they are gonna be attracted by this soluble protein.
And so, they are not gonna attach to the virus itself,
because they're being attached to the soluble protein.
And so the virus can then be released into the environment, and go and
infect another cell.
So the virus has several mechanisms that allow it to be getting to the cell,
stop the cell from producing substances that will stop the virus from growing.
And produce more virus, but produce extra other proteins that attract antibodies.
And so, the virus can go from one cell and infect another,
and reproduce in other cells.
Some studies that have been quite interesting
have studied what we call cytokines.
So, cytokines are substances that are body producers,
are lymphocytes and are cells that are fighting the virus.
Like interferon, alpha, interleukin-6, or interleukin-8.
All the substances that we call cytokines.
They have been measured in people that have had Ebola infections.
So in some of these people, those that have survived usually have very
high responses of interferons, IL-6, IL-8.
While people that end up having fatal disease, or fatal outcomes,
are those that have very high interleukin-10.
And this is important, because then you can sort of define
who are gonna be the survivors, and who are not gonna be the survivors.
So these are interesting studies that have shown us how the survivors actually
respond to the virus, and what they do to basically survive the disease.
So the other thing that people have been doing is what we call animal models.
So they take different, either mice or monkeys, and infect them
with Ebola to see how they respond and how what I've been describing about,
the interferon, and the interleukins and all this.
How it's working In mice or in primates,
to define how we can do better, and how we can create vaccines.
And what are the good things that, for the proteins, that we can put into a vaccine
so that they can help the person survive.
So what is interesting is that the rodent models,
like those done in hamsters or mice or guinea pigs, when you put Ebola,
the one that is circulating right now, they don't produce disease.
You have to adapt the Ebola virus to be able to cause disease in mice.
And this, if you remember, there was the case in Spain,
where there was the nurse had a dog.
And she got infected by one patient that they had over there.
And there was a big uproar because the authorities wanted to kill the dog.
Because they knew that the dog was infected.
Well, the dogs, the same way as mice, they can be infected but don't show disease.
So that's something that is interesting that in some animals,
the what we call the wild virus, the virus that is circulating,
doesn't really cause disease, they need to be adapted to the animal model.
That is not the case with primates.
With monkeys, baboons, macaques, you can use the wild type virus, the one that is
circulating, and it will cause basically the same disease that it causes in humans.
And actually it's really interesting, if you look at the literature and
veterinary literature, there's been some huge outbreaks of Ebola
in colonies of monkeys and gorillas that have
really decimated those populations because they obviously don't have the treatments.
And these animals die, and
the whole colony could be wiped out due to infections with Ebola.
So in pathology, these animal models, where do we see the Ebola virus?
Using what we call immunohistochemistry,
where you take an antibody against the virus.
And this antibody has some color that you can see under the scope.
We've seen the virus in many different organs and tissues.
We see them in the liver.
We see them in the spleen.
We see them in lungs.
We see them in kidneys.
So we see it basically all over the body in these animal models and
there's necropsies.
Now, in humans, we really have not done autopsies in those patients.
There was a study that where they've taken liver biopsies,
like fine needle liver biopsies, and a little
piece of adrenal came off on one of those needle biopsies, and they've taken skin.
And they've found the virus in many places.
They found it in macrophages.
They found in lots of liver cells.
And the liver cells, the machinery of liver cells,
is basically taken over by the virus.
And instead of working in favor of the human creating
the things that it needs to create, like albumin and the different things that we
need to survive, basically that machinery's completely gone.
Taken over by the virus.
And the virus just keeps on producing more and more virus.
And you can see the nucleus of a cell.
You can see the cytoplasm of the cell.
And you can see these big blobs of what we call eosinophilic,
or translated into pink material.
That is all, when you look at it under eletromicroscopy,
it's all viral particles that are being mass-produced by the liver cells.
So eventually, those liver cells die.
But in the meantime, they've already produced so many viruses
that have infected other cells that include the other liver cells, but also
macrophages that will take the virus from the liver into other parts of the body.
However, we've never been able to, since we've never done
a whole full autopsy in these patients, we don't know which other parts of the body.
We assume that it's gonna be very similar to what we see
in the necropsies from primates.
Where you can see the lung, and the spleen and the kidney, and all these other parts.
So, as I said, the study with immunohistochemistry,
also looked at skin biopsies.
And you can it see in the skin biopsies, you can see it in the blood vessels,
inside the blood vessels, in the endothelial cells.
You can see it, even in the proteins, are in the matrix itself of the tissue.
So it's very impressive, the amount of virus,
or material that this virus produces in the patients that have died.
And this, the skin biopsies, if you have a patient that died,
and you weren't able to do the testing using the PCR with blood,
you can take a skin biopsy, and
then test it using immunohistochemistry, or using PCR.
And then, see if the patient had died or not of Ebola.
And what is nice about using the scheme biopsies,
and doing immunohistochemistry, is that you don't need to have fresh tissue.
Fresh tissue is very infectious,
because the biopsies are gonna be fixed in formalin.
And the formalin basically kills the virus.
You don't need to use infectious material to do the testing.
All of the testing is basically done after it's has been fixed.
It can be done in the open, and in any laboratory that does immunohistochemistry.
So what do we do with patients that have died?
Patients that are deceased?
Well, the handling should be kept to a minimum.
And as you know, that's one of the issues that has basically spread the disease so
much in the countries where the outbreak is going on right now.
It's that one of the rituals they had is as soon as the patient died,
they would bathe the body of the patient.
And then the people that had taken care of bathing the body,
they would bathe themselves with the water that had been used.
Well basically,
the people that died are the ones that have the highest amount of virus.
And then you bathe yourself with this amount of virus.
People then obviously, got infected just by doing that.
So one of the things that needed to be done,
is to basically stop that type of ritual.
And basically stop handling the patients.
It should be kept to a minimum.
And if the patient was intubated or if they put an intravenous needle to put
fluids in the body of this patient, they should keep the needles in place.
They should not be trying to take those things out or anything like that.
We don't want to take something out and create an aerosol.
And then you have to put that body in a plastic shroud.
And this plastic shroud has to be leak-proof and zipper.
And then you put the body in the bag, inside a second bag.
And in between those bags you put absorbent material with bleach.
So as you can see, even with a tube of blood,
as we talked about at the very beginning.
When you're taking that tube of blood and
you're sending it to the CDC for testing for
Ebola, or to the state, that you have to clean it and put it an a container.
And then clean the container, and put it into another container.
The same things happens with a whole body, that you have to
basically put the body without handling, no cleaning, just the body.
Put that into a bag, and then put it into another bag.
So taking many precautions so
that there's no leakage of material into the surrounding areas.
And then, one thing that should not be done is embalm,
which really hasn't been done in Africa.
They don't really embalm deceased people.
But they, in the States what they say is, cremate, which is the best way.
However, they don't have crematoriums so that's also a moot point, but
if a patient were to die here, we would cremate that body.
And the body needs to be buried as promptly as possible.
And handling it with the most, you know, the people that are handling that body.
Again, these are the most infectious patients.
So those bodies have to be handled with all the proper equipment.
The protective gear that needs to be done.
And when people ask if we should be doing autopsies on those patients, say if
a patient here in the United States that has Ebola died, would we do an autopsy?
We can do an autopsy, it would have to be a limited autopsy.
Basically using only the fine needle aspirates and skin biopsies.
But this would have to be in a very contained environment,
and knowing that autopsies can be quite bloody.
And so, you want to limit anything that is done there.
And you don't You don't want to do it just for doing the autopsy.
You want to do it in conditions that are very strict.
And you would do, if you are suspecting Ebola in a patient that dies, but
did not get a diagnosis, then probably the best thing would be to take a skin biopsy.
And then do the immunohistochemical assessment.
I hope this lecture has been useful.
We talked a lot about the different things things that you do with these patients,
the testing that needs to be done.
Not only for diagnosis, but also for
the metabolic diseases, or the metabolic arrangements that they have.
We talked also about parthenogenesis,
we talked about how to handle those specimens.
And I thank you very much for your attention.
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