Dimorphic Fungi: Coccidioidomycosis
12/1/2012
Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide
short discussion of current topics and may be helpful to you in your practice. Our
speaker for this program is Dr. Glenn Roberts, a Professor of Laboratory Medicine and
Pathology and Microbiology at Mayo Clinic, as well as a consultant in the Division of
Clinical Microbiology. Dr. Roberts discusses disease-causing dimorphic fungi and how
to identify them in culture. This presentation examines Coccidioidomycosis. Thank
you, Dr. Roberts. Thank you, Sarah, for that introduction.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
I have nothing to disclose.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
The diseases caused by the dimorphic fungi include the ones you see on this
particular slide. The first three are seen most commonly in North America and
Paracoccidioidomycosis is seen in Central and South America. The others that you
see here, Sporotichosis is seen in the US. Penicilliosis is seen pretty much in the Far
East. These are things that we will see in the clinical laboratory.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
They are caused by fungi that are called dimorphic fungi. And that is that these
organisms happen to have two growth forms. They have some particular features
that we will go through that are in common to all of them and the growth rates are
relative and that depends upon how much organism is present in the clinical
specimen that you start off with. It depends upon the culture medium that you use
for recovering the organism and it depends upon how much organism is present in
the clinical specimen as well as the metabolism of that particular organism. One of
the things that helps in some instances but not all, is blood enrichment and we
include a medium most of the time that have a blood enrichment source so that we
can pick up some of the fastidious organisms. The microscopic morphology, there is
one thing that’s in common with all of them and that is that they produce small
septate hyphae. There are a few other things like dermatophytes that can do the
same thing but by and large, the dimorphic fungi are the ones that produce these
hyphae. We do have molecular methods that are available for identifying these
organisms in addition to using traditional methods. We have nucleic acid probes, we
have nucleic acid sequencing, and MALDI-TOF that is now being used in the clinical
laboratory that will identify these organisms much more easily than traditional
methods.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This image shows you the small hyphae. It is hard to tell on here if they are septate
or not but they are septate and very tiny in size, like 0.5 microns.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
The dimorphic fungi have colonial morphologic features that vary depending upon on
the isolate and also upon the medium in which these organisms are recovered. So
then you can have one organism that you can place on two different media and they
will look differently on either.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
The colonies of Histoplasma and Blastomyces are pretty much indistinguishable from
each other. The colonial morphology sometimes is not helpful at all in terms of
identifying these organisms
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This particular discussion is going to focus on Coccidioidomycosis. We will talk about
the organism Coccidioides immitis/posadasii. Coccidioides immitis is an organism that
we have been dealing with for many, many years and it’s been found that there is
another strain that it genetically differs from Coccidioides immitis, it’s called
Coccidioides posadasii. And it doesn’t differ in terms of its colonial morphology or its
microscopic morphology but rather its nucleic acid makeup. Where do we find this
organism? It is found in the southwestern part of North America and also in Central
and South America.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This slide here shows you a rectangular box that includes primarily the areas where
you see Coccidioides immitis in the United States. It goes from western Texas to all
the way to northern California and that’s pretty much where you’ll find it. You don’t
generally find it any place else. There are times whenever you can recover the
organism from patients who happen to be in an area that is not endemic for
Coccidioides and it may have something to do with the fact that they travel there
during the winter months, came from a cold area and traveled there during there in
the winter months and came back home with it. Sometimes there are fomites that
have been sent from one part of the country where Coccidioides is endemic, sent to
another part of the country and someone opens a package up and they get exposed
to the spores of these organisms.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is a map that shows you kind of where it’s found in the world. Coccidioides is
found as you can see in Northern California all the way to Western Texas and down
into Mexico. There are a couple of little black dots in Central America. Honduras is
one of those places where you see it. I happen to visit there a few years ago and they
see it every day, people walk in with the disease. So we know it’s found in Central
America and we know it’s also found in South America, pretty much in every country
except Chile.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
Where do you find this organism? Well, we know where this organism is found. It’s
found in the soil of the areas that typify what’s called the Lower Sonoran Life Zone
where the conditions are semiarid and dry. The temperatures reach 100 degrees or
more Fahrenheit during the summer months and then in the winter, temperatures
falls all of the way down to freezing or sometimes even below that.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
The soil has a very high alkaline and salt content. And if you look what grows there
you will see that its vegetation is short stubby type shrubs like the creosote bushes,
mesquite trees that are not very big, cacti, and particularly the saguaro cactus and
yucca plants. It’s kind of the typical environment that you see in Arizona and
southern California. And a lot of times there are a number of animals, small rodents
that burrow down into the soil and sometimes they’re actually the culprits that bring
this organism to the top of the soil. They have let the wind carry it along or let us
pick it up whenever we happen to be working the soil.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is an example of the Lower Sonoran Life Zone. You can see that the saguaro
cacti, they are the kind you see in the old western movies and this is the type of
environment where you find Coccidioides. And people go out hiking in those areas,
they go out poking around in this soil looking for something or the ones that get it are
people who go on archaelogic digs are at high risk for acquiring Coccidioidomycosis in
an environment like this.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
What happens to this organism? Well, it disappears from the soil during the wet
months. It goes down a little bit deeper in the soil and then when the summer
months and spring months come, the soil begins to dry out and the organism sort of
comes to the top and then the winds disseminate these spores that are the infectious
particles of this organism around in the environment and they are there for us to
inhale for small rodents to inhale. Anything that happens to be around can actually
become infected by this organism. So anything that disturbs the soil including the
wind is what’s going to disseminate this organism throughout the environment and
make the spores available for us to inhale.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
What kind of activities are associated with acquiring this infection? We kind of
already talked about a little bit about that, but, construction out in areas where this
organism is endemic as a very substantial source for getting infection. The
archaelogic digs are another thing we talked about a few minutes ago, here, that it is
responsible for causing great number of infections. But, people work in agriculture in
parts of Arizona and California who are out picking all sorts of vegetables and so on
that disturb the soil and actually inhale this organism. And then one of the big things
that spreads this to huge numbers of people are dust storms that you see in the
western part of the country that blow these spores all the way up to northern
California from southern California and even further away than that.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is an example of an advancing dust storm. The photograph was taken in Kern
County, California and this is in northern California where they hadn’t seen this
organism. They woke up in the morning and they found their cars were just covered
with dust. Well, what they didn’t know was not only was it covered with dust but it
was covered with arthroconidia of Coccidioides. And so there were many cases of
Coccidioidomycosis in Kern County as a result of this dust storm that was seen there.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This schematic shows you kind of what happens. Lower left hand side what we see
are hyphae that are broken down into these arthroconidia. Artho means jointed and
you can see that the spores there after the first arrow going from about 11:00 o’clock
pointing to the right, you see these arthroconidia. What happens is the arthroconidia
are formed and as they grow, there becomes a dead space in between the
arthroconidia. They are called alternate arthroconidia. What happens is that the wall
behind each of these arthroconidia breaks apart from the dead cell between them.
And it gives the appearance of little wings on them and allows them to float in the
environment for a long period of time. So, on this schematic we don’t necessarily see
alternate arthroconidia but I will show those to you as we go along. But what you see
up there is saguaro cactus and so on is like the Lower Sonoran Life Zone. These
things are aerosolized by whatever source, whether it is the wind or whatever it is.
We take it into the lungs and you see on the right lung what happens inside there is
the spores come in, those arthroconidia come in. They then convert to round cells
that are not yeast cells like we see with all of the other dimorphic fungi but they
convert into rounds cells that are called spherules. And in time these cells began to
produce what are called cleavage furrows, you can see in the lower middle of the
diagram where you see these cleavage furrows and then in turn forms these
sporangia, some people call them sporangia spores but they are really called
endospores for the most part. And those are things that then begin to develop into
new spherules inside the host and the cycle goes on. And so when you take a biopsy
from one of these patients who has active disease with Coccidioides immitis as the
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
cause of agent, you see these spherules in all stages of developments and you will
see endospores in there in some of the cells you look at. At sometimes you see
spherules that are just simply empty. But you have to look at the whole slide.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
Here’s what happens, we have kind of already alluded to this. The hyphae begin to
break down into rectangular arthroconidia. These things are easy aerosolized and
once they are taken into the lung they begin to transform into these spherules and as
these spherules begin to develop they begin to replicate internally by a process called
progress cleavage and this divides this internal cytoplasm into what are called
endospores inside this spherule and then as time goes along and these spherules
begin to mature, they break open and the endospores are liberated and they form
more spherules. And the cycle goes on.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
What are we going select for culture and for direct microscopic examination? Well,
we have a number of sources that we can find this organism in. One’s a lower
respiratory tract like most all of the dimorphic fungi have where they cause a
pneumonia, most of them do, not all. We know that with Coccidioidomycosis that
sepsis is a common manifestation, disseminated disease. Coccidioides involves the
bone. It causes lesions in the brain. It can be found in a spinal fluid, meningitis is a
common feature of Coccidioidomycosis and it’s also a very serious one. You could
find it when the skin and also the mucous membranes, particularly the oropharynx.
You could find it causing arthritis and you can find it causing an abscess of the
subcutaneous tissues. So pretty much, you can find it almost any place.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
So what are you going to see if you look underneath a microscope at a specimen that
may have Coccidioides immitis in it? What you are going to see are these rounded
spherules, some of them may not have any endospores in them at all. Others may
look like what you see here using Calcuflour white, you see the large cell that is the
spherule and coming out of that ruptured spore are all of these endospores that if
left in the patient would then begin to turn into individual spherules in time.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is an example of a biopsy. This is the H&E stain showing you a large spherule
containing numerous endospores and the H&E stain shows them pretty well. You can
see it also on a methenamine silver stain well.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is a culture of Coccidioides. And one thing we do know about this organism in
terms of culture is that for the most part it grows more rapidly than all of the other
dimorphic fungi. It’ll grow up within two or three days and form arthroconidia in a
time or a little bit close to that time or a little bit longer than that. But the colony
begins to appear in two or three days. As it matures, it will form these arthroconidia
and I can’t give you a time as to how long it takes to form them but I would say four
to six days the arthroconidia are going to be there. And so this is the most infectious
of all the dimorphic fungi and all of the fungi period to handle in the clinical
laboratory. There have been numerous reports of cases of laboratory acquired
infections and death caused by this organism. So, it grows up as a fluffy white mold
for the most part but if you look at photographs of a hundred isolates of this
organism, you probably would see a hundred different morphologic features on these
colonies. They can be yellow, they can be brown, they can be pink, they can be red,
they can be white, tan, all sorts of colors. When you get through with this thing, you
may have said to yourself, “Geez, I wonder if I threw one of those things out,”
because, it does happen.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is another example of colonies of Coccidioides immitis or posadasii. What you
see here are two colonies side by side and they are heaped up in the center. And
that’s a common feature for some of these isolates that we see. They are domed
colonies.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is a colony on a blood-containing medium. And it almost looks like it has a little
bit of a green color to it. Sometimes you see that this blood enrichment is helpful for
recovering Coccidioides but not as helpful as for some of the other fungi.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is one growing on a blood agar. And you can see that in the center it’s domed.
And in the periphery is kind of more, more fluffy. And this is an example of those
domed colonies we talked about.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is another example of what we see with Coccidioides and we kind of refer to
these things as cobweb type colonies. What happens is the center begins to kind of
domed up and the periphery begins to be more adherent to the agar. If you see a
colony like this, it’s pretty representative of Coccidioides immitis/ posadasii.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is an example of a plate where we used Cycloheximide to recover for the
organism. All the colonies there are Coccidioides except for the one at 3:00 o’clock
and that’s probably penicilium or some other saprophyte that we had happened to
get into the culture plate. You can see those domed colonies, you can see the kind of
cobweb type colonies that we were talking about and this is common to find these.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This next slide shows you a plate of blood agar that was used in the bacteriology
laboratory. Those three colonies are Coccidioides immitis. What you don’t want to
do is open this plate up and the bacteriology laboratory without being inside of a
biological safety cabinet. For the most part, the colonies may not have had time to
mature when they begin to look like this. On the other hand, I wouldn’t trust that
even if I said it to myself, because I think that there are exceptions to all of this and
what you don’t want to do is to have a laboratory acquired infection and get a big
dose of inoculum from opening up a plate like this. What you do is you open up
underneath a biological safety cabinet.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is chocolate agar showing you the colonies of Coccidioides immitis that grew up
in a bacteriology laboratory. The rule of thumb is that no cultures of molds should be
opened up in the room. They should be opened up but not on the bench, they
should be opened up in the biological safety cabinet.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
These are some colonies I am going to show you that vary and they are just different
isolates. This one looks almost grey. Can you see this center is kind of domed up?
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
And this is an example here of some of the colonies you have already seen but you
show them on three different medium. The top two plates containing blood
enrichment and the bottom one is Sabouraud’s dextrose agar. And they look different
depending on the three plates. They are all the same isolate.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is the example of four different culture media, same isolate. And you can see the
two blood containing media look about alike and also the two non containing blood
medium look a little bit alike. You have to become familiar with what these
organisms look like on different culture media to be able to recognize them, at least
suspicion of what they are.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
Now the next slides I am going to show you are maybe a little bit surprising to some
of you who are looking at this because there are many colonial variance of
Coccidioides immitis/posadasii. I’m just going to go through them and you can look at
them. This one looks very glabrous. Not really, yeast-like but just kind of
membranous.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This slide shows you again another one of these colonies that looks membranous and
this is Coccidioides immitis and it doesn’t look like what we talked about earlier.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This one including all of the colonies except for one, the large colony about 3:00
o’clock is all Coccidioides immitis. It doesn’t look white and fluffy or membranous
either.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This one looks like something you would probably throw away except for the tiny
colonies there that are bacteria. This is all Coccidioides.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
And this one except for the large colonies there that are yeast, its Coccidioides. It
almost blends in with the culture medium.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
And this is Coccidioides. It doesn’t even look like anything that’s described in
textbooks.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
There are a lot more variance of Coccidioides that we could show you and again, I
keep saying this but you wonder sometimes how many of these did we throw away
because we thought they didn’t look like anything that we should worry about. How
are we going to identify Coccidioides immitis? We are going to look underneath the
microscope at the microscopic features of this organism from the colonies and you
are going to start off by noticing one thing that’s in common with all of these
dimorphic fungi and that is they have small delicate hyphae present. With
Coccidioides, we find some structures in there that we don’t see with some of the
others. They are called racquet hyphae and they look like tennis racquets. It looks
like a swollen area that may be at the end of the hyphal strand or in the middle of a
hyphal strand. It’s produced in these young cultures. And they are just called racquet
hyphae. What we see though, that are characteristic for this organism are alternate
arthroconidia. The arthroconidia that join its spores, they produce an arthroconidium
and they produce a dead space which is called a disjunctor cell and then another
arthroconidium and it just keeps on going. So they are called alternate arthroconidia.
And one thing that is important to look at because there are lots of these fungi that
produce arthroconidia, one thing to look at is to look at the size of the hyphae that
are producing these arthroconidia. The hyphae are a whole lot smaller than these
arthroconidia, so the hyphae that produce the arthroconidia are smaller. These
arthroconidia can be barrel shaped and a lot of times they are that. They can be
rectangular or even rounded in shape. You generally do not see spherules or
endospores on culture medium. You see these alternate arthroconidia
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
On this image you can see these are the racquet hyphae. These are the alternate
arthroconidia that we are talking about but these structures here are the racquet
hyphae.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
There are racquet hyphae on here that are not as good and you can see that they’re
the kind of racquet shaped structures on there but they don’t all appear to be
textbook perfect, I can tell you.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is another example of racquet hyphae and you can see them scattered
throughout there.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
This is an example, now, of the early development of the arthroconidia. If you look at
the hyphae of Coccidioides in there, you notice that the hyphae themselves are very
small. But look at what’s beginning to develop in those. And those are your
arthroconidia that are kind of barrel shaped. They are much larger than those hyphae
that produced them and that’s what you look for when you are looking for
Coccidioides, that and the fact they produce these alternate arthroconidia.
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Dimorphic Fungi: Coccidioidomycosis
12/1/2012
Now this slide here shows you the alternate arthroconidia. The arthroconidia are the
rectangular cells and then there is a clear space in between them. The clear space is
called a disjunctor. What happens is the cells break apart and that clear space and as
they do that, they take a part of the wall of the cell behind, the disjunctor cell behind
them and on either side actually, and it just adds a little piece of disjunctor cell on
either side of it, the arthroconidium, and it allows it to give some lift to these spores
as they float around in the environment.
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Dimorphic Fungi: Coccidioidomycosis
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And this slide, you see a lot of arthroconidia. But you don’t necessarily see a real
prominent clear space in between them. So you have to look at everything and you
have to look at them closely to see if you have alternate arthroconidia or not because
there are lots of organisms that actually are found in the soil and that part of the
country where you find Coccidioides that may be a bit confusing.
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Dimorphic Fungi: Coccidioidomycosis
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Here’s a very good example. It’s an old slide from the Centers for Disease Control
that shows these alternate arthroconidia on the strand at the bottom you see there’s
a rectangular spore, some that look a little barrel shape, there’s a clear space
between them. That is what we talk about with mature alternate arthroconidia.
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Dimorphic Fungi: Coccidioidomycosis
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And here you can see that this disjunctor cell and then the arthroconidia on either
side.
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Dimorphic Fungi: Coccidioidomycosis
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