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Identification of Melanized (Dematiaceous) Fungi Part 3

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Published: December 2013

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Melanized fungi, those with dark pigments in their hyphae or conidia, cause several significant diseases including phaeohyphomycosis, chromoblastomycosis, and mycetoma. Correct identification of the causative fungi is critical to appropriate treatment. This is the final presentation of a 3-part Hot Topic in which Dr. Roberts describes the characteristics of melanized fungi and the steps necessary to identify the specific fungi present. Part 3 focuses on polymorphic fungi.

Presenter: Glenn D. Roberts, PhD

  • Professor of Laboratory Medicine and Pathology and Microbiology at Mayo Clinic

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This discussion will be about the melanized fungi, part 3 the polymorphic fungi.  These primarily represent the slower growing of these melanized fungi and just to recap what these are, these are the fungi that contain a melaninlike pigment and they’re pigmented fungi because of this and the pigment may range anywhere from very light pigmentation to some that’s dark brown to dark black, and even the hyphae and the conidia can be producing this dark pigment.  And you see kind of dusky brown to dark brown hue to those.  Sometimes it is very difficult to find this pigmentation but I think for the most part you would be able to recognize it.  They’re both rapidly and slow growing organisms in this group and we’re going to talk about the slow growing ones here, and the conidia vary in appearance from single to multicelled.  And you will see in this group, they even arrange to do some different things because they have more than 1 spore.  Polymorphic means they have more than probably 2 types of spore production.  So, you have to get used to looking at this group.  And one thing I think we need to remember again, and it's kind of tough to do that, but you look at this organism and if you see hyphae that are pigmented and there are some that are hyaline, it’s considered to be a hyaline mold and it’s not going to be dematiaceous and you aren’t going to see all of these spores that we keep talking about.

So, we’re going to look at the organism, look at the growth, we’re going to look at them underneath a microscope, we’re going to first of all, we’re going to look to see if it is slow growing or rapidly growing and you can tell that by the colonies and the time that it takes for them to grow.  Look at them underneath a microscope make sure that you see the conidia and how the conidia produced.  And then there’ll be times when we’re going to need to do some other things.  We’ll have to do some ancillary tests on a couple of them to be able to tell what the organism is, maybe even some temperature tolerant studies.  And we need to correlate these organisms with a clinical specimen that they came from.  If you can, you can hear about the clinical scenario to know if they’re significant or not and really if it’s possible, what you can do is to get the biopsy and take a look at it and you’ll see these pigmented hyphae in the biopsy.  They’re pretty obvious for the most part.

This organism here is Scedosporium Complex.  The name has been changed so many times that it’s hard to even remember what they are.  So what we’re going to do is we are going to be just kind of generic.  We’re going to say that this organism used to be called Scedosporium apiospermum and I still have that name on a couple of them, but there are some other species of Scedosporium and we’re going to call it Scedosporium Complex.  What we’re looking for with this organism are conidia that are pear-shaped, that are born on short to long stalks.  If you look down in the lower right-hand side about maybe 5 o’clock you will see up in there, there is a conidium that is sitting on the top of a long stalk all by itself.  And that’s what we’re looking for.  These are the conidia of Scedosporium and when you look at them underneath the microscope, sometimes you’ll notice there is a little vacuole and a little bit of brown pigment sitting inside of them when you see them.  So, you’re looking for conidia that are long to short stalks and they’ll be pretty obvious when you see them.

Here is a better view of it.  And these conidia are pigmented and they are pear-shaped.  Some will come right off the sides of the hyphae and some of them come off of a stalk.  Let’s take a look at them.  The conidiophore there is called something else.   It’s a conidiophore alright but it’s called an annellophore.  What it means is that when this spore is produced, it leaves a growth ring on the conidiophore down below it and that’s called an annelid, just like with the earth worm.  You saw the rings on an earth worm.  These are rings on the conidiophore.  And they’re called annelids.  And this structure is called an annellophore.  And so, what we do is to tell you, how do you look for an annelid on these things and what we have done in the past is we say close your eyes and look real hard.  They are really hard to see.  But I have a photograph here that we took with some ultra-high resolution and we’ll show you a little bit what an annelid does look like.  And at least you will have an idea of what you might see.  So these are conidia.  Look at the pear-shaped nature of the conidia.  A lot of these come off the sides of the hyphae and there is one about 9 o’clock, that’s where it’s sitting on the top of a long stalk. 

And here you can see these conidia being produced on the long stalk and there are branching conidiophores and you see some free conidia flowing out there.  This belongs to Scedosporium Complex.  Scedosporium causes a number of things.  It causes brain abscess, it causes mycetoma, pulmonary disease, just a lot of different clinical entities.  It’s a very significant organism to find and so when it’s there, it needs to be reported.  Typically, when you live in areas where there is lots of farming going on, farm-related injuries that get this organism into the tissue, are really difficult cases to treat.  This organism is tough to treat to start off with.

Look at this one, this one shows you what the conidia look like.  These are not exactly pear-shaped.  In fact, they are a little bit swollen.  But they’re sitting in there, down on the left-hand side about maybe 8:30, you see this one at the end of a long stalk.  If you just look around in there, you’ll see some that sitting along the sides of the hyphae and you may even see just a tiny stalk coming off a hyphae.  There’s one at about maybe 9 o’clock over there towards the center.  That’s a very short stalk.  Usually, these spores are kind of pear-shaped.

And here’s a good example of what they look like when they’re pigmented.  You can see a little bit in a large version of them.  And a lot of these are just coming off alongside the hyphae and there is one in the center almost to the left of center that’s on a short stalk.  So I don’t think you’ll have any problem finding it.

Now here’s what the annelids look like.  See those little rings there.  At the end of the arrow, there are 2 of them.  Those are called annelids.  And every time the spores popped out of that thing, it leaves that spore and it forms a growth ring and those rings are called annelids and that structure there that you see sitting there with those annelids on it is called an annellophore.  It’s not called a conidiophore, it’s called an annellophore, but you know you can call it a conidiophore if you want because it’s the same as in all the rest of them except that you have the annelids on there and it’s hard to see those annelids.  But at least you know what an annelid looks like now. 

There are times when this organism produces other structures.  It is polymorphic.  It has more than one form.  It has a graphium form.  A graphium anamorph form here where you’re looking at it, where you see clusters of conidiophores all stuck together and they’re producing conidia.  The conidia here look elongated rather than pear-shaped.  But if you look there, there’s the arrow with one, with 2 annelids on it and it’s producing the spore right at the top and on the right-hand side where it’s enlarged, you can see the annelids in the center.  There are 2 of them.  This is called a graphium state.  There are lots of organisms that have a graphium state that looks like this and you can’t tell what this organism is by just looking at this.  You have to see the other forms, one of the other forms to be able to tell you what it is.  What we’re going to be talking about here in all this group is organisms that have more than 2 forms.  And so you have to know what they all look like to be able to put it together.  But you look at everything before you make an identification.  So this just happens to be the graphium form.  If you saw this form and you saw the form we just looked at a minute ago, this Scedosporium Complex form, which is conidia, you could pretty much say it’s going to be Scedosporium.

Now this organism also produces cleistothecia.  Cleistothecia are big black sacs made up of organized tissue that have asci and ascospores inside of them.  So, they produce asexual ascospores inside.  These big sacs do not have an ostiole in them at all.  So what happens is the whole big structure breaks open and then the ascospores are produced on the outside.  If you look closely, and you can’t tell much of it in the background, all that background area in there are conidia.  And you see these big structures, these cleistothecia, and then in the background would be conidia.  So you’d have 2 forms.

This is a cleistothecia that has ruptured and you can see the ascospores that are coming out of that area there, they’re in a big cluster.  So this is one of the stages of this organism.

Now, this is showing you an enlarged view of this organism.  And now look at the name of it.  When you see the sexual form of these things, you call it another name.  In this case, instead of calling it Scedosporium Complex it’s called Pseudallescheria boydii.  And you can see there all the ascospores that are coming out of that big sac that has been popped open.  So we’re talking about the same organism but in a different name and a different structure.  Another structure added to it.

And this is cleistothecia, again.  Another big one that has been popped open and those are the ascospores that are coming out of there and it doesn’t look so black.  So this is what Pseudallescheria boydii looks like and you would call it that.  So if you see just the spores, the conidia, you would call it Scedosporium Complex.  If you see these big structures you call the organism Pseudallescheria boydii.  That’s what people like to tell you to do.  Some people decide if you see the structures like this, you can just call it Pseudallescheria boydii to be done with but I think you have to kind of decide how you are going to do it in your own laboratory.  This is a complex group of things to talk about.

This is what Scedosporium part of it would look like.  The culture of this organism would be a mousy gray.  This is what one looks like that we had here at Mayo and these organisms were introduced into the patient by trauma, for the most part, at least in the skin cutaneous ones.  And the spores are out in the environment and so the spores form the Scedosporium part are what probably got in there or maybe the hyphae.  And this is Scedosporium Complex.  And this one we call Scedosporium apiospermum because we knew that’s what it was.

This is another example of one that’s kind of gray.

This one actually turned out to be tan, a little bit brown.

So, there’s another Scedosporium that we have to deal with.  And it’s one called Scedosporium prolificans.  It’s different from the other Scedosporium and we can’t, I don’t think, include it in the Scedosporium Complex because it’s totally different.  It is an organism that is totally drug resistant.  And it’s causing a lot of disease right now, particularly sinusitis and osteomyelitis.  And it is not one that you want to get, but you need to be able to identify it.  And it does something different.  It produces 2 types of conidia.  Numerous dark brown conidiophores or annellophores with inflated bases and that’s why we used to call it Scedosporium inflatum because the base of the conidiophore was swollen and now it’s called Scedosporium prolificans.  The conidiophores are arranged singly or branched clusters and the conidia may have pigment in them or not. 

This is what the culture looks like.  You can see that it has some pigmentation to it.

And here you have to look very closely.  The hyphae are delicate and they are not produced on a short stalk or a long stalk.  They’re produced on the tip of the stalk that is very long and narrow.  But if you follow it down where it is attached to the hyphal strand, what you will see is that it’s swollen right down below the base where the conidia is.  And that’s what Scedosporium prolificans looks like.  It’s hard to tell.  I’ll have a good one to show you in just a minute so you will see.

But you can see it better here.  The conidia is attached at the top up there at about 12 o’clock.  There’s 1 big spore up there and if you follow it down, you’ll see the conidiophore is tapered and then all of a sudden it gets swollen.  It looks like a vase.  So this is where the Scedosporium inflated conidiophore comes from.  It’s that very bottom part. 

This slide shows you better what it looks like.  And there’s one where the base is inflated that you can see the narrow top of it and then look at the conidia.  You see the conidia has some brown pigmentation to them.  Some of them are pigmented and some of them are not pigmented.  And that’s what you see with Scedosporium prolificans.  It’s totally different than the other Scedosporium.  It’s that inflated base that makes it different.  And also the conidia that are pigmented and some of them are not.  So this is Scedosporium prolificans, one that you need to be able to identify. 

Now we’re going to get to the very slow-growing one that we see primarily on CAP surveys and not so much from patients because we’re not in the part of the world where we see all of these things, but we still may see them, part of the floor of the environment.  So we will talk about Phialophora, Exophiala, Fronsecaea, Rhinocladiella, and Cladosporium, and Cladophialophora

So we’ll go here to talk about Phialophora verrucosaPhialophora verrucosa produces flask-shaped phialides.  Nothing more than a conidiophore that produces spores and it’s flask-shaped.  The spores are produced from withinside of that phialide and they come out in a cluster and they retain there in a gelatinous mass.  It looks like a vase with the top of a bunch of conidia at the top in that slimy cluster.  And as these conidia are produced, they leave some of the material back behind and at the top of this flask-shaped phialide is produced 2 areas, well, all the way around the side and the top of it there.  What you see is these conidia are produced as a retention of some of the slimy material that’s produced by these, all the way around the top of this conidiophore.  It will be then producing what we call a collarette around the top of this thing.  And so you see a phialide with a collarette and that is characteristic of Phialophora verrucosa

And if you just see that, that’s pretty much what it’s going to be. Here you can see it’s dark and it takes a long time to grow.

This is one, that is the cause of chromoblastomycosis.  Chromoblastomycosis is a disease that is acquired by environmental floor and people pretty much work in Central and South America.  They work outside in agriculture and they don’t wear heavy clothes and they will just wear a t-shirt or something and they often times will be exposed to things in the environment.  They may be barefoot, primarily.  They may get stuck by a thorn or some vegetation and they get it, the organism introduced into the tissue, and then it starts to grow.  Well, what happens is it takes a long time for this infection to start to progress.  But as it does, it forms these lesions that are pigmented.  They’re kind of blue to purple to red and so that’s where the chromo part comes from and mycosis means the fungus infection.  And it forms cauliflower-like lesions and with time they get to be a real mess to deal with.   And these are significant infections.  We don’t see them much in this part of the world.

You can see that all these

Colonies that we have looked at here are pigmented. 

So we notice dematiaceous. And there is the phialide, in the center here. 

And there is a base to it.  I don’t know exactly what the bottom part is.  I don’t know why it is formed that way but the vase shaped thing, the flask-shaped thing is the phialide and you can see at the top, there is a spore coming out of that thing and it’s just sitting up there.  And at the top is that collarette just looking at it in through dimension, here, that’s the collarette.  So there is the conidiophore inside will be the conidia.  And I’ll show you a good example of how it’s being produced in just a second.

This is where it’s being produced and you can see the conidia is still retained back by the gelatinous mass.  Look at the conidiophore.  It doesn’t exactly look like a flask.  It looks elongated.  But the conidia are produced and you notice at the top is that brown collarette where the materials been left behind and then you can see it is just like a collarette that goes all the way around this whole conidiophore and the conidia are still in the cluster in this one.

This is one where you can see that the conidia have been produced and pushed out and you can see, maybe see one sitting there at the top of the thing and there’s the collarette around the outside.  This is Phialophora verrucosa.  If you see that, you know what it looks like.  This is exactly what it is.

Now this is one that we took with some really high resolution photomicrography and this is Phialophora verrucosa and you can see the conidia inside of this thing.  And notice you can see the collarette, and you can see the dimension to it, it looks like it’s coming off of the right-hand set, coming off all the way around not just on the side sticking out.  But look inside of it.  There are the conidia that are produced and are pushed out and these are not retained here in a gelatinous mass, but you can see they’ve been pushed out.  So this is Phialophora verrucosa and exactly what you would like to see.  So if you remember what this looks like, it will help you to, I think, recognize what Phialophora verrucosa looks like.

The next one is Exophiala jeanselmei and you call up a clinician and you say we recovered Exophiala jeanselmei from the skin lesion and they’d say, “What did you say?”  Well, it’s a long name for a brown fungus, but it is a significant one.  It produces darkly pigmented yeast cells with annelids on them, so the first thing you are going to see is a yeast form growing of this thing.  So, this is not what we’ve seen before the rest of these.  Darkly pigmented yeast and if you look real closely, you might even be able to tell if it has annelids on it.  And then it begins to produce brown slender, kind of flask-shaped annellophores or conidiophores with a long, tapered tip on them.  And they’re formed that way in a filamentous culture. And the conidia may be produced on peg-like projections and this organism can’t grow at 42 degrees and we use that when we think we have one of these things.  It is not tolerant to 42 degrees centigrade and it uses nitrates.  So you would use a nitrate test to be able to tell that.

This the yeast form of this thing.  This looks like a big mass.  It’s a mass of yeast cells and it’s very difficult to tell if they have annelids on them.  But it is a pigmented yeast and you see there are a couple of these that have this and you have to distinguish between the 2 of them.

This is what it looks like underneath a microscope.  In the very center, it’s a long, tapered tip to this conidiophore or annellophore and then there are the conidia that have been pushed out of it and they’re sitting all around in there and they’re kind of in a cluster a little bit.  And it’s the long, tapered tip on this thing that is the feature that people look for.

If you look here, this is not a very good slide but if you look within the cluster of those conidia, you can see that it’s narrowed down, very finely to a tapered tip.  And that’s what we are looking for.  The cluster of conidia being formed in a tapered, tipped conidiophore or annellophore.

The next organism is Exophiala dermatitidis.  And this is another one that has a pigmented yeast present and the phialides on this one, you will see it produces phialides that are more blunt at the top and they don’t have a collarette at all.  The conidia that are produced are produced from hyphae or pegs along the side of the hyphae.  And the conidia are produced in slimy clusters at the tips of those conidiophores that have that flattened top and don’t have a collarette on them.  This one grows well at 42 degrees.  It’s a difference between Exophiala and jeanselmei.  And it does not use nitrate.  And it does not usually cause much in the way of clinical disease but we have seen a few cases of this.

Now this is black yeast form.  This is what it would look like for Exophiala jeanselmei and Exophiala dermatitidis.  A black yeast, something that you don’t see very often and you don’t see except in this group.  So this is a helpful thing.  When you see the black yeast, you should know that you are going to be dealing pretty much with Exophiala jeanselmei or Exophiala dermatitidis.  So you start with this.

Then as time goes along, it begins to get more like a mold.  Notice the outside edge of this thing is getting to be fuzzy and in the center it looks almost leather-like.  Well, some of these fungi look this way as they get older. 

And this is going to be the mold form. And as time goes along, you’ll see it gets to be more mold-like.

And now you see on this one, these have individual colonies here.  This is a very slow-growing one.  Individual colonies here it would look like a mold.

These are not all from the same culture.  This is from the yeast form.  This is some of those black yeast that you saw.  This is what they would look like underneath a microscope.  One way to describe the yeast form of these things is kind of slimy.  That’s how they look.  So you’d see the yeast form first, you let it grow for a while to see if you are going to see a mold form.

Then you look underneath the microscope and there on the left-hand side is a hyphal strand with spores sitting all around it.  And then you follow it all the way to the top and there are a bunch of conidia sitting up there that looks like they’ve been produced and they kind of slid down the side of that long conidiophore.  Look to the right-hand side, we kind of took a picture of the top of that thing to see if it’s tapered or not and it’s not.  It’s flat and if its Exophiala jeanselmei, it’s going to be long and narrow and tapered and if its Exophiala dermatitidis, it’s going to be more blunt and flat at the top.  And that’s what this one looks like on the right-hand side.  This is not a very good example but it is the best I could find and it shows what Exophiala dermatitidis looks like. 

So, you’re going to have to distinguish between those 2 and you can use the nitrate test and the temperature tolerance ability to be able to do that. The next one is Cladophialophora bantiana, another name, fairly new name.  Cladophialophora means that this organism can produce Cladosporium type or the Phialophora type of sporulation, probably both.  And so they called it Cladophialophora bantiana.  This is the one that we call neurotropic.  The conidiophores are not all that well differentiated from the hyphae.  The conidia are kind of elliptical and they’re in delicate, long chains.  The chains of conidia don’t branch all that often.  And this one will grow up to 43 degrees centigrade.  So we use temperature tolerance as a way to identify this one, too. 

If it came from a brain, let’s say, we would probably do a temperature tolerance test if we grew it up.  This is where you can see 3 of those small elliptical cells still in a chain.  They break apart.  We’ve talked about Cladosporium before and how delicate it is.  Well, this is one that is like Cladosporium.  It breaks apart just like that. 

Now here you see the long, delicate chains of Cladophialophora bantiana and spores stay in contact with each other instead they are in long chains.  This is from a scotch tape prep.  If you made a wet mount out of this, the chains would break apart and then you would have more difficult time trying to recognize what it is.

This is Cladophialophora bantiana and what you are seeing here are the chains of conidia.  And these are the chains of conidia that resemble the Cladosporium type of sporulation just like you’ve seen with Cladosporium.  You might look around and find phialides in here and then that would be a second type of sporulation.

That’s what we would see with that particular organism.  Now with Cladosporium, we’ve talked about Cladosporium with another presentation, but it produces conidiophores that are brown and have long chains of conidia that branch and they have those black scars, they’re called disjunctors, that allow you to see where they have been attached.  And every once in a while what you see is a cell that has a, looks like a shield, it has 3 spots on it where there are 3 disjunctors on it. 

That’s how you can recognize a Cladosporium form just like we have talked about in one other presentation.  On the right-hand side the elongated cell is the shield cell. 

So, the type, that Cladosporium we talked about, and you probably are familiar with, the reason I show Cladosporium here is that we have an organism called Fronsecaea pedrosoi that does what it wants to do.  It produces Cladosporium type sporulation, it may produce Phialophora type of sporulation and it may produce one called Rhinocladiella.  It does anything.  So you look at it and you will find more than one type of sporulation.  So we’ll start off by talking about the fact that it produces brown one-celled conidia in short chains just like Cladosporium.  And these short chains that are produced by a successive conidial production and the conidia may be sometimes produced on short little bumps or denticles along the side and that’s called Rhinocladiella type.  And some isolates may produce phialides and sometimes you see one that does all 3.

This is an example of Fronsecaea pedrosoi that just produces Cladosporium type right there.  You would have to go along and find another type of sporulation to be able to call it what it is. 

The next one shows you just still the Cladosporium type of sporulation where you see just chains of budding cells.  So you will see that with this organism.

And there you might see the Rhinocladiella type.  They’re long conidiophores that have kind of little, short denticles sticking out and there’s a conidium on each one of those things that go all the way around the conidiophore and that’s called a Rhinocladiella type of sporulation.  There is an organism named Rhinocladiella and this is called a Rhinocladiella type of sporulation.  If you look on the right hand side you can see the little bumps there where the conidia are produced.  So you might find this type of sporulation.

Then you may find that along the sides of the hyphae there is a stalk and at the top there’ll be one spore and the next thing that happens, one is produced on the top of it and another is produce on the top of it and you get successive conidial production as this thing grows it just keeps pushing them up to the top and it produces almost like a cluster, or a head, of conidia.  And so this is successive conidial production and I don’t know if there’s a name for this cluster or not.  I don’t remember that there is one.

And here you see an example on the left-hand side.  You see Cladosporium type of sporulation and you look on the right-hand side and you successive conidial production being produced on the right-hand side.  And so you have 2 types of sporulation in the same field.  And you might even find a phialide some place.  Let’s look on here and see if we see a phialide.  No, this particular one, I don’t see one.

But here’s a cluster of phialides without collarettes that you might find in this organism, its Fronsecaea pedrosoi.