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Identification of Melanized (Dematiaceous) Fungi Part 2

Click CC box for captions; full transcript is below.

Published: November 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 Part 2 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 2 focuses on rapidly growing fungi.

Presenter: Glenn D. Roberts, PhD

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

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Now just to recap what the melanized or dematiaceous fungi are, they are the organisms that have high concentrations of melanin or melanin-like compounds in their cell wall causing them to be pigmented.  Sometimes the pigmentation is very slight and it’s kind of hard to see.  Most of the time it’s dusky brown, dark brown, even to black in the hyphae and maybe even in the conidia cell walls.  There are 2 distinct groups, the ones that grow slowly and ones that grow very rapidly and there is good distinction between those, you could look at the cultures and tell that verses time.  Conidia vary in their appearance from single cell to multicelled.  And some of them, this is kind of something people have difficulty with.  Some of these organisms may have melanized and hyaline, or not pigmented hyphae, in that same mount and they consider these to be hyaline molds rather than pigmented molds.  Both kinds are there so that’s something to keep in your mind.

We examine these fungi to quickly look at the hyphae, look at the growth to see if it’s pigmented and it’s pretty obvious that you can see the dark pigmentation on them, and if it’s there. You determine if it’s slow growing or rapid growing and then you look underneath the microscope to find the spores and the conidia, the way the spores are produced is very important and you can tell that by looking at the mount of the thing.  And you may have to perform a few ancillary tests with some of these to be able to definitively identify them.  And, more importantly, when you recover one of these organisms, you need to correlate that with the type of specimen that’s sent in and that it came from and then the clinical scenario and the histopathology if you can have access to it.

So, we are going to look at a group of fungi that produce, that are multicellular, or they don’t have just single cells.  They are cells that are multicellular conidia.  They have transverse and longitudinal septations within in them and you can see on the right hand side where the arrow is, there are septations within that pigmented cell, they go both directions and so what we’re talking about here and to the left of that cell at 9 o’clock you will see both horizontal and longitudinal septations within that spore or that conidia.  It’s what we’re going to talk about in this group.  There are some that are probably very familiar to you.  Alternaria is the one.  That is a very common organism that you see in the clinical lab every day.  It’s an allergen.  It rarely causes disease in patients, but every once in a while it can do that.  Any of these are capable of causing disease, but most of the time they’re just environmental organisms.  Epicoccum is another that we will talk about.  Pithomyces, Ulocladium, and Stemphylium, and people get a little bit confused when you start talking about this group, but I think if you look at the way the spores are produced and you know that they produce horizontal and longitudinal septations, you have to then concentrate on how they are produced.  That’s tells you what the organism is. 

Now with Alternaria, this one is a fairly easy one.  This is one you can recognize.  Probably all of you can recognize this by sight.  It is an organism that produces chains of pale brown, kind of club-shaped conidia.  They’re in long chains.  And they have horizontal, longitudinal septations.  The spore has a long beak on it at the distal end.  And my friend, Dr. Elmer Koneman, kind of says that he thinks this organism looks like a thing you get in the grocery store called a drumstick filled with ice cream.  We’ll look at that in a few minutes and we’ll maybe be able to see that.

Here’s a culture that has some pigmentation to it.

And now underneath the microscope, you’re looking at spores.  These are the conidia and they have horizontal and longitudinal septations.  The one in the center that is enlarged at the right shows you that it has both types of septations.  And the beak is at the top end of it about maybe 1 o’clock in the large view.  And you notice that some of these are very dark.  You can’t even see those septations in them.  This organism produces the same kind of conidia, but they look different depending upon the isolate sometimes.  Actually, they don’t stain well enough because there is so much melanin in the cells of these that they are so dark, it’s very difficult to tell what they are. 

There are times when we look at one like this where the cells are just that brown pigment and they’re not so dark and you can see the long beak on these and you can see that the septations are horizontal and longitudinal, but it’s hard to tell that except on the lower chain on the left-hand side, the last cell, you can see the longitudinal and horizontal septations.  You have to look at all of the slide to be able to find these, probably.  And so you have to examine every field that you can.

This next slide shows you Alternaria and there some of them beginning to chain and here, it is a little bit more difficult to tell if there are both kinds of septations in there.  So, what you would end up doing is looking around the whole slide to see what the predominant presentation of these conidia would be and I would bet that you would find areas where you would see horizontal and longitudinal septations.  These are probably young conidia. 

And here on this one, a little difficult to tell, this is sometimes difficult whenever these things get over stained with lactophenol aniline blue, but on the top cell, I think that you can see the horizontal and longitudinal septations.

Now in this one basically, we see mostly just the horizontal septations, but if you look at the one at the far left at the upper left corner, you’ll notice that there is a vertical area there.  And so that is the longitudinal septation.  So, they’re not always so obvious.  You can look at the morphology.  You can see the things where they have a beak.  Then at the right side, down about 3 o’clock, you’ll notice there are about 3 in a chain and you don’t see the long beak.  This organism has different morphologic features.  There’s a book that is written on the dematiaceous or pigmented fungi and Alternaria has many different types of presentations for conidia.  So they don’t all look like we tell you. 

Here you can see this one, you can see the long chain in the center here and you need to look carefully to look and see if you can see the horizontal and the longitudinal septations, but they’re in there.  And you can notice there is a little dark area where they’re connected together at the beak.

And then here’s one that you actually can see what Dr. Koneman was talking about.  It looks like a drumstick with ice cream in it.  But it does have the horizontal and longitudinal septations.  Sometimes these cells are rough-walled and most of the time they’re smooth. 

Sometimes, they are very elongated like you see here, and then that’s why I say that the morphology varies a lot because they don’t all look exactly the same.  But if you look at the features, you’ll be able to tell.  So, Alternaria is a very common one to see in the clinical laboratory.  And probably most of you can recognize it by sight, but you will encounter some of those ones sometimes that will be a real challenge to you because they don’t look quite the same.

This is Epicoccum that you may or may not be familiar with.  It produces conidiophores that are short and not well-differentiated from the hyphae and the conidia are grouped together to form kind of like a nest where the conidia can be produced.  And all the conidiophores are grouped together right in 1 spot and they form this structure called a sporodochia.  This is a totally different thing.  The conidia are brown, they are rough-walled.  They have horizontal and longitudinal septations because that’s the group that we are in and as they get older, they get kind of crusty looking and then you can’t see that very well.  So, we’re going to look at that. 

One of the hallmarks of this organism is that it produces an orange diffusible pigment and if you turn it over on the backside and take a look at it, you can see it pretty well.  The culture is dark, kind of a brownish color.

And there you see all these things, whatever they are sitting there all clumped together.  Well, that’s called a sporodochia.  That’s where there all the conidiophores are together and all the conidia are produced in the same area.  If you look at the large one on the left-hand side, you can see the horizontal and the longitudinal septations.  Maybe it’s a little bit more oblique rather than longitude, but this is EpicoccumEpicoccum is an environmental organism.  I don’t know that it has ever caused human disease.

Here you see an early growth of Epicoccum.  There’s where you see all the conidia are being produced in there side by side and after a little bit they’ll form more and more and they’ll just be all grouped together.  But if you look at the conidia themselves, you can’t see it on the young ones, there will be horizontal and longitudinal septations and they may even look like this. 

Sometimes, they are dark brown and crusty looking, rough-walled like that, but you still can look and see if they have the septations.  And then you look to see how they’re produced. 

And now you look at the old ones. These are the ones that are very mature.  You can’t tell much about it because they’re all clumped together and they’ve gotten kind of crusty looking.  But if you see a whole cluster of these in the sporodochia, you will know that you are dealing with Epicoccum, in addition to looking for that orange diffusible pigment.

The next one is one called Pithomyces.  In this situation, we are looking at conidia that kind of come right off the hyphae.  They’re not well differentiated from the hyphae.  There is no real structure there to show you they are produced by something.  They’re produced along the sides of the conidia.  The conidia have horizontal and longitudinal septations because that’s what group we are in.  They’re kind of elliptical, kind of club-shaped, kind of pear-shaped.  They may be smooth to rough, and they are produced singly. 

Now here’s a culture, you can tell that it has some dark pigment to it. 

And here are the conidia.  And you notice in the center is one that’s attached to the hyphal strand.  There is no real structure underneath it to show you how it’s produced.  It’s produced right from that hyphal strand.  There are conidia sitting around there and if you look at the top of some of them, then you see the horizontal and longitudinal septations.  So, you’re looking at conidia that are not produced on any kind of a structure, just along the sides of the hyphae.  They’re produced singly and they do have the 2 types of striations, the longitudinal and the horizontal.

The next one is, Ulocladium.  This is one that produces dark brown conidia with the horizontal and longitudinal septations.  We’re going to have to learn a couple of new words here now.  The conidia are produced sympodially, which means they are produced on one side and the other of a conidiophore.  And the conidiophore as it grows begins to bend to one side or the other and the conidia are produced on those bends.  The conidiophore, when it does that is called a geniculate conidiophore.  It’s like a series of bent knees.  The conidia may be smooth or rough and you may even see some secondary conidia or a short beak on these. 

But here’s the culture and you see the dark pigment in the center.

And this is what it’s like underneath the microscope.  See the rough-walled nature of this thing, but you can’t really tell on a young culture exactly if it’s pigmented or not.  So you are going to have to wait until it matures and then you are going to have to look around the whole culture, the whole organism, the whole slide. 

Here you see Ulocladium and if you look on the right hand side, there is a piece of hyphae.  Notice these come off one side and then the other.  Well, basically, what’s happened is, if you look really closely, you will notice that conidiophore, which looks like a piece of hyphae, is kind of turned to the left and then it goes up and it’s going to be turned to the right.  That’s the geniculate conidiophore, the beginning of one. 

Let’s look at the next slide and there you can see even more.  You can see that conidiophore, is actually sitting there at the top and it’s going, and it kind of twists and turns a little bit.  Look on the bottom that conidiophore twists and turns and the conidia are produced on one side and then the other and that’s the whole sympodial production.  It’s a little bit difficult sometimes to tell.  You are going to have to look at some of these a few times to be able to see what they look like.

Stemphylium, we’ll show you again what a geniculate conidiophore is because there is a good one in here, I think.  I may be wrong.  It may be one of the others that we’re going to cover but we’ll look and see.  The conidiophores are brown, they’re single.  They’re slightly branched.  The conidiophore is swollen at the top and the conidia are single, dark brown, smooth to rough and they have horizontal and longitudinal septations.  And you’re not going to see that conidiophore I talked about. 

What you are going to see here, someone described this to me one time.  They said it looks like a bale of hay on a long stalk.  I don’t know that I understand that but there you see a conidia, a big spore.  It has horizontal and longitudinal septations that are very obvious and if you look at the one on the left-hand side on the bottom, you will see that it is resting on top of a conidiophore that is swollen.  It’s swollen at the very top of the tip.  So that is Stemphylium.

So now, we’re going to conidia that have perpendicular septations.  No longitudinal ones and the conidia are multicelled.  And there is a group of these here that we will talk about Bipolaris, Dreschlera, Curvularia, Exserohilum and Helminthosporium.  The first one is BipolarisBipolaris is one that is the most common one that you will see in the laboratory of this group, pretty much.

It produces large, oval, brown-pigmented, multiseptate conidia.  So it has many segments to it.  This is where you will see what a geniculate conidiophore looks like.  It produces conidiophores that are twisted and turned and on each side of that twist and turn produced a conidia.  So that’s sympodial conidia production that we talked about before, you will see a good example here.  Conidia produce up to 6 septate, so there is a long conidia with all these sections.  And the way you confirm the organism is you put it in some distilled water and let it sit for a long time, for about 24 hours, and then you look at it to see if it has germinated.  And if it’s germinated, it will be germination at either end.  It’s called bipolar germination, so each end would be polar, and then the germ tube and I’ll show you in a minute, grows parallel to the longitudinal axis of the spore. 

So, let’s look.  This is a good example of Bipolaris.  On the right-hand side is that twisted and turned conidiophore, I talked about.  The conidia are produced everywhere it twists and turns.  On one side and then the other, and that’s called sympodial conidia production.  If you look at the right-hand side where the arrow is, you can see that that’s happened.  And there are the conidia and there’s a dark area where they are connected.  And that dark area is called a hilum.  And that becomes important to look at.  I’ll show you why.  The hilum does not protrude, it just kind of sits at the top of kind of like a bowl that got down in the conidium.  Down at the bottom is a good example of its twisted and turned one, which is enlarged on the right-hand side and you can see in the middle, down at the bottom of there, that area you can see the conidium, you can see the left-hand side, you see the dark area where it’s been attached.  And that does not protrude.  It’s kind of continuous with the wall of the cell.  This is another one with the Bipolaris, and notice that the spores are produced on one side and then the other and that’s called sympodial conidia production.

And notice this one has many segments to it.  You can see and count them where they have picked up the stain.

And here’s another one where you can see that it’s smooth-walled in this case and you can see that it is multiseptate and it comes off one side and then the other.

This is one that has been associated with causing fungal sinusitis and they found that it’s actually in things like marijuana, and cocaine, and it gets into the nose and it causes severe sinusitis. 

This is Bipolaris again, not a very pretty one, but look at the lower right-hand side.  There is a conidium and all of those are conidia. 

But look at that conidiophore how it just twists and turns slightly.  You can see it’s not just smooth, it just goes one way and goes the other, back and forth, back and forth.  And those conidia are produced first on one side and then on the other then back and forth, and back and forth, and this is what Bipolaris looks like. 

And if you put it in distilled water to germinate, you will see that there is a germ tube coming out at either end and they are parallel to the axis of the conidia.  And that is how you would tell what Bipolaris looks like.

And here’s another example of a germ tube.

The next one is one that we don’t see very often in the clinical lab, but it sure looks a lot like the one you just saw.  It is called Dreschlera.  It has conidiophores that are brown and geniculate, so they go back and forth.  There is no prominent hilum on the thing.  There is no dark area where it connects to the conidiophore.  The conidia are multiseptate.  And they may produce germ tubes, but when the germ tubes produce, it’s perpendicular to the axis and they can come from any segment within that spore.  So, it’s totally different than Bipolaris.  The problem is that it rarely does produce many spores.  So you are not going to see it very often.

Here you can see the example of Dreschlera and on the right hand side there is the area where the hilum is where it’s dark, where it’s connected to the conidiophore and then it has the conidiophore that twists and turns a little bit.

And we go to the next slide and this is what it looks like and the conidia are just, almost the same as Bipolaris, when you start to look at it. 

And here it just looks the same as Bipolaris.  You see the conidiophore in the center there that it twists and turns back and forth.  The conidia are produced on either side.  So that’s what you would do, is you would do the germ tube test on it and you would see that the germ tube is produced perpendicular to the axis of the cell and that the germ tube can be produced from any cell, not just the end cells.

Well, the next one is Curvularia and most of you, I am sure, are familiar with Curvularia.  This is one that produces conidia that are kind of chestnut brown and they are kind of obvious with the colors are very pretty with these.  It’s curved.  It usually has 3 septations and it’s curved because the center cell grows faster than the other cells and it causes the thing to kind of curve.  So, it’s asymmetrical when you look at it and the central cell, where the growth is darker and 2 end cells are lighter in color.  And if you look at it, it looks like a boomerang. 

This is there, there it is right here.  There are the septations and notice the big, with the central cell is bigger.  That’s the one that grew more rapidly and the rest of it stayed about the same size.  So it’s very curved.  That’s why they call it Curvularia

You can see here what it looks like.  It’s produced in same kind of conidiophore that all the other that we have talked about are.  It’s just linked to the conidiophore.

And here you see the cells.  The one on the right-hand side looks like a boomerang at 3 o’clock.  The other cells on there have been produced on one side and then the other of this twisted and turned conidiophore.

This is just a cluster of conidia.  We had one organism here that produced so many conidia that you could hardly believe it.  This is 1 field.  What you see in there are the cells that have a large central cell and lighter staining end cells.  So this is Curvularia.

The next one is one that we don’t see all that often.  It’s called Exserohilum.  It produces very large conidia that have brown pigmentation.  They are multiseptate.  The conidia have 7 to 9 septations and the hilum on these protrudes and it’s very prominent, it’s obvious. 

This is Exserohilum and you see where the arrow is, there is a protrusion from the end cell.  That is the hilum and they named this Exserohilum.  The hilum protrudes in these cells and none of the others that we have talked about has a hilum that does this, it sticks out like that.  The others are usually continuous with the cell wall.  This is an easy one to recognize because it does have so many cells to it and it has that small protrusion coming in there.  Right by the arrow, you can see the best view of it. 

And here on this slide, you can see on the upper cell, you can see the dark protrusion at the end.  And you can see where there are 2 cells together on the right hand side.  You notice that the bottom one has a, looks like a black peg sticking out there, that’s the hilum.   So Exserohilum is one that you don’t see all that often.

The next one is very pretty but it’s one that is very uncommon.  Kind of the rule we talked about in Mycology from my other sessions is if they look really nice and you think you can identify them, you generally can’t do it, it’s the rule.  It’s too tough.  The next one is HelminthosporiumHelminthosporium has conidiophores that are brown and they’re erect and rigid and the conidia are kind of round at one end and tapered at the other end and they are multiseptate.  And they are produced along the sides of the conidiophore and they just stay there. 

And you can see them right here.  They all are attached by the swollen, the large end and the tip of the conidiophore has a beak on it.  This is Helminthosporium.  This is the one that you see on the dead leaves that you rake outside.

There you can see the hilum on each one of these, the dark area, it continues to the cell wall and it grows on either side of that conidiophore and sometimes it has a long beak and sometimes you can see it doesn’t.

Now we get to something that has been a name change occurring with this organism.  It used to be called Phialophora richardsiae and now it’s called Pleurostomophora richardsiae.  And that is going to confuse some of us.  But, it is an organism that produces phialides and phialides are flat-like structures that produce conidia.  In this case, this organism is one that causes phaohyphomycosis fairly commonly.  Thick-walled brown phialides are produced.  The tips are tapered but on the tip, there is a flared area that looks like a saucer.  It just sits up there.  You may find the pigmented conidia and you may find some that aren’t pigmented at all. 

This is a view of Pleurostomophora.  The right-hand side shows you the phialides, which is the kind of flat-like structure.  The tip of that looks like a saucer sitting on there, that’s where the conidia are produced and are pushed out and they’re kept in a mucilaginous type clutch together. 

And there you can see the top of those that looks like it has a saucer sitting on it.  So, this is what this organism looks like.