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Identification of Melanized (Dematiaceous) Fungi Part 1

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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. In this 3-part Hot Topic, Dr. Roberts describes the characteristics of melanized fungi, the steps necessary to identify the specific fungi present, and provides a visual overview of some of the common melanized fungi. Part 2 focuses on rapidly growing fungi, and Part 3 on polymorphic fungi.

Presenter: Glenn D. Roberts, PhD

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

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This presentation will deal with a melanized or the dematiaceous fungi.  These are the pigmented fungi and they have high concentrations in the cell walls sometimes even in the conidia.  They cause the cells to be pigmented where they are either lightly pigmented or very deeply pigmented.  They produce dusky brown to dark brown pigment and even a black pigment in the hyphae or in the spores or the conidia and these organisms can kind of be divided up into groups of 2.  One the rapidly growing one, the other the slow growing ones and they are distinctly different.   And the conidia vary in appearance from single cells, all the way out to some that have multicelled.  You will see as we go along that some of them have more than 5 or 6 cells.  Some of the fungi in this group have both melanized and hyaline hyphae.  And the rule is that they are considered to be hyaline molds if they have both of those. 

So the way you look at these organisms is to examine the hyphal growth.  Look at them close to see if you see pigmentation, determine if it is slow growing or rapid growing.  You can do this fairly easily.  Put it under the microscope, take a look at it, look at the conidia, see how the, look at the conidiophores, look to see how the conidia are produced and there may be a few times where you have to do some additional testing but for the most part that’s how you do it.  And you correlate, when you recover one of these organisms, these pigmented fungi, you need to correlate it with a specimen type, with a clinical history and histopathology if you have it available because these organism are environmental and they may not always cause clinical disease but many times they do, particularly in the immunocompromised host. 

Let’s look at the back side of one of these cultures and see what the pigment looks like.  And you can see here, this is Alternaria that someone has written on the back of the culture dish.  Look how dark the pigmentation is on the reverse side of the plate.  So that tells you that this is one of the dematiaceous or melanized fungi. 

You look under the microscope and you see sometimes a culture like this.  This happens to be Aspergillus niger and you know from before niger, it means black, well, the spores here are conidia are dark black and the conidiophore is dark black, but the hyphae actually are not pigmented.  So this is not considered to be a melanized fungus. 

Here is the difference between the slow growing and rapidly growing molds in this group.  You can see that the colonies on the left side are slow growing and compact and they are not very big.  And they take weeks sometimes to grow.  With the rapidly growing ones, you see in the culture dish is filled in a few days.  So there is no problem with the distinction between these 2 groups. 

We need to look underneath the microscope, basically, with this traditional approach to see what we can tell about how the spores are produced, what they look like and so on.  So we look to see what kind of cells are there.  Well, with these pigmented fungi have cells that has produced single cells, they make a conidiophore that’s produced in just a simple manner where there is just a little conidiophore that is long or short and there may be a complex conidiophore that is twisted, and turned and produced different structures.  Even sometimes with these fungi, you will see that they produce black yeasts.  And so, we have to take all of this into consideration when we are trying to make an identification.  Once in a while you’ll see one of these dematiaceous or melanized fungi that produce 2-cell conidias, not very often.  More often you see cells that are produced that are multicellular.  There may be many, many segments to them. 

This is a slide that shows melanized hyphae where the arrow is, but look at the left hand side to the left of that and you will see that those hyphae are not pigmented.  So in this situation, you see both hyaline hyphae, which are the ones on the left, the hyphal strands on the left hand side.  It doesn’t show any pigmentation and on the right where the arrow is, you see that it’s a melanized or pigmented piece of hyphae.  So melanized hyphae are what we are going to be talking about with this group. 

This happens to be an example of one of the organisms in the group that has multiseptate conidia.  And you can notice how brown pigmented they are, they are brown and this is a very good example of a melanized fungus. 

These organisms are like I said, environmental but they cause some very serious infections.  Now one of these is called Phaeohyphomycosis, which just simply means that it’s an infection caused by a brown fungus.  And we have seen a brown fungi causing fungal brain abscess and this happened not very long ago in a lady who was in an automobile accident here from Mexico and she was sent up here from Mexico and she ended up getting this organism in her brain because the brain actually was exposed to the soil after the accident.  And eventually, this organism, despite all of the therapy, it killed this lady.  These are not benign organism that we are talking about here today.  In the immunocompromised host, they are susceptible to most anything and for some reason or other, there is an entity called a Phaeohyphomycotic cyst.  It is just a skin cyst down below the skin and the subcutaneous tissue where the organisms grow and most of the time these brown or melanized fungi are the ones that cause that.  There is a group of infections that are called Chromoblastomycosis that’s seen in the more temperate parts of the world.  And it causes severe lesions in the skin and these occur primarily in patients who don’t have access to good medical care.  Mycetoma is something that we see in this part of the world, but the tropical areas of the country see more or often even in Africa all the time, where the organism is introduced by trauma to the skin and it produces a sulfur granule just like you see with Actinomycosis, except that it is caused by an organism that may be a fungus or bacterium. And then, miscellaneous infections, sometimes these pigmented fungi get into the blood stream caused by a line-related infection and then sometimes they are in tropical parts of the world.  They are very superficial skin infections and even some of these look like melanoma.

So we will start off with single cell conidia being produced alone and that’s all we are going to see are single cell conidia.  So we will start off with, well, let me just say here that there is another group of these organisms that we are going to talk about that are a bit different than all of the rest of them and then we’ll continue on to talk about in another series about the ones that are multicelled. 

There is a group of fungi that produce single spores or even special structures that are totally different from anything else that you will see and these overlap a little bit. 

So we are going to talk about these and we will talk about Nigrospora and this organism produces conidiophores that are kind of clear, they are hyaline, they are not really pigmented.  They produce short conidiophores that are inflated.  You can, I will show you an example of that.  The most prominent feature is that they produce these jet black, oval to elliptical, horizontally flattened conidia.  And what that means is, if you think about a basketball and if you’ve tried to flatten the basketball out a little bit from the top to the bottom, that is what horizontally flattened looks like.  And you will notice that we see these.  These are very smooth walled, they are produced in the top of these inflated conidiophores.  The culture itself is described as being dirty white. 

And that’s what you see here. 

And here.  And the back side does not look like it has any pigmentation in it. 

This is Nigrospora and it’s pretty easy to see those conidia, those are those jet black, horizontally inflated conidia.  And if you look right down below the conidia, you will see there is a conidiophore, it’s kind of a swollen area, that’s what this organism looks like.  Maybe you can see better, towards the bottom left hand side, you can see an inflated conidiophore

and you can see the horizontally flattened jet black conidia.

So I’m just going to show you some more slides of it here and you will get a real good idea of what it looks like.  Here’s the horizontally flattened conidia.  And these conidia are smooth-walled.  There’s no roughness to them at all. 

Here you can see the same thing. 

Unfortunately, this is something that we don’t see all of the time.  But we talk about it because you might.  And when you look at it, depends upon the angle, if you look at it from the top, which is what we are doing here, it looks round, totally round without being flattened.  The moral of the story is, you have to look at everything on the slide to get an idea of the organisms are in and what they look like because 1 or 2 fields may not tell you the whole story. 

Well, the next one is AureobasidiumAureobasidium is common, ordinary mildew.  This is the one you find growing on your shower curtain if you don’t clean things very often.  And it likes to grow in moist areas.  It produces yeast-like conidia that arise in groups from hyphae that are down below the agar and the older hyphae as they age begin to produce a dark pigment.  And these look like arthroconidia, the rectangular cells and they are brown pigmented.  And something that is kind of important to remember is that when a colonies first start to grow, they may resemble a yeast.  And I’ll give you an example of some of the trap you might fall into.  We ended up having a culture one time that was nice and mucoid and kind of off white.  It looked like Cryptococcus and we could not identify it at that time so we sent it away.  A few days after we sent it away, it started to turn black and all of a sudden then we knew what the thing was.  The embarrassing thing was because we sent a culture to someone, and it looked like we didn’t know what we were doing. We figured it out because as time goes along it turned dark. 

Here’s one where the culture is older now and it is pigmented now that you see the melanin is black in this one and that’s what Aureobasidium would look like.  And that’s a fairly mature colony.  You can see it covers a lot of the plate. 

This is what you would see.  These are rectangular cell.  Some of those cells and you see the smaller of the larger cells are the cells in the hyphae.  The smaller cells that you see sitting around there are the conidia. 

And let’s look at the next slide.  This is where you see the real story.  All of the blue colored small spores are the conidium.  And if you look down you will see the brown Arthroconidia, down to the right of the center, you will see a strand of hyphae with 6 dark cells in it.  In the center cell, has a raised area.  That’s where those conidia are produced.  They are produced within the cell and they are pushed out of that opening there, you can see that raised area.  The conidia come from the cells that are down in the hyphae and there is a small one at the top and they are hyaline.  They are not pigmented at all.  But you can see that the other cells of Arthroconidia are definitely pigmented. 

This is another example.  These are pigmented cells.  These are the Arthroconidia and if you look to the far left, almost to the edge you will see an area where it is kind of indented.  That’s the area where all of those things in the background, all of those colorless things, those are the conidia are produced.  And if you look to the right hand side to the third cell from the right, you will notice that there is one with a little protrusion.  That’s where some of the conidia would be produced from too.  This is a very high resolution photograph to show you what the little pore looks like.  Ordinarily, it is not so easy to see.  These conidia are produced all at one time and they are called synchronous conidia production.  And this is what it looks like.  This is ordinary old mildew. 

We are going to talk about now, Cladosporium.  And Cladosporium is a common organism.  It is probably one of the second or third most common organisms you are going to see in the clinical lab.  It’s found out in the environment and this and in the autumn time of year, it’s really prominent on the leaves and some of the things that we see outside.  They produce conidiophores that are brown and the septate, and the whole thing though that makes it easy to recognize is that there are chains of Blastoconidia.  These are spores or conidia, they are kind of oval shaped and are produced in long chains.  They are connected by a little black area, which looks like a scar.  And if you try to make them out of one of these Cladosporium, you will notice that the spores begin to break apart.  You don’t see the chains any more.  So you end up using a scotch tape preparation in order to be able to see the exact morphology of the slide culture.  Sometimes in there is you look at the mount, what you will see will be some cells that look like the medieval shields that we have seen in literature where they have 3 points on them and each one of those is a dark cell or shield cells and what we know about Cladosporium is that 9 times out of 10 when we make a mount of this organism, always see your single cells sitting all around.  It just falls apart and you don’t see how it’s produced unless you use a cellophane tape preparation. 

So, we’ll look at Cladosporium, this is a culture.  This is kind of typical for Cladosporium.  A very nice velvety appearance and the color is kind of yellow and sometimes it is even green, sometimes it is even black with other and even different colors.  This is not exactly what you would see. 

These are conidia and it is hard to see on this slide but there are chains of small, oval conidia produced there.  And if you were to make a mount of that with a wet mount all of those conidia would just break apart.  And they would just be floating around.  If you choose the scotch tape preparation, you would see something like this but with a common variety that we see which is called Cladosporic herbarum is a common ordinary one that we see all of the time produces larger conidia and they are much more dark and easy to see.  This is the one that causes brain abscess here and I’ll show it to you better in just a little bit. 

So this is a mount of Cladosporium that if you used a wet mount, this is what you would see.  All of the cells would be disrupted, they would be floating around in there, they wouldn’t be in chains any more, they might be in groups of chains or 2 or 3.  But what do you notice in there is that some of those cells have dark areas where they are connected.  And that’s called a disjunctor.  We can look at this and we can say “Ah, this is Cladosporium,” and after we get done, you will be able to do the same thing because this is what would happen if you made a wet mount and that’s all you did, you would have to look at all of these cells and figure out if this is going to be Cladosporium or not.  You see all of those darkened areas in there where the cells are attached, those are the disjunctor cells and that’s what Cladosporium looks like. 

Now, this is one on the left-hand side where the culture is produced all of these spores and they are all on chains and they have this darkened area between them.  You see on the right hand side where you can see a better view of what this looks like and they break apart there.  That’s where all of those cells break apart there.  These are produced by budding cells and chains.  So this is Cladosporium.

Now this is a large view of some of the spores.  On the right hand side, the largest thing you see the cluster of 3 there.  The long cell on the right is the shields cell and there the conidia attached to the 2 top arch where the disjunctors are.  And if you look at those conidia, you can see where they have broken apart and where there is a darkened area so that is what Cladosporium looks like. 

And here’s one that is dark black, so they vary a lot in their morphology. 

You can see here some are even brown, green and they produce conidia but they are hard to recognize sometimes unless you make a scotch tape preparation.  

If you don’t see it very often in your lab, you might have a problem, but I think it is one that you will recognize because it is a very common one.

The next group that we are going to talk about have some specialized structures that we haven’t seen yet.  The first one has a structure called a perithecium structure and perithecia are produced and inside them are produced asci and ascospores.  The asci are the structures that give rise to the ascospores.  These are the sexual spores.  So you are looking at a product of sexual reproduction here with this organism.  And the name of the organism is called Chaetomium.  And you will see what I am talking about in the next few slides. 

Chaetomium has large, brown to black perithecia and a perithecia are these big sack-like structures that have an opening in them and the opening is called an ostiole.  Within that big sack, it’s kind of like tissues, like organized tissue but inside of that big sack are produced ascospores.  And sometimes we call these big structures, these perithecia ascocarbs because they just produce ascospores.  It is covered with these dark interwoven hyphae and there are a lot of spikes sticking out of this thing and those spikes are called satae (s-a-t-a-e) and if you were to kind of push on one of these organisms with a pencil eraser you are looking underneath a microscope, it would pop open and what you would see is ascospores coming out that are kind of lemon-shaped and these are the product of sexual reproduction of this group. 

Now, this organism is one that I bet you’ve seen before.  Not necessarily underneath a microscope.  Have you ever been in a furnace room or a store room or someplace and you had some newspaper laying on the floor and it’s been there for a while and the cement gets moist you will see that it’s starting to turn a little bit of black.  You will notice that there are black spots all over the whole thing.  Well, this is what Chaetomium does is it grows on paper.  That’s one of the sources.  It grows on lots of things out in the environment and our common environment that we have at home, this is what you see growing on an old newspaper.  It doesn’t have a lot of dark pigment on the back, but you can see that on the top here, it’s some black dots so we get to see what they are.

This is a culture.  It does have brown pigmentation to it so it’s a melanized fungus

And you look at it underneath the microscope and it looks bizarre.  It looks like something you have never seen before or almost looks like some sort of insect or something.  But there are the black sacks.  Those are the perithecia and you see all of those stiff hyphae coming off of those, those are the satae and inside of those structures are ascospores. 

This is a larger view showing you what they look like.  And there’s an opening in each one of those things where those ascospores can come out and that’s the ostiole.

Here is a large view of one.  Here you can see the spines sticking out the satae sticking out of there.  It’s difficult to see the opening, these stain very darkly and they are black.  So it’s hard to see the ostiole in there.

But if you look on the left-hand side there, there are some there, I don’t see anywhere where the ostiole is present but on the right-hand side we super-imposed what the ascospores look like.  They are kind of lemon-shaped and they pushed out of that perithecium, this is what you would see on the right hand side. 

The next slide shows you the same thing except they are brown and they are a little bit larger view.  This is what they look like.  And so the only way you are going to tell what Chaetomium is going to look like for sure is to look at the structures underneath the microscope and then define those ascospores coming out of there. And, if they don’t see the ascospores, you end up just kind of having to push on them with a pencil eraser up on the coverslip or the scotch tape to push them out and then you will see them without any problem. 

The next organism has an entirely different structure.  It has one of these big sack-like structures too.  It kind of has a beak on it.  And rather than produce ascospores inside of it, it produces conidia, the product of asexual reproduction and that’s a unique way for this organism, or for any organism, to produce conidia inside of a sack-like structure.  This also has an opening on it.  Now, these structures are called pycnidia and the organisms name is Phoma

And Phoma has a little bit of pigment to it on the culture when you look at it but it’s underneath the microscope where you’ll see what the structures look like and the pigment is going to come pretty obvious to you. 

So you have these light-brown pycnidia produced that are containing conidia, there is an ostiole present where they are pushed out and the conidia are kind of oval to elliptical, and colorless and single celled.

This is Phoma.  This is a pycnidium you are looking at and at the very tip top of the thing, what you see is the ostiole, that beak like area and the conidia are produced and pushed out of that ostiole. 

Now this slide you can see that the conidia are much smaller than ascospores.  They look the same as most conidia and they are produced on structures inside the cell the cell if you pop that open, you can see all of that.  But this is what Phoma looks like.