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Hot Topic: Identification of Hyaline Filamentous Fungi - Part 2 (Unusual Zygomycetes)



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Summary

Published: April 2014

Dimorphic fungi cause several common diseases including histoplasmosis, blastomycosis, and coccidioidomycosis. Dr. Roberts discusses the distribution and ecology of these fungi, as well as their pathogenesis and cultural characteristics. This is part 2 of a 6-part series.

Presenter: Glenn D. Roberts, PhD

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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.  In this series, focusing on hyaline fungi, Dr. Roberts discusses perhaps the most common fungi that you will see in the clinical laboratory and a significant cause of disease in immunocompromised patients including transplant patients.  This module examines unusual Zygomycetes.  Thank you Dr. Roberts.

Thank you, Sarah for that introduction.  I have nothing to disclose.

First, we’re going to discuss how to make mounts for a fungal culture so that we can make an accurate identification.  This image you see here actually is representative of what happens in the laboratory many times where a plate is contaminated with many organisms.  And you need to figure out how to identify those that are present. The next image shows you a schematic of what you might see.  This is a drawing with fungi exhibiting all of the different kinds of spores that might be produced, may be not all but a lot of them.  It gives you an idea of what you might expect to find with certain of the cultures.  And it’s got, I kind of call it the universal fungus because it has everything there.  You can notice in the center there is a tall stalk with a round sac at the top and this is a sporangium of a Zygomycetes.  Then we go from there down to Penicillium, which is like about 2 o’clock, which has different types of sporulation and you can just look around in there and see that many of these fungi sporulate differently so we will begin to look at some of these as we go along.

Scotch Tape Preparation

The first preparation that can be used in the clinical laboratory and probably the most widely used is the scotch tape preparation. Basically, what you do is to take a piece of scotch tape and tear off a piece and fold it up so the adhesive side is facing downward.  And what you do is you touch the colony with that piece of scotch tape, you stretch it out and place it on a slide that has a drop of lactophenol aniline blue on it.  This scotch tape will then stick to the slide and it will allow the fungus to be stained with the dye that you see in the center there. 

This is an example of where you might end up taking a scotch tape prep from the wrong place.  Many times if you take it from the very center of the colony, the oldest part of the culture and that’s where it sporulates the most heavily.  In this case what happened, you see all of these spores in there and it’s difficult to see because there are so many of them.  The bottom line is what you need to do to make it a good mount is to make the mount from an area that is in between there, the outside advancing edge of the culture and the center of the culture growing up so it’s kind of in the middle. 

This is an example of what you would like to be able to see.  This is an organism that has all of the spores attached to the conidiophore the way they were grown up.  This is what the scotch tape prep allows you to be able to do.  If you get it from the right place in the culture, you will see the spores that are attached just as they were growing in the culture.  They’re attached to the scotch tape and then you can see exactly how they are produced and exactly how they look and in this way you can be able to get an idea of what it is that you are dealing with. 

Wet Mount

Another kind of time honored preparation is the wet mount. This is where you take a little bit of the colony and you cut it out of the agar with a wire that’s been bent at a right angle and you take a little bit of the colony along with some of the supporting agar and you place that on a slide with some lactophenol aniline blue. This is an example here of where you can see the piece of agar that has been taken up along with the culture.  One of the things you have to remember is that it’s easy to get too much of the supporting agar on the slide.  If you do that, when you put the coverslip on, if it’s too large what it will do is it will fly out from underneath the culture slip onto the top of the bench where you are working and that’s not what you want it to do.  So you have to take a smaller piece. 

Here you can see the coverslip is going on there and it will be flattened out unless it is too large and you’ll be able to see the culture kind of as it’s been growing but the problem is with a mount like this is the spores don’t stay connected to where they were attached.  The pressure that you put down with a pencil eraser or some other object on there to flatten that out causes them to disassociate from the hyphae or the conidiophore that they are produced on.

Slide Culture

Probably the scotch tape prep is the most universal one right now and the wet mount may be second, and then as a last result, we have in the past used what’s called a slide culture, a micro slide culture. This is an example of what that is.  Basically, when you have a problem with a culture you need to see how it produces the spores in detail.  What you do is to take a plate of 2% agar, it’s just water agar, and you place a glass rod that’s sterile in there or you can just lay a slide on top of the agar like you see here.  The slide’s sterile and what you do is take a little bit of the culture and you cut out like a circle or like a square with a wire or with a sterile test tube, place the agar plug on the slide in 2 places, either end.  And then you inoculate the 4 quadrants of the plug with the culture.  Then you put a coverslip on top of it and as it grows, it produces spores just the way it does in the culture but they’ll be underneath that coverslip and what you can do when you think the culture is mature, is you can remove the coverslip, take it off, put it on a slide with some lactophenol aniline blue and look at it underneath the microscope and you probably will see the spores just as they have been produced underneath that coverslip.  Sometimes you happen to look at it too early so that you don’t see things that you need to see and that’s why we have a second plug on there.  You can go back and put a coverslip on top of that first plug and let it grow longer if you like to.

Here’s where you take the coverslip off and put it on the slide with a drop of lactophenol aniline blue and then take a look at it underneath a microscope. This is the cheap way to do it.  It works well.  You take a piece of filter paper, put it in a sterile petri dish, break an applicator stick that’s sterile in 2 and then put the slide on there with a couple of agar plugs and inoculate it, put a coverslip on top of it and let it grow.  And you put some water in the bottom so there enough humidity in there and that filter paper will absorb the water.

Unusual Zygomycetes

This presentation will focus on some of the uncommon or the unusual Zygomycetes that we see in the clinical laboratory. 

Apophysomyces elegans

One of those that we see is called Apophysomyces elegans.  An Apophysomyces is, we are starting to see it more often as a cause of infection and it’s an organism that has 1 very distinct structure.  It produces these straight to slightly curved sporangiophores that are produced as singly.  And it produces sporangia at the tip.  And these sporangias are kind of pear-shaped and they’re grey looking.  When you look at that sporangium, you will find that the base of the sporangium is a funnel-like structure called an apophysis.  That’s very obvious when you look at it with Lictheimia, you also find an apophysis is present.  It’s not quite as obvious as it is with Apophysomyces, that’s where the name of Apophysomyces comes from.

And here is an example of an organism, and if you look at the dark structure there that looks like a funnel, that is called the apophysis.  That whole thing would have been a sporangia, it would have been a sac full of spores but the top part of it is broken off and you can see the spores that are still in there so there’s part of the sporangium and then at the base of it is that apophysis.  And Apophysomyces is one you wouldn’t have to work at identifying. This is a culture of a Zygomycetes, not necessarily Apophysomyces, but it looks wooly, very rough in texture.

And here’s another one that we see infrequently and it’s called Circinella.  And it produces curved sporangiophores.  These sporangiophores are always kind of curved and toward the inside part of the hyphae.  I have never seen, I don’t think, this organism be associated with disease.  But it’s an easy one to recognize if you ever see it.  The problem with identifying these Zygomycetes is that if they are easier to identify, you don’t see them very often.

Circinella

So with Circinella, you have these sporangiophores that are distinctly curved downward and you have this sporangiophores produced within these sporangia. And this is the example here.  A larger view of Circinella and you can see the sac-like structure and notice that the sporangium or the supporting structure is not straight, it’s curved.  And you might see some septations in there as well. And here you see a better one of Circinella. And very large and full of spores.

It’s another Zygomycetes or Mucorales showing just a culture itself. This one produces spores in a whole different way.  This one produces a kind of a vesicle at the tip of the sporangiophore and there may be some secondary ones being produced.  And they have this structure that is dark.  You see there that is kind of swollen and it’s called a vesicle.  And then if you notice the spores are produced here under kind of a thread-like attachment to that vesicle, and the whole head of those things is covered with those, and they’re called sporangiola. 

Cunninghamella

If you notice that this organism is different from the ones that you’ve seen and it does produce those sporangiola.  This is one that you might see as a cause of disease particularly in a patient who has been burned.  And the organisms name is Cunninghamella.  The sporangiophore ends in kind of an oval terminal vesicle, which you saw a minute ago, and in some of those vesicles may produce some secondary ones in there and it kind of produce in a whirl.  The sporangiophores are smooth, sometimes they are kind of rough, and you can see that on these mounts.  The sporangiophores are produced on these short denticles over the surface of these vesicle and we call those sporangiola.

This is an example of a very high-powered view of these sporangia spores and if you look at the one almost to the left of center and notice that it is light in color but if you look closely you will see little tiny spikes coming out of that thing and it’s rough-walled.  You can see it if you focus up and down.  If you look at one at about eight o’clock down at the bottom, you’ll see that it’s darkly stained but it has those spikes coming off of it, very finely roughened spores. And this is an example of how those spores are produced and sometimes they just fall apart.  So what you end up having to do is look around and try and find all of the structures that are there to get an idea of what it is.  In the center, you can see that there is a long sporangiophore but it’s difficult to tell any more than that so it would be hard to know if this is what this is or not. And on here, the same thing holds true.  These are more like chlamydoconidia.

And this is an example of one where you actually can see the 2 on the right-hand side.  Those are the kind of vesicles that are produced.  And on the left-hand side, those sporangiola are attached to the vesicle that is in the middle of that.  It’s hard to see because it is so dark and then the spores are attached to the tip of that structure that adheres them to the vesicle. Here’s where you can see it well.  The left-hand side there is a vesicle and on the right-hand side, if you look down deep you can see the vesicle but you can see the little tiny pegs attached to it and then there is the spore at the tip of that.  Those are called sporangiola and the spores are what you see at the tip of that.  And that’s a good example of Cunninghamella.

Now this organism here is just another Zygomycetes that is wooly in texture and you end up having to make a mount of this to get an idea of what it is, but you certainly can’t tell from looking at the culture. Here is another one that shows you the kind of a brown pigmentation. This one is a different looking organism. This is one called Syncephalastrum. Some people say that when they look at it under low power, it reminds them of Aspergillus.  Well, that may be the case, but then what you do is you look under a higher power.  So we’ll look at some of those and see if we can figure out what the organism is.

Syncephalastrum

Syncephalastrum, there is a terminal vesicle that’s produced just like you saw the Cunninghamella but around that vesicle there are some cylindrical structures called merosporangia that contain the spores.  So the merosporangium is the sac-like structure that holds the spores but it’s not round, it is like a tube-like structure and so the sporangiospores are formed in its linear fashion within this merosporangia, which looks like a tube that surrounds this kind of vesicle.  And you may find a rhizoid with this too. This is an example here of the tube-like structure of this sporangia that you see called merosporangia and you can see the spores inside of there and they are arranged in a linear fashion.

This is Syncephalastrum and you can see the central vesicle there and surrounding it would be all of these merosporangia that are kind of tubular in fashion inside of those are the sporangiospores. And here’s one where you see a very large hyphal strand and if you look above at the dark structure you’ll see that there’s a round vesicle and then up around that vesicle are the cylindrical merosporangia containing all of the spores in that’s Syncephalastrum and that’s what it would look like. This is just another one showing you a better view of what it looks like.  Sporangia actually are those cylindrical structures.

This is another Zygomycetes or Mucorales.  It’s kind of brown. And this is an unusual organism to see except that we are starting to see it more often than we have in the past.  And it’s one that produces a different looking sporangium.  This organism is called Saksenaea vasiformis.  And it produces a sporangium that is kind of flask shaped or vase shaped and has a long beak on it.  Notice in the center we just start off about 4 o’clock, there are some rhizoids on this thing.  There are rhizoids, there’s a sporangiophore at the base and all of a sudden it swells up and becomes kind of vase shaped and it has a long beak on it.  And you notice at the tip of the beak, there are spores that are coming out of that thing. And here is just a culture.

Saksenaea vasiformis

To look at Saksenaea vasiformis in some detail is what you see is the distinctive long, flask-shaped sporangium with a long beak present and you find rhizoids that may be brown. Here is that sporangium.  You can see that it is kind of vase shaped at the bottom on the right-hand side facing 4 o’clock and look at the left-hand side going up at about 11 o’clock.  That is where the beak was that has broken apart and all of these spores are coming out of it and they are rhizoids that you see at the base there.  So this is Saksenaea vasiformis.

And here’s one that’s intact.  You see the vase-shaped part of the sporangium and you see the long beak on this and this is an unusual one to see, but it’s been associated with brain infection and a number of other things and we had a case of osteomyelitis associated with this organism.  It’s found in the environment.  It’s found in the soil. Another one of these Mucorales or Zygomcetes.  And they pretty much all look alike in culture.  You have to rely on a microscopic identification to be able to identify these organisms.


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