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Clinical Mycology: Direct Examination Series


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Published: October 2012

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Direct microscopic examination of fungi in clinical specimens relies on both bright-field and phase-contrast microscopy, as well as multiple stains to optimize visualization of the organism. This presentation includes an extensive collection of specimen photographs to assist you in identifying these organisms. Each presentation in this 11-part series addresses 1 or more genus or group.

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. Dr. Roberts discusses the features of specific organisms under direct microscopic examination using multiple preparations. This module examines zygomycetes. Thank you, Dr. Roberts.

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

The next slide shows you that this is an ongoing presentation that focuses on the individual or groups of organisms as seen in the direct microscopic examination of clinical specimens.

The next slide shows some of the stains that are used in clinical microbiology and in pathology for detecting microorganisms; but, not necessarily the fungi; however, the fungi can be detected if one thinks clinical microbiology when you are looking at the slides.

The next slide shows you a continuation of those stains and the different stains that are used in the same manner and examples would be the Pap smear used in cytotechnology area where you see the Pap smears for respiratory tract specimens and you might find an organism in there you might not expect to see but you can see fungi very well.  And then histopathologic sections used in methenamine silver stain, the PAS and H&E stains, those are designed for detecting for tissue architecture as well as detecting organisms.  And so you just need to be aware of what might be present and just keep that in your mind when you are looking at these stains because you might find a fungal organism there that you don’t expect to be there. 

The next slide shows you what we are going to talk about wide, pauciseptate hyphae that could be present in a clinical specimen if you happened to see one in your laboratory.  And if you saw that you would think about a group of fungi called the Zygomycetes and now they have decided to call this group the Mucorales.  Name changes have been frequent with this group.  Now I think Mucorales might be the better term.  And the name of the infection used to be called Zygomycosis, prior to that it was called Mucomycosis and now they would like to call it Mucomycosis again.  So as long as you understand what we are talking about it really doesn’t make much difference what you call it, in my opinion.  In terms of what you see in a direct microscopic examination with these pauciseptate hyphae you notice that many times in a biopsy for example, the hyphae fragment into smaller pieces.  You can find some septae in some of these and that is why we call them pauciseptate because there are just a few septations present.  If you happen to see a dressing or something where the organism can grow unrestricted, you will find that they look ribbon-like and another thing that I think is important to remember, is that Aspergillus flavus, one of the organisms that causes disease in leukemic patients can actually have hyphae that are pauciseptate and large just like the Zygomycetes.  And so when you look at a direct examination the odds are that it is probably going to be one of the Mucorales but it can be Aspergillus flavus and what you do is you try to look to see what the frequency of the septations is and if there are more septations then you would expect to see with a Zygomycete.  It is probably going to be Aspergillus flavus.  Some of the Mucorales that we talk about that we see in the clinical laboratory are Rhizopus, Mucor, and Absidia and now the name has been changed for Absidia is called   Lichtheimia and so there are a number of name changes going on here as a result of some of the molecular taxonomy and as a result of people looking back in the literature and finally determining that some of these organisms were named something else long before they were called what we have commonly called them.  And so they go back to the original name and start there. 

The next slide shows you a direct examination of a piece of material taken from a dressing from a young person who was riding a motorcycle and had an accident and skidded down a gravel road and the arm was actually damaged and the skin was removed from the surface of the arm.  It was debrided and then the dressing was placed on there, the gauze dressing was placed on there and then it became infected.  The dressing was taken off and some of the material from the dressing was put on a slide and this is what you see here.  The reason for the long ribbon-like hyphae is because they were growing on that gauze dressing unrestricted.  If you look at about 1:00 o’clock, maybe it is hard to tell on this, it’s the upper right corner there is a vertical hyphae that has a septation in it.  And that is why we call them  pauciseptate hyphae.  When we see these long branching fungi like this that are wide, they are twisted and ribbon-like.  And often times they sometimes will fragment into smaller pieces but when they are unrestricted, you see it like this.  This is not something you are going to see all of the time.  They are sparsely septated or pauciseptate and sometimes again you have to think about Aspergillus flavus

The next slide shows you a blood vessel that actually has the whole outer wall of the artery filled with hyphae of a Zygomycete.  This is probably an immuno-compromised patient.  And if you look at the interior of that blood vessel, you will see that it contains a large number of platelets in there and what happens is these hyphae actually project into the artery and the platelets and the white and red cells all aggregate together along with the hyphae and a clot or a thrombus forms in there and then basically what happens is the blood flow distal to that stops and the whole area down there becomes necrotic and so the hallmark of the Zygomycetes is they cause a lot of necrosis particularly in the sinuses.  And diabetics are people who are very prone to getting infections with these Zygomycetes.  And this happens to be probably from a case of a dissmeniated disease when you see it in an artery like this.

The next slide shows you the hyphae in there that are large and there are a number of septations in there.  This one actually came from an eye.  In a case of endophthalmitis where the interior of the eye was totally infected and the eye was removed because there is no real effective treatment for that disease.  But you can see how large the hyphae are and look at the septations there are a few in there and notice also they defragmented into smaller pieces, so some of the hallmark features we talked about are showing on this slide.

The next slide shows you the kind of branching that you might see.  But it also shows you, this is a biopsy, and it shows you that this organism fragmented into many pieces.  And sometimes it can branch, usually not at a forty-five degree angle mostly at a ninety-degree angle but it can branch pretty much any way.  But I think what you are seeing here are mostly fragmented pieces of these large hyphae.

The next slide, this is a silver stain, a methenamine silver stain, and every once in a while you will see that the silver stain will deposit silver around the perimeter of some of these hyphae but they don’t stain all that well with a silver stain at least in our hands here at Mayo.  And the stain that we have used in the past has been one that has been here for many, many years and so what we did is we use it as diagnostic of Zygomycetes.  If we saw a Zygomycete that really didn’t stain very well, we would pretty much know that is what it was.  If it stained, it probably turned out to be something like Aspergillus.  Well, evidently that was something that occured with the stain that we used.  We have now changed methods in the laboratory and the Zygomycetes now stain with the silver stain.  So basically, what you are seeing in here are hyphae that have stained with a silver stain.  They don’t appear to be septate, except, well.  There is one maybe at about 6:00 o’clock up from the bottom where you can see a clear septum there.  This is what you would see, fragmented pieces of hyphae and an occasional septum. 

The next slide shows you essentially the same thing except there are lots of fragmented pieces in there and these didn’t stain with the silver stain.  This is from the old stain.  It was very helpful to have that because we could distinguish the Zygomycete from an Aspergillus.  And I don’t think anyone here remembers what stain we used, how we use it, and why we use it, and why they changed it but nevertheless that is the way it worked. 

This is a frozen section from the lung of a young boy, a fifteen year old boy who had a history of leukemia, it was in remission and he came out of remission and developed pulmonary infection and this was a part of the biopsy and is what we saw in the frozen section and there are large hyphae in there that have occasional septations and it is fragmented.  This turned out to be a Zygomycete.  This young boy ended up having this organism grow into and surround the pulmonary artery and did not survive the episode because there was just too much involvement.

The next slide shows you another vessel and in the interior of this vessel you can see all of the hyphae and you can see the cross-section of hyphae in there.  And again what would happen is a thrombus would develop inside there and eclude that artery.  And then distal to that would be no blood flow and a large area of necrosis where you would probably see the organism.  These Zygomycetes or Mucoralesproduce a lot of necrosis.

And the next slide shows you tissue from a brain.  This is a silver stain from the brain and you see what look like hyphae in there.  And it is a bit disturbing to see this in a brain biopsy because it’s usually you don’t think about it being there except in cases of patients who are diabetics and develop brain abscess.

The next slide shows you essentially the same thing.  You can see the filaments are very long in there.

And then the next slide shows you something different.  These are actually capillaries that are seen in the brain.  And if you look at the interior of those capillaries, you will see that there are red cells that are stacked in there.  That is called rouleau or rouleau formation.  And those cells are stacked in there are red cells and what we have been looking at are capillaries found in the brain stained by the methenamine silver stain, it didn’t really take up the stain but they look like hyphae at first glance.  So you might think about then when you are looking at brain biopsies.

The next slide is a Pap smear of a respiratory tract specimen and you can see that there are large hyphae in there. One in there is branching at a right angle.  The hyphae are not of standard size, they are not of any particular size; some are very large and some are narrower.  So they look different when you start to look at them.

And here is an example of one where there is a distorted filament.  A portion or at least a segment of one and it has a septum by it.  And you can see there is a lot of variation with the Mucorales.  They don’t all look the same every time when you look at different specimens.  They sometimes will form some unusual forms.

The next slide shows you the same thing.  This is a Mucorales with large hyphae and in here I see one area which might show a septum up about maybe 11:00 o’clock other than that it is just filaments.  This is a Pap smear.

This is another Pap smear showing you hyphae that are just very large and you notice they narrow down to a a smaller size and it just shows you the variation of things that you can see with these Zygomycetes.  Again you need to look at the whole slide.