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Clinical Mycology: Direct Examination Series

Paracoccidioidomycosis and Penicilliosis

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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 Paracoccidiodes and Sporothrix. Thank you, Dr. Roberts.

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

This is an ongoing presentation that focuses on the individual groups of organisms as seen in the direct examination of clinical specimens.

The next slide shows a number of the methods that are used for detecting bacteria and other things.  And fungi can be also detected in those particular stains if one just simply looks for them.  So it is a matter of recognition and being aware that you would have to think clinical microbiology when you start looking at all of these things.

The next slide shows a continuation of different methods that can be used like the acid fast stain and the Pap smear used in pathology and histopathologic sections that where you can see different fungal organisms. 

The next slide shows you an organism that is not commonly seen in the United States but, nevertheless, it is seen in Central and South America so we are apt to see it at some time.  It is Paracoccidioides brasiliensis.  The hallmark of this organism is the yeast form.  This is a dimorphic fungus.  It has both a yeast and a mold form.  The yeast form is a large budding yeast, 8-40 microns and sometimes even larger than that.  There are multiple buds around the parent cell that make it resemble a mariner’s wheel like you see on a ship.  And the buds come off in all dimensions, the top, the bottom, and the sides and when you put a cover slip on it, it appears they are only on the sides.  But it is a round ball and you will see on the next slide

What I am talking about.  You can see some in the background there.  This is a yeast that is taken from a culture.  Kept at 35-37 degrees Centigrade and it is typical for what you would see if you were looking for the textbook perfect picture of an organism.  Unfortunately, that is not what you see often times. 

The next slide shows you what you might see and this is not anything that would tell you that this is Paracoccidioides brasiliensis except that it is.  We know that by culture.  There are a number of yeast cells in there.  There is one in the upper, kind of the top portion of the slide that shows a bud on the left hand side.  You would expect to find multiple buds but they are not present here.  What you do see about maybe 5:00 o’clock is there are two cells that are sitting adjacent to each other and they in some way resemble Blastomycetes.  This organism used to be called Blastomycetes brasiliensis because it mimicked Blastomyces.  And so that is why I want to point this out.

The next slide shows you, I think it’s a Gram stain of a specimen that I acquired when I was in Honduras, and what you can see are the numerous cells in the background that appear to be stained with a Gram stain.  And you have stippling in the center.  And if you look around on the left hand side off the center, you can see there are a number of cells of different sizes.  None of them really show multiple buds and so you would continue to look around in all of the fields to try to find that feature.

The next slide shows you just another field of that same section but if you look on the right hand side, there is a cell exhibiting multiple buds coming off all the way around the side and you don’t see them on the top there but that is what you probably would see.  It’s not textbook perfect but you don’t see those sort of things all that often. 

The next slide shows you a blood film and basically in there are all these yeast cells of Paracoccidioides but very little budding at all.  So, again, it just points out that you are going to see different forms and you need to look around and make sure that you are not missing something that’s in another field.

The next slide shows you histopathologic section with almost in the center, a little right of the center, you can see a cell that has some buds coming off around the periphery and that’s what you would like to see in a tissue section.  Sometimes when your processing is going on those buds are knocked off.  The other thing that happens sometimes is that when you see small forms of Paracoccidioides brasiliensis, as small as Histoplasma, as small as two microns in size with little tiny buds coming off the outside and it’s difficult to recognize those so you just might be aware that it can occur.

This slide is an over stained silver stain but on about 3:00 o’clock you can see one that has a central cell with all of the buds coming off around the perimeter.  So this is what Paracoccidioides brasiliensis would look like.  In the lower left hand corner, you see a cell that has a small cell attached to it at the top.  Some people might say, “Well, that looks like the beak that you see with Blastomyces.”   But if you look around in that field and you will happen to see up about 10:00 o’clock, you see another one with multiple buds.  You would have no problem recognizing this.  I think one thing that is important to remember as well as other things is that when you look for these organisms; you look for the common organisms first.  Then you look for an uncommon manifestation of common organisms next and then you start looking for all of the uncommon things.  And so when you sit down and you look at one of these things and somebody says, Ahh, that is going to be Paracoccidioides, the odds are it is not going to be and you probably didn’t even need to bring it up.  So what you do is you look at all of the all of the whole slide and you get a consensus of what’s there and then generally, you will find that this common thing or some uncommon manifestation but, nevertheless, you can see some of these and you just have to astute enough to remember what they look like.

This next organism that we are going to discuss in the next slide here is Sporothrix schenckii.  Sporothrix schenckii is something that we see not infrequently.  It is a small; it is another dimorphic fungus that has both a yeast and a mold form.  And the yeast form is what we are seeing here.  These are small budding yeast cells in the same size range as Histoplasma anywhere from 2-6 microns, generally.  They’re oval to elongated and you can see that some of them look like cigars.  And they are called cigar bodies just for that reason.  Some of them will have a bud or two attached that haven’t detached whenever the slide happened to be made.  Ordinarily, they just have a single bud.  But it’s the elongated yeast cell that is the hallmark of this organism.  And when you see that, you pretty much know what it is.

The next slide shows you a PAS stained slide with all of the elongated cells in there.  A major difficulty with diagnosing Sporothrix schenckii in a clinical specimen using direct microscopic examination is that you just don’t see them.  This happens to be one where you did and it is a rare occurrence that you see one that stains this well.  Most of the time you can grow it in culture and you can’t see it on direct examination.  And so if you did see it, it would be these elongated cells like you see here.  Some of them can be round and some of them can be oval. 

The next slide shows you some of the cells that are elongated up in the upper left part of the slide and then that is pretty much what’s there.  They’re few in number on this particular one, but again, most of the time you just don’t see it.  And no one knows the reason for this.

The next slide shows you an enlarged view of the first slide that you saw.  This organism, this is from a bronchoalveolar lavage.  And it was diagnosed by just simply looking in there and seeing what we see, elongated yeast cells and the only one that we ever see that is elongated is Sporothrix.  So, they stuck their neck out on a limb and said, “Okay, this is Sporothrix.”  And it turned out it grew that organism up.  So that is the hallmark of the organism, the elongated budding yeast cells.