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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 Blastomyces. Thank you, Dr. Roberts.

Thank you, Sharon for that introduction. I have nothing to disclose. This is a series on the direct examination of clinical specimens an ongoing presentation that focuses on the individual groups of organisms in the direct microscopic examination for clinical specimens, the detection of fungi. There are a number of methods that one can use for detecting these fungi and basically it just depends on what you have available at your own institution. In the next couple of slides we’ll show you each of those particular methods that can be used with some utility. We have covered those in a previous discussion.

This slide shows you some additional ones including bright-field microscopy which can be used by reducing the amount of light going through the condenser to give you some contrast. Phase-contrast microscopy is an older method. It is not used in many laboratories and probably would not be much value to most of you. But the others you can see are stains that are used in microbiology or in pathology. They can also have the ability to detect fungi in clinical specimens as well as histopathologic sections.

In terms of trying to detect the fungi in specimens, the size of an organism is important and whether or not it happens to be a yeast or a mold is also critical in trying to decide on which pathway to use to identify specifically the organism. Histoplasma capsulatum and Sporothrix schenckii are both two of the smaller ones that you see the size range is anywhere from 2-6 microns. They both have small budding cells Sporothrix schenckii will have elongated cells. Cryptococcus neoformans is sort of intermediate in size range, it is between 2-15 microns and the cells there are round and vary in size. Blastomyces is 8-15 microns in size. It has broad-based buds with a retractile wall which we will see in subsequent slides. And Paracoccidioides brasiliensis is one of the larger ones and the cells can be really anywhere from 2-60 microns. It says 5 here, it can be that size as well and it produces multiple budding cells.

So let’s start off with a discussion of Blastomyces dermatitidis. It is one of the larger ones that you see. Again it is 8-15 microns in size about the size of the smaller end of the scale would be about the size of a red cell to about twice the size of a red cell. If you look at the wall of the organism, you will see that it appears to be double-contoured and maybe looks like a double wall. And I think that the distinction characteristic is it has what is called broad-based buds. We remember the three b’s standing for what we just mentioned.

This is a slide that is a little difficult to interpret. It is a potassium hydroxide preparation from the skin lesion from a patient. If you look very carefully, you can see the two cells in there that have a flat base between the two of them or a broad base between the two cells. Both of those are a Blastomyces dermatitidis and they are not the best representation of what this organism looks like but in reality it can look just like that and not be so typical.

Next one is a phase-contrast photo micrograph and I think it is included here because if you look at the cell that is in about the 8:00 o’clock position, you will notice that there is a flattened area at the top of that cell and that is the place where the broad-based bud happened to be and sometimes you will see that if the cells don’t have buds on them, you will see individual cells. You might see the flattened area where the broad-based bud was. 

The next one, this next slide is actually almost a picture perfect representation of a Blastomyces dermatitidis should look like. It has a double-contoured wall. You can see that the cytoplasm appears to be constricted away from the cell wall and the daughter cell and the parent cell are connected to each other by a flattened area in between the two.  And so the broad-based bud is the characteristic and also another thing that you see is you see a lot of inter-cytoplasmic granular material within some of those cells. So this is a typical one. This is what it would look like if you were looking for the perfect one and if you saw this it would be no doubt that’s Blastomyces.

This next slide shows you a number of single cells sitting in a clump. Somewhat difficult to interpret but if you look carefully, you will notice that the cells have a double-contoured wall and there may be a flattened base on maybe one or two of them. But in this case you would have to look around further to try to decide if this is what this organism really is because there is nothing there that is typically characteristic for that organism in there. But they are not always seen as typical with a typical presentation.

This is a Gram-stain in the next slide showing you a cell with a daughter cell with the bud on it and it is connected to the parent cell by that flat base you can see at the top.

The next slide is another Gram-stain and there it appears to be that the crystal violet is precipitated around some of the cells but the one we are most interested in is the one in the very center of the slide and there you see two cells and it is difficult to see the broad base between them but if you look closely you can kind of get an idea of just what that is and that would be a cell of Blastomyces. It is not so good.

And in the next slide it shows you essentially the same thing, but you can see the broad-based bud between the two.  It is where the crystal violet is over stained most everything in the field and you see some stippling there where the cells are.

The next slide is a Gram-stain and you can see the broad-based bud and the parent and daughter cell and this mirror of an exit of a skin lesion and it shows on the Gram-stain this is what it would look like. The problem with a Gram-stain is that often times everything stains in there and you notice that this cell happens to be sitting in there and there is some other materials sitting around it making it a bit more difficult to see. And so you would have to scan this slide but the whole trick to detecting this organism as well as all of the others is when you look at the Gram stain, you have to think clinical microbiology so that you are looking for things other than bacteria, which you normally use the Gram stain for. And so you would have to look hard and have an astute eye to be able to find some of these things.

Next slide shows you another Gram stain with several cells of Blastomyces in there. If you look around, they are actually all over and they are not necessarily so typical. So what you would do is to look around and get a consensus of what you see in the whole slide until you convince yourself that either it is or isn’t Blastomyces. And when you find Blastomyces in a clinical specimen, a definitive diagnosis can be made if you see the typical morphology. So it is important to look around and try to find something that is typical so that you can make the diagnosis so that treatment and can be started as quickly as possible.

This is a Pap smear and not a very easy one to interpret but all of the round cells on this slide show a double-contoured wall and that is about what you see. You notice it looks like the cytoplasm is constricted away from the cell wall and it looks almost like an eyeball and so this is what you see of Blastomyces. But what you would like to do is look around and find one that is typical and sometimes you simply can’t find one, and you have to just say that it is suggestive of. 

Another Pap smear of a sputum and you can see where the arrow is that is a typical cell of Blastomyces with the double-contoured wall with a flat base between the two. So the broad-based bud is there. And so if you saw this, I think you would be able to call it and say it was Blastomyces.

This is a Pap smear as well, stained a little bit differently and this cell does not look as good but you still can see the parent cell which is the round one and the one on the bottom is elongated and is the daughter cell. And there is a flat space between them. I think when you see something like this you would like to continue to look around on the slide and see what else is there.

The next slide comes from a methenamine silver stain of a sputum sample that a Pap smear has been formed from. And you see the big cell which is the parent cell and the smaller one which is the daughter cell and in between there is a broad-base and it is not textbook perfect but that is what it might look like and we see them like this periodically.

The next slide shows you a silver stain showing you the very same thing, a broad-based bud and this is a very good one. You can call it Blastomyces based on the morphology that you see here.

The next slide shows you a number of cells sitting around that are some one area where two cells sitting adjacent to each other or may be attached to each other it is hard to tell.  The other two are individual cells. If you look at the one where the two cells are together you can notice that the cytoplasm has actually constricted away from the wall of the top cell and it appears to be a broad-base between the two cells and so we happen to know this came from a case of Blastomycosis so it is Blastomyces dermatitidis.

This is a biopsy showing you a cell, more then one cell actually. Three separate areas there where there are cells and on the left hand side at about 8:00 o’clock there is a single cell in the middle; there is a cell with a bud which does not show a typical broad-base to it, and in an area about 2:00 o’clock is another cell that looks like the one in the middle except turned at a different angle. And there the cell does not have a broad base between it and the parent cell. The cytoplasm, however, is constricted away from the cell wall so this turns out to be Blastomyces dermatitidis and this is one of those situations where you would like to look around some more and see what you can see. But this is what you might find and not everything you have to remember is textbook perfect. There is some that don’t present the way we think they should.

And the next one is another tissue section. You can see the cell and it almost looks like the bud has a pinched off appearance to it. But the connection between the two is sort of flattened.

The next one is what I would describe as just an ugly cell as we look at it. It has a parent cell and a broad-based bud and the daughter cell sitting at the top is kind of elongated and it doesn’t look textbook perfect by any means but it is in the middle of an abscess and if you look closely you can see the wall of the abscess and the periphery and you can see that is filled with neutrophils so it is an abscess, micro abscess. And this is where you find Blastomyces.

And this is another one showing you the wall of an abscess all the way around the top.  Neutrophils are in the center filling the abscess and then there are three cells of Blastomyces within that abscess and they are not the typical presentation so you would want to continue looking around. But the tissue response is typical.

This is one, a silver stain of Blastomyces that shows, not actually a broad-base between the two but a narrow base between the two and this is not typical but we have seen it a few times over the years.

And the next slide shows you a larger view of that same cell showing you that it looks almost like a weight set, we have a dumbbell. You see the little narrow connection between the two ends, do don’t be surprised if you happen to see one like this, it looks like a dumbbell and it could be Blastomyces as well.

This is a histopathologic section. The PAS stain stains the carbohydrates in the cell wall and if you look around in there, there are numerous cells of Blastomyces you can see the cytoplasm is constricted away from the cell wall and you notice that the nuclear material that appears to be sort of vacuolated and the one on the right about maybe 2:30 shows a broad-based bud between the two cells. So this is what it can look like in a biopsy.

This is a methenamine silver stain and there are numerous cells. This is a touch prep from a lung. One spot on the lung, this is the one field under the microscope from a lady that had a disseminated blastomycosis. In the cell about 8:00 o’clock, is typical. It has the broad-based bud on it and it shows you that it is not quite as broad as some of the others, it almost looks like that dumbbell appearance. But look around in there, you will see that there are lots of cells that are round, some of them have a flat base on them and at about maybe 4:00 o’clock sort of getting towards the center, you can see the cell where you see the cytoplasm constricted away from the wall. This is an over stained silver preparation and everything under here appears to be very dark. It is hard to tell what the morphology of the interior of the cells looks like.

The next one is just simply another methenamine silver stain and it shows you one in the center is a broad-based bud. There is one up to the left of 6:00 o’clock; halfway toward the middle you can see a cell, it is a large cell with a small bud on the bottom of it. Often times you will see what we call a small beak. It is just a tiny projection that starts off where the bud is going to be and you will see those in Blastomyces. The other thing is you will notice that some of the cells are cup shaped in there and that means that they have collapsed and we see those cells in with some of the other fungi as well. But the size range of this one is different from the others and so I think you can sort them out.

The next slide shows you a broad-based budding cell with a cytoplasm constricted away from the wall and notice that the nucleus is right in the very center of the larger of the two cells. And you can see it also at the top of the daughter cell. So, all of these cells are Blastomyces. 

And all of these next slides are all cells of Blastomyces. You can see different stages of development in there. You can see some with flat bases; you can see some just look like round cells. You can see one in the very center there that has a broad-based bud on it and so, pretty much all of the different forms are represented in this one.

Next slide shows you on the right hand side a small cell with a beak that we referred to and then the cell on the left with a bud on it and has a broad base on it. It is pretty apparent on that one. And if you look at the lower cell and both cells, as a matter of fact, you can see how the cytoplasm has pulled away from the cell wall.

So, this is a synopsis of what Blastomyces dermatitidis looks like microscopically in a clinical specimen and also in a biopsy as well. So, I think if you can recognize it you might be able to be of some assistance to your pathology colleagues who might have some difficulty recognizing these organisms.