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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 Cryptococcus. 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 or groups of organisms as seen in a direct examination of clinical specimens.

The next two slides show you the different methods that can be used for detecting these fungi in clinical specimens and some of those are stains that may not be used for staining fungi but they can be found there if one has the ability to remember to look for fungi in those particular stains.

So the next slide just shows you a continuation of the methods that we can use in clinical microbiology for seeing fungi.

The next slide shows you an example of Cryptococcus neoformans. ‘Crypto’ means hidden and ‘coccus’ as you know refers to a round cell. When they first described Cryptococcus neoformans, they had never seen anything like it because it was this round cell that was surrounded by what looked like a capsule, and so they called it ‘Crypto’ because it was a cell, it was ‘coccus’ that was hidden by a capsule. At that time, they had never seen a cell like that before so they called it ‘neoformans’ which means new form. So Cryptococcus neoformans has a hallmark of producing cells that vary in size anywhere from 2-15 microns, and with Cryptococcus gatti, the cells can be as large as 60 microns. The cells generally as a rule are nice and round, they are spherical, they may or may not have a capsule, the buds, if they are present, have narrow necks, and you may find pseudohyphae present with some of these cells of Cryptococcus. So in this particular slide, you can see in the background the size variation is apparent, and that’s the hallmark of identifying Cryptococcus neoformans along with the budding cells that have narrow necks on them.

So, the next slide is a phase contrast photomicrograph and you can see a big, thick gelatinous capsule around a perimeter of the central cells. The central cell is the yeast cell of Cryptococcus and the other part is the polysaccharide capsule of Cryptococcus.

The next slide shows you the very same thing. The capsule is around the perimeter, the central cell is round, and then there is a bud that appears to be pinched off and narrow-necked, and that’s what you see with Cryptococcus neoformans.

This is just a larger view, showing you next slide, essentially the same thing. Sometimes you see some granular material within the cell but this is the narrow-necked bud and the capsule, if it happens to be present, is helpful.

This is calcofluor white on the next slide showing you that the cells vary in size; they show up very well in this fluorescent stain. This stain is a fluorescent brightener, it stains the chitin in the cell walls, it makes the cells fluoresce, depending on the filter, either blue-white or yellow-green as you can see here. And these cells vary in size, you can see the very large ones and the small ones, and on the lower right-hand side about maybe 5:00 o’clock you notice that there is a cell that is budding and has a pinched-off bud present. The variation in sizes shows the narrow-necked buds that are helpful in identifying this organism.

The next slide is Calcofluor white showing you essentially the same thing and, in this case, it’s hard to tell that this is Cryptococcus neoformans you see at about maybe 2:00 o’clock or 1:30 there is a collapsed cell, it looks cup-shaped and you notice some of the other cells just appear to be round, and some of them are a little bit larger than others, but you would have to look around on the whole slide to get a consensus of what is there.

The next slide is an acid-fast stain, the Kinyoun acid-fast stain that we use for staining mycobacteria. This happens to be the counter stain, methylene blue, in the background that does not stain mycobacteria, but in this case you see numerous cells with Cryptococcus neoformans and they are primarily in the center, and you can see some there with maybe about 6 or 7 cells in there, one has chained to three of them sitting together, and if you didn’t think about it as microbiology in general, you would miss those cells. So in this case, you have to look around a bit more and decide if it’s going to be Cryptococcus or what it’s going to be, but if you look at the cells, the top cell, the top two cells in that cluster on the right inside, you notice that around those cells, there is something that appears to be kind of feathery around the perimeter, and that’s the polysaccharide capsule with Cryptococcus neoformans. It’s around, actually around all those cells. This is kind of feathery around the outside.

This next slide showing the same thing except that these appear to be the beginning of a pseudohyphal strand, and certainly with Cryptococcus you can see that. There are some other cells in there at about 6 o’clock and about maybe 5:00 o’clock where you can see round cells of Cryptococcus neoformans that have this capsule and it looks almost like they are projections sticking out from the sides of that. It’s just the capsule where it’s constricted away from the space. So this is what Cryptococcus neoformans can look like.

Now, this particular slide is a PAS-stained slide, and all the cells of Cryptococcus neoformans here are about the same size, probably right around 2 microns, and you notice there’s a clear space around the outside of the yeast cell. That’s the capsule. So here you would certainly have to think about Cryptococcus neoformans.

The next slide is a phase contrast photomicrograph of a pleural fluid from a patient with HIV, and the patient had a pneumonia and a lot of pleural effusion, and they actually removed some of the effusion, looked at it underneath the microscope and what we see are all the cells in there and the larger part of the cell is the capsule and the central cell, the central part that’s round is the cell of the yeast of Cryptococcus neoformans.

Same thing here.

And then in the next one you can see what looks like a germ tube in the one at the top coming out of this cell, and you do see that with Cryptococcus neoformans.

Here you see about maybe 5:00 o’clock you see what looks like a germ tube coming out of there and the other cells are pretty much single cells with a capsule around them.

This is a Wright stain from a bone marrow, and it’s not uncommon to find Cryptococcus neoformans in bone marrow or in blood or other body fluids when there’s disseminated disease. And here you see in the center, you see the cells that vary in size. There is a very large one of the lower cell is very large, compared to the others, about twice the size of the other two, but you would need to move around and look at all the fields to see what’s out there instead of just looking at this one field, but these cells are nice and round.

And here is another bone marrow, and you can see the cells that have a space around the outside and they are pretty much in the center. You can see the cells are the darker stained ones and there is a space around the outside. Notice that the cells do not touch each other and the reason for that is the capsule separates them. If you look at the lower part of the slide right about 6:00 o’clock, all those are platelets in there that stained with the Wright stain.

This is another example of the Wright stain. You can see the cells at the top and along the left-hand side, and actually all around in all the whole slide, at the left-hand side at right about 8:00 or 9:00 o’clock is where you see the yeast cells with a big space around the outside and those are encapsulated yeasts, and if you look at the top you’ll see there are the four of them up there and those are the cells, they are encapsulated, that’s what the clear areas are around those, and that is what Cryptococcus neoformans would look like.

And this next slide is just the same thing, and you see the bud on those two at the center where it looks pinched off, and so this is the hallmark of Cryptococcus - the capsule around the outside if it’s there. It may not always be there, and then that narrow-necked bud.

The next slide shows you essentially the same thing without the narrow-necked buds on it, you see some cells that are surrounded by a clear space a little bit and that’s what Cryptococcus would look like. The only place you see them on this slide is in the center.

And then this happens to be what we call a cell count of a spinal fluid, where they centrifuge the spinal fluid on the cytospin centrifuge and then they stain it with the Wright stain. All these cells are Cryptococcus neoformans, all of them. And you notice they have buds on them, they have narrow-necked buds, and some of the cells are larger than others, and when you see that hallmark, those are two hallmark features of Cryptococcus.

This is a Gram stain. The Gram stain appears to be overly colorized. It’s red, the safranin shows the red color but you can see the cells have a space around the outside and that’s Cryptococcus neoformans.

Same thing on the next slide. Very hard to recognize for sure if that’s what it is, so this is just part of a whole slide that was looked at.

The next slide shows you a Gram stain showing you that it’s been over-stained with the crystal violet. The cell in there is somewhere, you can’t even see it, actually on the left hand side, and on the right-hand side you can see two cells but all the capsule material is stained with the crystal violet in this over-stain.

The next slide shows you another slide where the crystal violet has stained the Cryptococcus neoformans yeast cell but not over-stained it. And you can see the difference, the different morphologic features, you can see that some of the cells are larger than others, and you can see that there’s a space around them the lighter red is the capsule, and that’s what the Cryptococcus neoformans would look like on a Gram stain. And it’s a very good one there.

This next slide is another example showing you the same thing.

This next slide shows you a couple of yeast cells in there stained with a Gram stain.

The next slide shows you something that we see more often than people think and this is on the feathered edge of a Gram stain and this central cell is Cryptococcus neoformans. If you notice in the center of it, you’ll see something that looks like basophilic stippling. Basophilic stippling is something that you see that is characteristic of Cryptococcus neoformans.

This slide shows you something that referred to a basophilic stippling. There are some very difficult to recognize spots on this slide that look like basophilic stippling with a clear space around it. In each one of those areas where you see that, one is about maybe 1:30, towards the top of the slide, one about maybe, I don’t know, it’s hard to describe exactly where they are, but if you look in the slide, you will see there are clear spaces with purple dots in them. That’s the basophilic stippling that you see with inside the cell of Cryptococcus neoformans.

The next slide shows you this even better. Look at the clear space with all the dots in there. It’s something that you see that more often than people recognize. You simply have to look for it, and when you ask people if they’ve ever seen it before, often times they’ll say, “Yes, but I didn’t know what it was.” Well, it’s Cryptococcus neoformans exhibiting basophilic stippling within the cell that did not stain, so you see the purple dots within that unstained cell.

This next slide is a PAS stained slide that shows you the polysaccharide capsule of Cryptococcus neoformans in a very massive amount of organism on a slide. And you can see the yeast cells, and if you look closely you can see that there is a yeast cell in the center of the slide with the perimeter is this purple looking capsule and the cells vary in size inside of that capsule. That’s Cryptococcus.

This is a Pap smear, and if you look closely, you will notice that there are some cells that have a space around them. One about 5:00 o’clock, one about 9:00 o’clock, about maybe 11:30, and inside of that clear space there’s a sort of a brownish looking cell that’s kind of round, and that’s Cryptococcus neoformans. That’s a Pap smear.

The next slide is a Pap smear and those cells are also present, except that they are brown in here and these don’t exhibit the space around the outside as much as the previous slide did. The brown cells in this slide are Cryptococcus neoformans without a capsule.

The next slide shows you the same thing, and there in the center is a cell, there are several cells in there that are sort of lightly brown pigmented, those are Cryptococcus neoformans. You would need to look at the whole slide to be able to decide that that’s what it is before you made a decision. This is a Pap smear.

This next slide is a Pap smear, and there you can see more of those cells, they are easy to see here, and all the brownish looking cells are Cryptococcus neoformans, and they vary in size, and that’s a Pap smear.

The next slide is a Pap smear showing you much better where you can see the round cells in the center of a big round cell. The large part of the cell that is surrounding that brown cell is the capsule, the interior of the cell where it’s brown is the yeast cell, and on the one, you can see there’s a bud beginning to form on the left-hand side. And at the top right, maybe 2:00 o’clock, there’s one that’s beginning to bud too.

The next slide shows you essentially the same thing. These cells vary in size. You can see one almost looks like it has a germ tube on it.

The next slide shows you one that does have a germ tube on it, and the cells vary in size on that one, and it’s not on that uncommon to find that.

On the next one shows you a cell that is totally encapsulated with a germ tube that is actually branching. So that whole thing is surrounded by the capsule. And that’s a Pap smear.

So, another thing that’s been used by many laboratories for a long time is the India ink preparation.

Where you simply take a drop of India ink and you place it with some sediment from centrifuge spinal fluid. If you saw a situation like this, the number of cells in there would be much too great to be causing infection. This happens to be a collection of bacteria that are probably found in the India ink bottle and contaminated the India ink bottle.

The next one shows you talc crystals from the glove powder from a clinician who has done the lumbar puncture.

The next slide shows you red cells and you can see on the left-hand side it looks a little bit concave.

The next slide shows you red cells and then there are two white cells that are very apparent in there.

And the next slide shows you yeast cells. If you had a patient with meningitis that contained this many yeast, it would be much more of a problem that what you imagine, and so this is probably more likely representing contamination of the India ink bottle again with the yeast rather than a clinical infection.

The next slide shows you talc crystals in the background. And one cell that is bright white and it’s over-exposed. Within that white area, that clear area, is supposed to be a yeast cell, and that’s Cryptococcus neoformans.

The next slide will show you what it might look like a little better on another slide. This is where there is only one cell seen in the sediment of spinal fluid from this patient’s spinal fluid. So you see this cell is round, and has a capsule around the outside. That is the only thing that perhaps would look like that.

The next slide shows you phase contrast photomicrograph of yeast cells, there are four of them in there, and they have a space around the outside and they vary in size.

The next one is an India ink preparation where the light has been turned up way too high, and the carbon particles in there that are black look almost red. But basically what you see are the yeast cells in the center and the capsule that’s polysaccharide is around the perimeter of that cell, and so this looks like a big, round headlight. So that’s Cryptococcus neoformans.

Same thing here on this one but the hallmark of this one is that you see cells that vary in size. There are some that are very small in the background, and there are some that are very large in there, and they appear to be granular and the cells appear to be granular internally.

And the next one shows you the pseudohyphae, and that’s a good example of what pseudohyphae look like. You can see they look like they’re yeast cells, they would be elongated and remain attached to each other and have constriction at the point where they look like they are attached.

The next slide shows you, this happens to be from a bronchoscopy specimen and all of these cells are just about the same size, right around 2-4 microns in size. Not all that usual to see one with this consistency in size, but this is, nevertheless, a case that we saw a few years ago that shows the cells are small, probably around 2 microns, and they have that capsule around the outside.

This is just another field over, the next slide showing you the very same thing. So that’s Cryptococcus neoformans.

This is a H&E stained slide from bone marrow, and in the very center you see one cell that’s sitting there with a capsule around the outside of it and the cell is in the center, and that’s Cryptococcus.

This is another one H&E stained slide, and you can see many cells of Cryptococcus neoformans in there. Some of them are round, some of them are football-shaped, and occasionally they appear to be football-shaped when they start to collapse.

Next slide shows you an H&E and you can see that there are several that are football-shaped in here.

The next slide shows you an H&E, showing you some collections of these cells, and notice that they vary in size, and they are not all necessarily so round, so they’re supposed to be nice and spherical but they don’t always have to appear that way. But, it’s the variation in size that you see and the space around the outside of them, and you notice in some of those the cells do not touch each other, and the reason for that again is because the capsule is present.

There you can see in the next slide a better view of that. You can see that none of the cells are touching each other because of that capsule keeps them separated.

The next one shows you a touch prep from a lung, and if you happen to look at the cells in there, you look, you can see some in the background that look extremely small and some that are very large. That’s what the hallmark is. You don’t have to look any further once you see the variation in size like that, you think of Cryptococcus neoformans.

The next slide shows you one that is not so clear but it shows you the very same thing. It varies in size of cells, and notice that none of them touch.

The next slide shows you a long piece of pseudohyphae in there and the yeast cells of Cryptococcus in the background.

So this is the discussion of Cryptococcus neoformans, and I’ve shown you lots of slides there with the different morphologic features of Cryptococcus and lot of variation upon those features to give you an idea that they all don’t look the same.