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Published: October 2012Print Record of Viewing
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
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 Candida and Malassezia. 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 organisms as we see them in the direct examination of clinical specimens.
In other presentations, we have discussed the different staining methods that are useful for detecting fungi and they are listed on the next two slides. And they are all useful if taken in the right context, so I think the thing people need to remember is, that if you are looking at a stain that is not designed to detect fungi, that you have to think clinical microbiology as I have said many times before so that you look for fungi in the stain that is not designed to detect them, but you can see them.
The next slide is a discussion of Candida species. With Candida, there is something that you can use that is very helpful for trying to make identification for Candida in clinical specimens. And that is that you have to remember that it is a yeast but it also is a polymorphic yeast. It is a yeast that has more than probably three forms, at least more than two forms and if you see any combination of forms that we talk about here you know that you are dealing with Candida. One of the Candida species and there are a huge number of species within that particular genus. So, one of the things that you would see would be a yeast and/or pseudohyphae or hyphae. It is a yeast cells by themselves, anyone of these three by themselves or you can see a combination of any of these. So yeast and pseudohyphae or yeast and hyphae would be a combination you that could see or you might find pseudohyphae and hyphae and so any combination of those. Yeast and pseudohyphae are a little bit difficult to recognize in some of the ways because the yeast cells as you know reproduce by budding. Pseudohyphae are nothing more than yeast cells that have elongated and remained attached to each other and they have constricted the ends. If you look at the end of the cells, they are kind of rounded down where they connect and they look like links of sausages. And then with septate hyphae, this makes it even much more difficult. If you don’t see any other form because it could be just another mold and in fact you are dealing with a yeast. But Candida can produce septate hyphae and generally it produces one of these other forms as a yeast and pseudohyphae along with hyphae. One of those two or both with septate hyphae. In the cases of thrush, you might find just septate hyphae. So you can have any combination of those and that is the trick is to learning and recognizing the different forms.
This is the example here of Candida. Showing budding yeast cells and if you look on the right hand side you see individual cells that are sitting out there. This happens to be a PAS stain slide and you can see the budding yeast cells kind of elongated and some of them are round and they have buds on them. And then there are pseudohyphae that are in the center of the slide where the cells are kind of elongated and they are attached to each other. It almost looks like there is a space between them but there is really not it is just the way the ends are constricted down, narrowed down. And then you may find just true septate-hyphae as we have mentioned before or any combination thereof.
This is an example of what looks like micro colonies of Candida in a sputum sample. This happens to be a phase-contrast microscopy photograph and that is what you are really looking at are just small, minute colonies of Candida and those are hyphae that are being produced in there so you would want to look around and try to see what more you could find when you are looking at the specimen.
The next slide is from urine. You can see the epithelial cells in the background and probably some casts in there. And if you look in there you will notice there are some cells that are budding and have a bud on the end of them. There are some hyphae in there and those are two features of Candida. And looking to see, I don’t really see what looks like pseudohyphae in there so it is a combination of hyphae and some budding cells. This would be enough to make it an identification of Candida. And you can’t tell which species of Candida that you are looking at as they pretty much all look alike in a clinical specimen. But it would certainly be a member of the genus Candida.
Next slide came from a patient with thrush and basically what you are looking at are septate hyphae in there. And so, you see there is a large filament in there in the center and it is going up and down. There are two cells about maybe 3:30 o’clock within the middle of the field. It looked like they have elongated and they kind of taper down. Those might be the beginning of two pieces of pseudohyphae but for the most part, all of those things are hyphae in there even in the background amongst the squamous cells.
The next slide shows you virtually the same thing. Those are hyphae in there and on the left side at maybe 9:00 o’clock and going towards the center of the field, you see two cells that are constricted down and kind of elongated and those are probably the beginning of pseudohyphae. This is just one thing that you might see with Candida. I would look at several fields before I made an identification.
This next slide shows you nothing more than just a bunch of budding yeast cells sitting in there. And then, it is a little difficult to tell; I think those are probably pseudophypae in there. You have to look very closely. Notice that the cells taper down by the point where they are connected to each other. They look almost like hyphae. So any combination of those is the trick.
The next slide is a calcofluor white stain of Candida and what you see in there is some buds coming off the sides of what look like hyphae. And you probably see some septations in those hyphae and once you see the buds in the septate hyphae, that is simply enough to call it a Candida.
This is just a slide, the next slide here shows you a budding yeast cells and a couple of them are elongated and the one in the middle towards the top a little bit is elongated and has a bud on it. This might be all that you see. In this case it would be difficult to say it belongs to the genus Candida because there is nothing there that tells you that it is a Candida species. There are lots of other yeasts that have budding cells and so this one I would have to look around a lot more before I would be able to call it a Candida.
The next one shows you what you have seen before and there are budding yeast cells in there and there are septate hyphae in there. You can see what looks like the beginning of pseudohyphae almost in the middle of the field you see one of the pieces of pseudohyphae that have kind of constricted down and maybe almost in the center of the slide. Mostly you are looking at budding yeast cells in hyphae.
This is a Gram stain of urine and you are seeing budding yeast cells and pseudohyphae. So that is good enough to call it Candida.
The name for the budding cells is Blastoconidia, the plural term. This is a slide that you saw earlier and it simply shows you budding yeast cells and there are pseudohyphae in there as well.
This is a PAS stained slide. The next one is the very same, showing you there what looks like hyphae. You can see the long filaments in there that don’t have any narrowing between the cells there. It is hard to see if they are septate, they should be but it is hard to see on this particular slide. But the bottom line is you have two different forms in here and this would be a member of genus Candida.
This next slide just shows you a Gram stain showing you some random budding yeast cells in there and in the center is what looks like pseudohyphae. And if you look at between the segments of this thing, you will notice that they are narrowed down, I wouldn’t say pointed, but it looks like there is a V in between the two cells top and bottom and more towards the center of the cell.
These are just yeast cells of Candida but looking at that it is all you have to see. You might be concerned that it is some other organism besides a Candida.
This next one is a blood film. This is from a patient who has fungemia. In this particular slide all you see are budding yeast cells. For the most part in patients who have Candidemia or fungemia, and let’s just say fungemia, most of the time it is caused by Candida. And so based on the odds, even though you don’t see any other forms in there, it is going to be a Candida. It is not very often that you see some of the other things but it does happen. But the most common thing would be Candida. So it would be Candida fungemia.
Same thing in this next slide, it just shows you budding yeast cells and that is about all you can say.
The next slide is from a kidney. This is a silver stain and you can see in there, that there is some budding, it is a little hard to see, but there are some what looked like a lot of pseudohyphae in there and in looking to see, everything is pretty small. There are some budding yeast cells in there. So this would be a Candida and if you had it on a higher power you would be able to tell a little bit more about the morphology but you have more than one form in there so it belongs to Candida.
Same thing in the next slide, it is a bit more difficult to see on this one. This one is a silver stain and it is pretty over stained and you can see it is hard to tell what the features of the organism really are.
The next slide shows you a silver stain and the counter stain in the background is a dye called light green. And you can see single cells in there with buds on. Those are budding yeast cells and then there are some pseudohyphae in there at about maybe 2:30 o’clock. You can see that there are some that look like sausages and pieces like that all around and in the field.
The next slide is a silver stain; again, I think it is a silver stain. I am not quite convinced that is what it is. But if you look and see the darkly stained slide areas in there, those would be some pieces of hyphae and if you look in the background, notice there are a number of cells that didn’t take up the dark stain. And this actually could be a Gram stain and I think maybe this is more what it is because you see some filaments that happen to be sort of a blue and some of them that happened to be stained in red in the background. So, this is probably a Gram stain of Candida from a piece of tissue. So, it is not easy sometimes to make an identification of Candida when you don’t have everything that is typical.
So that is how you would recognize at least some of the species of Candida and most of the time Candida albicans is the one you are going to be seeing in a clinical specimen. The next yeast that we are going to talk about is one called Malassezia furfur and the hallmark of this organism is that it is the smallest yeast that one would see in a clinical specimen. In the skin it would have yeast and pseudohyphae or hyphae in present in the skin. And, if you have a look at some blood you would see a small yeast in there and that is all. And it may form pseudohyphae sometimes, not all that often. And then when there are a lot of cells of this organism compacted together, it does not form a mosaic. A lot of times with Candida the cells will be in a closed space, pushed together and they take on the shape of whatever the space is. They get pushed together; the sides flatten between the two cells. With Malassezia the cells are rigid and so there is no mosaic there. The cells are round; they stay round even when they are pushed together. And that is something that we use sometimes when we are trying to distinguish Candida from Malassezia in skin.
This happens to be Malassezia in blood. And the cells are very, very, small. They are 1 to 4.5 by 2 to 4.5 microns in size. They bud at one end so they exhibit what is called monopolar budding. The bud is separated by a septum and there is a fission plane in there. They almost look like a small Blastomyces- very tiny. And whenever the bud is produced, it leaves a scar behind. It looks like a small collar that you see with Blastomyces. Malassezia furfur is one of a number of yeasts that belong to that genus. And it happens to be one that is lipophilic. Lipophilic just means that it has to have a lipid source before it can grow. So the hallmark of this organism is that you might recover it from a patient that has fungemia, it might be in the blood, and you recover it from a blood culture and then you try to subculture it to get a pure culture to identify it and it won’t grow. And the reason it won’t grow is because there is no lipid there in the culture medium. The lipid is present in the blood because the patient is getting an infusion of lipid for replacement therapy.
This happens to be from skin. Tinea versicolor is the name of the infection and you see budding cells and you see hyphae. And, that is what you see. This infection, tinea versicolor is a fairly common thing to see and if you just take a piece of skin, this is what you would probably recognize.
With Malassezia, you may just find just hyphal fragments but I think you would have to look around the whole field, the whole slide to find other morphologic forms and there would be yeast cells and again, there is no mosaic pattern with this one.
This happens to be from skin and if you look closely in the background you will see all of the yeast cells that are nice and round and they are rigid in structure. And then you see in the background pieces of hyphae sitting in there. Some people describe a way to remember this is to describe it as spaghetti and meatballs. And that is what it looks like.
Another one where you move it over a field and all you see are hyphae. And with that you couldn’t tell what it is. You would have to look around more to be able to decide if you are going to find yeast cells in there.
The next slide is a PAS stain slide and there you are seeing just hyphae and again you cannot say, these are septate-hyphae, you cannot say this is Malassezia. You would have to look further and see if you see budding cells in there as well.
And on this one, this is a PAS stained slide. You can see the hyphae in there and on the right hand side at about 4:00 o’clock. You see all of the budding yeast cells sitting in there; the round cells. That is typical Malassezia furfur particularly in skin lesions.
And the next slide shows you the same thing with the budding cells as well as the hyphae just sitting in the background.
This next slide is actually from a lung biopsy. And in tissue that is deep and including blood, you will find that the yeast cells of Malassezia will be found and not the hyphae or the pseudohyphae. You will find just yeast cells that are small, the smallest yeast that you probably have ever seen. And they look like small Blastomyces yeast cells. So this is a silver stain from a lung biopsy.
And this is a skin electron micrograph just showing you what the hyphae look like in there and you would find yeast cells sitting around amongst some of those as well.
So this is how you would recognize Malassezia. You could tell it pretty much from the clinical condition that it produces and if it is a skin lesion. Tinea versicolor you could expect to find the hyphae and pseudohyphae. With other things that are deeper, you would expect to find the yeast cells and probably not the hyphae and pseudohyphae. So this is a discussion of Candida and Malassezia furfur.