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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 Coccidioides, Geotrichum, and Trichosporon. 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 and used in the direct microscopic examination of clinical specimens.
The next two slides show you the different methods that can be used for detecting fungi in those clinical specimens as well as in biopsies. It’s important to know that not all of these are specific fungal stains so that when you are looking at these stains, you have to be thinking about looking for fungi as well.
The next slide is just a continuation of those stains.
The next slide shows you the direct microscopic examination for the detection of Coccidioides immitis or Coccidioides posadasii. They cannot be distinguished from each other microscopically. In this case you would be looking for something that would be different from all of the other things you would be looking for. With Coccidioides immitis and Coccidioides posadasii, you’re looking for spherules that may or may not contain endospores. If they contain endospores, these cells do not produce any buds. They might be mistaken for budding yeast cells but the bud would be absent. Coccidioides is one of those ones that is different from all of the others morphologically.
The next slide shows you a couple of spherules. These are large structures that reproduce by progressive cleavage and if you look at the cell on the left hand side you could notice there are lines that surround the periphery of that whole cell. Those are called cleavage furrows. This whole thing reproduces into endospores as a result of progressive cleavage. The cell at the bottom on the right hand side is a bit different, hard to tell what stage of development it really is in. You can just see the spherule wall there and it may not be at a point where it’s getting ready to divide yet.
The next slide shows you two very large spherules you’ve seen in a sample material used in a potassium hydroxide prep. The larger one, actually, there are three cells in there, two large ones and a smaller one about maybe 1:30, 2:00, the smaller cell is. You don’t see anything internally in those cells. You would look around to try to find if there are cells that contain endospores within them. But generally when you find these cells they are not containing endospores. It’s just kind of a given, that it’s going to be difficult to find the typical cell full of endospores.
This next slide shows you different stages of development of these spherules and some of those you can see actually contain endospores inside of them. There’s a cell at about maybe 11:00 o’clock up there that looks like two cells sitting adjacent to each other. It looks like for all the world it is a Blastomyces dermatitidis cell. This happens with Coccidioides, where two adjacent spherules stick together and that means you have to look around at all the fields to get a consensus of what’s there and you would find that most of the cells do not look like those two that are adjacent to each other. They look like the cells you see elsewhere on this slide, round cells that contain or may not contain the endospores that exhibit no budding.
The next slide shows you a gigantic spherule containing endospores. Inside there are the endospores that have no buds on them at all and they are produced inside this cell by progressive cleavage. This spherule ruptures, endospores pour out in the tissue, they in turn enlarge and form spherules again and they after that produce endospores and the cycle just keeps on going like that until it’s broken.
The next slide shows you two cells, two spherules that are sitting adjacent to each other that look just like slides that you see of Blastomycetes. You can almost see a broad based bud between them. In that case, you would have to continue looking around to see if you could see something that is either better or it tells you that it is not Blastomycetes.
The next slide shows you what you might want to see. The top cell has some endospores in it. The bottom one doesn’t really show much but the wall looks almost like it is double contoured. Not quite as much as Blastomyces, though. But you have difficulty sometimes in distinguishing two adjacent spherules from Blastomyces.
This is another one showing you the very same thing. The top cell, actually, shows you what a cell that is ruptured and the endospores are pouring out. The bottom cell is also ruptured and you don’t see much coming out of it but you see endospores in the top one and that tells you that it is not Blastomyces.
This is a very nice spherule that is ruptured and the endospores are pouring out. This is calcofluor white, if you saw that, you would know that you are dealing with Coccidioides.
In a clinical specimen that’s been submitted for culture, often times if you look at it microscopically, and it’s been sitting out at room temperature, you might find that the spherule in there has germinated. When it does germinate, it will form these germ tubes coming off at random places throughout this spherule. You can do this even if you have a, let’s say you take a clinical specimen and you think you see these spherules in there, you can take a drop of that, and put it with a drop of water and put it on a slide with a cover slip, put it out at room temperature and let it incubate for a day or two and look at it and you will see that those germ tubes will form there. When you see that, there is nothing else that really does that besides Coccidioides.
The next one shows you what happens as well if it sits for a little longer, the hyphae begin to form where those germ tubes are and they branch dichotomously at 45 degree angle and that’s what you see sometimes in a cavitary lesion with Coccidioides. So, these things have the capability of producing septate branching hyphae. And this is what it would look like in a clinical specimen that had, were it germinated. So this would be helpful to make a diagnosis particularly in a sample that has been submitted that has been in the mail for a few days.
This slide shows you spherules and endospores in all different stages of development. On the right hand side you see the spherules that are just very large and contain numerous endospores. Towards the left side you see some of them that are hollow. You see some that are very small. Those are the spherules that may contain no endospores or just a few or they may just be starting to develop them. So, you see all sorts of stages of development in there. And actually, if you look closely you can see with some of those ones on the left hand side, you can see basically what look like cleavage furrows in there and so it’s dividing up the spherules into endospores.
Same thing on the next slide showing you different stages of development of Coccidioides. Some of those are very tiny. All the way to the ones that are up to as large as maybe 60 microns.
The next one shows you a larger view of some of the spherules and you can notice that on the one about 8:00, the periphery of that cell shows you what looks like dividing endospores that are being produced by progressive cleavage.
The next one shows you one field over from the last slide that you saw. This happens to be the edge of a cavitary lesion that has communication with an airway and you saw a minute ago that slide that showed dichotomously branching hyphae out of the side of an endospore, or a spherule, this is showing you exactly what happens within the cavitary lesion where there is a connection with an airway. The organism can produce the spherules and endospores but it also can produce acute angle branching just like Aspergillus, just like any of the other molds. And if you didn’t look around and you weren’t able to find anything else, you would not know what mold it would be, but in this case if you looked around, you would find those spherules and endospores sitting adjacent to this area and you would know you would be dealing with Coccidioides. This is an unusual finding but it’s not uncommon.
This slide is an over stained silver slide. The left hand upper part of this slide shows you at maybe about 11:00 shows you a big spherule with endospores on the inside. The others appear to be hollow but they are so darkly stained, it is hard to tell.
The next slide is a silver stained slide showing you a better representation of what the different stages of spherule development and endospore production would look like. Some of those on the right hand side are big spherules that have ruptured and released endospores you see coming out in the tissue. And on the left hand side there are some of the spherules that are hollow and then there are some that actually contain endospores. So this is what Coccidioides looks like in different stages of development. If you saw that you would have no doubt that’s what it is.
The next slide shows you an H&E stained slide with a big spherule in the center. And it’s ruptured and the endospores would be coming out. But notice it’s surrounded by neutrophils. And a lot of times when the spherules ruptures, the endospores start to come out, you will see an influx of neutrophils around the perimeter of these spherules.
This is a silver stain slide showing you on the left hand side about maybe 8:00 o’clock, a big spherule. That’s hard to tell what’s in the inside. If you look over on the other side of the slide you will see there is a smaller one in the background. They are not always textbook perfect and it does keep re-emphasizing that you need to continually look at everything you can find to make a diagnosis because a lot of times one field is just simply not going to tell you enough.
The next part of this presentation would be the discussion of hyaline septate hyphae that produce arthroconidia. And when you see a slide, let’s say you happen to have a clinical specimen and basically all you see are septate hyphae and you see arthroconidia. Then, what do you think about? Well, you think about at least two organisms and two in particular Geotrichum and Trichosporon. With Geotrichum basically all you are going to see are arthroconidia. These are the rectangular cells that hyphae produce that break down of hyphae into rectangular fragments and Trichosporon shows you the same thing except in addition to the arthroconidia you may find budding cells present. And they are not going to be there in great numbers and they are going to be hard to find.
This next slide came from a biopsy from a patient that we saw with leukemia and we found in this biopsy these rectangular arthroconidia. The ends of some of them might have been a little bit rounded but we saw a lot of septate hyphae and that’s all that we saw. And basically, all you can say is that it’s going to be Trichosporon or Geotrichum because you may or may not be able to detect the budding cells in there. And Geotrichum is the one that has no buds and Trichosporon has some budding cells. The Trichosporon is the one that causes disease the most often and particularly I leukemia patients.
This is a phase contract photomicrograph of hyphae that are broken down to arthroconidia. You can see the rectangular segments sitting around all over everywhere and that’s what Geotrichum would look like. Trichosporon would look the same unless you were to find an area where you found a few budding cells. Then you could call it Trichosporon.
This slide here shows you some rectangular arthroconidia. And if you look around then you could then almost imagine that you see some budding cells here and there. And if you did, that would be Trichosporon and if you didn’t that would be Geotrichum. So you just have to continue to look to see what the consensus is. And that’s what you would have to do with most everything is look to see what’s there and get a consensus of what the morphology is going to be.
This happens to be from the same biopsy, the next slide, the first one I showed you there and in there, there are pieces of hyphae that are broken down into rectangular arthroconidia. And in this case it’s very difficult to tell if there are any budding cells in there or not. And this turned out to be Trichosporon. And so it’s supposed to have a few budding cells but maybe not any or maybe just a very few, and so, you would not be able to primarily detect it from Geotrichum based on the microscopic morphology here.
This is discussion of the cells that you see with Coccidioides and also with Geotrichum and Trichosporon.