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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 Histoplasma. Thank you, Dr. Roberts.

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

This is another in a series of ongoing presentations that focus on individual or groups of organisms that one might see when looking directly at a clinical specimen, whether it’s a biopsy or another type of clinical specimen.

The next slide, the next two slides actually show you the different stains that we have mentioned in the past that are useful for detecting fungi, however, they may not be designed specifically for that purpose.

The next slide is just a continuation of that list.

The next slide shows you an example of Histoplasma capsulatum within a mononuclear cell. If you look on the right-hand side of the photograph, you will see that those are very small budding yeast cells sitting inside of a mononuclear cell. They are about 2-5 microns in size, they are usually spherical to oval, and a lot of times they are intracellular within those mononuclear cells or they may not be. They may just simply be out away from those cells and so they’re described as being intracellular parasites but they may not be present like that.

This next slide shows you a phase contrast microscopy photograph of a small budding yeast cell to give you an idea that it would be difficult to try to recognize Histoplasma in a direct examination in something from a lung or from a respiratory tract. They are so small, hard to find, and even if you saw them, they could be confused with other yeasts. So it’s difficult to recognize these things in a respiratory tract specimen.

In the next slide is one that I photographed from a respiratory tract specimen under oil immersion showing you the different morphologic forms that you might see of Histoplasma in one of those samples, and it’s amazing that there are more features there than I ever knew about. What you are basically supposed to see are budding yeast cells, 2-5 microns in size. If you notice in here you see some budding yeast cells that are oval, you see some that have more, cells that have more than one bud on them, you see some cells that are in chains, you see some cells that appear to have a capsule around them, and Histoplasma capsulatum does not have a capsule. It has a pseudo-capsule, a sustaining artifact, but if you look in the background, some of them aren’t even all that apparent when you look at them but they’re oval shaped. So there is a lot of variation with the Histoplasma that we never really thought about.

The next slide shows you a calcofluor white photograph of just one single yeast cell that is not what you usually see, but it’s an oval cell with a tiny, pinched-off bud on it.

The next slide shows you the same thing, calcofluor white.

The next slide shows you a larger view of the slide you saw at first with the mononuclear cell containing all those intracellular cells of Histoplasma capsulatum. And if I saw something like that, I might be inclined to call it Histoplasma, otherwise I probably would not be because they’re so difficult to distinguish from other things, particularly Candidia glabrata or the other yeasts that are small.

Now, in this situation in this next slide, this came from an elbow. It came from a patient who was on Itraconazole therapy, and with Itraconazole therapy, you’re not supposed to take antacids because it activates the antibiotic. This person continued taking an antacid so the therapy was not effective and they removed some fluid from the elbow and this is what it was filled with. This is all Histoplasma capsulatum. All those cells in there, most of them are 2-5 microns, they have single little buds on them, and on calcofluor white, you can’t tell if they have a standing artifact or a shrinkage artifact around the outside or not because you don’t see the evidence of what looks like a capsule. But this is Histoplasma capsulatum and you can see from sheer number there are lots of cells in that exudate.

This next slide is just from this same patient. In a case like that, you see lots and lots of cells. And it’s from a sterile site so it means that it more than likely is going to be one of these pathogenic organisms because it’s in a sterile source.

The next slide shows you the intercellular nature of some of these cells of Histoplasma and you can notice it appears that there is a capsule around the outside. This is a false halo around the outside and they are very difficult to see if they are in small numbers like this, but in large numbers like you see here, you can recognize them. This is an H&E stained slide, so H&E or Wright stain. I think it’s a Wright stained slide of a bone marrow, actually it is, so that you can see those cells in the background that are filling up with mononuclear cells. But if they were there in small numbers, it would be very difficult to find them.
Now this is the example here of another slide, this next slide where you see these cells in the center. They have what look like a space around the outside; it’s a shrinkage artifact and those are small cells of Histoplasma capsulatum.

This next slide shows you a lung where you can see the stained cells of Histoplasma and they appear as small dots in the background in the cytoplasm of these cells, and when they are present in large numbers like this, you don’t have difficulty recognizing that they are Histoplasma but if they are there in very small numbers on a stain like this, you would probably never see them. So these are present, they look like tiny dots all in the background in the cytoplasm of all these cells; these are all cells of Histoplasma.

And the next slide shows you what they look like at a higher power view in just one portion of that slide. You can see all the cells in there with the space around the outside, and all of those are about 2-5 microns in size and they are all Histoplasma.

And the next slide is the PAS stained slide, and you can see the very same thing. You can see the red dots in the background. All those are cells of Histoplasma and you notice around the outside the perimeter of a lot of those, there’s a space that’s staining artifact from Histoplasma.

The next slide is a silver stained slide showing you the cells that appear to be round to oval shaped, and that’s how they look. Many of them are just oval shaped, but some appear to be spherical, and they may or may not be inside of other cells.

The next slide is from a lung, this is a silver stain showing you essentially the same thing but the cells there are present there in greater numbers. They are all 2-5 microns in size, and if you look around, you can find a bud on one of them here and there, but for the most part they are oval shaped cells and they are characteristic of Histoplasma.

So this is the description of Histoplasma that you would find in usually sterile sites from patients who have Histoplasmosis, disseminated Histoplasmosis.