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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 Mycetoma. Thank you, Dr. Roberts.

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

This is another in an ongoing presentation and it focuses on individual or groups of organisms that may be seen under direct examination of clinical specimens.

The next two slides show you the different stains that can be used in microbiology and in pathology to recognize organisms in clinical specimens. Some of them have specific uses, but we’re looking for fungi, and actually the use of these stains sometimes is not for that, but nevertheless, you still can detect fungi there if you’re looking for them.

So the next slide just is another slide showing the remainder of the list of stains that can be used.

The next slide shows you the direct microscopic examination of clinical specimens, and as a result, we’re looking for the detection of different structures that we’ve talked about before. These are granules or sulfur grains that have been found and present and found in clinical specimens. It’s a cause of Mycetoma. Mycetoma is an infection that is acquired by traumatic implantation of an organism to the skin and subcutaneous tissues and the organisms that cause Mycetoma may either be bacteria or fungi, in which case you would be looking for bacterial filaments, anaerobic Actinomycetes in the case of bacterial Mycetoma, or septate hyphae in the case of fungal Mycetoma.
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The next slide, this is a cross-section of a grain or sulfur granule of a case of Mycetoma, and the interior of that grain will contain the organism itself and the perimeter of the outside of that grain is in a sea of neutrophils making up an abscess. These sulfur granules are millimeters in size, you can see them with the naked eye, they are various colors depending on if no stain is used you can recognize the color of these grains. Sometimes when you look at the outside edge or the perimeter of these granules, you’ll see there are some clubbing or swollen cells present depending on the organism that causes the Mycetoma.                                                                                         

The next slide shows you a granule that has been released from a draining sinus tract of a case Mycetoma. It is a collection of organism and a bunch of host material that surrounded the organism that will allow you to determine that it’s a granule. Then what you have to do is to look at it using a potassium hydroxide preparation or section it using a microtome like you do in pathology for a tissue and look at the interior to see what it is composed of.

This is a potassium hydroxide preparation and this is a picture of a piece of a sulfur granule that has been placed in the presence of KOH for an extended period of time, and that softens the granule up, allows you to put a cover slip on top of it, then you can spread it out and you can see what it’s made of. In this case, it’s made up of bacteria. And if you look at the perimeter, you will see there are at least one or two cells, more than that actually, that shows swollen areas that are called, it’s called clubbing. This would be a case of bacterial Mycetoma.

This is the example of an H&E stain section, of a cross-section of material taken from a draining sinus tract in a case of Mycetoma, and if you look at around at the whole slide, you will notice it’s a collection of neutrophils so there’s an abscess formation there, and then in the center you see the cross-section of a granule, and you have to look closely to see what that granule is made up of. And in this case you notice that the perimeter of the cells are all swollen, and that would be a case of bacterial Mycetoma.

The next slide shows you one that is a little bit over-exposed, but at the interior, the whole granule is made up of bacterial filaments, and if you look at it around the perimeter of that on the right side you see that they are all neutrophils so again, it’s in an abscess.

And if you look closely at the sulfur granule, you would see that it is indeed made up of bacteria, the filamentous bacteria, most of the anaerobic Actinomycetes like Nocardia and Streptomyces.

The next one shows you a case of fungal Mycetoma, and if you look closely in the background, it’s kind of difficult to see on this one, all that material you see there that is brown is a mass of host material that has surrounded the hyphae of a fungus. And in the center if you look closely, you see that there is a piece of hyphae in the center of this thing and down below the center. This is a case of fungal Mycetoma.

And the grain happens to be black. The grains vary in color depending on the organism that causes the infection. They can be anywhere from white to pink to yellow to gray to many colors. In our part of the world, we don’t see Mycetoma very often, but in other parts of the world where they see Mycetoma often like in Africa, they can tell you what the organism is based on the color of the granule. In this case, this is a black grain Mycetoma. It is caused by an organism that we know produces these kinds of grains and seen usually in Africa. This happens to be one that we actually saw at Mayo Clinic.

The next slide shows you the anterior of that sulfur granule and you see these round swollen cells. That is one of the things you might see along with hyphae.

This is a cross-section of one of those grains and if you look at the whole perimeter, you see it’s just full of neutrophils. So it’s in a sea of neutrophils, it’s an abscess, and the cross-section doesn’t show you very much at all, you have to get into a little higher power to recognize what it is.

And if you looked at it, in this next slide, you see it’s made up of hyphae, and at the perimeter you notice that the cells that are there are swollen. So there are swollen cells at the perimeter, with hyphae in the middle, and this is a fungal Mycetoma.

This is a case, another case of fungal Mycetoma, this one caused by Pseudallescheria boydii, and you notice that there are hyphae in the grain, and in the left outer side, at the perimeter, these cells tend to be swollen. I don’t know what to mechanism is for making these cells swollen, but nevertheless, they’re part of the hyphae that are just enlarged of the organism that causes Mycetoma.

In the next one here, is a case of Mycetoma where you see a grain that is made up of filamentous bacteria and you can see in the background there are bacteria all throughout that whole thing.

So this is something that is uncommonly seen in the United States, but nevertheless, it does, it is acquired usually in the summertime when people have a lot of outdoor exposure and they implant an organism into the skin by a thorn, by a splinter, some other means of traumatic implantation into the tissue, and then this begins to invade the skin and the subcutaneous tissue, and then they form draining sinus tracts that come out to the skin, they may go all the way down to the bone and there are cases of osteomyelitis caused by these organisms that cause Mycetoma because they produce osteolytic enzymes in the patient. So this is discussion of Mycetoma.