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Clinical Mycology: Direct Examination Series

Uncommon Organisms

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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 unusual organisms. 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 the direct microscopic examination of clinical specimens. 

And the next slide shows you a number of the stains that can be used in clinical microbiology and pathology for staining organisms not necessarily the fungi.  However, the fungi may be recognized if one is looking for them in those particular stains. 

And the next slide shows you a continuation of different methods that can be used in the same manner.  For example, the Pap smear used in pathology or cytopathology can be very useful for detecting fungi even though it is not designed for that Pap smear.  Respiratory tract specimens will show fungi very easily but it is a matter of just “thinking clinical microbiology” when you are looking at all of these things. 

The next slide shows you something that is not very commonly seen in the United States or North America.  It is an organism that produces what we call dematiaceous sclerotic bodies.  And in some publications you will see they are called muriform bodies.  They are brown, kind of gold in color.  They look like copper pennies.  They reproduce by fission.  You can see the fission planes.  And the organisms that produce these would be Cladosporium, Phialophora, and Fonsecaea.

This next slide shows you an example of these copper pennies or muriform bodies.  They have thick septal walls which you can see in two of them, at least more than two where you can see the cross walls are very thick.  They are darkly pigmented particularly in the center and they are 5-20 microns in size. So you can see them microscopically.  And these are found in cases of chromoblastomycosis.  These occur in patients who have skin lesions that involve the skin and subcutaneous tissues.  Usually, the organism primarily all of the time is introduced by trauma into the subcutaneous tissues where it then begins to replicate.  People come in with these lesions that have been present for years, sometimes months to years.  They look almost cauliflower like in appearance.  The skin is all distorted and they are different colors.  Sometimes purple, some red and some brown and that is where the term chromo comes from and blasto means budding and mycosis stands for fungal infections.  So it appeared they were budding but these are not budding cells.  These are cells that reproduce by fission.  So chromoblastomycosis is commonly seen in parts of the world where the tropical areas exist.  Temperatures are moderate and patients will come in and will have these lesions that have been there for five, ten, or fifteen years because they had no access to medical care.  And they are very difficult to treat. 

Next slide shows you a biopsy and at about 4:00 o’clock or 5:00 o’clock, you can see there is a very large cell that contains these muriform bodies or “copper pennies” inside of that.  They are not as obvious as what you saw in the previous slide but, nevertheless, if you look around you would see probably some that are better than this.  Again, you need to look at the whole slide before you make a decision.

This next slide shows you a biopsy and you can see the “copper pennies” in there very readily.  This is an H &E stained slide.  Some of those cells are larger than others.  And you can see that some of them are round, hence the term “copper pennies”.  That is kind of what they look like.  At the top right hand corner about maybe 1:00 o’clock you can see the fission plane in the middle of those two cells.  So this is the muriform or sclerotic bodies of chromoblastomycosis. 

Now a number of pigmented or dematiaceous fungi cause infection and the name of the infection is called phaeohyphomycosis.  And it is just a general term for infection caused by pigmented fungi.  There are a lot of them and you cannot recognize the specific organism by looking at it in a biopsy or in a direct microscopic examination.  But it does help to know if the organism is dematiaceous or not in terms of directing therapy for treating these infections.  Some of the organisms that we would see that would produce dematiaceous septate hyphae are Alternaria, Bipolaris, Curvularia, Exophiala, Wangiella and it has now been changed to Exophiala so Exophiala would be the major genus for those two groups right there and a number of others, a whole lot of others.  And we have seen such common things as someone coming in with a splinter and if you look at the biopsy, you will see that there is a wooden splinter and around the perimeter of the splinter would be an organism growing there surrounding the whole splinter would be pigmented and that is what would be causing the infection.  Often times we see abscesses in the skin from patients who are transplant patients who are on corticosteroid therapy and they happen to be come in contact with a dematiaceous fungi and many times there is no history of trauma to the area.  One interesting thing that occurred a few years ago in a paper in The New England Journal of Medicine was a number of patients were in a hospital and who had the organism Alternaria causing these skin and subcutaneous abscesses.  And they determined that the gurneys actually were contaminated that they used to take the patients to the operating room.  And the patients that were immunocompromised ended up getting these Phaeohyphomycosis  cysts from being on the gurney. 

The next slide shows you what you would see in one of those.  These are nothing more than hyphae that are septate and they are brown, they have pigment, brown pigment to them and that means they are dematiaceous or melanized fungi.

The next slide shows you an organism that is really is not a fungus.  It is an achlorophillic alga.  But it is seen often times in the fungus area of the laboratory because it looks like a yeast to people who don’t have a lot of experience.  Prototheca does not bud.  There are no budding cells at all.  Even though it might look like a yeast, it does not reproduce by budding.  It has what we call sporangiospores that are produced by round sporangia.

The next slide shows you two species of Prototheca,; Prototheca wicherhamii and Prototheca zopfii

The next slide shows you what it would look like.  This is from a culture.  And basically, you see all of the single cells sitting there in the whole area of the field there.  But then there are some larger cells and you see they are kind of clumped together.  Well, there is a central cell and then there are cells that surround that, and that is a sporangium.  And they look sort of mulberry like.  That is the description the textbook gives them.  And when you see a sporangium like this with those sporangiospores inside of it, this is probably going to be ProtothecaPrototheca, one of the things that it causes is olecranon bursitis.  The elbow gets involved and sometimes there might be a history of trauma and sometimes not.  And you find the bursa is infected and if you drain that area, you culture or you look at it underneath a microscope, you will see these sporangia of Prototheca.

The next slide shows you phase-contrast photomicrograph from the same thing.  Single cells hide in the background but then you can see those sporangia and you can see those sporangiospores all around the perimeter of that cell.  And so this is what Prototheca looks like.  It is something that you don’t see very often but, nevertheless, it requires different therapy than some of the things and it is important you recognize it.