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Dimorphic Fungi



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Published: December 2012

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Dimorphic fungi cause several common diseases including histoplasmosis, blastomycosis, and coccidioidomycosis. Dr. Roberts discusses the distribution and ecology of these fungi, as well as their pathogenesis and cultural characteristics, in this 5-part presentation.

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 disease-causing dimorphic fungi and how to identify them in culture. This presentation examines Blastomycosis. Thank you, Dr. Roberts.

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

The diseases caused by the dimorphic fungi are Histoplasmosis, Blastomycosis, Coccidioidomycosis, Paracoccidioidomycosis, Sporotrichosis, and Penicilliosis.

The dimorphic fungi are different from most of the other fungi that we have talked about and will talk about in that the growth rates are relative.  It depends upon the organism.  In textbooks, you notice that dimorphic fungi require any where from a few weeks to a month or so for growth to appear.  And the growth rate is relative.  It depends upon how much organism is present in the clinical specimen, and also the culture medium in which it’s grown and probably the metabolism of each individual organism.  Blood enrichment actually enhances the recovery of these fungi in certain instances but not always, however, it does decrease sporulation so it becomes necessary to subculture an organism that is recovered on blood containing medium to allow it to sporulate.  The microscopic morphology shows very small septate hyphae which is in contrast to most of the fungi that we deal with.  And we do have some tools to allow us to identify these organisms besides just the microscopic morphologic features and that would be nucleic acid probes, nucleic acid sequencing, and MALDI-TOF, a relatively new tool for identification. 

This image that you see here shows the small hyphae I was talking about that is seen with the dimorphic fungi.  It’s also seen with some of the dermatophytes like Trichophyton rubrum as well but more commonly to the dimorphic fungi.

The colonial morphologic features of the dimorphic fungi vary depending upon the individual organism and also on the medium that’s used for recovery.  You can see that several cultures of an organism may appear different on a different media and so what is necessary is that you become familiar with the morphology of these organisms on different culture media so that you can ensure that you are not going to throw one of these organisms away.  The colonies of Histoplasma capsulatum and Blastomyces are virtually indistinguishable.  So, the colonial morphology there doesn’t really play a role in making an identification. 

This presentation will focus on Blastomyces dermatitidis and we will talk about

The geographic distribution of where it’s found and some of the morphologic features and how we identify the organism.  The geographic distribution includes North America, primarily the states that include Ohio, Missouri, and the Mississippi river valleys, and the primary states that are where you see it most often are Kentucky, Missouri, Wisconsin, Iowa, Arkansas, Louisiana, Tennessee, Illinois, and Minnesota.  It’s also found in Canada in the provinces of Manitoba, Ontario, Alberta, and Quebec.  So, it’s seen primarily in North America, however, it is seen in other parts of the world. 

It’s been reported in Africa, in South America, India, Eastern Europe, Israel, Saudi Arabia, and Mexico and there are some questions about whether or not those have been actual recoveries or whether they’ve actually been exposed to the organism within those countries.  Sometimes these organisms are transmitted by fomites that have been packed and mailed someplace.  So, this may be an instance where some of these have occurred like that.  There have been a number of outbreaks of Blastomycosis in North America, primarily in the US, and some of them are very interesting.  They’ve occurred in Minnesota, and Wisconsin and Illinois, primarily. 

This next image shows you a map that is a rough illustration of where this organism is found in the US and you can see the states that are included within this circle of the diagram. 

In terms of where we find this organism in the environment, actually, is a pretty important thing.  When you are taking a history, we talk about where patients have been and what they have been exposed to, but with Blastomycosis, it’s a little bit different.  The ecology of where this organism is in the environment has not been well defined.  It has been recovered from several soil samples but not repeatedly. And we know that there is some association with soil, wood, and areas near water, like in the north woods of Minnesota and Wisconsin, but it’s never been proven that there is one source that is linked to the presence of this organism.

One of the questions is to ask during the history and physical examination is, what kind of activities have you been doing and any activity associated with aerosolization of this organism from soil or wood is probably where it’s acquired.  But again, it’s unknown at this point. 

This diagram shows you what we think happens in the environment at the start of the left hand side, where you see on the bottom right above the word Blastomycosis, you see the hyphae that have kind of broken down into arthroconidia, and you see a few small spores or conidia.  Those are found, again, some where in the area where there’s and where there’s water and whether or not it’s an animal or a human this organism is probably aerosolized from the soil and it’s inhaled and taken down into the lungs, down into the alveoli of the lungs, the conidia are, or even the small portions of the hyphae and what happens within the lung, you see in that little, that square box, there in the lung, in the left lung, is that the organism, whether it’s a hyphal fragment or whether it is a conidium, it begins to convert into a budding yeast cell, a large budding yeast cell, much larger than what entered the lung.  Up to eight to fifteen microns in size and once that happens the organism then begins to replicate within the lung and the cycle starts all over again and what we recover in the laboratory is this yeast form that is recovered from the patient.  And this is where we get the term dimorphic fungi because it has two forms.  It has a form that is found out in the environment at twenty five to thirty degrees centigrade referring to the hyphal form you see on the left hand side of the slide or a yeast form that is found within the host or incubation at thirty five to thirty seven degrees centigrade. 

In terms of the pathogenesis, we’ve kind of touched on that already but it’s acquired by inhalation.  We know that it occurs by that because most of the infections are pulmonary in origin.  However, dissemination does occur as the result of hematogenous spread .  And for years we’ve thought this happened and never been proven and we ended up seeing a case some years ago.  A lady from Iowa who ended up having disseminated disease she had never left the state of Iowa for travel and her only history of exposure was that she was digging in her garden and we figure that she probably ended up getting pulmonary infection because if you had seen the X rays of the lungs, you would see why I say that and within a very short time she disseminated. 

This is an example of the kind of environment that we have seen Blastomyces infect people after been exposed by this environment.  It’s where you see wood and water, whether it is decayed wood, I have no idea what the water has to do with it, I have no idea about that either.  But this happened in Minnesota where two fishermen ended up fishing in the same place and didn’t even know each other and they both came down with Blastomycosis.  They had been at the very same place.  And walked through all of these areas here and so it had to be in that environment. 

In terms of what do we culture and what do we submit for direct microscopic examination.  Well, we know the lower respiratory tract is involved because pneumonia is a feature of Blastomycosis.  This organism causes disease of the long bones, like the femur.  One of the manifestations of disseminated disease is skin lesions, production of skin lesions and lesions in the mucous membranes.  Sometimes, even overlooked by people whenever the patients wear dentures and the dentures are not removed.  Often times you can remove those dentures and you will see all sort of lesions that look like carcinoma and then these turn out to be Blastomycosis.  Prostate in males is also a site where you can find Blastomycosis and also cerebrospinal fluid, meningitis is not necessarily the major clinical presentation in the nervous system but it has more to do with brain lesions, brain abscess and these are substantial infections and the patients don’t do well even on therapy. 

The direct microscopic examination may provide the earliest signs of diagnosis.  And most of the time you can actually make a definitive diagnosis because this organism is large enough to be seen under the microscope, its eight to fifteen microns.  It has broad-based budding yeast cells on it.  And we think about the three B’s, the broad-based buds that we see under the microscope in sputum or sometimes some skin lesions even from brain lesions.  This is an organism that is fairly easy to recognize, there is an exception to this and that is that there are some cells of Blastomyces that are around two microns in size.  It ‘s called the small form Blastomyces dermatitidis and this one is not so easy to recognize and often times it is confused with Cryptococcus neoformans but that’s not the rule that’s the exception. 

This is a potassium hydroxide Calcuflour white preparation showing Blastomyces dermatitidis in a sputum.  You can see the two cells touching each other and that area where they touch is kind of representative of the broad-based bud.  Sometimes you see them a bit more obvious than you see in this one.

The gram stain often times is helpful in making a diagnosis of Blastomycosis because the organism is large enough to be seen easily and you can see in the center here you can see the two cells touching each other and there is a broad-based connection between the two cells. 

In terms of the histopathology.  This disease presents differently in terms of tissue response compared to all of the other dimorphic fungi.  This thing causes a suppurative response and that is, it forms abscesses with a large number of neutrophils present.  And that’s most often seen and sometimes you can see granulomas as well but not, it’s not that common, most of the time it’s an abscess. 

This is an example of microabscess formation in tissue.  And if you look, you can see the walls of those abscesses and they actually are filled with neutrophils.  And if you look closely, in the one about 4:00 o’clock, you will see that there are some round cells in there and those are the cells of Blastomyces.  And the other cells are all neutrophils. 

This is an example of what Blastomyces dermatitidis will look like in lung tissue.  These are cells that have another feature that we did not talk about and that is a double contoured wall.  These cells appear to have a cytoplasm shrink away from the cell wall and when that happens, it gives the appearance of a double contoured wall.  The two cells together at about 9:00 o’clock show you a typical broad-based bud of Blastomyces dermatitidis.

This is a colony of Blastomyces and it is nothing more than a fuzzy white colony.  It could be just about anything.  The growth rate is in text books, it says it takes up to maybe four or five weeks for this to grow.  The lady I mentioned a minute ago that was from Iowa ended up having Blastomyces recovered from blood in two days.  And if you had seen the amount of organisms in those blood cultures you would see why it just took two days.  So, it’s all relative to the things that we talked about earlier.  There is no set time on these things and they don’t all grow slowly. 

This is another colony of Blastomyces.  These actually look like colonies of Histoplasma, too.  So you can’t tell them apart.  This is a colony that is a bit bigger and probably took longer to grow. 

This is one where you see the colony appears to be almost appears to be yeast-like, it’s very flat, it’s not fluffy at all.  It’s more urea into the agar.  It looks almost dried.  Sometimes, some of these colonies look a little bit like dermatophytes. 

This is one where it looks almost like a yeast colony on a blood containing medium but if you look closely, you’ll notice there are little tufts of hyphae sticking up in the center of some of those colonies.  And this is what some of the dimorphic fungi look like on a blood containing medium, not just Blastomyces but it can be Histoplasma as well and maybe even Coccidioides

But this image here shows you three different culture mediums having the same specimens inoculated in the same amount to each plate.  If you look at the one on the left hand side, upper, about 10:00 o’clock, you’ll notice that same type of colony we just talked about that sort of glabrous, almost yeast-like.  The one down at about 6:00 o’clock is white and fluffy and the one that you see at about 2:00 o’clock is another one that looks almost yeast-like with those hyphaes sticking up in tufts.  This is the same amount of specimen, the same culture on three different media and you need to become familiar with what these things look like on different culture media because they do appear to be different as you can see. 

This glabrous colony shows you one that is not fluffy and is varied here into the agar. It appears to have some embedding into the agar surface actually when you look at it.

This is an example of what a dimorphic fungus might look like among a bunch of yeast colonies.  And all of the white colonies that you see that are small and kind of shiny.  Those are all yeast colonies, probably of Candida albicans.  The colony about maybe 7:00 o’clock that is white and fluffy is Blastomyces dermatitidis sitting around those colonies of yeast cells. 

This is an example of what would happen if you took a colony of a mold form of Blastomyces dermatitidis and you put it at thirty five to thirty seven degrees centigrade and incubated it at that temperature for a few days, it would convert from the mold form to the yeast form.  And this is what the yeast form looks like.  It’s waxy.  It’s kind of sometimes heaped up and very wrinkled.  It’s not something that we do these days all that often because we have new tools that we can use to identify the organism but some people still do this because it’s cheaper. 

This slide here shows you an example of what Blastomyces might look like growing on a blood containing medium.  And all of the colonies of Blastomyces, except the one at maybe about 8:00 o’clock towards the center, it has a very smooth circumference, and it’s not kind of a fuzzy around the edge like you see with the rest of them. 

This is an example here of Blastomyces present in the amount that you can hardly can see in the specimen from a patient that has Blastomycosis.  This is confluent growth of Blastomyces and this patient had to have become very ill having to have this amount of organism in the clinical specimen.  The specimen is from the sputum. 

What would we be looking for under the microscope if we looked at a colony of Blastomyces?  What would we be looking for?  It’s not necessarily what we are going to find every time but it’s what we would be looking for.  We would be looking for small delicate septate hyphae that we talked about.  We would be looking for, in this case, spherical to pear-shaped or pyriform-shaped conidia produced at the tips of long or short conidiophores.  And some people refer to these conidias as looking like lollipops.  The conidia can sometimes be confused by some people with Scedosporium apiospermum or Trichophyton rubrum and there has been a name change for Scedosporium apiospermum and we will talk about that in another series but we’re going to continue to use this name because of the familiarity with it.  The colonies again, can look like Histoplasma.

This image shows you the conidia.  Those are the pear-shaped to round cells that you see all in this image and they are attached to a long stalk or a short stalk that is attached then to a hyphal strand within the culture.  It’s these small spores that probably get down into the alveoli of the lung and cause that to disease.  It’s also the break down of these tiny hyphae into smaller pieces that probably are aerosoled as well and inhaled by the person and then acquires Blastomycosis.

If you were to look at this image, do you think that it would be possible to distinguish Blastomyces dermatitidis from Histoplasma capsulatumHistoplasma capsulatum has two types of spores and we will get to that.  They have macroconidia that are rough and wart-like and very large and we have conidia like this that are small and almost look like Blastomyces, in fact they look like Blastomyces.  You could not tell these two apart by looking at this image because you would have to look at the whole slide and make sure that you didn’t happen to find one of the larger spores of Histoplasma capsulatum before you decided to put a name on it and then you still might miss the identification because it might not produce those large spores in the area where you looked.

 Blastomyces dermatitidis in the past has been converted to its yeast form on a medium called cottonseed agar that was incubated at thirty five to thirty seven degrees centigrade.  And what happens is the mold form actually begins to, the hyphae begin to enlarge and in time form these large budding yeast cells that are eight to fifteen microns in size that have those broad bases on them.  And some of the cells we mentioned earlier may be 2 microns in size.  Those yeast colonies we saw a while ago that’s heaped up and wrinkled and waxy in appearance. 

This is another example here of the yeast form of a Blastomyces in a large tube and you can see the colonies are kind of heaped up and appear to be a little waxy. 

And this is the beginning of a conversion of the mold form to the yeast form.  In the background, are the hyphae that this thing started with, very small hyphae.  And if you look at it over a period of days, what you would see would be the production of some round cells that are large and in time you would find some that would begin to bud. 

The next slide shows you the beginning of budding cells.  And you can see they don’t exactly look like round cells of Blastomyces but they do have that double contoured wall and they have a space between them, and a flat base between them.  If you look at the one about maybe 5:30 kind of at an angle you will see that it has a cell and a broad-based, starting at the top and a bigger cell then there is this flat base and there is a smaller bud connected to it.  That one looks pretty good for Blastomyces but it doesn’t have the double contoured wall, so what you do is you look around for everything and try to make sure you find some that are as typical as you can find.  You don’t always find them. 

This is a better example of what happens with time.  These cells begin to convert further and they begin to form cells that are round and have this double-contoured wall.  And here I don’t see any, but broad-based buds that are very good yet but the next one shows you further.

Development of these yeast cells and if you look around in there you can see some that have broad-based buds on them and they have a double contoured wall and this is what Blastomyces dermatitidis looks like.

This next image shows you what ideally they would look like.  This is what is a textbook perfect picture of this organism.  You don’t always find them looking this way.