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Dimorphic Fungi

Paracoccidioidomycosis and Penicilliosis

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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 Paracoccidioidomycosis and Penicilliosis. Thank you, Dr. Roberts. Thank you, Sarah, for that introduction.

I have nothing to disclose.

The diseases caused by the dimorphic fungi are as you see here, Histoplasmosis, Blastomycosis, Coccidioidomycosis, Paracoccidioidomycosis, Sporotrichosis, and Penicilliosis.  Paracoccidioidomycosis and Penicilliosis are infections that are generally not found in the United States.  All of the others are found in North America for sure.

In terms of the dimorphic fungi, they all have some features that are similar and they include the growth rate.  And the growth rate is relative.  It depends on the organism, it depends on the culture medium, it depends on how much organism is present in the clinical specimen and textbooks have the tendency to say that there is a fixed amount of time for them to grow, like it takes four to six weeks or something like this.  There are times when you see organisms and belong to the dimorphic fungi that will grow in a period of two days because of the sheer amount of organism present in the clinical specimen.  Another thing that’s in common with all of the dimorphic fungi is that some of them require some blood enrichment for recovery.  And so, we always include a medium containing blood so that we pick up that occasional fastidious organism.  In terms of the microscopic morphology, these dimorphic fungi are made up of small septate hyphae.  And so, when you see that, it is a pretty good indication that’s what you are dealing with, although, it’s not an absolute.  And we have nucleic acid probes and sequencing, nucleic acid sequencing and now MALDI-TOF that will help us to identify these organisms and so that we don’t have to use traditional microscopic morphology features for identification.  But for many of us that’s still the way to identify them and it probably will be the way they will be identified for many years.

This image shows you the small hyphae that is seen in the dimorphic fungi.  It’s difficult to see the septations in here but they are septate. 

The dimorphic fungi have colonial morphologic features that vary depending upon the isolate that you recover and also the medium on which they are recovered.  So, what you need to become familiar with what an organism looks like on a certain medium.  Colonies of Histoplasma and Blastomyces are pretty much indistinguishable from each other and so there is a problem there but for some of the others, it’s not so much of a problem.  You can kind of get an idea of what the organism is by looking at the colonial morphologic features but it’s not absolute either. 

This discussion will center on Paracoccidioidomycosis and also Penicilliosis.  They are found in two different parts of the world.  Dimorphic fungi usually has specific geographic niches in the world and these are no exceptions.  Paracoccidioidomycosis is found in Central and South America and Penicilliosis is found in places like Vietnam and Thailand.  We will discuss these two organisms and the infection that they cause.

And this is an example of a colony of Paracoccidioides brasiliensis.  Not a very good example but it’s a colony of white organism that turns a little bit tan with age and it may look like Blastomyces.  In fact, it used to be called South American Blastomycosis. 

This is a subculture.  You can see how slow growing it is and the colonies here are kind of white, or off-white.

This is a culture that we had here at Mayo, the only positive culture that we’ve ever had from a patient with Paracoccidioides infection.  The colonies appear to be yeast-like and they are kind of donut-shaped like one of the slides we showed with Blastomyces yeast form.  But these are seen with a primary culture of Paracoccidioides.

This is another plate of the same culture.  This plate contains a blood enrichment and you can see these colonies are kind of wrinkled up and almost donut-shaped.  

Here you see the same culture on another medium and there the colonies are bacteria in the background that are shiny, but all of the other colonies that are kind of brownish colored that are heaped up and wrinkled are all colonies of the yeast form of Paracoccidioides, not things you see discussed in the literature. 

You can see another one here where you see the primary recovery and notice the difference in morphologic features of this one.  This is all Paracoccidioides.

And these are four plates showing you the difference in the morphologic features of Paracoccidioides on these different media.  And it’s hard to see the one with blood containing medium because they are so dark. 

With Paracoccidioides, what are you going to look for to try and make an identification?  Well, it’s not that easy for us in the United States to make an identification of Paracoccidioides because we very rarely ever see it, although, patients come here with that infection and so you may see it.  The hyphae are generally small just like we talked about and may contain numerous chlamydoconidia.  It’s often sterile.  Sporulation doesn’t happen very often.  The conidia whenever they are produced are borne on the sides of hyphae or in short conidiophores.  They are kind of oval to pear-shaped.  And you may find arthroconidia.  It looks like just a mishmash of all sorts of things.

If you suspicion that this is what this is based on the history of travel of the patient, you will look at it and you’ll probably try and put it at thirty five to thirty seven degrees Centigrade as well to see if it will convert to the yeast form.  Right here you see an image with a massive hyphae and partially converted yeast cells. There are some round cells in there and there are pieces of hyphae sitting around in there.  It is kind of nondescript.  Everything’s nondescript in there. 

What happens is that once you convert to the yeast form, you see yeast cells that are two to thirty microns in size.  That’s a pretty broad range.  And they produce narrow-necked buds all the way around the perimeter of a yeast cell.  It looks like what we call a mariner’s wheel, like on a boat.  The colonies are off-white.  They are wrinkled and folded and they can look like those of Blastomyces and the mold to yeast conversion may be accomplished but there is something else that we have found out over the years that we can use, and that’s a Blastomyces probe that’s available commercially that can be used to identify Paracoccidioides if you have other features to go along with it because the Blastomyces probe cross reacts with Paracoccidioides.  Some people see this as a detriment of this particular probe.  We see it as an advantage in being able to help identify that organism even though it’s uncommonly seen.  Now, the Blastomyces probe cross reacts with a couple of other things as well and so the people feel that the probe is not specific enough, but in our experience it has been. 

This is an incomplete conversion of Paracoccidioides brasiliensis.  And if you look carefully in there, what you are seeing are yeast cells.  That one in the center is kind of cup-shaped, it’s collapsed and they really are kind of nondescript.  A couple of them, there is on about 10:00 o’clock down towards the center is in chains, so really not a lot there to tell you that it is anything in particular. 

This next image, it looks like a moth eaten image but basically what’s in there in the center, a little bit right to the center is a big round blue cell that has these tiny buds coming off all of the way around the outside.  And basically, in three dimension those little tiny buds come off all the way around the whole cell.  And that’s what Paracoccidioides looks like.  It has these multiple buds all the way around the perimeter of this central yeast cell.

And sometimes they are not as pretty as textbooks show them.  The real world is not the textbook world. 

Here you can see a couple of cells in there and the one in the center that’s large has little tiny buds coming out from about four places.  Not the best one but when you start looking through the thing, this is just to show you, just browsing through all the fields, this is what we ran across.

And then you look around in there and you say, “Well, I see some buds coming off the one at about 4:00 o’clock down there about three or four buds coming off there, but it sure doesn’t look all that good.” 

And then you look up here and you see a big cell with some other cells coming off and that still doesn’t look all that good. 

And then you get down to this one and you start to see multiple budding cells on some of those in there. 

You get a larger view.  There is what it looks like.  The buds are coming off all the way around the perimeter of the central yeast cell that’s large.  And that’s Paracoccidioides brasiliensis

And there is probably the world’s largest one. 

And this is another example here.  Look at all of the yeast cells that are coming off all the way around that central yeast cell itself. 

This is a scanning electron micrograph to show you what it really does look like in a high power view.  The buds do come off all the way around in three dimension. 

The next image that you see here is Penicillium marneffeiPenicillium marneffei is a dimorphic fungus but it is different from all the rest of them.  The others are called what we refer to as thermally dimorphic.  Their conversion from one form to another is dependent upon the temperature.  This one doesn’t necessarily depend upon that.  This is a colony of Penicillium marneffei.  It looks like a nondescript colony.  It is a colony of Penicillium, it can be green,

and it will have a red reverse, a red back on it.  And if you look at it underneath a microscope, you see there is a head or Penicillus, has divergent phialides on there and they give rise to chains of Penicillium-like structure just like another Penicillium.  And not all Penicillium cultures that are red are Penicillium marneffei. Most of the time you’re going to be able to make the identification

by looking at a clinical specimen that has the organism in it.  But in terms of looking at the morphology, what you are going to see are small conidial heads on short stalks.  You can have three to five divergent metulae, these are the branches that are produced before the phialides are produced that give rise to the chains of conidia.  The conidia are produced in long divergent chains with prominent disjunctors between the conidia.  You’ll see a space between them.  And a conversion shows irregular hyphal fragments and cylindrical arthroconidia and once you do it, it converges. You can take brain heart infusion broth and try to convert this organism from its mold form to its yeast form

and when it’s converted, you will see that the hyphal cells will become shorter and septated and branched and form these rectangular kind of arthroconidia looking things that have a cross septation in the middle.  Some of them look a little bit sausage-shaped and reproduction is by fission.  And it’s kind of prominent.  You will see what it looks like.

But it’s kind of hard to find.  This is a yeast cell of Penicillium marneffei in a patient who had infection.  This is blood and hard to see and in the very center is a rectangular cell that has a septum in the center.  There’s one that looks a little bit sausage-shaped.  You’d have to enlarge these tremendously to be able to see them because they are about the same size as Histoplasma, two to five microns.  In fact, some people say that they are easily confused.  I don’t think they are.

This next image shows you a cluster of Penicillium marneffei yeast cells.  If they are yeast, or they are yeast-like cells, notice in there they are elongated and they have a central septum in them.  And they are divided up in half by this septum.  You can see the one on the right hand side about 2:00 o’clock on that one, that one cluster.  And that’s what you see in clinical specimen with Penicillium marneffei

and they are awfully small.  You can see on this one the same thing.  There is a central cell in that one that is about 2:00 o’clock in the center.

And you can see on this one the top cell in the center has that distinct septum.  This is what Penicillium marneffei looks like. 

This is a biopsy and you can see in here it’s a little bit out of focus but there are cells like about at 2:00 o’clock that is the parallel to the longitudinal access to this thing.  You see one with a septum in it.  So this is what Penicillium marneffei looks like.