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Dimorphic Fungi

Histoplasmosis

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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 module examines Histoplasmosis. Thank you, Dr. Roberts. Thank you, Sarah, for that introduction.

I have nothing to disclose.

The diseases caused by the dimorphic fungi are the ones that you see on this slide and one of the hardest things about the dimorphic fungi is the pronunciation of the names of the infections because they get longer and longer as you look at them.  Histoplasmosis, Blastomycosis, Coccidioidomycosis, Paracoccidioidomycosis, Sporotrichosis, and Penicilliosis.  So, we are going to cover Histoplasmosis in this discussion.

Before we begin, we will talk a little bit about the dimorphic fungi including Histoplasma because there are some features that are in common with all of the infections for the most part.  And that is the growth rate.  The growth rate in textbooks is defined as being most of the time slow, weeks to a month or so or maybe even six weeks for the colonies to appear.  We know that for a fact that the growth rate is relative to a number of things.  It depends on how much organism is present in the clinical specimen, the more that is present, the faster it grows.  Culture medium has a lot to do with the growth rate and also how much organism is in that specimen and a metabolism of that organism.  So, we know that the growth rate varies a lot.  Blood enrichment is something that we use for being thorough to try to recover these dimorphic fungi and that is that we add blood to a certain of the culture media and we know that a few of these organisms and a few isolates will require blood to be able to grow.  And we also know if it does grow under blood containing medium, whichever the organism is, its probably not going to sporulate very well and it will need to be subcultured onto a medium that does not contain blood.  The microscopic morphology shows one thing in common with all of them and that’s a small septate hyphae that are present.  We have some molecular tools that we can use to identify these dimorphic fungi.  We have nucleic acid probes that are commercially available.  We have nucleic acid sequencing, and MALDI-TOF now that is being used for identification of some of these organisms. 

This image shows you the small hyphae that is seen in the dimorphic fungi.  It’s difficult to tell that they are septate here without going to a higher power but these hyphae are really, very small. 

The dimorphic fungi have features that varies.  We have already mentioned depending on the medium and on the isolate itself.  We know that colonies of Histoplasmaand Blastomyces are almost indistinguishable, some of them.  So that we don’t have access to being able to identify these things by looking at a colony and saying this is what it is because they often look alike.

With Histoplasmosis, we know that the etiologic agent.  The primary one that we see in the United States is Histoplasma capsulatum variety capsulatum.  There is an African strain that is called Histoplasma capsulatum variety duboisii and we’re going to focus our attention Histoplasma capsulatum because that’s the one that we see.

Geographic distribution is pretty much the same as seen with Blastomycosis or Blastomyces dermatitidis out in the environment.  It’s in Mississippi, Ohio, and Missouri river valleys.  The states with the highest prevalence include Missouri, Kentucky, Arkansas, Tennessee, Ohio, Illinois and Indiana and there are another of other states that have Histoplasmosis as well but the instances may not be quite as high.  We also know that this organism that causes Histoplasmosis is found in 55 different countries around the world.

This is a diagram showing you where it’s found in the United States.  It goes all of the way from Minnesota down to Louisiana, all the way over to Alabama and to Ohio.  It’s been commonly found in Ohio.  And I suspect it’s been found in a few other places as well.  This map is just a gross schematic of where it’s been found. 

Where is it found?  Well, we do know where it’s found.  This organism in contrast with Blastomyces is found in the soil.  It can be repeatedly recovered from the soil.  We know that the soil is contaminated with avian excrement.  It’s found where birds roost, like starlings and blackbirds.  Those kinds of birds are the ones that produce excrement that is alkaline and it causes the soil to become alkaline where this organism can grow.  We also know that Histoplasma capsulatum is found in caves where bats roost and in old trees where the bats roost as well.  The environment is perfect for it to grow there.

This happens to be a photograph of a tree containing a number of blackbirds.  And this is the kind of environment I am talking about.  The excrement that enriches the soil, I think it more contaminates the soil than enriches it, but anyway, it’s present there.  It causes the organism to be able to grow in the soil and that’s where we find it. 

Where do you get it and how do you get it?  One of the prime places that people used to get it and occasionally, that still happens today is people buy an old wild farm out in the country.  And the first thing they do is begin to try and clean things up and they clean out an old barn or chicken house and the organism is found and the manure environment is found.  Also where chicken coops are, some people use chicken manure for fertilizer.  It’s actually been sold as fertilizer and people will come down with Histoplasma infection just from the aerosolization of the organism in that substance.  Cleaning out an old bird roosting site, earth day, there have been some earth day cases written up where people go out and rake underneath old trees and so on and there is one particular case that is kind of noteworthy where the kids were outside at a grade school cleaning up the school yard on earth day and they raked up underneath the tree and it turned out the air intake to the ventilation system in the school was right near where they were raking.  And the whole school came down with Histoplasmosis.  There have been instances where people have cut down dead trees infested with bats.  Excavation sites where the organism is found in the soil and then people spelunking in caves often times can come down with Histoplasmosis because that’s an environment where you will find it.

This schematic shows you kind of what we have been talking about.  But it goes a little bit further than that.  Let’s say we start on the right hand side where you see a bat cave and you see a tree that’s underneath the tree is the avian excrement, what happens is the organism grows in the soil and they produce two types of spores and maybe three types of spores.  One is the spore you see on the upper left hand side that is rough kind of, these rounded areas around the perimeter of it.  And then there are small spores coming off another place and there are hyphae that break down into arthroconidia.  It’s any of those particles probably that are the infectious ones, well, with one exception.  And that is that the large spore which is called a macroconidium, is called tuberculate because it has these rough, these wart-like projections off all the way around the perimeter of it.  But it’s too large to get down into the alveoli of the lungs so it’s the small spores called microconidia or it’s pieces of hyphae that are broken apart that are aerosolized and once that happens, they are taken into the lungs, they go down into the lung itself and within the lung they begin to transform into yeast cells.  The particles that are inhaled are actually engulfed by macrophages and then within the macrophages they are converted to the yeast form and then they pop out of the macrophages and that’s what we culture in the clinical laboratory are these small yeast cells.

This is repetitious but we know that the microconidia, the small spores are the hyphal fragments probably are the things that cause infection.  They get down into the alveoli of the lungs, are taken in by macrophages and converted to the yeast forms, which we just said and what happens too is the lymphatics spread the organism to the mediastinal lymph nodes in the chest and to the systemic circulation where the mononuclear phagocyte system is infected.  And that means the lungs, liver, spleen.  It’s the old term we used to call the reticuloendothelial system.  And you can recover it from the lungs, from the liver and from the spleen and people have disease whether or not they actually have active infection versus disease because this is, what happens here happens just like with tuberculosis.  If you happen to get exposed to the organism, it always gets into the blood, it gets into the lymphatic system, it gets into the blood and you become infected but you may never develop active disease if you are a normal, healthy host.  If you happen to culture a person for some odd reason, at a time when this organism is being spread throughout the system in circulation, you can recover the organism and the patient may not even be ill and that has happened.  We’ve actually done that here at Mayo Clinic and it just is a circumstance but it is not something that we ordinarily think about.  You can recover the organism from blood, for example, because it does get into the mononuclear phagocyte system and blood cultures are often times positive. 

So what happens is, once the organism gets into the systemic circulation, into the lungs delivered to the spleen, granuloma was formed.  The organism is trying to be walled off by the host.  The host forms granulomas, walls it off, these granulomas calcify and then the organism is there sort of in a latent stage, it just sits there.  It doesn’t ordinarily progress any more if you are a normal healthy host, it never seems to do anything, but it doesn’t die.  So what happens is, if that person who has become infected but doesn’t develop disease becomes elderly and becomes immunocompromised or becomes immunocompromised by some other process like the administration of corticosteroids, then what happens is the person gets what’s called reactivation Histoplasmosis.  Those granulomas break down and then the organism is still alive and a person gets infected all over again and develops disease.  And disease is common in immunocompromised patients.  That is primarily the population that we see it in or in patients who get an overwhelming dose of inoculum from the environment where their immune system is overcome by this organism.

What are we going to use for culturing and direct microscopic examination?  Well, on the other infections, the same is true here.  The lower respiratory tract because most all of them cause pneumonia, you can find it from the sputum, from the bronchoscopy specimens, the respiratory tract specimen.  With Histoplasmosis, it’s common to have it recover from the blood and from the bone marrow.   Bone marrow is a very good source for recovery.  We know that Histoplasma meningitis is a manifestation that is commonly seen and sometimes you actually can recover it not only from the spinal fluid but from the brain.  In certain patients, it’s from the liver and from the spleen.  And you may see it from the liver and spleen biopsy and mucous membranes just like we saw with Blastomycosis and with Coccidioidomycosis.  And if the patients wearing dentures and those happen to be removed, you may find a lesion underneath there that resembles a carcinoma.  And once it’s biopsied they see the organism in there then you know that you are dealing with Histoplasmosis. 

Terms of the direct microscopic examination, one thing we know for sure is it is not a very useful thing for detecting Histoplasma capsulatum.  And the reason for that is there are all sorts of yeasts in clinical specimens and some of them are small.  One of them Candida glabrata is small just like Histoplasma capsulatum and sometimes can be mistaken for this organism.  Not that it happens very often, but I think that we see them in the respiratory tract where we don’t dare say that’s what the organism is because it could be something else.  This organism may be seen in peripheral blood smears or an impression smear of an oropharyngeal lesion or from the bone marrow and we pretty much know that’s what it is.  What we see are yeast cells that are two to five microns in size that have a single bud on them and they may be found inside of mononuclear cells and when you look at them on an H&E stain section, they are very difficult to see unless the numbers are great.  With the methenamine silver stain you can see them well. 

This is an image of a yeast form in a bone marrow.  And what you see here are cells that appear to have a capsule around the perimeter.  This actually is a staining artifact and it is not a true capsule but the name capsulatum is there.  When they first described this organism, they said it was found in cells, histo refers to finding it inside of cells and plasma then refers to a cell that resembles that of the malaria parasite and that’s what they thought back in that time when they identified it for the first time.  It was like a malaria parasite inside of a cell and it had a capsule around it.  What we know now that it does not resemble the plasmodium parasite and we know now that it doesn’t have a capsule.  And we know that you can see it in a bone marrow and it’s a, what looks like a capsulated small yeast cell.

This is tissue response that you would see in a patient with Histoplasmosis, a granuloma.  This is a very large granuloma.  On the right hand side you can see the wall of the granuloma, all of the material on the left hand side all the way towards the upper left hand corner is actually a granuloma.  And the granuloma, this is a necrotizing granuloma and it means that all of the cells within that granuloma are destroyed.  There is no tissue architecture there at all and you would find the organism sitting inside that environment inside of a necrotizing granuloma. 

This is what you would see using a methenamine silver stain.  It’s an over stained silver stain.  It is very dark but you can see individual yeast cells sitting there and at about maybe 8:00 o’clock you can see a big mononuclear cell that is just filled with them.  Those are all cells two to five microns in size, pretty much most of them will have a bud on them, hard to tell on this slide.  But they can have single buds on them.  Every once in a while you will see two or three yeast cells connected together. It’s where they haven’t separated.  And that’s something that is not necessarily seen very often.

In terms of culturing this organism, we know that we can grow it well.  It is easy to grow for the most part.  It produces small delicate septate hyphae as we talked about before.  It produces two types of spores.  It produces rough-walled, tuberculate, we call them, macroconidia, large spores produced on short, lateral hyphae.  And we also know that we find small conidia that are usually round to pear-shaped that may be produced on short, lateral pegs.  The microconidia resemble the conidia of Blastomyces dermatitidis.  And you can’t tell them apart.  In some instances, they will fail to sporulate and this happens with Blastomyces, it also happens with Histoplasma and it makes it very difficult to identify the organism based on traditional microscopic morphologic features.

We, in the past, used to try to convert this organism from the mold form to the yeast form, it’s too difficult to do and now we have molecular probes.  We have other tools that we can use to identify this organism and so there is no need to try to convert it from the mold form to the yeast form.  But if you took a mold and put it in a medium, that has some moisture in it that’s enriched and put it at thirty five to thirty seven degrees Centigrade and you put a little bit of the mold culture in that environment, it would convert to the yeast form.  It’s two to five microns in size.  The colonies become kind of a creamy yeast-like and non pigmented but it’s difficult to do because they don’t all convert well and it may take a bunch of transfers to be able to get that thing to convert and so it’s not worth the trouble to do that. 

This is a colony of Histoplasma.  It could be a colony of Blastomyces.  It starts off sometimes it’s white to tan colony and can be even towards a little bit more brown color.

Here you can see one that has the perimeter is lighter but the center is dark.  And sometimes they are fluffy and sometimes they are kind of powdery.  And the more powdery they are, the more spores they produce. 

Here you see one that’s fluffy and a little bit grey.  This one probably would not sporulate as well. 

Here is another one.  You are going to see that these colonies vary tremendously depending upon the isolate.

This one actually turns out to be Histoplasma.

All of these colonies are Histoplasma.  And in some specimens, once in a while you will see that the Histoplasma produces two types of colonies.  It’ll produce one that is white and it’ll produce one that is brown.  And the one that’s brown will produce conidia spores and the one that’s white doesn’t produce anything except just hyphae. 

This is an example, here of what happens commonly.  The kind of tan colonies are, all yeast cells of colonies of actual Candida albicans and the lighter colored colonies you see sitting on top of and around in there are all Histoplasma capsulatum colonies.  They grow after the yeast grows up.  There is something in the literature sometimes you see that refers to the fact that the acidic conditions around the colonies of Candida albicans lacks inhibit to grow the Histoplasma capsulatum.  I don’t think that’s true, not based on what I see here. 

These colonies you see in here are Histoplasma capsulatum with the exception of some of the flat round ones, lighter colored ones that are probably some sort of yeast. 

This is an atypical colony.  It almost looks like the yeast colony of Blastomyces but it turns out to be Histoplasma. 

This is Histoplasma glabrous colony, doesn’t sporulate much but it forms these kind of wrinkled up colonies.

This is another one.  This one is almost red.

All these are different types of colonies of Histoplasma.  This is a glabrous colony. 

If you look in here you see lots of colonies of yeast but towards the center of the colony there are some kind of opaque-looking colonies.  And there is some towards 2:00 o’clock and 1:00 o’clock in there that we see commonly on a blood containing medium because they don’t often produce the white fluffy colonies on a blood containing medium.  Most of the time they are kind of opaque-looking colonies, almost look like brown glass. 

And on this one right here, this one is a little bit difficult to see.  All of the colonies that you see that are prominent there are yeast colonies.  But if you look in the background the solid over growth in there of a film, and that is all Histoplasma capsulatum.  And if you could imagine what kind of disease that person has, probably severe because this is almost confluent growth of Histoplasma. 

This is a grey colony.  The colonies in the center there is a probably, a bacterium, it’s kind of irregular but the other colonies are fluffy and grey.  And I remember this one because this organism killed this patient.  You don’t take these things lightly. 

This is all Histoplasma capsulatum with the exception of some bacteria.  I think that most of it is all Histoplasma.  It’s mostly the fluffy white in there and there is also some brown in there.  Hard to recognize that is really Histoplasma.

These are four different plates showing you the organism and it’s very difficult to see these but there is a lot of growth on those plates.  And on the ones containing blood and not quite as obvious to see but the ones that don’t contain blood you can see the white colonies that are beginning to appear, those are young colonies. 

And the same here, these are all colonies of Histoplasma.  This came from bone marrow.

This is culture from sputum that’s confluent growth, Histoplasma capsulatum with about three places where you see bacteria.

This is confluent growth again on a blood containing medium.

All this is Histoplasma except for those colonies of yeast.  This gives you an idea that of these patients inhaled a tremendous quantity of this organism.

And here you can see in the background these brown looking colonies, some which look a little bit red looking around the perimeter.  Those are all Histoplasma.  The others are all bacteria. 

And the colony towards about 2:00 o’clock where you see the white fuzzy growth around the yeast colonies is Histoplasma and up about 12:00 o’clock. 

And here you see bacterial colonies that are prominent and in the background all those tiny colonies are Histoplasma.

Same thing here, these are yeast colonies and one bacterial colony about 6:00 o’clock and then all that stuff in the background is all confluent growth of Histoplasma.

And these tiny colonies that look like brown glass are Histoplasma in contrast to the yeast that are there.

And this is something that we see with some of the dimorphic fungi. It’s called a “prickly” stage.  It’s where you see those tiny tufts of hyphae sticking up and that’s common to find in some of these cultures.

Occasionally, you see these heaped up colonies of Histoplasma and donut-shaped type colonies.

 I’m thinking by this time you are getting the impression that there is a lot of variation with Histoplasma capsulatum and you are seriously going to wonder how many you threw away. 

You look underneath a microscope.  What are you looking for?  You are looking for the conidia.  The large ones are the, what we call the tuberculate, the macroconidia.  The problem with that is you look at a young culture, they don’t appear to be tuberculate or have rough-walls on them.

They appear to be just round and smooth on a long to short stalk like you see here.  And then as time goes along, they begin to get a little bit rough-walled and if you look closely, in the middle you will see one that’s beginning to get a little rough-walled, little spikes sticking out of it.

And here you can see that the wall of these are getting a little bit rough, not so much.

And here you can see the rough walls are a little bit more prominent with the spike sticking out.  These are called tuberculate macroconidia.

And then here you can see some in here that are prominently producing these rough walls.  And these are called tuberculate macroconidia.  These are too big to get down into the lung to get into the alveoli to cause infection but they are kind of like a diagnostic form for this organism.  There are some other things that could look like this.

These are tuberculate macroconidia and you can see that all of these are rough-walled.

This just gives you an enlarged view of what they look like and they are not always just spikes sticking out, there are kind of some rounded protuberances on there too.

And here you can see one that is really, gives you an idea that we are talking about three dimensional images here.  These spikes are coming out of these rough-walls or rough projections are coming out all the way around this organism.  This looks like back in medieval times where they carried the chain and the handle and they had this big brown ball with spikes on it that they beat people in the head with.  This is what this looks like.

Okay, here’s something else that you might see as you go along and looking at these macroconidia and that is some times they are in chains.  This is a case on this image right here, something that you might encounter.

And then you may find that some of them are kind of pear-shaped and not necessarily so round.

And here is another example, of that are some that are young and you don’t see the rough walls yet. 

And on here you see a chain of four.  You see all kinds of variations with Histoplasma.

Here you see some of three’s. 

And here’s one with more.

This is one that shows you the large tuberculate macroconidia that are dark blue and then the center you see the small pear-shaped spores that come off the sides of a hyphal strand.  These are probably infectious agents and this came from a blood culture.

And here you see the large spores and you see small spores intermingled in there.  Those are the microconidia and the big ones are the macroconidia. 

And the same thing is true here.  Here the predominance of microconidia with one large tuberculate macroconidia.  And if I move that image over so that you couldn’t see that macroconidia, you would be hard pressed to tell me that wasn’t Blastomyces

And this is just some more here.  What are these?  These are large tuberculate macroconidia that you see in this image.

It used to be, as I mentioned before, that we used to culture this organism and try to grow it up in-vitro and convert it up into a mold form to the yeast form.  The left tube shows you the mold form and the right image in the tube there, it’s hard to see, is the conversion to the yeast form.  This took a long time to do.

It’s a lot of trouble.  This is what the yeast colony would like of Histoplasma if you converted it.  I wouldn’t advise trying that.

This is what it would look like underneath a microscope if you converted it to the yeast form.  The same yeast cells we saw a while ago, that are two to five microns in size.


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