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Published: December 2012Print Record of Viewing
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
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 Coccidioidomycosis.Thank you, Dr. Roberts. Thank you, Sarah, for that introduction.
I have nothing to disclose.
The diseases caused by the dimorphic fungi include the ones you see on this particular slide. The first three are seen most commonly in North America and Paracoccidioidomycosis is seen in Central and South America. The others that you see here, Sporotichosis is seen in the US. Penicilliosis is seen pretty much in the Far East. These are things that we will see in the clinical laboratory.
They are caused by fungi that are called dimorphic fungi. And that is that these organisms happen to have two growth forms. They have some particular features that we will go through that are in common to all of them and the growth rates are relative and that depends upon how much organism is present in the clinical specimen that you start off with. It depends upon the culture medium that you use for recovering the organism and it depends upon how much organism is present in the clinical specimen as well as the metabolism of that particular organism. One of the things that helps in some instances but not all, is blood enrichment and we include a medium most of the time that have a blood enrichment source so that we can pick up some of the fastidious organisms. The microscopic morphology, there is one thing that’s in common with all of them and that is that they produce small septate hyphae. There are a few other things like dermatophytes that can do the same thing but by and large, the dimorphic fungi are the ones that produce these hyphae. We do have molecular methods that are available for identifying these organisms in addition to using traditional methods. We have nucleic acid probes, we have nucleic acid sequencing, and MALDI-TOF that is now being used in the clinical laboratory that will identify these organisms much more easily than traditional methods.
This image shows you the small hyphae. It is hard to tell on here if they are septate or not but they are septate and very tiny in size, like 0.5 microns.
The dimorphic fungi have colonial morphologic features that vary depending upon on the isolate and also upon the medium in which these organisms are recovered. So then you can have one organism that you can place on two different media and they will look differently on either.
The colonies of Histoplasma and Blastomyces are pretty much indistinguishable from each other. The colonial morphology sometimes is not helpful at all in terms of identifying these organisms.
This particular discussion is going to focus on Coccidioidomycosis. We will talk about the organism Coccidioides immitis/posadasii. Coccidioides immitis is an organism that we have been dealing with for many, many years and it’s been found that there is another strain that it genetically differs from Coccidioides immitis, it’s called Coccidioides posadasii. And it doesn’t differ in terms of its colonial morphology or its microscopic morphology but rather its nucleic acid makeup.
Where do we find this organism? It is found in the southwestern part of North America and also in Central and South America.
This slide here shows you a rectangular box that includes primarily the areas where you see Coccidioides immitis in the United States. It goes from western Texas to all the way to northern California and that’s pretty much where you’ll find it. You don’t generally find it any place else. There are times whenever you can recover the organism from patients who happen to be in an area that is not endemic for Coccidioides and it may have something to do with the fact that they travel there during the winter months, came from a cold area and traveled there during there in the winter months and came back home with it. Sometimes there are fomites that have been sent from one part of the country where Coccidioides is endemic, sent to another part of the country and someone opens a package up and they get exposed to the spores of these organisms.
This is a map that shows you kind of where it’s found in the world. Coccidioides is found as you can see in Northern California all the way to Western Texas and down into Mexico. There are a couple of little black dots in Central America. Honduras is one of those places where you see it. I happen to visit there a few years ago and they see it every day, people walk in with the disease. So we know it’s found in Central America and we know it’s also found in South America, pretty much in every country except Chile.
Where do you find this organism? Well, we know where this organism is found. It’s found in the soil of the areas that typify what’s called the Lower Sonoran Life Zone where the conditions are semiarid and dry. The temperatures reach 100 degrees or more Fahrenheit during the summer months and then in the winter, temperatures falls all of the way down to freezing or sometimes even below that.
The soil has a very high alkaline and salt content. And if you look what grows there you will see that its vegetation is short stubby type shrubs like the creosote bushes, mesquite trees that are not very big, cacti, and particularly the saguaro cactus and yucca plants. It’s kind of the typical environment that you see in Arizona and southern California. And a lot of times there are a number of animals, small rodents that burrow down into the soil and sometimes they’re actually the culprits that bring this organism to the top of the soil. They have let the wind carry it along or let us pick it up whenever we happen to be working the soil.
This is an example of the Lower Sonoran Life Zone. You can see that the saguaro cacti, they are the kind you see in the old western movies and this is the type of environment where you find Coccidioides. And people go out hiking in those areas, they go out poking around in this soil looking for something or the ones that get it are people who go on archaelogic digs are at high risk for acquiring Coccidioidomycosis in an environment like this.
What happens to this organism? Well, it disappears from the soil during the wet months. It goes down a little bit deeper in the soil and then when the summer months and spring months come, the soil begins to dry out and the organism sort of comes to the top and then the winds disseminate these spores that are the infectious particles of this organism around in the environment and they are there for us to inhale for small rodents to inhale. Anything that happens to be around can actually become infected by this organism. So anything that disturbs the soil including the wind is what’s going to disseminate this organism throughout the environment and make the spores available for us to inhale.
What kind of activities are associated with acquiring this infection? We kind of already talked about a little bit about that, but, construction out in areas where this organism is endemic as a very substantial source for getting infection. The archaelogic digs are another thing we talked about a few minutes ago, here, that it is responsible for causing great number of infections. But, people work in agriculture in parts of Arizona and California who are out picking all sorts of vegetables and so on that disturb the soil and actually inhale this organism. And then one of the big things that spreads this to huge numbers of people are dust storms that you see in the western part of the country that blow these spores all the way up to northern California from southern California and even further away than that.
This is an example of an advancing dust storm. The photograph was taken in Kern County, California and this is in northern California where they hadn’t seen this organism. They woke up in the morning and they found their cars were just covered with dust. Well, what they didn’t know was not only was it covered with dust but it was covered with arthroconidia of Coccidioides. And so there were many cases of Coccidioidomycosis in Kern County as a result of this dust storm that was seen there.
This schematic shows you kind of what happens. Lower left hand side what we see are hyphae that are broken down into these arthroconidia. Artho means jointed and you can see that the spores there after the first arrow going from about 11:00 o’clock pointing to the right, you see these arthroconidia. What happens is the arthroconidia are formed and as they grow, there becomes a dead space in between the arthroconidia. They are called alternate arthroconidia. What happens is that the wall behind each of these arthroconidia breaks apart from the dead cell between them. And it gives the appearance of little wings on them and allows them to float in the environment for a long period of time. So, on this schematic we don’t necessarily see alternate arthroconidia but I will show those to you as we go along. But what you see up there is saguaro cactus and so on is like the Lower Sonoran Life Zone. These things are aerosolized by whatever source, whether it is the wind or whatever it is. We take it into the lungs and you see on the right lung what happens inside there is the spores come in, those arthroconidia come in. They then convert to round cells that are not yeast cells like we see with all of the other dimorphic fungi but they convert into rounds cells that are called spherules. And in time these cells began to produce what are called cleavage furrows, you can see in the lower middle of the diagram where you see these cleavage furrows and then in turn forms these sporangia, some people call them sporangia spores but they are really called endospores for the most part. And those are things that then begin to develop into new spherules inside the host and the cycle goes on. And so when you take a biopsy from one of these patients who has active disease with Coccidioides immitis as the cause of agent, you see these spherules in all stages of developments and you will see endospores in there in some of the cells you look at. At sometimes you see spherules that are just simply empty. But you have to look at the whole slide.
Here’s what happens, we have kind of already alluded to this. The hyphae begin to break down into rectangular arthroconidia. These things are easy aerosolized and once they are taken into the lung they begin to transform into these spherules and as these spherules begin to develop they begin to replicate internally by a process called progress cleavage and this divides this internal cytoplasm into what are called endospores inside this spherule and then as time goes along and these spherules begin to mature, they break open and the endospores are liberated and they form more spherules. And the cycle goes on.
What are we going select for culture and for direct microscopic examination? Well, we have a number of sources that we can find this organism in. One’s a lower respiratory tract like most all of the dimorphic fungi have where they cause a pneumonia, most of them do, not all. We know that with Coccidioidomycosis that sepsis is a common manifestation, disseminated disease. Coccidioides involves the bone. It causes lesions in the brain. It can be found in a spinal fluid, meningitis is a common feature of Coccidioidomycosis and it’s also a very serious one. You could find it when the skin and also the mucous membranes, particularly the oropharynx. You could find it causing arthritis and you can find it causing an abscess of the subcutaneous tissues. So pretty much, you can find it almost any place.
So what are you going to see if you look underneath a microscope at a specimen that may have Coccidioides immitis in it? What you are going to see are these rounded spherules, some of them may not have any endospores in them at all. Others may look like what you see here using Calcuflour white, you see the large cell that is the spherule and coming out of that ruptured spore are all of these endospores that if left in the patient would then begin to turn into individual spherules in time.
This is an example of a biopsy. This is the H&E stain showing you a large spherule containing numerous endospores and the H&E stain shows them pretty well. You can see it also on a methenamine silver stain well.
This is a culture of Coccidioides. And one thing we do know about this organism in terms of culture is that for the most part it grows more rapidly than all of the other dimorphic fungi. It’ll grow up within two or three days and form arthroconidia in a time or a little bit close to that time or a little bit longer than that. But the colony begins to appear in two or three days. As it matures, it will form these arthroconidia and I can’t give you a time as to how long it takes to form them but I would say four to six days the arthroconidia are going to be there. And so this is the most infectious of all the dimorphic fungi and all of the fungi period to handle in the clinical laboratory. There have been numerous reports of cases of laboratory acquired infections and death caused by this organism. So, it grows up as a fluffy white mold for the most part but if you look at photographs of a hundred isolates of this organism, you probably would see a hundred different morphologic features on these colonies. They can be yellow, they can be brown, they can be pink, they can be red, they can be white, tan, all sorts of colors. When you get through with this thing, you may have said to yourself, “Geez, I wonder if I threw one of those things out,” because, it does happen.
This is another example of colonies of Coccidioides immitis or posadasii. What you see here are two colonies side by side and they are heaped up in the center. And that’s a common feature for some of these isolates that we see. They are domed colonies.
This is a colony on a blood-containing medium. And it almost looks like it has a little bit of a green color to it. Sometimes you see that this blood enrichment is helpful for recovering Coccidioides but not as helpful as for some of the other fungi.
This is one growing on a blood agar. And you can see that in the center it’s domed. And in the periphery is kind of more, more fluffy. And this is an example of those domed colonies we talked about.
This is another example of what we see with Coccidioides and we kind of refer to these things as cobweb type colonies. What happens is the center begins to kind of domed up and the periphery begins to be more adherent to the agar. If you see a colony like this, it’s pretty representative of Coccidioides immitis/ posadasii.
This is an example of a plate where we used Cycloheximide to recover for the organism. All the colonies there are Coccidioides except for the one at 3:00 o’clock and that’s probably penicilium or some other saprophyte that we had happened to get into the culture plate. You can see those domed colonies, you can see the kind of cobweb type colonies that we were talking about and this is common to find these.
This next slide shows you a plate of blood agar that was used in the bacteriology laboratory. Those three colonies are Coccidioides immitis. What you don’t want to do is open this plate up and the bacteriology laboratory without being inside of a biological safety cabinet. For the most part, the colonies may not have had time to mature when they begin to look like this. On the other hand, I wouldn’t trust that even if I said it to myself, because I think that there are exceptions to all of this and what you don’t want to do is to have a laboratory acquired infection and get a big dose of inoculum from opening up a plate like this. What you do is you open up underneath a biological safety cabinet.
This is chocolate agar showing you the colonies of Coccidioides immitis that grew up in a bacteriology laboratory. The rule of thumb is that no cultures of molds should be opened up in the room. They should be opened up but not on the bench, they should be opened up in the biological safety cabinet.
These are some colonies I am going to show you that vary and they are just different isolates. This one looks almost grey. Can you see this center is kind of domed up?
And this is an example here of some of the colonies you have already seen but you show them on three different medium. The top two plates containing blood enrichment and the bottom one is Sabouraud’s dextrose agar. And they look different depending on the three plates. They are all the same isolate.
This is the example of four different culture media, same isolate. And you can see the two blood containing media look about alike and also the two non containing blood medium look a little bit alike. You have to become familiar with what these organisms look like on different culture media to be able to recognize them, at least suspicion of what they are.
Now the next slides I am going to show you are maybe a little bit surprising to some of you who are looking at this because there are many colonial variance of Coccidioides immitis/posadasii. I’m just going to go through them and you can look at them. This one looks very glabrous. Not really, yeast-like but just kind of membranous.
This slide shows you again another one of these colonies that looks membranous and this is Coccidioides immitis and it doesn’t look like what we talked about earlier.
This one including all of the colonies except for one, the large colony about 3:00 o’clock is all Coccidioides immitis. It doesn’t look white and fluffy or membranous either.
This one looks like something you would probably throw away except for the tiny colonies there that are bacteria. This is all Coccidioides.
And this one except for the large colonies there that are yeast, its Coccidioides. It almost blends in with the culture medium.
And this is Coccidioides. It doesn’t even look like anything that’s described in textbooks.
There are a lot more variance of Coccidioides that we could show you and again, I keep saying this but you wonder sometimes how many of these did we throw away because we thought they didn’t look like anything that we should worry about. How are we going to identify Coccidioides immitis? We are going to look underneath the microscope at the microscopic features of this organism from the colonies and you are going to start off by noticing one thing that’s in common with all of these dimorphic fungi and that is they have small delicate hyphae present. With Coccidioides, we find some structures in there that we don’t see with some of the others. They are called racquet hyphae and they look like tennis racquets. It looks like a swollen area that may be at the end of the hyphal strand or in the middle of a hyphal strand. It’s produced in these young cultures. And they are just called racquet hyphae. What we see though, that are characteristic for this organism are alternate arthroconidia. The arthroconidia that join its spores, they produce an arthroconidium and they produce a dead space which is called a disjunctor cell and then another arthroconidium and it just keeps on going. So they are called alternate arthroconidia. And one thing that is important to look at because there are lots of these fungi that produce arthroconidia, one thing to look at is to look at the size of the hyphae that are producing these arthroconidia. The hyphae are a whole lot smaller than these arthroconidia, so the hyphae that produce the arthroconidia are smaller. These arthroconidia can be barrel shaped and a lot of times they are that. They can be rectangular or even rounded in shape. You generally do not see spherules or endospores on culture medium. You see these alternate arthroconidia.
On this image you can see these are the racquet hyphae. These are the alternate arthroconidia that we are talking about but these structures here are the racquet hyphae. There are racquet hyphae on here that are not as good and you can see that they’re the kind of racquet shaped structures on there but they don’t all appear to be textbook perfect, I can tell you.
This is another example of racquet hyphae and you can see them scattered throughout there.
This is an example, now, of the early development of the arthroconidia. If you look at the hyphae of Coccidioides in there, you notice that the hyphae themselves are very small. But look at what’s beginning to develop in those. And those are your arthroconidia that are kind of barrel shaped. They are much larger than those hyphae that produced them and that’s what you look for when you are looking for Coccidioides, that and the fact they produce these alternate arthroconidia.
Now this slide here shows you the alternate arthroconidia. The arthroconidia are the rectangular cells and then there is a clear space in between them. The clear space is called a disjunctor. What happens is the cells break apart and that clear space and as they do that, they take a part of the wall of the cell behind, the disjunctor cell behind them and on either side actually, and it just adds a little piece of disjunctor cell on either side of it, the arthroconidium, and it allows it to give some lift to these spores as they float around in the environment.
And this slide, you see a lot of arthroconidia. But you don’t necessarily see a real prominent clear space in between them. So you have to look at everything and you have to look at them closely to see if you have alternate arthroconidia or not because there are lots of organisms that actually are found in the soil and that part of the country where you find Coccidioides that may be a bit confusing.
Here’s a very good example. It’s an old slide from the Centers for Disease Control that shows these alternate arthroconidia on the strand at the bottom you see there’s a rectangular spore, some that look a little barrel shape, there’s a clear space between them. That is what we talk about with mature alternate arthroconidia.
And here you can see that this disjunctor cell and then the arthroconidia on either side.