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Superficial Fungal Infections: Dermatophytes

Part 2

Click CC box for captions; full transcript is below.

Published: July 2012

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Dermatophytes infect the skin, hair, and nails in humans. Some are transferred from contact with soil, some from animals to humans, and some only infect humans. Dr. Roberts shares his lifetime of experience to assist you in differentiating the organisms under consideration in your patient’s differential diagnosis. Part 2 provides additional clinical presentations with a focus on individual species. (See Part 1 for an overview of the dermatophytes and their ecology and microscopic and clinical features.)

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 most commonly seen dermatophytes in clinical specimens in North America. This is Part 2 of a 2-part presentation. Thank you, Dr. Roberts.

Thank you, Sharon, for that introduction.

I have nothing to disclose.

This part will be the dermatophytes part 2 where we continue our discussion of dermatophytes and we mentioned on the previous presentation that these dermatophytes involve the hair, skin, or the nails. They may involve a normal host and they also can involve an immunocompromised host if things are just in the right arrangement.

The next slide shows you a schematic for identifying Microsporum canis. And when I did these schematics or these flow charts, I tried to put the relevant features in there so that you wouldn’t have to read so much. And Microsporum canis is characterized by having these large, rough-walled microconidia. And the organism grows well on Mycosel agar and it sporulates sometimes there and it also sporulates well on cornmeal agar. And it does produce those macroconidia that are large, that are rough walled, you can see the spikes on them, and there are many cells. Sometimes though, they produce microconidia and sometimes you are not expecting to see that and it complicates things. Well, it’s just a normal thing for some isolates. These macroconidia of Microsporum canis are spindle shaped and if you look at the distal tip, the tip where they have not been attached, you will notice that it actually curves. Sometimes you’ll see some rough projections on that for part of it. It has a curved beak. And then, that is what we use for recognizing Microsporum canis. And that is one of the easiest ones to recognize.

Generally, you will find the hyphae are hyaline and septate and then here are the features you see on this slide. They are rough-walled, spindle-shaped, multicelled of macroconidia with a curved beak and just what we mentioned a minute ago. So these are the features that you would be looking for if you saw Microsporum canis before you put a name on it.

Let’s go to the next slide. This is an example of Microsporum canis. All of these are macroconidia but look in the background. You see some small things in the background. Those are microconidia of Microsporum canis. These have multiseptate macroconidia. It’s a little difficult to recognize the rough-walled projections on this particular slide.

Let’s go to the next slide. And you see the tip of this macroconidium on the upper right-hand side, it’s a bit curved and it’s just a little bit rough. It’s not easy to see the rough-walled macroconidia in this. You see lots of microconidia. So you have to look at more than 1 field. You have to kind of scour the whole culture to be able to put a name on these organisms sometimes, because they don’t sporulate the way you think they should.

The next slide shows you the information that relates to the cultural morphology. Young colonies produce this cottony to kind of floccose appearance. They are white to buff color. And then as cultures begin they get older, they turn brown in the center and then the reverse side turns orange and, you can see yellow kind of goes through the front of the culture and the back is orange and that’s what Microsporum canis looks like.

The next slide shows you an early culture of Microsporum canis and, the way I describe this is the culture, is kind of has a feathery appearance at the periphery. And you can see that it is pretty obvious on this one.

The next slide shows you another flow chart as to how to identify Microsporum gypseum, another one that belongs to the genus Microsporum. And it will grow well in Mycosel agar and it surely, it will grow better on cornmeal agar. It produces the same thing as I said a while ago, macroconidia that are large and rough walled, has many cells, and it has microconidia once in a great while. But what is different about it between this one and Microsporum canis is the end of the macroconidium that is rough walled, is the rounded end. They’re not spindle-shaped, they don’t have a point or a curved beak, they have a round or kind of blunt end. And that’s Microsporum gypseum.

The next slide shows you kind of a review of what we just talked about with the rough-walled macroconidia and I mentioned the clavate or club-shaped with Epidermophyton floccosum. This one happens to be the same kind of shape, kind of club shaped, but they’re rough walled.

The next slide shows you Microsporum gypseum and there you see all of the macroconidia and you can see the rough wall if you look close. If you notice at the end of the macroconidia, on the bottom one actually, what is in the center bottom of the slide, on the left-hand side you can see the end where it was attached to a hyphal strand because there is a little piece sticking out there. And you look at the opposite end and you see that it’s rounded, that is characteristic of Microsporum gypseum. And often times the center cell will grow a little bit faster than the others and you can see it's larger. So, this is what Microsporum gypseum looks like. It’s not something that you see all of the time. But the rule in mycology is if they are easy to recognize you don’t see them.

The next slide shows you Microsporum gypseum and there are lots of small microconidias in the background. They are kind of club-shaped. And, if you look, the macroconidia are there and if you look at the wall of those, the outside wall of the macroconidia, you can see that they are just a little bit rough. They are multicelled and they are rounded on the end. I keep repeating myself, but these are the features of Microsporum gypseum.

The culture, though, is something that you would recognize and it would probably tell you it is probably going to be Microsporum gypseum. It produces a floccose to powdery, cinnamon-talcum kind of appearance of the colony, and these colonies do the same thing that the Epidermophyton floccosum does with the pleomorphic in the center with these white hyphae coming out of a tan colony.

You will see, this is an example here of Microsporum gypseum and there are the feathery type colonies and you can see that in the center they are kind of tan colored, kind of a cinnamon-brown. And you see those fingerlike projections coming out of there. That’s kind of what Microsporum gypseum really does look like, powdery for the most part.

The next slide shows you the same kind of colonies, but in the center there of one of those colonies, you will see that there are white hyphae that are growing right out of the center of the colony and those hyphae are pleomorphic and if you subcultured that for any purpose, whether it is to try and identify the culture or not, you would see nothing but white sterile hyphae. So, this is what Microsporum gypseum does look like.

The next slide is a schematic for Trichophyton mentagrophytes and sometimes it’s very difficult to sort this organism out from Trichophyton rubrum. That’s why it says Trichophyton at the top and it doesn’t specifically specify which organism. You will see that the next one is just kind of a part, next part of the same kind of flow diagram. So, these organisms grow well on Mycosel, but the sporulate better on cornmeal agar and what you would see with many of these Trichophytons are nothing more than small microconidia and many of them are sometimes are elongated. You don’t generally see macroconidia hardly at all. Sometimes you see and you think you might recognize what the culture is you can put them on different agars like potato dextrose agar like cornmeal agar. In this case, this organism if it was placed on potato dextrose agar it would produce kind of star-shaped colonies. This is Trichophyton mentagrophytes that we are talking about and we know that this one produces urease. So urea agar would be in the schematic and if it turned urea agar produced ureas and turned it pink, you would know that you are dealing with Trichophyton mentagrophytes. The other reason that you would know it was Trichophyton mentagrophytes would be the microscopic morphology. If you looked at the microscopic morphology, what you would see are many, small, round clusters of microconidia. They look almost like clusters of grapes. Sometimes you will see hyphae in there that are actually spiral. They look like springs setting in there, particularly present in cornmeal agar. So this is what Trichophyton mentagrophytes would be, as how you would identify that.

And this is just kind of a review of what I just said. You can look at this next slide and it just mentions that the macroconidia are not commonly produced, but they are cylindrical to cigar shaped when they are. But most of the time, you are going to see those microconidia and those spiral hyphae.

The next slide shows you urease and on the left side, and actually both of these, I think the left one, is the one, is the control. The right-hand one is pink, you can see the organism actually produced a red color, a pink color. And so, this is what you would use for Trichophyton mentagrophytes to distinguish it from Trichophyton rubrum.

The next slide shows you the microscopic morphology of the organism. And there you see the round grapelike, grape-shaped conidia, microconidia. And I think some of them are in clusters, but also there are some hyphae that are spiral shaped in there that look like coiled up springs. And that’s what you see sometimes with Trichophyton mentagrophytes.

The next slide shows you better these microconidia and you see that they are in clusters and they’re round. And that’s what the characteristics of this organism are and we generally don’t see that much with any other dermatophytes.

The next slide just kind of shows you what the culture is going to look like with Trichophyton mentagrophytes. And it may be fluffy, it may be granular, it may be even be velvety, and as a general rule, any of these dermatophytes, if they produce a powdery like colony, that’s the kind of colony that’s going to produce the spores or the conidia. Now some of these colonies can be white to yellow, or pink, and some may be kind of a red-brown mahogany color, and this is what Trichophyton mentagrophytes would look like.

This is an example here of a Petri dish, where the specimen came over in the Petri dish, you can see a scalpel blade sitting in kind of in the middle of those colonies there. It came from dermatology, they scraped some skin scales off, sent it over right in the plate and you can see the colony is fluffy, it’s downy and for the most part, you probably would not see many spores at all in that culture.

The next slide shows you a powdery one and you can see the feathery nature of the colony and probably can, it looks a little bit like the colonies of Microsporum. So, you generally can’t tell from looking at the culture morphology what the organism is going to be. So, in this case here, this one here has some pink to it.

This next slide shows you the flow diagram that we are going to use for identifying Trichophyton rubrum, the one that we are trying to distinguish Trichophyton mentagrophytes from. It will grow up on Mycosel agar and on cornmeal agars and sporulate well on cornmeal agars. What it does is produce a lot of microconidia, that are smooth, or elongated, and most of the time they are sitting on the sides of the hyphae. As one of my colleagues put it, it looks like a bunch of birds on a fence. And that’s probably what it does look like. You can put this organism on urea agar and it will not produce urease at all, it is urease negative. If you put it on cornmeal agar it will produce a red pigment, on early it will produce kind of a yellow pigment, but in time it turns red, it’s diffusible pigment. And you will see these tear, kind of tear-shaped, elongated microconidia along the sides of the hyphae. And this is what Trichophyton rubrum would look like.

So the next slide reviews what we just said. These microconidia are pear-shaped and easy to spot. Usually they are there in great numbers. And the macroconidia are not usually there. If they are, they are kind of smooth walled and pencil shaped, that we talked about that earlier. That’s only if you see them and they are not there very often.

The next slide shows you an example of Trichophyton rubrum. This is where these pyriform or pear-shaped little microconidia are lined up along the sides of the hyphae. When you see that you pretty much know that’s Trichophyton rubrum if you recovered that from the skin or the hair or the nails.

And this is it again. You can see those pear-shaped conidia come right along the sides of the hyphae. One thing that is kind of interesting is the hyphae with the dermatophytes are very small. The hyphae of the dimorphic fungi are very small and if you look at the relatedness in their molecular profiles, you will notice that they actually are related. These are not systemic pathogens, but they look, they kind of remind you of that when you look at them because of the small hyphae.

Next slide talks about the colonial morphology of the Trichophyton rubrum. The colonies are usually a fluffy white, sometimes they can get kind of granular in appearance. The reverse side starts off as being yellow, and then with time the red diffusible is produced as we have mentioned a minute ago. And you can then tell you are dealing with the Trichophyton rubrum.

The next slide shows you a colonies, 3 colonies actually, that already have produced the red pigment, probably on the back and the front, and you can see there is some white in those colonies too. So the red pigment is very obvious and the rubrum refers to red, so that’s where Trichophyton rubrum gets it’s name and then

The next slide shows you the red diffusible pigment on the back side. It takes a little while for this to be produced, but this gives you an idea of what Trichophyton rubrum can do.

The next slide presents another organism that we deal with fairly infrequently. It grows up well on Mycosel agar. It sporulates better on cornmeal agar. It produces a lot of microconidia. It does not produce urease. It doesn’t produce a red pigment. What it does do, is produce pleomorphic microconidia. Some of them are round, they call balloon-forms, some of them are pear-shaped. Some of them are kind of just elongated. They are pleomorphic, they don’t have a particular shape to them and they are produced right along a hyphal strand. And they are generally recovered from the scalp of a child. This is Trichophyton tonsurans. This one has kind of a little bit of a tan color to the colony, and it is a little bit more slow-growing.

The next slide reviews what you might see with this organism. Hyphae are small, they are septate, they don’t have any pigment and microconidia are pleomorphic, and like I said there are some that are round that we call balloon forms, they kind of clue us in. The microconidia look bizarre.

The next slide shows you Trichophyton tonsurans as it’s growing. And you can see that if you look really closely along the sides of the hyphae, are these small microconidia and occasionally, you see some that are really long, produced and if you look closely, you see some that are round.

The next slide shows you some of the rounded cells of Trichophyton tonsurans, and some of the pear-shaped conidia coming off on the right-hand side of the slide. So it produces a whole variety of morphologic forms when you look at it.

Next slide talks about colonial morphology of Trichophyton tonsurans, it produces these kind of very smooth, kind of velvety colonies that are kind of creamy yellow and the reverse side may even be brown or mahogany in color. So they look different from the ones, this one looks different from the ones that we have talked about before.

The next slide shows you a culture of Trichophyton tonsurans and you can see this one has grown for a while. And it’s folded up in the center, but it’s very velvety in appearance. And it’s kind of powdery, and it’s kind of off white.

The next slide will show you, I think, one of the most uncommon ones that we see, but nevertheless, it’s around, if you live in the Midwest, you more than likely will see it. This is an organism called Trichophyton verrucosum and it grows slowly on Mycosel, even on cornmeal agar, very slow. And you generally, don’t see conidia. You may see just small hyphae, because it does take it so long to grow, it just doesn’t sporulate. The colony may be white and wrinkled, it might even be kind of ochre colored or amber colored, and that’s all it may be. But it’s really slow growing, and it stays on the culture plate so long that it actually caused the culture medium around it to crack. The colonies are very slow growing, they are heaped up. Sometimes, they submerge themselves in the agar and you can put (I’m not sure if we can get Trichophyton agar number 4 any more), but actually, it’s a medium that is enhanced by the presence of thiamine. And so this organism can be enhanced by the growth of thiamine and or inositol to prove that’s what it is.

And then in the next slide shows you what the culture may show. What I was going to mention was, there are times when you may see these long chains of densely compacted chlamydoconidia or there are swollen cells that are produced. And that occurs at 37 degrees centigrade and that’s something that separates this organism from all of the others is a production of these. You may find macroconidia, very rarely, and if you do they look like a rat tail. That’s the way they are described in the literature.

The next slide shows you those chains of chlamydoconidia that are produced at 37 degrees centigrade. No other dermatophyte that I know of does this. So when you see this feature you pretty much know that you are dealing with Trichophyton verrucosum.

Next slide mentions what we just said. The colonies are very slow growing, they are heaped and often times partially submerged into the medium. They may be white, grey-white, or yellow, or kind of even not even have any particular color to it. And they again require thiamine and or inositol and you can, if you can still get those agars that just cinches what it is, but if you see the morphology with those chlamydoconidia, you pretty much know that’s what it is.

The next slide shows you Trichophyton verrucosum and look at the culturing in this. It’s actually pulled away from the sides of the Petri dish because it has been incubated for so long. And the colony size is not all that big. So they take a long, long time to grow.

The next slide shows you some colonies of Trichophyton verrucosum and you can see that we have dug into some of those to try and find something that would tell us what it is. And in this case it produces some white hyphae on the colony surface. And you would be looking for anything to tell you what it is.

The next slide shows you what we call the Trichophyton agars that were used for many years. It had thiamine and inositol, or thiamine by themselves. And so, you could look at the one on the left-hand side, probably has thiamine and inositol in it, and you can see it grows better on the right-hand one. The left-hand side is the control and it doesn’t have either 1 of those compounds in it, so the growth is enhanced by those compounds. And that’s how we used to identify Trichophyton verrucosum.

The next slide shows you something here that is probably not done much anymore. With Trichophyton mentagrophytes there have been a lot of discussion over the years about trying to separate Trichophyton rubrum from Trichophyton mentagrophytes easily. And we came up with the urease test, which I think does it. And other times people said, oh well, we use something called hair perforation where we take a little yeast extract and we put it in some water and we get some hair and we put the organism on the surface of the hair, and what happens is if it’s Trichophyton mentagrophytes, it grows down into the hair and it forms these plugs. Trichophyton rubrum will not do this. This is an old technique and I don’t advocate to using it, but I just mentioned it for historical purposes. But, you can see this hair here really has no invasion.

The next slide shows you, you can see in the center of the hair some circularly, that’s where the hair is actually growing right in the, the dermatophyte has grown right into the, hair and this is called hair perforation. And Trichophyton mentagrophytes does this, but Trichophyton rubrum will not. And we used to use this routinely.

This is the end of our discussion on dermatophytes. It’s a very quick coverage, but at least it will give you a ballpark idea of how we identify dermatophytes in the clinical laboratory.