Steve A McClain, MD discusses the use of microscopy to aid in the diagnosis and treatment of common foot diseases. Dr McClain reviews several cases and focuses on the treatment of fungal infections to heal chronic wounds.
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Steve McClain, MD
Adjunct Professor Dermatology and Emergency Medicine
Stony Brook, LI
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Male Speaker: Just a little bit. We’re going to look at some pathology of some common foot diseases. Giving us insight into is Dr. Steve McClain. Dr. McClain has been a member of our faculty over the number of years, he is extremely entertaining, he is energetic, he is motivating, and he is bright. He’s a dermatopathologist. Grace, the only thing I ever remember about anything related to dermatology, if it’s wet dry and if it’s dry wet, by all means, don’t touch it. That was it. In any event, he’s going to share some important facts that we need to be aware of. I want you to please welcome Dr. Steven McClain.
Dr. Steve McClain: Thank you. You told me there would be a standing ovation, what’s going on? There’s nobody standing and that wasn’t much of an ovation. What can I say? I am Steve McClain from Long Island. I’ve been there 21 years. Actually, today starts the 12th year of McClain Laboratories. We started on May 15th in 2004. Those of you who have those handy dandy little notebooks, I want you to make three notes, if you will. The first one is the spelling of my last name, firstname.lastname@example.org. If you want to contact me, that’s the place to go. I’ve got several objectives, but mainly I want you to biopsy more. I want you to use the laboratory to guide your treatment. I want to talk about how normal feet are communities of microbes, they’re called microbiomes. I want to make the point about delayed healing and chronic wounds are frequently due to fungal and bacterial loads in these wounds, and talk a little bit about pathology, especially how fungi grows atop almost any foot skin pathology. Let me repeat that. Fungi growing on top of the pathology, and it hides it. So you sometimes have to scrape away or otherwise remove the callus in order to get down to where the real pathology lies. I’ve got a couple of controversial subjects. One is about dystrophic nails and the other is the notion of fungi having a role in the development of IPKs and corns. Here’s the other two points I want you to write down. Write down this first reference by Alexis Carrel. It’s available free on Google Books. You just go to advanced book search. Alexis Carrel “Treatment of Infected Wounds”. It’s almost a hundred years old. It’s literally the bible on the first part about infected wound healing. The second is this paper by Keisha Findley. It’s in Nature. This is available free if you go into ncbi.nlm.nih.gov or PubMed, topographical diversity of fungal and bacterial communities in human skin. I’m going to show some slides from hers. Mainly, I want you to anticipate that there are hidden fungal components in common foot diseases and to treat them. Whenever people talk about bioburden and biofilm, they always talk about bacteria and they forget about the fungus. I want you to think about fungus for a little while and begin to treat it. Some of the antibiotics you’re using to kill bacteria are actually antifungal as well. Okay, a little bit about me. I did get my first microscope at Christmas in 1960, trained at the University of Missouri, residency in Vermont. I did a fellowship in dermatopathology with Bernie Ackerman at NYU. Went to Stony Brook in 1994 and I began collaborating on a number of wound healing projects. Mesenchymal stem cell activation, diabetic healing, burn healing, PDGF and so on, over the years. When it came time to start my own lab, I found that by making a diagnosis and showing the surgeon the photographic proof, they were more likely to treat it, and so I’m hoping some of my pictures will entice you to do the same. Here’s an example of one of my reports. I had cut off the top part, so we’re HIPAA-compliant. Podiatrist plucked this bone out of a gangrenous ulcer and sent it to me about three years ago. I think it’s a proximal phalanx, the first phalanx. I noticed right away, there were fungi growing all over the thing.
Not only that, on the upper right, you can see grains that are gram positive. They look like
gram-positive cocci. On the bottom part, there are other grains that are PAS-positive. It began my search as what’s going on with this bone. Fungus was destroying bone. It had grains like Madura foot and yet it occurs today in my county. We don’t talk about Madura foot or mycetoma in today’s literature and yet it is really common. This is the reference I told you to write down. All surgeons must know the treatment of infected wounds. The bible was written on this, by Alexis Carrel in 1917, and they didn’t have antibiotics in those days. They only had disinfectants. Fortunately, Henry Drysdale Dakin was the chemist who was in charge of part of the laboratory and making up the antimicrobials, the disinfecting agents. This is from that same book. Normal healing follows a geometric curve when we’re taught there’s a lag phase and then a contraction phase or an inflammatory phase and then the contraction phase. But there’s a part about chronic wound healing that everyone doesn’t talk about and that’s the infected phase and I’m going to go into that a little bit. The first point that Carrel made was that wound healing is not dependent on wound size. A bigger wound doesn’t necessarily heal slower than a smaller wound, okay? If you graph it out, it will make a steady progression. What it does depend on is an absence of microbes. You have to get the microbes down to what is considered a surgically sterile or aseptic. It’s not bacteriologically aseptic but you have to get it down to reduce the bacterial load and the fungal load, for that matter, down to a minimum. So we’re taught that there’s two phases, there's lag and contraction or inflammatory phase and then the proliferative and contraction phase. But in chronic wounds, the lag phase overlaps with the infection. And until you clear the infection, those things will not close. Here’s a graph out of same part. On the upper left, you’ll see the wound. This is wound size, and it’s getting bigger up until you see the red cross. Then it begins to get smaller. If we look at the bacterial counts, and these guys really did it properly. They measured wound area by tracing out over cellophane, okay? They measured wound area every three days. But bacterial counts, they did every single day. They incorporated the laboratory into their wound healing even in World War I. You’ll see the graph at the bottom where the red cross is, the bacteria fell down and where the cross hatching changes, that’s one bacterium per high-powered field. More than two was considered infected. And so, when you see the curve on the upper curve flattening out, there’s a little peak on the bottom curve, and so there was a reinfection. And they up the disinfection. Mainly, they increase the concentration and then it finally closed. Okay. I’ve done a lot of animal wound research, measured thousands and thousands, more than 20,000 wounds so far. Diabetic animal wounds all heal in 28 days. Now, we don’t have the neuropathy, we don’t have the vascular comprise, and they’re not walking on them, okay? But diabetic animals all heal in 28 days, even with blood glucoses of 600 milligrams per deciliter. I would like to talk about this a little bit because the normals, they reepthelialize within about a week. They have thicker scars, more collagen, which is the darker blue in the wound area. I can’t really show that to you, and they’re more contracted. But on contrast, the diabetic wounds, they reepthelialize later. It’s more like two weeks, two and a half weeks. Thin weak scars, 50% less collagen, and there’s an excess of MMP-9 or collagenases. This is where the measurement part comes in. That wound on the upper and lower arrows in blue are set to the same level on the diabetic and the normal rat skin. And so, you see that the wounds are quite thin in diabetes. They don’t fill up and they down contract. Okay? They’re broad, they’re thin, they break open more easily. And so diabetic healing is weak and delayed healing. Okay? There’s less collagen and they’re thinner in general. Well, I promised you some pathology, so I’m going to shift gears a little bit to a tattoo granuloma.
Now, this is a young woman, she’s not so young actually, she’s about 45. But for me, that would be a younger woman, tattoo granuloma. And so, you see the red part of her tattoos. Her tattoo there has gotten thicker. Now, this is the tattoo that she did not have redone. Here’s the one she had retattooed about six months ago. This was a ying and yang symbol and now the thing is erupting with these miniature keratoacanthomas and then granulomatous response. All because of the red tattoo, it was redone. So this is a tattoo redo. You like the hearts on your feet? These are really pretty gross, right? Microscopically, she developed these keratoacanthoma-like lesions. In the bottom photographs, there’s the reddish brown pigment on the left bottom. On the right, it’s pulverized which makes it fluoresce like coral. Okay. I mentioned about fungal bioburden and we see these all the time. I’m sure you see these everyday but we don’t talk about the burden of the fungus on the foot. These are taking energy away from the foot. Fungal nails to me indicate bioburden. When you see a fungal nail or multiple fungal nails, especially in diabetes, they’re at risk for all kinds of other fungal infections on the foot and leg, and I’ll talk about those. This is the biofilm. I’ve stained the biofilm here blue. Ooh, that’s kind of cutoff. Whoa, there it was. Elysian blue stains it blue. It stains the mucinous part of this blue and I didn’t realize that fungi produce biofilm. Then I started finding it in other places. The carbohydrates here are stained in red. I don’t know how red that looks to you but on my screen, it’s pretty red. That cross-shaped hyphae structure there and the blue, they’re intermittently related. When I learned this, I began staining with PAS and Elysian blue on the fungal nails. To me, nail dystrophy quickly became a myth. Podiatrists are right about 99% of the time on the nails they send in. If you’re getting a diagnosis of nail dystrophy, many podiatrists who experience will just treat nail dystrophy as if it’s fungal, and they have success. Now, they’re not going to use an oral medication. They don’t have their 007 on that yet because they don’t have a pathology report. But to me, nail dystrophy is a myth. It’s a misdiagnosis. It’s a failure of the laboratory to stain fungus. Here’s a chunk of a nail which we stained with Elysian blue and this is what I call a cheese nail. It looks like stinky cheese. We stained it with Elysian blue and the [indecipherable] [13:07] actually diffuse into the cheesy part of the nail and turned it green. To me, fungal biofilm is really common. More than 50% of fungal infections I see have fairly abundant biofilm to them. Here’s a nail we dipped into Elysian blue, cheese nail. The definition is if it’s thicker than a millimeter and it looks like stinky cheese, it’s infected until proven otherwise. I’ve got about 16,000 cases which support that and maybe a hundred in which I couldn’t find the fungus. When it’s repeated, you find it again on a later sample. Okay. There are three zones in an infected nail in nail fungus. There’s an upper hard plastic one and that is virtually impenetrable. Xylene won’t penetrate that. Acetone doesn’t penetrate it. Very little penetrates that upper, what I’ve stained yellow in this. In the middle zone are cornified cells that are derived from the nail bed. All that’s part of the cheesiness of the nail. What I didn’t know initially was that the infected squamous part is also infected, the red inclusions at the bottom on the H&E. All zones need treatment. The reason that debridement and topicals work is you can get the topical medication down to where the medicine needs to go. Some doctors will either pulse with lasers or pulse with Lamisil or pulse for a month with another medication to get coverage into the squamous part of the nail bed. But you’ve got to get your treatment all the way down to the base. Maceration. All the podiatrists taught me that maceration, it means too much water.
Yes, it means too much water but it means too much water on a fungal foot, and maceration is all fungal. Look at all those vines growing in there. Can you see them? The clumps of yellow stuff, all fungal. It’s a fungal proliferation. It’s gone like mad because you’ve watered it. Whether the water came from a wet work environment or the water came from a wound that was oozing, doesn’t really matter to the fungus. If there’s water and food there, the fungi will go crazy and grow. Whoops, what happened here? This is a heel ulcer. Notice all the flakes around the ulcer. At the center and the bottom is where a punch biopsy was done. Harvey Lemont taught me that all dry skin is fungal. Now, I don’t know exactly whether that means that it begins fungal, but if you have something where cornified cells begin to accumulate, the fungi will see that as food. They will find their way into it. Okay. I’m going to skip those too. Polymerase chain reaction, everyone has probably heard about this. You can take and define a specific DNA sequence by amplifying it, by creating a new DNA set, and then getting it to split apart in releasing. You can amplify DNA so it becomes detectable. People have used it to study in a research since in onychomycosis and some labs are beginning to promote it now. The question is do we need a thousand-dollar test to diagnose nail fungus? Well, if you look at the sensitivities [indecipherable] [16:46], it’s not as sensitive as microscopy. You’d think that PCR would be more sensitive, right? A thousand-dollar test? But in fact, it’s not. It may be more specific but in my opinion, it’s still a research test. This is from the Quest Laboratory. This is reference number six. Down the PA, you’ll see the number of cases, the biopsy by PAS, by KOH, the various sensitivities. The PAS is still the gold standard even with the molecular methods. This is a study in the British Journal of Dermatology from 2010. Now, look at the bottom. Fifty two percent were positive for KOH and PCR. Okay? Fifty four percent were positive by PAS alone. In conclusion, PCRs are specific relatively sensitive test for onychomycosis when used in conjunction with the microscopic test of KOH. So I’m not sure what the value is of PCR in onychomycosis. Maybe it’s calming, I don’t know but AETNA doesn’t pay for it yet. I don’t know though that I really want to go into any more fights with insurance companies. So I haven’t adapted PCR because I found that basically PAS and Elysian blue works about 99% of the time. Okay. In conclusion, that’s an expensive research tool and it reveals many bacteria and fungi. It’s a questionable clinical utility. I mentioned a microbiome. A microbiome is the microbes that are on a particular environment including the body are a part of this. This is the second reference I asked you to write down. This is a phenomenal paper. I’ve gone back to this. I probably read this 30 times and it’s worth you reading as well. These microbiomes can include fungi, algae, bacteria, plankton, you name it, amoebae. Some of the wound I’ve seen in Florida, they look green. They look like a swimming pool that’s gone bad. So when I go to Florida, all the wounds look green down there to me. It could include insects, right? This wasn’t maggot therapy. This was maggot therapy that happened on its own. Okay? So that’s part of the microbiome. Now, why am I bringing this up? Here’s Keisha Findley’s data. Here's the point I wanted to make. The feet have a lot of fungi. If we look at the left hand axis, that’s the fungal axis, that’- the one TSI median richness, and it’s a reflection of the diversity or the numbers of different fungi on the feet. Here was the surprising part. The top number one at 80 was the plantar heel, had more than the toe web. Does that surprise you? It did me. The toe web had more than the toenails. On the right horizontal axis, you’ll see that the hypothenar palm, volar forearm, and antecubital fossa have more bacteria.
A little bit of a surprise. Your hands and your feet are two of the dirtiest sides around. Okay? And the feet have far more diverse fungal populations. They did the fungi and the bacteria. Basically, the foot on the left is blue and it’s all over the map. In the right, the moist environment is also kind of bluish purple of the darker color for bacteria, and they’re all over the map. So these are normal feet that were sterilized and then they did scrapings. Okay? So this is what they found. Now, what do we find on the microscope that corresponds to this? We begin talking about IPKs for a minute. The pink callus network corresponds to kind of the blue areas. On the right is the roadmap. The yellow is the hard callus part of a really hard dense part in the middle of the IPK and the dark purple is where the eccrine coils have all these microbes in them. Okay? So they actually live in eccrine ducts. Not only that, the catalyst itself seems to be a partly fungal product. Let’s look at this. There are three basic parts to an IPK. Number one is the hard core in the center. Number two is the infected callus. You can recognize a foot that has a fungal callus on it in part because the dermatoglyphs, the fingerprint-like things look excessively deep. They look too beautiful. They’re too prominent. The reason they’re too prominent is that they’re laden with fungi. They’re a hard part. Those were where the eccrine ducts are. And so, those tend to stand up even more where all the rest of it wears away in between. The third part is the infected eccrine epithelium. My point is you’re not going to be able to fix an IPK if you just scoop out that hard part in the center. I think podiatrists have proven that amply to themselves and maybe don’t need me telling them about it. But maybe number two that there is an infected callus over on the far right where the number two is. Number three, at the bottom, it actually goes down into the eccrine epithelium. That hyperplasia is part of it. So this surgeon actually undertook to take the entire thing out, treated with topical antifungals for six weeks, and thus far hasn’t had a recurrence, surprising. It will be surprising if it lasts because these things are pressure lesions. They’re caused by the pressure on the skin, on the fungally-infected skin. I think what happens is that the IPK on this will be on the left from sort of at a diagonal. On the upper part, you see the fungally-infected eccrine ducts. This is a PAS stain and those are layers of fungi in that hard dense conical part of the IPK. A podiatrist heard my lecture and said, “Well, Steve, I saw this case and I scooped out the IPK, and I listened to what you said, so I went deeper and I shaved off some more. And I found there was this little brown ball underneath the IPK, and so I went down even more.” What he found in there was this granulation tissue. It looked a little bit like a pyogenic granuloma and yet it wasn’t covered with epithelium especially. Here was the surprising part to me. Around those vessels didn’t exactly look normal or healthy. They’re hyperchromatic but there’s this pink kind of globules. I thought that’s kind of a strange appearance. Anyway, I stained it up with everything I had, including this immunostain I’ve been working on for trichophyton. The red on the bottom image is actually a fungal protein which is in that fiber-like material. So there’s evidence of fungi growing within that pyogenic granuloma-like lesion or the granulation. These are black hyphae in the callus, just to show they really were fungi. Okay. So the conclusion is that these hard fungal pathologies are not only due to high pressures but they can also exert high pressures down at the point. Into the dermis, they can actually infect the dermis, and that was a light on moment for me to understand that.
Let’s see. What do we have here? Let’s see. This is a picture off the internet. This is not my own work, but I probably should have cited. I wish I had. The point I wanted to make is that we talk about the normal skin around the ulcer if it’s normal and it’s not. In fact, this young woman is more normal. She bathes everyday. Most of your patients with foot ulcer haven’t bathe in weeks. She has normal skin and normal legs. I’d be willing to bet that but I don’t have a picture of those to show you. Now, why am I showing you this? There’s a second point, and that is that fungi produce dermatitis. There’s an id reaction. There’s an allergy to fungus that it happens in many patients with fungi. And you’ll see the little red band around the fungally-infected heels there, the fissured and callused heels, and it’s pretty common. This leg dermatitis I found is so common it’s nearly 100%. Now, what’s the point, Steve? The point is if that’s a patient who has an ulcer and you’re worried about that ulcer, you’re thinking they may ultimately end up in an amputation. Look how high that dermatitis goes. If that’s fungal, all those flakes and things on there, that patient is at risk of recurring even in the amputation stump, as sometimes happens, they develop these hyperkeratoses. Okay. Bleach bath. It’s so hard to treat these patients with hyperkeratotic lesions and leg dermatitis. I looked this up. This is from the Mayo Clinic. It’s from their website. You can find out about it. Just look up bleach bath and eczema and you’ll find this. A half a cup of regular bleach to a full bath of water and you put them in it for 10 minutes, 15 minutes, like three times a week. And it says, oh, you can use other medications. Well, you’re the doctors, you need to know what medications would you use. In my case, I’d use an antifungal of some kind or an antifungal combined with a steroid. One of the trade names is Lotrisone, okay, to treat both the allergy and the infection. If you can calm it down and quit making it ooze so much, the fungi won’t grow as much. There is one treatment that you may not think about in terms of a bleach bath, and you can apply it to a broad area. Okay. I got two more cases I’d like to show you. One is a squamous cell carcinoma with a fungal callus. This was almost a disaster because it was a delay in diagnosis. It was six months went by before the real diagnosis was made. This was in 2013. And we sliced it and diced it in the laboratory and you can see the layers. These are layers. You can practically make out the fungi in there, the kind of starry sky. Fungi like to layer. They layer, they grow, and they layer. They’re part of that callus. Fungi will grow on top of almost anything. “Well, we treated fungal infection,” he says. “Call me back.” “It’s still red.” And I said, “Well, you need to biopsy deeper.” So he did, not exactly then. There’s the fungi, you can see. Fungi, you can see. But there's the squamous cell carcinoma that’s growing underneath so the oozing from the squamous cell carcinoma gave rise to a fungal callus in this patient. So it’s possible to mistake and treat the fungal part and miss the carcinoma underneath. You think that happens as an isolated thing. This is the typical cells of the squamous cell carcinoma, too much fungi. How about an amelanotic melanoma beneath the fungus on the heel? That sounds kind of tricky, amelanotic, this fungus growing on top. This is from the excision. On the lower part of that, you’ll see that part of it looks like a fungal callus and it is. In the center was where a prior biopsy was made. At the top part, there’s a black suture. So if you need orientation, it’s important when you biopsy or you excise to give the pathologist some indicator. That way I can make a roadmap especially as I slice it. Now, what this means is that the top part near the suture was marked in blue ink and the bottom part was marked in black ink. Then we sliced it and diced to in the laboratory. Okay? Each one of those numbers represents a different slide, a different cassette that we made into a slide.
Here’s the melanoma in blue. You can see the volar skin and the cornified layer. That’s a
10-millimeter scale bar there. The melanoma measured almost millimeters in thickness. This melanoma grew for a while before even being diagnosed. This is a MART-I stain, red staining melanocytes. These are the pagetoid cells and pagetoid pattern. The melanocytes grow in a buckshot pattern. So let me show you the original punch biopsy which was inadequate but better than nothing. Part of the reason it was inadequate was that the podiatrist didn’t label the specimen. This is a no-no. Label every specimen before it gets away. Punch biopsy. Here’s a two-millimeter thick melanoma and yet we can’t even demonstrate invasion because this only contains the cornified layer and the epidermis. There’s no dermis in this punch biopsy. Buckshot pattern, pagetoid melanocytes and pagetoid pattern. This is the MART-I stain again and melanocytes are in red. Not to label the specimen bottle is a recipe for disaster. And to rely on a single punch biopsy, you should probably consider doing multiple. Okay? I’m going to stop there. There’s plenty more we could talk about. There’s fungal paronychia, commonly gives rise to ulcerations and granulations, which are not unlike that other case that I showed you but I’m going to stop. I think there is a role for noting the fungus in fungal paronychia and in the
so-called granulomas around the toenail. Don’t forget we have a biopsy workshop tomorrow at 10:00 a.m. and I’ll be happy to take your questions then. Thank you for your time.