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Laboratory Diagnosis of Fungal Infections

The Last Course

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Published: September 2011

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In November 2011, Mayo Medical Laboratories will host the Laboratory Diagnosis of Fungal Infections: The Last Course. This education conference introduces basic clinical mycology to those not acquainted with the field. In this presentation, Dr. Roberts, the Course Director for the conference, discusses 2 case studies that illustrate the type of information participants encounter in the course. Information about registering for the program is provided at the end of the presentation.

Presenter: Glenn D. Roberts, PhD

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Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Glenn Roberts, a Professor of Laboratory Medicine and Pathology, and Microbiology as well as a consultant in the Division of Clinical Microbiology at Mayo Clinic in Rochester, Minnesota.

In November 2011, Mayo Medical Laboratories will host the Laboratory Diagnosis of Fungal Infections: The Last Course. This education conference introduces basic clinical mycology to those not acquainted with the field. In this presentation, Dr. Roberts, the Course Director for the conference, discusses 2 case studies that illustrate the type of information participants encounter in the course. Information about registering for the program is provided at the end of this presentation. Thank you, Dr. Roberts.

Laboratory Diagnosis of Fungal Infections: The Last Course

The laboratory diagnosis of Fungal Infections: The Last Course is a conference that we will put on here at Mayo Clinic November 16-18, 2011. It will be given here at Rochester, Minnesota. We have given this course over 26 times that I have counted and this is the last time it will be given. It is a course that is designed to introduce you to basic clinical mycology. It is to use case presentations to illustrate how we work between the clinical staff and the laboratory staff to pass the information along. It is also the course that is there to teach you how to select appropriate specimens, how to culture, how to identify organisms and how to do all of this within a rapid period of time so that relevant clinical information can be given to the clinical staff in as accurate fashion as possible. At the end of the presentation we will give you some information regarding the registration.

Laboratory Diagnosis of Fungal Infections

I think we need to remind ourselves sometimes of exactly why it is we work in the clinical laboratory and in the case of microbiology, we know that the laboratory provides the final diagnosis or the definitive diagnosis of infectious diseases, primarily by culture most of the time or by molecular means and we work together with the clinician to allow the information to be used to treat the patient so that patient can recover from illness thoroughly and faster than if we didn’t work together so whether we really know it or not it is a team approach to make the diagnosis and treating a patient. That is one of the strong points that we will emphasize throughout this course. It is important to remind ourselves about why we do what we do. And we hopefully are working in the clinical laboratory to benefit the patient. And case studies will reinforce this point as we go throughout this presentation. We have 2 cases that will illustrate exactly what I have just mentioned, how the laboratory contributes to patient care.

Case Study #1

The first case study is that of a 24-year-old male forestry student, actually from Rochester, Minnesota, who was in school at Washington State. He developed an infection in his left third finger that drained some serous fluid and shortly thereafter he noticed he developed some painful hyperpigmented nodules that went up the dorsal left arm.

And on examination, he had two nonfluctant masses that were palpable along that same line and then noticed that there were two pustules present over the left biceps muscle.

There was no axillary lymphadenopathy at that time at all and he was seen by a physician in Washington and the diagnosis of staphylococcal cellulitus was made and he was treated with some antibacterial antibiotic for staph infection and the lesions did not resolve.

He came back to Rochester, Minnesota for his sister’s wedding and went directly from the airport to St. Mary's Emergency Department. He was seen there by one of the staff and after taking a thorough history from him, he mentioned that he had a history of handling sphagnum moss because he was a forestry student. Well, the consultant that saw him in the emergency room knew there was an ongoing outbreak of sporotrichosis that was occurring throughout the country as a result of contaminated sphagnum moss from Wisconsin and so he suspected this but was not absolutely sure.

But it looked pretty certain that is what he had. They aspirated the nodule and the exudate was sent to Microbiology for culture and he placed the patient on itraconazole therapy and let him go back home after the wedding with follow up.

Organism Recovered After 3 Days Incubation

Well, after three days, this is what the culture plate looked like from culturing the exudate from one of those nodules and there is a lot of organism there. There is almost some stellite or starlike colonies on this blood containing medium, so it grew very quickly within three days.

Microscopic Examination: Transparent Tape Preparation

This is transparent scotch tape preparation we use for looking at microscopic morphology of molds. You will notice by the arrow, there is an organism that shows very small hyphae. At the tip of the arrow, you see it looks like a flowerette at the tip of a long stalk. This is the first glance at the culture.

As the culture got just a little bit older it began to turn dark and as it did, the microscopic morphology shows you why. There are a number of canidia or spores that are being produced along the sides of the hyphae not in a flowerette arrangement but along the sides of the hyphae. You will notice that they are turning black. This is because of the melanin pigment that is produced by this organism. You can see a flowerette on one on the left hand side on the top of this illustration.

Microscopic Examination: Culture

Well, we took some of this mold culture, put it on a blood containing medium, put it at 37° C, let it incubate over night, came in the next morning, took some material out of the culture tube, looked at it and here is what we saw. These are small, elongated budding yeast cells. They look almost like cigars, some of them do and some of them are still oval and some of them are round because they haven’t totally converted to the cigar body-type form.

Microscopic Examination: Biopsy

This would be what it would look like in a biopsy. It is difficult to see the organism in a biopsy from patients but this is an instance where it was seen. You can see this is stained with Gomori methanamine-silver that is used in pathology for staining fungi and these show the very same thing. These elongated budding yeast cells that we define as cigar bodies. And this is characteristic of sporothrix schenckii.

This happens to be a PAS stained slide showing the very same thing and I think you can see the cigar bodies even more clearly on this particular slide. So the organism was identified as sporothrix schenckii. The diagnosis was correct. The laboratory diagnosis didn’t require any more than probably five or six days.

Case Study #1

The clinician was called and the patient was told he was on the correct therapy and to just to be followed up. While he was away, an antibody titer came back that was significant for positive result for sporothrix antibody. We know the cultures became positive after three days. And a follow-up telephone conversation with the person’s mother three weeks later, found that the patient’s lesions were slowly resolving and he was getting better. And he had not been seen again for follow up and remains well and the lesions have totally resolved. So that is the case for sporothichosis. It gives you an idea, that the laboratory may confirm diagnosis but the clinician made the diagnosis initially based on a thorough medical history. So it took the laboratory and the clinician to get the diagnosis absolutely defined.

Case Study #2

Well, this is a case study number two. This is an unusual case of a 77-year-old lady who had a porcine valve implanted in September of 2005. This is a valve from a pig source that is implanted and they seem to last for a long time and are very successful implants. She also during that same time had a coronary artery bypass surgery and this is a lot of surgery for a 77-year-old lady. She did well. She had no problems and no evidence of infection and she went home and did fine.

Well, about a year later, she returns. She developed shortness of breath and came to see her physician. She had an ECHO cardiogram which was able to look at the heart valves and tissue and revealed that she had stenosis or occlusion of the valve; this was thought to be responsible for the shortness of breath that she was having. So they decided they would take her to the operating room and they saw something on the ECHO cardiogram that looked like a blood clot in the valve and they said, “We will go in and we will remove this porcine valve and replace it.” And that was in April of 2006.

This is the porcine valve that was removed. This is the top of the valve and if you notice, you will see all of the white material on the inside of the valve that happens to be a vegetation from an organism growing on that valve. This is not a blood clot at all.

If you look at the next slide, you will see this is the underside of the valve and you can see that vegetation occluding the valve. If you look up through there, you will see that not much blood is going to come down there through the valve because the vegetation is blocking the blood flow. So this is the reason that she had the major problem.

Pathology of Vegetation

This is a cross section stain of Gomori methanamine-silver and you can see the arrows there, there is some fibrin around the outside and the black area happens to be all of the fungal area. And then the green on the bottom where it says valve tissue, you can see is a cross section of the valve and on the inside of that valve is the darkened area where that vegetation happened to be.

Methenamine-Silver Stain of Vegetation

Well, this is a methanamine-silver stain of the vegetation and I was approached by a cardiac pathologist and a resident with a slide and they said, “Can you tell us what this is?” So I looked at it and I said, “I don’t know what it is. It is a fungus, but I can’t tell you what it is just by looking at this.” But basically what we saw here were hyphae, swollen round cells in there and they were all methanamine-silver positive which indicated it was truly fungal.

Case Study #2

So we knew the patient had a diagnosis of fungal endocarditis. We saw the vegetation, we saw the hyphae in there and that was the diagnosis. And that was as far as we could go hoping that the culture that had been taken would grow and give us some information.


Well, as it turns out, the cultures were done and the organism was recovered after a few days.

I asked the laboratory if they knew what it might be and they said it looked like Acremonium. And I asked if I could look at the slides so this is the example of what I saw and what they saw that in some ways does resemble Acremonium. You see kind of a shortened stalk which is the conidiophore with what looks like cluster of conidia at the tip which is pretty much looks like Acremonium.

Well, I looked at some further areas of the slide and noticed that there were some conidiophores that didn’t look like Acremonium and they weren’t produced in the same way. So you have to look at the entire slide when you try to make an identification. That’s one important thing to remember about all mycology slides is to look at the whole slide and get a consensus of what you see. Well, in this particular slide, I noticed that the conidia were elongated and you don’t see that with Acremonium. And there were none of those in clusters like I saw in that part of the slide over there.

The next image has the enlarged view of what we just saw and you can see the elongated conidia but you don’t see them being produced in clusters. Well, I had no idea what this organism was.

I started looking at it and there are times when you look at something and you think to yourself, I think this looks like something but I am not sure of that but maybe I will guess that is what it is. I looked at it and I thought this looks a little bit like some kind of an aspergillus in a way or something to do with aspergillus. Well, I didn’t think it was aspergillus, but I remembered looking up some things in a book which is what we do some times.

And I ran across the name of organism that had aspergillus in the name and it was called Hormographiella aspergillata. I had no idea what this organism was. So I looked it up in the text book that we have with some nice photographs and it actually matched what I was looking at. We confirmed this by nucleic acid sequencing.

And I told them that it was Hormographiella aspergillata knowing fully well they would just gasp and ask me, “What in the world is it?” Well, I said it is an anamorph or Coprinus cinereus. And I got kind of a pause from the clinicians, from the pathologist, he said, “What’s that?” Well, here is what it is.

Coprinus cinereus

These are mushrooms that grow commonly in your backyard. They are called inky caps. They are white, turned gray and in time turn black and fall over in the ground and they are jet black.

The next one shows you the inky caps that are started to get kind of frayed at the top and they would eventually fall over. And the spores are underneath the cap that you see on the top of that mushroom. That is the spore that entered this lady’s heart and grew in the mold form and formed hypae on her valve and caused endocarditis. Coprinus cinereus is a very common organism to see. And I looked in the literature to see if there were any cases of this and there have been a number of cases of Hormographiella aspergillata that have caused endocarditis and no one really knows how they occurred or anything and the thought is that perhaps the spore fell into the operating field while the surgery was going on and it could have been on the shoes of the person. If could have been anywhere. It could have been in the air handling system, whatever it happened to be.

Case Study #2

But this lady was treated with Voriconazole and Amphotericin B and was sent home and we heard later that she had problems again and I don’t know the follow-up for sure on this lady but this is an unusual case that came along and you know it is something that just shows you that you can see almost anything when you work in the clinical laboratory.