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Introduction to Clinical Mycology

Part 1

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Published: November 2011

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This is Part 1 of a 4-part introduction to clinical mycology, including the culture and identification of organisms encountered in the clinical practice.

Presenter: Glenn D. Roberts, PhD

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Introduction

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 presenterfor 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. Dr. Roberts provides a 4-part introduction to clinical mycology, including culture and identification of organisms encountered in the clinical practice. This is Part 1 in the series. Thank you, Dr. Roberts.

Thank you, Sharon, for that introduction.

Introduction to Clinical Mycology

Introduction to Clinical Mycology. This is the first in a series of four presentations on Clinical Mycology. Part 1 discusses what the clinical laboratory does to make the diagnosis of a fungal infection, discusses the classification of fungal infections and fungi related to them, and also discusses the general features of the fungi, their importance and basic morphologic features of the molds.

Laboratory Diagnosis of Fungal Infections

I think we periodically need to think of why we are working in the field that we are in and that is to support patient care. A lot of activities go on behind the scenes that we are not aware of for the most part. One is that a patient with some symptoms of something, whether it is a fungal infection or something else, and for our purposes we will say it is a fungal infection, will come in with signs and symptoms of infection. The clinician has to decide what he or she suspects might be the problem, ask pertinent questions like a history of travel and then investigate the immune status of the person because fungal infections take advantage of people who are immunosupressed. They then have to decide if they are going to culture something and they select the appropriate specimen for that. They have to order a direct microscopic examination if they think it is appropriate so that we can make a rapid diagnosis in some instances. And then sometimes it even goes a lot further than that where they involve an invasive procedure like bronchoscopy or a fine needle biopsy or something like that.

Other things that can be done are serologic tests for antigen, antibody and some metabolites of some of these fungi that assist in making a diagnosis prior to maybe doing an invasive procedure. If a culture is ordered and something grows, they will perhaps order an antifungal susceptibility test and then the laboratory sometimes is involved in interpretation of those results whether it is a serologic test or whether it is an antifungal susceptibility test. And then the most important thing, and we will reemphasize this again in just a moment, is communication with the physician. The laboratory must communicate with the physician and not be afraid to call the clinician up and give him the results of patient care for about the patient result so that we don’t delay therapy for these patients. Often times we also have to go back and communicate with others in the laboratory to make sure we have all of this information straight.

Clinical Mycology: Laboratory Involvement

What is the laboratory involved with? Often times we are asked about the collection of specimens by the clinician and we need to be aware of what those rules are. We need to be proficient at the direct microscopic examination of specimens. We know about culturing. We identify about yeast and molds in the laboratory. We either refer out or do antifungal susceptibility tests and fungal serologic testing. We are involved in reporting results and again here we are communicating with physicians again and this is a key part of what we do: communication with our colleagues and with our physicians.

Fungal Infections: General Features

What are fungal infections? What are some general features? First of all, these fungi may affect normal and immunocompromised patients. Many of them are chronic in duration. They last for years like ringworm or some things like that but in the immunocompromised patient, these can mean acute infections and progress very quickly to death. They are not transmitted from patient to patient. They are not susceptible to usual antimicrobial agents that we know about. However, they are susceptible to several groups of antifungal drugs called polyenes, azoles, and some others and the therapy for treating fungal infections is not easy for the patient, very difficult. The side effects are substantial and we need to make sure that we make an accurate diagnosis before these patients are placed on therapy. And again these infections may range from being just chronic type things to some that are acute and progress to rapid death. We need to be thinking about rapid reporting of results as we do our work.

Fungal Infections: Clinical Classification

We try to classify fungal infections into groupings and it is hard to do because it is an artificial thing. But we divide them up into superficial infections, subcutaneous, systemic and opportunistic infections.

Fungal Infections: Classification

The superficial infections you can remember by just thinking about the fact they involve the keratinized tissue: the hair, the skin, and the nails. The dermatophytes are good examples of those and some others. Subcutaneous infections involve the skin and contiguous subcutaneous tissues that include the lymphatic vessels and most of the infections that are involved in the subcutaneous tissues are acquired by trauma to a sight usually an extremity. Systemic infections can involve any organ system and generally they are caused by certain groups of fungi that have specific geographic niche out of the environment and they live in certain locales within the world that we know where those are and so the clinician asks appropriate questions. The organisms that are involved in this, actually, can infect any organ system and actually do that. The patients may be totally asymptomatic and unaware of this, or they may be very, very ill if they happen to be immunocompromised. And then the last group is a very important one, opportunistic fungal infection. These are infections caused by fungi that normally don’t cause disease in humans. They are environmental floras and if we have a break down of our immune system and we happen to be in the wrong place at the wrong time, then we acquire these infections and they progress very quickly, particularly in transplant patients. So we need to be aware of what organisms cause these infections.

Superficial Infections: Examples

The hardest part of Mycology is the terminology. I am just going to go through these very quickly. A superficial infection, dermatophytes involved primarily causing these infections. Another organism, however, that does cause infection, that causes Tinea versicolor, is Malassezia furfur.

Fungi Related to Superficial Infections

The dermatophytes belong to three groups Trichophyton, Microsporum and Epidermophyton.

Subcutaneous Infections: Examples

Subcutaneous infections: Sporotrichosis, Mycetoma, Chromoblastomycosis and Phaeohyphomycosis...

Fungi Related to Subcutaneous Infections

...are caused by Sporothrix schenckii, Pseudallescheria boydii, Phialaphora verrucosa and Cladophialophora carrionii, respectively. You can see the names get a little complicated sometimes, but they are not that hard once you learn them.

Systemic Infections: Examples

Terms of Systemic Infections: Histoplasmosis, Blastomycosis, Coccidioidmycosis and Paracoccidioidomycosis are the infections that are grouped into this particular heading. There actually is another one which is not listed here. I will mention it to you in a minute.

Fungi Related to Systemic Infections

The fungi that involved and causing those infections are Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis and one called Penicillium marneffei that causes Penicilliosis in places like Cambodia, Vietnam and Thailand. We don’t see it in this country very often but we do see it.

Opportunistic Infections: Examples

Opportunistic Infections: Examples are Candidiasis, Cryptococcosis, Aspergillosis, Zygomycosis and a whole lot more. The list could go on and on because we see organisms, that we have never thought would cause disease, cause disease in patients here and cause substantial infection, and often that leads to death.

Fungi Related to Opportunistic Infections

Some of the organisms related to that Candida albicans, Cryptococcus neoformans, Aspergillus, Rhizopus, Mucor, and a number of the other Mucorales, Fusarium, Acremonium, Trichosporon, and many others.

Fungi: General Features

Let’s talk about the fungi in general for just a moment. They are Eukaryotic cells. They have a cell wall. They have a cell membrane. The cell wall is made up of a poly-n-acetylgucosamine. The cell membrane contains ergosterol in contrast to mammalian cells which contain cholesterol and the reason that I am mentioning this is that they are certain there are anti-fungal agents that actually bind to the cell membrane that has ergosterol in it. The Hetertrophic break down into organic matter to get energy sources for themselves, decaying matter.

They are not susceptible again to the usual antimicrobials. They reproduce sexually or asexually. Most of the time in the laboratory we see the result of asexual reproduction with spores. They may be monomorphic which means they have one form, a yeast or a mold form. They may be dimorphic in that they may have a yeast and a mold form or another type of form. And then some of them actually produce more than two or three forms and they are called polymorphic. So reproduction by these is usually by asexual spores and that is what we see all of the time when we look at these organisms underneath a microscope.

Examples of Fungi

Examples of the fungi that you would be familiar with include yeast and molds, which we will talk about, mushrooms, puffballs, shelf fungi, the morels, which I am sure some of you have collected, plant pathogens, and animal pathogens. And some of these are very substantial things that are involved in causing disease of plants and animals.

Importance of Fungi

One of the things that the fungi have a very important role in doing is keeping the carbon cycle going. They break down organic matter and decompose it into carbon. They cause disease in trees for example like Dutch elm disease, which is a devastating thing for a population of trees and we lose them all whenever they get Dutch Elm. Crop diseases in wheat, corn, barley: corn smut is one of those examples of that and we actually have seen patients who have had disease caused by corn smut. Wood and timber degradation: if you look at wood, you will see that there are some purple to blue materials sometimes on there where the wood gets broken down by these fungi and it damages it. Another very important thing is these fungi can cause spoilage of food, particularly grain in developing countries, where grain is the primary staple; the grain will get infected with a fungus and will produce a toxin called aflatoxin, if it happens to be infected by aspergillus, and actually cause substantial disease in these patients and often times liver cancer. And where would we be without the fungi in the brewing and baking industry? We would be nowhere because the yeasts are the things that causes fermentation so they play a major role in that.

What Are Fungi?

Well, what are these fungi in general? We mentioned Hetetrophic. They break down organic matter. They have chitin in the walls. They have ergosterol in the cell membrane. They have an organized nucleus. They reproduce by asexual spores again, most often. And if it happened to be a mold, they produce hyphae which you can see on this left hand photograph, they are nothing more than filaments that look like garden hoses underneath the microscope. And if it happens to be a yeast, they produce single cells that reproduce by budding and often times a few more structures in there.

Fungi: Morphology of Molds

So just to give you an idea of some of the terms, some more language if you will, to learn, we have terms that we use to help us describe what we see under the microscope to make it a little easier for others to understand. If you look at a mold on a culture plate, you will see a colony there, a fluffy colony. In our laboratory, we call them fuzzies. If you look underneath a microscope, you will find they are made up of these hyphae. These are filaments that have parallel walls; they look like small garden hoses. The collective name for the colony is mycelium and I don’t think many people use the term mycelium anymore but it is there for you to see. The hyphae, these garden hose type structures, may be divided up into compartments by some structures called septae. And so we talk about septate hyphae and we talk about nonseptate hyphae which don’t have any of these division, these compartments in them and sometimes we just refer to nonseptate hyphae as pauci-septate because certainly, these fungi that are thought to be nonseptate have a few of these septations and I will show you shortly here. Spores. Most of the fungi that we deal with produce spores that are called Conidia. They are produced on specialized structures whether it is a short or a long stalk or a really elaborate structure called a Conidia form. Some of the spores are small. Some of them are large. So we define them by being macro Conidia or micro Conidia. Sometimes these spores on a hyphae may be pigmented or they may not be and that leads us to different groups.

General Terms Used in Clinical Mycology

Well, just some more terms, the hyphae we talked about, these are the structures that make the mold colony. The septae are the cross walls that break down the hyphae into compartments. Nonseptate means that they are lacking septae like we said a while ago and hyaline is another term we haven’t talked about yet and it is the hyphae that may be non pigmented. In other words, when you look at them underneath the microscope without any stains at all, they will be clear. If you stain them with a dye like we do in the laboratory, lactophenol aniline blue, you can see they will turn blue with the dye but they still don’t have any definable pigment to them. So they are called hyaline.

Then we have other fungi that are dematiaceous. These fungi are the ones that contain a dark pigment either a chestnut brown pigment or a very black. They belong to a whole group of different fungi. Sometimes, some of which are very difficult to identify. There are a lot of structures that we have to deal with. Some of them are just a nonspecific kind of things that don’t tell you anything about the organism. One of those is called a chlamydoconidium. It is a big round spore found right within the hyphal strand or on the end of the hyphal strand and its primary function is to protect the organism so if it becomes under adverse conditions, it will round up and form these spores so it can survive. We see it nonspecifically in lots of organisms. And then the Conidia. The conidia are those asexual spores we mentioned produced by molds that have septae. And the reason I mention with septae is that there are a whole group of fungi that do not produce asexual spores and have septate hyphae. They happen to have nonseptate hyphae and the spores are produced in a different way and we will talk about that.

Typical Mold Colonies

This is a typical mold colony. Colonies are things that we see that we have to work from, to make them out in the laboratory to be able to identify the organism; however, looking at a culture plate sometimes gives you a little information but many times doesn’t help you at all. The colonies can be multi colored; they can be white, off white, brown, tan, green, yellow, pink, brown, or black. They can be all sorts of colors and you may get a ball park idea of what kind of organism is but it is not going to tell you specifically what it is.

Glabrous Mold Colonies

This is another one where you can see the colonies are smooth adherent to the agar. They have different morphologic forms when you start looking at these colonies they look either very fluffy or very adherent to the agar and other appearances as well. So if you look at molds you will be surprised as it is a variety of things that you see. With experience you can tell sometimes which of the ones are going to be important.

Hyphae

Now this is an example of the hyphae. This is an electron micrograph where you can see these garden hose structures I told you about and there is a septum. That cross wall that divides that organism up and there would be another septum probably down below it there. It allows that organism to be compartmentalized so that if it happens to break apart, then each compartment can then grow and start a new colony. It's a mode of survival for these organisms.

Septate Hyphae

This is what it looks like under the microscope. When you really look at it you can see the septum there to the right of the arrow that divides it up and they are not usually so hard to see as this one is but sometimes they are.

Dematiaceous Septate Hyphae

This is an organism that is dematiaceous. In other words, it is pigmented. One of the things that you do in the laboratory is recognize these molds is by looking at the spores produced, you look at the spores to recognize the size and the shape of the spores and so on. You can look at this one and say, "Ah! I know what this is. This is Bipolaris!" because of the features you will be familiar with as we go through with a few other sessions. The arrow here shows those septae and you can see that this hyphal strand at the bottom left hand corner is divided up into compartments so that each one of those is a separate unit and can survive and grow and produce a new colony.

Nonseptate Hyphae

The image here is one of nonseptate hyphae. These are very large. They are not compartmentalized at all. When the laboratory processes something that is suspected to have one of these organisms that have nonseptate hyphae, if you grind it up like people do in the laboratory, that hyphal strand will pop open, all of the cytoplasm will leak out and it will die. So, what you have to do is be very careful. You end up having to not grind the culture up, instead you have to cut pieces of whatever it is, tissue or whatever it is, so that you can allow survival for this organism. These hyphae are nonseptate and occasionally they will have septations and we call them pauciseptate.

Introduction to Clinical Mycology

This completes part 1, the Introduction of Clinical Mycology. Future presentation will be part 2, reviews the basic structures of molds and yeasts and presents a brief introduction to the direct microscopic examination of clinical specimens and also media that are useful for culture. Part 3 presents specific information on the culturing and incubation of cultures for the optimal recovery of fungi and part 4 presents methods for identification of fungi, primarily molds, and some helpful hints for working within the clinical laboratory.

 


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