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Published: March 2012Print Record of Viewing
An introduction to the field of clinical mycology. Dr. Roberts reviews the basics of mycology, including culture and identification of organisms encountered in the clinical practice. This is Part 4 in the series of 4.
Presenter: Glenn D. Roberts, PhD
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. Dr. Roberts provides a 4-part introduction to clinical mycology, including culture and identification of organisms encountered in the clinical practice. This is Part 4 in the series. Thank you, Dr. Roberts.
Thank you, Sharon, for that introduction.
This is the introduction to clinical mycology, the final presentation in a series of 4 in clinical mycology. Part 1 deals with the diagnosis, classification, and general features of the fungi and fungal infections. Part 2 dealt with the basic structures of molds and yeasts and a brief introduction to culturing. Part 3 presents specific information on the culturing and incubation of cultures for the optimal recovery. In part 4, we will present methods for identification of fungi, primarily molds, and some helpful hints for working in the clinical laboratory, which we will cover today.
We are going to review some terms again that we have covered in previous presentations that still have application to what we are going to cover. A sporangium is a sac-like structure that produces spores found in molds particular in Zygomycetes. And these have few or no septae in the hyphae. Blastoconidia are budding yeast cells found in yeast. Pseudohyphae are chains of blastoconidia that have elongated and remained attached to each other like links of sausages. Arthroconidia are rectangular cells that are formed within hyphae. Spherules are round, sac-like structures found in the tissue of patients who have coccidioidomycosis that is caused by the organism Coccidioides immitis. These spherules contain numerous spores called endospores. And then, dichotomous branching is nothing more than acute angle branching at 45 degree angles and we happen to use this particularly when we are looking at descriptions for hyphae that are found in tissues.
This happens to be a typical mold colony. It’s a fluffy colony. Sometimes they are powdery, sometimes they are fluffy, sometimes they are yeast-like a little bit. But, they nevertheless have what are called aerial hyphae. They grow upward in the culture dish and they have different colors. There are many, many, colors of molds.
Identification of molds is made up of several criteria. When we sit down to try to identify a mold, one of the things that we first start off with is to see if it is an organism that has hyphae that are septate or nonseptate or pauciseptate (having few septations). The next thing we look for, is this organism pigmented or not. If it is pigmented, it is called dematiaceous, if it is not, it is called hyaline. This happens to be pigment in the hyphae or the spores.
The other thing we look for is to see if the organism is monomorphic or dimorphic, not always easy to tell at first glance. Sometimes, you will see, you have to work at trying to get the second form to be there. But you have criteria that you look at, for example, the spores, the type of spores that are produced, and the arrangement of those spores, gives you an idea of what the organism might be in terms of the dimorphic fungus too. And then the growth rate – the growth rate is something that we seem to rely on probably more than we should. The growth rate in text books says, for example, the dimorphic molds like Histoplasma or Blastomyces are slow growing and may take up to 4 to 6 weeks and so on. We actually have seen things like Blastomyces grow up in a shorter period of time, in just 2 days. Well, the reason for that is the growth rate is influenced by how much organism happens to be in the initial specimen that is cultured. If there are a few organisms in the specimen, the culture is going to grow very slowly; you won’t see it for several days or several weeks. If there are lots of cells in there in the original inoculant, the organism will grow very quickly and you would be able to make an identification much earlier. So the growth rate is not an absolute criterion for making an identification, but it is helpful.
In terms of how to look at a mold and try to make an identification, there are several ways to do that. One is to make what is called a simple wet mount, where you open up the culture dish and you take a wire that is bent at a right angle, it is sterile wire that you flamed, and you take a small part of the supporting agar along with the culture and you place it on the slide that contains a dye that we use for staining fungi.
Lactophenol cotton blue or lactophenol aniline blue are 2 dyes that are used commonly. Lactofuchsin is another dye that is not used very often. It stains the organisms red. Lactophenol cotton blue and aniline blue will stain it blue.
So what you do is you take a little bit of the supporting agar along with the organism and you put it on the slide like you see here. It contains a drop of the dye.
And then, you place a cover slip on that. Then you can take something like a pencil eraser and push down on that and allow that to disperse the supporting agar and the organism on there so that you can see it better. There is one thing you will learn very quickly and that is that when you put too much organism on the slide you put the coverslip on there and you push down with the pencil eraser, that piece of agar will shoot across the table. What you have done is contaminated your work site and you have lost the specimen that you were looking for. So pick a moderate amount before you put it underneath that coverslip.
Then you look at it underneath the microscope and, if you can recognize the spores, you can make an identification. If you don’t recognize the spores or how they are arranged, then you have to figure out what the organism is if you are going to base the identification on a morphologic features. And, in most cases, this means trying to go to a key or look in through some books until you find the photograph that matches what you are going to try and identify and which you have in your culture. Today, with nucleic acid sequencing, it is much easier to identify organisms without having to spend all of the time looking at the morphologic features, but it is a costly process.
We use what is called a Scotch tape preparation for identifying cultures in the laboratory more often than a wet mount. The reason for that is that it allows the spores to remain attached the way they are growing in the culture, so you can see how they are produced this way. Basically, what you do is you take a piece of transparent tape. You fold it over, the sticky side down. You touch it to the surface of the colony, preferably, in between the center and the periphery, and once you have done that you push down on there and you stretch it out and put it on a slide that contains a drop of lactophenol aniline blue like this. If you use the right kind of tape, you can actually look right down through the tape and be able to look at the morphologic features and you will see the spores produced just as they were growing in the culture like you see this.
This happens to be an organism called Helminthosporium, which we don’t see very often, but it is kind of pretty and it shows you the spores are still attached just the way they were produced. So that is a way to rapidly look at some of these molds and be able to help make an identification.
Another way to do this is when you have difficulty getting it to sporulate, and you can’t tell how those spores are produced, and you want to be able to tell that before you put the final name on it, that is to make a microslide culture. Basically, you choose a medium that you think it will grow best on and start off by taking a plate of just water agar as your incubation chamber. You take that plate, set it down on the table that you are going to be working on, and you take a sterile test tube and you cut out plugs of agar on the medium you are going to use to try to grow the organism.
You take the plug, put it on a sterile slide on top of the sterile water agar...
...and then you inoculate the 4 quadrants with an organism.
Once that is done, you take a coverslip and place that on, and you can do this on either end. This gives you 2 opportunities to be able to look at a coverslip that contains an organism growing underneath it. So basically, you inoculate, you put a coverslip on each plug, let it incubate.
And then, when it is time, you remove 1 of the coverslips from 1 end of the slide, put it on top of a drop of lactophenol aniline blue and look at it underneath the microscope. What you do not want to do, is to take the slide out of the incubation chamber, that water agar plate, and take that out and look at it underneath a microscope. Because, if you happen to have a pathogen growing there and you take that open system out and look at it like that, you are apt to get yourself infected and the laboratory infected and it has happened.
This is a cheap way to make a microslide culture. Basically, take a piece of filter paper, put in the bottom of a sterile Petri dish, take an applicator stick, break it in 2, put the slide with the 2 plugs on top of it with a coverslip on there, put a little water in there and let it incubate. And, it works fine. You can then take off the coverslip, put it on top of the slide with the dye and be able to look at the morphology right through that slide.
Well, in terms of what you might find in the laboratory, this is just a partial list of some of the things that we see in the clinical laboratory. Probably the most common things: Aspergillus fumigatus, Mucor, Rhizopus, Penicillium, Fusarium, Scopulariopsis, Paecilomyces, Acremonium, and you will notice that one of the things that is a problem in mycology is the language and the pronunciation of some of these words. And my philosophy is that as long as somebody understands what you are talking about, and if you happen to mispronounce the name, that is probably alright just as long as you know what you are talking about.
Others, Bipolaris, Curvularia, Alternaria, Cladosporium, Epicoccum, Chaetomium, Graphium, and Pseudallescheria boydii. These are all dematiaceous fungi. The previous ones are all hyaline fungi. They have no pigment.
Some more, Scedosporium apiospermum, Aureobasidium and then the dimorphic fungi, Histoplasma, Blastomyces, Coccidioides, Sporothrix, Paracoccidioides, and Penicillium marneffei. So we see a lot of organisms, and there are a lot more than this. There are over 500,000 different fungi, and thank goodness we don’t see them all.
Now, in terms of what we have available in the clinical laboratory to help make a rapid diagnosis, is the direct microscopic examination of clinical specimens, and that is an old tool, as we talked about. It still is rapid, and it is still very helpful, but it is not 100% sensitive. We have an antigen test for cryptococcal antigen so that we can detect that in body fluids and make a rapid diagnosis in terms of cryptococcal meningitis or disseminated diseases in probably two thirds of the patients. So that is something that is rapid. We still don’t have a lot of real rapid tools yet in mycology. We have nucleic acid probes for identification of dimorphic fungi. We have Real-time PCR identification for things like Histoplasma and Coccidioides that are very useful. And we have nucleic acid sequencing that we can use for identifying organisms, and now we have mass spectroscopy, which is a new tool that is being introduced right now for identifying organisms. And databases are being built for fungi, for dermatophytes, for yeasts, for molds, for trying to identify these and the costs for consumables are minimal and the time for identification is something like 5 minutes. It is a very useful tool. It is going to be out for a lot of promise and that is the way things are probably going to go at least at this time. And then we have PNA-FISH probes. They can be used for detecting fungi in blood cultures and they are very helpful for being able to distinguish one particular type of Candida from another, so that therapy can be started right away. Now mass spectroscopy is being used already in the bacteriology laboratory for identifying bacteria directly from blood cultures. And my guess is what is called MALDI-TOF will be used in the same way for identifying the yeast in fungal blood cultures. And fungi and blood are a very serious event when they get Fungemia, so these PNA-FISH probes and MALDI-TOF are going to play a big role in helping to minimize the death rate due to sepsis due to the some of the yeasts.
Well, let’s talk a little bit about mycology and where it is done and some things that we can do to make it better. Where do you do mycology in the clinical lab? Well, clinical microbiology laboratories, some of the larger ones, certainly have an area where mycology is done. It is not usually a very large area. Reference laboratories generally do a lot of the work for smaller laboratories. Where cultures are sent, specimens are sent for culture because of their experience. And there are a small number of mycology laboratories, for example, the one here at Mayo Clinic where they have a specific laboratory and a dedicated staff of about 35 people who work strictly in the mycology and mycobacteriology combined.
So we find ourselves in a different environment these days. We find that we have working pressures that are different from ever before, we have to work with fewer people for the most part because of cost containment, we want shortened turnaround times. Our staff insists that we do the work and try to get rapid results, there with fewer people, we don’t use as many expendables, and so on, as we used to. So we try to reduce the expense in the laboratory. We are supposed to use rapid tests to get rapid turnaround times. We are supposed to get accurate results, and you combine all of this, it makes it difficult. But people are able to do that by just working together.
In terms of patient care, I think in the laboratory we often have a tendency to forget about this. We are busy doing our own work and we don’t think much about the patient. The patient is not just a number. I think sometimes you really ought to sit there and think about it and say, “What if I was the patient who had this specimen submitted and it was a bronchoscopy, what would happen then?” Well, you be very uncomfortable. So you need to put yourself in the circumstances of the patient sometimes to remind yourself of why we are doing the work. You also need to know that the work needs to be done in a prompt manner. Don’t put it off, because there is a clinician waiting for that result for that patient so that they can start the right therapy or make the right diagnosis. The other thing is that when you are working in a clinical laboratory and you don’t know the answer to something, just go find out. Ask anywhere you can to get the answer. It doesn’t make a difference whether it is someone in-house or if you have to pick up the phone and call them, you pick up the phone and you say, “I need some help with this.” You can call me, you can call whoever it happens to be because the needs of the patient come first. That is the philosophy here at Mayo Clinic and we don’t hesitate to talk to each other or to talk to anyone on the outside when we need to, because we know it is the patient that we are dealing with and it is the patient that is important and not our ego.
I think something else that we need to remember and that is that we have rapid ways of detecting things in clinical specimens, for example the Gram stain. We read a Gram stain, and we have mentioned this before, you need to think clinical microbiology because you can find fungi in Gram stains, you can see other things in there as well. When you are reading bacterial cultures, look there and see if you are going to pick up something like a fungus or maybe even another bacterium like Nocardia that the bacteriology lab might look at. When you are reading acid-fast smears, don’t just look for a mycobacteria, look in the background and see if you see a fungus. In terms of histopathology, the pathologists are primarily looking for tissue morphology. They are looking for tumors, looking for infection, looking for a lot of things. They often times don’t have the time to spend looking at a biopsy. And the laboratory can help them in certain circumstances because the organisms that you see in a direct examination of a clinical specimen look the very same as they look in a biopsy, the tissue morphologies, the tissue is stained and the organism looks the same.
There are times you can be a resource to not only the pathology, but to others in the clinical laboratory. And, for example, in the hematology laboratory where they read peripheral blood smears, bone marrow smears, CSF cell counts, they see fungi in those specimens. And you can tell them, your colleagues there. “Hey, I know what things look like in a clinical specimen, and if you have something you see in one of those, give me a call if you think it is a fungus and I will come and help you look at it.” In cytology, looking at pap smears for respiratory track secretions, you can actually see the organisms there and make an identification. Talk to your people in the Cytotechnology laboratory and tell them you can help them. And pathology again, you can tell them that "I would be willing to help you if I can to identify organisms that are found in tissue and body fluid."
So we need to all work together to do the best job that we can. One of the biggest problems is communication. A lot of times you don’t feel like you can pick up the phone and call somebody or take a slide up to see someone. Well, you need to learn to communicate better if you can. Within your own laboratory, you probably don’t have a problem. You can talk to people there without any difficulty at all, but when it comes to talking to somebody in another lab or in a reference laboratory, you have to take the initiative to pick up the phone and call them and say, “I need help. Can I send you a photograph? Can I tell you about this?” or whatever. It doesn’t make any difference who you have to call to get the help, just do it. In terms of calling physicians, I think you need to always feel free to call about patient results because they are interested in taking care of the patient and you are interested in trying to help get them the results that are going to make the patient be treated in the right way and to convalesce to get them home.
Once you learn information in the clinical laboratory, it is one thing to keep it to yourself and do a good job, but it is something else to share it with those around you so that they can do the same. You need to share your information readily. You need to share it within your own laboratory. You need to teach anywhere you can. And if you have the urge, you can volunteer to teach in workshops or regional groups around and try to show them what you have learned and let them pass it along to others. And that way we all learn to help our patients.
Safety is a big item in the hospital setting these days and it is also an item in the laboratory where you need to concentrate. Use common sense when you work. Consider all specimens to be infectious and potentially pathogenic. Consider all fungi to be pathogenic, and you work with anything like that inside of a biological safety cabinet particularly just the molds, so you don’t infect someone within the laboratory. Because you never know in your own clinical laboratory who is immunosuppressed and who is not. And, if you are careless and you end up contaminating the laboratory, someone in there could become ill and, if they are highly immunosuppressed, they could become very sick and there have been patients who have died from a laboratory-acquired infection.
Another thing is that when you are working in the laboratory, you have to consider the endemic organisms, like Coccidioides or Histoplasma, might be one of the things that you are going to see despite the fact you don’t live in an area where it happens to be because patients travel from one area to another and they are apt to come from an area where it is endemic to where you are. Let’s say that you have a person who picked up Histoplasma in Florida and you happen to live in Washington and you don’t happen to see Histoplasma in Washington state. Well, the patient comes back home to Washington, the Histoplasma is in your laboratory, you need to be able to recognize it. In your lab you need to perform a risk assessment to make sure that you are working safely in there. You need to develop a biosafety plan for spills in case something is dropped. And when you are teaching in a teaching hospital you need to make plans for caution for teaching rounds because there have been laboratory-acquired infections that have occurred from teaching rounds and not handling the organisms correctly.
And, just remember, that when you are working with fungal cultures it is best to work inside a biological safety cabinet to prevent organisms from contaminating the laboratory.
And I think one thing you need to remember and that is, that it is up to you. The institution where you work is made up of probably wonderful people, but it is your institution and when you are working in circumstances that are not right, you need to say something. You look at a result from a culture and you say, “This just doesn’t make any sense.” Then, you need to pick up the phone and call someone or call a clinician and say, “Here is what we have, but this just doesn’t seem right.” You are the one that is the “stakeholder” in there and you need to be assertive and work for your institution to do the best job you can for patient care.
So, some guiding principles that I put together that I try to follow as I go is always keep the patient in mind. Think clinical microbiology when you work in the laboratory no matter what area it happens to be in. Utilize all resources you have available to you to help your work. Enhance your communication skills and make sure you feel comfortable talking to people when you need to be. Be a volunteer and help those others who don’t know the material that you know, and work as safely as you can and question the results when things don’t fit.
These are just some common sense things, but are important because in the clinical laboratory, we are there for the patients. You work for the benefit of others and not yourself, you discourage competition and foster collaboration with others to try and do the best job you can. There are no secrets. You share information willingly and you encourage collegiality among all of your colleagues.
Well, in summary, this was the final presentation in this introduction to mycology series. All 4 parts can be accessed online at any time. This current topic is useful as an orientation to clinical mycology. The next series of presentations will provide detailed information regarding the detection and identification of fungi found in clinical specimens.