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Fungal Meningitis

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Published: November 2012

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The outbreak of fungal meningitis has resulted in anxious patients, concerned physicians, and a rush to identify affected patients. With the number of infected patients rising, as well as the number of deaths, it is critical to appropriately identify the causative organism so that treatment can be initiated. This presentation will review the cause and source of the infection, discuss the nature of the fungi involved, and offer guidance on the correct tests to utilize in symptomatic patients.

Presenter: Glenn D. Roberts, PhD

  • Elitza Theel, PhD

    • Assistant Professor of Laboratory Medicine and Pathology, and Microbiology
    • Director of the Infectious Disease Serology Laboratory
    • Consultant in the Division of Clinical Microbiology at Mayo Clinic in Rochester, Minnesota
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    Welcome to Mayo Medial Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Our speakers for this program are 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 and Dr. Elitza Theel an Assistant Professor of Laboratory Medicine and Pathology and Microbiology at Mayo Clinic and Director of the Infectious Disease Serology Laboratory. In this presentation they will cover the history of the recent fungal meningitis outbreak, the current status of infections diagnosed in the United States and the appropriate laboratory testing for diagnosis of this fungal infection.

    Thank you, Heidi, for that introduction. We have nothing to disclose.

    This presentation deals with fungal meningitis, a problem that has appeared in this country recently, and is now increasing in numbers. I think most of you probably have been following in the news and the newspaper or on the internet to see that there are many cases of fungal meningitis that have occurred. What happened to cause this problem to occur? Well, I am going to give you a little bit of background. In 1997, congress passed some legislation that established some compounding pharmacies and the regulations for those pharmacies. And we will talk about a compounding pharmacy in just a moment but, over time, there has been some questionable oversight for these laboratories and the oversight has kind of gotten lost between the state and the federal government and so, consequently, there has not been a whole lot of oversight for these laboratories and so this, I think, is one of the problems of this situation here. The New England Compounding Laboratory in Framingham, Massachusetts prepares some single-dose vials for injection containing methylpresnisolone acetate without preservatives, and notice without preservatives, and this is a key thing.

    These are vials that were prepared. They’re single 1-mL vials used for injection and this is partly the reason for putting it in a compounding pharmacy. The compounding pharmacy prepares compounds that are pretty much custom made for clients and, for example, if someone can’t swallow a capsule, then a compounding pharmacy will make it into a liquid form. And make it easier for those patients, in particular pediatric patients, to be able to take some of these medications, so, they custom make things. Well, they custom made these 1-mL vials of methylpresnisolone. It turns out that now it looks like there is another compound that is made, another steroid that perhaps may be contaminated, but this is the primary one here.

    Well, here’s the problem. This company, the New England Compounding Center, made 17,676 vials of this steroid, in 1 mL amounts without preservatives. As a result, these were shipped all over the country and, it turns out that 14,000 patients were injected with the steroid from these vials beginning May 21 of this year 2012. As of October 17, 2012, 245 patients have developed fungal meningitis and there have been 19 deaths due to this. There also have been 2 patients with joint space infections because these compounded steroids have been used to inject knees, shoulders, hips, and so on. There were 3 lot numbers that we know of that were contaminated. First lot was contaminated with Aspergillus and it’s later been determined that it was contaminated with Exserohilum, kind of an unusual organism and uncommon one for the clinical laboratory to see.

    It turns out there are some more problems that are kind of evolving along the way here. There has been a report that 2 transplant patients who had heart surgery have developed Aspergillus infection after the use of a cardioplegic solution that’s used to stop the heart to prevent damage during a cardiac surgery. And all of these were produced by the same company. It’s now been shown that Aspergillus has been discounted as the cause of infection in 1 of these patients. The FDA is asking people to follow the patients along who have been in any contact with these compounds, and all the centers are being notified that have received all of these compounds, and there are a huge number as you can tell. People have to be notified that have been injected with this steroid. This particular compounding laboratory makes over 2,100 compounds, and now they have found that ophthalmic steroid preparations that are used in ophthalmic surgery may also be contaminated. So this is a big problem, it is an evolving problem, and it is going to continue to evolve until the incubation period is over for this organism.

    You can see on this map where the greatest numbers of infections have occurred, and you can see that Tennessee has the greatest number with 61, followed by Michigan and Indiana. And the cases are localized to pretty much that area, except Virginia, but look there is 1 in Idaho, 1 in Texas, Minnesota has had 7 cases. So, they are kind of distributed here and there throughout the country and the people have been injected with the steroids, particularly in the back, are sitting there waiting for something to happen and it’s a terrible problem for them and a problem for the physicians as well.

    This diagram will show you kind of what happened. If you look at where the needle is on the lower part of the diagram, the steroid was injected into the epidural space and this allows the steroid to relieve some of the inflammation associated with back pain and as I was looking at this, I decided that with 14,000 people injected, probably most of them in the back, we are a nation of bad backs! But, I think this is a situation that has occurred that has never happened before that we know of. So this is a common procedure for people to have whenever they have back pain, even other joint pains as well. So, this is the situation that’s happened.

    And now, how do we make the diagnosis in these patients, because a diagnosis needs to be made as quickly as possible.

    Thank you, Dr. Roberts. The diagnosis of fungal meningitis or joint infection during this outbreak is dependent on both the presence of clinical signs of infection among patients who have received the implicated medication, as well as on the presence of certain laboratory findings.

    With regards to laboratory testing, it is important to remember that the currently recommended laboratory methods for diagnosis do not differ from those routinely performed for the identification of fungal meningitis or joint infection.

    These routine studies should include obtaining both aerobic and anaerobic bacterial blood cultures in addition to drawing fungal blood cultures.

    Currently, the CDC also recommends that CSF be collected from suspected cases of meningitis and synovial fluid be obtained from suspected cases of joint infection and be submitted to the laboratory for routine testing. These routine studies should include a Gram stain, in addition to a fungal stain if possible. The stains should be evaluated for the presence of bacteria or fungi, since meningitis or joint infections due to either of these classes of microorganisms cannot be distinguished solely based on patient presentation alone. Detection of infectious agents by this method however, is largely dependent on the amount of organism present in the sample and therefore the sensitivity of a Gram or fungal stain can vary greatly. Importantly, a negative stain does not rule-out infection. However, the presence of hyphae, as indicated by the arrow on the Gram stain to the right, is strongly suggestive of a fungal infection.

    Following direct staining, the specimen should be plated to appropriate media for both bacterial and fungal culture. As Dr. Roberts indicated earlier, the predominant fungus isolated from the CSF of infected individuals during this outbreak has been Exserohilum rostratum, a darkly pigmented, dematiaceous fungus, which will be described in greater detail shortly. Currently, the best laboratory method for isolation and identification of this organism is by fungal culture. Therefore, CSF specimens and synovial fluid from suspected cases should be plated to the appropriate media as soon as possible to ensure efficient recovery of the organism. All isolated colonies should be identified by the primary laboratory or submitted to a reference lab for further assistance.

    Identification can be done by evaluation of both the macroscopic appearance of the colony and by microscopic characterization of the sporulating structures, which Dr. Roberts will define next. Additionally some reference laboratories may also offer isolate identification by DNA sequencing or by specifically designed polymerase chain reaction or PCR assays.

    Following these routine studies, the CDC recommends that CSF specimen be submitted to them for further analysis by DNA sequencing or PCR for detection of fungal DNA directly from the sample. Importantly, the CDC indicates that samples should be submitted only if certain criteria are met, which includes a minimum volume requirement of 1 mL. Additionally, the CSF should be from a patient that meets the current outbreak case definition, and the CSF profile should be notable for pleocytosis, regardless of protein or glucose levels. Important to remember, however, is that despite the high sensitivity of these molecular techniques, a negative result does not rule-out infection, and cultures should continue to be monitored for the duration of their incubation. For more information on whether the CSF specimen meets the required criteria or how to properly submit CSF samples for testing by the CDC, please contact your local public health laboratory.

    Finally, many questions have arisen from providers regarding the utility of the Aspergillus galactomannan antigen assay. As a brief background, this test is designed to detect galactomannan, which is a polysaccharide component of the Aspergillus cell wall, though it may be found in a number of other fungi as well. This antigen is released during fungal growth and can be detected by an enzyme-linked immunosorbent assay or ELISA. There is currently 1 FDA-approved assay for detection of galactomannan from serum samples only, though a number of case studies indicate that this test may also be useful for detection of galactomannan from CSF specimens in cases of fungal meningitis due to Aspergilllus.

    However, due to the majority of confirmed fungal meningitis cases being caused by Exserohilum, the galactomannan assay is no longer recommended by the CDC as an additional test to perform in suspected cases of meningitis during this outbreak.

    In summary, during this outbreak, providers and laboratories should continue to follow their previously established protocols for diagnosis of fungal meningitis. This includes performing routine bacterial and fungal cultures from CSF specimens or from synovial fluid in cases of suspected joint infection. All culture plates should be monitored for growth for at least 2 weeks and any colonies should be fully identified with the results reported to the provider as soon as possible. Excess CSF specimens can also be submitted to the CDC for further molecular testing through your local public health laboratory.

    Well, the organism that was identified from all of these cases of meningitis is an organism called Exserohilum and just what is this Exserohilum. It is an uncommon organism that we’ve seen in clinical laboratories not very often. It’s a pathogen of grasses and easily aerosolized, and what I have done now is to list some of the cases that have been reported caused by Exserohilum, and there are not a whole lot of them in terms of gross numbers, but this gives you an idea of the spectrum of this disease. Invasive fungal sinusitis is one of those, and another one is allergic fungal sinusitis with intracranial abscess, and so it is spread actually into the brain. Lower extremity invasive disease has occurred, joint space infection, and there are 2 of these in this series that we’re talking about specifically. Keratitis with the corneas involved, disseminated disease, and phaeohyphomycosis. Phaeohyphomycosis is an infection caused by a brown fungus, usually on the extremities, and we actually have seen a case here at Mayo Clinic where it was on a hand. I’ve only seen it once as a cause of disease at Mayo Clinic, so it’s not a very common organism to see in the clinical laboratory. But it’s always mentioned in courses because it has a unique presentation in terms of its morphology.

    But, it is not unlike all the other dematiaceous fungal cultures. It starts off by being a colony that’s kind of wooly and grows up in a fairly short period of time, in several days. It’s usually kind of brown to olive to grey. The hyphae in the colony are kind of wooly to hairy, and are a little bit coarse.

    This happens to be a melanized fungal colony, not one of Exserohilum, but one that could be Exserohilum. And, you can see the nature of that, that the back side is on the top of this image, and the back side will be dark, and the front side will be pigmented as well.

    Exserohilum has a couple unique features. One is that the conidia that are produced are extremely large. They are brown, and they are pigmented, and they are multiseptate with what looked like pseudoseptae. The conidia that are produced on a geniculate conidiophore, and we’ll show you a good example of that in just a second, and the number of septae that are produced, are pseudoseptae, produced within the conidium varies anywhere from 7 to 9, which makes it a very long conidium. And the hallmark of this organism turns the microscopic morphology is that there is a very prominent dark hilum that protrudes. And this is an area at the base of the conidium where it’s been attached to the conidiophore. I’ll show you an example of that and that is how you can recognize that Exserohilum. There are 3 species of this organism and I don’t know that you need to be involved in trying to key it out to the species level. The CDC or some other reference laboratory can do that.

    This is an example of a geniculate conidiophore. This happens to be a Bipolaris. If you look at the kind of wavy structure here that is darkly pigmented and you will see the conidium or spore down at the bottom there and right above it is this conidiophore that is attached to a hyphal strand you can see in the background. What happens with this conidiophore is that it grows one way and then it grows back and forth, back and forth, like a series of bent knees. Bipolaris is very closely related to Exserohilum. Another thing that is important to note, is that if you look at the conidium at the bottom of the slide, you will see on the left-hand side there is a dark area where the conidium was attached to the conidiophore and notice that it does not protrude, and it is continuous with the cell wall. That’s in stark contrast to Exserohilum.

    Now Bipolaris produces conidia at the apical end, the top end of this long conidiophore that goes, twists itself around back and forth, back and forth, and it’s called the sympodial arrangement of conidia. When they go back and they grow one side and then the other. This is Bipolaris and Exserohilum will do the same thing. So this happens to be the apical end, the same as the end of the conidiophore that you’re going to see with Exserohilum.

    Now, here is the conidia of Exserohilum. Notice where we put an arrow where it says hilum, that is not continuous with the cell wall of that conidium. It’s a peg-like structure that is very prominent, dark, and protrudes. Notice how long these conidia are. If you count those areas that are stained blue in there it will give you an idea of just how many cells are within that particular conidium.

    This next one shows you the same thing with a whole lot more conidia present in the slide. And you can see the protruding hilum on many of them. And that is the hallmark of this particular organism and kind of the rule in mycology is if they’re easy to recognize, you don’t see them very often. And I think that, you know if you are going to look for the extended length of the conidium and you look for the prominent hilum, then you should be able to recognize Exserohilum.

    Also, in this cardioplegic solution, Aspergillus has been found in at least 1 patient. And I think many of you are familiar with Aspergillus, and there are over 200 species and there are only a few of those that we deal with on a routine basis that cause disease. At least in smaller laboratories, an implication is made based on the microscopic morphology. And you look at the fruiting heads, and you can get an idea of what it is and the common species are identified and, if it goes beyond that, it’s usually unnecessary to go to that trouble to try to key them out to particular species.

    So, the cultures of Aspergillus vary a lot and the one we are going to look at today is going to be blue-green. And the species can’t be generally recognized by looking at colony. You can look at a blue-green colony like Aspergillus fumigatus and it might be Penicillium, it might be something else, so you have to rely upon the microscopic morphology. Aspergillus fumigatus has a single row of phialides on a hemispherical vesicle. In other words, this vesicle, this swollen area at the top is kind of half round. It looks like it’s been round and flattened out, and it’s called a vesicle and, it has, some of the organisms of Aspergillus, will have a branch and then a bottle like structure called a phialides produced that then in turn produce little chains of conidia. Aspergillus fumigatus does not have a branch. It has these phialides or these bottle like structures that are formed right on this hemispherical vesicle and then, in turn, the conidia or the spores are produced within that bottle like structure and then they are extruded out and they remain in long chains. And when we make mounts of these, we can see the results of those chains.

    You can see Aspergillus fumigatus culture is blue-green. Sometimes, it’s white so you have to rely upon, again, the microscopic morphology.

    And this is just an example of what Aspergillus fumigatus looks like. If you look at the long stalk called the conidiophore, that’s where you begin to start looking and you follow it on up to the top, and the round structure that you can see, about 4 o’clock is called the vesicle. And that is the structure that we see with its swollen end of the conidiophore. And if you look at the tip of that structure, it’s actually about 5 o’clock, and it goes straight up and you’ll see this fan-like structure, if you look at the top of that swollen area, you will see there are these bottle like structures, these are called phialides. And in turn, they produce conidia, and then they’re kept in chains. They are produced and pushed out in a chain. And we see this hemispherical vesicle and we see the conidia, we know this is pretty much Aspergillus fumigatus. If you want to go further, what you can do is you can put it at 45°C and it’s thermotolerant and it will grow at that temperature, but this is not the problem with this situation that we are dealing with currently. The Exserohilum is a problem, and so you need to be careful and look at your cultures daily and make sure that you don’t miss anything, because there are a lot of patients out there who actually are waiting to see if they are going to develop disease. And our hearts go out to the families of those who have lost family members and we hope that this situation does improve.

    As you know the numbers for these cases changes every day and we have been monitoring these numbers as well, and that will be posted here on a future update on MayoMedicalLaboratories.com and it will be updated very frequently, so that you can keep track of all the number of cases that have happened during this outbreak.


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