CDC Responds: Coping with Bioterrorism—The Role of the Laboratorian
(November 9, 2001)
(View the webcast on the University of North Carolina School of Public Health site.)
Segment 5 of 9
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Michael, now that we’ve seen the whole structure of the LRN, can you detail for us exactly what the responsibilities of the Level A laboratories are?
Dr. J. Michael Miller:
I think so, Fred. I believe we all know that the clinical microbiologists are really the cornerstone of this laboratory system.
This first graphic actually shows the two simple things that the Level A laboratory must be able to do. First of all, our job is to rule out Bacillus anthracis by simple observation. Ruling in implies an identification process that the Level A lab is not necessarily going to do. So we need to know and actually practice the safety precautions that are recommended for BSL-2 labs. Certainly know microbiology
observation criteria; in other words, be proficient at plate reading. And then know the limitations and the capacity of your own laboratory.
Secondly, it’s very important to know when and how to refer any of these suspicious specimens to a higher-level laboratory. Knowing when to stop working on an isolate is many times more important than knowing when to start. Know where that next level is, whether it’s the state public health laboratory and in what part of the state, and especially knowing the appropriate packing and shipping protocols is very important. It’s not dangerous to work with clinical specimens if we follow those BSL procedures, and anthrax is no different. Anthrax as a disease is not contagious person-to-person. It’s a BSL-2 agent, and the clinical specimens would be considered BSL-2 specimens. Now, if you’re going to work with this agent in pure culture, if you’re going to work with the spores in large numbers, then it becomes a BSL-3, but we’re talking about clinical specimens at the Level A procedures. There’s a low risk there, and if we follow the rules of BSL-2, we’re going to be all right. Remember that spores of anthrax are usually not produced in the body or in tissue. And so, therefore, we will likely not be infected with spores by working with this tissue, particularly if we use the biological safety cabinet at the setup area as we are supposed to do. It lowers and eliminates most all of the laboratory risk that might be associated. Now, using 10% bleach as a disinfectant is what we recommend, and that bleach is going to kill most of the vegetative cells—in fact, it will kill all the vegetative cells and most of the spores.
The specimens we’re most likely to receive from inhalation anthrax are likely to be blood, which would be the specimen of choice (that’s the specimen that’s most likely to be positive), and then sputum. Although sputum is not ideal specimen for anthrax, remember, anthrax is not a true pneumonia, so we’re really looking for blood as our specimen of choice.
The laboratory handling really just needs to follow some very simple procedures. Keep in mind that working in the community hospital laboratory, we do not need anthrax vaccination; in fact, we don’t need antimicrobial prophylaxis, even if we isolate an isolate of Bacillus anthracis. If we follow the BSL rules by wearing laboratory coats and gloves, particularly at the setup bench, and if you find a suspicious agent in the laboratory, that plate and all of its associated materials should go directly into the biological safety cabinet and further work should be done right there inside the cabinet. That would include preparing slides for Gram stain or motility, and certainly wearing gloves inside the hood if you’re preparing the slide for motility. Washing hands is the classic procedure for safety, whether it’s in the laboratory or outside the laboratory. So when we leave that laboratory area, make sure we wash our hands.
The specimens we’re most likely to receive will be associated with the type of disease that’s being presented. Anthrax may appear as inhalational disease, in which case blood would be the specimen of choice. Sputum may be requested, but still, blood would be our choice. Cutaneous anthrax is most likely to be isolated from vesicles and eschars, taken with swab specimens underneath the eschar. Gastrointestinal anthrax—blood again would be the specimen of choice along with stool to make sure that we have this organism.
For specimens that we have received a lot of questions about, let me mention one thing about nasal swabs as a screening test. This really should not be used for routine testing. It is not a valuable test, particularly for making decisions regarding patient care. The nasal swabs were designed to be used with support for epidemiologic teams who are in the field, or to evaluate known, documented exposures. So they are not to be used routinely. We don’t even know how sensitive or specific the nasal swab is for detecting the spores of anthrax, so that’s why we really do not want to use it. But you may be asked in some cases, and many of you have already, to work up a nasal swab (or a nares swab) for the spores of anthrax. If you do, just remember to use a noncotton fiber, use swabs either Dacron, rayon, or calcium alginate. Resist Gram stain requests on these specimens, because it’s not going to be helpful in looking for spores of the agent. With a nares swab, if you are asked to work these up, we found that placing these swabs into phosphate-buffered saline, about 1½ ml, heat shocking them at 65 degrees for 30 minutes, and then plating on blood agar is an excellent way to determine if spores for this agent are present.
Now, there’s another question that we’ve gotten quite often also, and I know you’ve faced it, too. It has to do with environment samples. What do we do with people who ask us to sample the environment? Let’s talk about superficial surface swabs and separate that from other types of environmental samples. A simple superficial surface swab, just a tabletop or the top of a telephone, looking for anthrax swab may be acceptable in the Level A laboratory. There’s no reason (for technical reasons) why you would not be able to do this. And I would recommend that if you are asked by your administrators to sample, for instance, your own mailroom or certain facilities within your hospital, that you could accommodate that, and it would be handled much the same way as you would a swab from the nose. A moistened swab over a specified surface area, take that swab in a small amount of saline and heat shock it and plate it. Very similar to processing nares cultures. But there’s no indication that a community hospital should be in the business of sampling powders or bulk samples (such as office supplies, computer keyboards, water or clothing). Leave that to the authorities. If you have a question, call your state health laboratory and get information from them on what they recommend.
But what are you going to do in your laboratory? It’s very simple. Tanja has alluded to this earlier. We need to do a Gram stain to know that this is a gram-positive rod. Understand the growth characteristics on blood agar where we’re looking for a nonhemolytic bacillus colony. These organisms will sporulate in air. They are nonmotile, and in some cases you may want to look for the capsule by India ink or special stain. It’s very simple. That’s it; we do no more. If we get a suspicious isolate, we forward it to the next level laboratory for confirmation.
Now, let’s take a look at the Gram stain morphology of an organism. The Bacillus anthracis cell is going to be a gram-positive rod that’s 1-1½ microns wide and 3-5 microns long, and you may actually see them in chains. And, yes, you can view the Gram stain outside of the biological safety cabinet. These spores are oval, they are central to subterminal, and they do not significantly swell the cell. Remember, the spores are not going to be seen in a body specimen. Right out of fresh blood or out of tissue you’re likely not to see a spore-forming bacillus. It’s going to have to be incubated in air.
Here you see the picture of a typical Gram stain of a Bacillus anthracis. The colony morphology is very much like some of the other bacilli you may see. After 18-24 hours at 35 degrees, you’re going to see in well-isolated colonies those that are about 2-5 mm in diameter. Keep in mind, this is really a rapidly growing organism. You don’t have to wait for 24 hours to actually see the colony morphology. They are flat colonies, they are slightly convex, irregularly round, and the edges may be slightly undulate and often have little, curly, tailing edges, as you can see on this slide. Colonies have a ground glass appearance and kind of a sticky consistency. Now, this organism grows very well on blood agar and chocolate agar, even Martin-Lewis agar, but it does not grow on some of the inhibitory media, such as colistin-nalidixic acid agar (CNA); it does not grow in XLD, Salmonella-Shigella agar or MacConkey, and it does not grow on PEA.
This is an illustration of how the colony we describe as being sticky can be lifted with your inoculating loop. The edge of a colony can be lifted, and the colony many times will remain erect as you remove the inoculating needle or loop from under the edge of the colony.
So to summarize, here’s an algorithm that illustrates how the Level A laboratory should respond to a request for culture for Bacillus anthracis. We’re looking for large, aerobic, gram-positive rods, and there’s only two simple tests we need to be concerned about after that. Is it hemolytic? If the answer is yes (and in this graphic, yes goes to the right and no goes down), if this organism is hemolytic, it’s not going to be Bacillus anthracis; just report it as a Bacillus species. Motility can be done either using motility medium or you can use a slide motility. If the organism is motile, it will not be Bacillus anthracis. If it’s not motile and it’s nonhemolytic, then you’ll certainly want to forward this isolate as suspicious to your state public health laboratory.
The safety issue is one that is absolutely critical, and you want yourself and everyone in your laboratory to understand the safety rules of working at the BSL-2 level. There are a number of Web sites that are listed on this slide that can be very helpful to you, both from CDC and items on packing and shipping. If you do have questions, the first source you might try is going to be your state public health department.
Michael, thank you very much.
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