Webcast Transcript
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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Dr. Tenover:
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.
Dr. Tenover:
Michael, thank you very much.
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