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 3 of 9
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Dr. Fred Tenover:
Tanja, let’s begin with you. Tell us about the recent laboratory
activities at CDC involving anthrax.
Dr. Tanja Popovic:
Certainly, Fred. Over the past 5 weeks during the ongoing
anthrax investigation, professional lives of many laboratorians
at 1600 Clifton Road have been drastically changed, and what I would
like to do today is share with you an insight into those lives,
into their long hours, into their struggles, obstacles, and the
successes. I would also like to share with you an overview of the
CDC’s laboratory capabilities at this time, starting with very basic
microbiology procedures to state-of-the-art molecular diagnostic
methods.
For us, it all started on October 3, 2001, with reports of suspected
Bacillus anthracis isolates in Florida. And the very next
day, the first CDC team, consisting of epidemiologists, laboratorians,
and support staff was deployed to Florida. Two days later, as shown
on this slide, 11 boxes with a couple hundred clinical and environmental
samples came our way. This indeed was our “Saturday Night Live”
for all of us who were there that evening. You can see our colleagues
unloading these boxes and then taking them into our laboratories
for analysis. One of many challenges that faced us at that time
was to ensure very accurate tracking of all of these samples and
the results that were done on them.
Here are some of our staff working on rolls of the CDC ID numbers,
and that very first night we also had to set up our initial database.
These pictures were e-mailed to us from the Miami Public Health
Laboratory, and they show the results of the gamma phage testing
and India ink staining testing for one of the first suspect isolates.
Shown here are the results done on the very same isolate at the
CDC the next day, and you can see the results of the DFA (direct
fluorescent assay) where we have confirmed the presence of both
cell wall polysaccharide and capsule antigens.
This slide shows our initial organizational setup. All specimens
were received at our Rapid Response and Advanced Technology Laboratory.
There they were screened using rapid biodetection assays, such as
PCR and time-resolved fluorescence. Then, specimens are forwarded
to the anthrax laboratory for isolation, confirmatory testing, and
molecular characterization. Biopsy materials and tissues, on the
other hand, go to the infectious disease pathology activity, where
recently developed immunohistochemistry assays specific to Bacillus
anthracis were used.
I thought that it might be of interest to you to see what an average
day in an anthrax lab looks like. So here we go. Specimens are first
processed, and that means they’re aliquotted—part
of the specimen is used for molecular characterization and DNA is
extracted first, while the other part is used for the standard microbiological
procedures. Next is the work with the cultures themselves once the
organism is growing, and that means performing presumptive and confirmatory
testing. All records of the results and testing need to be sent
out of the laboratory either by scanning the paperwork or by faxing
it out so that nothing leaves our biosafety containment. Once that
information is received outside of the Biosafety Level 3 laboratory,
that information is entered into our databases, and then we can
have reports and spreadsheets available to be shared.
Here is the work-up of a typical clinical material that came our
way, and many of you who are working in Level A laboratories will
be familiar with these methods. Initial work on clinical specimens
includes Gram staining and India ink staining of blood for demonstration
of the capsule. Once the organism is growing, it is very important
to observe the typical colony morphology and especially the lack
of hemolysis. Finally, B. anthracis is not motile, and that
characteristic is extremely helpful for differential diagnosis.
Now, here are the tests for confirmatory identification of an isolate
as Bacillus anthracis. Lysis by gamma phage in conjunction
with demonstration of capsule is the first confirmatory approach.
Capsule can be demonstrated in several ways: by growth on bicarbonate-supplemented
medium in enhanced CO2 environment or by incubation in
horse blood. Each then can be followed by M’Fadyean stain or India
ink stain, as demonstrated on the slide. The second confirmatory
approach is to detect both cell wall and capsule antigens by the
DFA.
Work on the environmental specimens is somewhat different. Specimens
can be directly examined for the presence of spores, either by the
wet mount or malachite green stain. They can also be directly examined
by the DFA for both of the earlier mentioned antigens. And finally,
and understandably, microbiological media will be also immediately
inoculated.
In addition to confirmatory testing of an isolate, a whole number
and range of molecular approaches have been taken during this investigation.
I have mentioned briefly that rapid detection assays such as PCR
and time-resolved fluorescence are used in our Rapid Response and
Advanced Technology Laboratory, and the PCR approach is also used
directly on clinical specimens and on the cultures in the anthrax
lab itself. Molecular subtyping of Bacillus anthracis is
carried out by a method called MLVA (it stands for Multilocus VNTR
Typing). It’s a method that focuses on a number of specific targets
in the Bacillus anthracis chromosome and two of its plasmids.
It allows for identification of a particular pattern that can have
association with geographic, temporal, or other relevant epidemiological
designations, and this method has been extremely useful in the ongoing
investigation. Finally, we’re also conducting sequencing of the
gene, coding for the 16S ribosomal RNA and a couple of other genes
specific for Bacillus anthracis.
In addition to all of these activities, it became very soon quite
clear that we needed to expand even further. So, the basic structure
that we initially had was kept with Rapid Response Laboratory being
the triage laboratory and doing the initial screening of samples.
Most of the samples go into the anthrax lab for the isolation confirmatory
testing and molecular typing, and then subsequently on for antimicrobial
susceptibility testing. However, we have added activities in our
microbial pathogenesis and immune response laboratory, where actually
hundreds of sera were tested by serologic assays. And only last
week, we have opened a surge capacity laboratory, our newest addition,
so that large numbers of environmental specimens that are coming
our way can be rapidly screened. To date, over 2,000 specimens have
been analyzed at the CDC. From them, over 100 isolates have been
confirmed as Bacillus anthracis. In many of these of specimens, presence of Bacillus anthracis was demonstrated by
immunohistochemistry methods, and as I mentioned, many sera were
shown to be reactive.
As you can imagine, a number of organizational and logistical issues
had to be dealt with quite in a hurry, and some of them are the
same ones that you are probably dealing with. If that is not the
case, then this list might be very helpful for you to be prepared.
As we moved on with our work, we had to develop protocols for collection
of specimens that we never thought we would have to deal with, specimens
like air filters, keyboards, clothing. And we had to develop protocols
for collection, shipping, and packaging of these specimens. Also,
we rapidly had to expand from working a comfortable 5 days a week
for 8 hours a day to working 7 days a week, 24 hours a day. You
can envision that a number of additional staff had to be rapidly
trained. We had to learn how to efficiently communicate with each
other as well as with everybody else who wanted to communicate with
us.
Finally, we had to learn how to keep the enormous amount of data
that we were generating all the time in an understandable and manageable
manner. As we continue to face new challenges, at the same time
we also continue to be extremely encouraged by the strength of our
laboratory network for which Level A laboratories serve as the wide
and strong basis. The index Bacillus anthracis isolate was detected
in one such laboratory and was then forwarded to the Level B laboratory
in Florida where confirmatory testing was carried out. Shown in
this picture is our colleague from that particular Level B laboratory,
and the picture was taken exactly a year ago during the first of
four bioterrorism preparedness laboratory trainings that we have
conducted for laboratorians from throughout the country. As you
can see, we have now made a full circle here, from isolation of
a suspected isolate in a Level A laboratory, to talking to you today,
again, about ever-increasing and important role that Level A laboratories
play in the fight against bioterrorism. Thank you.
Dr. Tenover:
Tanja, thank you very much.
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- Page last updated November 20, 2002
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