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.
Tanja, thank you very much.
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