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 6 of 9
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Ms. Rayam:
I would like to thank you all for your very timely and very important information contributing to this very important discussion on the effectiveness of the laboratory, safety precautions, and testing. Dr. Tenover, I’d like to take this a step further with yet another question that might spark some more discussion. How is the CDC determining what antibiotics are effective for treating these anthrax infections?

Dr. Tenover:
Lisa, it’s very important to note that there is no standard method yet defined for Bacillus anthracis testing. So here at CDC we’re using the reference broth microdilution method as defined by the National Committee for Clinical Laboratory Standards. So far, of the 15 clinical isolates of anthrax that we tested from the U.S., all have been susceptible to penicillin, doxycycline, and ciprofloxacin. At this time, however, we are not recommending that other laboratories, including state health departments, do this testing, and there’s really 3 reasons: the first one is one of safety. It’s very important to recognize that susceptibility testing potentially could produce aerosols. So we do this testing in a Biosafety Level 3 laboratory in a biological safety cabinet.

The second one is a scientific one. That is, we are still understanding the ways that this organism can become resistant to antimicrobial agents, and the optimal method for testing has not yet been determined. So we hope to work out these procedures, and at that point we will reconsider disseminating the methods to other laboratories.

The last one really is the practical issue, though, and that is we have evaluated alternate methods of susceptibility testing that potentially could be done within the safety cabinet. However, at this point none of them reflect the methods and the results that we get by the broth microdilution reference method. So right now, again, that is the method we are using here at CDC.

It raises several other questions, though, that we’ve received during the past week, and I’d like to share some of those with our panel. Again, Tanja, let me start with you. We’ve had a lot of discussion about typing, molecular subtyping of this organism. Can you comment on the use of pulsed-field gel electrophoresis or ribotyping or other molecular methods?

Dr. Popovic:
Certainly. I think we can all appreciate the importance of the issue of molecular typing at this time, because that is the approach that allows us quite frequently to trace the origin of organisms in question. Let me start by saying that Bacillus anthracis is extremely homogeneous. A number of methods, including those that you have mentioned, have been tested and tried. Unfortunately, it seems that all organisms—all Bacillus anthracis organisms—when tested by these methods look either identical or very much alike. So it does not appear to be a lot of differentiation potential. As I have briefly mentioned, therefore, currently the method of choice is multilocus VNTR typing approach, in which we focus on a number of chromosomal and plasmid targets, and we come out with a pattern that can then later be associated with temporal or geographic or other relevant epidemiological markers, and this method has been used again in a real-time manner throughout this investigation, and we have found it to be extremely useful.

Dr. Tenover:
Good, thank you. Richard, in your presentation you mentioned that some states have more than one Level B laboratory. So how do we know which specimen to send to which laboratory?

Mr. Kellogg:
Well, yes; in general, most states have more than one B level laboratory. For example, Texas (for Bacillus anthracis) has eight. There are B level laboratories, though, not only at the state level facilities, but also at large city and county public health laboratories, and federal and military facilities as well. By contacting your state public health laboratory director, your closest B level facility can be identified in advance. Always keep in mind that the designation, the rating is agent-specific.

Dr. Tenover:
Good, thank you. Michael, we received a lot of questions from Level A laboratories about how do we disinfect our benches, and do we need to do special autoclaving to get rid of these samples? Can you comment about using bleach versus maybe a quaternary ammonium compound for disinfecting lab surfaces?

Dr. Miller:
Sure. A lot of people are really concerned about how to take care of their disinfectant issues here. We recommend a 10% solution of household bleach, and that’s simply made by one part bleach into 9 parts of water. This takes care of virtually all vegetative cells. While it’s not by definition a sporicide, it actually reduces the number of spores 3 to 5 logs. Now, quaternary ammonium compounds, alcohol, other normal hospital disinfectants are not effective against these spores, so we are recommending the 10% bleach solution.

Now, what about autoclaving? In our laboratories, we for our own purposes use a one-hour autoclave time, but that’s because we have large loads and large amounts of potential anthrax inside those loads. But the clinical laboratory need not vary from the routine 15-minute autoclave time that they’re using now. That should adequately kill the spores of Bacillus anthracis.

Dr. Tenover:
Good, thank you. Tanja, in the news there’s been a lot about hand-held devices for finding spores in the environment. Recently we heard about a new PCR test from the Mayo Clinic. I wonder if you could comment about these procedures?

Dr. Popovic:
Well, this is certainly not an unexpected question and I’ll be happy to comment. I’d like to make a distinction between the hand-held devices that are primarily used to detect spores of Bacillus anthracis in the environment and those PCR-based tests that are used to detect genetic material of Bacillus anthracis, either directly, in clinical specimens, or on the growing culture. Regarding hand-held devices, CDC is not recommending use of these hand-held devices for testing environmental samples. Data that is currently available and provided by the manufacturers of these devices suggests that the number of spores necessary for the test to be positive is very large, and they go up to 10,000—in the range of 10,000. And while that might be of value in heavily contaminated areas or samples, we might actually miss areas where that level of contamination is not so high. So CDC has been asked to assist in validation and evaluation of these assays. As soon as the studies are underway and completed, the results will be shared.

The second is the comment about the PCR assay such as that as reported by the Mayo Clinic. I’d like to say, over the past 2 years, we have worked with a number of partners and have developed a PCR-based assay that has proven to be extremely sensitive and very specific that has been used for the past 5 weeks intensively, primarily in our Advanced Technology Laboratory that serves for that screening purpose. And, specifically, about the Mayo Clinic assay: just like any other new assay, it does need to be compared to the assays that are already available, and until such tests are done, it is very difficult to talk about specificity, sensitivity, and appropriateness of a general use of these assays.

Dr. Tenover:
Good, thank you. Michael, maybe we can talk a little bit about environmental sampling at the Level A laboratory level. What are sort of the boundaries if the laboratory director is approached by the hospital administrator and wants to have their mailroom cultured? How would you advise these people, the laboratory directors?

Dr. Miller:
Well, the good point is that you’re approached by your own management, and I believe if that’s the case, where you have been asked to sample an area within your own hospital where there’s a low risk or no risk, then probably it’s okay to provide this type of sampling. And all this would mean would be using, again, a non-cotton swab that has been moistened, rubbed over a specified area of a tabletop or a mailbox or whatever you’re sampling within your institution, taken to the laboratory and heat shocked in 1½ ml of saline, and then plated. The heat shock takes place at 65°C for 30 minutes, then you plate 100 microliters. That’s what we do. Now, I don’t think at this point that hospitals at all should be taking on specimens from which there really may be a credible threat, or we just don’t want to bring into the hospital laboratory (or into the facility where our patients are) specimens that may likely be contaminated with anthrax spores. So if we’re going to do environmental sampling, it probably needs to be done within the institution, and management certainly needs to be involved in that decision.

Dr. Tenover:
Good, thank you. Our last question goes to you, Richard, and that is sort of a follow-up to what Michael just said. What problems would you see in performing Level B type of activities in a Level A laboratory?

Dr. Kellogg:
Well, I really do want to reiterate what Mike has said, and that for Bacillus anthracis, the LRN does not recommend that clinical labs, especially those located in patient care facilities, pursue LRN B level status and high-risk environmental sample testing. The LRN has particular concerns about potentially high-risk environmental samples, such as spore powders, especially those that could further contaminate and cause contamination problems in a facility. Other environmental samples not related to a credible threat assessment or established area of exposure may be done at Level A labs. But again, this should be very low-risk work, often taken to calm people’s fears, and again, at the discretion of the laboratory management.

Dr. Tenover:
Good. Thank you all for your responses.

Ms. Rayam:
Again, thank all of you. Very timely responses during a very critical time in America. Thanks to you all.

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