The Search for Anthrax
The Search for Anthrax
A letter addressed to Senator Patrick Leahy was found by FBI and Environmental Protection Agency (EPA) hazardous materials personnel on Friday, November 16. The letter was in one of the 280 barrels of unopened mail collected from Capitol Hill after an anthrax-contaminated letter to Senator Daschle was first discovered. An innovative protocol was developed by scientific and forensic experts to analyze these congressional mail bags for anthrax contamination. The new method eliminated the need for hazardous materials teams to sift through each piece of mail in the 635 trash bags to find additional contaminated mail.
Safety was the primary consideration of the entire operation. Although the level of contamination was not known beforehand, the work space and sampling procedures were designed so that the risk of exposure would be minimized even if the mail were heavily contaminated. And, everyone in the containment area would wear personal protective equipment and respirators as seen in the figures. The mail was sampled and sorted in a containment facility constructed within a large warehouse.
To ensure that bacterial spores would not escape into the surroundings, the containment facility was maintained with negative air pressure, and intake and exhaust air was filtered with high efficiency particulate air (HEPA) filters, which trap essentially all particles the size of anthrax spores. Negative air pressure was achieved by exhausting air faster than it was let in, which means that, aside from the exhaust fans, the air always flowed into the room through any small openings that were present. Work began only after the structural integrity of the containment facility was approved by inspectors.
Air samplers were used to monitor the air both inside and outside the containment area, also called the "hot zone." These samplers help estimate the quantity of airborne spores as well as when spores are in the air. No spores were detected for the entire operation in air samples collected in the decontamination area, or the "cold zone," which includes the outer work space and outside the air exhausts. On the other hand, spores were detected from all four samplers within the hot zone whenever a particularly "hot" bag was handled. The exposure of workers was further monitored by taking samples directly off of their clothing. In general, people did not become contaminated unless a hot bag was handled, and then contamination was sometimes quite high. This graphically demonstrated the ease with which the spores become airborne, and underscores the respect with which contaminated material must be handled.
Seemingly, the most straightforward way to find a letter that looks like another one is to simply sort through the mail by hand. However, such an approach would have drawbacks. First, it might unnecessarily expose workers to high concentrations of spores if a letter was actually present. Second, it would be time-consuming and extremely tedious, which might decrease the attention to detail that would be needed for a thorough search. And third, if an anthrax-loaded letter were present that did not resemble those sent to Sen. Daschle and Mr. Brokaw, it might be missed.
The strategy for finding an anthrax-loaded letter in the mail was also designed to minimize risk. In this operation, simple was best. The methods used were classic microbiology techniques and the plan to search through the bags of mail was based on some very simple ideas. The known behavior of the Daschle letter suggested that all of the above drawbacks might be overcome by looking for spores rather than for letters. It was clear that the Daschle letter made numerous people sick and contaminated large areas wherever it went. Therefore, it seemed almost inconceivable that a trash bag containing a similar anthrax-loaded letter would not contain an overwhelming number of spores.
The sampling plan to find the Leahy letter was accomplished by teams of hazardous materials (Hazmat) workers from the FBI and EPA Criminal Investigative Division. Each bag, sampled in turn, was jostled around to mix up any spores present. A swab was inserted into a small hole in each bag and wiped around the inside. After the swab was withdrawn, the hole was sealed with duct tape, and the swab was used to inoculate a Petri dish containing a particular solid growth medium, which was sent to the Naval Medical Research Center (NMRC) for analysis. Colonies resulting from the growth of Bacillus anthracis (the anthrax bacterium) on this culture medium could be easily identified by the experienced laboratory workers at NMRC.
This plan had a number of advantages. Workers would have to sample only 635 bags rather than examine many thousands of individual letters. Sampling through small holes minimized the release of spores into the work space. The swabbing technique was simple and easily mastered by the workers. Normally, swabs are packaged and transported to the laboratory where they are then used to inoculate the culture medium. Inoculating the medium at the scene greatly reduced the workload on the laboratory. In addition, the time needed to obtain results was reduced because the bacteria started growing as soon as they were put in the Petri dish, producing visible colonies by early the following morning.
Contamination was detected in only about 60 bags. The swabs from about 50 bags revealed only trace contamination. Seven produced greater than 100 bacterial colonies from a swab and were considered "hot." The inoculation of Petri dishes with swabs is a very sensitive technique that can theoretically detect a single spore. However, the maximum number of spores that can be identified with this method is about 300. If more are present, the resulting colonies become too crowded to count. This leaves very little room to distinguish samples that produced 100 colonies on Petri dishes from those that may have contained many more than 300. Therefore, a second sampling technique was used to determine whether or not there were large differences in contamination levels between bags.
For the second sampling method, air was drawn out of selected bags for two minutes each and bubbled through water. The water accumulates spores over time, which can then be counted, no matter how many there are. This method proved to be much less sensitive than direct plating onto Petri dishes, but it turned out to be more discriminating. Spores were detected in the air of only three bags. One bag produced about 100 spores over the two minute sampling time, another, 300, and a third between 19,000 and 23,000. The difference between this last bag and all of the others made it clear that, if there was just a single anthrax-loaded letter, it must be in that bag. To be cautious, all bags that produced greater than 20 spores from the swabbing method were sorted by hand. These bags were handled in biological safety cabinets because of the increased risk associated with opening the bags and handling contaminated mail. Safety cabinets use directed airflow to prevent contamination from escaping while work is being done inside of them.