Surface sampling methods for bacillus anthracis spore contamination - Bioterrorism-Related Anthrax

Emerging Infectious Diseases, Oct, 2002 by Wayne T. Sanderson, Misty J. Hein, Lauralynn Taylor, Brian D. Curwin, Gregory M. Kinnes, Teresa A. Seitz, Tanja Popovic, Harvey T. Holmes, Molly E. Kellum, Sigrid K. McAllister, David N. Whaley, Edward A. Tupin, Timothy Walker, Jennifer A. Freed, Dorothy S. Small, Brian Klusaritz, John H. Bridges

During an investigation conducted December 17-20, 2001, we collected environmental samples from a U.S. postal facility in Washington, D.C., known to be extensively contaminated with Bacillus anthracis spores. Because methods for collecting and analyzing B. anthracis spores have not yet been validated, our objective was to compare the relative effectiveness of sampling methods used for collecting spores from contaminated surfaces. Comparison of wipe, wet and dry swab, and HEPA vacuum sock samples on nonporous surfaces indicated good agreement between results with HEPA vacuum and wipe samples. However, results from HEPA vacuum sock and wipe samples agreed poorly with the swab samples. Dry swabs failed to detect spores >75% of the time when they were detected by wipe and HEPA vacuum samples. Wipe samples collected after HEPA vacuum samples and HEPA vacuum samples collected after wipe samples indicated that neither method completely removed spores from the sampled surfaces.

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The Brentwood Mail Processing and Distribution Center in Washington, D.C., was extensively contaminated with Bacillus anthracis spores after two letters containing spores were processed at this facility on October 12, 2001 (1). Subsequently, inhalational anthrax developed in four postal workers. An investigation in late October 2001, using surface wipe and HEPA vacuum sock sampling techniques, showed widespread B. anthracis spore contamination inside the building. Spore concentrations were particularly high around Delivery Bar Code Sorter (DBCS) machine no. 17, which had processed the letters, and in the government mail area, where the letters had been processed before being distributed.

This report describes the results of sampling for B. anthracis spores in an investigation conducted December 17-19, 2001, by the Centers for Disease Control and Prevention (CDC), the Agency for Toxic Substances and Disease Registry (ATSDR), the U.S. Postal Service (USPS), and a USPS contractor. At the time of this investigation, technical issues regarding sampling and analyses for B. anthracis spores remained unresolved, such as which technique for surface sampling (swabs, wipes, or HEPA vacuum socks) is most appropriate for collecting spores in specific situations, how the different types of surface sampling methods compare, and how effectively the sampling methods collect spores from contaminated surfaces. Surface sampling to determine the presence of B. anthracis spores in an environment is essential for determining extent of contamination, assessing potential for exposure and need for medical treatment, and guiding clean-up and reentry efforts.

Sampling methods (swabs, wipes, rinses, direct agar contact, and vacuuming) have been evaluated for collecting microorganisms from surfaces (2-7), primarily in laboratory settings. B. subtilis spores, which may behave much like B. anthracis spores, have been frequently used as the microbiologic agent sampled. Substantial variation in sample recoveries was observed for the various methods. In addition, the methods have not been validated specifically for collecting and analyzing B. anthracis spores in environmental samples. The primary objective of our survey was to compare the levels of B. anthracis spores in side-by-side samples obtained by the surface swab, wipe, and HEPA vacuum sock methods to evaluate their relative effectiveness.

USPS representatives and a USPS contractor had conducted clean-up operations at the Brentwood facility since late October. However, much of the facility had not been cleaned and was believed still contaminated with B. anthracis spores. Even though the DBCS machine (no. 17) that processed the contaminated letters had been cleaned by HEPA vacuum, washed with a 10% sodium hypochlorite solution followed by neutralization with a sodium thiosulfate solution, and rinsed with water, this machine was reportedly still contaminated with B. anthracis spores (8). For these reasons, the Brentwood facility was thought to be a good location to compare surface sampling and analytical methods.

Methods

Surface sampling was conducted by using swabs, wipes, and HEPA vacuum socks. To compare the sampling techniques, we selected locations where the three types of samples could be collected adjacent to each other on nonporous surfaces, with an emphasis on locations believed to be still contaminated with B. anthracis spores. The locations sampled included the surfaces of selected DBCS machines (particularly machine no. 17), return air ducts, tops of the window boxes along the postal inspector walkways, and the tops of mail sorting bins in a secure area approximately 23 m from DBCS machine no. 17. The order in which the samples were collected varied in a randomized fashion from location to location; each site was assigned a location number and sampled according to a predetermined, randomized plan. We used the randomized sampling plan to reduce sampling biases that might be caused by nonuniform distribution of spores across surfaces and affected by the order in which samples were collected.

Seven swab, six wipe, and five HEPA vacuum sock samples were collected as control samples; that is, these samples were handled in the same way as others but not used to sample any surfaces. The purpose of these control samples was to evaluate the potential contamination of sample media, unrelated to actual sample collection.

Nine additional blank HEPA vacuum sock samples were collected to estimate cross-contamination by inserting them into the vacuum nozzle after a HEPA vacuum sock sample had been collected and the nozzle cleaned; these socks were then withdrawn and placed in a sterile conical tube for laboratory analysis.

Investigators were given written instructions for collecting samples at each selected location (Figure 1). The surface areas sampled by each technique were intended to be comparable, but not necessarily equal. In particularly dirty areas, swabs and wipes could not cover as large a surface area as the HEPA vacuum sock samples without becoming overloaded; investigators were instructed to avoid overloading the samples by reducing the size of the surface sampled.