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This work was initiated to address the gaps identified by Congress regarding validated biothreat environmental sampling and processing methods. Nine Laboratory Response Network-affiliated laboratories participated in a validation study of a cellulose sponge wipe-processing protocol for the recovery, detection, and quantification of viable Bacillus anthracis Sterne spores from steel surfaces. Steel coupons (645.16 cm(2)) were inoculated with 1 to 4 log(10) spores and then sampled with cellulose sponges (Sponge-Stick; 3M, St. Paul, MN). Surrogate dust and background organisms were added to the sponges to mimic environmental conditions. Labs processed the sponges according to the provided protocol. Sensitivity, specificity, and mean percent recovery (%R), between-lab variability, within-lab variability, and total percent coefficient of variation were calculated. The mean %R (standard error) of spores from the surface was 32.4 (4.4), 24.4 (2.8), and 30.1 (2.3) for the 1-, 2-, and 4-log(10) inoculum levels, respectively. Sensitivities for colony counts were 84.1%, 100%, and 100% for the 1-, 2-, and 4-log(10) inocula, respectively. These data help to characterize the variability of the processing method and thereby enhance confidence in the interpretation of the results of environmental sampling conducted during a B. anthracis contamination investigation.

After the 2001 anthrax incidents, surface sampling techniques for biological agents were found to be inadequately validated, especially at low surface loadings. We aerosolized Bacillus anthracis Sterne spores within a chamber to achieve very low surface loading (ca. 3, 30, and 200 CFU per 100 cm(2)). Steel and carpet coupons seeded in the chamber were sampled with swab (103 cm(2)) or wipe or vacuum (929 cm(2)) surface sampling methods and analyzed at three laboratories. Agar settle plates (60 cm(2)) were the reference for determining recovery efficiency (RE). The minimum estimated surface concentrations to achieve a 95% response rate based on probit regression were 190, 15, and 44 CFU/100 cm(2) for sampling steel surfaces and 40, 9.2, and 28 CFU/100 cm(2) for sampling carpet surfaces with swab, wipe, and vacuum methods, respectively; however, these results should be cautiously interpreted because of high observed variability. Mean REs at the highest surface loading were 5.0%, 18%, and 3.7% on steel and 12%, 23%, and 4.7% on carpet for the swab, wipe, and vacuum methods, respectively. Precision (coefficient of variation) was poor at the lower surface concentrations but improved with increasing surface concentration. The best precision was obtained with wipe samples on carpet, achieving 38% at the highest surface concentration. The wipe sampling method detected B. anthracis at lower estimated surface concentrations and had higher RE and better precision than the other methods. These results may guide investigators to more meaningfully conduct environmental sampling, quantify contamination levels, and conduct risk assessment for humans.

The survival of the biothreat agent Burkholderia pseudomallei on the surfaces of four materials was measured by culture and esterase activity analyses. The culture results demonstrated that this organism persisted for <24 h to <7 days depending on the material, bacterial isolate, and suspension medium. The persistence determined by analysis of esterase activity, as measured with a ScanRDI solid-phase cytometer, was always longer than the persistence determined by culture analysis.

Seven species of bacterial select agents were tested for susceptibility to monochloramine. Under test conditions, the monochloramine routinely maintained in potable water would reduce six of the species by 2 orders of magnitude within 4.2 h. Bacillus anthracis spores would require up to 3.5 days for the same inactivation with monochloramine.