In early 2005, a systematic study of laboratory-scale decontamination of five porous surfaces (carpet, ceiling tile, cinder block, painted wallboard, and unpainted wood) and one nonporous surface (painted I-beam steel) was initiated by the U.S. EPA in collaboration with the U.S. Army Edgewood Chemical Biological Center (ECBC). The overall objective of this collaborative study was to systematically investigate the abilities of fumigants to effectively decontaminate building materials contaminated with anthrax spores. This unprecedented systematic investigation involved the determination of efficacy (or log reduction in the number of viable spores) as a function of fumigant technology, technology operating parameters (e.g., fumigant concentration and exposure time), environmental conditions (temperature and relative humidity [RH]), and building material types. The magnitude and scope of this study required that new methods be developed to incorporate the use of complex materials in sporicidal efficacy testing and the processing of an unprecedented number of complex samples.
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Current standardized sporicidal test methods include the Association of Official Analytical Chemists (AOAC International) sporicidal activity of disinfectant test (AOAC Official Method 966.04) (4) and the American Society for Testing and Materials (ASTM) 2414-05 (3) and quantitative carrier test (QCT) (2). All of these methods are based on testing hard-surface carrier-based spores, which are submerged in a disinfectant for a desired contact time, followed by the addition of a neutralizer and enumeration of viable spores recovered from the carrier. Almost all standard test methods for liquid disinfectants use small coupons, e.g., 5- by 5-mm squares or 1-cm discs, on which 1 million to 10 million (6 to 7 log) spores are inoculated. While AOAC Official Method 966.04 is qualitative, the other two test methods are quantitative and provide log reduction estimates. Currently, demonstration of a >6-log-unit inactivation of B. anthracis or an appropriate surrogate spore (e.g., Bacillus subtilis) using a quantitative test method, such as QCT, which is also known as ASTM 2197-02, or the three-step method (TSM), also known as ASTM 2414-05, by a decontaminant is a requirement for product registration as a sporicidal agent against spores of B. anthracis Ames (18).
Sporicidal efficacy of CD gas (9,000-ppmvh CT) as a function of spore challenge levels. Five replicate coupon types were exposed to 3,600 ppmv of CD gas for 3 h. Spores were extracted and enumerated. Log reductions were computed as described in Materials and Methods. The error bars indicate standard deviations.
Efficacy of VHP (870-ppmvh CT) as a function of spore challenge levels. Five replicate coupons were exposed to 290 ppmv of VHP for 180 min, and spores were extracted. The log reductions were computed as described in Materials and Methods. The error bars indicate standard deviations.
Effect of fetal bovine serum on the efficacy of CD gas (9,000-ppmvh CT). Five replicate coupons were exposed to 3,600 ppmv of CD gas for 3 h, and spores were extracted. The log reductions were computed as described in Materials and Methods. The error bars indicate standard deviations.
Effect of bioburden inclusion on spore recovery from building surfaces using CD gas fumigant. Spore suspensions were prepared with different serum concentrations. Five replicate coupons were inoculated with an aliquot of 50 ml spore suspension and dried as described in Materials and Methods. The log difference was computed by subtracting the log CFU recovered from the coupons after drying and log CFU estimated in the aliquot spotted on coupons The error bars indicate standard deviations.
Because of a negligible effect of 0.5% serum on the sporicidal efficacy of CD gas and the spore recovery results, a low level of protein serum, 0.5%, was included with the spore preparation used in future fumigation studies to increase the sporicidal challenge for the fumigant (unpublished data). Further work is required to determine the nature (organic or inorganic) and the amount of burden that should be included in laboratory- or building-scale studies. It may be reiterated here that in many of the published studies of disinfectant testing, no bioburden was included (12, 13, 17).
The results summarized here were obtained using a spore suspension dried on various surfaces. In contrast to the use of aerosolized spores for determining the efficacies of swabs and wipes for spore recovery (5, 10), recovery of dried spores on environmental surfaces is even more challenging. This challenge is likely due to the increased adhesion of spores to the material surface and/or penetration of the spores within the pores of complex building materials.
Since only one strain (avirulent B. anthracis) was used in the present study, the relevance of the results obtained with STEM in the context of other strains or surrogates must be addressed. Recent work by Sagripanti et al. (14) clearly established similar or comparable sensitivities of spores from B. anthracis strains and other commonly used simulants, such as B. subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus atrophaeus, and Bacillus megaterium, for a number of different disinfectants, e.g., Clorox, Decon Green, and Sandia DF 100 and DF 200. Even though the results presented here were derived from the use of only one strain of B. anthracis, we have confirmed high spore recovery and fumigant efficacy with the STEM protocol with spores from three other simulants of B. anthracis, i.e., B. subtilis, B. atrophaeus, B. thuringiensis, and Geobacillus stearothermophilus (results not shown). 2ff7e9595c
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