The sponge wipe method has not previously been evaluated for recovery of B. anthracis
spores from surfaces. Other researchers have evaluated the efficiency of gauze wipes to sample for B. anthracis
spores on surfaces. Brown et al. (2
) found that polyester-rayon blend gauze wipes recovered 35% (SD, 12%) of the B. atrophaeus
spores that had been deposited on a 25-cm2
steel surface by settling from aerosol dispersion. This gauze wipe study was conducted in one laboratory with 20 samples using a surrogate organism for B. anthracis
at a spore concentration range of 100 to 1,000/645-cm2
surface area. Estill et al. (7
), using a rayon gauze wipe, recovered 18% of the aerosolized B. anthracis
Sterne spores from 929-cm2
steel surfaces when the inoculum level was 270 CFU/100 cm2
. The Estill study was conducted with B. anthracis
spores, and processing was conducted in three independent laboratories, but the number of gauze wipes sent in each shipment was limited to three. Lewandowski et al. (14
) used a foam spatula to recover aerosolized B. atrophaeus
spores from surfaces and obtained a median efficiency of 9%. Though the authors recognize that in a B. anthracis
contamination event, the spores would most likely be deposited on a surface by the settling of spores in air, in a laboratory setting, depositing spores on a surface in a uniform manner is difficult, especially at low concentrations (7
). Edmonds et al. (6
) compared the recovery of spores deposited as a liquid (in water) to the recovery of spores deposited as an aerosol and found that neither deposition resulted in consistently better or worse recovery of spores. We therefore chose the liquid deposition method for better inoculum size accuracy.
In preliminary work, limited in-house evaluations (n
= 10) of several sampling devices and premoistening solutions were conducted. All sampling devices were inoculated and processed using the same method as the sponge wipe used in this study. The results of those in-house evaluations () indicated that other products may yield similar results, since all of the devices recovered between 26% and 36% of the spores. In order to be confident in the data collected with these alternate sampling devices, more extensive evaluations need to be conducted to better characterize the performance of these devices before use in a contamination event. During the preliminary investigations, heat shock times and temperatures were also investigated. Even with low heat shock times and temperatures (65°C, 15 min), a significant loss of viability was observed (data not published). This reduction in the number of viable spores after heat shock treatment was also seen by Turnbull et al. (18
). Considering that environmental sampling of dry, nonporous surfaces would most likely be cocontaminated with other Bacillus
sp. spores or fungal spores that would not be reduced by a heat shock step; we eliminated heat treatment from the protocol.
Evaluation of the sampling efficiency of alternate wipes and alternate premoistening fluidsa
In order to investigate the effect of shipping conditions on environmental samples, an extensive in-house evaluation of the stability of inoculated macrofoam swabs stored at various temperatures was conducted. The results demonstrated that environmental samples maintained at 5 or 20°C would not suffer loss of viability or germinate and multiply if processed within 24 h of sampling (unpublished data). These data may be applicable to B. anthracis spores on cellulose sponge sticks as well but should be confirmed with additional testing.
This sponge wipe method is capable of sampling a larger surface area than a previously validated swab sampling and processing method described by Hodges et al. (11
). There may be instances when a swab sample is appropriate, such as when sampling irregularly shaped or hard-to-reach areas. However, sampling of larger areas can often provide a better assessment of the contamination of a room or building (5
). The %R (SE) of the swab sampling (25.8 cm2
) and processing method is 55.0 (5.2), 27.9 (3.9), and 42.0 (4.6) for the 1-, 2-, and 4-log10
CFU inoculum levels, respectively, if dust and background organisms are present (10
). Though the %R from swab sampling may appear to be slightly better than that found for the sponge wipe, we cannot directly compare them since the surface areas sampled were different. Further work is needed to determine the effect of surface area on the sampling efficiency of a given sampling device.
This sponge wipe sampling study found that the sampling efficiency of the sponge wipe method varied with the inoculum level sampled and that the SE was greater at the lowest inoculum level. The between-lab, within-lab, and total variabilities, as measured by %CV also varied with the inoculum level, with the greatest variability seen at the lowest inoculum level. The sensitivity of the method was 100% when ≥2 log10 spores were present on the surface and 84% when 1 log10 spores were present. The specificity in the presence of a well-characterized dust containing a consortium of other organisms (103 CFU/10 mg) was 100% (if the one case of a malfunctioning biosafety cabinet is omitted).
Though the mean %R varied from 24.4% to 34.6% for each of the inoculum levels, it should be noted that these values represent recovery on the same order of magnitude. When sampling after the anthrax contamination of a room, building, or area, obtaining the same order of magnitude is still helpful in determining the source of the contamination or in characterizing the extent of the contamination.
Enriching the spores remaining on the sponge wipe by placing the sponge wipe into TSB and incubating it did not help lower the level of detection as we thought it might. The other Bacillus spp. present in the dust appeared to outcompete the low levels of B. anthracis remaining on the sponge, and B. anthracis colonies were isolated only a few times at the low inoculum level. The presence of the background consortia of organisms, the cellulose from the sponge wipe, and/or the ingredients of the TSB may have contributed to inhibition of the PCR reagents when attempting to detect B. anthracis DNA directly from TSB. Further work is needed to investigate whether an alternate DNA preparation method would enhance detection by blocking inhibitors.
Because this work was meant to focus on the variability of the processing method in multiple laboratories, only two individuals conducted the actual surface sampling in this study. In an actual environmental sampling event, additional variability may be introduced by multiple individuals conducting the sampling. In addition, the data do not address the potential for variability between runs (multiple shipments) at the same inoculum level since each lab processed only a single run for each inoculum level.
These data provide performance characteristics for a sponge wipe-processing method for use when investigating environmental B. anthracis
contamination. If validation is defined as stated in the GAO report, as “a formal, empirical process in which an authority determines and certifies the performance characteristics of a given method” (9
), we consider this method validated, with the LRN review committee as the oversight authority. This controlled study brought to light the large variability inherent in environmental sampling and processing, especially when dealing with low numbers of organisms. Nonetheless, these data help to establish acceptance criteria and characterize the variability of the method and thereby enhance confidence in the interpretation of the results of environmental sampling after a B. anthracis