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Based on the ANOVA results, we found that two of the independent experimental factors were significant with a p-value less than 0. The surface materials were selected in order to give a range of recovery efficiencies, so this significant difference was expected. Regardless of the compositing method, vinyl and drywall had the lowest RE of the tested coupon materials. However, the material with the greatest magnitude difference in RE using the different composite methods was drywall.

Of the materials tested, ceramic tile and stainless steel had the highest recovery efficiency and vinyl tile had the lowest RE. The process controls, MP-Discrete, data are included for comparative purposes. This was the case whether all coupons were contaminated or just one, as well as if 4 samples were composited or 8 samples composited.

Although the same number of sponges must be extracted using the MM-MPC as the MP-Discrete method, the number of samples to be analyzed at the end point of the method refer to Fig 2 is reduced. The four scenarios tested were 4 samples composited with A all or B one random coupon contaminated and 8 samples composited with C all or D one random coupon contaminated. The process control, MP-Discrete, data is included for comparative purposed for the composite methods. This interaction is shown in Fig 6. At 10 CFU target per coupon, the all coupons contaminated composite samples had a higher RE than those composite samples with only one coupon contaminated.

However, at 25 CFU and 50 CFU target per coupon, the composite samples with only one coupon contaminated had a higher RE than those with all coupons contaminated. The random order of the one coupon contaminated samples could be responsible for this; however this study was not designed to determine the effect of the random coupon swipe order. Therefore, no further testing was done to investigate this interaction. The means are plotted as open circles and the error bars are representative of two standard deviations. For this study, we focused on the comparison of the number of locations to composite after sampling: 4 individual locations, 8 individual locations, or 16 individual locations at one time.

The data set used for these results was obtained from a collaboration of test matrices 41—44 and a subset of the data from matrices 1—40 S2 Table. We obtained a balanced data set with 24 values where an analysis of 4, 8, and 16 locations could be compared. To obtain this balanced set of data, the data set was restricted to two CFU target values 10 and CFU , two types of coupon materials ceramic tile and stainless steel , and sampling only with the MM-MPC method. Both deposition locations of contamination all coupons locations or only one random coupon location were tested with this set of data.

Using this subset of data, an ANOVA was conducted as previously described with four factors and the results are shown in Table 3. The results of the ANOVA revealed zero significant F tests for any independent experimental factor or any two way interactions. However, for all other tests, RE was neither improved nor reduced when 4, 8, or 16 locations were composited, and RE were similar to those obtained using the MP-Discrete method.

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These data indicate that increasing the number of locations composited to 16 locations using the MM-MPC method does not adversely affect RE. The experiments were conducted using the MM-MPC methodology with 1, 4, 8, or 16 locations composited. The data for ceramic and stainless steel coupons are provided together in the MP-Discrete data set.

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The test design described for the full factorial study with clean coupons was recapitulated with the grime coated coupons. The raw data from these experiments are provided in S4 Table. An ANOVA was performed in order to compare the differences of the experimental factors and their variations. The significant interaction between material and grime presence Table 4 is plotted in Fig 8. This inconsistency, as shown by the intersecting lines in Fig 8 , is why the interaction was significant. The presence of grime is an independent significant factor on RE.

The interaction between material type and grime presence is also significant for RE. The FNR for the different coupon materials tested stainless steel, vinyl, and ceramic are shown in Fig 9. The gray line in each plot demarcates the 0. The limit of detection LOD at the 0. LOD 90 is calculated by using a value of 0. These values are shown in Fig 7. Coupons were contaminated in four or eight locations for the both composite methods.

These results are consistent with reduced RE for ceramic coupons coated with grime Fig 8. Using this sampling approach, we investigated the effects of composite methodology, number of locations composited, deposition location of the spores, coupon material, and number of spores deposited on recovery efficiency Table 1.

We applied ANOVA to our experimental results in order to compare the significance of these experimental factors and their interactions. We identified two significant factors and one significant interaction from the analysis Table 2. Our RE are consistent with findings from different groups that have conducted spore recovery studies from different materials in that nonporous surfaces have been reported to have higher RE compared to painted or rough surfaces.

Probst et al. As the number of spores used to contaminate the coupons increases, so does the RE; therefore, this difference between our work and reports by others was expected.

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These results indicate that in situations where the spore concentration is unknown, and the hot spots could contain low spore numbers, the MM-MPC method could be considered as a candidate method for sampling to reduce the number of analyses without sacrificing RE achieved with MP-Discrete samples. This is because the MP-Discrete method requires each sponge to be extracted in 90 mL of buffer. Thus, for 16 coupons, there would be 16x90 mL solutions to process post extraction using the MP-Discrete method.

For example, instead of centrifuging 32 to 48 sample tubes 16 coupons x 2 to 3 tubes per coupon generated for 16 coupons with the MP-Discrete method, only 3 tubes are centrifuged and processed for the same number of coupons with the MM-MPC method. In addition, rather than 48 plate counts for 16 coupons with the MP-Discrete method, only 3 plate counts are required for the same number of coupons sampled with the MM-MPC method.

The reduction in number of tubes centrifuged, number of tubes combined into a single sample, number of samples plated on agar, and number of plates requiring counting saves a significant amount of time, labor, and resources. Our results are in agreement with the France et al.

In their study, it was reported that the composite method was significantly different. However, their test materials contaminated dust vary greatly from coupon surfaces [ 3 ]; therefore, the relative agreement between our study and the France et al. We tested four different scenarios: 4 or 8 locations composited with only one or all coupons contaminated. The MP-Discrete method was conducted in parallel for comparative purposes.

A consistent RE for the MP-Discrete method is expected based on previous work we have conducted as well as reports by others, which further bolsters these experimental results [ 5 , 11 — 13 , 21 , 25 ]. Previous work by Tufts et al. Spores were deposited on only one coupon location, but the location of the contaminated coupon in the test varied location one or location 4, with 4 locations composited.

The results from the Tufts study indicated that most of the surface bound spores were recovered by the sponge in the first pass across the coupon surface, and therefore multiple passes could potentially be unnecessary [ 9 ]. However, this difference could be attributed to the lower spore concentrations used in our study compared to high concentrations used in the Tufts study, or could be attributed to spore application method wet vs.

In addition, the Tufts study only examined stainless steel coupons. In this study, stainless steel coupons as well as the other materials ceramic tile, vinyl, and drywall which have varying RE associated with these surface types were used. The data from our study suggests that multiple passes across the coupon surface would be required in order to maximize spore recovery. The results indicate that the difference between all or one coupon being contaminated varies with the CFU target.

However, a consistent trend between CFU target and number of coupons contaminated is not readily apparent.

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We postulate that the random order of the contaminated coupon is a contributing factor for this variation at the lowest CFU target tested. However, this study was not designed to determine the effect of the random coupon swipe order on RE. The study conducted by Tufts et al. Future studies are needed to determine the impact of contamination at different locations. The purpose of these experiments was to determine if doubling the number of locations composited from eight to 16 would cause a reduction in the RE from ceramic or stainless steel coupons. We used 10 or as the CFU target and compared these data to data sets from four and eight samples composited at the same CFU targets.

ANOVA results from the experimental data did not show any significant differences between four, eight, or 16 individual locations composited. These results are similar to those reported by France et al. Taken together, these data suggest that compositing up to 16 samples using the MM-MPC method with a low spore concentration would not vary significantly from the results achieved using the MP-Discrete method.

As stated previously, there is significant savings with respect to time and resources when using the MM-MPC method compared to the MP-Discrete method, and accuracy of RE is not sacrificed. We also investigated the effect of grime coating on spore RE using ceramic, stainless steel, and vinyl coupons.

In these experiments, we focused on the experimental factors that were significant: coupon material, composite method, and the two-way interaction of CFU target number and number of contamination locations. When comparing data from these experiments to those done with clean coupons, we only compared data sets using the same CFU target numbers i. We conducted tests with spores deposited on either one or all coupons.

Interestingly, the RE from ceramic tiles decreased when grime was present We postulate that this change in RE is due to unknown features of the ceramic surface, and not due to a biological effect from the grime on the Bacillus spores. This idea is further supported by the ANOVA results which show that the interaction between the presence of grime and material type is significant.

These results are consistent with our previous work and reports by other groups demonstrating that coupon surface characteristics are a significant factor with respect to RE [ 11 , 13 , 20 ]. The false negative rates FNR and limit of detection LOD 90 were calculated from the results of the experiments conducted with clean and grime coated coupons. Ceramic tile and stainless steel coupons had lower FNR and LOD values compared to vinyl tile, which is consistent with the results reported by Krauter et al.

In conclusion, we have tested three composite sampling approaches at low CFU targets with four different coupon materials. Our results demonstrate that the multi-media multi-pass composite sampling MM-MPC methodology is a more effective sampling campaign at low spore concentrations compared to other composite sampling strategies tested.

The advantages of the MM-MPC method are reduced time, labor, and material requirements as described above. This approach could improve response times to biological events, reduce costs and labor requirements, and is an excellent candidate method for further testing and verification by other laboratories. PNNL is a multiprogram national laboratory operated for the U. We would like to thank Staci Kane for providing grime samples. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

National Center for Biotechnology Information , U. PLoS One. Published online Oct Becky M.

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Hess , Brett G. Amidan , Kevin K. Anderson , and Janine R. Brett G. Kevin K. Janine R. George-John Nychas, Editor. Author information Article notes Copyright and License information Disclaimer. Competing Interests: The authors have declared that no competing interests exist.

Conceptualization: JH BA. Methodology: JH BA. Software: BA KA. Received Jun 14; Accepted Sep This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This article has been cited by other articles in PMC.

Associated Data Supplementary Materials S1 Fig: Plot of theoretical versus sample quantiles of the residuals shows the data has a normal distribution. S2 Table: Test Matrices for the full factorial composite study.

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S3 Table: Results from the full factorial study. S4 Table: Description of each experimental run using grime coated coupons with results. Abstract Restoring all facility operations after the Amerithrax attacks took years to complete, highlighting the need to reduce remediation time.

Introduction The process for decontaminating a facility after anthrax contamination incident can be described in five phases: 1 environmental sampling, 2 laboratory sample analysis, 3 hazard mapping and assessment based on laboratory results; 4 decontamination, and 5 decontamination sampling to validate decontamination efforts [ 1 — 3 ]. Surface Materials Eighteen gauge stainless steel sheets L were cut to the desired coupon size of Coupon Grime Coating A grime mixture was applied to clean test coupons to mimic background dust.

Coupon Inoculation Clean coupons were inoculated following the cleaning process described above. Open in a separate window. Fig 1. Pictorial overview of the sampling methods used in the study. Fig 2. Pictorial overview of the sample process and analysis method for each sampling sponge. Test Design Experiments were conducted in two stages: clean coupons, then grime coated coupons. Statistical Analysis A plot of the theoretical versus sample quantiles QQ-plot of the residuals theoretical quantiles versus sample quantiles was used to confirm the data was normally distributed S1 Fig as previously described [ 17 ].

Table 1 The six experimental factors used in the study to evaluate recovery efficiency. Coupons surfaces were clean, coated with grime alone, or coated with grime containing the biological component refer to Materials and Methods. Fig 3. Testing clean coupon surfaces, full factorial study The test matrices for the full factorial composite study are provided in S2 Table Matrices 1 through ANOVA results investigating experimental factors and their interactions ANOVA was performed in order to compare the differences of the experimental factors Table 2 and their interactions.

Table 2 ANOVA results showing statistical significance for the tested factors and their interactions for clean coupon materials. Fig 4.

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Coupon material and compositing methodology are significant experimental factors on the response variable, recovery efficiency. Fig 5. The composite methodology is significant, and the MM-MPC method yields the highest RE when considering the number of locations composited and number of coupons contaminated. Fig 6. Increasing the number of locations composited after sampling does not reduce RE For this study, we focused on the comparison of the number of locations to composite after sampling: 4 individual locations, 8 individual locations, or 16 individual locations at one time.

Fig 7. Increasing the number of locations composited from 8 to 16 does not reduce RE. Table 4 ANOVA results show that the presence of grime and the interaction between coupon type and grime presence are significant for RE. Fig 8. Grime coating on coupons is associated with an increased RE for stainless steel and vinyl coupons, but RE is reduced when grime is present on ceramic coupons. Fig 9. Fig Supporting Information S1 Fig Plot of theoretical versus sample quantiles of the residuals shows the data has a normal distribution.

TIF Click here for additional data file. XLSX Click here for additional data file. S2 Table Test Matrices for the full factorial composite study. S3 Table Results from the full factorial study.