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Title: False-Negative Rate and Recovery Efficiency Performance of a Validated Sponge Wipe Sampling Method

Abstract

Recovery of spores from environmental surfaces varies due to sampling and analysis methods, spore size and characteristics, surface materials, and environmental conditions. Tests were performed to evaluate a new, validated sponge wipe method using Bacillus atrophaeus spores. Testing evaluated the effects of spore concentration and surface material on recovery efficiency (RE), false-negative rate (FNR), limit of detection (LOD), and their uncertainties. Ceramic tile and stainless steel had the highest mean RE values (48.9 and 48.1%, respectively). Faux leather, vinyl tile, and painted wood had mean RE values of 30.3, 25.6, and 25.5, respectively, while plastic had the lowest mean RE (9.8%). Results show roughly linear dependences of RE and FNR on surface roughness, with smoother surfaces resulting in higher mean REs and lower FNRs. REs were not influenced by the low spore concentrations tested (3.10x10^-3 to 1.86 CFU/cm^2). Stainless steel had the lowest mean FNR (0.123), and plastic had the highest mean FNR (0.479). The LOD90 (>1 CFU detected 90% of the time) varied with surface material, from 0.015 CFU/cm^2 on stainless steel up to 0.039 on plastic. It may be possible to improve sampling results by considering surface roughness in selecting sampling locations and interpreting spore recovery data. Further,more » FNR values (calculated as a function of concentration and surface material) can be used presampling to calculate the numbers of samples for statistical sampling plans with desired performance and postsampling to calculate the confidence in characterization and clearance decisions.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1076709
Report Number(s):
PNNL-SA-86204
400904120
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Applied and Environmental Microbiology, 78(3):846-854
Country of Publication:
United States
Language:
English
Subject:
Anthrax; Surface sampling; false negative; recovery efficiency; statistics

Citation Formats

Krauter, Paula, Piepel, Gregory F., Boucher, Raymond, Tezak, Matthew S., Amidan, Brett G., and Einfeld, Wayne. False-Negative Rate and Recovery Efficiency Performance of a Validated Sponge Wipe Sampling Method. United States: N. p., 2012. Web. doi:10.1128/AEM.07403-11.
Krauter, Paula, Piepel, Gregory F., Boucher, Raymond, Tezak, Matthew S., Amidan, Brett G., & Einfeld, Wayne. False-Negative Rate and Recovery Efficiency Performance of a Validated Sponge Wipe Sampling Method. United States. doi:10.1128/AEM.07403-11.
Krauter, Paula, Piepel, Gregory F., Boucher, Raymond, Tezak, Matthew S., Amidan, Brett G., and Einfeld, Wayne. Wed . "False-Negative Rate and Recovery Efficiency Performance of a Validated Sponge Wipe Sampling Method". United States. doi:10.1128/AEM.07403-11.
@article{osti_1076709,
title = {False-Negative Rate and Recovery Efficiency Performance of a Validated Sponge Wipe Sampling Method},
author = {Krauter, Paula and Piepel, Gregory F. and Boucher, Raymond and Tezak, Matthew S. and Amidan, Brett G. and Einfeld, Wayne},
abstractNote = {Recovery of spores from environmental surfaces varies due to sampling and analysis methods, spore size and characteristics, surface materials, and environmental conditions. Tests were performed to evaluate a new, validated sponge wipe method using Bacillus atrophaeus spores. Testing evaluated the effects of spore concentration and surface material on recovery efficiency (RE), false-negative rate (FNR), limit of detection (LOD), and their uncertainties. Ceramic tile and stainless steel had the highest mean RE values (48.9 and 48.1%, respectively). Faux leather, vinyl tile, and painted wood had mean RE values of 30.3, 25.6, and 25.5, respectively, while plastic had the lowest mean RE (9.8%). Results show roughly linear dependences of RE and FNR on surface roughness, with smoother surfaces resulting in higher mean REs and lower FNRs. REs were not influenced by the low spore concentrations tested (3.10x10^-3 to 1.86 CFU/cm^2). Stainless steel had the lowest mean FNR (0.123), and plastic had the highest mean FNR (0.479). The LOD90 (>1 CFU detected 90% of the time) varied with surface material, from 0.015 CFU/cm^2 on stainless steel up to 0.039 on plastic. It may be possible to improve sampling results by considering surface roughness in selecting sampling locations and interpreting spore recovery data. Further, FNR values (calculated as a function of concentration and surface material) can be used presampling to calculate the numbers of samples for statistical sampling plans with desired performance and postsampling to calculate the confidence in characterization and clearance decisions.},
doi = {10.1128/AEM.07403-11},
journal = {Applied and Environmental Microbiology, 78(3):846-854},
number = ,
volume = ,
place = {United States},
year = {Wed Feb 01 00:00:00 EST 2012},
month = {Wed Feb 01 00:00:00 EST 2012}
}
  • The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in × 2 in) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest formore » vinyl tile (50.8% with BAS and 40.2% with BG) and the highest for glass (92.8% with BAS and 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG; values increased as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results will be discussed in a subsequent article.« less
  • Recovery of spores from environmental surfaces is known to vary due to sampling methodology, techniques, spore size and characteristics, surface materials, and environmental conditions. A series of tests were performed to evaluate a new, validated sponge-wipe method. Specific factors evaluated were the effects of contaminant concentrations and surface materials on recovery efficiency (RE), false negative rate (FNR), limit of detection (LOD) - and the uncertainties of these quantities. Ceramic tile and stainless steel had the highest mean RE values (48.9 and 48.1%, respectively). Faux leather, vinyl tile, and painted wood had mean RE values of 30.3, 25.6, and 25.5, respectively,more » while plastic had the lowest mean RE (9.8%). Results show a roughly linear dependence of surface roughness on RE, where the smoothest surfaces have the highest mean RE values. REs were not influenced by the low spore concentrations tested (3 x 10{sup -3} to 1.86 CFU/cm{sup 2}). The FNR data were consistent with RE data, showing a trend of smoother surfaces resulting in higher REs and lower FNRs. Stainless steel generally had the lowest mean FNR (0.123) and plastic had the highest mean FNR (0.479). The LOD{sub 90} varied with surface material, from 0.015 CFU/cm{sup 2} on stainless steel up to 0.039 on plastic. Selecting sampling locations on the basis of surface roughness and using roughness to interpret spore recovery data can improve sampling. Further, FNR values, calculated as a function of concentration and surface material, can be used pre-sampling to calculate the numbers of samples for statistical sampling plans with desired performance, and post-sampling to calculate the confidence in characterization and clearance decisions.« less
  • The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in. × 2 in.) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest formore » vinyl tile (50.8% with BAS, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm 2, where CFU denotes ‘colony forming units’). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results will be discussed in a subsequent report.« less
  • The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in × 2 in) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest formore » vinyl tile (50.8% with BAS, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2, where CFU denotes ‘colony forming units’). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results are discussed in a separate report.« less
  • Two concerns were raised by the Government Accountability Office following the 2001 building contaminations via letters containing Bacillus anthracis (BA). These included the: 1) lack of validated sampling methods, and 2) need to use statistical sampling to quantify the confidence of no contamination when all samples have negative results. Critical to addressing these concerns is quantifying the probability of correct detection (PCD) (or equivalently the false negative rate FNR = 1 - PCD). The PCD/FNR may depend on the 1) method of contaminant deposition, 2) surface concentration of the contaminant, 3) surface material being sampled, 4) sample collection method, 5)more » sample storage/transportation conditions, 6) sample processing method, and 7) sample analytical method. A review of the literature found 17 laboratory studies that focused on swab, wipe, or vacuum samples collected from a variety of surface materials contaminated by BA or a surrogate, and used culture methods to determine the surface contaminant concentration. These studies quantified performance of the sampling and analysis methods in terms of recovery efficiency (RE) and not PCD/FNR (which left a major gap in available information). Quantifying the PCD/FNR under a variety of conditions is a key aspect of validating sample and analysis methods, and also for calculating the confidence in characterization or clearance decisions based on a statistical sampling plan. A laboratory study was planned to partially fill the gap in PCD/FNR results. This report documents the experimental design developed by Pacific Northwest National Laboratory and Sandia National Laboratories (SNL) for a sponge-wipe method. The study will investigate the effects on key response variables from six surface materials contaminated with eight surface concentrations of a BA surrogate (Bacillus atrophaeus). The key response variables include measures of the contamination on test coupons of surface materials tested, contamination recovered from coupons by sponge-wipe samples, RE, and PCD/FNR. The experimental design involves 16 test runs, to be performed in two blocks of eight runs. Three surface materials (stainless steel, vinyl tile, and ceramic tile) were tested in the first block, while three other surface materials (plastic, painted wood paneling, and faux leather) will be tested in the second block. The eight surface concentrations of the surrogate were randomly assigned to test runs within each block. Some of the concentrations will be very low and may present challenges for deposition, sampling, and analysis. However, such tests are needed to investigate RE and PCD/FNR over the full range of concentrations of interest. In each run, there will be 10 test coupons of each of the three surface materials. A positive control sample will be generated prior to each test sample. The positive control results will be used to 1) calculate RE values for the wipe sampling and analysis method, and 2) fit RE- and PCD-concentration equations, for each of the six surface materials. Data analyses will support 1) estimating the PCD for each combination of contaminant concentration and surface material, 2) estimating the surface concentrations and their uncertainties of the contaminant for each combination of concentration and surface material, 3) estimating RE (%) and their uncertainties for each combination of contaminant concentration and surface material, 4) fitting PCD-concentration and RE-concentration equations for each of the six surface materials, 5) assessing goodness-of-fit of the equations, and 6) quantifying the uncertainty in PCD and RE predictions made with the fitted equations.« less