Evaluation of sampling tools for environmental sampling of bacterial endospores from porous and nonporous surfaces

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 ( -an integrated network of state and local public health, federal, military, and international laboratories that can respond to bioterrorism, chemical terrorism, and other public health emergencies. However, results from surface samples were inconsistent. In some cases, contamination at a given location within a facility was not detected with initial samples, and subsequent samples were required to detect the contamination (19). A Government Accountability Office (GAO) investigation following the 2001 anthrax incident concluded that validated sampling methods and statistical sampling designs were needed to provide confidence that there is no contamination when all sample results are negative (17, 18). This conclusion strongly reinforces the need for validated sampling methods to effectively respond to biothreats and ensure public safety.Following the 2001 anthrax incident, several research teams developed and investigated (in laboratory studies) the performance of sampling methods using swab, wipe, and vacuum collection devices for Bacillus anthracis or surrogate contaminants on different surfaces (1,2,3,4,5,6,7,13,14,15,16,22,23,25,28,34,38,40,44,45). In addition, the CDC has conducted formal validation studies on two methods for sampling nonporous surfaces: macrofoam swabs (23) and cellulose sponge wipes (39).A review of the cited laboratory studies identified numerous gaps in the data on method performance (30). For example, none of the studies quantified the false-negative rate (FNR) for the sampling and analysis methods investigated. The term "false negative" refers to a failure to detect contamination from a sample collected at a contaminated location. A false negative can occur because of inefficiencies (i.e., biases) and uncertainties (i.e., imprecision) at any step of the sampling and analysis process (sample collection, storage/transportation, processing/extraction, and analytical). False negatives can occur during preliminary screening or characterization sampling at low contamination levels and during clearance sampling following the decontamination process. A better understanding of FNRs and how they are infl...

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

Krauter

Piepel

Boucher

et al. 2012

“…Preliminary investigations were conducted to select the optimum swab type and the optimum processing method (10,11,16). Others have evaluated the efficiency of various swab types for conducting surface sampling and processing methods for the swabs (1,4,8,15,19). However, using a swab to sample an area larger than 25.8 cm 2 may result in reduced recovery efficiency, so an alternative device for sampling of larger surface areas is needed.…”

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confidence: 99%

“…However, using a swab to sample an area larger than 25.8 cm 2 may result in reduced recovery efficiency, so an alternative device for sampling of larger surface areas is needed. Though other researchers have evaluated the efficiency of gauze wipes (2,7,19), a foam spatula (14), or the Biological Sampling Kit (BiSKit; Quicksilver Analytics, Inc., Abingdon, MD) (3) for sampling of B. anthracis or surrogates for B. anthracis from larger surface areas, none of these methods were evaluated to the extent required to provide a validated method. No acceptance criteria for environmental sampling currently exist; hence, one goal of this study was to investigate the variability of the method and establish acceptable limits for environmental sampling and processing protocols.…”

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confidence: 99%

National Validation Study of a Cellulose Sponge Wipe-Processing Method for Use after Sampling Bacillus anthracis Spores from Surfaces

Rose

Hodges

O’Connell

et al. 2011

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.

“…As a result, over the last decade needs for quantitative assessment capabilities have driven research on the relative effectiveness of different sampling processes as well as the development of validated surface sampling and sample processing methods (27,49). Notably, the vast majority of these studies have utilized B. anthracis spores (13,20,23,27,28,48) or spores of a surrogate organism (4,5,8,9,18,58). Comparatively little effort has been applied in recent years toward improved surface sampling of viruses (31) and vegetative bacterial cells that represent likely biothreat (BT) agents (or surrogates thereof) (7,29,39,60).…”

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Impact of Processing Method on Recovery of Bacteria from Wipes Used in Biological Surface Sampling

Downey

Silva

Olson

et al. 2012

ABSTRACTEnvironmental sampling for microbiological contaminants is a key component of hygiene monitoring and risk characterization practices utilized across diverse fields of application. However, confidence in surface sampling results, both in the field and in controlled laboratory studies, has been undermined by large variation in sampling performance results. Sources of variation include controlled parameters, such as sampling materials and processing methods, which often differ among studies, as well as random and systematic errors; however, the relative contributions of these factors remain unclear. The objective of this study was to determine the relative impacts of sample processing methods, including extraction solution and physical dissociation method (vortexing and sonication), on recovery of Gram-positive (Bacillus cereus) and Gram-negative (Burkholderia thailandensisandEscherichia coli) bacteria from directly inoculated wipes. This work showed that target organism had the largest impact on extraction efficiency and recovery precision, as measured by traditional colony counts. The physical dissociation method (PDM) had negligible impact, while the effect of the extraction solution was organism dependent. Overall, however, extraction of organisms from wipes using phosphate-buffered saline with 0.04% Tween 80 (PBST) resulted in the highest mean recovery across all three organisms. The results from this study contribute to a better understanding of the factors that influence sampling performance, which is critical to the development of efficient and reliable sampling methodologies relevant to public health and biodefense.