EPA/600/R-21/124 | May 2021
www.epa.gov/emergency-response-research
United States
Environmental Protection
Agency
oEPA
Understanding Detection Limits
Within the Interim Clearance Goal for
Bacillus anthracis Contamination
Office of Research and Development
Homeland Security Research Program
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EPA/600/R-21/124
May 2021
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus
anthracis Contamination
U.S. Environmental Protection Agency
Cincinnati, OH 45268
May 20, 2021
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development's
Homeland Security Research Program, funded and managed this evaluation. The document was prepared by
Battelle Memorial Institute under EPA Contract Number EP-C-16-014, Task Order 68HERC19F0089. This
document was reviewed in accordance with EPA policy prior to publication. Note that approval for
publication does not signify that the contents necessarily reflect the views of the Agency. Mention of trade
names or commercial products does not constitute endorsement or recommendation for use of a specific
product.
Questions concerning this document or its application should be addressed to:
Erin Silvestri
Center for Environmental Solutions and Emergency Response
Office of Research and Development (NG16)
U.S. Environmental Protection Agency
26 West Martin Luther King Drive
Cincinnati, OH 45268
513-569-7619
Silvestri .erin@Epa.gov
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the Nation's land,
air, and water resources. Under a mandate of national environmental laws, the Agency strives to formulate
and implement actions leading to a compatible balance between human activities and the ability of natural
systems to support and nurture life. To meet this mandate, EPA's research program is providing data and
technical support for solving environmental problems today and building a science knowledge base
necessary to manage our ecological resources wisely, understand how pollutants affect our health, and
prevent or reduce environmental risks in the future.
The Center for Environmental Solutions and Emergency Response (CESER) within the Office of Research
and Development (ORD) conducts applied, stakeholder-driven research and provides responsive technical
support to help solve the Nation's environmental challenges. The Center's research focuses on innovative
approaches to address environmental challenges associated with the built environment. We develop
technologies and decision-support tools to help safeguard public water systems and groundwater, guide
sustainable materials management, remediate sites from traditional contamination sources and emerging
environmental stressors, and address potential threats from terrorism and natural disasters. CESER
collaborates with both public and private sector partners to foster technologies that improve the effectiveness
and reduce the cost of compliance, while anticipating emerging problems. We provide technical support to
EPA regions and programs, states, tribal nations, and federal partners, and serve as the interagency liaison for
EPA in homeland security research and technology. The Center is a leader in providing scientific solutions to
protect human health and the environment.
Following an environmental contamination incident involving Bacillus anthracis, assessments will be needed
to determine the extent of contamination, efficiency of remediation, and the risks that might be present if
remediation is not 100% effective or if low levels of contamination are still present at the edges of the
contaminated area. Multiple lines of evidence are applied in this determination, among them results from
environmental sampling. Uncertainties in sampling and analysis results complicate decision making, namely
that a non-detect result may not truly be indicative of no contamination and possible false negative results
and detection limits from the methods themselves need to be considered. In this report, sampling and analysis
method ranges of recovery efficiency, limit of detections, and false negative rates for environmental matrices
and analysis methods were summarized and used to illustrate their effects on the interpretation of sampling
results in regard to clearance decisions. The current detection limits and considerations discussed in this
report will help to define the inputs to these risk considerations and could be used by decision makers and the
environmental unit when characterizing and clearing a site.
Gregory Sales, Director
Center for Environmental Solutions and Emergency Response
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Acknowledgments
This document was developed by the U.S. Environmental Protection Agency's (EPA) Homeland Security
Research Program (HSRP) within EPA's Office of Research and Development. Contributions of the following
individuals and organizations to this report are gratefully acknowledged:
US Environmental Protection Agency (EPA) Project Team
Erin Silvestri (Principal Investigator)
John Archer
Worth Calfee
Keely Maxwell
US EPA Technical Reviewers of Report
Jill Hoelle
Sanjiv Shah
US EPA Quality Assurance
Ramona Sherman
Battelle Memorial Institute
Ryan James
Ann Murdock
Technical Editing
Marti Sinclair, General Dynamics Information Technology, Inc.
External Peer Review
Marshall Gray, Exposure Reduction Consultants
Catharine Mattioli, Centers for Disease Control and Prevention
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
List of Abbreviations
AIMS
automated immunomagnetic separation
Ba
Bacillus anthracis
BaS
Bacillus anthracis Sterne
Be
Bacillus cereus
Bg
Bacillus atrophaeus (formerly Bacillus atrophaeus subsp. globigii)
Btk
Bacillus thuringiensis var. kurstaki
CARC
chemical agent resistant coating
CDC
Centers for Disease Control and Prevention
cfu
colony forming units
C102
chlorine dioxide
C02
carbon dioxide
CT
cycle threshold
DMEM
Dulbecco's Modified Eagle's Medium
DTIC
Defense Technical Information Center
ELISA
enzyme-linked immunosorbent assay
EPA
U.S. Environmental Protection Agency
FNR
false negative rate
HDIAC
Homeland Defense and Security Information Analysis Center
HPV
Hydrogen Peroxide Vapor
HVAC
heating ventilation and air conditioning
LOD
limit of detection
MCE
mixed cellulose ester
MDL
method detection limit
MPS
massively parallel sequencing
mRV-PCR
modified rapid viability polymerase chain reaction
NaDCC
sodium dichloroisocyanurate
NIOSH
National Institute for Occupational Safety and Health
NRC
National Research Council
PBST
Phosphate buffered saline with 0.02% Tween® 80
PCR
polymerase chain reaction
ppm
part per million
PTFE
polytetrafluoroethylene
RE
recovery efficiency
RT-PCR
Real-time polymerase chain reaction
RV-PCR
Rapid viability polymerase chain reaction
SAP
sampling and analysis plan
SCRD
sample collection recovery device
VSP
Visual Sampling Plan
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Table of Contents
Disclaimer ii
Foreword iii
Acknowledgments iv
List of Abbreviations v
Executive Summary vii
1. Introduction 1
2. Literature Search Methods and Quality Assurance 3
3. Contributing Factors to Clearance Risk Estimates 5
3.1 Overall Recovery Efficiency for Ba and Ba Surrogates 5
3.1.1 Surface Material Properties 6
3.1.2 Sampling Method 7
3.1.3 Transport, Storage, and Lab Variability 16
3.1.4 Surface Inoculation Methods 17
3.2 False Negatives Rates 17
3.2.1 Sample Quantity 18
3.2.2 Concentration of Contaminant 19
3.3 Limits of Detection 21
3.4 Other Factors Affecting RE, LOD, and FNR 23
3.4.1 RE for Dirty Samples 24
3.4.2 RE for Extraction Kits 24
3.4.3 RE and Surrogates 24
4. Evaluation of the Risk of Residual Contamination 25
5. Considerations for Communication of Risk to the Public 31
6. Recommendations for Further Research 32
7. References 33
Appendix A. Literature Search Keywords 37
Appendix B. Detailed Limit of Detection Summary 41
Appendix C. Recovery Efficiencies 49
Tables
Table 1. Summary of Literature Search Results 4
Table 2. Common Microbiological Sampling Methods and Parameters (11) 8
Table 3. Summary of Recovery Efficiency Ranges for Bacillus spores for Swab Sampling 10
Table 4. Summary of Recovery Efficiency Ranges for Bacillus spores for Wipe (Sponge-Sticks and
Guaze Wipes) Sampling 12
Table 5. Summary of Recovery Efficiency Ranges for Bacillus spores for Vacuum Sampling 14
Table 6. Summary of Recovery Efficiency Ranges for Bacillus Spore for Air Sampling 16
Table 7. Summary of False Negative Rates for Sample Analysis Results 20
Table 8. Summary of Environmental LOD Ranges 23
Table 9. Summary of Clearance Considerations for Example Scenario 30
Figures
Figure 1. Sampling and Analysis Inputs Flowchart 28
Figure 2. Clearance Decision Flow Chart 29
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Executive Summary
The clearance of a site after a contamination incident involves assessments of the extent of contamination,
efficacy of remediation, and the evaluation of potential risk of exposure to the contamination. Multiple
lines of evidence are applied in this determination. Among them results from environmental sampling
might not always be 100% accurate due to inefficiencies associated with sampling and analysis, possible
false negative results, and the detection limits from the methods themselves. In this report, sampling and
analysis method ranges of recovery efficiency (RE), limit of detections (LOD), and false negative rates
(FNR) for environmental surface and air samples were summarized and used to illustrate their effects on
the interpretation of sampling results in regard to clearance decisions. The scope of this document does
not cover other environmental matrices such as soil or water. A previous literature review by Herzog et al.
(2009) investigated how instrument and environmental limits of detection (LOD) for Bacillus anthracis
(Ba) analysis methods affected determination of risk to human health1. This literature search looked to
expand on that search by focusing on journal articles, reports, guidance documents, book chapters, and
conference proceedings since the 2009 article, focusing on LOD, RE, and FNR for sampling and analysis
methods for Ba and its surrogates. The resulting data from this search was used to generate this report.
Environmental sampling for site evaluation is composed of several factors that determine the reliability
and uncertainty in the reported data. The primary factors include RE of sampling methods, sampling and
analysis LOD, and FNR, and with respect to the numerous sample materials and matrices that would be
present at a contaminated site. False negatives occur when there is a failure to detect a target analyte from
a sample where the analyte is present and can be caused by biases and imprecision in any of the sampling
and analysis processes2. FNR is affected by the concentration of the contaminant, sampling processes
(overall RE and LOD), contaminant deposition methods, inhibition from co-collected organisms,
reduction in sensitivity from matrix components, and sampling methods. Using FNR to calculate the
probability of detection can be one way to analyze the risk from a certain method, procedure, or analysis.
Software tools such as the Department of Energy's Visual Sampling Plan (VSP) use inputs such as FNR
in the development of a Sampling and Analysis Plan3. Higher FNR equals less confidence in
contamination detection and clearance decisions. As surface contamination concentration increases, the
FNR decreases and is impacted by RE and LOD. Limits of detection as low as 2 colony forming units
(cfu)/sample for swabbed stainless-steel surfaces were experimentally observed but also ranged to greater
than 1 x 107 cfu/sample for recovery of spores from soil. Recovery efficiencies have a similarly large
range based on matrix type and sampling method.
Due to lack of information on human infectious doses for either inhalational, ingestional, or cutaneous
anthrax, there are no federal guidelines or documented threshold limits for cleanup of B. anthracis spores
1 Herzog, A.B., S.D. McLennan, A.K. Pandey, C.P. Gerba, C.N. Haas, J.B. Rose, S.A. Hashsham. (2009). Implications of limits
of detection of various methods for Bacillus anthracis in computing risks to human health. Appl Environ Microbiol, 75 (19):
6331-6339.
Krauter, P.A., G.F. Piepel, R. Boucher, M. Tezak, B.G. Amidan, W. Einfeld. (2012). False-negative rate and recovery
efficiency performance of a validated sponge wipe sampling method. Appl Environ Microbiol, 78 (3): 846-854.
Calfee, W., S. Lee, T. Boe. (2017). A review of biological agent sampling methods and application to a wide-area incident
scenario to characterize time and resource demands. U.S. Environmental Protection Agency, Washington, D.C.
Vll
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
below which there is acceptable low threshold of risk to human health4. Risk estimates indicate that any
detectable level of B. anthracis constitutes a tangible risk of exposure1, thus EPA and CDC have
recommended setting a clearance goal of no detection of viable spores5. Uncertainties in sampling and
measurement results complicate decision making, namely that a non-detection result might not truly be
indicative that no spores are present. The current detection limits and considerations discussed in this
report will help to define the inputs to these risk considerations. In addition, a flow chart is presented in
Figures 1 and 2 that summarize the steps involved in the clearance process and links to information in the
text that informs these decisions.
4 Rastogi, V.K., L. Wallace. (2019). Environmental sampling and bio-decontamination-recent progress, challenges, and future
direction, p. 195-208, Handbook on Biological Warfare Preparedness. Academic Press: London.
5 EPA, U.S., CDC. (2012). Interim clearance strategy for environments contaminated with Bacillus anthracis. U.S.
Environmental Protection Agency and the Centers for Disease Control and Prevetion.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
1. Introduction
Environmental agencies (local, state, federal) must determine potential risk of exposure—be it
radiological, chemical, or biological—when considering whether to reopen a site that had been
contaminated for public use. A 1997 National Research Council (NRC) book on risk-based methods for
environmental cleanup at Navy facilities states the following, "Although 'safe ' has not been found to
necessarily mean zero risk (State of Ohio v. EPA 997 F.2d 1520, 1533, D.C. Cir. 1993), the courts have
not provided (1) a risk level above which risk management action must occur, (2) specific guidance as to
what might be done to determine whether a risk is acceptable, or (3) workable definitions of acceptable,
safe risk levels. The EPA currently "endorses " a risk range from 1(T6 (one in a million) to Kf4 for one's
lifetime risk from exposure to carcinogens and a hazard quotient of 1.0 for noncarcinogens" (1). While
these ranges may be defined for carcinogens, contaminants of biological origin present a unique
combination of risks. Site evaluation and clearance in the event of a biological contamination event has
evolved significantly in the almost 20 years since the anthrax mailings and contamination events of 2001.
The resulting gaps in site remediation and clearance that were identified from that incident have sparked
many studies to better understand the factors that influence all aspects of disaster management. Despite
advancements, there are still no federal guidelines or documented threshold limits for cleanup of
hazardous bacterial spores below which there is no risk to human health (2). In 2009, Herzog (3) and
coworkers described a method for quantifying the risk of mortality from inhalation of Bacillus anthracis
(Ba) that used instrument limit of detection (LOD) (the lowest concentration of the analyte that can
reliably be detected and distinguished from a known background with a given confidence, evaluated using
pure cultures) and environmental LOD (the lowest concentration of the analyte that can reliably be
detected and distinguished from a known background with a given confidence, evaluated using
cells/culture spiked into an environmental matrix) ranges in an extensive literature search. They
concluded that their risk estimates indicate that any detectable level of Ba constitutes an unacceptable risk
(3). Thus, current recommendations for no detection of viable spores in order to clear a site (2, 4) are
fraught with complications and inherent risks, namely that a non-detect result may not truly be indicative
of no contamination.
Sampling strategies, sampling and analysis methods, understanding LODs and false negative rates (FNRs
- the proportion of samples containing contamination that are reported as non-detect), risk calculation and
communication, and other numerous aspects that are involved in site remediation and clearance all factor
into decision making when a contamination event occurs. Many individual studies have been published
that review common sampling and analysis methods. This report, however, attempts to summarize as
many of the recent developments and current technologies as possible that have been published since the
2009 Herzog review (3) so that decision makers can thoroughly consider all impacts of sampling and
remediation in the event of a biological incident. In this report, ranges of recovery efficiency (RE - the
detected response obtained from an amount of the analyte added to and extracted from the biological
matrix, compared to the detected response obtained for the true concentration of the pure authentic
standard or control). LOD. and FNR for environmental surface and air sampling techniques and analysis
methods are summarized and used to illustrate their effects on sampling results that are used to support
clearance decisions. In order to fully use these data to evaluate the clearance decision risk associated with
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
the various method limitations, a sensitivity analysis (computational analysis of the effect of changes in
input values or in assumptions on the output of a model or prediction) or a model that incorporates the
impact of each of these factors would need to be developed. This report has included much of the data
that would be required to perform such a sensitivity analysis or build such a model, but execution of this
logical next step was outside of the scope of this project.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
2. Literature Search Methods and Quality Assurance
A literature search of current journal articles, reports, guidance documents, case studies, etc. was
completed in March 2020. Because an extensive literature search was previously conducted in 2009 by
Herzog and coworkers (3), this search focused on articles published after 2009 or that were not included
in that review. The information sources were collected from existing data primarily in peer-reviewed
documents, including journal articles and government reports. The literature search included searches in
the STN® database, which offered a breadth of premier resources covering scientific and technical
information in chemistry, biomedical, pharmaceutical, intellectual property, and engineering disciplines.
STN searches numerous databases including: EMBASE™, MEDLINE®, CABA, SCISEARCH, BIOSIS,
PQSCITECH, CAPLUS. In addition, searches were also conducted in the Homeland Defense and
Security Information Analysis Center (HDIAC) managed by the Defense Technical Information Center
(DTIC), Google Scholar™. Identification of U.S. Environmental Protection Agency (EPA) research
reports that were in varying stages of completion was also undertaken.
A list of keywords was generated to encompass various sampling methods, environmental matrices,
analysis methods, site clearance and risk communication. A list of keywords used in the literature search
can be found in Appendix A. As a necessary part of limiting the scope of this report, searching was
limited to the organism of interest, Ba, and its surrogates.
Literature searches returned 374 references that contained valid search terms. The title and abstract of
each article were initially reviewed to eliminate non-relevant articles and duplicates, and to identify
highly relevant articles. During the literature search, information and secondary data sources was
qualitatively assessed according to source type to determine the trustworthiness of the
information/secondary data contained therein, based on general professional judgment of each source
type. Sources that were not peer-reviewed or government sources were to be assigned a literature review
assessment factor, however no articles were included in this review that were not peer-reviewed or
government sources. After this evaluation, two hundred and seventeen articles were identified as
potentially relevant.
Once potentially relevant references were identified, data mining was performed by reviewing the
citations listed in each source as well as cited references to identify any newer relevant publications. Data
collected from these sources were summarized and tabulated in Microsoft® Excel spreadsheets to be used
as a general reference for this report. Caution should be employed when trying to directly compare the
data mined from the literature due to: some literature not reporting how FNR, RE, and LOD were
calculated; slight differences being noted in how the calculations were accomplished; differences in
surface or material types used during the reported study; differences in pathogen used during the reported
study; and differences in overall objectives reported for the study. The citations for the literature were
recorded using the reference managing software Endnote. Electronic copies of the references were linked
to the citations. Table 1 shows a summary of the search results from the literature review.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Table 1. Summary of Literature Search Results
Type of Article/Data
Number of Resources
Sources Identified as Having High Potential Relevance
217
Journal Articles
185
Review Articles
9
Govt. Reports/Publications
16
Books/Book Chapters
12
Conference Proceedings
2
Relevant Articles Reviewed
63
Data mined from Articles with Recovery Efficiency Data
21
Data mined from Articles with Limit of Detection Data*
15
Data mined from Articles with False Negative Rate Data*
3
*Studies did not differentiate a limit of detection or false negative rate for sampling separate from the analysis method
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
3. Contributing Factors to Clearance Risk Estimates
Clearance, with regard to environmental contamination cleanup, is the process of determining whether an
outdoor area, semi-enclosed structure, indoor facility or water system is ready for reuse or reoccupancy
based on the best possible scientific evidence, including environmental samples and other data, implying
that the determination of risk to the public is at an acceptable level. Clearing a site without considering all
contributing safety factors presents risks to the public and the authorities clearing the site. Clearance
decisions are based upon numerous contributing factors, and one main factor is analytical results from
environmental sampling of the site, performed for site evaluation prior to remediation and subsequent
clearance. Environmental sampling for the detection of biological organisms like Ba may be
accomplished using methods such as swab or wipe sampling (for non-porous surfaces), the use of vacuum
cassette filtration (for porous surfaces), air sampling (for detection of initial contamination or
reaerosolization), or direct matrices sampling, such as soil collection. These samples are sent for
quantitative or semi-quantitative laboratory analysis that often include the use of viable culture or
polymerase chain reaction (PCR) to detect the presence or amount of a contaminating organism. Ideally,
the return of a non-detect result would indicate that the sample does not contain the organism in question,
but a truly critical question that all decision makers face is: What does it really mean to have no growth,
or no amplification detection results?
Undetectable contamination is not necessarily indicative of absence of contamination due to several
factors in sample processing and analysis that determine the reliability and uncertainty in the reported
data. One primary factor is the probability that a sample will produce a non-detect result when
contamination is actually present, otherwise known as a false negative result. False negatives can be
caused by the limits of the analysis method's ability to detect a target analyte at low levels, otherwise
known as LOD. LOD is also affected by the presence of possible inhibitors, or inefficiencies in processes
or methods in extracting the target organism from the environmental matrix. The sum of the effect that
these inefficiencies/inhibitors have on the ability to detect or quantify a target analyte from its
environmental matrix is quantified and compared with other methods using RE. Thus, the quantification
of FNRs, sampling and analysis LODs, and RE of sampling methods with respect to the myriad of sample
materials and matrices that would be present at a contaminated site have a vital impact on sampling and
analysis data supporting clearance decisions. The following sections describe these factors and the
expected limitations of current capabilities in providing information to guide the site clearance process.
Summaries of LOD and overall RE for the studies in this literature review can be found in Appendix B
and C, respectively.
3.1 Overall Recovery Efficiency for Ba and Ba Surrogates
Overall recovery rates or RE for Ba and Ba surrogates, as defined by a 2012 Piepel et al. report (5),
include all steps in the sample analysis process including: sample method (collection/processing/
extraction), analysis, and transportation/storage. RE influences FNR and LOD and is the factor most often
investigated in studies related to environmental sampling. FNR can be affected by all factors contributing
to the sampling process including the recovery efficiency of the sampling method (6). In 2013, Da Silva
and co-authors (7), illustrated the various losses that can occur during the recovery processes by
performing a mass balancing experiment using fluorescence microscopy and culturable plate count to
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
examine recovery rate of Ba Sterne (BaS) off of glass sides using rayon wipes, the following equations
were used to determine the overall RE and RE from the glass slide surface and the extraction tube. For
details of all calculations used refer to Da Silva et al. (2013)(7).
Where: "NdeP is the number of spores initially deposited on the coupon surface, N„ipe is the number of
spores remaining on the wipe, and Ntube is the number of spores adhered to the tube in a given extraction
solution (PBST) and was determined by inoculating the spores directly into the extraction solution. Nb+C is
the number of spores remaining on the glass slide surface after wiping. The coupons were designated 'b'
and 'c' for sampled and receiving coupons, respectively. Nsurf is the number of spores extracted from the
surface substrate obtained by placing an inoculated coupon in the extraction solution. Nsoin is the number
of spores extracted from the wipe." A correction factor, Cp/m, was included in the equation and
represented the difference in absolute cell count between plating counting (viable spores) and microscopy
(fluorescent spores) (7).
It was determined that the overall recovery efficiency was 57%, while examination of the extraction steps
illustrated that, of the unrecovered spores, 2.8% remained on the glass slide and 39.9% remained on the
wipe. For control samples (glass slide extracted in buffer in a tube), 5.8% remained on the surface of the
slide, and 1.2% remained on the extraction tube (7). A summary table of all reviewed RE data can be
found in Appendix C. The following sections discuss the variables that influence overall RE.
3.1.1 Surface Material Properties
Numerous studies have examined the RE from various surfaces and determined that the material
properties of the surface can have a large effect on RE, LOD and FNR depending upon the sampling
method used (8-10). Surfaces are usually defined as either porous or non-porous with pore size, shape,
volume, and depth all effecting sampling recovery efficiency. Examples of porous surfaces are paper,
carpet, unprocessed wood, fabric, concrete, and asphalt (11). Examples of non-porous surfaces include
stainless steel, glass, and ceramic. A 2012 study by Krauter and coworkers (8) demonstrated with wipe
sampling of Bacillus atrophaeus (Bg: formerly Bacillus atrophaeus subsp. globigii) on various surfaces
(stainless steel, vinyl, ceramic tile, primed wood paneling, faux leather, and plastic lighting panels) that
there was a roughly linear relationship between surface roughness and extraction efficiency with recovery
being more efficient on smoother surfaces (8). In 2016, Hess and coworkers (12) demonstrated with
cellulose sponge wipes that RE of Bg was highest from ceramic tile (41%), followed by stainless steel
(37%), drywall (29%), and with the lowest RE from vinyl tile (24%) (12). These results were consistent
with other studies that have suggested that nonporous surfaces have higher recovery than rough or painted
surfaces (12). Hutchison et al., 2018 (10), studied recovery of BaS and Bg using macrofoam swab
samples on glass, stainless steel, vinyl tile, and plastic ceiling tile, analyzed by both modified rapid
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
viability PCR (RV-PCR) and culturable plate count (10). Hutchison et al., 2018 (10), showed a
significantly higher FNR for vinyl tile than glass (10). It is difficult to separate the effect of material from
sampling method and, as such, the effect of surface materials on RE will also be reviewed in the
following discussion of sampling methods.
3.1.2 Sampling Meth od
Environmental samples collected from a contaminated site vary widely and depend upon the specific
materials present at the site, be it an office building, park, stadium, subway terminal, or other location. As
such, the matrices that would need to be analyzed are widely varied and could include soil, cloth, non-
porous surfaces (plastic, metal, glass), porous surfaces (carpet, wood, concrete), paper, liquid, or air.
Likewise, the sampling methods used to analyze these matrices could be equally varied, although over the
last decade certain methods have emerged as standards that are widely used including swabbing, wiping,
matrix extraction (e.g., for soil, miscellaneous solids), vacuum sampling, and air sampling. Each of these
methods has strengths and limitations. Digel et al., 2018 (11), presented a summary table, recreated in
Table 2 below, that summarizes, with detailed descriptions and pictures, the commonly used
microbiological surface sample methods and the parameters that are usually associated with them (11).
In a study to compare different sampling methods on various surfaces, Frawley et al., 2009 (13),
determined RE of BaS spores on both porous (cloth, carpet, brick, and wood) and non-porous surfaces
(plastic, glass, formica, metal) using swabs, wipes (moist and dry), sample collection and recovery
devices, trace evidence collection filters, and contact plates. As expected, RE was larger for hard non-
porous surfaces. They found that contact plates, where a petri dish filled with agar is directly placed onto
the sampling location, were significantly more efficient than swabs for porous surfaces. Moist wipes were
a factor of 4 to 7 more effective than dry wipes and were 1.75 times more effective than moist swabs.
Sample collection and recovery devices (sample collection recovery device [SCRD] paddles-ASD
BioSystems, Danville, VA) were 2 to 4 times more effective than trace evidence collection filters (3M,
Saint Paul, MN), and contact plates were 3 times more effective than moist swabs (13). As demonstrated
in this study, choosing which sampling method to use will be dependent in part on which types of
surfaces need to be sampled.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Table 2. Common Microbiological Sampling Methods and Parameters (11)
Sampling
Method
Common
Material/Tool
Process
Typical
Surface Type
Wet or
Dry
Typical target
surface size
Swabbing
Swab
Swabbing, brushing
Smooth
surfaces
Dry/Wet
Localized,
< 25 cm2
Contact
Plate
Typical/modified
plates
Printing, impression
Rough and
smooth non-
porous
Dry/rare
wet
> 25 cm2
Sponge
Sponge/Spatula
Swabbing, brushing,
sweeping
Rough and
smooth non-
porous
Dry/rare
wet
> 100 in2
Wipe
Wipe
Wiping
Smooth non-
porous
Dry/Wet
Variable,
usually
> 144 in2
Rinse
Water Solution
Rinsing, immersion
Rough and
smooth non-
porous
Dry/Wet
Variable,
usually
>100 cm2
Bulk
Tweezer
Excision
Rough and
smooth
porous and
non-porous
Dry/Wet
> 20 cm2
Scraping
Scraper, Scoop
Scraping/grinding
Rough and
smooth non-
porous
Wet, rare
dry
Variable,
usually
>100 cm2
Vacuum
Vacuum filter
Vacuuming
Rough porous
Dry
Variable,
usually
> 1000 cm2
Sonication
Ultrasonic
generator/transdu
cer
Sonication
Rough porous
Wet/rare
dry
Localized,
< 25 cm2
Brushing
Brush
Brushing, sweeping
Rough non-
porous
Mostly
dry
>100 cm2
While sampling methods are usually evaluated to include the removal of contaminants from surfaces, the
inherent extraction efficiency of material itself is important to take into consideration when analyzing RE
results. For example, Hodges et al., 2010 (14), determined higher extraction efficiency of BaS with
directly inoculated swabs versus swabs sampled from stainless steel surfaces. REs from inoculated swab
heads were approximately 69% across all concentrations tested versus 24% from swabbed stainless steel
coupons (14). Likewise, Rose et al., 2011 (15), saw higher RE with wipes directly inoculated with BaS
than wipes used to sample stainless steel surfaces (average of approximately 63.5% for directly inoculated
wipes versus an average of 29% for wipe sampled) (15).
3.1.2.1 Swab Sampling Method
Swab sampling is one method that has been used for site clearance and has been validated and published
by the Centers for Disease Control and Prevention (CDC) (14). Digel et al., 2018 (11), discussed several
common swab materials in their review including fibrous swabs (cotton, rayon, nylon, polyester,
Dacron™), spongeous swabs (macrofoam, sample collection and recovery device, polyurethane) or
hydrogel swabs (calcium alginate), and it has been shown that their composition can significantly affect
8
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
the uptake and release of bacteria (11). Swabs are best used for smaller sampling areas on surfaces that
are relatively smooth, and are advantageous for sampling hard-to-reach and irregular surfaces. Some
limitations of swab sampling methods are that only a limited sampling area is covered and that there is a
relatively low recovery efficiency on rough porous surfaces (11). Swab REs ranged from 0% for low
levels (50 spores) of BaS spores on cloth (13), to greater than 95% for high levels (1 x 106) ofBaS spores
on plastic, stainless steel, and vinyl (16). This large range of RE covers different swab types/analysis
methods, and surfaces, highlighting the effect these have on RE. For example, Probst et al., 2010 (17),
determined lower RE of Bg (5.9 and 8.8%) with cloth samples (two types of Vectran™ fabric) using
flocked nylon swabs stored in PBST and processed using vortexing and plating on R2A agar plates (17).
Whereas RE was 35.4, 45.2 and 62% for sampling on carbon fiber-reinforced plastic, roughened carbon
fiber reinforced plastic and stainless steel respectively using the same sampling methods and analysis
(17). Direct inoculation of the flocked nylon swabs with 100 cfu Bg, stored in sterile water, and extracted
using vortexting and sonication prior to culture plating resulted in a RE of 80.1% (17). Edmonds et al.,
2009 (18), used various swab types to recover Bg spores from hard non-porous surfaces with RE ranging
from 42.5 to 88.3% (18). Hong-Geller et al., 2010 (16), determined an RE of Ba Ames spores for stainless
steel and glass of 74 and 35%, respectively, but recovered just 2 and 4% for vinyl tile and plastic using
swabs. This study did not reveal the same difference in sample collection method RE between matrix type
for BaS spores, and the RE for BaS spores was higher across all materials as well (> 90%) (16). These are
just some examples of RE with swab sampling methods; Table 3 summarizes the RE ranges reviewed and
a detailed summary of RE can be found in Appendix C.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Table 3. Summary of Recovery Efficiency Ranges for Bacillus spores for Swab Sampling
Type of Matrix
Target Analyte
Inoculation
Concentration
(cfu/sample)
Analysis Method*
Overall
Recovery
Efficiency,
%**
Reference***
Bacillus atrophaeus (Bg)
lxlO2
Culturable Plate Count
5.9-8.8
(13, 17, 19)
Cloth
Bacillus anthracis Sterne (BaS)
1 x 102-1 x 105
Culturable Plate Count
0.6
(13)
Virulent Strains (Average of Bacillus
anthracis [Ba] Ames, Ba Vollum, Ba
LSU 158, and Ba LSU 62 strains)
50
Culturable Plate Count
0
(13)
BaS
3-2 xlO2
Culturable Plate Count
12-14
(19)
Carpet
Virulent Strains (Average of Ba Ames,
Ba Vollum, Ba LSU 158, and Ba LSU
62 strains)
50
Culturable Plate Count
2
(13)
Brick
Virulent Strains (Average of Ba Ames,
Ba Vollum, Ba LSU 158, and Ba LSU
62 strains)
50
Culturable Plate Count
2
(13)
Bg
2-1 x 1010
Culturable Plate Count
42.1-92.7
(18,20)
Glass
BaS
2-1x106
Culturable Plate Count,
real time quantitative
polymerase chain reaction
PCR (RT-QPCR)
74.9->90
(16,20)
Virulent Strains (Ba Ames; Average
BaAmes, Ba Vollum, BaLSU 158,
and Ba LSU 62)
50-1x106
Culturable Plate Count,
RT-QPCR
15-35
(13, 16)
Bacillus cereus (Be)
5 x 103
Culturable Plate Count
6-17
(9)
Metal
(Stainless Steel,
Bg
2-1 x 1010
Culturable Plate Count
42.5-65.5
(17, 18, 20)
tin plate,
CARC Painted
Steel)
BaS
2-1x106
Culturable Plate Count,
RT-QPCR
3.4->95%
(14, 16, 19,
20)
Virulent Strains (Ba Ames; Average
BaAmes, Ba Vollum, BaLSU 158,
and Ba LSU 62; Ba Pasteur II)
50-1x106
Culturable Plate Count,
RT-QPCR
14-75
(9, 13, 16)
Be
5 x 103
Culturable Plate Count
9
(9)
Plastic (plastic,
Formica,
polycarbonate,
polypropylene)
and plastic
laminate)
Bg
2-1 x 1010
Culturable Plate Count
35.4-88.3
(17, 18,20)
BaS
2-1x106
Culturable Plate Count,
RT-QPCR
5.5->95
(13, 16,20)
Virulent Strains (Ba Ames; Ba Pasteur
II; Average Ames, Vollum, LSU 158,
and LSU 62)
50-1x106
Culturable Plate Count,
RT-QPCR
4-15
(9, 13, 16)
Bg
2-1 x 1010
Culturable Plate Count
34.7-72.0
(18,20)
Vinyl
BaS
2-1x106
Culturable Plate Count,
RT-QPCR
50.4->95
(16,20)
Virulent strain (Ba Ames)
lxlO6
Culturable Plate Count,
RT-QPCR
2
(16)
Be
5 x 103
Culturable Plate Count
9
(9)
Wood
BaS
1 x 102-1 x 105
Culturable Plate Count
0.3-2.5
(13)
Virulent strain (Ba Pasteur II)
5 x 103
Culturable Plate Count
34
(9)
Swab Head
(direct
inoculation)
BaS
26^1.2 xlO4
Culturable Plate Count
53.8-96.7
(14,21)
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Ba, Bacillus anthracis; BaS, B. anthracis Sterne; Be, B. cereus; Bg, B. atrophaeus; CARC, chemical agent resistant coating; cfu,
colony forming units
* All analysis for studies in this table was completed using culturable plate count with the exception of the following: Hong-
Geller et al., 2010 (16), quantified of BaS and Ames from wipes using real-time quantitative polymerase chain reaction PCR (16)
"The only study to calculate FNR in the references reported in this table was Piepel et al., 2016 (20). FNR rates are listed in
Table 7 of this report.
* "References are found in the reference list at the end of the report.
3.1.2.2 Wipe Sampling Me thod
Wipes, including gauze and cellulose sponge sticks, are also one of the most often used devices for
environmental sampling, and environmental sampling protocols using these devices are also validated by
CDC. Wipes are usually employed for smooth, non-porous surfaces, usually on areas greater than 100
cm2 (11). The two main advantages of wipe sampling are that they can be employed over a larger surface
area than swabs and can be used for hard-to-reach and/or irregular spaces. Also, highly standardized
sampling procedures and data evaluation have been developed for their use. In a national validation study
of cellulose sponge wipe-processing methods, Rose et al., 2011 (15), determined the RE of BaS from a
stainless steel surface using cellulose sponge wipe sampling was 24.2 to 32.4% (15). Gahan et al., 2015
(22), demonstrated a higher RE of 88.7% with wipes sampling Bacillus thuringiensis var. kurstaki (Btk)
from a non-porous surface analyzed by PCR (22). One main drawback of wipe sampling is that it is labor
intensive for both sampling and analysis (11). Extraction usually requires use of a paddle blender (such as
the Stomacher® blender), centrifugation, vortexing and sonication. Advances in extraction methods may
help reduce processing steps. For example, Abdel-Hady et al., 2019 (23), compared the CDC-validated
cellulose wipe sampling and extraction method to a "fast" version of the CDC method (faster by
elimination of centrifugation step) using Btk sampled from glazed ceramic tiles. A significant difference
was determined between the two methods with the CDC method producing an overall RE of 39.9%
compared to the "fast" method at 54.5%. Incidentally, the "fast" method was also shown to be twice as
fast and produce half as much waste (23). Wipe RE varied greatly based on surface material and
concentration level. Two of the lowest REs from wipe samples were from a low (100 colony forming
units [cfu]) concentration of BaS sampled from wood with a RE of 0.2% (13), and no recovery was
achieved with low (5 cfu) concentrations of Bg on acrylic lighting panels (8). Alternatively, Hong-Geller
et al., 2010 (16), recovered greater than 95% of BaS from plastic vinyl and stainless steel; however, this
recovery was from a much higher initial concentration (1 x 106 cfu) (16). A recent study by Calfee et al.,
2019 (24), investigated RE from samples collected from outdoor and subway surfaces and sampled using
sponge sticks (24). Surfaces included floor tiles, concrete floors, wall tiles, glass, electronic display
panels, light fixtures, signs, and a metro card machine, and other surfaces found in and around subway
stations. Environmentally sampled sponges were then spiked with 3 concentrations (15, 150, and 1,500
cfu) of BaS spores and extracted. RE varied based on surfaces sampled and in general ranged from 10 to
50%, with no trend related to challenge concentration (24). The authors noted that background flora, due
to the environmental nature of the samples, may have obscured the target organism resulting in false
negatives for some samples. Although this study did not find any false positives when analysis was
completed for sponge-stick samples using RV-PCR, false positives were noted following analysis via
culture (24). These false positives were attributed to material being present in the sample following
sampling (24). False positives can occur for low spike levels of spores in samples. Since these sponges
were challenged directly with BaS and, thus, the removal of BaS from surfaces was not addressed, these
results are included as "wipe (direct inoculation)" in the Type of Matrix column of the summary table
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
below. For a detailed discussion on RE from these surfaces, see Calfee et al., 2019 (24). Table 4
summarizes the RE ranges seen for wipe sampling for several different surfaces. A detailed list of REs
can be found in Appendix C.
Table 4. Summary of Recovery Efficiency Ranges for Bacillus spores for Wipe (Sponge-Sticks and
Guaze Wipes) Sampling
Type of Matrix
Target Analyte
Inoculation
Concentration
(cfu/sample)
Analysis Method*
Recovery
Efficiency,
%**
Reference***
Carpet
Bacillus anthracis
Sterne (BaS)
3-2 xlO2
Culturable Plate Count
21-120
(19)
Ceramic
(glazed and
unglazed)
Bacillus atrophaeus
(Bg)
2-1.2 xlO3
Culturable Plate Count
15.17-75.5
(8, 12)
Bacillus
thuringiensis (Btk)
50-5 x103
Culturable Plate Count
36.8-64.2
(23)
Cloth
BaS
1 x 102-1 x 105
Culturable plate count
0.9^1
(13)
Drywall
Bg
5-1x102
Culturable Plate Count
3.6-29.4
(12)
Faux leather
Bg
5-1x102
Culturable Plate Count
4-76.9
(8)
Glass
BaS
1 x 102-1 x 106
Culturable Plate Count, real
time quantitative polymerase
chain reaction (RT-QPCR)
24-62
(7, 16)
Virulent strains
(Ames)
lxlO6
Culturable Plate Count, RT-
QPCR
44
(16)
Bg
5-1x102
Culturable Plate Count
0-25.8
(8)
Plastic
BaS
1 x 102-1 x 106
Culturable Plate Count, RT-
QPCR
0.9->95
(13, 16)
Virulent strains
(Ames)
lxlO6
Culturable Plate Count, RT-
QPCR
6
(16)
Bg
2-1.2 xlO3
Culturable Plate Count
24.27-63.3
(8, 12)
Stainless Steel
BaS
3-1x106
Culturable Plate Count, RT-
QPCR
18—>95
(7, 15, 16,
19)
Virulent strains
(Ames)
lxlO6
Culturable Plate Count, RT-
QPCR
49
(16)
Bg
2-1.2 xlO3
Culturable Plate Count
12.5-65
(8, 12)
Vinyl
BaS
lxlO6
Culturable Plate Count, RT-
QPCR
>95
(16)
Virulent strains
(Ames)
lxlO6
Culturable Plate Count, RT-
QPCR
4
(16)
Wipe
(direct
inoculation)
BaS
10-1x104
Culturable Plate Count, RT-
QPCR
0-110
(15,24)
Wood
Bg
5-1x102
Culturable Plate Count
10.4^19.4
(8)
BaS
1 x 102-1 x 105
Culturable Plate Count
0.2-6
(13)
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
BaS, Bacillus anthracis Sterne; Be, B. cereus; Bg, B. atrophaeus; Btk, B. thuringiensis var. kurstaki; FNR, false negative rate
* Analysis was completed using culturable plate count with the exception of the following: Hong-Geller et al., 2010 (16)
quantified of BaS and Ames from wipes using real-time qPCR (16) and Calfee et al., 2019 (24), compared recovery of BaS from
sponge-sticks using both culture and RV-PCR (24).
"The only study to calculate FNR in the references reported in this table was Krauter et al., 2012 (8). FNR rates are listed in
Table 7 of this report.
*** References are found in the reference list at the end of the report.
3.1.2.3 Vacuum Sampling Method
Vacuum methods are often the sampling process of choice for rough and/or porous surfaces and can be
used over a wider surface area, commonly greater than 1,000 cm2 (11). Vacuum based methods include
vacuum filter cassettes and socks, analysis of HVAC filters from existing building systems, and even
robotic vacuum samplers. Advantages of vacuum methods include the ability to sample large areas,
suitability for high-dust locations, ability (in the case of sampling robots) for autonomous sampling, and
the ability to sample from porous surfaces (11). The RE of various vacuum sampling methods was
determined by Calfee et al., 2013 (25), in recovering aerosol-deposited Bg spores from carpet, concrete,
and upholstery. RE was calculated by comparing vacuum samples to recovery of Bg from stainless steel
coupons by wipes (25). Relative recoveries for vacuums compared to wipes were seen from the
upholstery matrix (3.5 to 35%) (25). Relative recoveries for vacuums compared to wipes for carpet and
concrete were reported as 15.1 to 64.1% and 23.8 to 124.2%, respectively (25). In a recent study by
Calfee et al., 2019 (24), RE from vacuum filter cassettes that had been used to sample surfaces from a
subway and surrounding outdoor areas, and that were subsequently spiked with 15 to 1,500 BaS spores
and analyzed via culturable plate count, was generally less than 10% for all surfaces tested (24).
Robotic vacuum samplers have the advantage of being able to cover a much larger sampling area with
less involvement from sampling personnel. Thompson et al., 2018 (26), used various robot vacuums to
sample Bg from carpet, polyvinyl chloride, and laminate surfaces, and demonstrated low RE for all
surfaces (from < 1 to 17.1%) (26). Lee et al., 2013 (27), performed a similar study with robotic vacuum
samplers and determined higher RE of Bg from laminate and carpet surfaces (2.4 to 61.7% for laminate,
and 25.8 to 91.9% for carpet); however, the methods used for control sample comparison differed and
may be partially responsible for these discrepancies (27). An overall summary of vacuum sampling RE
ranges is presented in Table 5. For a detailed summary of vacuum sampling RE refer to Appendix C.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Table 5. Summary of Recovery Efficiency Ranges for Bacillus spores for Vacuum Sampling
Type of Matrix
Target Analyte
Inoculation
Concentration
(cfu/sample)
Analysis Method
Recovery
Efficiency,
%*
Reference*
Carpet
Bacillus anthracis
Sterne (BaS)
3-1 xlO2
Culturable Plate Count
3.7-6.3
(19)
Bacillus atrophaeus
(Bg)
1 x 106—1.94 x 109
Culturable Plate Count
<1-161.5
(25-27)
Cloth/Upholstery
Bg
9.29 xl07-2.79x 10s
Culturable plate count
3.5-35
(25)
BaS
1 x 102-1 x 105
Culturable plate count
0.7-1.7
(13)
Concrete
Bg
9.29 xl07-2.79x 10s
Culturable Plate Count
23.8-124.2
(25)
Laminate
Bg
1 xl06-1.94 xlO9
Culturable Plate Count
<1-61.7
(26, 27)
Plastic
Bg
1.94 xlO9
Culturable plate count
<1-7.3
(26)
BaS
1 x 102-lx 105
Culturable plate count
1.0^1.1
(13)
Stainless Steel
BaS
3-2 xlO2
Culturable Plate Count
3.7-5.5
(19)
Wood
BaS
1 x 102-lx 105
Culturable plate count
0.1-3.6
(13)
BaS, Bacillus anthracis Sterne; Bg, B. atrophaeus
* References are found in the reference list at the end of the report.
3.1.2.4 Air Sampling Method
Thus far only surface sampling methods have been discussed, however, another useful component of
environmental sampling after a contamination event includes air sampling. Collection of aerosol samples
can be used to detect aerosolized spores either from the main contamination event or reaerosolization
from surface contamination. Air sampling can be accomplished by using a vacuum pump to draw air
through a filter (or other aerosol sampler) specifically for the collection of aerosols for sample analysis or
by using existing heating ventilation and air conditioning (HVAC) filters, subway platform filters, or air
quality filters (e.g., native air filters) from buildings in a contaminated area. Using native air filters is one
way that wide area sampling can be accomplished, especially for evaluation of the spread of
contamination. Calfee et al., 2014 (28), examined mechanical and electrostatic HVAC filters for RE of
Bg spores sampled by both extraction of the HVAC filter material directly and sampling using vacuum
socks or 37 mm mixed cellulose ester (MCE) filters. Direct extraction of the filter material produced
higher RE (15 to 34%) than the vacuum sock or MCE filter methods (2 to 19%) (28). Factors impacting
RE of vacuum samples include vacuum time, suction pressure, size of surface area to be collected and the
density/concentration of spores across that area, direction, and extraction protocols, and vary widely
depending on the sampler used. Vacuum sock RE ranged from 2.7 to 64.1% across multiple surfaces.
Robot vacuum methods had a large range (<1 to > 100%) over all surfaces tested. A recent study by
Calfee et al., 2019 (29), investigated the use of different types of native air filters as a method to
characterize the extent of contamination after a biological contamination event using both culture and
RV-PCR analysis methods (29). RE from PM2.5 air quality filters of varying ambient particles loads
inoculated with 150 to 1,500 cfu of BaS spores were generally 40 to 80% for the average to high ambient
particle loading. With new filter material RE for the same range was 35 to 45% (29). In contrast, RE for
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
PM10 air quality filters were generally 5 to 20% although the authors point out that one reason for this
could be the differences in particle adhesion to the different filter materials (Teflon® for PM2.5 and
quartz fiber for PM10), or different levels of ambient particulate loading to start (12-16 (.ig/cnr for PM2.5
vs. 41-118 (ig/cm2 for PM10) (29). Native air filters from a bus engine, building HVAC, subway
platform, and subway rolling stock ranged from 5.3 to 63% RE (29). However, false negative and false
positive results were noted for both culture and RV-PCR analysis methods used. False negatives were
attributed to either competition of background organisms or the 0 spike scenario for analysis using culture
and particulate matter recovered in the sample or poor recovery of spores from the filter for analysis using
RV-PCR (29). Several presumptive positives for BaS following analysis via culture were determined to
be false positives following PCR confirmation due to background flora having similar colony
morphology, thus making determination of the true recovery efficiency difficult (29). The false positives
for the RV-PCR were attributed to suspected cross-contamination (29).
The U.S. EPA 2013 (30) detailed an additional aerosol sampling technique termed aggressive air
sampling that involves aggressive agitation of particles from a surface to resuspend the particles in an
aerosol form followed by aerosol collection by high volume air samplers (30). The concentration of
aerosols generated from the contaminated surface in the respirable size range can be determined and used
to assess risk of inhalation exposure, an important factor in clearance decisions (30). In this study, carpet,
laminate, and drywall surfaces were tested to determine if aggressive air sampling is an effective
technique for sampling of Ba from surfaces. A leaf blower was used to resuspend particles from coupon
surfaces that were sampled by high volume air samplers. RE from carpet using the aggressive air
sampling technique compared to the standard vacuum sock technique was approximately 1% (30).
Recovery from laminate and drywall using the aggressive air sampling technique compared to a standard
sponge wipe technique was 0.37 to 5.84% and 0.4 to 1.13% respectively (30). A 2017 EPA (31) report
examining the use of aggressive air sampling in a field environment determined that although it shows
promise, it needs more development as a sampling technique for a field environment (31).
Estill et al., 2011 (32), examined polytetrafluoroethylene (PTFE) membrane filters, gelatin filters and an
Anderson Cascade Impactor (ACI) to determine RE of aerosolized BaS spores (32). RE for all three
samples was similar and ranged from 22 to 25% (32). An overall summary of aerosol sampling RE ranges
is presented in Table 6. For a detailed summary of air sampling RE refer to Appendix C.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Table 6. Summary of Recovery Efficiency Ranges for Bacillus Spore for Air Sampling
Type of Matrix
Analyte
Inoculation
Concentration
(cfu/sample)
Analysis
Method
Recovery
Efficiency, %
Reference*
Aerosol
Bacillus anthracis
Sterne (BaS)
l.lx 102-1.9x 103
Culturable
Plate Count
22-25
(32)
Aerosol
(High Volume Aerosol
Samplers)
Bacillus atrophaeus
(Bg)
1 x 103-1 x 104
Culturable
Plate Count
0.37-5.84
(30)
2.5 Air Quality Filter
(direct inoculation)
BaS
1.5 x 102—1.5 x 103
Culturable
plate count
33-81
(29)
PM10 Air Quality
Filter (direct
inoculation)
BaS
1.5 x 102—1.5 x 103
Culturable
plate count
5.1-24
(29)
Bus engine, Building
HVAC, Subway
Platform, Subway
rolling stock
(direct inoculation)
BaS
1.5 x 102—1.5 x 103
Culturable
plate count
5.3-63
(29)
BaS, Bacillus anthracis Sterne ; Bg, B. atrophaeus; PM10, particulate matter 10 micrometers or less
* References are found in the reference list at the end of the report.
3.1.2.5 Miscellaneous Sampling Method
Although not all of these methods have been reviewed here, other sampling methods include contact
plates/tape/gel, bulk sampling of matrix, scraping, sonication, brushing, and rinsing (11). One method not
widely used is hydrogel. Smith et al., 2016 (33), used biohydrogel as a sampling method to remove Ba
Delta Sterne from pinewood, polycarbonate, painted steel, and entire screws. The biohydrogel showed
over 80% RE from pinewood and screw matrices, and 15 to 60% for polycarbonate and painted steel (33).
It could be a promising sampling method for hard-to-sample surfaces, but needs more investigation.
3.1.3 Transport, Storage, and Lab Variability
Other factors that affect REs are sample losses due to transport, storage conditions, and inconsistency
between analysts and between laboratories. Results from Rose et al., 2011 (15), on cellulose sponge sticks
determined that for BaS spores, environmental samples stored at 5 or 20°C would not lose viability nor
germinate if processed within 24 hours of sampling (15). In a 2012 review by Piepel et al. (5), who
examined 20 laboratory studies to determine RE, LOD and/or FNR of Ba/surrogate contamination
sampling methods, one major data gap that was noted was the availability of RE data on transport and
storage conditions for surface sampling methods like swabs, wipes, and vacuum samples (5).
Additionally, Perry et al., 2013 (21), showed that swabs pre-moistened with neutralizing buffer stored at -
15°C and 5°C and processed using phosphate buffered saline with 0.02% Tween® 80 (PBST) exhibited a
higher RE than swabs stored at 21 or 35°C for up to 7 days when inoculated with BaS. Swabs held at 5°C
for up to 4 days demonstrated no differences in RE from day 0 to day 5 (21). REs were above 89% for all
storage conditions, with -15, 5, and 21°C all being > 94% (21). The optimal storage conditions based on
this study (shipping in secondary containment, storing at -15°C or 5°C, and processing within 4 days)
were in line with CDC/NIOSH recommended protocols of storage and shipping at 4°C with analysis
within 48 hours. Perry et al., 2013 (21), cited a difference in recovery of up to 0.80 logio for conditions
outside these parameters (21). A novel approach of storing sampled spores on biohydrogel was also
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
investigated and was shown to be stable at 4°C for up to 5 weeks, however recovery decreased when
stored at 25 or 37°C due to spore germination and the bactericidal properties of the hydrogel (33).
Uncertainty can also be introduced from variability between different labs/analysts. Rose et al., 2011 (15),
saw total variance (including % coefficient of variation (CV) between labs and within the same labs
combined) for wipe analysis as high as 76% at low inoculum concentrations. At higher inoculum
concentrations total variance was only -30% (15). Similarly, Hodges et al., 2010 (14), determined overall
variance in swab analysis to be between 56 and 60% (14).
3.1.4 Surface Inoculation Methods
It should be noted that the deposition method for the contaminant can have effect on RE as well.
Depending upon the contamination event, surfaces could be exposed to either aerosol or liquid deposition,
however Edmonds et al., 2009 (18), noted that liquid deposition might not be well-suited for a real-world
event (18). Studies investigating RE have inoculated coupons using both methods; however, liquid
deposition methods are by far the most common as it is easier to control the inoculation concentration.
Edmonds et al., 2009 (18), investigated the effect of liquid versus aerosol deposition of Bg spores on the
ability of various swab types to sample contamination from glass coupons, chemical agent resistant
coating (CARC)-painted steel, polycarbonate, and vinyl tile (18). Depending on the swab type and surface
material, significant differences in RE were seen depending on whether the coupon had been inoculated
with an aerosol or liquid deposition (18). Lee et al., 2013 (27), saw significantly higher REs of Bg spores
from laminate and carpet surfaces using robot vacuums than Thompson et al., 2018 (26), using the same
surfaces, analyte, and vacuums; one major difference being that Lee et al. (27) challenged coupons with
an aerosol deposition and Thompson et al. (26) used a liquid deposition (26, 27). However, the calculation
method of positive control coupons has played a part in these differences as well. Lee et al. (27) sampled
positive controls using a vacuum-based method while Thompson et al. (26) used a water-soluble tape
method that may have resulted in approximately 103 better recovery on control samples (26). This further
highlights the cumulative effect that each sampling and analysis choice has in the calculation of RE.
Further specific investigation of the effects of liquid versus aerosol deposition are needed.
3.2 False Negatives Rates
Sample analysis false negatives rates are another important factor in calculating risk and developing
sampling and analysis plans (SAPs) for clearance. False negatives, or the return of a negative (or non-
detect) result for a surface that is actually contaminated, can be caused by biases and imprecision at any
point of the sampling and analysis process (8). Using FNRto calculate the probability of detection can be
one way to analyze the risk from a certain method, procedure, or analysis. For example, software tools
such as Visual Sampling Plan (VSP) use inputs such as FNR in the development of a SAP (6). When
discussing FNR, a FNR of 0 indicates no negative results were obtained for a sample or test coupon at a
specific concentration, while an FNR of 1 indicates that all results were negative for a sample or test
coupon at that concentration (10). Higher FNR equals less confidence in contamination detection and
clearance decisions. Piepel et al., 2016 (20), give the following equation for the relation of FNRto surface
concentration (20):
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
FNRhijkm = 1 - 0 (Yifc + Sik In
Where: subscripts are h = block, /' = species,/ = target surface concentration, k = material, m = coupon.
chij = mean of control coupon RE (served as reference value), 0) (yik and Sik are regression estimates which represent
shape parameters), and Aik(>()) (scale parameter that represents the surface concentration (CFU/cm) at
which FNR reaches the zero asymptote) depend on the species and surface material, and come from
experimental data for each combination of species and surface material using nonlinear weighted-least-
squares regression. See Piepel et al., 2016 (20), for a detailed description of determination of y, 8, and
coefficients using experimental data (20).
As explained by Piepel et al., 2016 (20), the equation can be used to calculate an estimate of the surface
concentration in cfu/cm2 at which the equation predicts a FNR of 0.05, or alternatively, a 95% probability
of detection if the contaminant is present (the LOD95) for each combination of species and surface
material (20). Because FNR is affected by the concentration of the contaminant, sampling processes
(overall RE), contaminant deposition methods, surface material properties (roughness, porosity, etc.),
sampling methods, and sample processing and analysis method used (5, 8, 10, 12, 20), these types of
calculations require inputs from experimental data that determine RE for the variables being evaluated. In
addition to the above discussion on RE, the following sections discuss these factors in more detail.
3.2.1 Sample Quantity
In situations where research indicates that FNRs may be high (low levels of contamination and
matrix/sampling method combinations that are known to have low RE), one way to offset FNR is by
taking more samples (5, 8). Increased sampling can cause complications with available resources (lab
analysis capacities, sampling method reagents and supplies, manpower, etc.) and the availability to
quickly turn around sample results. Rather than taking discrete samples from each sampling location,
composite sampling is one way to increase sample numbers and reduce the amount of sampling needed to
clear an area (31, 34). The decision to collect composite or discrete samples should be based on the size
of the area requiring sampling, laboratory throughput, and the applicability of composite sampling to
effectively meet the data quality objectives for the sampling event (35). During some incidents, both
composite and discrete sampling (taking from separate sampling locations) may be appropriate.
Composite sampling methods could include using one sample kit or collection device to take samples
from multiple locations as the composite sample, or to take several discrete samples and physically
combine them into a homogenous sample for extraction and analysis (35)(34). Composite sampling can
be used in conjunction with probabilistic, judgmental, or combined judgmental and probabilistic sampling
(35)(31).
Composite sampling is most cost effective when the costs for sample analysis are larger compared to the
costs for sampling (35). However, there should not be potential biases or safety hazards present when
composite sampling is employed (35). Non-target debris within the sample could potentially inhibit
detection and impact RE. The composite sampling approach allows for a dramatic reduction in sampling
needs of large areas and the number of samples needing to be processed and analyzed (12, 35, 36).
However, a major disadvantage to composite sampling is that if the sample comes back positive, then the
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
locations would need to be revisited/samples re-analyzed to determine where the contamination is located
or the entire area would need to be decontaminated and re-sampled since the composite sample covered
multiple areas (34-35). While compositing can significantly reduce the samples that need to be analyzed,
one of the drawbacks of this approach is the dilution effect that occurs for each sample as it is combined
with others and the subsequent effect dilution has on LOD if the concentration of the combined samples
are not uniform (34-36). For example, if one or a few of the sample locations were at very low, but still
detectable levels, and the remaining samples in the composite were not detectable, those low detect
samples could be diluted and appear as non-detectable (35). One solution is to restrict collection of
multiple sample locations to the same decision area (within relatively the same area) so that all samples
within that same decision area are treated with the same decision and reduce the need to determine which
or how many of the discrete samples in that location were positive (35). An additional disadvantage is
potential contaminant transfer of spores from a contaminated area to an uncontaminated area when using
the same collection device for multiple locations (34). Tufts et al., 2014 (34), suggested using only one
side of the sampling device per surface to be sampled to help mitigate this effect (34). Efficacy of
innovative composite sampling techniques such as aggressive air sampling and the robotic floor vacuum
has largely been tested in indoor or subway type settings (31, 34, 35), but more information is needed to
see how these techniques can be applied to large scale outdoor settings.
France et al., 2015 (36), examined the use of filtration to concentrate composited samples and the effect it
had on LOD (36). After samples were combined, they were filtered through a 0.2 |a,m filter and the filter
was then washed and suspended in a smaller volume. Results demonstrated that the filtration method
itself did not cause sample losses (36). Further, dilution of the sample to simulate a 333-sample composite
had the same result, suggesting that dilution effects in composite samples can be sufficiently addressed
(36). Experiments extracting B. subtilis spores from Arizona Test Dust and potting soil illuminate another
benefit of the composite analysis, namely that by combining samples that contain low concentrations (that
as a single sample would be below the LOD) those samples may be detectable if combined to form a
larger sample before filtering/concentration (36). Although the geographic resolution would be reduced,
the FNRs would be lowered as well (36). Hess et al., 2016 (12), also determined that FNRs and LOD90
were lower for composite samples than for single samples alone (12). They investigated composite
sampling but using solid surfaces sampled with cellulose sponges and found that combining several
individual sponge samples after individual extraction produced higher REs than using one sponge over
several samples (12). This type of compositing would decrease FNR but not the number of samples that
need to be collected and processed.
3.2.2 Concentration of Contaminant
The concentration of the contaminant on the surface or in the matrix that is contaminated has been shown
to affect FNRs (8, 10, 20, 37). A contaminated site would have a variety of concentration ranges from
potentially very high at the site of release (a hot spot) to very low farther from the release site or in areas
where contamination may have been spread. FNRs for wipe samples on several surfaces at varying
concentrations were calculated by Krauter et al., 2012 (8), using recovery efficiencies from experimental
data (8). Statistical analysis allowed for the relation of FNR to contaminant concentration for each
material type. LOD95 values were then calculated using the FNR data. The authors suggest that the FNR-
concentration equations developed could then be used in a real-world scenario to aid in predicting FNR.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
As discussed previously, FNR input is required for statistically determining the sample number required
to obtain the desired confidence in characterization and clearance decisions (8). Krauter et al., 2012 (8),
results indicated no dependence for RE based on concentration; however, at lower concentrations, results
were more variable. FNR decreased with increasing concentration for all surfaces, however, the
concentration threshold at which FNR reached 0 was different for different surfaces. The lowest mean
FNR was found in stainless steel (0.12) and was highest for plastic lighting panels (0.48) (8). Piepel et al.,
2016 (20), and Hutchison et al., 2016 and 2018 (10, 37), also used experimental REs and FNR to
determine LOD for collection of Bg and BaS using macrofoam swabs on various surfaces. Like others,
they demonstrated that FNR is strongly dependent on surface concentration and also surface roughness
(10, 20, 37). As inoculation concentrations increased from 2 cfu/coupon to 100 cfu/coupon, the FNR
dropped from a range of 0.5-0.83 to 0 for Bg and BaS sampled with macrofoam swabs on stainless steel,
glass, vinyl, and plastic ceiling tile (20). FNRs determined by Hutchison et al., 2018 (10), for macrofoam
swab samples of glass, stainless steel, vinyl tile, and plastic ceiling tile, analyzed by both modified RV-
PCR (mRV-PCR) and culturable plate count showed decreasing FNR with increase in inoculation
concentration (10). When using mRV-PCR, FNR dropped to < 0.2 for all surfaces at 20 cfu/coupon for
both BaS and Bg (inoculation concentrations ranged from 2 to 500 cfu/coupon). Modified RV-PCR
FNRs ranged from 0 to 0.917 for BaS and 0 to 0.875 for Bg, and no statistical difference between BaS
and Bg was observed. Hess et al., 2016 (12), observed a FNR of 0 at 100 cfu/coupon for all solid surfaces
tested using cellulose sponges (12). A summary FNR from reviewed sources is presented in Table 7.
Table 7. Summary of False Negative Rates for Sample Analysis Results
Organism
Sampling
Method
Analysis
Method
Type of
Matrix
Sample
Concentration
/Inoculum
(cfu/sample)
Average
False
Negative
Rate3
Concentration
at which FNR
drops and
stays at 0
(cfu/sample)
Max
FNRac
Reference"1
Bg
Wipe
Culture
stainless
steel
2-1200
0.12
10
0.6
(8)
ceramic
0.18
25
0.8
vinyl
0.26
100
0.93
faux leather
5-100
0.14
25
0.93
wood
0.20
25
0.73
plastic
0.48
50
1
BaS, Bg
Swab
Culture
glass
2-500
0.22, 0.24
100
0.59,0.57
(20)
plastic
2-500
0.28,0.30
100
0.83,0.74
stainless
steel
2-500
0.24, 0.26
100
0.75,0.79
vinyl
2-500
0.36,
0.36
100
0.83,0.81
mRV-PCR
glass
2-500
0.17,0.19
N/Ab, 20
0.79,0.83
(10)
plastic
2-500
0.18,0.27
N/Ab, 100
0.69,0.88
stainless
steel
2-500
0.29, 0.24
500, 100
0.92,0.71
vinyl
2-500
0.25,0.33
500, 100
0.92,0.88
BaS, Bacillus anthracis Sterne; Bg, B. atrophaeus; cfu, colony forming unit(s); FNR, false negative rate; mRV-PCR, modified
rapid viability polymerase chain reaction; N/A, not available
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
aWhere two numbers are present, first number is for BaS, second is for Bg. bFNR was variable and dropped to 0 at 20 cfu and
achieved 0.01-0.03 FNR at 500 cfu. cMax FNR typically occurred at the lowest concentration tested. References are found in the
reference list at the end of the report.
3.3 Limits of Detection
The sample analysis LOD is a variable involved in analyzing results in site evaluation and clearance and
itself is the accumulation of several inputs including sample extraction and REs, method accuracy and
specificity, and instrument limitations. It is also a main input into FNR calculations, which are important
for defining risk and making site clearance decisions. LOD can be defined several ways; most commonly
in sampling studies an empirical yes/no at a certain tested level is used to quantify the LOD. One other
common method is to calculate either the LOD90 (the lowest concentration that is reliably detected 90% of
the time) or LOD95 (the concentration for which there is a 95% probability of correct detection) (8). The
following sections describe some common analysis methods and their LODs for both control samples
(instrumental LOD which looks at pure cells or culture of the pathogen of interest) and environmental
matrices (environmental LOD where cells or culture are spiked into an environmental matrix and
subsequently processed and analyzed). A detailed summary of LOD values can be found in Appendix B.
If the LOD for a given analysis method is examined with respect to environmental matrices, the resulting
LODs can be quite different. In general, the addition of a matrix, whether it be introduced as a remnant of
the sampling method (e.g., wetting and extraction liquids) or integral to the sample itself (e.g., soil) often
increases the detection limit and is referred to as the environmental detection limit (38, 39). The LOD for
a specific method (extraction or analysis method) without the addition of environmental matrices is
defined as the method detection limit (39). Hospodsky et al., 2010 (39), determined that the method
detection limit for Bg, a surrogate for Ba, was as low as 5 cells per filter when in buffer alone (39).
However, when Bg was collected on aerosol filter material and DNA and filter extraction efficiencies
were accounted for, the detection limit increases to approximately 2000 to 3000 cells per filter (39).
Similarly, detection of Ba via PCR in buffer was as low as 5 colony forming units (cfu) but ranged from
500 to 5,000 cfu when extracted from cotton swabs, baking soda, and talcum powder (38). Gahan et al.,
2015 (22), didn't quantify the difference between controls and environmental samples, but in the absence
of sampling matrix, their PCR assay was able to detect Ba surrogates, Bacillus anthracis Sterne (BaS) and
Btk from purified DNA extract, as low as 9 and 4 gene copies, respectively (22).
Gulledge et al., 2010 (40), used PCR to determine the LOD of Ba Pasteur and Sterne strains in soil
samples using five different extraction kits combined with or without pretreatment or enrichment steps
before extraction (40). They found LOD ranging from as low as 10 spores/sample to over 107
spores/sample with sample sizes ranging from 0.1-0.5g. They determined that enrichment in growth
media overnight increased the detection of low numbers of spores (40). For a detailed review on soil
extraction methods, LODs, REs, refer to Appendix B and the 2013 EPA review on this topic (41).
Rapid viability PCR (RV-PCR) involves conducting a PCR assay on an initial sample and again after a
broth culture incubation period, and then comparing the cycle threshold (CT) values of the two. As
described by Kane et al., 2009 (42), RV-PCR has been shown to detect approximately 10 spores of Bg per
wipe sample when 2 in. by 2 in. rayon-polyester blend wipes were directly inoculated with Bg (42). This
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
method is advantageous in that it detects only viable cells (a delta Ct > 9 with an endpoint PCR Ct of <36
for the T9 aliquot relative to the TO aliquot, represents an increase viable cell concentration) and there is
no interference with live environmental background. In the scenario where a decontamination event has
occurred, but dead lysed spores may still be present, those non-viable spores would not interfere with
detection levels of viable spores. Kane et al., 2009 (42), demonstrated detection of 1 to 10 viable spores in
a background of 1 x 106 chlorine-dioxide-killed spores (42). Letant et al., 2011 (43), confirmed this by
adding 1 / 10" autoclaved Ba spores to wipes before inoculation with a 10 cfii/wipe sample viable target
concentration of Ba spores (43). The 10 cfu/sample LOD was not affected by the addition of the killed
spores in this experiment. Letant et al., 2011 (43), used RV-PCR to detect B. anthracis Ames on wipes,
air filters, and in water samples (clean, with added dust/dirt/PCR inhibitors, with added dead Ba Ames
spores, and added Bg and Pseudomonas aeruginosa bacteria) as low as 10 cfu/sample (43). During this
study, matrices were directly inoculated with organisms so extraction efficiencies may not have been
equivalent to an environmentally collected sample. Likewise, Calfee et al., 2019 (24), used RV-PCR to
detect BaS from sponge sticks inoculated with 15 to 1,500 cfu after being used to sample various surfaces
from a subway station and surrounding outdoor areas, and determined the LOD was near 15 cfu (24).
Conversely, results from the same study but using vacuum filter cassettes did not consistently detect
positives until the 1,500 cfu inoculation concentration was reached (24). A study by Hutchison et al.,
(2018) (10), using macrofoam swabs to recover BaS and Bg spores from stainless steel, glass tile, vinyl
tile, and plastic ceiling tile compared culturable results with those of mRV-PCR and produced calculated
LOD95 values (10). LOD95 values were lowest for stainless steel (11.1 cfu for BaS) and highest for vinyl
tile (23.7 cfu for BaS) and were in general also lower than the LOD95 values determined by culturable
analysis (17.5 to 26.4 cfu for BaS) (10).
Makdasi et al., 2020 (44), coupled a magnetic bead immunoassay (multiplexed to detect Ba specific lethal
factor, edema factor, and protective antigen) with a 5-hour incubation of the sample in Dulbecco's
Modified Eagle's Medium (DMEM) supplemented with 10% serum in the presence of carbon dioxide
(CO2) to detect Ba in soil at concentrations as low as 300 cfu/mL (3,000 cfu/mL were detectable with a 2-
hour incubation) (44). Detection of Ba in buffer alone was as low as 100 cfu/mL (44). Magnetic beads
coupled to antibodies specific to Ba spores as part of the automated immunomagnetic separation (AIMS)
method were used to extract spores from various soils which demonstrated a LOD via culturable plate
count of 100 spores/g (45). Similar beads have also been used in concert with mass spectrometry
(immuno-liquid chromatography-tandem mass spectrometry) to detect BaS spores in milk and soil with
LODs of 7,000 spores per mL of milk or 10 mg of soil (46).
In summary, review of the literature reveals that overall method RE was more often investigated than
analysis LOD, although it is difficult to decouple these two factors when analyzing environmental
samples. The LOD values identified for Bacillus spores from swab sampled surfaces ranged from 2 to 190
cfu/sample. The lowest LOD for swab sampling method was reported for stainless steel surfaces swabbed
with a flocked nylon swab and analyzed via culturable plate count technique (17). The highest LOD using
the swab sampling method was for moist foam swabs on stainless steel (19). Wipe sampled surfaces
returned LODs with a smaller range, from 9.7 to 25.2 cfu/sample, with the lowest from stainless steel,
ceramic tile, and faux leather analyzed via culturable plate count. Acrylic lighting panels had the highest
LOD at 25 cfu/sample for wipe samples (8). Soil and powder matrix LODs were accomplished with
various extraction kits/methods and were mostly analyzed by PCR. LOD ranges for soil/powder were 100
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
to 50,000 cfu/sample. Liquid LOD was mainly determined for buffer samples as a control for other
matrices and a range of 5 to 500 cfu/sample was mainly determined by PCR. While low LODs are
possible with genomic techniques and pure DNA samples, the lowest LOD from environmental matrices
found in this review still remains culturable analysis.
A previous review by Herzog et al., 2009 (3), summarized the state of the science at the time and the
current review focused on subsequently published literature. In the more than 10 years since that review
researchers have explored and expanded on the methods and matrices discussed, however, with few
exceptions, the basic extraction and sampling methods remain the same. The current literature review
revealed, as also found by Herzog et al., 2009 (3), that the two main analysis methods continue to be
culturable plate count methods and PCR. The development of RV-PCR, a combination of enrichment
culture and PCR, has lowered the LOD using swab sampling. When comparing detection limits from the
previous review to this one results are similar but in general with lower detection ranges for the newer
studies. The previous review by Herzog et al., 2009 (3), found the most sensitive LODs for environmental
samples were 10 cfu/sample (0.1 cfu/g) for soil (using PCR-enzyme-linked immunosorbent assay
[ELISA]), 17 cfu/L for aerosol samples (using an ELISA biochip system), 1 cfu/L for water (via culture
methods) and 1 cfu/cm2 for stainless steel (via culturable methods) (3). Table 8 summarizes LOD ranges
identified in the reviewed articles as well as ranges provided by Herzog et al., 2009 (3).
Table 8. Summary of Environmental LOD Ranges
Organism(s)
Sampling
Method
Matrices
Analysis
Methods
Limit of
Detection
(cfu/sample)
Limit of
Detection
(cfu/unit of
measure)
Limit of
Detection
(Herzog et
al.) (3)
References*
Various
Bacillus, BaS,
Bg
Swab
Acrylic
lighting panel,
Carpet, Glass,
Stainless Steel,
Vinyl Tile
Culturable
Plate Count,
mRV-PCR
2-190
0.08-1.9
cfu/cm2
1-20
cfu/cm2
(10, 17, 19,
20, 38)
Bg, BaS
Wipe
Stainless steel,
Ceramic Tile,
Faux leather,
Carpet, Primed
wood paneling,
Vinyl Tile,
Acrylic
Lighting Panel
Culturable
Plate Count
9.7-25.2
0.015-0.039
cfu/cm2
90-105
cfu/cm2
(8, 15, 19)
BaS, Bacillus anthracis Sterne; Bg, B. atrophaeus; cfu, colony forming units; mRV-PCR, modified rapid viability polymerase
chain reaction
* References are found at the end of the report.
3.4 Other Factors Affecting RE, LOD, and FNR
This section discusses additional factors that should be considered when evaluating data for use in risk
calculations and that can have an effect on RE, LOD, and FNR. This section briefly discusses RE of clean
vs. dirty samples, use of extraction kits, and the use of surrogates.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
3.4.1 RE for Dirty Samples
In a true environmental sample, surfaces are not likely to be as clean as found in a laboratory setting. To
explore this, Hess et al., 2016 (12), investigated the effect of dirty coupons versus clean coupons. Dirty
coupons were coated with "grime" consisting of 94 g Arizona test dust (ISO 12103-1, Al Ultrafine Test
Dust, Analysis 5430, Powder Technology Inc.), with and without the addition of other biological
components such as 3 grams of metro grime (collected Bay Area Rapid Transport car grime) (12). The
researchers saw that, depending on the surface material, the addition of grime either increased (vinyl tile,
stainless steel) or decreased (ceramic tile) RE with the rougher surfaces being negatively affected by the
addition of grime (12). Hodges et al., 2010 (14), added grime (PBS+0.02% Tween-80 + Arizona Test
Dust + lxlO4 spores/mL Bg + lxlO4 spores/mL Staphylococcus epidermidis) to pre-moistened swabs and
found REs using dirty swabs to be larger with the addition of grime (24.2% RE overall clean versus
41.6% RE overall dirty) (14). Calfee et al., 2019 (24), also investigated the effect of the addition of
environmental material using sponge sticks that were used to sample surfaces from a subway terminal and
surrounding outdoor area and found less accuracy (77% correct identification of BaS inoculated onto
sponges) using traditional/commonly used microbiological plate culture methods, while RV-PCR analysis
was greater than 97% (24). Thus, the amount of surface dirt/grime at a contaminated site should be
carefully considered when evaluating sample results.
3.4.2 RE for Extraction Kits
As mentioned in previous sections, the sample processing method itself can be optimized to increase RE.
This might include improving recovery from the sampling device through direct and indirect processing
and extraction of nucleic acids if PCR will be utilized (49). Several studies have examined the
effectiveness of use of various DNA extraction kits prior to analysis (38, 48). Dauphin et al., 2009 (38),
used three different commercially available extraction kits to extract DNA from swabs and powder
(baking soda, corn starch, and talcum powder). No one extraction kit was better for all matrices, which
highlights the variability in extraction methods and the importance of optimized extraction in the analysis
process. Of the three kits tested with Ba Ames spores, the UltraClean kit resulted in the lowest
concentration (500 cfu) by Real-time PCR (RT-PCR) for swabs. All three extraction kits (NucliSens®,
QIAamp®, and UltraClean®) resulted in detection of 500 cfu of Ba spores in cornstarch but for baking
soda and talcum powder the NucliSens and UltraClean kits returned 500 and 5,000 cfu, respectively (38).
Molsa et al., 2016 (48), tested four kits for the extraction of Btk DNA from potato flour and icing sugar
and found that three of the four kits had detection limits of 30 cfu (QIAamp, RTP Pathogen Kit, and
genesig Easy DNA/RNA extraction kit), while one kit (ZR Fungal/Bacterial DNA MiniPrep™ kit) had a
significantly higher LOD of 3000 cfu (48). These data highlight the differences in LOD that can occur
just from DNA extraction processes.
3.4.3 RE and Surrogates
The majority of studies reviewed in this report used surrogates for Ba. For summary tables and ranges of
RE and LOD data, all Ba strains and surrogate organisms (all Bacillus spores) were grouped together.
When using surrogate studies to inform clearance decisions it is important to note that the organisms
under question may play a large role in the efficiency calculations. This report focuses on Ba and its
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
surrogates; however, even within Bacillus strains there is variation and several studies attempted to
evaluate species differences in terms of its effect on RE. Probst et al., 2010 (17), used seven Bacillus
strains to investigate swab REs and saw that Bg, a common simulant for Ba, had a 2-fold higher RE than
the other six Bacillus strains tested (17). BaS showed marked differences in REs for vinyl and plastic
recovered via swab and wipe sampling when compared to Ba Ames in a study by Hong-Geller et al., 2010
(16), with the Sterne strain RE greater than 95% compared to Ames at 2 to 6%. Results of the two strains
on stainless steel (Ames 49-75%; Sterne > 95%) and glass surfaces (Ames 35-44%; Sterne 62-90%)
were not as substantially different but still significant (16). Conversely, Fujinami et al., 2015 (9), using
rayon swabs on various surfaces, did not see a significant difference in RE between Bacillus cereus (Be)
and Ba Pasteur II (9). Enger et al., 2018 (50), examined Ba, Be, Btk, and BaS strains for their persistence
on laminate, steel, and polystyrene over 1,038 days and found that Bg on steel had the largest
inactivation, at 56% ,and that Btk had the most similar behavior to BaS (50). The lack of depth of
information for Ba is caused by the hazards and restrictions involved with performing work with this
CDC select agent. Further studies with virulent Ba in comparison to its surrogates would be beneficial.
4. Evaluation of the Risk of Residual Contamination
In order for a surface, room, or building to be cleared for reoccupancy, no culturable organisms of interest
can be present in the selected matrix, whether from an area that was never actually contaminated or from
an area sampled post-decontamination. Knowledge of the RE, LOD, and FNR following post-
decontamination phase sample analysis ultimately supports clearance decisions. With an ideal sample
analysis plan, sampling and analysis REs would be 100% with a FNR of 0, non-detect results (no
culturable organisms) would be obtained from all samples, and there would therefore be confidence in the
subsequent decision to clear the site. However, as described above, inefficiencies and uncertainties in all
these areas are present and, thus, risk cannot be eliminated from clearance decisions. So, when a surface
sample is determined to be non-detect for Ba spore presence, what confidence can be placed upon
clearance of the site using the result? Many factors come into play when defining the values that factor
into these calculations.
Following an accidental or intentional contamination event, a site is evaluated to determine the amounts
and locations of contamination; based on these results, remediation is performed to address the
contamination and decisions are made regarding site clearance. In order to clear the site, results from
environmental sampling and evaluation can be applied to generate risk-based evaluations and inform
officials of the efficacy of the remediation and the safety of the site to the public. Risk-based approaches
to site clearance involve defining the levels of acceptable risk based on known variables. A risk level of
10"4to 10"6 (a one in 10,000 to one in one million chance of exposure) would indicate extremely low
levels of risk (1, 51). Several biological contamination event risk estimation methods are available that
include common inputs to risk-based calculations including the probability a sample is positive when not
indicated as such (or FNR) and the concentration of contaminant (taken from sampling results) (6, 51,
52). These methods can not only examine the risk inherent with current methods but can also inform
SAPs so that a known level of risk is obtained by appropriate sampling strategies.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
For example, Calfee et al., 2017 (6), used the Department of Energy's software tool VSP, to calculate
sampling criteria, number of samples, and needed resources to collect samples for a scenario including an
outdoor area of 5.75 x 107 ft2 to identify ahotspot of 10 ft2 with 95% probability of detection . It was
determined that approximately 3.6 million samples (vacuum and soil samples were the selected sample
types) would be needed to accomplish the sampling objective. An indoor scenario with similar parameters
would take approximately 26 million samples (swab/wipe/gauze and vacuum samples were the selected
sample types) (6). Of the various inputs to VSP, the estimate of required sample number was most
sensitive to sampling area and that FNR had a relatively low impact on the number of samples that needed
to be collected (6). The authors acknowledge that these estimates of sample number needed are likely
unrealistic in terms of actual implementation. However, many factors can reduce or change the number of
estimated samples including multiple lines of evidence, on-site conditions, and learning through sampling
results, etc. (6). Estimates such as these do inform the need for different sampling methods and
approaches that focus on wide area sampling, such as the use of HVAC filters from buildings in an
exposed area (6). The authors note that the RE of a sampling method is an integral part of FNR, although
VSP does not directly consider the variation in RE, as RE is not a separate input (6,8). For example,
sampling from smoother surfaces result in higher recovery efficiencies and lower FNR (8). Therefore the
RE will affect the overall FNR and, thus, does have an influence in VSP estimates (6).
Price et al., 2009 (52), presented a visual estimation tool designed to understand the factors that go into
determining what risk is acceptable and the number of samples required to support the acceptable risk
determination for a contaminated region or building (52). The inputs for this tool include the risk from 1
hour of exposure in a contaminated building, the reaerosolization of spores per square meter, the
probability a sample is a positive, and the spores/m2 (52). Using the most conservative variables, such as a
10"8 acceptable risk (from 1 hour of exposure), and high risk per dose and resuspension rate, the authors
concluded that even 0.1 spores/m2 would be an unacceptable level of contamination, and that thousands of
measurements could be made without finding one positive result (52).
Hong et al., 2010 (51), detail an approach for calculating environmental concentration standards for risk
mitigation in the event of a Ba contamination event by linking environmental sampling results with
human health risk. Calculations based on their approach generated concentration standards for both
retrospective and prospective risk mitigation as well as perfect (calculated recovery with no clumping of
spores) and imperfect (simulated recovery based off distribution of different spores clumping sizes)
sampling recovery (51). Retrospective risk is defined as the risk to which occupants were exposed in the
past, as opposed to prospective risk, which is defined as risk that occupants would be exposed in the
future. A key component in this calculation is the LOD and RE, which is informed by available
experimental data. For example, the authors used their calculations to determine the concentration
standard (for a 10"6 retrospective inhalation risk from 1 (.un particles) for floors at 0.035 spores/m2 (51).
Surface sampling to demonstrate this would require 485 m2 be sampled at 100% collection efficiency
(51). At a risk target of 10"4, the required sample area would drop to 4.9 m2 (assuming 100% recovery)
but increase to 36 m2 (which could not be covered by a single sample) if literature estimates of recovery
(median recovery = 0.38) are used (51).
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
While these examples of risk-based calculations could be used to make decisions about sampling plans
based on acceptable risk and resource availability, the standard of "no detection of viable spores" is still
the recommended criteria for clearance for the purpose of reoccupancy. Because of this, knowledge of the
sample collection, processing, and analysis RE (where available) and analysis LOD (which is determined
by RE through sample processing and analysis accuracy and specificity, and instrument limitations) is
critical to clearance decision making. Flow charts summarizing the clearance decision making process are
presented below in Figures 1 and 2. The flow chart in Figure 1 depicts determination of the sampling and
analysis inputs that will need to be considered in order to interpret the sampling and analysis results that
will be used for decision making. The sampling and analysis inputs can be affected by each of the four
pathways depicted in the flowchart including: the LOD, FNR, and RE for the sampling techniques; the
sampling matrix involved; the efficacy of the decontamination method used; and the available analysis
methods that can be used. Once the sampling and analysis inputs are determined, they can be applied to
the flow chart in Figure 2 to interpret the results. Interpretation of the results in the flow chart is split into
two pathways; one for detected analytical results and the other for non-detected analytical results. The
two pathways step the user through determining: if the site can potentially be cleared for reuse or
reoccupancy; if additional samples might need to be taken; if analysis might need to be repeated; or if
additional decontamination efforts might be necessary.
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Figure 1. Sampling and Analysis Inputs Flowchart for Surfaces and Air Samples
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Sampling and Analysis Inputs
Interpretation of
results
Non-detect
*
Clearance goal set at
no detection of viable
spores
Clearance goal set as a
threshold
Most sensitive analysis
method used?
Detection results
below threshold
Clear/reoccupy
Detection results
above threshold
Sample pooled from
wider area results?
Yes, and method
sensitivity can meet
clearance goal or
threshold
Yes, but method can't
meet clearance goal or
threshold
Clear/reoccupy
Re-sample area to
further define
contamination if
needed
Repeat
decontamination and
sampling
Repeat analysis with
more sensitive means
possible
non-detect
Repeat
decontamination and
sampling
Clear/reoccupy
• Sampling and analysis inputs inform the results from clearance sampling
o If the sample results in a detection, is the level above or below the clearance thresholds?
above threshold - perform more sampling if needed and repeat decontamination
below threshold - site can be cleared/reoccupied
o If the sample results in a non-detect, was the most sensitive analysis method used (as indicated by sampling and analysis
inputs)?
Yes, and the method is sensitive enough to meet clearance goal or threshold - clear the area
Yes, and the method is not sensitive enough to meet clearance goal or threshold - discuss next steps with
decision makers
No - repeat with most sensitive means possible
• Still non-detect - clear building
Figure 2. Clearance Decision Flow Chart
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
To further illustrate the effect RE, LOD and FNRhave on clearance decision, consider this example: if a
decision maker received the result that a macrofoam swab sample from a stainless-steel countertop
returned a detection of 30 cfu via culturable plate count analysis, what does that result mean when
sampling limitations are taken into consideration? If decontamination of the surface is completed and
resampled with a non-detect result, what is known about the confidence in the non-detect result? Taking
into account the RE for macrofoam swabs on stainless steel which ranges from approximately 16% to
85%, we can estimate that the initial contamination may range from 35 to 187.5 cfu. Subsequently, a non-
detect after decontamination must consider FNR, LOD, and RE. The limit of detection for macrofoam
swabs on stainless steel has been reported at approximately 21 cfu. Since the lowest reported RE for
macrofoam swabs is 16%, there is potential that the non-detect was false and spores could have been left
on the surface. However, the FNR for macrofoam swabs on stainless steel is low at contamination levels
of 25-100 cfu (0.028-0, although this is based on a limited data set from one study) so there is a potential
that the non-detect is reliable. However, if the concentration was reduced but not completely eliminated,
for example to 2 cfu, the post-decontamination sample may fall below the LOD when combined with RE
and the FNR as the FNR at 2 cfu is much higher (0.75) lending less credibility to the non-detect result.
This example highlights the many factors that contribute to interpretation of clearance results and is
summarized in Table 9.
Table 9. Summary of Clearance Considerations for Example Scenario
Clearance Step
Result
Considerations
Possible effects
Next Step
Site Characterization Sampling
Detect
RE of macrofoam swab on
Initial Concentration may
Decontamination
Results
(30 cfu)
stainless steel (15.8% to
be higher than 30 cfu
of surface
(Swab of stainless-steel countertop)
>85.2%)
(35 to 187.5 cfu)
Decontamination
Decontamination
Decontamination of
Contamination at low
Sampling for
(HPV decon of room)
results show
stainless-steel coupons with
levels, a 6-log reduction
clearance
> 6 log
HPV has been shown to be
should eliminate
determination
reduction
effective in > 6-log
reduction in contamination
contamination
Clearance Sampling Results after
Non-Detect
RE of analysis method
Possibility that if RE was
Clearance
decontamination
(0 culturable
(15.8 to 85.2%)
on the low end of range,
decision for
(Swab of stainless-steel countertop)
cfu)
non-detect is false
(~ 2 cfu remaining)
countertop
surface based on
FNR of analysis method
contributing
1. For stainless-steel at
1. Greater probability
factors
levels of 25-100 cfu,
that the non-detect is
FNR is low (0.028 to
reliable
0)
2. Lower probability
2. For stainless-steel at
that the non-detect is
low levels of 2 cfu,
reliable
FNR is higher (0.75)
LOD of analysis method
Contamination was likely
(20.8 cfu)
above LOD, so higher
confidence in result.
cfu, colony forming unit(s); FNR, false negative rate; HPV, hydrogen peroxide vapor; LOD, limit of detection; RE, recovery
efficiency
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
5. Considerations for Communication of Risk to the Public
The World Health Organization (WHO) defines risk communication as, "the real-time exchange of
information, advice, and opinions between experts, community leaders, or officials and the people who
are at risk" (53) and communication of risk analysis results from remediation efforts to the public and to
authorities presents a complex subject. Literature reviewed for this report did not, in general, present
many proposed solutions, rather a statement of factors involved in public communication. Minimal
literature pertaining to actual communication of technical issues to the public, such as the risk inherent
with LOD, RE, and FNR, was identified. Both of these topics would be good areas for further
investigation.
One major theme in the literature that was identified was the importance of the public's perceived trust in
the governing bodies providing information. This trust was built through caring, empathy, competence
and expertise, dedication and commitment, and honesty and openness in communication (54). A 2015
study by Malet and Korbitz (55) examined risk communication in a simulated release of anthrax spores
into a municipal water supply (55). They state that, "most local and state government officials who
participated in this study expressed unfamiliarity with agents and precautions related to bioterrorism
containment, demanded the most extensive available decontamination treatments even when they were
described as providing only marginally reduced risk despite maximal disruption to affected communities,
and expressed cynicism about the information presented to them by Federal government partners" (55).
The contaminants and the decontamination chemicals were largely unfamiliar to respondents in both
panels, and it did not appear that most participants had the knowledge or interest to weigh probabilities of
contamination or determination of levels of risk (55). This highlights the need for clear information
dissemination not just on the analysis of the risks but of general information (contaminant and
decontamination methods) so that the public and authorities that are likely not familiar with these factors
can understand the discussion, communicate effectively, and built trust and credibility in their
community.
In a study examining postal workers response to communications after the 2001 anthrax attacks,
participants reported that as awareness of their risk increased, they wanted assurance for their safety and
they pursued other sources of information when official sources were not enough (54). Openness and
honesty in reporting risks and evolving information (and following up when that information was not
immediately available) was one of the most critical factors identified from postal workers after the
anthrax attacks in 2001 (54). Although aimed at communication of risks during public health
emergencies, the 2017 WHO publication (53), Communicating Risk in Public Health Emergencies, also
identifies establishing trust and engaging with affected populations as key issues in risk communication
efforts (53).
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
6. Recommendations for Further Research
While progress has been made in filling in information gaps for environmental sampling and analysis,
there are still several significant gaps remaining. The below recommendations detail identified areas
where further information would allow for a more complete assessment of risk and the ability to
communicate that risk to the public.
• A majority of studies focused on determination of RE and did not always calculate FNR and LOD
and the few studies that focused on determination of FNR used mainly swabs and wipes. While
RE and FNR are closely related, more research on how RE and FNR are interrelated for sampling
methods other than swabs and wipes is needed.
• RE determined for various traditional sampling methods by Calfee et al., 2014 (28), is valuable
data. REs were no higher than 34% with direct extraction of the filter material, and as low as 2%
when vacuum filter methods were used (28). In addition, REs and FNRs determined by two 2019
Calfee et al. publications (23, 24) provide useful information pertaining to the RE and FNR of
both real-world air filters (HVAC, air quality sample, and vehicle) and surface wipe and vacuum
samples. Similar data for innovative sampling techniques in additional real-world settings would
aid in filling in data that could be used for risk estimates and SAP determination calculations. A
sensitivity analysis or model that evaluates the risk associated with the various method limitations
(using the data ranges discussed in this report) would be a valuable tool for risk estimation and
the effect these factors have on the risk calculations. The first step in execution of such a
sensitivity analysis would be identification or development of a model that predicts meaningful
outcomes (e.g., reduction in risk associated with clearance, cost) based on inputs for the
parameters of interest reviewed in this report. The ranges of parameter values identified in this
report can be used to construct a set of reasonable parameter value permutations that can be used
to predict outcomes from the model. The outcome results from the model based on the
permutations of these input parameter values can be utilized to assess which of the identified
parameters have the highest impact on potential outcomes, by analyzing the results for
permutations that vary one parameter while keeping the others constant. If an acceptable risk
threshold was determined in the future, this analysis could be used to inform future investments in
improvements to setting limits of detection on decontamination clearance decisions.
• Literature reviewed for this report did not, in general, present many proposed solutions for public
communication of risk. Minimal literature pertaining to actual communication of technical issues
to the public, such as the risk inherent with LOD, RE, and FNR, was identified. It seems this area
of research is underrepresented in the literature and therefore additional fundamental research in
this area may be warranted.
• Hong et al., 2010 (51), considered aerosol particle size in their estimates, which is something that
has not been well studied in the reviewed papers, but the possibility that an intentional
contamination event would occur via aerosol route is high (having happened previously). Most
laboratory studies use liquid deposition to inoculate test surfaces and the studies that do use
aerosol deposition have not investigated the effect of different particles sizes on REs or LOD.
Additional research to determine LODs and FNRs and varying particle size of aerosol
contamination may be valuable.
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Studies, 17 (1): 5-10.
Molsa, M., L. Kalin-Manttari, E. Tonteri, H. Hemmila, S. Nikkari. (2016). Comparison of four
commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked
powder samples. J Microbiol Meth, 128:69-73.
35
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
49. Silvestri, E.E., S.D. Perkins, D. Feldhake, T. Nichols, I. F. W. Schaefer. (2015). Recent literature
review of soil processing methods for recovery of Bacillus anthracis spores. Ann Microbiol, 65
(3): 1215-1226. DOI 1210.1007/sl3213-13014-10932-x
50. Enger, K.S., J. Mitchell, B. Murali, D.N. Birdsell, P. Keim, P.L. Gurian, D.M. Wagner. (2018).
Evaluating the long-term persistence of Bacillus spores on common surfaces. Microbial Biotech,
11 (6): 1048-1059.
51. Hong, T., P.L. Gurian, N.F.D. Ward. (2010). Setting risk-informed environmental standards for
Bacillus anthracis spores. Risk Anal, 30 (10): 1602-1622.
52. Price, P.N., M.D. Sohn, K.S.H. Lacommare, J.A. McWilliams. (2009). Framework for evaluating
anthrax risk in buildings. Environ Sci Technol, 43 (6): 1783-1787.
53. World Health Organization (WHO). (2017). Communicating risk in public health emergencies. A
WHO guideline for emergency risk communication (ERC) policy and practice. World Health
Orginization, Geneva.
54. Quinn, S.C., T. Thomas, C. McAllister. (2005). Postal workers' perspectives on communication
during the anthrax attack. Biosecur Bioterror, 3 (3): 207-215.
55. Malet, D., M. Korbitz. (2015). Bioterrorism and local agency preparedness: Results from an
experimental study in risk communication. JHomel Secur EmergManag, 12 (4): 861-873.
36
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Appendix A. Literature Search Keywords
Literature Review - List of Search Terms
Initial literature searching attempted to be inclusive of a wide selection of applicable terms. The
subsequent search results, however, returned a large majority of articles not relevant to the scope of this
report. Search terms were simplified, and additional searches conducted based upon related relevant
articles. The below tables provide the initial overarching search topics (as indicated in the performance
work statement), core terms based on the focus of the task order, and Boolean logic of the core terms as
well as terms that were anticipated to be used during first and second level searching using a Boolean
"AND". For example, the first level search was done using the core terms AND any of the Level 1 terms.
Additional narrowing of the search, the Level 2 search will take the Level 1 search result and return
documents that also include any of the Level 2 terms.
An example search string used is: ( TITLE-ABS ( anthra* ) AND TITLE-ABS ( fomite OR soil OR
environ* ) AND (TITLE-ABS (detect* OR sampl* OR contam* OR extract* OR recovery) W/3
( method OR limit OR efficiency ))) AND NOT TITLE-ABS ( food* OR biosensor OR sludge
OR wastewater OR anthracene). This string returned 175 results of which the most relevant based on
title/abstract were exported for further review.
Search Topic 1. Instrument and environmental detection limits for selected pathogen sampling and
analysis methods
Core Term
Level 1 Search "AND"
Level 2 Search "AND"
Bacillus anthracis
Sampling methods
Pathogen
OR
Sample processing
Detection limit
Microbiological
Detection Limits
Fomite
OR
Decontamination
Environmental
Pathogen
Detection technologies
Specificity
Identification or verification
Immunoassay
Acceptance criteria
Microarrays
Statistically significant
Biosensor
post-decontamination efficacy
Electrochemiluminescence
Decontamination assurance
Quantitation limit
Culturable
Sequencing
Environmental detection limit
Instrument detection limit
Method detection limit
Background
Environmental matrix spike
Fomite spike
Interferences
37
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Search Topic 2. Interpretation of no growth and no amplification detection results for selected
analysis methods
Core Term
Level 1 Search "AND"
Level 2 Search "AND"
Bacillus anthracis
Sampling methods
Pathogen
OR
Sample processing
No amplification
Microbiological
Detection limits
Non-detect
OR
Decontamination
Detection limit
Pathogen
Detection technologies
Culture-negative
Identification or verification
Clearance assay
Industry standard
Countable range
Acceptance criteria
No growth
Statistically significant
Viability
post-decontamination efficacy
Decontamination Assurance
Search Topic 3. Expected ranges of recovery at each sampling, processing, and analysis step for
several environmental matrices, sampling methods, and analysis methods of interest.
Core Term
Level 1 Search "AND"
Level 2 Search "AND"
Bacillus anthracis
Sampling methods
Pathogen recovery
OR
Sample processing
Extraction
Microbiological
Detection limits
Organism recovery
OR
Decontamination
Sampling methods
Pathogen
Detection technologies
Environmental
Identification or verification
Samples
Industry standard
Matrix
Acceptance criteria
Efficiency
Statistically significant
Matrices
post-decontamination efficacy
Environmental matrix
Decontamination assurance
Fomite
Microorganism recovery
Percent recovery
Collection efficiency
38
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Search Topic 4. How sampling and analysis results and recovery efficiency affect calculation of
exposure risk.
Core Term
Level 1 Search "AND"
Level 2 Search "AND"
Bacillus anthracis
Sampling methods
Recovery methods
OR
Sample processing
Recovery efficiency
Microbiological
Detection limits
Risk of exposure
OR
Decontamination
Risk of illness
Pathogen
Detection technologies
Exposure
Risk communication
Risk
Public concerns and preparations
Residual contamination
Identification or verification
Urban
Industry standard
Residue
Acceptance criteria
Microorganism recovery
Statistically significant
Pathogen recovery
post-decontamination efficacy
Reaerosolization
Decontamination assurance
Resuspension Rates
Resuspension fraction
Particle size
Inhalation
Search Topic 5. Determination of how recovery efficiency and detection limit limitations could
affect site clearance decisions
Core Term
Level 1 Search "AND"
Level 2 Search "AND"
Bacillus anthracis
Sampling methods
Re-occupancy
OR
Sample processing
Decision support
Microbiological
Detection Limits
Recovery efficiency
OR
Decontamination
Detection limit
Pathogen
Detection technologies
Risk of exposure
Risk communication
Risk of illness
Public concerns and preparations
Site clearance
Identification or verification
Acceptable risk
Industry standard
Perceived risk
Acceptance criteria
Statistically significant
post-decontamination efficacy
Decontamination assurance
39
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Search Topic 6. Impact of the above-mentioned topics on communication of risk to the public
Computing risk to human health
Core Term
Level 1 Search "AND"
Level 2 Search "AND"
Bacillus anthracis
Sampling methods
Communicating risks
OR
Sample processing
Public health risk messaging
Microbiological
Detection limits
Risk communication
OR
Decontamination
Acceptable level of risk
Pathogen
Detection technologies
Acceptable risk
Risk communication
Right to know
Public concerns and preparations
Site clearance
Identification or verification
Scientific uncertainty
Industry standard
Risk perceptions
Acceptance criteria
Public concerns (health, stigma, trust,
right to know)
Statistically significant
Evacuation decisions
Post-decontamination efficacy
Reoccupancy decisions
Decontamination assurance
40
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Appendix B. Detailed Limit of Detection Summary
Table B-l. Analytical and Environmental Limits of Detection
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size
Type of
Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Limit of
Detection
(cfu/sample)
Swabs
Probst et al.
(2010)
B. alrophaeus,
B. anthracis
Sterne 34F2,
B. megaterium
2cl ,B.
thuringensis
E24, and B.
safensis
Cotton swab
104.8
24 cm2
Stainless steel
Culturable
Plate
Count
water
Vortex/Sonication
7.6E+00
Hutchison et
al. (2018)
Bacillus
alrophaeus
Macrofoam
Swab
2-500
25.8
cm2
Stainless Steel
mRV-PCR
PBST
Vortex
8.8E+00
Hutchison et
al. (2018)
Bacillus
anthracis
Sterne
Macrofoam
Swab
2-500
25.8
cm2
Stainless Steel
mRV-PCR
PBST
Vortex
1.1E+01
Hutchison et
al. (2018)
Bacillus
anthracis
Sterne
Macrofoam
Swab
2-500
25.8
cm2
Plastic
Ceiling Panel
mRV-PCR
PBST
Vortex
1.3E+01
Hutchison et
al. (2018)
Bacillus
anthracis
Sterne
Macrofoam
Swab
2-500
25.8
cm2
Glass Tile
mRV-PCR
PBST
Vortex
1.6E+01
Hutchison et
al. (2018)
Bacillus
alrophaeus
Macrofoam
Swab
2-500
25.8
cm2
Plastic
Ceiling Panel
mRV-PCR
PBST
Vortex
1.6E+01
Hutchison et
al. (2018)
Bacillus
anthracis
Sterne
Macrofoam
Swab
2-500
25.8
cm2
Glass Tile
Culturable
Plate
Count
PBST
Vortex
1.8E+01
Hutchison et
al. (2018)
Bacillus
alrophaeus
Macrofoam
Swab
2-500
25.8
cm2
Glass Tile
mRV-PCR
PBST
Vortex
1.8E+01
Hutchison et
al. (2018)
Bacillus
anthracis
Sterne
Macrofoam
Swab
2-500
25.8
cm2
Plastic
Ceiling Panel
Culturable
Plate
Count
PBST
Vortex
2.1E+01
41
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size
Type of
Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Limit of
Detection
(cfu/sample)
Hutchison et
al. (2018)
Bacillus
atrophaeus
Macrofoam
Swab
2-500
25.8
cm2
Stainless Steel
Culturable
Plate
Count
PBST
Vortex
2.1E+01
Hutchison et
al. (2018)
Bacillus
anthracis
Sterne
Macrofoam
Swab
2-500
25.8
cm2
Vinyl Tile
m RV-
PCR
PBST
Vortex
2.4E+01
Hutchison et
al. (2018)
Bacillus
anthracis
Sterne
Macrofoam
Swab
2-500
25.8
cm2
Vinyl Tile
Culturable
Plate
Count
PBST
Vortex
2.4E+01
Hutchison et
al. (2018)
Bacillus
alrophaeus
Macrofoam
Swab
2-500
25.8
cm2
Vinyl Tile
mRV-PCR
PBST
Vortex
2.4E+01
Hutchison et
al. (2018)
Bacillus
atrophaeus
Macrofoam
Swab
2-500
25.8
cm2
Glass Tile
Culturable
Plate
Count
PBST
Vortex
2.5E+01
Hutchison et
al. (2018)
Bacillus
anthracis
Sterne
Macrofoam
Swab
2-500
25.8
cm2
Stainless Steel
Culturable
Plate
Count
PBST
Vortex
2.6E+01
Hutchison et
al. (2018)
Bacillus
atrophaeus
Macrofoam
Swab
2-500
25.8
cm2
Plastic
Ceiling Panel
Culturable
Plate
Count
PBST
Vortex
3.1E+01
Hutchison et
al. (2018)
Bacillus
atrophaeus
Macrofoam
Swab
2-500
25.8
cm2
Vinyl Tile
Culturable
Plate
Count
PBST
Vortex
3.8E+01
Piepel et al.
(2016)
Bacillus
anthracis
Sterne
Moist Foam
swab
2-500
25.8
cm2
Glass
Culturable
Plate
Count
PBST
Vortex
1.8E+01
Piepel et al.
(2016)
Bacillus
anthracis
Sterne
Moist Foam
swab
2-500
25.8
cm2
Acrylic
lighting panel
Culturable
Plate
Count
PBST
Vortex
2.1E+01
Hodges et al.
(2010)
Bacillus
anthracis
Sterne
Moist Foam
Swab
49^1.2E4
26 cm2
Stainless steel
Culturable
Plate
Count
PBST
Vortex
2.1E+01
Piepel et al.
(2016)
Bacillus
atrophaeus
Moist Foam
swab
2-500
25.8
cm2
Stainless steel
Culturable
Plate
Count
PBST
Vortex
2.1E+01
Piepel et al.
(2016)
Bacillus
anthracis
Sterne
Moist Foam
swab
2-500
25.8
cm2
Vinyl Tile
Culturable
Plate
Count
PBST
Vortex
2.4E+01
42
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size
Type of
Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Limit of
Detection
(cfu/sample)
Piepel et al.
(2016)
Bacillus
atrophaeus
Moist Foam
swab
2-500
25.8
cm2
Glass
Culturable
Plate
Count
PBST
Vortex
2.5E+01
Piepel et al.
(2016)
Bacillus
anthracis
Sterne
Moist Foam
swab
2-500
25.8
cm2
Stainless steel
Culturable
Plate
Count
PBST
Vortex
2.6E+01
Piepel et al.
(2016)
Bacillus
atrophaeus
Moist Foam
swab
2-500
25.8
cm2
Acrylic
lighting panel
Culturable
Plate
Count
PBST
Vortex
3.0E+01
Piepel et al.
(2016)
Bacillus
atrophaeus
Moist Foam
swab
2-500
25.8
cm2
Vinyl Tile
Culturable
Plate
Count
PBST
Vortex
3.8E+01
Estill et al.
(2009)
Bacillus
anthracis
Sterne
Moist Foam
Swab
3-200
100 cm2
Carpet
Culturable
Plate
Count
BBT
Vortex/Sonication
5.0E+01
Estill et al.
(2009)
Bacillus
anthracis
Sterne
Moist Foam
Swab
3-200
100 cm2
Stainless steel
Culturable
Plate
Count
BBT
Vortex/Sonication
1.9E+02
Probst et al.
(2010)
B. atrophaeus,
B. anthracis
Sterne 34F2,
B. megaterium
2cl ,B.
thuringensis
E24, and B.
safensis
Nylon
Flocked
Swab (2
protocols)
104.8
24 cm2
Stainless steel
Culturable
Plate
Count
PBST
Vortex/Sonication
2.0E+00 -
2.2E+00
Dauphin et
al. (2009)
Bacillus
anthracis
N/A
1-1E5
1 swab
head
Cotton Swab
RTPCR
PBS
V ortex/UltraC lean
extraction kit
5.0E+02
Dauphin et
al. (2009)
Bacillus
anthracis
N/A
1-1E5
1 swab
head
Cotton Swab
RTPCR
PBS
Vortex/NucliSens
extraction OR QIAamp
extraction kit
5.0E+03
Wipes
Krauter at al.
(2012)
Bacillus
atrophaeus
Cellulose
Sponge
Wipe
2-100
645.16
cm2
Stainless
steel, ceramic
tile, faux
Leather
Culturable
Plate
Count
PBST
Vortex/Sonication
9.7E+00
43
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size
Type of
Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Limit of
Detection
(cfu/sample)
Krauter at a I.
(2012)
Bacillus
atrophaeus
Cellulose
Sponge
Wipe
2-100
645.16
cm2
Primed wood
paneling
Culturable
Plate
Count
PBST
Vortex/Sonication
1.5E+01
Rose et al.
(2011)
Bacillus
anthracis
Sterne
Cellulose
Sponge
Wipe
10-1E4
645 cm2
Stainless steel
Culturable
Plate
Count
PBST
Stomacher® paddle
blender/ centrifugation
2.0E+01
Krauter at a I.
(2012)
Bacillus
atrophaeus
Cellulose
Sponge
Wipe
2-100
645.16
cm2
Vinyl tile
Culturable
Plate
Count
PBST
Vortex/Sonication
2.0E+01
Krauter at a I.
(2012)
Bacillus
atrophaeus
Cellulose
Sponge
Wipe
2-100
645.16
cm2
Acrylic
Lighting
Panel
Culturable
Plate
Count
PBST
Vortex/Sonication
2.5E+01
Estill et al.
(2009)
Bacillus
anthracis
Sterne
Moist Wipe
3-200
100 cm2
Carpet
Culturable
Plate
Count
BBT
Vortex/Sonication
9.9E+00
Estill et al.
(2009)
Bacillus
anthracis
Sterne
Moist Wipe
3-200
100 cm2
Stainless steel
Culturable
Plate
Count
BBT
Vortex/Sonication
1.5E+01
Letant et al.
(2011)
Bacillus
anthracis
Ames
N/A
10 or 100
N/A
Wipe, Air
Filter
mRV-PCR
70% of 0.25 mM
KH2PO4/0.1%
Tween 80 and
30% ethanol
Vortex
1.0E+01
Kane et al.
(2009)
Bacillus
atrophaeus
N/A
NG
25.8
cm2
Rayon-
polyester
Wipes (Clean,
with added
ATD, and
with added
killed Ba
spores)
mRV-PCR
0.25 mM
KH2P04, pH
7.2; 30% ethanol;
0.01% Tween 80
vortex
1.0E+01
Aerosol/V acuum
Estill et al.
(2011)
Bacillus
anthracis
Sterne
Air-25
mm, 3 |xm
Gelatin
Filter
1.2E2-1.4E3
120 L
Aerosol
Culturable
Plate
Count
N/A
N/A
4.7E+00
Estill et al.
(2011)
Bacillus
anthracis
Sterne
Air-37
mm, 1 |xm
PTFE filter
1.2E2-1.4E3
120 L
Aerosol
Culturable
Plate
Count
N/A
N/A
4.2E+00
44
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size
Type of
Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Limit of
Detection
(cfu/sample)
Estill et al.
(2011)
Bacillus
anthracis
Sterne
Air-
Anderson
Single stage
Impactor
1.1E2-1.9E3
112 L
Aerosol
Culturable
Plate
Count
N/A
N/A
4.0E+00
Hospodsky
et al. (2010)
Bacillus
atrophaeus
N/A
NG
1 cm2
Aerosol
Filters: Quartz
glass fiber
filter, PCTE
membrane
filter
RT-PCR
NG
MoBio extraction kit
2.1E+03 -
2.9E+04
Estill et al.
(2009)
Bacillus
anthracis
Sterne
Vacuum
Sock
3-200
100 cm2
Carpet
Culturable
Plate
Count
N/A
Shaking/ Centrifugation
2.8E+01
Estill et al.
(2009)
Bacillus
anthracis
Sterne
Vacuum
Sock
3-200
100 cm2
Stainless steel
Culturable
Plate
Count
N/A
Shaking/ Centrifugation
4.4E+01
Soil/Powder
Bradley et al.
(2011)
Bacillus
anthracis,
34F2
N/A
10-1E4
lg
Potting Soil,
Minnesota
Loam, Sand,
ATD
Culturable
Plate
Count
lOxPBST
AIMS
1.0E+02
Gulledge et
al. (2010)
Bacillus
anthracis
Sterne and
Pasteur
N/A
5-1.33E7
0.1-0.5
g
Florida Sand,
Texas Sand,
Potting Soil
PCR
Sodium
pyrophosphate,
EDTA, Tris-Cl
PLET enrichment,
MoBio UltraClean™
Soil DNA isolation kit
1.0E+02
Gulledge et
al. (2010)
Bacillus
anthracis
Sterne and
Pasteur
N/A
5-1.33E7
0.1-0.5
g
Florida Sand,
Texas Sand,
Potting Soil
PCR
Sodium
pyrophosphate,
EDTA, Tris-Cl
MagNaPure® LC
> 1.0E+07
Gulledge et
al. (2010)
Bacillus
anthracis
Sterne and
Pasteur
N/A
5-1.33E7
0.1-0.5
g
Florida Sand,
Texas Sand,
Potting Soil
PCR
Sodium
pyrophosphate,
EDTA, Tris-Cl
PLET enrichment,
Epicentre
SoilMaster™DNA
extraction kit
1.0E+01 -
1.0E+02
45
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size
Type of
Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Limit of
Detection
(cfu/sample)
Gulledge et
al. (2010)
Bacillus
anthracis
Sterne and
Pasteur
N/A
5-1.33E7
0.1-0.5
g
Florida Sand,
Texas Sand,
Potting Soil
PCR
Sodium
pyrophosphate,
EDTA, Tris-Cl
Fast DNA® SPIN
1.0E+01 -
1.0E+02
Gulledge et
al. (2010)
Bacillus
anthracis
Sterne and
Pasteur
N/A
5-1.33E7
0.1-0.5
g
Florida Sand,
Texas Sand,
Potting Soil
PCR
Sodium
pyrophosphate,
EDTA, Tris-Cl
Qiagen® BioRobot M48
workstation
1.0E+01 -
1.0E+02
Bielawaska-
Drozd et al.
(2008)
Bacillus
anthracis,
34F2, 211
N/A
10-1E8
100 g
Soil
Nested
PCR
NG
PLET
enrichment/Genomic
DNA Prep Plus kit
1E2-1E3
Chenau et al.
(2011)
Bacillus
anthracis
Sterne
N/A
1E3-1E8
10 mL
or mg
Milk, Soil
LC-
MS/MS
analysis
(HEPES/BSA)
solution
immunocapture of
spores, 80% TFA
extraction, enzymatic
digestion, and LC-
MS/MS analysis).
7.0E+03
Dauphin et
al. (2009)
Bacillus
anthracis
N/A
1-1E5
0.025 g
Baking
Powder
RT PCR
PBS
Vortex/NucliSens
extraction OR
UltraClean extraction kit
5.0E+02
Dauphin et
al. (2009)
Bacillus
anthracis
N/A
1- 1E5
0.025 g
Baking
Powder
RT PCR
PBS
V ortex/ QIAamp
extraction kit
5.0E+03
Dauphin et
al. (2009)
Bacillus
anthracis
N/A
1-1E5
0.025 g
Com Starch
RT PCR
PBS
Vortex/NucliSens OR
UltraClean OR QIAamp
extraction kit
5.0E+02
Molsa et al.
(2016)
Bacillus
thuringiensis
ssp. kurstaki-
aizawav strain
GC-91
N/A
30, 3E3, and
3E5
lg
Icing sugar,
Potato Flour
RT-PCR
N/A
QIAamp DNA Mini Kit,
RTP Pathogen Kit, Easy
DNA/RNA Extraction
Kit
3.0E+01
Molsa et al.
(2016)
Bacillus
thuringiensis
ssp. kurstaki-
aizawav strain
GC-91
N/A
30, 3E3, and
3E5
lg
Icing sugar,
Potato Flour
RT-PCR
N/A
ZR Fungal/Bacterial
DNA MiniPrep genesig
3.0E+03
Dauphin et
al. (2009)
Bacillus
anthracis
N/A
1-1E5
0.025 g
Talcum
Powder
RT-PCR
PBS
Vortex/NucliSens
extraction OR
UltraClean extraction kit
5.0E+03
46
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size
Type of
Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Limit of
Detection
(cfu/sample)
Dauphin et
al. (2009)
Bacillus
anthracis
N/A
1-1E5
0.025 g
Talcum
Powder
RT-PCR
PBS
Vortex/QIAamp
extraction kit
5.0E+04
Liquids/Controls
Dauphin et
al. (2009)
Bacillus
anthracis
N/A
2-2E5
200 uL
PBS
RT-PCR
N/A
NucliSens, QIAamp,
UltraClean extraction
kits
5.0E+00
Dauphin et
al. (2009)
Bacillus
anthracis
N/A
2-2E5
200 uL
PBS
RT-PCR
N/A
Puregene® extraction kit
5.0E+01
Dauphin et
al. (2009)
Bacillus
anthracis
N/A
2-2E5
200 uL
PBS
RT-PCR
N/A
Charge Switch
extraction kit
5.0E+02
Letant et al.
(2011)
Bacillus
anthracis
Ames
N/A
10 or 100
NG
Water
mRV-PCR
N/A
N/A
1.0E+01
Hospodsky
et al. (2010)
Bacillus
atrophaeus
N/A
NG
1 cm2
Extraction
Buffer
RT-PCR
NG
MoBio extraction kit
5.0E+00
AIMS, Automated Immunomagnetic separation; ATD, Arizona Test Dust; BBT, Butterfieldbuffer; HEPES /BSA, 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic
acid / bovine serum albumin; NG, not given; PBS, phosphate buffered saline; lOx PBST, 10X PBS + 0.05% Tween 80); PBST, PBS + 0.02% Tween 80; PTFE,
polytetrafluoroethylene; mRV-PCR, modified rapid viability- polymerase chain reaction [PCR]; PCTE, polycarbonate; RT-PCR, real time PCR.
Appendix B References
Bielawska-Drozd, A., M. Niemcewicz, M. Bartoszcze. (2008). The evaluation of methods for detection of Bacillus anthracis spores in artificially
contaminated soil samples. Polish J Environ Studies, 17 (1): 5-10.
Bradley, M.D., M.J. Arduino, J. Noble-Wang, L.J. Rose. (2011). Biological sample preparation collaboration project: Detection of detection of
Bacillus anthracis spores in soil: Final study report. U.S. Environmental Protection Agency and the Centers for Disease Control and
Prevention.
Chenau, J., F. Fenaille, E. Ezan, N. Morel, P. Lamourette, P.L. Goossens, F. Becher. (2011). Sensitive detection of Bacillus anthracis spores by
immunocapture and liquid chromatography-tandem mass spectrometry. Anal Chem, 83 (22): 8675-8682.
Dauphin, L.A., B.D. Moser, M.D. Bowen. (2009). Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus
anthracis spores and comparison of DNA yields from spores and spiked environmental samples. JMicrobiolMeth, 76 (1): 30-37.
Estill, C.F., P.A. Baron, J.K. Beard, M.J. Hein, L.D. Larsen, L. Rose, F.W. Schaefer Iii, J. Noble-Wang, L. Hodges, H.D.A. Lindquist, G.J. Deye,
M.J. Arduino. (2009). Recovery efficiency and limit of detection of aerosolized Bacillus anthracis Sterne from environmental surface samples.
Appl Environ Microbiol, 75 (13): 4297-4306.
47
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Estill, C.F., P.A. Baron, J.K. Beard, M.J. Hein, L.D. Larsen, G.J. Deye, L. Rose, L. Hodges. (2011). Comparison of air sampling methods for
aerosolized spores of B. anthracis Sterne. J Occup Environ Hyg, 8 (3): 179-186.
Gulledge, J.S., V.A. Luna, A.J. Luna, R. Zartman, A.C. Cannons. (2010). Detection of low numbers of Bacillus anthracis spores in three soils
using five commercial DNA extraction methods with and without an enrichment step. JApplMicrobiol, 109 (5): 1509-1520.
Hodges, L.R., L.J. Rose, H. O'Connell, M.J. Arduino. (2010). National validation study of a swab protocol for the recovery of Bacillus anthracis
spores from surfaces. J Microbiol Meth, 81 (2): 141-146.
Hospodsky, D., N. Yamamoto, J. Peccia. (2010). Accuracy, precision, and method detection limits of quantitative PCR for airborne bacteria and
fungi. Appl Environ Microbiol, 76 (21): 7004.
Hutchison, J.R., G.F. Piepel, B.G. Amidan, B.M. Hess, M.A. Sydor, B.L. Deatherage Kaiser. (2018). Comparison of false-negative rates and limits
of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture. J Appl
Microbiol, 124(5): 1092-1106.
Kane, S.R., S.E. Letant, G.A. Murphy, T.M. Alfaro, P.W. Krauter, R. Mahnke, T.C. Legler, E. Raber. (2009). Rapid, high-throughput, culture-
based PCR methods to analyze samples for viable spores of Bacillus anthracis and its surrogates. J Microbiol Meth, 76 (3): 278-284.
Krauter, P.A., G.F. Piepel, R. Boucher, M. Tezak, B.G. Amidan, W. Einfeld. (2012). False-negative rate and recovery efficiency performance of a
validated sponge wipe sampling method. Appl Environ Microbiol, 78 (3): 846-854.
Letant, S.E., G.A. Murphy, T.M. Alfaro, J.R. Avila, S.R. Kane, E. Raber, T.M. Bunt, S.R. Shah. (2011). Rapid-viability PCR method for detection
of live, virulent Bacillus anthracis in environmental samples. Appl Environ Microbiol, 11 (18): 6570-6578.
Molsa, M., L. Kalin-Manttari, E. Tonteri, H. Hemmila, S. Nikkari. (2016). Comparison of four commercial DNA extraction kits for the recovery
of Bacillus spp. spore DNA from spiked powder samples. J Microbiol Meth, 12869-73.
Piepel, G.F., B.L. Deatherage Kaiser, B.G. Amidan, M.A. Sydor, C.A. Barrett, J.R. Hutchison. (2016). False-negative rate, limit of detection and
recovery efficiency performance of a validated macrofoam-swab sampling method for low surface concentrations of Bacillus anthracis Sterne
and Bacillus atrophaeus spores. J Appl Microbiol, 121 (1): 149-162.
Probst, A., R. Facius, R. Wirth, C. Moissl-Eichinger. (2010). Validation of a nylon-flocked-swab protocol for efficient recovery of bacterial spores
from smooth and rough surfaces. Appl Environ Microbiol, 76 (15): 5148-5158.
Rose, L.J., L. Hodges, H. O'Connell, J. Noble-Wang. (2011). National validation study of a cellulose sponge wipe-processing method for use after
sampling Bacillus anthracis spores from surfaces. Appl Environ Microbiol, 11 (23): 8355-8359.
48
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Appendix C. Recovery Efficiencies
Table C-l. Recovery Efficiencies
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfn/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Swabs
Hong-Geller et al. (2010)
Bacillus anthracis
Ames
Swab
1.00E+06
25.8
Glass
PCR
PBST
FastDNA® Spin Kit
for Soil
35
Hong-Geller et al. (2010)
Bacillus anthracis
Sterne
Swab
1.00E+06
25.8
Glass
PCR
PBST
FastDNA Spin Kit
for Soil
90
Hong-Geller et al. (2010)
Bacillus anthracis
Ames
Swab
1.00E+06
25.8
Plastic
PCR
PBST
FastDNA Spin Kit
for Soil
4
Hong-Geller et al. (2010)
Bacillus anthracis
Sterne
Swab
1.00E+06
25.8
Plastic
PCR
PBST
FastDNA Spin Kit
for Soil
>95
Hong-Geller et al. (2010)
Bacillus anthracis
Ames
Swab
1.00E+06
25.8
Stainless Steel
PCR
PBST
FastDNA Spin Kit
for Soil
75
Hong-Geller et al. (2010)
Bacillus anthracis
Sterne
Swab
1.00E+06
25.8
Stainless Steel
PCR
PBST
FastDNA Spin Kit
for Soil
>95
Hong-Geller et al. (2010)
Bacillus anthracis
Ames
Swab
1.00E+06
25.8
Vinyl Tile
PCR
PBST
FastDNA Spin Kit
for Soil
2
Hong-Geller et al. (2010)
Bacillus anthracis
Sterne
Swab
1.00E+06
25.8
Vinyl Tile
PCR
PBST
FastDNA Spin Kit
for Soil
>95
Probst et al. (2010)
Bacillus
atrophaeus
Swab (Flocked
Nylon)
1.00E+02
25
Carbon Fiber-
Reinforced Plastic
Culturable
plate count
PBST
Vortex
35.4
49
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfn/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Probst et al. (2010)
Bacillus
atrophaeus
Swab (Flocked
Nylon)
1.00E+02
25
Roughened Carbon
Fiber-Reinforced
Plastic
Culturable
plate count
PBST
Vortex
62
Probst et al. (2010)
Bacillus anthracis
Sterne, Bacillus
atrophaeus,
Bacillus
megaterium,
Bacillus
megaterium 2cl,
Bacillus safensis,
Bacillus
thuringiensis,
Bacillus
thuringiensis E24
Swab (Flocked
Nylon)
1.00E+02
25
Stainless Steel
Culturable
plate count
PBST
Vortex
35 (average
for all strains
tested)
Probst et al. (2010)
Bacillus anthracis
Sterne, Bacillus
atrophaeus,
Bacillus
megaterium,
Bacillus
megaterium 2cl,
Bacillus safensis,
Bacillus
thuringiensis,
Bacillus
thuringiensis E24
Swab (Flocked
Nylon)
1.00E+02
25
Stainless Steel
Culturable
plate count
PBST
Vortex/Sonication
33.4 (average
for all strains
tested)
Probst et al. (2010)
Bacillus
atrophaeus
Swab (Flocked
Nylon)
1.00E+02
25
Stainless Steel,
punched and
grounded
Culturable
plate count
PBST
Vortex
45.2
Probst et al. (2010)
Bacillus
atrophaeus
Swab (Flocked
Nylon)
1.00E+02
25
Vectran Fabric
Type A
Culturable
plate count
PBST
Vortex
5.9
50
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Probst et al. (2010)
Bacillus
atrophaeus
Swab (Flocked
Nylon)
1.00E+02
25
Vectran Fabric
Type B
Culturable
plate count
PBST
Vortex
8.8
Probst et al. (2010)
Various Bacillus
spore strains
Swab (Cotton)
1.00E+02
24
Stainless Steel
Culturable
plate count
Water
Vortex/Sonication
11.4
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Cotton)
1.00E+10
10
CARC Painted
Steel(aerosol
deposition)
Culturable
plate count
PBST
Vortex
51.9
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Cotton)
2.00E+05
10
CARC Painted
Steel (liquid
deposition)
Culturable
plate count
PBST
Vortex
47
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Cotton)
1.00E+10
10
Glass(aerosol
deposition)
Culturable
plate count
PBST
Vortex
62.4
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Cotton)
2.00E+05
10
Glass (liquid
deposition)
Culturable
plate count
PBST
Vortex
88.7
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Cotton)
1.00E+10
10
Polycarbonate
(aerosol deposition)
Culturable
plate count
PBST
Vortex
65.1
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Cotton)
2.00E+05
10
Polycarbonate
(liquid deposition)
Culturable
plate count
PBST
Vortex
74.9
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Cotton)
2.00E+05
10
Vinyl Tile (liquid
deposition)
Culturable
plate count
PBST
Vortex
49
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Cotton)
1.00E+10
10
Vinyl Tile (aerosol
deposition)
Culturable
plate count
PBST
Vortex
60.3
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Dacron)
1.00E+10
10
CARC Painted
Steel(aerosol
deposition)
Culturable
plate count
PBST
Vortex
57.6
51
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfn/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
2.00E+05
10
CARC Painted
Steel liquid
deposition)
Culturable
plate count
PBST
Vortex
42.5
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
1.00E+10
10
Glass(aerosol
deposition)
Culturable
plate count
PBST
Vortex
64.9
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
2.00E+05
10
Glass (liquid
deposition)
Culturable
plate count
PBST
Vortex
82.1
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
2.01E+04
10
Glass (liquid
deposition)
Culturable
plate count
PBST
Vortex
42.1
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
1.84E+05
10
Glass (liquid
deposition)
Culturable
plate count
PBST
Vortex
61.1
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
2.21E+06
10
Glass (liquid
deposition)
Culturable
plate count
PBST
Vortex
75.8
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
2.34E+07
10
Glass (liquid
deposition)
Culturable
plate count
PBST
Vortex
92.7
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
1.00E+10
10
Polycarbonate
(aerosol deposition)
Culturable
plate count
PBST
Vortex
71.9
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
2.00E+05
10
Polycarbonate
(liquid deposition)
Culturable
plate count
PBST
Vortex
83.4
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
2.00E+05
10
Vinyl Tile (liquid
deposition)
Culturable
plate count
PBST
Vortex
62.2
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab (Dacron)
1.00E+10
10
Vinyl Tile (aerosol
deposition)
Culturable
plate count
PBST
Vortex
68.7
52
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfn/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab
(Macrofoam)
1.00E+10
10
CARC Painted
Steel(aerosol
deposition)
Culturable
plate count
PBST
Vortex
51.5
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab
(Macrofoam)
2.00E+05
10
CARC Painted
Steel (liquid
deposition)
Culturable
plate count
PBST
Vortex
55.7
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab
(Macrofoam)
1.00E+10
10
Glass(aerosol
deposition)
Culturable
plate count
PBST
Vortex
61.2
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab
(Macrofoam)
2.00E+05
10
Glass (liquid
deposition)
Culturable
plate count
PBST
Vortex
89.1
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab
(Macrofoam)
1.00E+10
10
Polycarbonate
(aerosol deposition)
Culturable
plate count
PBST
Vortex
75.5
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab
(Macrofoam)
2.00E+05
10
Polycarbonate
(liquid deposition)
Culturable
plate count
PBST
Vortex
88.3
Edmonds et al. (2009)
Bacillus
alrophaeus
Swab
(Macrofoam)
2.00E+05
10
Vinyl Tile (liquid
deposition)
Culturable
plate count
PBST
Vortex
72
53
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfn/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab
(Macrofoam)
1.00E+10
10
Vinyl Tile (aerosol
deposition)
Culturable
plate count
PBST
Vortex
67
Piepel et al. (2016)
Bacillus anthracis
Sterne
Swab
(Macrofoam)
2-500
25.8
Glass
Culturable
plate count
PBST
Vortex
82.1 (avg
over all
concentration
tested with
FNRof
0.2207)
Piepel et al. (2016)
Bacillus
alrophaeus
Swab
(Macrofoam)
2-500
25.8
Glass
Culturable
plate count
PBST
Vortex
74.9 (avg
over all
concentration
tested with
FNRof
0.2391)
Perry et al. (2013)
Bacillus anthracis
Sterne 34F2
Swab
(Macrofoam)
1.00E+02
1 swab
head
N/A-DI
Culturable
plate count
PBST
Vortex/ Sonication
(after storage
at -15°C for 7 days)
94.8
Perry et al. (2013)
Bacillus anthracis
Sterne 34F2
Swab
(Macrofoam)
1.00E+02
1 swab
head
N/A-DI
Culturable
plate count
PBST
Vortex/ Sonication
(after storage at 5°C
for 7 days)
96.7
Perry et al. (2013)
Bacillus anthracis
Sterne 34F2
Swab
(Macrofoam)
1.00E+02
1 swab
head
N/A-DI
Culturable
plate count
PBST
Vortex/Sonication
(after storage at 21°C
for 7 days)
111.8
Perry et al. (2013)
Bacillus anthracis
Sterne 34F2
Swab
(Macrofoam)
1.00E+02
1 swab
head
N/A-DI
Culturable
plate count
PBST
Vortex/ Sonication
(after storage at 35°C
for 7 days)
89.1
54
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfn/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Piepel et al. (2016)
Bacillus anthracis
Sterne
Swab
(Macrofoam)
2-500
25.8
Plastic ceiling light
cover
Culturable
plate count
PBST
Vortex
69.3 (avg
over all
concentration
tested with a
FNRof
0.2754)
Piepel et al. (2016)
Bacillus
atrophaeus
Swab
(Macrofoam)
2-500
25.8
Plastic ceiling light
cover
Culturable
plate count
PBST
Vortex
52 (avg over
all
concentration
tested with a
FNRof
0.2963)
Piepel et al. (2016)
Bacillus anthracis
Sterne
Swab
(Macrofoam)
2-500
25.8
Stainless Steel
Culturable
plate count
PBST
Vortex
68.5 (avg
over all
concentration
tested with a
FNRof
0.2384)
Piepel et al. (2016)
Bacillus
atrophaeus
Swab
(Macrofoam)
2-500
25.8
Stainless Steel
Culturable
plate count
PBST
Vortex
65.5 (avg
over all
concentration
tested with a
FNRof
0.2571)
Hodges et al. (2010)
Bacillus anthracis
Sterne
Swab
(Macrofoam)
49^1.2E4
26 cm2
Stainless Steel
Culturable
plate count
PBST
Vortex
24.2
Hodges et al. (2010)
Bacillus anthracis
Sterne
Swab
(Macrofoam)
26-3.1E4
26 cm2
Stainless Steel
Culturable
plate count
PBST-grime
Vortex
41.6
55
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfn/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Piepel et al. (2016)
Bacillus anthracis
Sterne
Swab
(Macrofoam)
2-500
25.8
Vinyl Tile
Culturable
plate count
PBST
Vortex
50.4 (avg
over all
concentration
tested with a
FNRof
0.3579)
Piepel et al. (2016)
Bacillus
atrophaeus
Swab
(Macrofoam)
2-500
25.8
Vinyl Tile
Culturable
plate count
PBST
Vortex
34.7 (avg
over all
concentration
tested
0.3634)
Estill et al. (2009)
Bacillus anthracis
Sterne
Swab (Moist
Foam Critical)
3-200
103
Carpet
Culturable
plate count
BBT
Vortex/ Sonication
12-14
Estill et al. (2009)
Bacillus anthracis
Sterne
Swab (Moist
Foam Critical)
3-200
103
Stainless Steel
Culturable
plate count
BBT
Vortex/ Sonication
5-6.5
Frawley et al. (2008)
Avg. of 4 strains
of B. anthracis
(Ames, Vollum,
LSU 158, LSU
62)
Swab (Moist
Polyester)
50
1
carpet, brick, cloth
Culturable
plate count
PBST
vortex
0-2
Frawley et al. (2008)
Avg. of 4 strains
of B. anthracis
(Ames, Vollmn,
LSU 158, LSU
62)
Swab (Moist
Polyester)
50
1
Plastic, glass,
Formica, tin plate
Culturable
plate count
PBST
vortex
8-15
Frawley et al. (2008)
B. anthracis
Sterne
Swab (Moist
Polyester)
1E2-1E5
1
Plastic, Wood,
Cloth
Culturable
plate count
PBST
vortex
2-5.5
56
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfn/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Frawley et al. (2008)
B. anthracis
Sterne
Swab (Dry
Polyester)
1E2-1E5
1
Plastic, Wood,
Cloth
Culturable
plate count
PBST
vortex
0.3-2.3
Fujinami et al. (2015)
Bacillus cereus
Swab
(Polyester)
5.0E+03
25
Stainless Steel
(mirror and hairline
finish), Luan
Plywood,
Polypropylene
Culturable
plate count
Water
Vortex
4-17.3
Fujinami et al. (2015)
Bacillus cereus
Swab (Flocked
polyamide)
5.0E+03
25
Stainless Steel
(mirror and hairline
finish), Luan
Plywood,
Polypropylene
Culturable
plate count
Water
Vortex
5-13
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Rayon)
1.00E+10
10
CARC Painted
Steel(aerosol
deposition)
Culturable
plate count
PBST
Vortex
53.1
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Rayon)
2.00E+05
10
CARC Painted
Steel liquid
deposition)
Culturable
plate count
PBST
Vortex
43.6
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Rayon)
1.00E+10
10
Glass(aerosol
deposition)
Culturable
plate count
PBST
Vortex
65.2
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Rayon)
2.00E+05
10
Glass (liquid
deposition)
Culturable
plate count
PBST
Vortex
87.5
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Rayon)
2.00E+05
10
Vinyl Tile (liquid
deposition)
Culturable
plate count
PBST
Vortex
58.3
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Rayon)
1.00E+10
10
Vinyl Tile (aerosol
deposition)
Culturable
plate count
PBST
Vortex
60.2
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Rayon)
1.00E+10
10
Polycarbonate
(aerosol deposition)
Culturable
plate count
PBST
Vortex
68.9
Edmonds et al. (2009)
Bacillus
atrophaeus
Swab (Rayon)
2.00E+05
10
Polycarbonate
(liquid deposition)
Culturable
plate count
PBST
Vortex
75.4
57
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Fujinami et al. (2015)
Bacillus cereus
Swab (Rayon)
5.00E+03
25
Luan Plywood
Culturable
plate count
Water
Vortex
9
Fujinami et al. (2015)
Bacillus anthracis
Pasteur II
Swab (Rayon)
5.00E+03
25
Luan Plywood
Culturable
plate count
Water
Vortex
34
Fujinami et al. (2015)
Bacillus cereus
Swab (Rayon)
5.00E+03
25
Polypropylene
Culturable
plate count
Water
Vortex
9
Fujinami et al. (2015)
Bacillus anthracis
Pasteur II
Swab (Rayon)
5.00E+03
25
Polypropylene
Culturable
plate count
Water
Vortex
34
Fujinami et al. (2015)
Bacillus cereus
Swab (Rayon)
5.00E+03
25
Stainless Steel
(hairline finish)
Culturable
plate count
Water
Vortex
13.7
Fujinami et al. (2015)
Bacillus anthracis
Pasteur II
Swab (Rayon)
5.00E+03
25
Stainless Steel
(hairline finish)
Culturable
plate count
Water
Vortex
19
Fujinami et al. (2015)
Bacillus cereus
Swab (Rayon)
5.00E+03
25
Stainless Steel
(mirror finish)
Culturable
plate count
Water
Vortex
6-17
Fujinami et al. (2015)
Bacillus anthracis
Pasteur II
Swab (Rayon)
5.00E+03
25
Stainless Steel
(mirror finish)
Culturable
plate count
Water
Vortex
31
Hodges et al. (2010)
Bacillus anthracis
Sterne
N/A
49^1.2E4
1 swab
head
N/A
Culturable
plate count
PBST
Vortex
69.1
Hodges et al. (2010)
Bacillus anthracis
Sterne
N/A
26-3.1E4
1 swab
head
N/A
Culturable
plate count
PBST-grime
Vortex
53.8
Wipes
Krauter et al. (2012)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge)
2-1.2E3
645.16
Ceramic tile
Culturable
plate count
PBST
Stomacher paddle
blender
32.1-75.5
(Avg. 25.6)
Abdel-Hady et al. (2019)
Bacillus
thuringiensis var.
kurstaki
Wipe (Cellulose
Sponge)
50
645.16
Ceramic Tile
(glazed)
Culturable
plate count
PBST
Stomacher paddle
blender (CDC
Method)
36.8
58
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Abdel-Hady et al. (2019)
Bacillus
thuringiensis var.
kurstaki
Wipe (Cellulose
Sponge)
5.00E+02
645.16
Ceramic Tile
(glazed)
Culturable
plate count
PBST
Stomacher paddle
blender (CDC
Method)
43
Abdel-Hady et al. (2019)
Bacillus
thuringiensis var.
kurstaki
Wipe (Cellulose
Sponge)
5.00E+03
645.16
Ceramic Tile
(glazed)
Culturable
plate count
PBST
Stomacher paddle
blender (CDC
Method)
39.9
Abdel-Hady et al. (2019)
Bacillus
thuringiensis var.
kurstaki
Wipe (Cellulose
Sponge)
50
645.16
Ceramic Tile
(glazed)
Culturable
plate count
PBST
Stomacher paddle
blender (Fast
Method)
45.2
Abdel-Hady et al. (2019)
Bacillus
thuringiensis var.
kurstaki
Wipe (Cellulose
Sponge)
5.00E+02
645.16
Ceramic Tile
(glazed)
Culturable
plate count
PBST
Stomacher paddle
blender (Fast
Method)
64.2
Abdel-Hady et al. (2019)
Bacillus
thuringiensis var.
kurstaki
Wipe (Cellulose
Sponge)
5.00E+03
645.16
Ceramic Tile
(glazed)
Culturable
plate count
PBST
Stomacher paddle
blender (Fast
Method)
54.2
Krauter et al. (2012)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge)
5-100
645.16
Faux leather
Culturable
plate count
PBST
Stomacher paddle
blender
4-49.4
(Avg. 25.5)
59
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Krauter et al. (2012)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge)
5-100
645.16
Plastic Lighting
Panel
Culturable
plate count
PBST
Stomacher paddle
blender
0-25.8
(Avg. 9.8)
Krauter et al. (2012)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge)
5-100
645.16
Primed Wood
Paneling
Culturable
plate count
PBST
Stomacher paddle
blender
10.4-49.4
(Avg. 30.3)
Krauter et al. (2012)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge)
2-1.2E3
645.16
Stainless Steel
Culturable
plate count
PBST
Stomacher paddle
blender
35.8-63.3
(Avg, 48.1)
Krauter et al. (2012)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge)
2-1.2E3
645.16
Vinyl
Culturable
plate count
PBST
Stomacher paddle
blender
12.5-65
(Avg. 48.9)
Hess et al. (2016)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge)
(multiple pass)
100
645.16
Ceramic Tile
Culturable
plate count
PBST-grime
Stomacher paddle
blender /
Centrifugation
27.3
Hess et al. (2016)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge)
(multiple pass)
100
645.16
Drywall
Culturable
plate count
PBST-grime
Stomacher paddle
blender /
Centrifugation
11.4
Hess et al. (2016)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge)
(multiple pass)
100
645.16
Stainless Steel
Culturable
plate count
PBST-grime
Stomacher paddle
blender /
Centrifugation
30.67
Hess et al. (2016)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge)
(multiple pass)
100
645.16
Vinyl Tile
Culturable
plate count
PBST-grime
Stomacher paddle
blender /
Centrifugation
56.67
Hess et al. (2016)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge) (Single
pass)
100
645.16
Ceramic Tile
Culturable
plate count
PBST-grime
Stomacher paddle
blender /
Centrifugation
15.17
Hess et al. (2016)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge) (Single
pass)
100
645.16
Drywall
Culturable
plate count
PBST-grime
Stomacher paddle
blender /
Centrifugation
3.6
Hess et al. (2016)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge) (Single
pass)
100
645.16
Stainless Steel
Culturable
plate count
PBST-grime
Stomacher paddle
blender /
Centrifugation
24.27
60
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Hess et al. (2016)
Bacillus
atrophaeus
Wipe (Cellulose
Sponge) (Single
pass)
100
645.16
Vinyl Tile
Culturable
plate count
PBST-grime
Stomacher paddle
blender /
Centrifugation
13.87
Rose et al. (2011)
Bacillus anthracis
Sterne
Wipe (Cellulose
Sponge)
10-1E4
645
Stainless Steel
Culturable
plate count
PBST (Pre-wet
with Butterfield
Buffer or Dey
Engle Broth)
Stomacher paddle
blender
26.8- 32.3
Rose et al. (2011)
Bacillus anthracis
Sterne
Wipe (Cellulose
Sponge)
10-1E4
645
Stainless Steel
Culturable
plate count
PBST
Stomacher paddle
blender
24.2-32.4
Frawley et al. (2008)
Bacillus anthracis
Sterne
Wipe (Dry
Gauze)
1E2-1E5
1
Plastic, Wood,
Cloth
Culturable
plate count
PBST
vortex
0.2-0.9
Frawley et al. (2008)
B. anthracis
Sterne
Wipe (Moist
Gauze)
1E2-1E5
1
Plastic, Wood,
Cloth
Culturable
plate count
PBST
vortex
4-6.6
Estill et al. (2009)
Bacillus anthracis
Sterne
Wipe (Moist)
3-200
929
Carpet
Culturable
plate count
BBT
Shaking/
Centrifugation
21-120
Estill et al. (2009)
Bacillus anthracis
Sterne
Wipe (Moist)
3-200
929
Stainless Steel
Culturable
plate count
BBT
Shaking/
Centrifugation
18-31
61
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfn/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Da Silvaetal. (2013)
Bacillus anthracis
Sterne (GFP
Labeled)
Wipe (Non-
woven
Polyester-
Rayon)
200
38.7
Glass
Culturable
plate count
PBS+.04%T
vortex
57.7
Da Silvaetal. (2013)
Bacillus anthracis
Sterne (GFP
Labeled)
Wipe (Non-
woven
Polyester-
Rayon)
100-200
38.7
Glass
Culturable
plate count
PBST, PBS,
T80, Water
vortex
48
Da Silvaetal. (2013)
Bacillus anthracis
Sterne (GFP
Labeled)
Wipe (Non-
woven
Polyester-
Rayon)
100-200
38.7
Stainless Steel
Culturable
plate count
PBST, PBS,
T80, Water
vortex
39
Hong-Geller et al. (2010)
Bacillus anthracis
Ames
Wipe
1.00E+06
25.8
Glass
PCR
PBST
FastDNA Spin Kit
for Soil
44
Hong-Geller et al. (2010)
Bacillus anthracis
Sterne
Wipe
1.00E+06
25.8
Glass
PCR
PBST
FastDNA Spin Kit
for Soil
62
Gahan et al. (2015)
Bacillus
thuringiensis var.
kurstaki
Wipe
NG
50
Non-porous surface
PCR
PBS
Vortex, QIAamp®
DNA extraction mini
kit
88.7
Hong-Geller et al. (2010)
Bacillus anthracis
Ames
Wipe
1.00E+06
25.8
Plastic
PCR
PBST
FastDNA Spin Kit
for Soil
6
Hong-Geller et al. (2010)
Bacillus anthracis
Sterne
Wipe
1.00E+06
25.8
Plastic
PCR
PBST
FastDNA Spin Kit
for Soil
>95
Hong-Geller et al. (2010)
Bacillus anthracis
Ames
Wipe
1.00E+06
25.8
Stainless Steel
PCR
PBST
FastDNA Spin Kit
for Soil
49
Hong-Geller et al. (2010)
Bacillus anthracis
Sterne
Wipe
1.00E+06
25.8
Stainless Steel
PCR
PBST
FastDNA Spin Kit
for Soil
>95
Hong-Geller et al. (2010)
Bacillus anthracis
Ames
Wipe
1.00E+06
25.8
Vinyl Tile
PCR
PBST
FastDNA Spin Kit
for Soil
4
Hong-Geller et al. (2010)
Bacillus anthracis
Sterne
Wipe
1.00E+06
25.8
Vinyl Tile
PCR
PBST
FastDNA Spin Kit
for Soil
>95
62
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Da Silvaetal. (2013)
Bacillus anthracis
Sterne (GFP
Labeled)
Wipe (Woven
Cotton)
100-200
38.7
Glass
Culturable
plate count
PBST, PBS,
T80, Water
vortex
50
Da Silvaetal. (2013)
Bacillus anthracis
Sterne (GFP
Labeled)
Wipe (Woven
Cotton)
100-200
38.7
Stainless Steel
Culturable
plate count
PBST, PBS,
T80, Water
vortex
23
Da Silvaetal. (2013)
Bacillus anthracis
Sterne (GFP
Labeled)
Wipe (Woven
Polyester)
100-200
38.7
Glass
Culturable
plate count
PBST, PBS,
T80, Water
vortex
24
Da Silvaetal. (2013)
Bacillus anthracis
Sterne (GFP
Labeled)
Wipe (Woven
Polyester)
100-200
38.7
Stainless Steel
Culturable
plate count
PBST, PBS,
T80, Water
vortex
29
Rose et al. (2011)
Bacillus anthracis
Sterne
Wipe (Sponge-
stick)
1.00E+04
645
Stainless Steel
Culturable
plate count
PBST
Stomacher paddle
blender
36.3
Rose et al. (2011)
Bacillus anthracis
Sterne
Wipe (Polyester
Foam sponge)
10-1E4
645
Stainless Steel
Culturable
plate count
PBST
Stomacher paddle
blender
26
Rose et al. (2011)
Bacillus anthracis
Sterne
Wipe (Rayon
Gauze)
1.00E+04
645
Stainless Steel
Culturable
plate count
PBST
Stomacher paddle
blender
30.8
Rose et al. (2011)
Bacillus anthracis
Sterne
N/A
10-1E4
1
Cellulose
sponge
N/A
Culturable
plate count
PBST
Stomacher paddle
blender
46.1 -77.9
Soil/Powder
France et al. (2015)
Bacillus subtilis
Direct
Inoculation, 10
sample
composite
2.10E+05
250 g
Arizona Test Dust
(ATD)
Culturable
plate count
BBT
Shake/Vortex
followed by
Filtration/ Digestion
99.5
63
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
France et al. (2015)
Bacillus subtilis
Direct
Inoculation, 20
sample
composite
2.10E+05
250 g
ATD
Culturable
plate count
BBT
Shake/Vortex
followed by
Filtration/ Digestion
82.9
France et al. (2015)
Bacillus subtilis
Direct
Inoculation, 4
sample
composite
2.10E+05
250 g
ATD
Culturable
plate count
BBT
Shake/Vortex
followed by
Filtration/ Digestion
91.3
France et al. (2015)
Bacillus subtilis
Direct
Inoculation,
Single Sample
2.10E+05
250 g
ATD
Culturable
plate count
BBT
Shake/Vortex
followed by
Filtration/ Digestion
86.7
France et al. (2015)
Bacillus subtilis
Direct
Inoculation, 4
sample
composite
2.10E+05
250 g
Potting Soil
Culturable
plate count
BBT
Shake/Vortex
followed by
Filtration/ Digestion
88
France et al. (2015)
Bacillus subtilis
Direct
Inoculation,
Single Sample
2.10E+05
250 g
Potting Soil
Culturable
plate count
BBT
Shake/Vortex
followed by
Filtration/ Digestion
96.6
Bradley et al. (2011)
Bacillus
anthracis, 34F2
N/A
10-1E4
lg
Sand, ATD, Potting
Soil, Minnesota
Loam
Culturable
plate count
lOxPBST
HSGS
0.75-9
Bradley et al. (2011)
Bacillus
anthracis, 34F2
N/A
1E4-1E6
lg
Sand, ATD, Potting
Soil, Minnesota
Loam
Culturable
plate count
lOxPBST
AIMS
15-68
Contact Plates
Frawley et al. (2008)
Bacillus anthracis
Sterne
Agar Contact
plates
1E-1E5
1
Carpet, Brick,
Synthetic Cloth
Culturable
plate count
PBST
vortex
3-5
64
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Frawley et al. (2008)
Bacillus anthracis
Sterne
Agar Contact
plates
1E2-1E5
1
Plastic, Glass,
Formica, Metal
Culturable
plate count
PBST
vortex
28-54
Air/Vacuum
Estill etal. (2011)
Bacillus anthracis
Sterne
Air - 25 mm, 3
|xm Gelatin
Filter
1.2E2-1.4E3
120 L
Aerosol
Culturable
plate count
N/A
N/A
22
Estill etal. (2011)
Bacillus anthracis
Sterne
Air - 37 mm, 1
|xm PTFE filter
1.2E2-1.4E3
120 L
Aerosol
Culturable
plate count
N/A
N/A
25
Estill etal. (2011)
Bacillus anthracis
Sterne
Air - Anderson
Single stage
Impactor
1.1E2-1.9E3
112 L
Aerosol
Culturable
plate count
N/A
N/A
25
EPA 2013
Bacillus
atrophaeus
Eligh Volume
Aerosol
Sampler
1E3-1E4
35.56 cm2
Carpet
Culturable
plate count
PBST
Stomacher paddle
blender
1.06-1.1
EPA 2013
Bacillus
atrophaeus
Eligh Volume
Aerosol
Sampler
1E3-1E4
35.56 cm2
Laminate
Culturable
plate count
PBST
Stomacher paddle
blender
0.37-5.84
EPA 2013
Bacillus
atrophaeus
Eligh Volume
Aerosol
Sampler
1E3-1E4
35.56 cm2
Drywall
Culturable
plate count
PBST
Stomacher paddle
blender
0.4-1.13
Hospodsky et al. (2010)
Bacillus
atrophaeus
N/A
NG
1 cm2
Quartz glass fiber
filter, PCTE
membrane filter
RT-PCR
NG3
MoBio extraction kit
3.4^1.8
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(i-robot Scooba
390)
1.94E+09
25000
Carpet
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
12
65
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(i-robot Scooba
390)
1.94E+09
25000
Laminate
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
< 1
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(i-robot Scooba
390)
1.94E+09
25000
PVC
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
7.3
Lee et al. (2013)
Bacillus
atrophaeus
Robot Vacuum
(Evolution
Robotics, Mint
4200)
1E6-1E7
5112.25
Laminate
Culturable
plate count
PBST
Mopping Cloth
stomached
61.7
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(Hoover
Robo.com2 RB
C009)
1.94E+09
25000
Carpet
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
< 1
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(Hoover
Robo.com2 RB
C009)
1.94E+09
25000
Laminate
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
4.5
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(Hoover
Robo.com2 RB
C009)
1.94E+09
25000
PVC
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
3
Lee et al. (2013)
Bacillus
atrophaeus
Robot Vacuum
(i-robot
Roomba 760)
1E6-1E7
5112.25
Carpet
Culturable
plate count
PBST
Shaken 300 rpm
25.8
Lee et al. (2013)
Bacillus
atrophaeus
Robot Vacuum
(i-Robot
Roomba 760)
1E6-1E7
5112.25
Laminate
Culturable
plate count
PBST
Shaken 300 rpm
8.1
66
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(i-Robot
Roomba 770)
1.94E+09
25000
Carpet
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
0.2
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(i-Robot
Roomba 770)
1.94E+09
25000
Laminate
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
11
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(i-Robot
Roomba 770)
1.94E+09
25000
PVC
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
< 1
Lee et al. (2013)
Bacillus
atrophaeus
Robot Vacuum
(i-Robot Scooba
360)
1E6-1E7
5112.25
Laminate
Culturable
plate count
PBST
Shaken 300 rpm,
Liquid collected
31.9
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(Moneual
MR6800)
1.94E+09
25000
Carpet
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
< 1
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(Moneual
MR6800)
1.94E+09
25000
Laminate
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
17.1
Thompson et al. (2018)
Bacillus
atrophaeus
Robot Vacuum
(Moneual
MR6800)
1.94E+09
25000
PVC
Culturable
plate count
PBMA
Swabbing of
collection bin
/stomaching of Filter
material
3
Lee et al. (2013)
Bacillus
atrophaeus
Robot Vacuum
(Neato robotics
XV-11)
1E6-1E7
5112.25
Carpet
Culturable
plate count
PBST
Shaken 300 rpm
161.5
67
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Lee et al. (2013)
Bacillus
alrophaeus
Robot Vacuum
(Neato robotics
XV-11)
1E6-1E7
5112.25
Laminate
Culturable
plate count
PBST
Shaken 300 rpm
10.9
Lee et al. (2013)
Bacillus
alrophaeus
Robot Vacuum
(P3
international P3
P4920)
1E6-1E7
5112.25
Laminate
Culturable
plate count
PBST
Shaken 300 rpm
2.4
Lee et al. (2013)
Bacillus
alrophaeus
Robot Vacuum
(P3
international P3
P4920)
1E6-1E7
5112.25
Carpet
Culturable
plate count
PBST
Shaken 300 rpm
91.9
Calfee et al. (2013)
Bacillus
alrophaeus
Vacuum - 37
mm MCE Filter
Cassette
9.29E+07
929
Carpet
Culturable
plate count
PBST
Sonication/
Vortex/Rinse/
Sonication
47.4
Calfee et al. (2013)
Bacillus
alrophaeus
Vacuum - 37
mm MCE Filter
Cassette
9.29E+07
929
Concrete
Culturable
plate count
PBST
Sonication/
Vortex/Rinse/
Sonication
124.2
Calfee et al. (2014)
Bacillus
alrophaeus
Vacuum - 37
mm MCE Filter
Cassette
~1E8
929
HVAC filter
(electrostatic and
mechanical)
Culturable
plate count
PBST
Sonication/
Vortex/Rinse/
Sonication
2-19
Calfee et al. (2013)
Bacillus
alrophaeus
Vacuum - 37
mm MCE Filter
Cassette
9.29E+07
929
Upholstery
Culturable
plate count
PBST
Sonication/
Vortex/Rinse/
Sonication
35
Calfee et al. (2013)
Bacillus
alrophaeus
Vacuum - 37
mm PTFE Filter
Cassette
9.29E+07
929
Carpet
Culturable
plate count
PBST
Sonication/
Vortex/Rinse/
Sonication
20.2
Calfee et al. (2013)
Bacillus
alrophaeus
Vacuum - 37
mm PTFE Filter
Cassette
9.29E+07
929
Concrete
Culturable
plate count
PBST
Sonication/
Vortex/Rinse/
Sonication
23.8
Calfee et al. (2013)
Bacillus
alrophaeus
Vacuum - 37
mm PTFE Filter
Cassette
9.29E+07
929
Upholstery
Culturable
plate count
PBST
Sonication/
Vortex/Rinse/
Sonication
12.9
68
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Estill et al. (2009)
Bacillus anthracis
Sterne
Vacuum sock
3-200
929
Carpet
Culturable
plate count
BBT
Shaking/
Centrifugation
2.7-6.3
Estill et al. (2009)
Bacillus anthracis
Sterne
Vacuum sock
3-200
929
Stainless Steel
Culturable
plate count
BBT
Shaking/
Centrifugation
3.7-4.5
Calfeeetal. (2013)
Bacillus
atrophaeus
Vacuum Sock -
Fast
2.79 E8
2787
Carpet
Culturable
plate count
PBST
Shaking
38.7
Calfee et al. (2013)
Bacillus
atrophaeus
Vacuum Sock -
Fast
2.79 E8
2787
Concrete
Culturable
plate count
PBST
Shaking
29.9
Calfeeetal. (2013)
Bacillus
atrophaeus
Vacuum Sock -
Fast
2.79 E8
2787
Upholstery
Culturable
plate count
PBST
Shaking
23.2
Calfeeetal. (2013)
Bacillus
atrophaeus
Vacuum Sock -
Slow
2.79 E8
2787
Carpet
Culturable
plate count
PBST
Shaking
64.1
Calfeeetal. (2013)
Bacillus
atrophaeus
Vacuum Sock -
Slow
2.79 E8
2787
Concrete
Culturable
plate count
PBST
Shaking
25.9
Calfeeetal. (2013)
Bacillus
atrophaeus
Vacuum Sock -
Slow
2.79 E8
2787
Upholstery
Culturable
plate count
PBST
Shaking
10.6
Calfee et al. (2014)
Bacillus
atrophaeus
Vacuum Sock
~1E8
929
HVAC Filter
(Electrostatic and
Mechanical)
Culturable
plate count
PBST
Shaking
4-10
Calfeeetal. (2013)
Bacillus
atrophaeus
Vacuum:
Forensic
Evidence Filter
9.29E+07
929
Carpet
Culturable
plate count
PBST
Stomacher paddle
blender /
Centrifugation
15.1
69
-------
Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
Reference
Organism
Sampling
Method
Inoculation
Concentration
(cfu/sample)
Matrix
size (cm2)
Type of Matrix
Analysis
Method
Extraction
Liquid
Extraction Method
Recovery
Efficiency,
%
Calfeeetal. (2013)
Bacillus
atrophaeus
Vacuum:
Forensic
Evidence Filter
9.29E+07
929
Concrete
Culturable
plate count
PBST
Stomacher paddle
blender /
Centrifugation
33
Calfeeetal. (2013)
Bacillus
atrophaeus
Vacuum:
Forensic
Evidence Filter
9.29E+07
929
Upholstery
Culturable
plate count
PBST
Stomacher paddle
blender /
Centrifugation
3.5
Frawley et al. (2008)
Bacillus anthracis
Sterne
Sample
collection and
recovery
devices
1E2-1E5
1
Plastic, Wood,
Cloth
Culturable
plate count
PBST
vortex
1.7-4.1
Frawley et al. (2008)
Bacillus anthracis
Sterne
Trace Evidence
Collection
filters
1E2-1E5
1
Plastic, Wood,
Cloth
Culturable
plate count
PBST
vortex
0.1-1
Calfee et al. (2014)
Bacillus
atrophaeus
Vacuum Sock
~1E8
929
HVAC Filter
(Mechanical)
Culturable
plate count
PBST
Shaking
26-34
Calfee et al. (2014)
Bacillus
atrophaeus
Vacuum Sock
~1E8
929
HVAC Filter
(Electrostatic)
Culturable
plate count
PBST
Shaking
15-22
Hydrogel
Smith et al. (2016)
Bacillus anthracis
Delta Sterne
BioHydrogel
1.00E+07
19.3548
Painted Steel
Culturable
plate count
PBS+.01%T
Rehydration
15-60
Smith et al. (2016)
Bacillus anthracis
Delta Sterne
BioHydrogel
1.00E+07
19.3548
Pinewood
Culturable
plate count
PBS+.01%T
Rehydration
>80
Smith et al. (2016)
Bacillus anthracis
Delta Sterne
BioHydrogel
1.00E+07
19.3548
Polycarbonate
Culturable
plate count
PBS+.01%T
Rehydration
15-60
Smith et al. (2016)
Bacillus anthracis
Delta Sterne
BioHydrogel
1.00E+07
19.3548
Screw
Culturable
plate count
PBS+.01%T
Rehydration
>80
AIMS, Automated Immunomagnetic separation; ATD, Arizona Test Dust; BBT, Butterfield buffer; HSGS, High Specific Gravity Sucrose Extraction; N/A-DI,
direct inoculation onto swab head; inRV-PCR, modified rapid viability- polymerase chain reaction [PCR]; NG, not given; PBMA, Phosphate buffer with Manucol
70
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Understanding Detection Limits within the Interim Clearance Goal for Bacillus anthracis Contamination
and antifoam; PBS+.01%T, PBS+0.01% Tween 80; PBS+.04%T, PBS = 0.04% Tween 80; lOx PBST, 10X phosphate buffered saline [PBS] + 0.05% Tween 80);
PBST, PBS + 0.02% Tween 80; PBST-grime, phosphate buffered saline+0.02% Tween-80 + Arizona Test Dust, + 1E4 spores/mL Bg, +1E4 spores/mL
Staphylococcus epidermidis; PCTE, polycarbonate; RT-PCR, real time PCR.
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Bradley, M.D., M.J. Arduino, J. Noble-Wang, L.J. Rose. (2011). Biological sample preparation collaboration project: Detection of detection of
Bacillus anthracis spores in soil: Final study report. U.S. Environmental Protection Agency and the Centers for Disease Control and
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evaluation of vacuum-based surface sampling methods for collection of Bacillus spores. J Microbiol Meth, 95 (3): 389-396.
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Hong-Geller, E., Y.E. Valdez, Y. Shou, T.M. Yoshida, B.L. Marrone, J.M. Dunbar. (2010). Evaluation of Bacillus anthracis and Yersinia pestis
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validated sponge wipe sampling method. Appl Environ Microbiol, 78 (3): 846-854.
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for Bacillus spores using commercially available cleaning robots. Environ Sci Technol, 47 (6): 2595-2601.
Perry, K.A., H.A. O'Connell, L.J. Rose, J.A. Noble-Wang, M.J. Arduino. (2013). Storage effects on sample integrity of environmental surface
sampling specimens with Bacillus anthracis spores. Biosafety, 2013 (Suppl 1): 002-002.
Piepel, G.F., B.L. Deatherage Kaiser, B.G. Amidan, M.A. Sydor, C.A. Barrett, J.R. Hutchison. (2016). False-negative rate, limit of detection and
recovery efficiency performance of a validated macrofoam-swab sampling method for low surface concentrations of Bacillus anthracis Sterne
and Bacillus atrophaeus spores. J Appl Microbiol, 121 (1): 149-162.
Probst, A., R. Facius, R. Wirth, C. Moissl-Eichinger. (2010). Validation of a nylon-flocked-swab protocol for efficient recovery of bacterial spores
from smooth and rough surfaces. Appl Environ Microbiol, 76 (15): 5148-5158.
Rose, L.J., L. Hodges, H. O'Connell, J. Noble-Wang. (2011). National validation study of a cellulose sponge wipe-processing method for use after
sampling Bacillus anthracis spores from surfaces. Appl Environ Microbiol, 11 (23): 8355-8359.
Smith, L.S., V.K. Rastogi, L. Burton, P.R. Rastogi, K. Parman. (2016). A novel hydrogel-based biosampling approach. Director, Edgewood
Chemical Biological Center: Aberdeen Proving Ground, MD
Thompson, K.A., S. Paton, T. Pottage, A.M. Bennett. (2018). Sampling and inactivation of wet disseminated spores from flooring materials, using
commercially available robotic vacuum cleaners. J Appl Microbiol, 125 (4): 1030-1039.
U.S. EPA. (2013). Systematic evaluation of aggressive air sampling for Bacillus anthracis spores: Assessment and evaluation report. U.S.
Environmental Protection Agency: Washington, D.C. EPA /600/R/13/068
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vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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