EPA/600/R-10/086 | August 2010 | www.epa.gov/ord
United States
Environmental Protection
Agency
Persistence Testing and Evaluation
of Fumigation Technologies for
Decontamination of Building
Materials Contaminated with
Biological Agents
Office of Research and Development
National Homeland Security Research Center
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of for
of
SHAWN P. RYAN
NATIONAL HOMELAND SECURITY
RESEARCH CENTER
OFFICE OF AND
DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION
AGENCY
MAIL CODE E343-06
RESEARCH TRIANGLE NC 27711
Office of Research and Development
National Homeland Security Research Center
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The U.S. Environmental Protection Agency, through its Office of Research and Development, funded and
managed this investigation through a Blanket Purchase Agreement under General Services Administration
contract number GS23F0011L-3 with Battelle. This document has been subjected to the Agency's review
and has been approved for publication. Note that approval does not signify that the contents necessarily
reflect the views of the Agency.
Mention of trade names or commercial products in this document or in the methods referenced in this
document does not constitute endorsement or recommendation for use.
Questions concerning this document or its application should be addressed to:
Shawn P. Ryan
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-0699
tyan.shawn@epa.gov
If you have difficulty assessing these PDF documents, please contactNickel.Kalliy@epa.gov orMcCall.
Amelia@epa.gov for assistance.
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Contributions of the following individuals and organizations to the development of this document
arc acknowledged.
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research Center
M. Worth Calfee
Joan Bursey (SEE)
Office of Research and Development, National Risk Management Research Laboratory
Timothy Dean
Office of Solid Waste and Emergency Response, Office of Emergency Management,
National Decontamination Team
R. Leroy Mickelsen
Battelle
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The Environmental Protection Agency (EPA) hold responsibilities associated with homeland security
events: EPA is the primary federal agency responsible for the country's water supplies and for
decontamination following a chemical, biological, and/or radiological (CBR) attack. The National
Homeland Security Research Center (NHSRC) was established to conduct research and deliver scientific
products that improve the capability of the Agency to carry out these responsibilities.
An important goal of NHSRC's research is to develop and deliver information on decontamination
methods and technologies to clean up CBR contamination. The research described here provides specific
information that will aid EPA and other stakeholders in choosing a decontamination method or technology
when addressing clean up of biological threat agents. NHSRC evaluated the effectiveness of several
fumigation technologies against a number of agents under various environmental conditions. In addition,
this document presents information on persistence of the agents in the absence of fumigation to help assess
the feasibility of clean up by natural processes.
NHSRC is pleased to make this publication available to assist the response community prepare for and
recover from disasters involving CBR contamination. This research is intended to move EPA one step
closer to achieving its homeland security goals and its overall mission of protecting human health and the
environment while providing sustainable solutions to our environmental problems.
—Gregory Sayles, Ph.D., Acting Director
National Homeland Security Research Center
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Executive Summary
The U.S. Environmental Protection Agency's National
Homeland Security Research Center (NHSRC) is helping
to protect human health and the environment from adverse
impacts resulting from acts of terror by identifying methods
and equipment that can be used for decontamination
following a terrorist attack in which chemical, biological,
or radiological agents are used and by investigating the
fate (e.g., persistence) of such agents in the absence of
decontamination. The persistence of biological agents is
influenced by environmental conditions and the materials
with which the biological agents are in contact. The
generation of scientifically defensible persistence data
is useful for the proper planning of decontamination
efficacy tests and helps formulate first response plans
in preparation for possible natural occurrences or
intentional releases of biological agents. In this current
effort, persistence data were generated for Brucella suis,
Francisella tularensis, vaccinia virus (a surrogate for the
variola virus that causes smallpox), and Yersinia pestis.
Additionally, four fumigation technologies (Sabre chlorine
dioxide [C1OJ, BIOQUELL Claras C HP [hydrogen
peroxide], BIOQUELL Claras S HP, and STERIS VHP®
[Vaporized Hydrogen Peroxide]) were evaluated for their
ability to decontaminate several materials contaminated
with an array of biological agents, including Bacillus
anthracis spores, B. suis, F. tularensis, vaccinia virus, or
Y. pestis.
The intent of the fumigant testing was to assess the
ability of the technology or decontamination process to
decontaminant materials at conditions consistent with
use in a facility. However, laboratory testing may present
a challenge when testing at a smaller scale than for
which the decontamination equipment was designed. For
the Sabre C1O2 testing, Sabre Technical Service, LLC.
provided a prototype unit designed for reproducing their
process in a smaller, lab-scale, environment (e.g., 317
L glove box). For the BIOQUELL hydrogen peroxide
fumgition, the initial intent was to test using the 317 L
glove box. In order to represent a typical room fumigation
with the BIOQUELL hydrogen peroxide fumigation
process, the temperature rise in the enclosed space due
to the fumigation equipment must be minimized. To
accomplish this in lab testing, BIOQUELL provided their
Claras S unit desgined for typical use in biological safety
cabinets. After testing with that unit, it was decided to
test at a larger scale (1275 L Biological Safety Cabinet),
utilizing one of their larger fumigation units (Claras C)
with an attempt to obtain a better representation of room-
scale fumigation. The STERIS VHP® system was a unit
of similar size and design parameters to the BIOQUELL
Claras C, and tested at the same scale (1275 L).
Persistence
Persistence (recovery of viable organisms) was
determined for B. suis, F. tularensis, vaccinia virus, and
Y. pestis spiked onto four materials (aluminum, keyboard
[computer keyboard keys], carpet, and joint tape [painted
paper joint tape]) and held under ambient environmental
conditions for up to 7 days. Persistence was determined
by the recovery of biological agents from the materials
at the completion of the exposure duration. The longest
exposure duration for the biological agents recovered
from the various materials was 7 days with the following
exceptions (the longest durations yielding viable agent
are noted in parentheses): B. suis on joint tape (4 hours
[hr]), F. tularensis on aluminum and joint tape (8 hr) and
carpet (4 hr), vaccinia virus on joint tape (3 days), and
Y. pestis on keyboard (3 days) and carpet (8 hr) (see also
Table ES-1).
Table ES-1. Longest Persistence Observed*
Biological
Agent
B. suis
F. tularensis
Vaccinia Virus
Y. pestis
Longest Duration with Viable Biological Agent
Recovered by Material
Aluminum
7 days
8hr
7 days
7 days
Keyboard
7 days
7 days
7 days
3 days
Carpet
7 days
4hr
7 days
8hr
Joint Tape
4hr
8hr
3 days
7 days
Testing was conducted for a maximum of 7 days.
SaJbre C/O2
The evaluation of Sabre C1O2 was conducted with 3,000
parts per million by volume (ppmv) C1O2 at both 40%
and 75% relative humidity (RH) against B. anthracis
spores. A lower C1O2 concentration (50-100 ppmv) was
used for the other biological agents (B. suis, F. tularensis,
vaccinia virus, and Y. pestis), which were also tested at
40% and 75% RH. Fumigation was also conducted at
60% RH with B. suis and vaccinia virus. All tests were
conducted with aluminum, keyboard, carpet, and joint
tape at 23 °C ± 2 °C and contact times ranged from 0 to
180 minutes (min).
Following 180 min of exposure to 3,000 ppmv C1O2,
viable B. anthracis spores were not recovered from
keyboard or carpet, but B. anthracis spores were
recovered from aluminum (at 40% RH and 75% RH). B.
anthracis spores were recovered from joint tape after the
180-min exposure at 40% RH but not after the 180-min
exposure at 75% RH.
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B. suis was not recovered from carpet or joint tape
exposed to 50-100 ppmv C1O2 (23 °C) for 120 min
at 40% RH, 60% RH, or 75% RH. However, B. suis
generally persisted on aluminum and keyboard. Only-
after 60-min or 120-min exposures to 50-100 ppmv CIO,
at 75% RH was B. suis not recovered from aluminum.
Interestingly. B. anthracis spores were most easily
decontaminated from keyboard, but B. suis was most
resistant to decontamination from keyboard.
K tularensis was not recovered from any of the four test
materials after 120-min exposure to 50-100 ppmv CIO,
(23 °C and 75% RH). When tested at a lower RH (40%),
F. tularensis was always recovered from aluminum and
keyboard, but not from carpet or joint tape. (F. tularensis
was not recovered from the carpet and joint tape positive
controls associated with the contact times tested at
the lower RH [40%]. Natural decontamination ofF.
tularensis, as spiked onto the materials, may occur over
time.)
Vaccinia virus was recovered from all four materials
following 120-min exposure to 50-100 ppmv at 23 °C and
40% RH. At 75% RH vaccinia was not recovered from
aluminum, carpet, or joint tape after 30-min exposure to
50-100 ppmv CIO,. Vaccinia virus persisted on keyboard
even following a 120-min exposure to 50-100 ppmv CIO,
at75%RH.
Y. peslis was not recovered from any of the four materials
tested when exposed to 50-100 ppmv CIO, at 40% RH
or 75% RH. Positive controls show high levels of loss of
viable Y. pesiis without fumigation.
C HP
The BIOQUELL Clarus C HP fumigation involved
exposure to HP generated using cycle parameters
(dehumidification, gassing phase, and dwell phase)
specified by the manufacturer. The evaluation was
primarily conducted with the following cycle: fumigate
10 min at 8 g/min and dwell at 0.8 g/min for contact
times of 180 min. Tests were conducted on aluminum,
keyboard, carpet, and joint tape with B. suis, Y. pesiis, and
vaccinia virus. Testing was also conducted with vaccinia
virus on glass and with B. anthracis spores on carpet,
laminate, ductwork, concrete, wood, glass, and ceiling
tile. Some level of efficacy was observed against all types
of biological agents on all surfaces after the 180-min
contact time.
No viable B. anthracis spores were recovered from
laminate, ductwork, ceiling tile, or glass. Viable B.
anthracis spores were recovered from carpet, concrete.
and wood after the 180-min exposure.
No viable B. suis, vaccinia virus, or Y. pestis was
recovered from any of the materials tested following
exposure to BIOQUELL Clams C HP fumigation. Neither
B. suis, vaccinia virus, nor Y. pestis was recovered from
aluminum, keyboard, carpet, joint tape, or glass (used for
vaccinia virus only) following a 180-min exposure to HP.
S HP
The BIOQUELL Clarus S HP fumigation involved
exposure of biological agents to various HP fumigation
parameters (e.g., HP volume of 15 mL to 50 inL over
injection times of 15 to 20 min) and contact times of 15
to 192 min. Testing was generally conducted at ambient
temperature (22 °C) under two initial RH conditions
(40% - 50% and 60% - 70%). Testing with B. anthracis
spores was conducted only at 45% RH. Testing was
conducted with 13. anthracis spores. B. suis. F. tularensis,
and Y. pestis on aluminum, keyboard, carpet, and joint
tape.
B. anthracis spores were not recovered from aluminum,
keyboard, or joint tape following a 75-min exposure to
HP (fumigate 50 mL with an injection time of 20 min
for a nominal concentration of 500 ppmv HP and a peak
concentration of 528 ppmv) at an initial RH of 45%, but
B. anthracis spores were recovered from carpet following
a 192-min exposure to HP (three fumigate cycles totaling
50 mL with dwell times between the 15 min injections of
approximately 45 min).
B. suis was generally recovered from aluminum,
keyboard, carpet, and joint tape following exposures to
HP (fumigate 15 mL with an injection time of 15 min) at
initial RH conditions of 40% - 50% and 60% - 70%. Two
exceptions with no B. suis recover}7 occurred following
a 30-min exposure to HP (actual peak concentration of
414 ppmv HP) at an initial RH of 45% on keyboard and a
60-min exposure to HP (actual peak concentration of 303
ppmv HP) on joint tape at an initial RH of 65%.
F. tularensis was exposed to BIOQUELL Clarus S HP
(fumigate 15 mL with an injection time of 15 min) at
initial RH conditions of 45% and 65%. K tularensis
was not recovered from any of the materials tested
(aluminum, keyboard, carpet, or joint tape) after 30-min
exposures to HP (442 ppmv peak concentration for all
materials) at an initial 45% RH or 30-min exposures to
HP at an initial 65% RH.
Y. pestis was generally recovered from aluminum,
keyboard, carpet, and joint tape following exposures
to HP (fumigate 15 mL with an injection time of 15
min) under initial RH conditions of 45% and 65%. Two
exceptions with no Y. pestis recover}' occurred following
a 30-min exposure to HP (392 ppmv peak concentration)
at an initial 45% RH on aluminum and a 60-min exposure
to HP (379 ppmv peak concentration) on joint tape at an
initial 65% RH.
VHP® HP
The STER1S VHP® HP fumigation was conducted at 500
ppmv or 200-250 ppmv HP for various contact times and
biological agent/material combinations. All biological
agents (B. anthracis spores, B. suis, F. tularensis, vaccinia
virus, and Y. pestis) were tested on aluminum and
keyboard. B. anthracis spores. B. suis and vaccinia virus
were also tested on carpet and joint tape, and B. anthracis
spores were also tested on laminate, ductwork, concrete.
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wood, glass, and ceiling tile.
Following a 4-hr exposure to the 500 ppmv HP
fumigation cycle, B. anthracis spores were not recovered
from any of the materials tested (i.e., aluminum,
keyboard, carpet, joint tape, laminate, ductwork, concrete,
and wood).
With a STERIS VHP® 500 ppmv HP fumigation cycle, B.
suis was not recovered from carpet or joint tape following
a 60-min exposure. B. suis was not recovered from
aluminum or keyboard following a 90-min exposure.
F. tularensis was not recovered from aluminum or
keyboard after a 90-min exposure to the 200-250 ppmv
HP fumigation cycle or after a 30-min exposure to the
500 ppmv HP fumigation cycle.
Vaccinia virus was not recovered from carpet or joint
tape following a 30-min exposure to the 200-250 ppmv
HP fumigation cycle, but vaccinia virus was recovered
from aluminum and keyboard. Vaccinia virus was not
recovered from keyboard following a 60-min exposure to
200-250 ppmv HP fumigation cycle or a 60-min exposure
to the 500 ppmv HP fumigation cycle. Viable vaccinia
virus was recovered from aluminum even after a 60-min
exposure to the 500 ppmv HP fumigation cycle.
Y. pestis was not recovered from any material tested
(aluminum and keyboard) following a 90-min exposure to
the 200-250 ppmv HP fumigation cycle.
Fumigation Summary
All fumigation technologies exhibited efficacy against
each biological agent with the level of efficacy being
dependent on decontamination parameters, e.g.,
concentration and time, and the type of material
inoculated with the biological agent. Based on incidents
requiring B. anthracis spore decontamination, the real-
world criterion for "adequate decontamination" tends
to be that no viable spores are recovered after extensive
sampling. With this "adequate decontamination" goal
in mind, Table ES-2 provides the minimum treatment
condition for each fumigation technology that resulted
in no viable biological agent being recovered from any
of the materials tested. Under the conditions tested,
each biological agent was completely rendered non-
recoverable by at least one of the technologies, and each
of the tested technologies was found to render at least
one biological agent non-recoverable.
IMPORTANT NOTE: The results in Table ES-2 are
derived from the specific tests, materials, methods
of biological agent preparation and application, and
conditions that were used in this investigation. The
results in Table ES-2 show the decontamination
conditions that were identified in which no viable
biological agent was recovered. These results should
not be interpreted as a comparison of decontamination
technologies; concentrations, contact times, or
environmental conditions different from those
investigated may yield different efficacy results for
the various decontamination technologies. Further,
demonstration that no viable biological agent was
recovered should not be generalized to other materials,
environmental conditions, or other methods of
application/dispersion of the biological agents.
Table ES-2. Overview of Fumigation Conditions Yielding
No Viable Biological Agent Recoveries on Any Tested
Material
Biological
Agent
B.
anthracis
spores
B. suis
F.
tularensis
Vaccinia
Virus
Y. pestis
Conditions Yielding No Biological Agent Recovery
Sabre
CIO,
2
NA
NA
50-100
ppmv
C1O2, 23
°C, 75%
RH, 120
min*
NA
50-100
ppmv
CIO 23
°C 40%
RHor
75% RH,
30 min*
BIOQUELL
Claras C
HP
NA
Fumigate 10
min at 8 g/
min; dwell
at 0.8 g/min;
180-min
contact time
Not Tested
Fumigate 10
min at 8 g/
min; dwell
at 0.8 g/min;
180-min
contact time
Fumigate 10
min at 8 g/
min; dwell
at 0.8 g/min;
180-min
contact time
BIOQUELL
Claras S HP
NA
NA
Fumigate 15
mL, initial
RH 45% or
65%, 15- or
30-min
contact time*
Not Tested
NA
STERIS
VHP® HP
500 ppmv
fumigation
cycle, 240
min
500 ppmv
fumigation
cycle, 90
min
200-250
ppmv
fumigation
cycle, 90
min
or
500 ppmv
fumigation
cycle, 30
min
NA
200-250
ppmv
fumigation
cycle, 90
min
or
500 ppmv
fumigation
cycle, 30
min
* Low/no biological agent recovered from the associated positive
control confounding the interpretation of the fumigant efficacy data
(the lack of biological agent recovery could be attributable to natural
degradation rather than fumigant efficacy).
NA = Not applicable, no fumigation test conditions used resulted in no
viable recovery on all tested materials.
Biological indicators were used in parallel with the
biological agent decontamination testing. The organisms
used as the biological indicators were B. atrophaeus
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spores (nominally 106 spores) on steel inTyvek®
packaging (Apex Laboratories, Apex, NC), for the Sabre
CIO, fumigation testing and G. stearothermophilus
(nominally 1 x 106 spores) on stainless steel in Tyvek®
packaging for the three HP technologies. The results from
qualitative evaluation of the biological indicators did not
correlate consistently with the results from quantitative
evaluation of viable biological agent remaining on
coupons of various materials after decontamination.
For example, the B. atrophaeus biological indicators
used for the Sabre CIO, fumigation were all positive for
growth after exposure to 3.000 ppmv CIO, for the 180-
min contact time (the longest time tested), indicative
of incomplete kills. The evaluation of decontamination
of biological indicators (on steel) is consistent with
decontamination of B. anthracis on aluminum where
viable spores were recovered under all treatment
conditions. However, no B. anthracis spores were
recovered from keyboard or carpet under the same
conditions. In contrast, the G. stearothermophilus
biological indicators were negative for growth (indicating
complete kill) after HP fumigation treatments in which
viable B. anthracis, B. snis, and vaccinia virus were
recovered from some of the materials tested. For these
hardy biological agents, observation of no growth of
G. stearothermophilus biological indicators cannot
be assumed to correlate to no viable biological agent
remaining on any material.
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Disclaimer iii
Acknowledgements iv
Foreword v
Executive Summary vii
Abbreviations and Acronyms xvii
1.0 Introduction[[[
2.0 Procedures[[[3
2.1 Biological Agents 3
2.1.1 Bacillus anthracis 3
2.1.2 Bruce/la suis 3
2.1.3 Francisella tularensis 3
2.1.4 Vaccinia Virus 3
2.1.5 Yersinia pestis 4
2.2 Test Materials 5
2.3 Spiking Coupons 5
2.4 Test and Control Chambers 5
2.4.1 Persistence Test Chambers 5
2.4.2 Decontamination Test and Control Chambers 5
2.5 Monitoring and Controlling Temperature and Relative Humidity 6
2.5.1 Persistence Test Environmental Monitoring and Control 6
2.5.2 Decontamination Test Environmental Monitoring and Control 6
2.6 Extracting and Quantifying Biological Agent 6
2.6.1 Method for Quantifying B. anthracis 6
2.6.2 Method for Quantifying/?, suis 7
2.6.3 Method for Quantifying F. tularensis 7
2.6.4 Method for Quantifying Vaccinia Virus 7
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3.0 Quality Assurance / Quality Control[[[ 13
3.1 Performance Evaluation Audit 13
3.2 Technical Systems Audit 14
3.3 Data, Quality Audit 14
3.4 QA/QC Reporting 14
3.5 Deviations from the Test/QAPlan 14
4.0 Recovery and Persistence Results[[[ 15
4.1 Recover}-Results 15
4.2 Persistence Testing 17
4.2.1 B. suis Persistence 17
4.2.2 F. tularensis Persistence 19
4.2.3 Vaccinia Virus Persistence 21
4.2.4 Y. pestis Persistence 23
4.2.5 Summary of Persistence Testing 25
5.0 Fumigation Technologies, Test Matrices, and Results..................................................27
5.1 C1O2 Fumigation (Sabre) 27
5.1.1 Description of Sabre C1O2 Technology 27
5.1.2 Test Matrix for Sabre C1O2 Fumigation 28
5.1.3 Sabre CIO, Fumigation Results 29
5.2 HP Fumigation (BIOQUELL Claras C) 48
5.2.1 Description of B1OQUELL Claras C HP Technology 48
5.2.2 Test Matrix for BIOQUELL Clams C HP Fumigation 49
5.2.3 BIOQUELL Claras C HP Fumigation Results 49
5.3 HP Fumigation (BIOQUELL Claras S) 55
5.3.1 Description of BIOQUELL Claras S HP Technology 55
5.3.2 Test Matrix for BIOQUELL Clams S HP Fumigation 55
5.3.3 BIOQUELL Clams S HP Fumigation Results 57
5.4 HP Fumigation (STERIS VHP®) 65
5.4.1 Description of STER1S VHP* HP Technology 65
5.4.2 Test Matrix for STERIS VHP® HP Fumigation 66
5.4.3 STERIS VHP® HP Fumigation Results 67
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Figure 2-1. Spiking Coupon Using a Multichannel Pipette 5
Figure 2-2. Glove Box for Decontamination Testing 6
Figure 2-3. B. anthracls Ames Colonies onTryptic Soy Agar. 7
Figure 4-1. B. suis Persistence 18
Figure 4-2. F. tularensis Persistence 20
Figure 4-3. Vaccinia Virus Persistence 22
Figure 4-4. Y. pestis Persistence 24
Figure 5-1. Sabre Bench-scale CIO,, Generator. 27
Figure 5-2. Temperature, RH, and C1O2 Concentration Dynamics
During aTypical Fumigation Cycle; "15 min Strip" and "5 min Strip"
Refers to Addition of CIO, to die Test Chamber. 29
Figure 5-3. Sabre fumigation results for B. anthracis at 3,000 ppmv C1O2
and 23 °C, line chart *.. 32
Figure 5-4. Sabre Fumigation Results for 13. anthracis at 3,000 ppmv CIO,
and 23 °C, Column Chart \ 32
Figure 5-5. Sabre Fumigation Results for B. suis at 50-100 ppmv C1O2
and 23 °C, Line Chart .". 36
Figure 5-6. Sabre Fumigation Results for B. suis at 50-100 ppmv CIO,
and 23 °C, Column Chart .". 37
Figure 5-7. Sabre Fumigation Results for .K tularensis at 50-100 ppmv C1O2
and 23 °C, Line Chart 40
Figure 5-8. Sabre Fumigation Results for F. tularensis at 50-100 ppmv C1O0
and 23 °C, Column Chart ". 40
Figure 5-9. Sabre Fumigation Results for Vaccinia Vims at 50-100 ppmv C1O2
and 23 °C, Line Chart ". 44
Figure 5-10. Sabre Fumigation Results for Vaccinia Virus at 50-100 ppmv C1O2
and 23 °C, Column Chart
("a" Indicates Initial Test; "b" Indicates Repeat Test) 44
Figure 5-11. BIOQUELL Claras C HP Vapor Generator.14 48
Figure 5-12. Typical Temperature (°C). RH (%). and HP Concentration (ppmv) Dynamics
During the Fumigation Cycle
(Fumigate 10 min at 8 g/min; Dwell at 0.8 g/min) 50
Figure 5-13. BIOQUELL Claras S HP Vapor Generator.15 55
Figure 5-14. Typical Temperature (°C). RH (%). and HP Concentration (ppmv) Dynamics
During the Fumigation Cycle (15 mL HP Injection) 57
Figure 5-15. Typical Temperature (°C). RH (%), and HP Concentration (ppmv) Dynamics
During the Fumigation cycle (50 mL HP Injection) 58
Figure 5-16. STERTS VHP® Generator Series 1000ED.16 65
Figure 5-17. STEMS VHP® Biodecontamination Cycle.16 65
Figure 5-18. Typical Temperature (°C). RH (%), and HP Concentration (ppmv) Dynamics
During a Fumigation Cycle (225 ppmv HP) with the STERIS VHP® Generator
Series 1000ED 67
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Figure 5-19. Typical Temperature (°C). RH (%). and HP Concentration (ppmv)
Dynamics During a Fumigation Cycle (500 ppmv HP) with the
STERIS VHP® Generator Series 1000ED 67
Figure 5-20. STEMS VHP® Fumigation Results for B. anthracis at the
500 ppmv HP Fumigation Cycle, Line Chart 71
Figure 5-21. STERIS VHP® Fumigation Results for B. anthracis at the
200-250 ppmv HP Fumigation Cycle, Line Chart 72
Figure 5-22. STERIS VHP® Fumigation Results for B. suis at the
200-250 ppmv and 500 ppmv HP Fumigation cycles. Line Chart 73
Figure 5-23. STERIS VHP® Fumigation Results for B. suis at the
200-250 ppmv and 500 ppmv HP Fumigation Cycles, Column Chart 73
Figure 5-24. STERIS VHP® Fumigation Results for Vaccinia Virus at the
200-250 ppmv and 500 ppmv HP Fumigation Cycles, Line Chart 75
Figure 5-25. STERTS VHP® Fumigation Results for Vaccinia Virus at the
200-250 ppmv and 500 ppmv HP Fumigation Cycles, Column Chart 76
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Table ES-1. Longest Persistence Observed vii
Table ES-2. Overview of Fumigation Conditions Yielding No Viable
Biological Agent Recoveries on Any Tested Material ix
Table 2-1. Test Materials 4
Table 3-1. Performance Evaluation Audits 13
Table 4-1. Biological Agent Recover}- 16
Table 4-2. B. raw1 Persistence 17
Table 4-3. F. tularensis Persistence 19
Table 4-4. Vaccinia Virus Persistence 21
Table 4-5. Y. pestis Persistence 23
Table 4-6. Summary of Persistence Results Calculated as Mean Log Reduction 25
Table 5-1. Test Matrix for Sabre CIO,, Fumigation 28
Table 5-2. Sabre CIO, Fumigation Results for B. anthracis 30
Table 5-3. Summary of Efficacy (Calculated as Mean Log Reduction)
of C1O2 against B. anthracis spores 33
Table 5-4. Sabre C1O2 Fumigation Results for 5. snis 35
Table 5-5. Summary of Efficacy (Calculated as Mean Log Reduction)
of CIO, against B. suis 38
Table 5-6. Sabre C1O2 Fumigation Results for A" tularensis 39
Table 5-7. Summary of Efficacy (Calculated as Mean Log Reduction)
of CIO, against F. tularensis 41
Table 5-8. Sabre C1O2 Fumigation Results for Vaccinia Virus 43
Table 5-9. Summary of Efficacy (Calculated as Mean Log Reduction)
of C1O2 against Vaccinia Virus 45
Table 5-10. Sabre CIO, Fumigation Results for Y. pestis 46
Table 5-11. Summary of Efficacy (Calculated as Mean Log Reduction)
of C1O2 against Y. pestis 47
Table 5-12. Test Matrix for BIOQUELL Claims C HP Fumigation 49
Table 5-13. BIOQUELL Clarus C HP Fumigation Results for G. stearothermophilus 50
Table 5-14. BIOQUELL Clarus C HP Fumigation Results for B. anthracis 51
Table 5-15. BIOQUELL Clams C HP Fumigation Results for B. suis 52
Table 5-16. BIOQUELL Clarus C HP Fumigation Results for Vaccinia Virus 52
Table 5-17. BIOQUELL Clarus C HP Fumigation Results for Y. pestis 53
Table 5-18. Summary of Efficacy (Calculated as Mean Log Reduction)
of BIOQUELL Clarus C HP Fumigation 54
Table 5-19. Test Matrix for BIOQUELL Clarus S HP Fumigation 56
Table 5-20. BIOQUELL Clams S HP Fumigation Results for G. stearothermophilus 59
Table 5-21. BIOQUELL Clarus S HP Fumigation Results for B. anthracis 59
Table 5-22. BIOQUELL Clarus S HP Fumigation Results for B. suis 60
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Table 5-23. BIOQUELL Clams S HP Fumigation Results for E tularemis 61
Table 5-24. BIOQUELL Claras S HP Fumigation Results for Y. pestis 62
Table 5-25. Summary of Efficacy (Calculated as Mean Log Reduction)
of BIOQUELL Claras S HP Fumigation Results 63
Table 5-26. Test Matrix for STERIS VHP® HP Fumigation 66
Table 5-27. STER1S VHP® HP Fumigation Results for G. stearothermophilus 68
Table 5-28. STER1S VHP® HP Fumigation Results for B. anthracis 69
Table 5-29. STERIS VHP® HP Fumigation Results for B. suis 72
Table 5-30. STERIS VHP® HP Fumigation Results for F. tularensis 74
Table 5-31. STERIS VHP® HP Fumigation Results for Vaccinia Virus 75
Table 5-32. STERIS VHP® HP Fumigation Results for Y. pestis 76
Table 5-33. Summary of STERIS VHP® HP Fumigation Efficacy
(Calculated as Mean Log Reduction) 77
Table 6-1. Summary of Sabre CIO, Fumigation 81
Table 6-2. Summary of BIOQUELL Claras C HP Fumigation 81
Table 6-3. Summary of BIOQUELL Clarus S HP Fumigation 82
Table 6-4. Summary of STERIS VHP® HP Fumigation 82
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and
BSC III Class III biological safety cabinet
°C degrees Celsius
CaSO,, calcium sulfate
CFU(s) colony-forming unit(s)
CI confidence interval
CIO, chlorine dioxide
cm centimeter
CoCl, cobalt chloride
CT contact time = concentration x time
CV coefficient of variation
EPA U.S. Environmental Protection Agency
g gram
g gravity
HEPA high-efficiency paniculate air
HP hydrogen peroxide
far hour
K2CO, potassium carbonate
L liter
min minute
mL milliliter
mm millimeter
mM millimolar
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
nm nanometer
O.D. optical density
PBS phosphate buffered saline
PFU(s) plaque-forming unit(s)
ppm parts per million
ppmv parts per million by volume
QA quality assurance
QC quality control
QMP quality management plan
RH relative humidity
rpm revolutions per minute
STS sodium thiosulfate
TSA technical systems audit
TTEP Technology Testing and Evaluation Program
uL microliter
-------
-------
1.0
Introduction
The U.S. Environmental Protection Agency's (EPA's)
National Homeland Security Research Center (NHSRC)
is helping to protect human health and the environment
from adverse impacts resulting from acts of terror. The
emphasis of NHSRC is directed toward decontamination
and consequence management, water infrastructure
protection, and threat and consequence assessment.
NHSRC is working to develop tools and information
that will help detect the intentional introduction of
chemical, biological, or radiological contaminants into
buildings or water systems, contain these contaminants,
decontaminate buildings or water systems, and dispose
of materials resulting from cleanups.
NHSRC's researchers work in partnership with
recognized testing organizations; with stakeholder
groups consisting of buyers, vendor organizations, and
permitters; and with the full participation of individual
technology developers in carrying out performance tests
on homeland security technologies. NHSRC evaluates
the performance of homeland security technologies
by developing test plans that are responsive to the
needs of stakeholders, conducting tests, collecting and
analyzing data, and preparing peer-reviewed reports. All
evaluations are conducted in accordance with rigorous
quality assurance (QA) protocols to ensure that data
of known and high quality are generated and that the
results are defensible. Such evaluations provide high-
quality information that is useful to decision makers
in purchasing or applying the tested technologies.
Potential users are provided with unbiased, third-party
information that can supplement vendor-provided
information. Stakeholder involvement ensures that
user needs and perspectives are incorporated into the
test design so that useful performance information is
produced for each of the tested technologies.
In the interest of expanding our national readiness
against highly-ranked threat scenarios, the NHSRC
is conducting tests to evaluate the performance of
products, methods, and equipment for decontaminating
contaminated materials. NHSRC is also investigating
the fate (i.e.. persistence) of biological, chemical, and
radiological agents in the absence of decontamination.
For biological agents, persistence reflects the extent
that viability or pathogenicity is retained over a defined
period of time. Some biological agents are unstable
and lose viability or pathogenicity within minutes
(min) of their release, thereby diminishing the risk to
human health and the environment and the need for
decontamination; other agents can remain viable or
pathogenic for weeks, months, or years. The persistence
of biological agents is influenced by environmental
conditions and the materials with which they are in
contact. The generation of scientifically defensible
persistence data is useful for the proper planning of
decontamination efficacy tests and helps formulate
first response plans in preparation for possible natural
occurrences or intentional releases of biological agents.
The investigation described in this report included three
elements:
* Persistence: investigating the change in the %
recovery of biological agents overtime from various
types of indoor building materials under controlled
environmental conditions;
* Decontamination efficacy: investigating the log
reduction in biological agent extracted from building
materials following an experimental fumigation
treatment compared to mean log reduction of
biological agent extracted from coupons in the
absence of the treatment (control); and
* Fumigant damage: observation of visual damage
to the surface of building materials caused by the
fumigation treatment.
For this report, the persistence of the biological threat
agents Brucella suis, Francisella tularensis, vaccinia
vims (a surrogate for the variola virus), and Yersinia
peslis spiked onto various materials (i.e., aluminum,
computer keyboard keys, carpet, and painted joint tape
paper) was investigated. Persistence was quantified as the
amount of biological agent recovered from the materials
following exposure to ambient environmental conditions
for up to 7 days. The experimental design allowed us to
test whether or not there was significant loss of biological
agent over time from Hie various materials.
This report also summarizes an evaluation of four
fumigation technologies: Sabre chlorine dioxide (C1Q,).
BIOQUELL Claras C hydrogen peroxide [HP],
B1OQUELL Claras S HP, and STER1S VHP® HP) that
were evaluated with regard to their ability to decontaminate
several materials that were spiked with various biological
agents including Bacillus anthracis spores, B. suis, F.
tularensis, vaccinia virus, and Y. pestis.
As used in this investigation, "efficacy" means that
the fumigation treatment had the desired effect, at a
statistically significant level, of decreasing the amount of
viable biological agent recovered from a material, given a
fumigant treatment, than from a corresponding untreated
positive control. Efficacy is quantified as log reduction.
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-------
2.0
Procedures
This section provides an overview of the procedures
that were used for the bench-scale investigation of the
persistence of biological agents on various materials
and the evaluation of fumigation technologies to
decontaminate biological agents from indoor surfaces.
Testing was performed in accordance with the peer-
reviewed and EPA-approved Test/[Quality Assurance]
QA Plan for Systematic Investigation ofFumigant
Technologies for Decontamination of Biological Agents
from Contaminated Building Materials1 and associated
amendments excepted as noted in the deviations
(Appendix A). The test/QA plan provides additional
procedural details that are not included in this report.
The general approach and methods, biological agents,
and types of materials used are summarized in this
section.
2.1 Biological Agents
2.1.1 Bacillus anthracis
B. anthracis Ames spores (Battelle culture: USAMRIID
M-BAA202) were prepared according to established
Battelle Biomedical Research Center procedures.2 A
primary culture of B. anthracis Ames from Battelle stock
was grown overnight (16-18hrat37°C)in nutrient
broth (BD Diagnostic Systems, Sparks, MD) on an
orbital shaker (Model 3827, Lab-Line Instruments,
Thermo Scientific, Pittsburg, PA) set at 150-200 rpm.
An aliquot was used as an inoculum for a scale-up
culture that was grown in nutrient broth for 6-8 hr at
150-200 rpm on the orbital shaker. Leighton-Doi Broth
(BD Diagnostic Systems, Sparks, MD) inside a BioFlo
fermentor (New Brunswick Scientific Co., Inc., Edison,
NJ) was inoculated with the scale-up culture and left
to grow for approximately 24 hr at 37 °C. Cultures
exhibiting >80% refractile spores were centrifuged
(fixed angle rotor) (Avanti J-26 XPI, Beckman Coulter,
Brea, CA) at approximately 10,000 - 12,000 xg for
15-20 min at 2 °C-8 °C. The resultant pellet was washed
twice, re-suspended in ice-cold sterile water, heat-
shocked (incubated at 60 °C for 45-60 min), centrifuged,
and washed at least twice to remove cellular debris.
The spore preparation was purified by centrifuging
through a gradient of ice-cold, sterile 58% Hypaque-76
(Nycorned Amersham, Princeton, NJ) at 9,000 x g for
2 hr at 2 °C-8 °C. The resultant pellet was washed and
re-suspended in ice-cold, sterile water and evaluated
by phase-contrast microscopy (LEICA CME, Leica
Microsystems, Bannockburn, IL). Preparations containing
>95% refractile spores with <5% cellular debris were
enumerated, diluted with sterile water to approximately
1.0 x 109 colony-forming units (CFUs)/mL and stored
at 2 °C-8 °C. Details of the method are published in the
Journal of Applied Microbiology.3
2.7.2 Brucella suis
B. saw biotype I (Battelle culture: BRU163) stock
solutions were prepared fresh in advance of each day that
coupons were spiked. Stock solutions were prepared by
transferring B. suis colonies from a streak plate (freshly
growing or stored at 2 °C - 8 °C for <2 weeks) into
10 mL of brain heart infusion broth (B 11059, Fisher
Scientific, Pittsburg, PA) that was then incubated at 37
°C ± 2 °C with shaking until an increase in turbidity was
observed (typically 3 days). The broth culture was added
to 40 mL of fresh brain heart infusion broth and incubated
overnight to achieve a suitable culture density (>1 x 10s
CFUs/mL). Alternatively, stock solutions were prepared
by transferring B. suis colonies from a streak plate
(freshly growing or stored at 2 °C- 8 °C for <2 weeks)
into brain heart infusion broth (~15 mL) that was then
incubated at 37 °C ± 2 °C with shaking until an increase
in turbidity was observed.
2.7.3 Francisella tularensis
A stock solution of F. tularensis LVS (Battelle culture:
OSU FTL361) was prepared fresh in advance of each
day that the coupons were spiked. Stock solutions were
prepared by transferring F. tularensis colonies from
a streak plate (freshly growing or stored at 2 °C - 8
°C for <2 weeks) into 10 mL of Muller-Hinton broth
(OXCM0405B, Fisher Scientific, Pittsburg, PA) (cation
adjusted plus IsoVitaleX™ [BD, Franklin Lakes, NJ]) that
was then incubated at 37 °C ± 2 °C with shaking until
an increase in turbidity was observed. The broth culture
was added to 40 mL of fresh Muller-Hinton broth (cation
adjusted plus IsoVitaleX™) and the incubation continued
for the time necessary to achieve a suitable density (>1
x 10s CFUs/mL). Alternatively, stock solutions were
prepared by transferring F. tularensis colonies from a
streak plate (freshly growing or stored <2 weeks) into
Muller-Hinton broth (cation adjusted plus IsoVitaleX™)
(-15 mL) and incubated at 37 °C ± 2 °C with shaking
until an increase in turbidity was observed.
2.7.4 Vaccinia Virus
Stock samples of vaccinia virus, WR (Mouse
Neurotropic) (New York City Department of Health,
strain (VR119, ATCC, Manassas, VA)) were propagated
in Vero cell monolayers (prepared by Battelle). Vero
cells were grown in tissue culture flasks at 37 °C ± 2 °C
in a 5% CO2 atmosphere in Eagle minimum essential
medium (SH30265.01, Fisher Scientific, Pittsburg, PA)
-------
supplemented with 5%-10% fetal bovine serum (30-
2020, American Type Culture Collection, Manassas,
VA). (Vero cells are typically passaged 20-25 times.
A new stock culture is then used.) Confluent Vero cell
monolayers were spiked with 1.0 mL of approximately
1 x 105 plaque-forming units (PFUs)/mL vaccinia
virus. The plates were rocked every 15 min during
the 60-min adsorption process. Following adsorption,
the Vero monolayers were overlaid with 0.8% methyl
cellulose (M0512, Sigma-Aldrich, St. Louis, MO)
containing fetal bovine serum, antibiotics (penicillin
and streptomycin [30-002-CI, Cellgro, Manassas, VA])
non-essential amino acids (M7145, Sigma-Aldrich, St.
Louis, MO) and L-glutamine (25030, Gibco, Carlsbad,
CA). The target seeding density was 4 x 105 cells/well
of a 12-well tissue culture plate (353225, BD, Franklin
Lakes, NJ). Following 2-6 days in culture, the Vero cells
were harvested and processed through 2-3 freeze-thaw
cycles to liberate the viral particles. Cell lysates were
centrifuged at approximately 800 x g for 10 min at 4 °C
± 1 °C to remove cell debris. The resultant supernatants
were separated into aliquots and frozen at -70 °C until
used.
Table 2-1. Test Materials
2.7.5 Yersinia pestis
A stock solution of Y. pestis CO-92 (Battelle culture:
M-YPO166) was prepared fresh in advance of each
day that the coupons were spiked. Stock solutions were
prepared by transferring Y. pestis colonies from a streak
plate (freshly growing or stored 2 °C- 8 °C for <2 weeks)
into 10 mL of Trypticase soy broth (Remel Inc., Lenexa,
Kansas, or Becton Dickinson and Company, Franklin
Lakes, NJ) and incubated at 27 °C ± 2 °C with shaking
until an increase in turbidity was observed indicative
of bacterial replication. The broth culture was added
to 40 mL of fresh Trypticase soy broth and incubated
until a suitable cell density (>1 x 10s CFUs/mL) was
achieved. Alternatively, stock solutions were prepared by
transferring Y. pestis colonies from a streak plate (freshly
growing or stored <2 weeks) into Trypticase soy broth
and incubated at 27 °C ± 2 °C with shaking until an
increase in turbidity was observed.
Material
Aluminum*
(finished)
Keyboard*
(computer keys)
Carpet*
(industrial)
Joint tape*
(painted joint tape
paper)
Laminate']'
(decorative)
Ductwork!
(galvanized metal)
Concrete!
(painted
concrete block)
Wood!
(unfinished and
untreated pine)
Glass!
Ceiling tile!
Lot, Batch, or Observation
Aluminum alloy 2024, 1.6 mm thick
Medium grey IBM" shell blanks, acrylonitrile,
butadiene, and styrene plastic
Style M 7978, color #9 10
SHEETROCK" joint tape
(paper tape without glue),
roller painted on one side using Martin- Senour paints,
one primer (#31-1185) and two finish (flat white, #22-
1101) coats
Grade 10 (nominal thickness 1.2 mm), matte white
finish
Industrial heating, ventilation, and air conditioning
standard 24 gauge galvanized steel
American Society for Testing and Materials
International C90 cinder block; brush and roller
painted all sides, one coat Martin- Senour latex primer
(#71-1185) and one coat Porter" paint latex semi-gloss
finish (#9 19)
Generic modeling
American Society for Testing and Materials
International C1036
Armstrong 954, classic fine textured
Manufacturer or
Supplier Name
Adept Products, Inc.,
West Jefferson,
OH, USA
DataCal,
Gilbert, AZ, USA
Carpet Corporation of
America, Rome,
GA, USA
United States
Gypsum Company
Chicago, IL, USA
Solid Surface Design
Columbus, OH, USA
Accurate
Fabrication
Columbus, OH, USA
Wellnitz
Columbus, OH, USA
West Jefferson Hardware
West Jefferson, OH, USA
Brooks Brothers Glass and
Mirror Service Columbus,
OH, USA
Armstrong
Columbus, OH, USA
Coupon Size,
Width x Length
1.9 cm x 7.5 cm
1.3 cm x 1.3 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
Material
Preparation
Autoclave
Gamma
irradiation
Gamma
irradiation
Gamma
irradiation
Gamma
irradiation
Autoclave
Autoclave
Gamma
irradiation
Autoclave
Gamma
irradiation
* Material used for persistence and decontamination testing.
! Material used only for decontamination testing.
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2.2 Test Materials
Building materials used for persistence testing and/or
fumigation experiments are described in Table 2-1. Most
of the testing was conducted with aluminum, computer
keyboard keys (keyboard), carpet, and painted joint
tape paper (joint tape). Test coupons of the materials
were cut to the sizes indicated in Table 2-1 from larger
pieces of stock material. Coupons were sterilized by
autoclaving or gamma irradiation. The selected approach,
as shown in Table 2-1, was based on cost-effectiveness
and minimization of physical alterations of the material.
Autoclaving was performed at Battelle according to an
internal standard operating procedure4, and gamma-
irradiation at 40 kilogray was conducted by STERIS
Isomedix Services (Libertyville, IL). Gamma-irradiated
coupons were sealed in 6 mL Uline poly tubing (Uline,
Chicago, IL) to preserve sterility until the coupons were
ready for use. Test coupons were each visually inspected
prior to being used in any experiment or test. Coupons
with anomalies on the application surface were discarded
and not used.
2.3 Spiking Coupons
The titer of the B. anthracis spores and the vaccinia
virus stock suspensions was determined prior to
use as described below. For the non-spore forming
bacterial species (Y. pestis, F. tularensis, and B. suis),
the stock broth cultures were incubated with shaking
until an increase in turbidity was observed indicating
bacterial replication. The growth curve was determined
by periodically taking samples and measuring the
absorbance at 600 nm, i.e., optical density (O.D. 600
nm), using a spectrophotometer (SPECTRAmax Plus
384, Molecular Devices, Sunnyvale, CA) and/or the
turbidity (McFarland unit) using the cuvette reader on
the spectrophotometer. The CFUs/mL of the sample were
simultaneously determined by serial dilution and culture
on solid media. A correlation between O.D. 600 nm and/
or McFarland units and Iog10 CFUs/mL was determined
by linear regression analysis. The linear equation was
used to estimate the CFUs/mL of the growing cultures.
The culture suspension was then diluted so that the stock
suspension was at the specified titer. The CFUs/mL were
determined for the stock suspension at the time of use.
Test and positive control coupons were placed flat and
spiked with approximately 1 x 107 viable organisms
(B. anthracis spores, B. suis, F. tularensis, or Y. pestis)
per coupon. A 100 uL aliquot of a stock suspension
of approximately 1 x 10s CFUs/mL of B. anthracis
spores, B. suis, F. tularensis, or Y. pestis was generally
dispensed using a multichannel micropipette (L12-200,
Rainin, Oakland, CA) applied as five 10 uL droplets in
each of two rows across the surface of the coupon (see
Figure 2-1). Only one type of organism was inoculated
onto a given test or control coupon. For vaccinia virus,
coupons were spiked with approximately 1 x 10s PFUs
per coupon; a 100 uL aliquot of a stock suspension
(approximately 1 x 109 PFUs/mL) of vaccinia virus
was dispensed using a multichannel micropipette. The
solution was applied as five 10 uL droplets in each of two
rows across the surface of the coupon.
Because of their small size, the keyboard keys were
spiked with a single 100 uL droplet, rather than the 10 x
10 uL droplets used with all of the other materials.
Figure 2-1. Spiking Coupon Using a Multichannel
Pipette.
2.4 Test and Control Chambers
2.4.7 Persistence Test Chambers
Bacterial persistence experiments were conducted
in scalable chambers (Chefmate® Covered Cake Pan
purchased from Target, Minneapolis, MM) with the
following dimensions: 34 cm (length) x 24 cm (width)
x 8 cm (height). The volume of each chamber was
approximately 6.5 L. A maximum of two materials
inoculated with one biological agent were placed into a
chamber for a given persistence experiment. The chamber
used for virus persistence testing is described below
(Section 2.4.2).
2.4.2 Decontamination Test and Control
Chambers
During testing of the Sabre and BIOQUELL Claras S
technologies, a Compact Glove Box Model 830-ABC
(Plas Labs, Inc., Lansing, MI; Figure 2.2), with a total
volume of 317 L was used to expose test coupons to
the fumigants (decontamination test chamber). For the
decontamination evaluations of the STERIS VHP® and
BIOQUELL Claras C technologies, a Class III biological
safety cabinet (BSC III) (SG603, Baker, Sanford, ME)
was used as the decontamination test chamber; this
chamber had a total volume of roughly 1275 L.
The chambers used for positive controls during
decontamination testing were generally comparable to
the test chambers having the same temperature and RH
present in the test chambers at the start of the fumigation
cycle. The control chambers were not exposed to
fumigants. For the Sabre and STERIS VHP® evaluation,
positive controls were placed in sealed vials inside the
decontamination chamber.
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Laompact
Glove Box
Figure 2-2. Glove Box for Decontamination Testing.
2.5 Monitoring and Controlling
Temperature and Relative Humidity
2.5.7 Persistence Test Environmental Monitoring
and Control
For bacterial persistence testing, each chamber used
contained an open 9 cm Petri dish (08-757-11YZ, Fisher
Scientific, Pittsburg, PA) containing a predetermined
quantity of the desiccant Indicating Drierite® (98%
CaSO4, 2% CoCl2, W A Hammond Co., Xenia, OH) and
an open 9 cm Petri dish containing saturated potassium
carbonate (K2CO3) (Sigma-Aldrich, St. Louis, MO) to
maintain the humidity at 35%-45% RH at 22 °C. The
Drierite® desiccant was removed from the chamber after
the humidity returned to the lower end of the target range
specified in the test/QAplan.1 The individual chambers
were placed in a low temperature incubator (Model
LR1201, Thermo Scientific, Pittsburg, PA) capable of
maintaining the temperature within the specified range.
The temperature and % RH within the chambers were
monitored using remote sensors (Model 14-648-53.
Fisher Scientific, Pittsburg, PA) that transmitted the data
back to a main data logging unit via radio frequency.
For vaccinia virus persistence testing, humidity in the
chamber was raised by pulling air from the chamber into
a fogging chamber (Battelle, custom manufactured). In
the fogging chamber, the air was humidified to -100%
RH using an ultrasonic fog generator (Battelle, custom
manufactured). The high humidity air passed out of the
fogging chamber through a water trap to remove any
liquid water and returned to the chamber. Mixing fans in
the chamber caused the humidity to rapidly equilibrate at
a higher RH. Drierite® desiccant was placed into the test
chamber to lower RH. The temperature and % RH within
the chambers were monitored using remote sensors that
transmitted the data back to a main data logging unit via
radio frequency.
2.5.2 Decontamination Test Environmental
Monitoring and Control
The temperature and RH in the test chamber immediately
prior to fumigation were at the levels specified for the
given trial (see Section 5.0 for technology-specific
details). To raise the humidity in the test and control
chambers, air from the test or control chamber was pulled
into a fogging chamber through an inlet. In the fogging
chamber, the air was humidified to -100% RH using an
ultrasonic fog generator. The high humidity air passed out
of the fogging chamber through a water trap to remove
any liquid water and returned to the test or control
chamber. Mixing fans in the test or control chambers
caused the humidity to rapidly equilibrate at a higher RH.
Drierite® desiccant was placed into the test chamber to
lower RH.
The temperature and RH were measured immediately
prior to initiating treatment and approximately every
20 min during the evaluation using a hygrometer/
thermometer (Model 14-648-53, Fisher Scientific.
Pittsburg, PA). While RH readings were taken during
fumigation, the RH levels during treatment may be
confounded by moisture introduced by the fumigant.
Efforts were made only to control the initial RH levels;
RH changes within the chamber during fumigation were
considered integral to the decontamination method.
2.6 Extracting and Quantifying
Biological Agent
For each biological agent, test, positive control, and
blank, coupons were placed into individual sterile 50 mL
conical vials to which 10.0 mL of sterile extraction buffer
was added. Phosphate buffered saline (PBS) (D8537,
Sigma, St. Louis, MO) was the extraction buffer for all
of the biological agents except B. cmihracis. PBS with
0.1% Triton X-100 (Sigma-Aldrich®, St. Louis, MO)
was the extraction buffer for B. anthracis spores. The
vials containing the coupons and extraction buffer were
agitated on an orbital shaker for 15 min at approximately
200 revolutions per minute (rpm) at room temperature.
The resulting liquid extract was removed and serially
diluted (typically 1:10 dilutions) in sterile water (up
to 10'7, as necessary), for subsequent quantification of
biological agent (i.e., CPUs orPFUs of biological agent
recovered as determined by a plating the serial dilutions).
2.6.7 Method for Quantifying B. anthracis
For B. anthracis, an aliquot (0.1 mL) of the undiluted
extract and each serial dilution were spread plated onto
tryptic soy agar plates (Remel Inc., Lenexa, KS, or
Becton Dickinson and Company, Franklin Lakes, NJ)
in triplicate. The cultures were incubated for 18-24 hr
at 37 °C ± 2 °C. Colonies were identified and counted
manually; well-isolated colonies of B. anthracis are
white, 2-5 mm in diameter. As shown in Figure 2.3, the
flat or slightly convex colonies are irregularly round
with undulating edges and a ground glass appearance.
The CFUs/mL in the extracts were determined by
multiplying the average number of colonies per plate by
the reciprocal of the dilution (typically based on plates
having colony counts between 30 and 300). The number
of detected viable spores extracted from a coupon was
calculated according to Equation 1, below.
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Equation 1. Total CPUs/coupon = [(mean CPUplate
count x 1/dilution factor)/plated volume] x (extraction
buffer volume)
Where:
Mean CPU plate count =
Plated volume
Dilution factor
Extraction buffer volume =
average number of
colonies counted in the
three replicate plates
volume that is applied
to each plate. In this
case 0.1 mLwas
applied.
portion of the total
extraction buffer used
to prepare the dilutions
volume of the
extraction buffer used
to extract the coupon.
In this case, 10 mL was
applied.
Figure 2-3. B. anthracis Ames Colonies onTryptic Soy
Agar.
2.6.2 Method for Quantifying B. suis
For B. suis, an aliquot (0.1 mL) of the undiluted extract
and each serial dilution were spread-plated onto brain
heart infusion agar (Becton Dickinson and Company,
Franklin Lakes, NJ) in triplicate. The cultures were
incubated for up to 72 hr at 37 °C ± 2 °C. Colonies
were identified and counted based on their growth
characteristics on the medium. After 48 hr, colonies of B.
suis are round, 1-2 mm in diameter, convex, pearly-white
when viewed from above, with smooth margins.5 The
bacteria recovered from coupons (enumerated as mean
CPUs/coupon) were determined in the same manner as
described for B. anthracis spores in Section 2.6.1.
2.6.3 Method for Quantifying F. tularensis
ForF. tularensis, an aliquot (0.1 mL) of the undiluted
extract and each serial dilution were plated onto
chocolate II agar plus IsoVitaleX (Becton Dickinson
and Company, Franklin Lakes, NJ) in triplicate. The
cultures were incubated for up to 72 hr at 37 °C ± 2 °C.
Colonies were identified and counted based on their
growth characteristics on the medium; F. tularensis
colonies after 48 hr are small (1-2 mm in diameter), flat
with a shiny surface, white to gray to bluish gray, opaque,
with a smooth, entire edge.6 The bacteria recovered
from coupons (enumerated as mean CPUs/coupon)
were determined in the same manner as described for B.
anthracis spores in Section 2.6.1.
2.6.4 Method for Quantifying Vaccinia Virus
For vaccinia virus, an aliquot (0.1 mL) of the undiluted
and each appropriate serial dilution of the stock
suspension and each coupon extract were plated onto
Vero (African green monkey kidney) cell monolayers
and allowed to adsorb for 1 hr. Following inoculation
and adsorption of virus to the Vero cells, 1.0 mL of 0.7%
methylcellulose was added to each well of the 6-well
plate. Plates were incubated for 24-48 hr at 37 °C ± 2
°C under 95% air and 5% CO2. The methylcellulose was
removed and 2.0 mL of 0.13% crystal violet (C6158,
Sigma-Aldrich, St. Louis, MO) was added and the
cells incubated for 30 min. The crystal violet was then
removed, the cells were washed with PBS, and the
plaques visualized and counted. The virus recovered from
the coupons (enumerated as mean PFUs/coupon) was
determined by multiplying the average number of PFUs
per plate by the reciprocal of the dilution. The PFUs/
coupon were calculated by multiplying the PFUs/mL by
the volume of the extraction buffer used for each coupon
(typically 10 mL per coupon).
2.6.5 Method for Quantifying Y. pestis
For Y. pestis, an aliquot (0.1 mL) of the undiluted extract
and each serial dilution were plated onto tryptic soy
agar (Remel Inc., Lenexa, KS, or Becton Dickinson and
Company, Franklin Lakes, NJ) in triplicate. The cultures
were incubated for up to 72 hr at 37 °C ± 2 °C. Colonies
were identified and counted based on their growth
characteristics on the medium. Small grey-white to pale
yellow colonies (1-2 mm) are observed at 48-72 hr. Early
colonies have a shiny surface described as "hammered
copper". Later colonies have an irregular "fried egg"
appearance.7 The bacteria recovered from coupons
(enumerated as mean CPUs/coupon) were determined in
the same manner as described for B. anthracis spores as
described in Section 2.6.1.
-------
2.7
Biological indicators were occasionally included in the
fumigation testing as specified in the test/QA plan.1
Specifically:
* For Sabre C1O2 fumigation B. atrophaeus (nominally
1 x 106 spores) on steel in Ty vek* packaging (Apex
Laboratories, Apex, NC) was used.
• For Clarus S, Clarus C, and STERIS VHP38 HP
fumigations Geobadlhts stearothermophihis,
nominally 1 x 106 spores on stainless steel in Ty vek®
packaging, was used.
Biological indicators were used in some preliminary
trials to verify that the selected fumigation cycles
were efficacious against G. stearothermophilus. The
presence of viable spores on the biological indicators was
determined using a qualitative method. The biological
indicators were aseptically transferred into individual
tubes containing 30 mL of tryptic soy broth culture
medium and capped. The tubes were cultured for 7 days
at 37 °C ± 2 °C to encourage viable spore germination
and subsequent proliferation of vegetative bacteria.
At 1 day and 7 days post-decontamination, the tubes
were visually assessed for cloudiness. A cloudy culture
medium may indicate "growth" of viable spores. Clear
culture medium indicates "no growth" and is consistent
with a complete kill of all spores on the biological
indicator. Data were presented as "growth" ("+") or "no
growth" ("-")• No additional testing (e.g., streak plating)
was performed on the biological indicators.
2.8 Calculations
Design
2,8.1 Percent Recovery
The amount of biological agent that can be recovered
may be dependent on the specific material to which it was
applied and the type of biological agent. Arithmetic mean
% recover)' at a given time (;') for a given material (/') was
calculated as:
Equation 2.
Where:
% Recovery, =
Biological Agent Applied
<100
-^ is the mean number of viable organisms (CPUs
ij or PFUs) recovered at the /'* contact time from
the/11 material.
The amount of biological agent applied to the material
equals the CFUs/mL or PFUs/mL measured in the
application control multiplied by the volume (mL)
applied to the coupon (i.e., the spike amount). The
application control is the enumeration of culturable
biological agent in the stock suspension determined by
serial dilution, plating, and enumeration.
Statistical analysis (analysis of variance) was used to
evaluate whether the mean recover}' of the biological
agent at a particular contact time on a particular test
material was statistically significantly different (p.
0.05) from the recovery of biological agent at time zero.
Both point estimates and corresponding p-valucs were
determined for each comparison.
2.8.2 Persistence
The persistence testing used a single group time scries
experimental design, diagrammed as:
R 0
0 0 0 0
where time passes from left to right and:
R = Random selection of the test coupons for each
time point and type of biological organism.
Ot = Mean measurement (observation) of biological
agent extracted from replicate coupons at time
(t) = 0 and five subsequent time periods, designated
by subscripts 1-5.
At a given point in time (t), the effect of time on
persistence is Ot - O0. The experimental design allowed
the following null (H0) and alternate (HA) hypotheses to
be statistically tested:
That is. the experimental design enables testing of the
null or alternate hypothesis that, given an equivalent
application of biological agent, the amount of biological
agent on the coupons was constant, or, alternatively,
decreased over time.
2.8.3 Decontamination Efficacy
Treatments for a given biological agent and building
material were defined in terms of the concentration
of the fumigant (C1O2 or HP), temperature, RH,
and contact time. To determine the efficacy of the
fumigation treatment on a biological agent, a pre-
test-post-test control group design was used for each
material, biological contaminant, and set of conditions,
diagrammed as:
R O. X O,
R
o,
o,
where time passes from left to right and:
R = Random selection of the test coupons for
control, experiment, and type of biological
organism.
O = Mean log reduction in measured biological agent
extracted from replicate coupons
[O
1 (Pretest)- 2 (Treatment)'' 3 3 (Control)" ^
X = Experimental variable, in this case the
decontamination process.
At a given point in time, the effect of the experimental
variable is (O, -Oj)-(O3 -O,), or simplified, the effect of
the experimental variable is O2 (Tniatment) - O3 (Con/ml). The
-------
experimental design was the following null (HQ) and
alternate (H4) hypotheses to be tested statistically:
H ' O — O =0
O' 2 (Treatment) 3 (Control)
i\' 2 (Treatment) 3 (Control)
For any particular material, the planned comparisons
included decontamination efficacy under given
fumigant CT (concentration x contact time), and given
environmental conditions (temperature and RH), for
a particular biological agent. The experimental design
enabled testing of the null hypothesis that there is no
difference, or the alternate hypothesis that there is an
increase, in the decontamination efficacy using the
treatment compared to the control. The design also
enabled comparison of rates of removal of biological
agents from different material types under specific CT.
Log reduction for a single positive control coupon and a
single test coupon is calculated as the difference between
the log (recovered biological agent) from the control
coupon and the log (recovered biological agent) from the
test coupon. At least three complications arise in the use
of log reduction to quantify efficacy:
• First, in tests where no viable biological agent is
recovered, the log of 0 is mathematically undefined.
By convention, a positive number is substituted
for the 0. Different substitutions yield different log
reduction results, and, when recoveries are low but
greater than zero on a coupon, average log reduction
values may be higher when viable organisms are
recovered than when no viable organisms are
recovered.
• Second, there are multiple methods for determining
the "average" log reduction when replicate control
and test coupons are used. These alternative
calculation methods do not yield the same log
reduction values or variance.
« Third, some biological agents naturally lose viability
over time, at rates that depend on environmental
conditions and the material in contact with the
biological agent. In determining efficacy, at least two
alternative reference points exist: positive controls
extracted immediately at time zero
(O, .„ ,, above), or positive controls (O, .„ r)
v 1 (Pretest? " " ^3 (ControTy
inoculated and extracted at the same time as the test
coupons (O0 „. ,).
" x 2 (Ireatmenty
If the time zero controls are used as the basis for
determining efficacy for a biological agent that loses
viability over time, the calculated log reduction value
includes both a "natural attenuation" component and
the effect of the decontamination technology. The effect
of the technology is not differentiated from the natural
attenuation. However, if the positive controls have the
same history as the test coupons (inoculated at the same
time, maintained in the same environmental conditions
for the same time), there are controls for "natural
attenuation" of the biological agent, but loss of biological
agent from the positive control coupons lowers the basis
for comparison, resulting in lower log reduction values
suggestive of lower efficacy, even if the technology
sterilizes the test coupons.
The log reduction complications were addressed in the
following manner:
« First, when no viable biological agent is recovered
from any coupon in a set, 1 was substituted for
the average recovered agent. Substituting a 1 for 0
results in the log reduction being numerically equal
to the mean log (recovered biological agent) from the
control coupon.
• Second, the log reduction was calculated using
two different methods. These calculation methods
are described below in this section. The first
calculation method was specified in the test/QA
plan1; the second calculation method is being used
in related testing and was included to enable ease of
comparisons across studies. The results of using two
methods for calculating log reductions are included
in this report.
* Third, for biological agents that may lose viability
with time, the log reduction was calculated based on
positive control coupons (O3 (rowlro/)) with the same
history (without treatment) as the test coupons.
Thus, the reported log reductions reflect only the
incremental contribution of the decontamination
treatment to the overall reduction in viable biological
agent recovered after a given treatment. Additional
information on the combined effect of "natural
attenuation" and the treatment technology was
provided in the text.
Decontamination efficacy using the method specified
in the test/QA plan1 was calculated as the log reduction
in viable biological agent recovered from coupons
(enumerated as CFUs or PFUs/coupon) after a given
treatment. For a given initial inoculum applied to a
coupon, the higher the decontamination efficacy (log
reduction) value, the less biological agent remains on the
material after a given treatment.
Efficacy was calculated for each individual coupon in
a given set of replicates. Efficacy was defined as the
extent (by log reduction) to which the biological agent
extracted from test coupons after fumigation was less
than what was extracted from positive control coupons
(not exposed to the fumigant) maintained at the same
temperature, RH. and time (test and control coupons
were spiked with the same amount and type of biological
agent). Efficacy was calculated for each test coupon
within each combination of contact time (/') and test
material (/) according to Equation 3, below.
-------
Equations.
where:
Efficacy^ = logl,C0- !og1BX" is calculated as the mean of the base-10 logarithm
of recovered agent from the control coupons minus the
mean of the base-10 logarithm of recovered agent from
the treated coupons except that "zero recovery" coupons
have a substituted recovered value of "1" (base-10 log
is 0). Since the log becomes an increasing negative
value below 1 and is undefined at 0, this substitution is
necessary and results in a lower bound on the mean log
difference, as indicated by the ">". The number of "zero
recovery" treatment coupons and the total number of
treatment coupons is shown in parentheses. The p-value
is from the non-parametric Kolmogorov-Smirnov test.20
A p-value less than 0.05 denotes less than 1 in 20 chance
that results as different as or more different than observed
would occur by chance if the distribution of the control
and treatment recoveries were truly identical.
2.8.4 CT Calculation
A measure of decontamination efficacy as a function
of fumigation treatment is often reported as a CT
curve. The product of the fumigant concentration and
the contact time of treatment was graphed against the
decontamination efficacy. The calculation of the CT
value is shown in Equation 8.
Equation 8. CT = Concentration x Contact Time
-------
The specific units for concentration and contact time
depend on the technology in use. For example, for C1O2
the concentration is in ppmv and the time is in hr. In the
investigation of the Clarus C and Clarus S efficacies, the
treatments were defined in terms of cycle parameters,
rat her than a fumigant concentration. Therefore the HP
concentration resulting from the specified fumigation
cycles is implicit rather than explicit in the CT; the
contact time is explicit. Results are reported as log
reductions after exposure for a given contact time to a
specified fumigation cycle.
2.9
The physical effect of the decontamination technologies
on the materials was also monitored during the
evaluation. The qualitative approach provided a gross
visual investigation of the damage to the various
materials caused by the decontamination technology.
Before and after decontamination of the test coupons,
the appearance of the decontaminated coupons was
visually inspected for any obvious changes in the color.
reflectivity, and apparent roughness of the material
surfaces. These comparisons were performed for each
material, before extraction of the decontaminated test
coupons.
-------
-------
3.0
Quality Assurance / Quality Control
Quality assurance/quality control (QC) procedures were
performed in accordance with the Quality Management
Plan (QMP)8 and the QA/test plan1 for the persistence
investigation and the technology evaluations. Quality
assurance/quality control procedures are summarized
below.
3.1 Performance Evaluation Audit
Performance evaluation audits were conducted by the
respective laboratory personnel to assess the quality
of the results obtained during these experiments.
For persistence testing, no performance evaluation
audits were performed for biological agents because
quantitative standards for these biological materials do
not exist. The controls, blanks, and method validation
efforts support the biological evaluation results. Table
3-1 summarizes the performance evaluation audits that
were performed.
Table 3-1. Performance Evaluation Audits
The PE audit for HP concentration compared the output
from the Analytical Technology HP gas sensor (B-12
2-Wire Toxic Gas Transmitter, Analytical Technology,
Collegeville, PA) to a titration using a Hach HYP-1 test
kit. The result of the PE audit met the required tolerance
of ± 10% and is shown in Table 3-1. In addition to the PE
audit, five additional comparisons of the instrumental
reading for HP concentration to the titration method
(Hach HYP-1) were performed. In two cases, the
comparison exceeded the desired tolerance of ± 10%.
These comparisons were not the required PE audit and
therefore do not represent a deviation in the test/QA plan,
but are provided for information only.
Measurement
CFU/PFU
Temperature
pH (meter)
Flow
(mass flow controller;
Sierra Instruments)
Time (stopwatch)
RH
CIO concentration
HP concentration
Optical density and
wavelength
Volume
Audit Procedure
Compare to independent count of colonies/plaques
Compared to independent thermometer (Model 14-648-
53, Fisher Scientific, Pittsburg, PA) value
Measure a standard buffer not used to calibrate the pH
meter
Compare against mini-BuckTM (National Institute of
Standards and Technology [NIST] traceable) primary
flow calibrator (mini-Buck M-30, Orlando, FL)
Compare against NIST official U.S. time at http://nist.
time.gov/timezone.cgi?Eastern/d/-5/java
Compared to independent hygrometer value
Titration of standard solution
Hach HYP-1 HP test kit (HYP-1, Hach, Loveland, CO)
Compare optical density measurement of the microplate
reader (SPECTRAmax Plus 384, Molecular Devices,
Sunnyvale, CA )to standard
All micropipettes were certified as calibrated at
time of use. Pipettes are recalibrated by gravimetric
investigation of pipette performance to manufacturer's
specifications every six months by supplier (Rainin
Instruments).
Allowable Tolerance
± 10%
±1°C
±0.1 pH units
±5%
± 1 sec/min
±5%
± 10%
± 10%
Optical density ± 1.0%,
Wavelength ± 1 nm
±5%
Actual Tolerance
0%
(100% accurate)
<1 °C for 6 of 6 instances
<0.1 pHunit
0.96%
0 sec/min for 44 of 44
measurements
<5% for 6 of
6 instances
<10%
4.2%
10 of 10 readings, all within
tolerance
<0.5% for 4 of 4 pipettes
-------
3.2 Technical Audit
Battelle QA staff conducted technical systems audits on
6/03/2008 and 10/02/2009 to ensure that the tests were
being conducted in accordance with the appropriate QA/
test plan1 and QMP.8 As part of the audit, test procedures
were compared to those specified in the test/QA plan;
and data acquisition and handling procedures were
reviewed. Observations and findings from the technical
systems audit (TSA) were documented and submitted
to the Battelle Task Order Leader for response. None of
the findings of the TSA required corrective action; only
minor issues were noted. TSA records were permanently
stored with the Battelle QA Manager.
3.3 Quality Audit
At least 10% of the data acquired during the persistence
investigation and decontamination technology
evaluation were audited by the Battelle QA Manager or
a designee. A Battelle QA auditor traced the data from
the initial acquisition, through reduction and statistical
analysis, to final reporting to ensure the integrity of the
reported results. All calculations performed on the data
undergoing the audit were checked.
3.4 QA/QC Reporting
Each assessment and audit was documented in
accordance with the test/QA plan1 and QMP.8 For
these tests, no significant findings were noted in any
assessment or audit, and no follow-up corrective action
was necessary. Copies of the TSA and assessment reports
were distributed to the EPA QA Manager and Battelle
staff. QA/QC procedures were performed in accordance
with the QMP8 and the test/QA plan1.
3.5 Deviations from the Test/QA Plan
Two deviations were documented, in compliance with
the QMP.8 B. anthracis testing with C1O2 fumigation,
was conducted by having the spiked coupons in closed
vials in the test chamber and opening them in sequence
so that an appropriate contact time for exposure to C1O2
was achieved without opening the test chamber which
could potentially have resulted in unacceptable variation
in the test condition.
The target ranges for application, recover}', and
coefficient of variation (CV) of recovery of vegetative
bacteria and virus proved very difficult to achieve.
Part of the challenge was that the vegetative bacteria
may exhibit a rapid loss of viability on one or more
of the materials. The materials may have cytotoxic or
inactivating properties. Further, unlike spores or virus,
vegetative bacteria were actively replicating before (and
possibly after) application. These opposing forces, loss of
viability and ongoing reproduction, confound efforts to
ensure accurate and precise biological agent recoveries at
time zero.
Methods used do not allow the bacteria being applied
to be precisely determined; therefore, applications
of biological agents outside of the target ranges were
observed in some cases. The actual inoculation amounts
were documented in this report.
Positive control recoveries at time zero were occasionally
outside the target recovery range. Further, during
persistence testing with B. suis, contamination of blanks
occurred for a limited number of trials.
There were no methods available to determine more
precisely, before the testing, the amount of vegetative
bacteria applied, to prevent loss of viable bacteria and
virus during drying on certain materials, or to reduce
the C V of recoveries from the various materials. Tests
exceeding the target ranges and/or CV were noted in
Appendix A and, where appropriate, in data tables; the
tests were not repeated.
The second deviation documents that trials were not-
repeated when:
* The amounts of biological agent inoculated onto
the coupons were outside of the acceptance criteria
(>10% - <120% for spores; 1 log for vegetative
organisms).
* Positive control recoveries were outside of the
acceptance criteria (>10% - <120% of the spores and
vegetative bacteria applied; >1 x 105 PFUs/coupon
for virus.
* Blank contamination occurred.
The deviations (tests not repeated) are provided in
Appendix A. Where applicable, the data impacted
by the deviations are noted in appropriate tables. The
higher than expected variability in the biological agent
applications, positive control recoveries, and the rare
contaminated blank coupons were believed to have had a
minimal impact on the test results.
-------
4.0
Recovery and Persistence Results
4.1
The amount of biological agent that can be recovered
may be dependent on the specific material to which
it is applied and the type of biological agent. For
combinations of biological agent and materials where
recoveries had not previously been determined by the
laboratory, method demonstration was performed to
determine the percent recoveries by extraction in PBS.
Results of the recovery demonstrations are presented in
Table 4-1. The recovery demonstrations included:
• B. suis from aluminum, keyboard, carpet, and joint
tape.
* B. cmlhracis from aluminum and keyboard.
* F. tularensis from aluminum, keyboard, and joint
tape.
• Vaccinia virus from aluminum, keyboard, carpet.
and joint tape.
* Y. peslis from aluminum, keyboard, carpet, and joint
tape.
In addition to the PBS extractions, two modifications of
the extraction solution were tested in an effort to improve
recoveries of F. tularensis and B. suis. For.B. suis and
F. tularensis, the extraction solution was modified by
addition of catalase (Roche, Indianapolis, IN) to the
PBS (PBS with 0.1% catalase). The hypothesis was
that the catalase might serve as an antioxidant to reduce
the rate at which F. tularensis and B. suis viability (and
corresponding recovery) declined. The addition of
catalase did not appreciably change the recoveries and
therefore calalase was not added to the methodology.
For F. tularensis, an additional extraction solution was
tested by amending the PBS with 100 mM trehalose
(Fisher Scientific. Pittsburg, PA). The hypothesis was that
trehalose might increase the persistence of F. tularensis as
had been demonstrated for Escherichia coli and Bacillus
fhuringiensis.9 Trehalose, likewise, did not substantially
improve recovery and, therefore, trehalose was not-
added to the methodology. Based on these extraction and
recovery results the Task Order Project Officer selected
the extraction methods described in Section 2.6.
For undetermined reasons, no B. suis was recovered from
keyboard keys in the first extraction and recovery from
aluminum was also low. However, in subsequent testing
B. suis was recovered from keyboard keys and aluminum
at useful levels (see Table 4-2).
For undetermined reasons, no F. tularensis was recovered
from joint tape. Attempts to improve recover}' by
incorporating catalase into the extraction buffer did not
yield any viable F. tularensis. Therefore, catalase was not
added to Hie extraction buffer for subsequent F. tularensis
persistence testing.
-------
Table 4-1. Biological Agent Recovery
Agent /
Material
Unique Aspects of Extraction
Approach
Replicate
Coupons
B. anthracis
Aluminum
Keyboard
Aluminum
Keyboard
None, enumerated 7/26/07
None, enumerated 7/26/07
None, enumerated 8/1/07
None, enumerated 8/1/07
3
3
3
3
B. suis
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Joint tape
Aluminum
Joint tape
None
None
None
None
Catalase, 0 hr
Catalase, 0 hr
Catalase, 5 hr
Catalase, 5 hr
3
3
3
3
5
5
5
5
F. tularensis
Aluminum
Aluminum
Aluminum
Aluminum
Aluminum
Aluminum
Aluminum
Keyboard
Keyboard
Aluminum
Joint tape
Aluminum
Joint tape
None
0 hr, no trehalose
0 hr with 100 mM trehalose
1 hr, no trehalose
1 hr with 100 mM trehalose
10 drops without trehalose
Single drop without trehalose
Single drop without trehalose
Single drop with trehalose
0. 1% Catalase, 0 hr
0. 1% Catalase, 0 hr
0.1% Catalase, 5 hr
0.1% Catalase, 5 hr
3
5
5
5
5
5
5
5
5
5
5
5
5
Vaccinia Virus
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
0-min dry time
0-min dry time
0-min dry time
0-min dry time
1 hr dry time
1 hr dry time
1 hr dry time
1 hr dry time
5
5
5
5
5
5
5
5
Y. pestis
Aluminum
Keyboard
Carpet
Joint tape
None
None
None
None
3
3
3
3
Spike Amount
CFU/coupon
9.87 x 10n
9.87 x 107t
9.17xl07t
9.17xl07t
CFU/coupon
1.71 x 10'
1.71 x 10'
1.71 x 10'
1.71 x 10'
4.10 x 10'
4.10 x 10'
4.10 x 10'
4.10 x 10'
CFU/coupon
6.80x10'
9.40 x 10'
1.11 x 10'
9.40 x 10'
1.11 x 10'
NA
NA
NA
NA
5.37x10'
5.37x10'
5.37 x 10'
5.37 x 10'
PFU/coupon
9.11 x 10'
6.38x10'
6.38x10'
9.11 x 10'
9.11 x 10'
6.38x10'
6.38x10'
9.11 x 10'
CFU/coupon
4.20 x 10'
4.20 x 10'
4.20 x 10'
4.20 x 10'
Agent Recovered
CFU/coupon*
3.79 ± 0.66 x 10'
4.25 ± 0.44 x 10'
4.18 ± 0.07 x 10'
4.61 ±0.62x10'
CFU/coupon*
1.75 ± 1.67x10'
0.00 ±0.00
2.45 ± 0.52 x 10'
1.85 ± 1.92x10'
3. 12 ±0.22x10'
2.49 ±0.53x10'
3.48 ± 0.58 x 10'
9.69 ± 0.88 x 10<
CFU/coupon*
2.70 ±2. 17x10'
7.95 ±2. 17x10'
1.25 ± 0.08 x 10'
2.31 ± 1.43x 10'
8.33 ± 1.60x10'
1.68 ± 0.10 x 10'
2.44 ± 1.51 x 10<
1.70 ± 0.24 x 10'
1.69 ± 0.53 x 10'
1.61 ±0.99x10'
0.00 ±0.00
1.19±0.70x 104
0.00 ±0.00
PFU/coupon*
6.68 ± 8.43 x 10'
3.97 ± 1.97x10'
3.44 ± 0.95 x 10'
7.34 ± 0.97 x 10'
3.46 ± 1.44x 10'
3.68 ± 1.51 x 10'
2.87 ±0.90x10'
7.03 ±3.41 x 10'
CFU/coupon*
3.75 ± 1.24x 10'
7.49 ± 21.6 x 10!
1.61 ± 1.86x10'
1.93 ± 1.67x10'
%*
6.39 ±0.67
7.18 ±0.44
7.60 ± 0.07
8.38 ±0.68
%*
1.70 ±0.98
0.00 ±0.00
23.9 ±3.02
1.80 ± 1.12
76.0 ± 5.47
6.07 ± 1.29
84.9 ± 14.2
2.36 ±0.21
%*
6.62 ±3. 18
84.6 ±23.1
113 ±6.95
2.46 ± 1.52
7.48 ± 1.44
NA
NA
NA
NA
0.30 ±0.18
0.00 ±0.00
0.22 ±0.13
0.00 ±0.00
%*
47.0 ± 9.26
62.3 ±30.8
54.0 ± 14.9
80.6 ± 10.6
38.0 ± 15.8
57.7 ±23.7
45.1 ± 14.1
7.71 ±3.75
%•
0.15 ±0.03
0.03 ± 0.05
0.06 ±0.04
0.01 ±0.00
NA = Not available.
* Data are expressed as mean ± standard deviation.
| Application was inadvertently about 1 log higher than the target 1 x 10' CPUs/coupon.
-------
4.2 Persistence Testing
Persistence results for each material and environmental
condition are summarized in Tables 4-2 through 4-5
and Figures 4-1 through 4-4. Persistence testing was not
conducted with B. anthracis spores, which are known
to survive for decades under ambient and adverse
conditions.9-10'u
Except as noted, in Section 4.2.1, no viable organisms
were recovered from any blank coupon.
4.2.7 B. suis Persistence
The persistence results obtained for B. suis are
summarized in Table 4-2 and Figure 4-1. B. suis
persisted on aluminum, keyboard, and carpet for the
longest exposure duration tested (7 days). Low levels of
B. suis were recovered after 4 hrs, but were not recovered
after 8 hr of exposure on joint tape.
Table 4-2. B. suis Persistence
Duration
Test
Temperature (oC)
Test
RH (%)
Spike Amount (CFU/
coupon)
Mean Recovered
B. suis (CFU/coupon)*
Mean (%)
Recovered
B. suis
Aluminum
Ohrt
2hr
4hr
8hr
3 days
7 days
NA
20-24
20-25
20-25
20-25
20-25
NA
24-38
25-41
24-37
26-42
27-42
3.30x10'
3.30x10'
3.30 x 10'
2.72 x 10'*
3.30x10'
3.30x10'
1.84 ± 0.15 x 10' t
8.82 ± 4.16 x 10' i
3.69 ± 1.36 x 10'
1.19±0.20x 10'
8.45 ± 1.47x10'
8.62 ±3.23x10'
56
27
11
44
2.6
2.6
Keyboard
Ohr|
2hr
4hr
8hr
3 days
7 days
NA
21-22
20-22
20-22
18-22
18-22
NA
40-51
40-51
35-52
35-60
37-60
2.30 x 10'
2.30 x 10'
2.30 x 10'
2.30 x 10'
2.30 x 10'
2.30 x 10'
2.45 ± 0.25 x 10'
2.45 ±0.11x10'
2.32 ±0.26x10'
1.39 ± 0.30 x 10'
2.96 ± 1.09 x 10"
1.06 ± 0.06 x 10'
106
106
101
60
13
4.6
Carpet
Ohr|
2hr
4hr
8hr
3 days
7 days
NA
20-24
20-25
20-25
20-25
20-25
NA
24-38
25-41
24-37
26-42
27-42
3.30x10'
3.30 x 10'
3.30 x 10'
2.72x10"
3.30x10'
3.30 x 10'
1.76 ±0.07x10'
3.23 ± 3.03 x 10'
4.91 ± 1.66 x 104
5.00 ± 4.92 x 10<
3.91 ±2.36x10'
4.66 ± 4.35 x 10!
53
<1
<1
<1
<1
<1
Joint Tape
Ohrt
2hr
4hr
8hr
3 days
7 days
NA
21-22
20-22
20-22
18-22
18-22
NA
40-51
40-51
35-52
35-60
37-60
2.30 x 10'
2.30 x 10'
2.30 x 10'
2.30 x 10'
2.30 x 10'
2.30 x 10'
2. 13 ±0.40x10'
1.60 ± 1.90 xlO!
3.26 ± 2.37 x 10'
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
93
<1
<1
0
0
0
NA = Not applicable.
Data are expressed as mean ± standard deviation of five replicates.
' 0 hr durations are positive control coupons that are spiked and extracted at time zero.
* 2.30 x 102 CPUs/coupon were recovered from the associated blank coupon.
§ 6.70 x 101 CPUs/coupon were recovered from the associated blank coupon.
Due to a spill associated with the 8 hr-test with aluminum and carpet, these materials were retested on a different day and therefore have spike
amounts different from associated tests.
^ 3.00 x 101 CPUs/coupon were recovered from the associated blank coupon.
-------
Figure 4-1. B. suis Persistence.
50
100
Duration (hi')
150
200
•Aluminum
•Kevboard
Carpet
•Joint tap?
-------
4.2.2 F. tularensis Persistence
The results obtained for persistence of F. tularensis are
summarized in Table 4-3 and Figure 4-2. F. tularensis
persisted 7 days (the longest duration tested) only on
keyboard. Persistence of F. tularensis on other materials
was: aluminum (8 hr), carpet (4 hr), and joint tape (8 hr).
Table 4-3. F. tularensis Persistence
Duration
Test
Temperature (°C)
Test
RH (%)
Spike Amount (CFUs/
coupon)
Mean Recovered
F. tularensis (CFUs/
coupon)*
Mean (%)
Recovered
F. tularensis
Aluminum
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
NM
NM
20-24
22-24
22-24
NA
NM
NM
25-45
21-42
22-41
4.47 x 10'
4.47 x 10'
4.47 x 10'
4.47 x 10'
4.47 x 10'
4.47 x 10'
5.37 ±0.75x10'
4. 40 ±3. 55 x 10'
1.95 ±0.81 x 10'
1.29 ± 1.04 x 10'
0.00 ±0.00
0.00 ±0.00
not
9.8
<1
<1
0
0
Keyboard
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
NM
NM
20-24
22-24
22-24
NA
NM
NM
25-45
21-42
22-41
4.47 x 10'
4.47 x 10'
4.47 x 10'
4.47 x 10'
4.47 x 10'
4.47 x 10'
5.40 ± 0.83 x 10't
5.04 ±3.02x10'
4. 12 ± 0.75 x 10'
6.05 ±4.17x10'
1.67 ±0.97x10'
3.98 ±3.66x10'
121
113
92
1.4
<1
<1
Carpet
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
NM
NM
20-25
22-24
22-23
NA
NM
NM
22-36
23-38
24-40
1.77x10'
1.77x10'
1.77x10'
1.77x10'
1.77x10'
1.77x10'
3.00 ± 1.02 x 10't
5.07 ±3.48x10'
4.54 ±3. 87 x 10'
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
169
<1
<1
0
0
0
Joint Tape
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
NM
NM
20-25
22-24
22-23
NA
NM
NM
22-36
23-38
24-40
1.77 x 10'
1.77 x 10'
1.77x10'
1.77x10'
1.77 x 10'
1.77 x 10'
1.29 ± 0.25 x 10'
6.00 ± 6.63 x 10'
6.00 ± 13.4 x 10'
6.00 ± 13.4 x 10'
0.00 ±0.00
0.00 ±0.00
73
<1
<1
<1
0
0
NA = Not applicable.
NM = Not monitored.
* Data are expressed as mean ± standard deviation of five replicates.
| 0 hr durations are positive control coupons that are spiked and extracted at time zero.
JExceeds target recovery of <120% of spike amount.
-------
Figure 4-2. F. tularensis Persistence.
50
100
Duration (hr)
150
200
•Aluminum
•Keyboard A Carpet Joint tape
-------
4.2.3 Vaccinia Virus Persistence
The results obtained for the persistence of vaccinia virus
are summarized in Table 4-4 and Figure 4-3. Vaccinia
virus persisted 7 days on aluminum, keyboard, and
carpet. On joint tape, vaccinia virus was recovered after
3 days but not after the 7-day duration.
Table 4-4. Vaccinia Virus Persistence
Duration
Test
Temperature (°C)
Test
RH (%)
Spike Amount (PFU/
coupon)
Mean Recovered
Vaccinia Virus (PFU/coupon)*
Mean (%)
Recovered
Vaccinia Virus
Aluminum
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
23-23
22-23
21-23
21-23
21-23
NA
37-45
37-45
37-45
37-45
37-45
1.14x10'
1.14x10'
1.14x10'
1.14x10'
1.14x10'
1.14x10'
1.19± 1.12x10'
1.57 ± 1.30x10'
8.15 ±6.72x10'
7.53 ± 17.5x10'
1.16±0.40x 10'
9.70 ± 7.69 x 10'
47
57
37
24
5.0
<1
Keyboard
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
23-23
22-23
21-23
21-23
21-23
NA
37-45
37-45
37-45
37-45
37-45
1.14x10'
1.14x10'
1.14x10'
1.14x10'
1.14x10'
1.14x10'
8.34 ± 1.10x10'
5. 13 ±2.08x10'
4.60 ±2.33x10'
3.33 ±0.59x10'
6.21 ± 1.67x10'
1.19±0.40x 10s
73
45
40
29
5.5
<1
Carpet
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
23-23
22-23
21-23
21-23
21-23
NA
37-45
37-45
37-45
37-45
37-45
1.14x10'
1.14x10'
1.14x10'
1.14x10'
1.14x10'
1.14x10'
5.08 ± 1.16x10'
2.94 ±2.26x10'
2.43 ± 0.41 x 10'
8.94 ±4.26x10'
9.66 ±2.07x10'
1.87 ± 1.34x 10'
45
26
21
7.8
<1
<1
Joint Tape
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
23-23
22-23
21-23
21-23
21 -23
NA
37-45
37-45
37-45
37-45
37-45
1.14x10'
1.14x 10'
1.14x 10'
1.14x10'
1.14x 10'
1.14x 10'
4.27 ± 1.09xlO't
1.46 ±0.71 x 10s
7.69 ±3. 14 x 10'
1.64 ±2.28x10'
2.01 ±2.75x10'
0.00 ±0.00
37
<1
<1
14
<1
0
NA = Not applicable.
* Data are expressed as mean ± standard dev
| 0 hr durations are positive control coupons
{Exceeded the positive control CV target of:
iation of five replicates.
that are spiked and extracted at time zero.
<25%.
-------
Figure 4-3. Vaccinia Virus Persistence
50
100
Duration (hi1)
150
200
•Aluminum
•Kevboard
Carpet
•Joint tape
-------
4.2.4 Y. pestis Persistence
The Y. pestis persistence results are summarized in Table
4-5 and Figure 4-4. 7 pestis persisted at relatively low
levels for 7 days on aluminum and joint tape. Y. pestis
persisted for 3 days on keyboard and 8 hr on carpet.
Table 4-5. 7 pestis Persistence
Duration
Test
Temperature (°C)
Test
RH (%)
Spike Amount (CFUs/
coupon)
Mean Recovered
Y. pestis (CFUs/coupon)*
Mean (%)
Recovered
Y. pestis
Aluminum
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
20-20
20-21
20-21
20-21
20-21
NA
58-64
52-62
36-62
36-59
36-61
2.90 x 10'
2.90 x 10'
2.90 x 10'
2.90 x 10'
2.90 x 10'
2.90 x 10'
6.49 ± 3.06 x 10'
1.45 ± 0.92 x 10'
1.23 ± 0.95 x 10'
5.92 ±7.31 x 10"
3.20 ± 4.09 x 10'
6.00 ± 13.4 x 10°
224J
50
42
<1
<1
<1
Keyboard
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
20-21
20-21
20-21
20-21
19-20
NA
49-75
51 -71
39-71
40-70
40-73
2.86 x 10'
2.86 x 10'
2.86 x 10'
2.86 x 10'
2.86 x 10'
2.86 x 10'
8.23 ± 1.26 x 10'
3. 89 ± 3.27 x 10'
2.52 ± 2.39 x 10'
2.12 ± 1.49 x 104
1.20 ± 1.64 x 10'
0.00 ±0.00
288J
136J
88
<1
<1
0
Carpet
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
20-20
20-21
20-21
20-21
20-21
NA
58-64
52-62
36-62
36-59
36-61
2.90 x 10'
2.90 x 10'
2.90 x 10'
2.90 x 10'
2.90 x 10'
2.90 x 10'
9.55 ± 0.84 x 10'
2.02 ± 0.79 x 10'
2.34 ± 5.23 x 10'
7.20 ± 12.8 x 10'
0.00 ±0.00
0.00 ±0.00
329J
70
8.1
<1
0
0
Joint Tape
Ohrf
2hr
4hr
8hr
3 days
7 days
NA
20-21
20-21
20-21
20-21
19-20
NA
49-75
51-71
39-71
40-70
40-73
2.86 x 10'
2.86 x 10'
2.86 x 10'
2.86 x 10'
2.86 x 10'
2.86 x 10'
4.30 ± 3.42 x 10'
8.69 ± 11. 4 x 10<
5. 14 ±8.28x10'
4.81 ±7.12x10'
1.20 ± 1.64 x 10'
3.34 ± 3.35 x 10'
150t
<1
<1
<1
<1
<1
NA = Not applicable.
* Data are expressed as mean ± standard deviation of five replicates.
| 0 hr durations are positive control coupons that are spiked and extracted at time zero.
JExceeds target recovery of <120% of spike amount.
-------
Figure 4-4. Y. pestis Persistence.
0
50
100
Duration (hi1)
150
200
-Aluminum
-Kevboard
Carpet
•Joint tape
-------
4.2.5 Summary of Persistence Testing
Table 4-6 provides a summary of persistence data,
calculated as the difference in the mean log of viable
bacteria (enumerated as CPUs/coupon) or virus
(enumerated as PFUs/coupon) recovered from coupons
at time zero and the mean log viable spores recovered
from coupons at a later specified time. The 95% CI and
p-value are also shown. Significant reduction in viable
biological agent was observed for most agent/material
combinations within 2 hr. Every biological agent
exhibited significant loss of viability within 2 hr from at
least one material.
Table 4-6. Summary of Persistence Results Calculated as Mean Log Reduction
Agent
Material
B. suis
F. tularensis
Vaccinia
Virus
Y. pestis
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Mean Log Reduction (95% confidence interval) and p-Value*
or Mean Log Reduction (# of persistence coupons with zero recovery /# of persistence coupons) and
p-Valuef
2hr
0.37 (0.09, 0.66)
p=0.0374
1.89 (1.47, 2.30)
p=0.0005
-0.00 (-0.05, 0.05)
p=0.9364
>5.63 (1/5)
p=0.0079
1.48 (0.48, 2.47)
p=0.0265
>5.24 (1/5)
p=0.0079
0.11 (-0.24, 0.46)
p=0.5107
>5.70 (1/5)
p=0.0079
-0.08 (-0.21, 0.05)
p=0.1879
0.32 (-0.02, 0.66)
p=0.0827
0.24 (0.04, 0.44)
p=0.0419
2.50 (2.22, 2.78)
p<0.0001
0.68 (0.18, 1.19)
p=0.0146
0.70 (0.51, 0.89)
p=0.0007
0.48 (-0.01, 0.96)
p=0.0833
2.80 (2.02, 3.58)
p<0.0001
4hr
0.72 (0.55, 0.90)
p=0.0004
2.57 (2.42, 2.72)
p<0.0001
0.02 (-0.04, 0.09)
p=0.4478
>6.05 (1/5)
p=0.0079
3.47 (3.26, 3.67)
p<0.0001
>6.10 (1/5)
p=0.0079
0.12 (0.01, 0.23)
p=0.0414
>6.81 (4/5)
p=0.0079
0.10 (-0.02, 0.22)
p=0.0934
0.32 (0.19, 0.45)
p=0.0008
0.30 (0.05, 0.56)
p=0.0446
2.76 (2.54, 2.99)
p<0.0001
0.73 (0.30, 1.16)
p=0.0040
4.49 (2.20, 6.78)
p=0.0106
0.63 (0.27, 1.00)
p=0.0138
>5.80 (2/5)
p=0.0079
8hr
0.19 (0.11, 0.28)
p=0.0030
2.68 (2.31, 3.05)
p<0.0001
0.25 (0.14, 0.36)
p=0.0022
>7.32 (5/5)
p=0.0079
4.73 (4.36, 5.10)
p<0.0001
>7.46 (5/5)
p=0.0079
2.09 (1.61, 2.57)
p=0.0005
>6.81 (4/5)
p=0.0079
>1.70(l/5)
p=0.0794
0.79 (0.54, 1.04)
p=0.0005
0.40 (0.30, 0.51)
p<0.0001
1.79(0.10,3.47)
p=0.0706
>6.02 (2/5)
p=0.0079
>6.89 (2/5)
p=0.0079
3.70 (3.30, 4.10)
p<0.0001
>4.60 (1/5)
p=0.0079
3 day
1.34 (1.26, 1.43)
p<0.0001
3.71 (3.47, 3.95)
p<0.0001
0.94 (0.75, 1.13)
p=0.0002
>7.32 (5/5)
p=0.0079
>7.73 (5/5)
p=0.0079
>7.46 (5/5)
p=0.0079
5.61 (5.21, 6.00)
p<0.0001
>7.10 (5/5)
p=0.0079
0.97 (0.87, 1.07)
p<0.0001
1.72 (1.57, 1.87)
p<0.0001
1.14 (1.00, 1.27)
p<0.0001
>6.14 (3/5)
p=0.0079
>6.75 (2/5)
p=0.0079
>7.98 (5/5)
p=0.0079
>7.32 (3/5)
p=0.0079
>6.86 (3/5)
p=0.0079
7 day
1.35 (1.19, 1.51)
p<0.0001
>5.51 (2/5)
p=0.0079
1.36 (1.31, 1.41)
p<0.0001
>7.32 (5/5)
p=0.0079
>7.73 (5/5)
p=0.0079
>7.46 (5/5)
p=0.0079
>6.42 (1/5)
p=0.0079
>7.10 (5/5)
p=0.0079
2.09 (1.96, 2.22)
p<0.0001
3.53 (3.15, 3.90)
p<0.0001
2.86 (2.71, 3.02)
p<0.0001
>7.62 (5/5)
p=0.0079
>7.45 (4/5)
p=0.0079
>7.98 (5/5)
p=0.0079
>7.91 (5/5)
p=0.0079
>6.41 (2/5)
p=0.0079
* Mean log reduction is the mean of the base-10 logarithm of recovered agent from the control (time zero) coupons minus the mean of the base-10
logarithm of recovered agent from the persistence time coupons. A 95 % CI for the difference is shown in parentheses. A p-value is provided for
the probability that the time zero and persistence time recoveries are the same. The p-value is from the two sample t-test with Satterthwaite's
method to allow for potentially different variances in the two groups. P-values less than 0.05 denote less than 1 in 20 chance that a difference
as large as or larger than observed would occur by chance if the time zero and persistence time means were truly identical. Comparisons with
p-values less than 0.05 (statistically significant at the 0.05 level) are bolded.
| One or more of the persistence time coupons had no recovered agent. The mean log reduction of the form ">X" is calculated as the mean of the
base-10 logarithm of recovered agent from the control (time zero) coupons minus the mean of the base-10 logarithm of recovered agent from the
persistence time coupons except that "zero recovery" coupons have a substituted recovered value of "1" (base-10 log is 0). Since the log becomes
an increasing negative value below 1 and is undefined at 0, this substitution is necessary and results in a lower bound on the mean log difference,
as indicated by the ">". The number of "zero recovery" persistence time coupons and the total number of persistence time coupons is shown
in parentheses. The p-value is from the non-parametric Kolmogorov-Smirnov test. P-values less than 0.05 denote less than 1 in 20 chance that
results as different as or more different than observed would occur by chance if the distribution of the time zero and persistence time recoveries
were truly identical. Comparisons with p-values less than 0.05 (statistically significant at the 0.05 level) are bolded.
-------
-------
5.0
Fumigation Technologies,
Test Matrices, and Results
The intent of the fumigant testing was to assess the
ability of the technology or decontamination process
to decontaminant materials at conditions consistent
with use in a facility. However, laboratory testing may
present a challenge when testing at a smaller scale
than for which the decontamination equipment was
designed. For the Sabre C1O2 testing, Sabre Technical
Service, LLC. provided a prototype unit designed
for reproducing their process in a smaller, lab-scale,
environment (e.g., 317 L glove box). For the BIOQUELL
hydrogen peroxide fumgition, the initial intent was to
test using the 317 L glove box. In order to represent a
typical room fumigation with the BIOQUELL hydrogen
peroxide fumigation process, the temperature rise in
the enclosed space due to the fumigation equipment
must be minimized. To accomplish this in lab testing,
BIOQUELL provided their Claras S unit desgined for
typical use in biological safety cabinets. After testing
with that unit, it was decided to test at a larger scale
(1275 L Biological Safety Cabinet), utilizing one of their
larger fumigation units (Claras C) with an attempt to
obtain a better representation of room-scale fumigation.
The STERIS VHP® system was a unit of similar size
and design parameters to the BIOQUELL Claras C, and
tested at the same scale (1275 L).
Various controls were included in all of the fumigation
testing described below. Application controls, positive
controls, and blanks were included with the test samples
in the experiments. An application control (i.e., spike
amount) is a quantification of the amount of biological
agent applied using a streak plate method described in
Section 2.6. A positive control is a coupon spike with
biological agent but not subjected to the test conditions.
A laboratory blank is a coupon spiked with diluents
without biological agent and not subjected to the test
condition. A procedural blank is a coupon spiked with
diluents without biological agent and subjected to the test
condition. A test coupon is spiked with biological agent
and subjected to the test condition.
No viable organisms were recovered from any blank
coupon.
5.1 CIO2 Fumigation (Sabre)
5.1.1 Description of Sabre CIO2 Technology
The Sabre technology (Sabre Technical Services, LLC,
Slingerlands, NY) in this evaluation uses C1O2 as the
active ingredient. C1O2 is unstable as a compressed
gas and, therefore, CIO gas must be produced on-site.
The Sabre decontamination technology includes the
equipment and chemicals for on-site generation, delivery,
removal, and neutralization of C1O2. The decontamination
technology was operated as specified in SOP MREF
X-13512 and summarized below.
The Sabre equipment included a 20 cm base onto which
was mounted a 15 cm square, 91 cm high sparging
column (see Figure 5-1). A 5-gallon container containing
15 L of an aqueous solution of 3 g/L of C1O2 plus 1,000
parts per million (ppm) of chlorite was prepared on-site.
The 5-gallon container was vented through a sodium
thiosulfate trap and placed in an over-pack for safety.
The C1O2 solution was pumped (using a peristaltic
pump) into a sparging column and air from the test
chamber was pumped into and through the column to
sparge C1O2 from the liquid into the air stream. The air
stream re-entered the glove box to establish the desired
gaseous C1O2 concentration. Liquid introduction from
the reservoir of ClO2/chlorite solution to the sparging
column was initially at the rate of 60 mL per min. When
the desired C1O2 concentration in the test chamber
was achieved, the liquid introduction into the sparging
column was decreased to 0 to 3 mL per min. As the C1O2
concentration dropped, additional gas was added to the
chamber by manually increasing the flow rate to achieve
the target concentration. The spent liquid exiting the
sparging column was collected in a reservoir. The air
from the chamber was recirculated into and out of the
sparging column.
Figure 5-1. Sabre Bench-scale C1O2 Generator.
At the end of the decontamination test the C1O2 in the
system was neutralized by pumping a 10% sodium
hydroxide solution (or a 10% sodium thiosulfate solution)
into the sparging column.
-------
The concentration of C1O2 in the test chamber was
monitored before and during (approximately every
20 min) an experiment using a modified titration
method based on the Standard Method 4500-C1O2 E
Amperometric Method II13 as recommended and used
by Sabre Technical Services and as specified in the test/
QA plan. For this titration method, air from the test
chamber is drawn through impingers (at a rate of 1 L/
min using an air mass flow controller) that contain
15 mL of 5% potassium iodide in phosphate buffer
(pH 7.0) solution. The pH of the impinger solution
was measured and recorded before use. Under these
conditions chlorine dioxide oxidizes iodide to iodine
and reduces chlorine dioxide gas to the chlorite ion
which dissolves in solution. Chlorite ion does not react
at neutral pH. After collection and reaction of the
chlorine dioxide gas, the impinger solution was acidified
and the chlorite was allowed to react further with the
iodide ion, forming additional iodine and reducing the
chlorite to chloride. The pH of the impinger solution was
measured and recorded immediately before titration.
The total resulting iodine was reduced to iodide when
titrated against standard 0.1 normal [equal to 0.1 molar]
sodium thiosulfate (STS). Molecular iodine appears
yellow-brown in aqueous solution. The titration endpoint
was determined when the color of the solution changed
from yellow-brown to colorless. The volume (mL) of
STS solution titrated is proportional to the amount of
iodine generated, which is proportional to the gas-phase
chlorine dioxide concentration in the air that passed
through the impinger. Using the formula in Equation
9 below, the concentration of chlorine dioxide was
calculated.
Table 5-1. Test Matrix for Sabre CIO Fumigation
Equation 9.
where:
CIO. (ppmv) =
V2(L)
-x-x24.45xlOOO
C1O2 = chlorine dioxide (ppmv in air)
Vj = volume of STS titrant (mL)
M = molarity (mol/L) of STS titrant (which for STS is
equal to its normality)
V2 = volume of air (at 25 °C, 1 atm) that passed
through impinger (L)
24.45 = ideal gas constant, L/mol, at 25 °C, 1 atm
1000 = conversion factor = 106 ppmv x 1 L /1000 mL
Certified NIST- traceable chlorite standards,
appropriately diluted in solution comparable to the
sampling solution, were titrated each day of chlorine
dioxide sampling to verify accuracy.
5.7.2 Test Matrix for Sabre CIO2 Fumigation
The tests performed with Sabre C1O2 are shown in Table
5-1. The experimental design tested decontamination
efficacy by determining whether there was a difference
between the log reductions in the viable biological agents
after fumigation compared to controls for aluminum,
keyboard, carpet and joint tape. These tests also assessed
whether there were any differences in efficacy at varying
RH levels and varying fumigation contact times. Critical
parameters impacting the viability of biological agents
included C1O2 concentration, fumigation contact time,
temperature, RH, and natural attenuation. An adaptive
management approach was used to incorporate new
knowledge into the testing as decontamination efficacy
results became available.
Biological
Agent
B anthracis
indicator
B. suis
F. tularensis
Vaccinia virus
Y. pestis
Material
Aluminum Keyboard
Aluminum, Keyboard
Aluminum, Keyboard
Aluminum^, Keyboard
Carpet ' , Joint tape
Aluminum, Keyboard
C1O2 Concentration, Temperature
3 000 ppmv
23 °C ± 2 °C
50-100 ppmv ± 25 ppmv,
23 °C ± 2 °C
50-100 ppmv ± 25 ppmv,
23 °C ± 2 °C
50-100 ppmv ± 25 ppmv,
23 °C ± 2 °C
50-100 ppmv ± 25 ppmv,
23 °C ± 2 °C
% RH ± % (full scale)
40% ± 5%
75% ± 5%
40% ± 5%
60% ± 5%
75% ± 5%
40% ± 5%
75% ± 5%
40% ± 5%
60% ± 5%
75% ± 5%
40% ± 5%
75% ± 5%
Contact Times (min)
0 20 40 60 90 180
0, 20, 40, 60, 90, 180
0, 30, 60, 120
0, 30, 60, 120
0, 30, 60, 120
0, 30, 60, 120
0, 30, 60, 120
0, 60, 120
0, 120
0, 30, 60, 120t
0, 30, 60, 120
0, 30, 60, 120
* Five B. atrophaeus on steel in Tyvek* packaging were exposed to Sabre C1O2 fumigation at the 40% RH at each contact time out to 180 min.
T Aluminum, carpet, and joint tape were not tested at 120 min at 75% RH.
-------
Five replicate test coupons (plus one procedural blank)
and five replicate positive control coupons (plus one
laboratory blank) were included at each set of conditions
and time points. Negative controls (procedural
blanks and laboratory blanks) were coupons to which
corresponding diluent, but no biological agent, was
applied.
Decontamination was halted by dropping the
atmospheric concentration of CIO, to near zero. The
coupons were placed in sealed vials and transferred
into a second BSC III, the vials were opened, and
the coupons were transferred into the extraction
medium. No quenching agents were added to stop the
decontamination reaction because residual CIO, was
assumed to be rapidly removed from the coupon through
convection, diffusion, and dilution.
5.1,3 CIO2 Fumigation
Figure 5-2 shows a graph of typical temperature, RH,
and C1O2 concentration during a fumigation cycle.
Figure 5-2. Temperature, RH, and CIO, Concentration Dynamics During aTypical Fumigation Cycle; " 15 min Strip"
and "5 min Strip" Refers to Addition of CIO, to the Test Chamber.
C1O2 Concentration
3500 r 100
h 90
_...-•-"" ""'"- •—„,.„„.. j
2500 / I 70
j p"
2000 ^
t: s *
1500 j 40 Ł*
j v
h 30
500 1
I- 10
0
*
cto2PPM ^— Temp
-------
B. atrophaeus
Biological indicators that were B. atrophaeus spores
(nominally 106 spores) on steel in Tyvek® packaging
were exposed during the Sabre C1O2 fumigation test with
B. anthracis spores at "3,000 ppmv C1O2, 23°C, 40%
RH". All biological indicators, including five replicates at
the 180-min contact time (the longest time tested), were
positive for growth, which is indicative of incomplete
kills. The biological indicators (on steel) are consistent
with B. anthracis on aluminum which retained viable
spores at all treatment conditions.
B. anthracis
Sabre C1O2 fumigation results for B. anthracis spores are
presented in Table 5-2 and Figures 5-3 and 5-4. For all
materials tested except aluminum, a 90-min contact time
at 3,000 ppmv C1O2, 23 °C, and 75% RH resulted in no
viable B. anthracis spore recovery. B. anthracis spores
were recovered from aluminum following Sabre C1O2
fumigation for all tests conducted (out to 180 min).
Log reductions in B. anthracis spores on keyboard were
> 6.57 for all tests conducted at both 40% and 75% RH.
Viable B. anthracis spores (6.6 CPUs/coupon) were
recovered from keyboard at only one condition (20-min
contact time, 3,000 ppmv C1O2, 23 °C, and 75% RH).
At the 3,000 ppmv C1O2, 23 °C condition, no viable B.
anthracis spores were recovered from carpet after the
180-min contact time at 40% RH or following the 90-
min contact time at 75% RH.
At the 3,000 ppmv C1O2, 23 °C condition, no viable B.
anthracis spores were recovered from joint tape after the
90-min contact time at 75% RH. At the corresponding
test condition except 40% RH rather than 75% RH, B.
anthracis spores were recovered at 180-min contact time
(the longest time tested).
Table 5-2. Sabre CIO Fumigation Results for B. anthracis
Contact
Time
Material
Spike
Amount CFU/
coupon
Mean Recovered B. anthracis (CFU/coupon)*
Positive
Controlf
Test
CouponJ
Mean Log
Reduction*
3,000 ppmv C1O2, 23°C, 40% RH
0 min
20 min
40 min
60 min
90 min
180 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
9.60 x 106
1.00 x 107
1.00 x 107
9.60 x 106
9.60 x 106
1.00 x 107
1.00 x 107
9.60 x 106
9.60 x 106
1.00 x 107
1.00 x 107
9.60 x 106
9.60 x 106
1.00 x 107
1.00 x 107
9.60 x 106
9.60 x 106
1.00 x 107
1.00 x 107
9.60 x 106
9.60 x 106
1.00 x 107
1.00 x 107
9.60 x 106
2.31 ± 0.07 xlO6
2.96 ± 0.31 xlO6
5.91 ± 0.40 xlO6
2.42 ± 0.10 x 106
3. 84 ± 0.78 x 106
3.71 ± 1.23 x 106
5.53± 1.20 x 106
3. 80 ± 0.37 x 106
3.84±0.78xl06
3.71 ± 1.23 xlO6
5.53±1.20xl06
3.80±0.37xl06
3. 84 ± 0.78 x 106
3.71 ± 1.23 x 106
5.53± 1.20 x 106
3. 80 ± 0.37 x 106
3. 84 ± 0.78 x 106
3.71 ± 1.23 xlO6
5.53±1.20xl06
3.80±0.37xl06
3.84±0.78xl06
3.71 ± 1.23 x 106
5.53± 1.20 x 106
3. 80 ± 0.37 x 106
Not applicable
Not applicable
Not applicable
Not applicable
2.82 ± 1.15 x 106
0.00 ±0.00
5.16±3.13x 104
4.21 ± 3.33 x 103
9.01 ± 2.48 xlO5
0.00 ±0.00
8.89±4.49xl03
9.50 ± 4.24 x 103
1.19±0.89x 106
0.00 ±0.00
1.20 ± 1.39 x 102
1.07 ± 2.07 x 104
2.61 ± 3.26 x 105
0.00 ±0.00
2.66 ± 4.91 xlO2
1.22 ± 1.47xl04
1.21 ± 2.39 xlO3
0.00 ±0.00
0.00 ±0.00
8.53 ± 11.1 x 103
Not applicable
Not applicable
Not applicable
Not applicable
0.17 ±0.23
6.57 ±0.00
2.22 ±0.61
3.28 ±0.80
0.64 ±0.12
6.57 ±0.00
2. 84 ±0.24
2.65 ± 0.26
0.58 ±0.25
6.57 ±0.00
5.41 ± 1.24
3.29 ±0.96
1.54 ±0.67
6.57 ±0.00
5.67 ± 1.49
2.68 ±0.42
4.88± 1.66
6.57 ±0.00
6.74 ±0.00
2.93 ±0.55
-------
Contact
Time
Material
Spike
Amount CFU/
coupon
Mean Recovered B. anthracis (CFU/coupon)*
Positive
Controlf
Test
CouponJ
Mean Log
Reduction*
3,000 ppmv C102, 23°C, 75% RH
20 min
40 min
60 min
90 min
180 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
5.73 x 106§
3.83 x 106§
5.73xl06§
3.83xl06§
5.73 x 106
3.83xl06§
5.73 x 106§
3.83 x 106§
5.73 x 106§
3.83 x 106§
5.73xl06§
3.83xl06§
5.73xl06§
3.83xl06§
5.73xl06§
3.83 x 106§
5.73 x 106§
3.83 x 106§
5.73 x 106§
3.83xl06§
7.92 ± 1.08 x 106*
8.91 ± 1.09 x 106*
3.89 ± 0.47 xlO6
3.89 ± 4.21 xlO6
6.64 ± 0.77 xlO6
1.41±0.77xl07*
4.45± 1.65 x 106
5.12 ± 1.44 x 106*
6.98 ± 1.07 x 106*
8.99 ± 0.48 x 106*
3.79 ± 0.16 x 106
2.54 ± 1.06 xlO6
6.27 ± 0.76 xlO6
9.07 ± 1.37 xlO6*
3.72 ± 0.66 x 106
3.61 ±0.31 x 106
7.16 ± 1.15 x W6
1.66 ± 1.46 x 107*
5.99 ± 1.11 x 106
3.16 ± 0.18 xlO6
1.13± 1.88x 102
6.60 ± 14.8 x 10°
9.99 ± 7.43 xlO3
1.07 ± 2.03 xlO2
3. 14 ± 4.46 x 102
0.00 ±0.00
2.39 ± 2.34 x 103
2.00 ± 2.99 x 101
9.01 ± 18.8 x 102
0.00 ±0.00
2.20 ± 2.56 x 102
4.02 ±3.67x10'
3.73 ± 6.45 xlO2
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
2.00 ± 2.99 x 101
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
5.95 ± 1.32
6.65 ±0.68
2.68 ±0.31
5.69 ±1.27
4.97±1.15
7.15 ±0.00
3.40 ±0.35
6.04 ±0.92
4.99 ± 1.29
6.95 ±0.00
4.52 ±0.57
5.31 ±1.00
5.35 ± 1.44
6.96 ±0.00
6.57 ±0.00
6.56 ±0.00
6.19 ±0.92
7.22 ±0.00
6.78 ±0.00
6.50 ±0.00
* Data are expressed as mean ± standard deviation of five replicates.
T Positive control coupons were spiked but not exposed to the fumigant.
* Test coupons were spiked and exposed to the fumigant for the contact time.
§ Application lower than the target range 7.5 x 106 - 1.25 x 10' CFU/coupon.
* Exceeds target recovery of <120% of spike amount.
-------
Figure 5-3. Sabre fumigation results for B. anthracis at 3,000 ppmv C1O2 and 23 °C, line chart.
8
•40% RH, Aluminum
75 %RH, Aluminum
-40°o RH, Keyboard
75% RH, Keyboard
-40% RH: Carpet
75%RHr Carpet
-4 0%RH, Joint tape
75 %RHr Joint tape
2000 4000 6000
CT, ppmv-hr
sooo
10000
Figure 5-4. Sabre Fumigation Results for B. anthracis at 3,000 ppmv CIO and 23 °C, Column Chart.
• 40% RH, Aluminum
75Do RH. Aluminum
• 40% RH: Keyboard
75%RH: Keyboard
• 40% RHr Carpet
75%RH, Carpet
• 40% RH: Joint tape
75 %RH: Joint tape
1000
2000
3000
CT. ppmv-hr
4500
9000
-------
Table 5-3 provides a summary of C1O2 decontamination
efficacy against B. anthracis spores, calculated as the
difference in the mean log of viable spores recovered
from coupons at time zero and the mean log of viable
spores recovered from coupons after fumigation for a
given contact time. The 95% CI and p-value are also
shown. Significant reduction in viable biological agent
was observed for all agent/material combinations with a
40-min contact time at both 40% RH and 75% RH.
Table 5-3. Summary of Efficacy (Calculated as Mean Log Reduction) of C1O2 against B. anthracis spores
Mean Log Reduction (95% CI) and p-value*
or Mean Log Reduction (# of treated coupons with zero recovery/# of treated coupons) and p-Valuef
Material
Aluminum
Carpet
Keyboard
Joint Tape
%RH
40
75
40
75
40
75
40
75
211 niin
0.17 (-0.085, 0.42)
p=0.1865
>5.94 (3/5)
p=0.0079
2.21 (1.57, 2.84)
p=0.0010
2.68 (2.36, 3.01)
p<0.0001
>6.55 (5/5)
p=0.0079
>6.64 (4/5)
p=0.0079
3.28 (2.45, 4.11)
p=0.0008
>5.51 (3/5)
p=0.0079
40min
0.64 (0.49, 0.78)
p<0.0001
>4.97 (1/5)
p=0.0079
2.83 (2.56, 3.11)
p<0.0001
3.38 (2.99, 3.77)
p<0.0001
>6.55 (5/5)
p=0.0079
>7.11 (5/5)
p=0.0079
2.65 (2.38, 2.93)
p<0.0001
>6.02 (3/5)
p=0.0079
611 niin
0.57 (0.30, 0.85)
p=0.0052
>4.98 (1/5)
p=0.0079
>5.40 (2/5)
p=0.0079
4.51 (3.92, 5.11)
p<0.0001
>6.55 (5/5)
p=0.0079
>6.95 (5/5)
p=0.0079
3.29 (2.30, 4.28)
p=0.0015
>5.27 (2/5)
p=0.0079
90min
1.54 (0.84, 2.24)
p=0.0066
>5.35 (2/5)
p=0.0079
>5.66 (3/5)
p=0.0079
>6.57 (5/5)
p=0.0079
>6.55 (5/5)
p=0.0079
>6.95 (5/5)
p=0.0079
2.68 (2.25, 3.11)
p=0.0001
>6.56 (5/5)
p=0.0079
180 min
>4.87 (2/5)
p=0.0079
>6.18 (3/5)
p=0.0079
>6.73 (5/5)
p=0.0079
>6.77 (5/5)
p=0.0079
>6.55 (5/5)
p=0.0079
>7.07 (5/5)
p=0.0079
2.93 (2.36, 3.49)
p=0.0003
>6.50 (5/5)
p=0.0079
* Mean log reduction is the mean of the base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm
of recovered agent from the treated coupons. A 95 % CI for the difference is shown in parentheses. A p-value is provided for the probability that
the control and treatment recoveries are the same. The p-value is from the two sample t-test with Satterthwaite's method to allow for potentially
different variances in the two groups. p-Values less than 0.05 denote less than 1 in 20 chance that a difference as large as or larger than observed
would occur by chance if the control and treatment means were truly identical. Comparisons with p-values less than 0.05 (statistically significant
at the 0.05 level) are bolded.
| One or more of the treatment coupons had no recovered agent. The mean log reduction of the form ">X" is calculated as the mean of the
base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm of recovered agent from the treated
coupons except that "zero recovery" coupons have a substituted recovered value of "1" (base-10 log is 0). Since the log becomes an increasingly
negative value below 1 and is undefined at 0, this substitution is necessary and results in a lower bound on the mean log difference, as indicated
by the ">". The number of "zero recovery" treatment coupons and the total number of treatment coupons is shown in parentheses. The p-value
is from the non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance that results as different as or more
different than observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with
p-values less than 0.05 (statistically significant at the 0.05 level) are bolded.
-------
B. suis
Sabre CIO, fumigation results for 13. suis are presented in
Tables 5-4 and 5-5 and in Figures 5-5 and 5-6. Persistence
tests reported in Section 4.2.1. as well as positive controls
used in these tests, showed that there was a differential
loss of viability of B. suis. depending on the coupon
type. Specifically, over the 1 hr drying time and multiple
hr contact times, there was a significant reduction in
viable B. suis on carpet and joint tape. As described in
the Introduction, the calculated log reductions reflect the
incremental impact of the fumigation technology.
B. suis on aluminum exposed to 50-100 ppmv CIO,
(23 °C) exhibited log reductions that increased with
increasing RH. Specifically, log reductions were low
(<2.0) for all contact times tested at low RH (40%). At
the same C1O2 concentration and temperature, the log
reductions increased but remained <4.0 at 60% RH. At
the same C1O2 concentration and temperature, the log
reductions were >5.0 with no B. suis recovered from
aluminum after 60-min and 120-min contact times at 75%
RH.
B. suis on keyboard exposed to 50-100 ppmv CIO, (23
°C) exhibited low log reductions at both 40% and 60
% RH (<1.0) for all contact times tested. At the same
CIO, concentration and temperature, the log reductions
were low (<2.66 up to 60 min), but reached a 5.63 log
reduction at the 120-min contact time at 75% RH. B. suis
was recovered from every test conducted with keyboard.
Because of their small size, the keyboard keys were
spiked with a single 100 uL droplet, rather than the 10 x
10 pL droplets used with all of the other materials. The
single droplet may result in a "'stacking" of bacteria that
may shield them from contact with decontamination
treatments, thus resulting in relatively lower efficacy
results.
There was generally a decrease in the recovery of viable
B. suis from carpet positive control coupons (not exposed
to fumigation) during the period of decontamination. The
decline in viable B. suis from carpet was consistent with
the ~2 log loss of viable B. suis from carpet after 2 to 4
hr observed in the persistence testing. During fumigation
testing with CIO,, the positive control coupons were
allowed to dry for one hr.and an additional period of
time (about 30 min) passes before the CT clock starts
for fumigation. Thus, after 120-min fumigation, the
positive controls may be at a time equivalent to 3.5 hr
in the persistence testing. There is a decrease in the
recovery of viable B. suis from carpet positive control
coupons (not exposed to fumigation) during the period
of decontamination that was consistent with the loss
of viable B. suis from carpet in the persistence testing.
Because the log reduction reflects only the incremental
impact of fumigation (controls for loss of viability
without treatment), overall efficacy must be interpreted
in the context of the loss of viability with treatment. For
example, carpet exposed to the fumigation treatment for
60 min at 60% RH shows a log reduction of only 2.11.
However, only about 102 bacteria are recovered from the
untreated carpet. (This recover}' results in a very low base
to which the treated carpet was compared.) Compared to
the amount of bacteria spiked onto the coupon (3.77 x 107
CPUs), the amount of bacteria recovered after the 60-min
treatment (6.00 x 101 CPUs/coupon) represents almost
a 6 log reduction in viable bacteria attributable to the
fumigation and the loss of viability from carpet arising
from other (unknown) causes.
There was a decrease in the recover}- of viable B. suis
from joint tape positive control coupons (not exposed to
fumigation) during the period of decontamination. (The
decline in viable B. suis from joint tape was consistent
with the >5 log loss of viable B. suis from joint tape after
2 hr observed in the persistence testing.) B. suis was only
recovered from joint tape after Sabre CIO, fumigation
from "50-100 ppmv CIO,, 23 °C, 40% RH" at contact
times of 30 min and 60 min; the associated log reductions
were <2.0. Compared to the amount of bacteria spiked
onto the coupon (5.90 x 107 CPUs for 40% RH test
and 8.23 x 107 CPUs for 60% RH test), the amount of
bacteria recovered after the 120-min treatment (0 CPUs/
coupon) represents >7.7 log reduction in viable bacteria
attributable to the fumigation and the loss of viability
from joint tape arising from other (unknown) causes.
-------
Table 5-4. Sabre CIO Fumigation Results for B. suis
Contact
Time
Material
Spike
Amount
(CFU/coupon)
Mean Recovered B. suis (CFU/coupon)*
Positive
Controlf
Test
CouponJ
Mean Log Reduction*
50-100 ppmv C1O2, 23°C, 40% RH
0 min
30 min
60 min
120 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
5.90 x 107
5.90 x 107
5.90 x 107
5.90 x 107
5.90 x 107
5.90 x 107
5.90 x 107
5.90 xlO7
3.37 x 107
3.37 x 107
3.37 x 107
3.37 x 107
5.90 x 107
5.90 x 107
5.90 x 107
5.90 x 107
4.65 ± 0.97 x 107
3.62 ± 1.26 x 107
7.29 ± 0.82 x 107
1.08±0.35xl04§
7.37 ± 1.24 xlO7
3.38 ± l.llxlO7
2.47 ± 1.01 x 107
4.40 ± 2.39 x 102§
3.38 ± 0.43 x 107
4.55 ± 1.81 x 107
5.53 ± 2.67 x 105
1.74 ± 1.30 x 104§
8. 18 ± 2.48 x 107
2.94 ± 0.21 xlO7
2.14± 1.39 xlO5
0.00±0.00§
Not applicable
Not applicable
Not applicable
Not applicable
3.83 ± 0.38 xlO7
2.47 ± 0.48 xlO7
6.60 ± 14.8x10°
2.51 ± 3.94 xlO3
1.61 ± 0.16 x 107
1.01 ± 0.27 x 107
2.55 ± 4.67 x 103
4.16 ±4.11 x 103
7.89 ± 2.57 xlO6
5.81 ± 1.90 xlO6
0.00 ±0.00
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
0.29 ±0.04
0.14±0.10
7.09 ±0.68
1.14±2.06
0.32 ±0.05
0.67 ±0.13
2.84 ±0.67
0.82 ±0.49
1.03 ±0.12
0.72 ±0.14
5.33 ±0.00
Not calculable
50-100 ppmv C1O2, 23°C, 60% RH
0 min
30 min
60 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
3.77 x 107
3.77 x 107
3.77 x 107
3.77 xlO7
8.23 x 107
8.23 x 107
8.23 x 107
8.23 x 107
3.77 x 107
3.77 x 107
3.77 x 107
3.77 x 107
8.85 ± 10.9 x 107
1.17±0.15x 107
1.16 ± 0.59 x 107
8.44 ± 3.36 x!03§
1.94±0.20xl07
5.69 ± 1.96 xlO7
4.94 ± 3.37 xlO6
1.28 ± 1.47 x 102§
1.19 ± 0.17 x 107
7.91 ± 0.67 x 106
8.81 ± 2.35 x 102
2.60 ± 1.66 x 102§
Not applicable
Not applicable
Not applicable
Not applicable
2.08 ± 0.55 xlO6
2.53 ± 0.79 xlO7
9.34 ± 10.6 xlO2
0.00 ±0.00
1.77 ± 1.74 x 106
4. 19 ± 0.42 x 106
6.00 ± 10.1 x 101
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
0.98 ±0.12
0.37 ±0.14
3.92 ±0.45
2.11 ±0.00
1.03 ±0.48
0.28 ±0.04
2.11 ± 1.16
2.41 ±0.00
-------
Contact
Time
120 min
Material
Aluminum
Keyboard
Carpet
Joint tape
Spike
Amount
(CFU/coupon)
8.23 x 107
8.23 x 107
8.23 x 107
8.23 x 107
Mean Recovered B. suis (CFU/coupon)*
Positive
Controlf
2.01 ± 1.13 x 107
2.09 ± 0.70 xlO7
1.17± l.OlxlO5
3.31 ± 2.75 x!02§
Test
CouponJ
4.77 ± 1.26 x 103
2.25 ± 0.49 x 106
0.00 ±0.00
0.00 ±0.00
Mean Log Reduction*
3.64±0.11
0.98 ±0.11
5.07 ±0.00
2.52 ±0.00
50-100 ppmv C102, 23°C, 75% RH
0 min
30 min
60 min
120 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
3.33 x 107
3.33 x 107
3.33 x 107
3.33 x 107
3.33 x 107
3.33 x 107
3.33 x 107
3.33 x 107
8.00 x 107
8.00 x 107
8.00 x 107
8.00 x 107
3.33 x 107
3.33 x 107
3.33 x 107
3.33 x 107
1.28 ± 0.89 xlO7
4.32 ±2. 10 x 107
1.01 ± 0.36 x 107
4.20 ± 0.64 x 104§
8.59 ± 1.98 x 106
3.25 ±2. 12 x 107
5.54±3.25xl06
7.29 ± 5.09 x!03§
3.22 ± 0.64 xlO7
6.31 ± 1.73xl07
1.61 ± 2.10 xlO6
8.66 ± 11. 5 x 101§
2.51 ± 1.37 x 107
9.60 ± 0.85 x 106
7.81 ± 16.7 x 105
3.80±3.62xl02§
Not applicable
Not applicable
Not applicable
Not applicable
6.87 ± 10.9 x 102
2.12 ± 1.78 x 105
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
7.96 ± 2.46 xlO6
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
8.63 ± 19.2 x 103
0.00 ±0.00
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
5.21 ± 1.61
2.66 ± 1.10
6.74 ±0.00
3. 86 ±0.00
7.51 ±0.00
0.92 ±0.14
6.21 ±0.00
1.94 ±0.00
7.40 ± 0.00
5.63 ±2.05
5. 89 ±0.00
2.58 ±0.00
Data are expressed as mean ± standard deviation of five replicates.
t Positive control coupons were spiked but not exposed to the fumigant.
^ Test coupons were spiked and exposed to the fumigant for the contact time.
§ Below target recovery of >10% of spike amount.
Figure 5-5. Sabre Fumigation Results for B. suis at 50-100 ppmv C1O2 and 23 °C, Line Chart.
S
—»—40% RHr Aluminum
60% RH7 Aluminum
75 %RHr Aluminum
—•—40% RH, Keyboard
60% RHr Keyboard
75%RHr Keyboard
—*— 40% RHr Carpet
60% RHr Carpet
75%RH; Carpet
X 40% RfL Joint tape
—*— 60% RHr Joint tape
75 %RHr Joint tape
20
40
60
120
SO 100
CT, ppmv-hr
* Log reduction not calculated for the 150 CT as B. suis was not recovered from either the test or associated control coupons.
140
160
-------
Figure 5-6. Sabre Fumigation Results for B. suis at 50-100 ppmv CIO and 23 °C. Column Chart.
PH
HH
RH,
RH,
RH;
RH,
RH,
RH.
RH,
RH,
RH,
RH,
«j
A 1ltt>i<*"'*^
. * iC -
-\i' 1111 1111 illl "
Aluminum „
Ke- 'board = "
Ker-*Doaro "S •*
Keyboard —
Carpet
T _
Carpet
Joint tape ,
Joint tape
Joint tape 0
ii - , _
150
CT, ppmv-hr
* Log reduction not calculated for the 150 CT as B. suis was not recovered from either the test or associated control coupons.
Table 5-5 provides a summary of CIO, decontamination
efficacy against B. suis, calculated as the difference in
the mean log of viable bacteria recovered from positive
control coupons and the mean log of viable bacteria
recovered from coupons after fumigation for a given
contact time. The 95% CI and p-value are also shown.
To control for history, the elapsed time from spiking
to recover}' was the same for the positive control and
test coupons. At the 120-min contact time efficacy was
demonstrated at all three RH conditions (40%, 60%,
and 75%) for aluminum, carpet, and keyboard. Because
of the temporal loss of viable bacteria from positive
control coupons at 40% and 120% RH, only the 60% RH
results confirm incremental efficacy from the fumigation
compared to the temporal loss in viability.
For aluminum and keyboard (hard, non-porous surfaces)
for the 120-min contact time, the efficacy was higher at
75% RH than at 40% or 60% RH.
-------
Table 5-5. Summary of Efficacy (Calculated as Mean Log Reduction) of CIO against B. suis
Mean Log Reduction (95% CI) and p-Value*
or Mean Log Reduction (# of treated coupons with zero recovery /# of treated coupons) and p-Valuef
or N/A (# of control coupons with zero recovery /# of control coupons # of treated coupons with zero recovery /#
of treated coupons) and p-ValueJ
Material
Aluminum
Carpet
Keyboard
Joint Tape
%RH
40
60
75
40
60
75
40
60
75
40
60
75
30min
0.28 (0.19, 0.37) p=0.0002
0.98 (0.85, 1.11) p<0.0001
>5.20 (2/5) p=0.0079
>7.06 (4/5) p=0.0079
3.85 (3.31, 4.39) p<0.0001
>6.68 (5/5) p=0.0079
0. 13 (-0.035, 0.29) p=0. 1090
0.35 (0.14, 0.56) p=0.0047
2.54 (1.34, 3.75) p=0.0046
>1.08(3/5)p=0.3571
N/A (1/5 5/5) p=0.0476
>3.78 (5/5) p=0.0079
611 niin
0.32 (0.25, 0.40) p<0.0001
1.02 (0.53, 1.52) p=0.0081
>7.50 (5/5) p=0.0079
2.79 (2.07, 3.52) p=0.0003
>2.09 (3/5) p=0.0079
>5.59 (5/5) p=0.0079
0.64 (0.42, 0.86) p=0.0002
0.28 (0.22, 0.33) p<0.0001
0.91 (0.71, 1.10) p<0.0001
0.70 (0.043, 1.35) p=0.0408
>2.35 (5/5) p=0.0079
N/A (2/5 5/5) p=0. 1667
120 min
1.02 (0.83, 1.20) p<0.0001
3.52 (3.04, 3.99) p<0.0001
>7.33 (5/5) p=0.0079
>5.22 (5/5) p=0.0079
>4.96 (5/5) p=0.0079
>4.81 (5/5) p=0.0079
0.72 (0.57, 0.87) p=0.0002
0.95 (0.76, 1.15) p<0.0001
>5.63 (3/5) p=0.0079
N/A (5/5 5/5) p=l. 0000
>2.29 (5/5) p=0.0079
N/A (2/5 5/5) p=0. 1667
* Mean log reduction is the mean of the base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm
of recovered agent from the treated coupons. A 95 % CI for the difference is shown in parentheses. A p-value is provided for the probability that
the control and treatment recoveries are the same. The p-value is from the two sample t-test with Satterthwaite's method to allow for potentially
different variances in the two groups. p-Values less than 0.05 denote less than 1 in 20 chance that a difference as large as or larger than observed
would occur by chance if the control and treatment means were truly identical. Comparisons with p-values less than 0.05 (statistically significant
at the 0.05 level) are bolded.
| One or more of the treatment coupons had no recovered agent. The mean log reduction of the form ">X" is calculated as the mean of the
base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm of recovered agent from the treated
coupons except that "zero recovery" coupons have a substituted recovered value of "1" (base-10 log is 0). Since the log becomes an increasingly
negative value below 1 and is undefined at 0, this substitution is necessary and results in a lower bound on the mean log difference, as indicated
by the ">". The number of "zero recovery" treatment coupons and the total number of treatment coupons is shown in parentheses. The p-value
is from the non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance that results as different as or more
different than observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with
p-values less than 0.05 (statistically significant at the 0.05 level) are bolded.
J One or more of both the control and the treatment coupons had no recovered agent. In this case, the log reduction is indeterminate and the
mean log reduction is identified as "N/A". The number of "zero recovery" control coupons and the total number of control coupons are shown in
parentheses followed by the number of "zero recovery" treatment coupons and the total number of treatment coupons. The p-value is from the
non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance that results as different as or more different
than observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with p-values
less than 0.05 (statistically significant at the 0.05 level) are bolded.
F. tularensis
Sabre C1O2 fumigation results for F. tularensis are
presented in Tables 5-6 and 5-7 and Figures 5-7 and
5-8. Persistence tests reported in Section 4.2.2, as
well as positive controls used in these tests, showed
that there was a differential loss of viability of F.
tularensis, depending on the coupon type. Specifically,
over the Ihr drying time and multiple contact times, a
substantial reduction in viable F. tularensis on carpet
and joint tape was observed at 75% RH. As described
in the Introduction, the calculated log reductions reflect
the incremental impact of the fumigation technology.
Viable F. tularensis was not recovered from any material
following the exposure to 50-100 ppmv C1O2 for a 120-
min contact time at 75% RH.
No viable F. tularensis was recovered from aluminum
and keyboard exposed to 50-100 ppmv C1O2 (23 °C) for
a contact time of 120-min at 75% RH. For aluminum
and keyboard, log reductions generally increased with
increasing CTs and log reductions were generally higher
at 75% RH than the associated test at 40% RH.
There was a decrease in the recovery of viable F.
tularensis from carpet and joint tape positive control
coupons (not exposed to fumigation) during the period
of decontamination. The decline in viable F. tularensis
from carpet and joint tape at 75% RH was consistent
with the >5 log loss of viable F. tularensis from carpet
and joint tape after 2 hr that was observed in the
persistence testing. During fumigation testing with
C1O2, the positive control coupons were allowed to dry
for one hr; and an additional period of time (about 30
min) passes before the CT clock starts for fumigation.
Thus, after 30-min fumigation, the positive controls
may be at a time equivalent to 2 hr in the persistence
testing. There was a decrease in the recovery of viable
F. tularensis from carpet and joint tape positive control
coupons (not exposed to fumigation) during the period
of decontamination that was consistent with the loss
-------
of viable F. tularensis from carpet and joint tape in the
persistence testing. Because the log reduction reflects
only the incremental impact of fumigation (controls
for loss of viability without treatment), overall efficacy
must be interpreted in the context of the loss of viability
with treatment. For example, joint tape exposed to the
fumigation treatment for 30 min at 75% RH shows a
log reduction of only 2.17. However, only about 102
Table 5-6. Sabre CIO Fumigation Results for F. tularensis
bacteria were recovered from the untreated carpet. (This
results in a very low base to which the treated carpet was
compared.) Compared to the amount of bacteria spiked
onto the coupon (6.77 x 107 CPUs), no bacteria recovered
after the 30-min treatment (0 CPUs/coupon) represented
about a 7.8 log reduction in viable bacteria attributable
to the fumigation and the loss of viability from joint tape
arising from other (unknown) causes.
Contact
Time
Material
Spike
Amount (CFU/coupon)
Mean Recovered F. tularensis (CFU/coupon)*
Positive Controlf
Test CouponJ
Mean Log
Reduction*
50-100 ppmv C1O2, 23°C, 40% RH
0 min
30 min
60 min
120 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
5.17xl07
5.17xl07
5.17xl07
5.17xl07
5.17xl07
5.17 x 107
5.17 x 107
5.17 x 107
1.05 x 10s*
1.05 x 10s*
1.05 x 10s*
1.05 x 10s*
1.05 x 10s*
1.05 x 10s*
1.05 x 10s*
1.05 x 10s*
1.68 ± 0.88 xlO7
2.47 ± 1.25 xlO6
3.76 ± 4.80 xlO6
3.07 ± 1.30 xlO2
1.88±0.87xl06
1.87 ± 0.70 x 106
0.00 ±0.00
0.00 ±0.00
4.28 ± 1.35 x 106
3.46 ± 0.23 xlO6
0.00 ±0.00
0.00 ±0.00
3.95 ± 0.78 xlO6
3. 15 ± 0.93 x 106
0.00 ±0.00
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
1.35 ± 0.58 xlO6
9.54 ± 1.58 x 105
0.00 ±0.00
0.00 ±0.00
6.22 ± 2.95 x 105
9.49 ± 3.73 xlO5
0.00 ±0.00
0.00 ±0.00
1.92 ± 0.60 xlO5
5.52± 1.75 x 105
0.00 ±0.00
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
0.19 ±0.23
0.30 ±0.07
Not calculable
Not calculable
0.92 ±0.36
0.60 ±0.23
Not calculable
Not calculable
1.34±0.18
0.78 ±0.17
Not calculable
Not calculable
50-100 ppmv C1O2, 23°C, 75% RH
0 min
30 min
60 min
120 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
6.77 x 107
6.77 x 107
6.77 x 107
6.77 x 107
6.77xl07§
6.77xl07§
6.77xl07§
6.77xl07§
1.15x 10s*
1.15x 10s*
1.15x 10s*
1.15x 108*
6.77 x 107
6.77 xlO7
6.77 xlO7
6.77 xlO7
5.39 ± 1.36 x 107
4.35 ± 0.78 x 106
2.22 ± 0.28 x 10s
0.00 ±0.00
1.93 ± 1.39 xlO7
3.11±0.26xl06
8.36 ± 4.48 xlO6
1.47 ± 2.22 xlO2
1.80 ± 1.43 x 107
9.45 ± 2.67 x 105
1.30 ± 0.89 x 107
5.32 ± 10.2 x 101
2.39 ± 1.41 x 106
3.86±0.82xl04
7.01 ± 9.14 xlO5
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
1.28 ± 1.69 xlO5
5.70±1.88xl04
0.00 ±0.00
0.00 ±0.00
4.31 ± 5.08 x 103
9.03 ± 4.35 x 104
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
2. 50 ±0.60
1.76 ±0.17
6.92 ±0.00
2. 17 ±0.00
5.00 ±2.08
1.06 ±0.21
7. 12 ±0.00
1.73 ± 0.00
6.38 ±0.00
4.59 ±0.00
5.85 ±0.00
Not calculable
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
§ The spike amount for the associated positive control was 1.15 x 10s CPUs/coupon.
# Exceeds target application of 1.0 x 10s CPUs/coupon.
-------
Figure 5-7. Sabre Fumigation Results forF tularensis at 50-100 ppmv CIO, and 23 °C, Line Chart.
S
•40% REL Aluminum
e -
75% REL Aluminum .z -
•40% RH. Keyboard =
3 4
75% REL Kevboard *
bl
•40% RH: Carpet J: 3
75%RH: Carpet
•40% REL Joint tape
75 %REL Joint tape 1
20 40 60 SO 100 120 140 160
CT, ppmv-hr
* Log reductions not calculated for some or all CTs as F. tularensis was not recovered from either the test or associated control coupons.
Figure 5-8. Sabre Fumigation Results forF tularensis at 50-100 ppmv CIO and 23 °C, Column Chart.
• 40% REL Aluminum
75 %RH; Aluminum .1 -"
•40% RH= Keyboard J
75 %RH; Keyboard
• 40% RHr Carpet
75 %RHr Carpet
•40% RHr Joint tape
• 75 %RH; Joint tape
I
38
CT. ppmv-hr
* Log reductions not calculated for some or all CTs as F. tularensis was not recovered from either the test or associated control coupons.
-------
Table 5-7 provides a summary of C1O2 decontamination
efficacy against F. tularensis, calculated as the difference
in the mean log of viable bacteria recovered from positive
control coupons and the mean log of viable bacteria
recovered from coupons after fumigation for a given
contact time. The elapsed time from spiking to recovery
was the same for the positive control coupons and test
coupons to control for history. The 95% CI and p-value
are also shown. At the 120-min contact time (75% RH),
no viable bacteria were recovered from any test coupons.
Because of the temporal loss of viable bacteria at the
120-min contact time statistically significant incremental
reduction in viable bacteria arising from the fumigation
treatment was demonstrated only for aluminum and
keyboard. However, for the carpet and joint tape, a 7.8
log reduction in viable bacteria was attributable to the
combined effects of fumigation and the loss of viability
over time arising from other (unknown) causes.
Table 5-7. Summary of Efficacy (Calculated as Mean Log Reduction) of C1O2 against F. tularensis
Mean Log Reduction (95% CI) and p-Value*
or Mean Log Reduction (# of treated coupons with zero recovery/# of treated coupons) and
p-Valuef or N/A (# of control coupons with zero recovery /# of control coupons # of treated coupons
with zero recovery /# of treated coupons) and p-ValueJ
Material
Aluminum
Carpet
Keyboard
Joint Tape
%RH
40
75
40
75
40
75
40
75
30min
0. 14 (-0.19, 0.48) p=0.3589
2.16 (1.44, 2.88) p=0.0003 s
N/A (5/5 5/5) p=l. 0000
>6.64 (5/5) p=0.0079 s
0.27 (0.097, 0.45) p=0.0132
1.53 (1.35, 1.71) p<0.0001 s
N/A (5/5 5/5) p=l. 0000
N/A (3/5 5/5) p=0. 1667 s
611 niin
0.90 (0.51, 1.30) p=0.0031
>4.80 (2/5) p=0.0079
N/A (5/5 5/5) p=l. 0000
>6.76 (5/5) p=0.0079
0.60 (0.36, 0.84) p=0.0042
1.05 (0.80, 1.29) p<0.0001
N/A (5/5 5/5) p=l. 0000
N/A(3/55/5)p=0.4444
120 min
1.33 (1.13, 1.53) p<0.0001
>6.32 (5/5) p=0.0079
N/A (5/5 5/5) p=l. 0000
N/A (2/5 5/5) p=0. 1667
0.76 (0.53, 0.99) p<0.0001
>4.58 (5/5) p=0.0079
N/A (5/5 5/5) p=l. 0000
N/A (5/5 5/5) p=l. 0000
* Mean log reduction is the mean of the base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm
of recovered agent from the treated coupons. A 95 % CI for the difference is shown in parentheses. A p-value is provided for the probability that
the control and treatment recoveries are the same. The p-value is from the two sample t-test with Satterthwaite's method to allow for potentially
different variances in the two groups. p-Values less than 0.05 denote less than 1 in 20 chance that a difference as large as or larger than observed
would occur by chance if the control and treatment means were truly identical. Comparisons with p-values less than 0.05 (statistically significant)
are bolded.
T One or more of the treatment coupons had no recovered agent. The mean log reduction of the form ">X" is calculated as the mean of the base-10
logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm of recovered agent from the treated coupons
except that "zero recovery" coupons have a substituted recovered value of "1" (base-10 log is 0). Since the log becomes an increasingly negative
value below 1 and is undefined at 0, this substitution is necessary and results in a lower bound on the mean log difference, as indicated by the
">". The number of "zero recovery" treatment coupons and the total number of treatment coupons are shown in parentheses. The p-value is
from the non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance that results as different as or more
different than observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with
p-values less than 0.05 (statistically significant at the 0.05 level) are bolded.
* One or more of both the control and the treatment coupons had no recovered agent. In this case, the log reduction is indeterminate and the
mean log reduction is identified as "N/A". The number of "zero recovery" control coupons and the total number of control coupons are shown in
parentheses followed by the number of "zero recovery" treatment coupons and the total number of treatment coupons. The p-value is from the
non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance that results as different as or more different
than observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with p-values
less than 0.05 (statistically significant at the 0.05 level) are bolded.
§ The inoculum concentrations for control and treatment coupons were different. Where reported, the mean log reduction is calculated as a
relative difference in log reduction for each of controls and treated coupons relative to their respective inoculum concentrations.
-------
Vaccinia Virus
Sabre CIO, fumigation of vaccinia virus was at 50-
100 ppmv C1O2 (23 °C). Three RH conditions, 40%,
60%, and 75%, were evaluated. Sabre CIO,, fumigation
results for vaccinia virus are presented in Tables 5-8 and
5-9, and Figures 5-9 and 5-10. No vaccinia virus was
recovered from aluminum, carpet, or joint tape after
30-min fumigation at 75% RH; viable vaccinia virus
was recovered from keyboard after 120-min fumigation
at 75% RH. The greater resistance to decontamination
may be attributable to the difference in application
on the keyboard due to its small surface: a single 100
tiL droplet was used rather than 10 x 10 uL droplets
on the other coupon materials. Vaccinia virus was not
recovered from carpet or joint tape (log reductions > 6)
following any Sabre CIO, fumigation treatment at 60%
and 75% RH, but was recovered intermittently from
aluminum (i.e., from the 120-min contact time at 60%
RH) and was generally recovered from keyboard under
all tested conditions. Log reductions attributable to the
decontamination treatment were generally higher at 60%
and 75% RH than at 40% RH.
As shown in Table 5-8 and Figure 5-9, at 40% RH the
efficacy against vaccinia appears to decline with longer
contact time. High variability in efficacy results was
often observed in the transition from low efficacy to
high efficacy. At 40% RH, both the 60-min and 120-
min contact times are in the high variability transition
range. There appears to be an unknown factor that
differs between the 60-min test and the 120-min test
that causes a decline in the number of viable vaccinia
recovered at the longer contact time. (The negative
slopes for aluminum and carpet at 75% RH reflect only
the difference in the spike amount between tests; no
viable virus was recovered from the test coupons at
either contact time.)
Decontamination testing was performed at 40% and
75% RH before the testing was performed at 60% RH.
Because vaccinia virus had survived at both 40% and
75% RH on keyboard after a 120-min contact time,
the observation that no virus was recovered from
keyboard after a 120-min contact time at 60% RH was
an unexpected result. The 120-min contact time at 60%
RH was repeated. The result of the second test (6.84
x 104 virus recovered from keyboard) was consistent
with previous test results. No reason was identified to
explain the anomaly. In parallel EPA testing (report
to be completed in May, 2010), a CT of 125 ppm-hr of
C1O2 (80% - 83% RH) resulted in no vaccinia virus
being recovered from any coupon of any of the seven
materials tested (glass, painted concrete, galvanized
metal, decorative laminate, cellulose insulation, particle
board, and industrial carpet). Because the application of
vaccinia onto keyboard was in a single 100 uL droplet,
rather than the 10 x 10 uL droplets applied to the other
coupons, the thickness of the dried droplet may provide
a protection for interior virus from decontamination
resulting in lower efficacy than was observed with the
other materials.
-------
Table 5-8. Sabre CIO Fumigation Results for Vaccinia Virus
Contact
Time
Material
Spike Amount (PFU/coupon)
Mean Recovered Vaccinia Virus (PFU/
coupon)*
Positive Controlf
Test CouponJ
Mean Log
Reduction*
50-100 ppmv C1O2, 23°C, 40% RH
0 min
60 min
120 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
1.98 x 107
1.98xl07
1.98xl07
1.98xl07
4.82 xlO7
4.82 xlO7
4.82 x 107
4.82 x 107
1.98 x 107
1.98 x 107
1.98xl07
1.98xl07
4.68 ± 1.68 x 107§
5.77 ± 2.70 x!07§
1.13±0.50xl07§
6.78±1.50xl06§
1.06 ± 0.90 x!08§
8.03 ± 4.91 x!07§
5.92 ± 2.12 x 106§
1.57 ± 1.19x 105§
4.64 ± 2.99 x 107§
3.75 ± 1.18x 107§
7.49 ± 3.05 x!06§
2.29 ± 0.62 x!06§
Not applicable
Not applicable
Not applicable
Not applicable
4.68 ± 0.20 xlO6
5.90 ± 0.84 xlO6
1.13±0.99x 10°
6.68 ± 14.9 x 10'2
4.48 ± 1.46 x 106
9.95 ± 3.33 x 106
2.68 ± 2.10 xlO4
1.27 ± 0.96 xlO4
Not applicable
Not applicable
Not applicable
Not applicable
1.35 ±0.02
1.14 ±0.06
6.70 ± 0.22
5.29 ±0.21
1.03 ±0.15
0.60 ±0.17
2.57 ±0.40
2.65 ± 1.04
50-100 ppmv C1O2, 23°C, 60% RH
0 min
120 min
(Initial)
120 min
(Repeat)
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
2.60 x 10s
2.60 x 10s
2.60 x 10s
2.60 x 10s
8.17xl08
8.17x 10s
8.17x 10s
8.17x 10s
2.60 x 10s
2.60 x 10s
2.60 x 10s
2.60 x 10s
5.34±1.58xl07§
6.95±5.19xl07§
2.01±1.80xl07§
3.01 ± 4.77 x!07§
1.83±0.23xl08§
1.44 ±0.71 x 108§
3. 16 ± 5.00 x 107§
2.29 ±2.71 x 106§
6.23 ± 4.83 x 107§
2.83 ± 2.05 x!07§
3.48±5.14xl07§
9.96 ± 2.95 x!05§
Not applicable
Not applicable
Not applicable
Not applicable
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
3. 55 ± 0.54 x 104
6.84 ± 2.14 xlO4
0.00 ±0.00
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
8.26 ±0.00
8. 16 ±0.00
7.50 ±0.00
6.36 ±0.00
3.25 ±0.07
2.63 ±0.14
7.54 ±0.00
6.00 ±0.00
50-100 ppmv C102, 23°C, 75% RH
0 min
30 min
60 min
120 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Keyboard
6.09 x 10s
6.09 x 10s
6.09 x 10s
6.09 x 10s
6.09 x 10s
6.09 x 10s
6.09 x 10s
6.09 x 10s
1.20 x 10s
1.20 x 10s
1.20 x 10s
1.20 x 10s
3.97 x 10s
8.54 ± 3.60 x!07§
1.04 ± 0.78 xlO8
2.70 ± 1.20 x 107§
5. 86 ± 2.73 x 106§
1.20 ± 0.79 x 108§
9.51 ±5. 18 x 107§
4.47 ± 4.87 x 107§
2.77 ± 4.05 x!06§
3.07 ± 0.83 x!07§
4.06 ± 0.94 xlO7
9.81 ± 2.86 x!06§
7.67 ± 1.93 x 106§
1.87 ± 0.77 x 108§
Not applicable
Not applicable
Not applicable
Not applicable
0.00 ±0.00
1.08 ± 0.35 x 106
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
1.33 ± 0.35 xlO5
0.00 ±0.00
0.00 ±0.00
4.13 ± 1.84 x 104
Not applicable
Not applicable
Not applicable
Not applicable
8.08 ±0.00
1.96 ±0.15
7.65 ± 0.00
6.44 ±0.00
7.49 ± 0.00
2. 50 ±0.11
6.99 ±0.00
6.89 ±0.00
3.69 ±0.21
Data are expressed as mean ± standard deviation of five replicates.
' Positive control coupons were spiked but not exposed to the fumigant.
* Test coupons were spiked and exposed to the fumigant for the contact time.
§ Exceeded the positive control CV target of <25%.
-------
Figure 5-9. Sabre Fumigation Results for Vaccinia Virus at 50-100 ppmv CIO, and 23 °C, Line Chart.
9
-40% RH: Aluminum
75% RH, Aluminum
-40% RH, Keyboard
75 %RH: Keyboard
-40% RH, Carpet
75 %RH, Carpet
•40% RH, Joint tape
75 %RH, Joint tape
40
60
120
140
SO 100
CT, ppmv-hr
Note: Log reductions associated with 60% RH are not shown as data were only generated at 150 CT, ppmv-hr.
Figure 5-10. Sabre Fumigation Results for Vaccinia Virus at 50-100 ppmv C1O2 and 23 °C, Column Chart ("a"
Indicates Initial Test; "b" Indicates Repeat Test).
• 40% RH, Aluminum
60% RH, Aluminum -a.
60% RH, Aluminum - b
75% RH, Aluminum
• 40% RH, Keyboard
60% RH, Keyboard - a
60% RH, Keyboard - b
75 %RH, Keyboard
• 40% RH, Carpet
60% RH, Carpet - a
60% RH, Carpet-b
75%RH, Carpet
• 40% RH, Joint tape
• 60% RH: Joint tape -a
60% RH, Joint tape-b
75% RH, Joint tape
160
75
150
CT, ppmv-hr
-------
Table 5-9 provides a summary of C1O2 decontamination
efficacy against vaccinia virus, calculated as the
difference in the mean log of viable (plaque-forming)
virus recovered from positive control coupons and the
mean log of viable virus recovered from coupons after
fumigation for a given contact time. The elapsed time
from spiking to recovery was the same for the positive
control coupons and test coupons to control for history.
The 95% CI and p-value are also shown. At the 30-min
and 60-min contact times (75% RH), no viable virus
was recovered from any aluminum, carpet, or joint tape
test coupons. Viable virus was recovered from keyboard
coupons at the 120-min contact time (75% RH). Efficacy
was higher at 75% RH than at 40% RH at all contact
times tested for aluminum, carpet, and joint tape test
coupons.
Table 5-9. Summary of Efficacy (Calculated as Mean Log Reduction) of CIO against Vaccinia Virus
Mean Log Reduction (95% CI) and p-Value*
or Mean Log Reduction (# of treated coupons with zero recovery /# of treated coupons) and p-Valuef
Material
Aluminum
Carpet
Keyboard
Joint Tape
%RH
40
60
75
40
60
75
40
60
75
40
60
75
30min
>7.98 (5/5) p=0.0079
>7.47 (5/5) p=0.0079
1.91 (1.61, 2.21) p<0.0001
>6.06 (5/5) p=0.0079
611 niin
1.21 (0.80, 1.62) p=0.0024
>7.48 (5/5) p=0.0079
>6.67 (1/5) p=0.0079
>6.98 (5/5) p=0.0079
1.07 (0.80, 1.35) p=0.0006
2.49 (2.33, 2.64) p<0.0001
>4.67 (4/5) p=0.0079
>6.87 (5/5) p=0.0079
120 min
0.97 (0.66, 1.27) p=0.0003
>5.70 (5/10) p<0.0001
2.55 (2.11, 2.99) p<0.0001
>7.10 (10/10) p<0.0001
0.58 (0.37, 0.80) p=0.0003
>5.29 (5/10) p<0.0001
3.64 (3.30, 3.99) p<0.0001
2.64 (1.56, 3.72) p=0.0045
>5.93 (10/10) p<0.0001
* Mean log reduction is the mean of the base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm
of recovered agent from the treated coupons. A 95 % CI for the difference is shown in parentheses. A p-value is provided for the probability that
the control and treatment recoveries are the same. The p-value is from the two sample t-test with Satterthwaite's method to allow for potentially
different variances in the two groups. p-Values less than 0.05 denote less than 1 in 20 chance that a difference as large as or larger than observed
would occur by chance if the control and treatment means were truly identical. Comparisons with p-values less than 0.05 (statistically significant
at the 0.05 level) are bolded.
| One or more of the treatment coupons had no recovered agent. The mean log reduction of the form ">X" is calculated as the mean of the base-
10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm of recovered agent from the treated coupons
except that "zero recovery" coupons have a substituted recovered value of "1" (base-10 log is 0). Since the log becomes an increasingly negative
value below 1 and is undefined at 0, this substitution is necessary and results in a lower bound on the mean log difference, as indicated by the
">". The number of "zero recovery" treatment coupons and the total number of treatment coupons is shown in parentheses. The p-value is from
the non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance results as different or more different than
observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with p-values less
than 0.05 (statistically significant at the 0.05 level) are bolded.
Y. pestis
Sabre C1O2 fumigation results for Y. pestis are presented
in Tables 5-10 and 5-11. Persistence tests reported
in Section 4.2.4, as well as positive controls used in
these tests, showed that there was a differential loss of
viability of Y. pestis, depending on the coupon type.
However, persistence was higher during the persistence
testing than was observed from positive controls. Both
the persistence testing and the positive controls for
the various fumigation tests demonstrate that viable Y.
pestis spores are rapidly lost from the various material
coupons under the conditions tested. As described in
the Introduction, the calculated log reductions reflect
the incremental impact of the fumigation technology.
Y. pestis was not recovered following any Sabre C1O2
fumigation treatment of 30 min or longer. Variable
amounts of Y. pestis were recovered from positive control
coupons. Y. pestis was often not recovered from the
associated positive controls; mean log reductions could
not be calculated for the Sabre C1O2 fumigation.
Because the log reduction reflects only the incremental
impact of fumigation (controls for loss of viability
without treatment), overall efficacy must be interpreted
in the context of the loss of viability with treatment.
Compared to the amount of bacteria spiked onto the
coupon (about 107 CPUs/coupon), no bacteria were
recovered after the 30-min treatment (0 CPUs/coupon)
at either the 40% RH or 75% RH condition. The loss
of viable bacteria represents >7 log reduction in viable
bacteria attributable to the fumigation and the loss of
viability over time from other (unknown) causes. The
causes of variability in the rates of decline in recoverable
Y. pestis bacteria from positive control coupons and in
persistence testing (no fumigation) are unknown.
-------
Table 5-10. Sabre CIO Fumigation Results for Y. pestis
Contact
Time
Material
Spike
Amount (CFU/coupon)
Mean Recovered Y. pestis (CFU/coupon)*
Positive Controlf
Test CouponJ
Mean Log Reduction*
50-100 ppmv C1O2, 23°C, 40% RH
0 min
30 min
60 min
120 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
2.30 x 10'
2.30 x 10'
2.30 x 107
2.30 x 10'
1.56xl07
1.56xl07
1.56 x 107
1.56 x 107
2.30 x 107
2.30 x 107
2.30 x 107
2.30 x 107
1.56 x 107
1.56 xlO7
1.56 xlO7
1.56 x 107
9.54 ± 5.52 x 103
6.16 ± 6.09 xlO3
5.04 ± 2.64 x 106
0.00 ±0.00
0.00 ±0.00
4.00 ±7.24x10'
1.05 ± 1.10 x 106
0.00 ±0.00
1.33 ± 1.23 x 102
2.59 ± 3.42 xlO3
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
Not calculable
1.60 ±0.00
6.02 ±0.00
Not calculable
2. 13 ±0.00
3.41 ±0.00
Not calculable
Not calculable
Not calculable
Not calculable
Not calculable
Not calculable
50-100 ppmv C1O2, 23°C, 75% RH
0 min
30 min
60 min
120 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
3.03 x 107
3.03 x 107
3.03 x 107
3.03 x 107
1.77 x 107
1.77 x 107
1.77 xlO7
1.77 xlO7
3.03 x 107
3.03 x 107
3.03 x 107
3.03 x 107
1.77 xlO7
1.77 x 107
1.77 x 107
1.77 xlO7
1.23 ± 0.79 x 10"
1.18±0.45x 10"
8.69 ± 2.60 xlO6
7.32 ± 10.6x10'
0.00 ±0.00
0.00 ±0.00
2.42 ± 2.58 xlO6
0.00 ±0.00
1.53 ± 1.37 x 102
1.05 ± 1.12 x 103
5.02 ± 7.23 x 10"
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
4.57 ± 9.75 x 10"
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
Not applicable
Not applicable
Not applicable
Not applicable
Not calculable
Not calculable
6.38 ±0.00
Not calculable
2. 19 ±0.00
3.02 ±0.00
4.70 ± 0.00
Not calculable
Not calculable
Not calculable
4.66 ± 0.00
Not calculable
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
-------
Summary Statistics for Sabre CIO2 Decontamination
Table 5-11 provides a summary of C1O2 decontamination
efficacy against Y. pestis, calculated as the difference in
the mean log of viable bacteria recovered from positive
control coupons and the mean log of viable bacteria
recovered from coupons after fumigation for a given
contact time. The elapsed time from spiking to recovery
was the same for the positive control coupons and test
coupons to control for history. The 95% CI and p-value
are also shown. At the 120-min contact time (75% RH),
no viable Y. pestis was recovered from any aluminum,
keyboard, carpet, or joint tape test coupons. Because
of the loss of viable Y. pestis from positive control
coupons due to unknown, time dependent causes, the
log reductions attributable only to the fumigation effect
could not be determined; the combined effects represent
>7 log reduction in viable bacteria attributable to the
fumigation and the loss of viability over time from other
(unknown) causes.
Table 5-11. Summary of Efficacy (Calculated as Mean Log Reduction) of CIO against Y. pestis
Surface Damage
The physical effect of the Sabre C1O2 fumigation on the
materials was evaluated qualitatively. The appearance
of the decontaminated coupons was visually inspected
for any obvious changes in the color, reflectivity, and
apparent roughness of the material surfaces. These
comparisons were performed for each material,
before extraction of the decontaminated test coupons.
No differences were observed between control and
fumigated coupons for any material except that at high
C1O2 for prolonged contact times some darkening of the
aluminum was observed.
Mean Log Reduction (95% CI) and p-Value*
or Mean Log Reduction (# of treated coupons with zero recovery /# of treated coupons) and p-Valuef
or N/A (# of control coupons with zero recovery/# of control coupons # of treated coupons with zero recovery/# of treated
coupons) and p-ValueJ
Material
Aluminum
Carpet
Keyboard
Joint Tape
%RH
40
75
40
75
40
75
40
75
30min
N/A (5/5 5/5) p=l. 0000
N/A (5/5 5/5) p=l. 0000
>5.79 (5/5) p=0.0079
>5.81 (5/5) p=0.0079
0.75 (3/5 5/5) p=0.4444
N/A (5/5 5/5) p=l. 0000
N/A (5/5 5/5) p=l. 0000
N/A (5/5 5/5) p=l. 0000
611 in in
>1.94 (5/5) p=0.0079
>2.02 (5/5) p=0.0079
N/A(5/55/5)p=1.0000
>3.81 (5/5) p=0.0079
>3.18 (5/5) p=0.0079
>2.80 (5/5) p=0.0079
N/A (5/5 5/5)p=1.0000
N/A (5/5 5/5)p=1.0000
120 min
N/A (5/5 5/5)p=1.0000
N/A (5/5 5/5)p=1.0000
N/A(5/55/5)p=1.0000
N/A (3/5 5/5)p=0.4444
N/A (5/5 5/5)p=1.0000
N/A (5/5 5/5)p=1.0000
N/A (5/5 5/5)p=1.0000
N/A (5/5 5/5)p=1.0000
* Mean log reduction is the mean of the base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm
of recovered agent from the treated coupons. A 95% CI for the difference is shown in parentheses. A p-value is provided for the probability that
the control and treatment recoveries are the same. The p-value is from the two sample t-test with Satterthwaite's method to allow for potentially
different variances in the two groups. p-Values less than 0.05 denote less than 1 in 20 chance that a difference as large as or larger than observed
would occur by chance if the control and treatment means were truly identical. Comparisons with p-values less than 0.05 (statistically significant
at the 0.05 level) are bolded.
| One or more of the treatment coupons had no recovered agent. The mean log reduction of the form ">X" is calculated as the mean of the base-
10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm of recovered agent from the treated coupons
except that "zero recovery" coupons have a substituted recovered value of "1" (base-10 log is 0). Since the log becomes an increasingly negative
value below 1 and is undefined at 0, this substitution is necessary and results in a lower bound on the mean log difference, as indicated by the
">". The number of "zero recovery" treatment coupons and the total number of treatment coupons are shown in parentheses. The p-value is
from the non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance that results as different as or more
different than observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with
p-values less than 0.05 (statistically significant at the 0.05 level) are bolded.
J One or more of both the control and the treatment coupons had no recovered agent. In this case, the log reduction is indeterminate and the
mean log reduction is identified as "N/A". The number of "zero recovery" control coupons and the total number of control coupons is shown in
parentheses followed by the number of "zero recovery" treatment coupons and the total number of treatment coupons. The p-value is from the
non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance that results as different as or more different
than observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with p-values
less than 0.05 (statistically significant at the 0.05 level) are bolded.
-------
5.2 HP Fumigation (BIOQUELL Clarus C)
5.2.7 Description of BIOQUELL Clarus C HP
Technology
The following is a description of the BIOQUELL Claras
C unit, based on information provided by the vendor. The
information provided below was not verified in this test.
The BIOQUELL Claras C unit is a hydrogen peroxide
gas generator (Figure 5-11) that uses a dual circuit
system. The first circuit provides high-efficiency
paniculate air (HEPA) filtration, dehumidification, and
hydrogen peroxide removal from the air stream via
catalytic conversion. The second circuit delivers high-
concentration HP and water vapors. During gassing,
the BIOQUELL Claras C unit recirculates the vapors
through the second circuit, constantly increasing the
concentration of HP and water vapor within the chamber
or area intended for decontamination. This recirculation
and vapor injection continues until the chamber reaches
saturation, and the process of microcondensation
begins. In microcondensation, a microscopic film of
aqueous hydrogen peroxide solution is deposited on all
surfaces. Once the gassing phase has been completed,
the BIOQUELL Claras C unit returns to the first
circuit and brings the chamber to a safe condition by
cataly tically converting the hydrogen peroxide to water
(humidity) and oxygen. Excess humidity is removed via
the refrigerant-based dehumidification system of the
BIOQUELL Claras C unit. To ensure that all essential
data are captured, the BIOQUELL Clarus C unit prints
out all critical parameters recorded throughout the cycle.
The BIOQUELL Claras C unit has a personal computer
connection for more in-depth cycle analysis.
The BIOQUELL Claras C unit was designed to
decontaminate enclosures of up to 200 cubic meters.
The unit weighs 128 kilograms and is 68 cm wide by 90
cm in depth by 106 cm in height. The dehumidification
system is designed to ran continuously. Because
there was no need for dehumidification regeneration
down-time, the BIOQUELL Claras C unit can operate
continuously, if required, from a normal (120 volts
alternating current) domestic power supply. The
BIOQUELL Claras C unit is controlled by a Siemens
programmable logic controller, which is complemented
by optional sensors (including a microcondensation
sensor), allowing repeatable validated decontamination
cycles.
For this verification test, the BIOQUELL Claras C
unit was attached to a test chamber that was a BSC III,
approximately 1275 L. The BIOQUELL Clarus C unit
and the glove box were connected by flexible supply
and delivery gassing hoses that contained in-line HEPA
filtration. A hydrogen peroxide sensor, HP sensor, and
pressure sensing tube were connected to the inside of the
test chamber and data were transmitted through the test
chamber wall to the BIOQUELL Clarus C unit.
The vendor provided on-site support for the installation
of the Clarus C unit and cycle development before
testing was begun. Testing was performed by Battelle
personnel only. Early tests were observed by a vendor
representative.
Figure 5-11. BIOQUELL Claras C HP Vapor Generator.14
HP measurements were made inside the test chamber
using an Analytical Technology Model B12 HP gas
sensor. In order to detect sudden degradation in the
sensor performance, the gas sensor measurements
were compared periodically to a titration measurement
of air samples drawn through a sampling train of two
impingers, each containing 20 mL of 5% sulfuric acid
(H2SO4). The 40 mL of solution from the impingers was
added to 150 mL of deionized water for a total of 190 mL
of solution. The solution was titrated with 0. IN or 0.02N
potassium permanganate (KMnO4). The total equation
can be expressed as: 5 H2O2 (hydrogen peroxide) + 2
KMnO4 + 4 H2SO4 -^ 2 KHSO4 (potassium sulfate) +
2 MnSO4 (manganese sulfate) + 8 H2O (water) + 5 O2
(oxygen). Prior to each fumigation cycle, air was drawn
through the impinger train and analyzed as a negative
control. In the event that the impinger method and the
sensor measurements differed by 10% or greater, standard
solutions of HP were prepared and titrated to ensure
that the titration method was accurate. The method was
adapted from the liquid analysis found at http://www.
h2o2.com/intro/highrange.html.
-------
5.2.2 Test Matrix for BIOQUELL Clarus C HP
Fumigation
The testing performed with BIOQUELL Clarus C is
shown in Table 5-12. The experimental design tested
decontamination efficacy by determining whether
there was a difference between the log reductions of
the viable biological agents after fumigation compared
to controls for various materials. These tests also
assessed whether there was any difference in efficacy
at varying fumigation cycles and fumigation contact
times. Critical parameters included fumigation cycle,
fumigation contact time, and the viability of the
biological agents. An adaptive management approach
was used to incorporate new knowledge into the testing
as decontamination efficacy results became available.
Table 5-12. Test Matrix for BIOQUELL Clarus C HP Fumigation
Trial
1
2
3
4
5
6a
6b
7a
7b
Biological Agent
B. anthracis
spores*
B. anthracis
spores*
B. anthracis
spores*
Y. pestis*
B. suis*
Vaccinia virus*
Vaccinia virus*
B. anthracis
spores*
B. anthracis
spores*
Material
Carpet
Carpet
Carpet
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Keyboard
Carpet
Aluminum
Joint tape
Glass
Laminate
Ductwork
Carpet
Concrete
Wood
Glass
Ceiling tile
Fumigation Cycle
Fumigate 10 min at
8 g/min; dwell at 0. 8 g/min
Fumigate 5 min at 8 g/min;
dwell at 0.8 g/min
Fumigate 10 min at 8 g/min;
dwell at 0.8 g/min
Fumigate 10 min at
8 g/min; dwell at 0. 8 g/min
Fumigate 10 min at
8 g/min; dwell at 0. 8 g/min
Fumigate 10 min at
8 g/min; dwell at 0. 8 g/min
Fumigate 10 min at
8 g/min; dwell at 0. 8 g/min
Fumigate 10 min at
8 g/min; dwell at 0. 8 g/min
Fumigate 10 min at
8 g/min; dwell at 0. 8 g/min
Contact Times (min)
15, 30, 60, 90, 120, 180
15, 30, 60, 90
60, 120, 180
180
180
180
180
180
180
* Biological indicators (G. stearothermophilus on steel in Tyvek envelopes) were also tested.
Five replicate test coupons (plus one blank) per time
point and five replicate positive control coupons were
included at each set of conditions. Negative controls
(blanks) were coupons to which corresponding diluents
but no biological agent were applied.
5.2.3 BIOQUELL Clarus C HP Fumigation Results
Figure 5-12 shows a graph of typical temperature,
RH, and HP concentration during a fumigation cycle
(fumigate 10 min at 8 g/min; dwell at 0.8 g/min). The HP
concentration peaked initially and then gradually drifted
lower during the dwell phase. The large spike in HP
concentration occurred when aeration began and without
the introduction of any additional HP. Although the spike
was consistently observed, understanding the cause of
the spike was beyond the scope of the project and the
cause was not determined.
-------
Figure 5-12. Typical Temperature (°C), RH (%), and HP Concentration (ppmv) Dynamics During the Fumigation
Cycle (Fumigate 10 min at 8 g/min; Dwell at 0.8 g/min).
Test Run #1 091409
-H202PPM
-Temp °t
%RH
G. stearothermophilus
The results for BIOQUELL Claras C HP fumigation
of biological indicators are shown in Table 5-13. The
biological indicators were G. stearothermophilus
nominally 1 x 106 spores on stainless steel inTyvek®
packaging (Apex Laboratories, Apex, NC, USA). In this
qualitative test, no growth was observed at any contact
time, including the shortest tested (15-min contact).
Table 5-13. BIOQUELL Claras C HP Fumigation Results
for G. stearothermophilus
Trial
1,3
2
4
5
6
7
Fumigation Cycle
Fumigate 10 min
at 8 g/min; dwell at
0.8 g/min
Fumigate 5 min at
8 g/min; dwell at
0.8 g/min
Fumigate 10 min
at 8 g/min; dwell at
0.8 g/min
Fumigate 10 min
at 8 g/min; dwell at
0.8 g/min
Fumigate 10 min
at 8 g/min; dwell at
0.8 g/min
Fumigate 10 min
at 8 g/min; dwell at
0.8 g/min
Contact Times (min)
Growth Positive/Total Biological
Indicators
15
1/10
15
0/10
30
0/5
30
0/5
60
0/15
60
0/5
90
0/5
90
0/5
120
0/10
180
0/10
180
0/10
180
0/10
180
0/30
180
0/10
No growth was observed on the biological indicators
following exposure to the fumigant, with the exception
of growth observed during trial #1 on one biological
indicator following 15 min of exposure to the fumigant.
The high efficacy, suggested by the completed kill of the
biological indicators, did not correlate with complete kill
of B. anthracis spores on other materials.
B. anthracis
BIOQUELL Claras C HP fumigation results for B.
anthracis spores are presented in Table 5-14.
The efficacy of the BIOQUELL Claras C HP fumigation
varied, depending on the type of test coupon. At 180 min
and fumigation cycle of 10 min at 8 g/min; dwell at 0.8
g/min, no viable spores were recovered from laminate.
ductwork, glass or ceiling tile. While some efficacy
against spores was observed for carpet, concrete, and
wood, viable spores were recovered from these materials
after fumigation with a 180-min contact time and
fumigation cycle of 10 min at 8 g/min; dwell at 0.8 g/
min. In one of three replicate trials for carpet for 180 min
and fumigation cycle of 10 min at 8 g/min; dwell at 0.8 g/
min, no viable spores were recovered.
-------
Table 5-14. BIOQUELL Clarus C HP Fumigation Results for B. anthracis
Trial
Contact
Time
Spike Amount
Material (CFU/coupon)
Mean Recovered B. anthracis (CFU/coupon)*
Positive Controlf
Test CouponJ
Mean Log
Reduction*
Fumigate 10 min at 8 g/min; dwell at 0.8 g/min
1
15 min
30 min
60 min
90 min
120 min
180 min
Carpet
Carpet
Carpet
Carpet
Carpet
Carpet
8.57 xlO6
8.57 xlO6
8.57 xlO6
8.57 xlO6
8.57 x 106
8.57 x 106
4.50 ± 0.74 xlO6
4.50 ± 0.74 xlO6
4.50 ± 0.74 xlO6
4.50 ± 0.74 x 106
4.50 ± 0.74 x 106
4.50 ± 0.74 x 106
8.03 ± 5.97 xlO5
2.40 ± 2.28 xlO5
0.00 ±0.00
5.68 ± 7.79 xlO4
2.39 ± 3.33 x 104
0.00 ±0.00
0.82 ±0.26
1.49 ±0.52
6.65 ± 0.00
4.59 ±2.82
4. 74 ±2.61
6.65 ± 0.00
Fumigate 5 min at 4 g/min; dwell at 0.8 g/min
2
15 min
30 min
60 min
90 min
Carpet
Carpet
Carpet
Carpet
8.17 x 106
8.17 x 106
8.17 x 106
8.17 xlO6
3.27 ± 1.07 x 106
3.27 ± 1.07 x 106
3.27 ± 1.07 x 106
3.27 ± 1.07 xlO6
1.85 ± 1.17x 106
1.12±0.23x 106
2.90 ± 2.60 x 105
3.84±1.90xl05
0.33 ±0.32
0.47 ±0.09
1.62 ± 1.28
0.99 ±0.27
Fumigate 10 min at 8 g/min; dwell at 0.8 g/min
3
60 min
120 min
180 min
Carpet
Carpet
Carpet
8.53 x 106
8.53 x 106
8.53 x 106
2.95 ± 0.26 x 106
2.95 ± 0.26 x 106
2.95 ± 0.26 x 106
1.32±1.81xl04
1.66 ± 1.67 xlO5
2.49 ± 2.44 xlO4
4. 14 ±2.26
3.22 ±2.97
3.71 ±2.52
Fumigate 10 min at 8 g/min; dwell at 0.8 g/min
7a
7b
180 min
180 min
Laminate
Ductwork
Carpet
Concrete
Wood
Glass
Ceiling tile
1.08 xlO7
1.08 x 107
1.08 x 107
1.08 x 107
1.08 x 107
1.08 x 107
1.08 xlO7
7.18±3.60xl06
2.86 ± 1.71 x 106
5.42 ± 0.75 x 106
8.51 ± 2.94 x 106
5.25 ± 1.46 x 105§
6. 17 ± 0.72 x 106
6.66 ± 1.63xl05§
0.00 ±0.00
0.00 ±0.00
8.63 ± 16.6 x 104
4.99 ± 10.6 x 103
7.95 ± 9.18 x 103
0.00 ±0.00
0.00 ±0.00
6.86 ±0.00
6.46 ± 0.00
4.68 ±2.84
5.46 ±2.07
2. 16 ±0.64
6.79 ± 0.00
5.82 ±0.00
* Data are expressed as mean ± standard deviation of five replicates.
T Positive control coupons were spiked but not exposed to the fumigant.
* Test coupons were spiked and exposed to the fumigant for the contact time.
§ Below target recovery of >10% of spike amount.
-------
B. suis
BIOQUELL Claras C HP fumigation results for B. suis
are presented in Table 5-15. No B. suis was recovered
from aluminum, keyboard, carpet or joint tape following
fumigation at a contact time of 180 min and a fumigant
cycle that was 10 min at 8 g/min; dwell at 0.8 g/min.
Table 5-15. BIOQUELL Claras C HP Fumigation Results for B. suis
Trial
Contact
Time
Material
Spike
Amount (CFU/
coupon)
Mean Recovered B. suis (CFU/coupon)*
Positive
Controlf
Test
CouponJ
Mean Log Reduction*
Fumigate 10 min at 8 g/min; dwell at 0.8 g/min
5
180 min
Aluminum
Keyboard
Carpet
Joint tape
3.50 xlO7
3.50 xlO7
3.50 xlO7
3.50 xlO7
1.50±0.64xl07
2.24 ± 0.93 xlO7
7.06 ± 1.30xl06
2.17 ± 0.25 xlO6
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
7.18 ±0.00
7.35 ±0.00
6.85 ±0.00
6.34 ±0.00
* Data are expressed as mean ± standard deviation of five replicates.
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
Vaccinia Virus
BIOQUELL Claras C HP fumigation results for vaccinia
virus are presented in Table 5-16. No vaccinia virus was
recovered from keyboard, carpet aluminum, joint tape, or
glass following fumigation at a contact time of 180 min
and a fumigant cycle that was 10 min at 8 g/min; dwell at
0.8 g/min.
Table 5-16. BIOQUELL Claras C HP Fumigation Results for Vaccinia Virus
Trial
Contact
Time
Material
Spike
Amount (PFUs/
coupon)
Mean Recovered Y. pestis (PFUs/coupon)*
Positive
Controlf
Test
CouponJ
Mean Log Reduction*
Fumigate 10 min at 8 g/min; dwell at 0.8 g/min
6a
6b
180 min
Keyboard
Carpet
Aluminum
Joint tape
Glass
3.52 xlO7
3.52 xlO7
9.64 xlO7
4.30 x 107
9.64 x 107
7.74±3.90xl05
3.93 ± 1.88xl04
1.59±0.59xl07
1.09±0.45x 105
1.60±0.54x 107
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
5.89 ±0.00
4.59 ±0.00
7.20 ± 0.00
5.04 ±0.00
7.20 ± 0.00
Data are expressed as mean ± standard deviation of five replicates.
Positive control coupons were spiked but not exposed to the fumigant.
Test coupons were spiked and exposed to the fumigant for the contact time.
-------
Y. pestis
BIOQUELL Claras C HP fumigation results for Y. pestis
are presented in Table 5-17. No Y. pestis was recovered
from keyboard, carpet aluminum, joint tape, or glass
following fumigation at a contact time of 180 min and a
fumigant cycle that was 10 min at 8 g/min; dwell at 0.8
g/min.
Table 5-17. BIOQUELL Clarus C HP Fumigation Results for Y. pestis
Trial
Contact
Time
Material
Spike
Amount (CFUs/
coupon)
Mean Recovered Y. pestis (CFUs/coupon)*
Positive
Controlf
Test
CouponJ
Mean Log Reduction*
Fumigate 10 min at 8 g/min; dwell at 0.8 g/min
4
180 min
Aluminum
Keyboard
Carpet
Joint tape
9.07 xlO6
9.07 xlO6
9.07 xlO6
9.07 xlO6
3.02 ± 0.71 xlO4
4.56 ± 1.53xl05
2.14 ± 0.93 xlO3
4.29 ± 2.76 xlO3
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
4.48 ±0.00
5.66 ±0.00
3.33 ±0.00
3.63 ±0.00
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
Summary Statistics for BIOQUELL Clarus C HP
Decontamination
Table 5-18 provides a summary of BIOQUELL Clarus
C HP Fumigation decontamination efficacy, calculated
as the difference in the mean log of viable bacteria
recovered from positive control coupons and the mean
log of viable bacteria recovered from coupons after
fumigation for a given contact time. The elapsed time
from spiking to recovery was the same for the positive
control coupons and test coupons to control for history.
The 95% CI and p-value are also shown. At the 180-
min contact time (fumigate 10 min at 8 g/min; dwell at
0.8 g/min), no viable Y. pestis, B. suis, or vaccinia were
recovered from any type of material tested. At the 180-
min contact time (fumigate 10 min at 8 g/min; dwell at
0.8 g/min), B. anthracis was recovered only from carpet,
concrete, and wood.
Surface Damage
The physical effect of the BIOQUELL Clarus C HP
fumigation on the materials was evaluated qualitatively.
The appearance of the decontaminated coupons was
visually inspected for any obvious changes in the color,
reflectivity, and apparent roughness of the material
surfaces. These comparisons were performed for each
material, before extraction of the decontaminated test
coupons. No differences were observed between control
and fumigated coupons for any material, except that the
coupons were visibly moistened by condensation during
fumigation.
-------
Table 5-18. Summary of Efficacy (Calculated as Mean Log Reduction) of BIOQUELL Claras C HP Fumigation
Trials
Agent
Material
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
Trial 6
Trial 7
B. anthracis
spores
B. anthracis
spores
B. anthracis
spores
Y. pestis
B. suis
Vaccinia
B. anthracis
spores
Carpet
Carpet
Carpet
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Glass
Keyboard
Painted Joint
Tape
Carpet
Ceiling Tile
Concrete
Glass
Laminate
Metal
Wood
Mean Log Reduction (95% CI) and p-Value*
or Mean Log Reduction (# of treated coupons with zero recovery /# of treated coupons) and
p-Valuef
15 min
0.82 (0.54,
1.10)
p=0.0015
0.31 (-0.06,
0.69)
p=0.1032
30min
1.48 (0.95,
2.02)
p=0.0027
0.45 (0.26,
0.64)
p=0.0014
611 niin
>6.65 (5/5)
p=0.0079
1.60 (0.27,
2.93)
p=0.0486
>4.14 (2/5)
p=0.0079
90min
>4.59 (3/5)
p=0.0079
0.97 (0.64,
1.29)
p=0.0003
120 min
>4.74 (3/5)
p=0.0079
>3.22 (2/5)
p=0.0079
180 min
>6.65 (5/5) p=0.0079
>3.71 (2/5) p=0.0079
>4.47 (5/5) p=0.0079
>3.30 (5/5) p=0.0079
>5.64 (5/5) p=0.0079
>3.54 (5/5) p=0.0079
>7.15 (5/5) p=0.0079
>6.84 (5/5) p=0.0079
>7.31 (5/5) p=0.0079
>6.33 (5/5) p=0.0079
>7.18 (5/5) p=0.0079
>4.55 (5/5) p=0.0079
>7.18 (5/5) p=0.0079
>5.85 (5/5) p=0.0079
>5.00 (5/5) p=0.0079
>4.67 (3/5) p=0.0079
>5.81 (5/5) p=0.0079
>5.43 (3/5) p=0.0079
>6.79 (5/5) p=0.0079
>6.81 (5/5) p=0.0079
>6.34 (5/5) p=0.0079
2.15 (1.48, 2.82)
p=0.0014
* Mean log reduction is the mean of the base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm
of recovered agent from the treated coupons. A 95 % CI for the difference is shown in parentheses. A p-value is provided for the probability that
the control and treatment recoveries are the same. The p-value is from the two sample t-test with Satterthwaite's method to allow for potentially
different variances in the two groups. p-Values less than 0.05 denote less than 1 in 20 chance that a difference as large as or larger than observed
would occur by chance if the control and treatment means were truly identical. Comparisons with p-values less than 0.05 (statistically significant
at the 0.05 level) are bolded.
| One or more of the treatment coupons had no recovered agent. The mean log reduction of the form ">X" is calculated as the mean of the
base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm of recovered agent from the treated
coupons except that "zero recovery" coupons have a substituted recovered value of "1" (base-10 log is 0). Since the log becomes an increasingly
negative value below 1 and is undefined at 0, this substitution is necessary and results in a lower bound on the mean log difference, as indicated
by the ">". The number of "zero recovery" treatment coupons and the total number of treatment coupons is shown in parentheses. The p-value
is from the non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance that results as different as or more
different than observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with
p-values less than 0.05 (statistically significant at the 0.05 level) are bolded.
-------
5.3 HP S)
5.3.1 Claras S HP
Technology
The B1OQUELL Clams S. shown in Figure 5-13, is a
compact and mobile HP vapor technology' designed to
bio-decontaminate laboratory equipment. BIOQUELL's
Claras S uses low temperature, residue-free hydrogen
peroxide vapor technology. The B1OQUELL Clams
S Hydrogen Peroxide Vapor Generation System was
operated in an automated cycle controlled by the
commercial unit using manufacturer's recommended
parameters. The Claras S unit controlled the cycle phases
and duration.
5.3.2 Test for Claras S HP
Fumigation
The testing performed with B1OQUELL Clams S
fumigation is shown in Table 5-19. The experimental
design tested decontamination efficacy by determining
whether there was a difference between the log
reductions in the viable biological agents after
fumigation compared to controls for various materials.
These tests also assessed whether there was any
difference in efficacy at varying RH and fumigation
contact times. Critical parameters impacting the
viability of biological agents amount of HP injected,
fumigation contact time, temperature, RH, and natural
attenuation. An adaptive management approach was
used to incorporate new knowledge into the testing as
decontamination efficacy results became available.
Figure 5-13. BIOQUELL Claras S HP Vapor Generator.15
The HP vapor in the test chamber was monitored using
an Analytical Technology HP gas sensor with liquid
crystal display, as described in Section 5.2.1. The
HP gas sensor was oriented in the test chamber at a
position distant to the HP vaporizer but in proximity to
test coupons. The display unit and power supply were
located outside the test chamber. The concentration
of HP vapor was documented approximately every 20
min during the gassing portion of the decontamination
cycle while test coupons are in the test chamber. The HP
vapor concentration was a non-critical measurement,
but the data from the sensor enabled monitoring of HP
concentration variability between fumigation cycles.
-------
Table 5-19. Test Matrix for BIOQUELL Claras S HP Fumigation
Trial
1
2
3
4
5
6
7
8
9
9a
Biological Agent
G. stearothermophilus
F. tularensis
F. tularensis
B. suis
B. suis
Y. pestis
Y. pestis
B. anthracis spores
B. anthracis spores
B. anthracis spores
Material
Biological
indicator: Steel
in Tyvek"
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Keyboard
Carpet
Carpet
Aluminum
Keyboard
Carpet
Joint tape
HP Fumigation Parameters
HP volume: 15 mL
Injection time: 55 min
HP volume: 15 mL
Injection time: 15 min
HP volume: 15 mL
Injection time: 15 min
HP volume: 15 mL
Injection time: 15 min
HP volume: 15 mL
Injection time: 15 min
HP volume: 15 mL
Injection time: 15 min
HP volume: 15 mL
Injection time: 15 min
HP volume: 15 mL
Injection time: 15 min
HP volume: 15 mL
Injection time: 15 min
Dwell 45: min
HP volume: 17.5 mL
Injection time: 15 min
Dwell 45: min
HP volume: 17.5 mL
Injection time: 15 min
Dwell 45: min
HP volume: 50 mL
Injection time: 20 min
Initial
Environmental
Condition
40% - 50% RH; 22 °C
± 3 °C (ambient)
40% - 50% RH; 22 °C
± 3 °C (ambient)
60% - 70% RH; 22 °C
±3°C
40% - 50% RH;
22 °C ± 3 °C
(ambient)
60% - 70% RH;
22 °C ± 3 °C
(ambient)
40% - 50% RH;
22 °C ± 3 °C
(ambient)
60% - 70% RH;
22 °C ± 3 °C
(ambient)
40% - 50% RH;
22 °C ± 3 °C
(ambient)
40% - 50% RH;
22 °C ± 3 °C
(ambient)
40% - 50% RH;
2 2°C ± 3 °C (ambient)
Contact Times
(min)
5,30, 120
30,60
15*, 30, 60
15,30
15, 30, 60
15,30
15, 30, 60
30,60
60, 126, 192
75
*Carpet and joint tape only.
-------
5.3.3 BIOQUELL Clams S HP Fumigation Results
Figure 5-14 shows a graph of typical temperature, RH,
and HP concentration during a fumigation cycle (15 mL
HP injection). The HP concentration peaked initially,
and then gradually drifted lower during the dwell phase
(starting at 15 minutes in Figure 5-14, after the injection
phase).
Figure 5-14. Typical Temperature (°C), RH (%), and HP Concentration (ppmv) Dynamics During the Fumigation
Cycle (15 mL HP Injection).
10
20
40
50
60
70
Time(min)
•Temp (2C)
-%RH -*-H202(ppm)
-------
Figure 5-15 shows a graph of temperature, RH, and HP
concentration during a high concentration fumigation
cycle (50 mL HP injection). The HP concentration
peaked initially, and then gradually drifted lower during
the dwell phase (starting at 20 minutes in Figure 5-14,
after the injection phase).
Figure 5-15. Typical Temperature (°C), PJ3 (%), and HP Concentration (ppmv) Dynamics During the Fumigation cycle
(50 mL HP Injection).
600 n
0 10 20 30 40 50 60 70 80
Tune (mm)
B-Temp (°C) H2O2(ppm)
-------
G. stearothermophilus
The results for BIOQUELL Claras S HP fumigation
of biological indicators are shown in Table 5-20. The
biological indicators were G. stearothermophilus
nominally 1 x 106 spores on stainless steel in Tyvek®
packaging. In this qualitative test, no growth was
observed at any contact time, including the shortest time
tested (5-min contact time).
Table 5-20. BIOQUELL Clarus S HP Fumigation Results
for G. stearothermophilus
Trial
1
Fumigation Cycle
HP volume: 15mL
Injection time: 55 min
Contact Times (min)
Growth Positive/Total Biological
Indicators
5
0/5
30
0/5
120
0/10
B. anthracis
BIOQUELL Claras S HP fumigation results for B.
anthracis spores are presented in Table 5-21. With an HP
volume of 15 mL injected over 15 min and a contact time
of 30 min, no viable B. anthracis spores were recovered
from keyboard (initial RH 40% - 50%). No viable B.
anthracis spores were recovered from aluminum or joint
tape with an HP volume of 50 mL injected over 20-min
and a 75-min contact time (initial RH 40% - 50%). Note
that 75 min was the shortest contact time tested with
aluminum and joint tape. In contrast, lower efficacy was
observed against B. anthracis spores on carpet; viable
spores were recovered from the carpet after a 192-min
contact time (with a cycle including multiple HP injections
totaling 50 mL, initial RH: 40% - 50%; see Table 5-19).
Viable B. anthracis spores were also recovered after a
75-min contact time with a fumigant cycle of 50 mL HP
injected over 20 min (initial RH: 40% - 50%).
No growth was observed on the biological indicators
following exposure to the fumigant. The high efficacy,
suggested by the completed kill of the biological
indicators, did not correlate with complete kill of B.
anthracis spores on other materials.
Table 5-21. BIOQUELL Clarus S HP Fumigation Results for B. anthracis
Trial
Contact
Time
Material
Spike
Amount (CFU/coupon)
Mean Recovered B. anthracis (CFU/coupon)*
Positive Controlf
Test CouponJ
Mean Log
Reduction*
Initial RH 45%; Fumigate 15 mL Injected Over 15 min
8
8
30 min
60 min
Keyboard
Carpet
Keyboard
Carpet
3.03 x 106§
3.03 x 106§
3.03 x 106§
3.03 x 106§
1.40 ± 0.44 x 106
5.88 ± 0.50 x 106
1.24 ± 0.33 x 106
3.94 ± 2.24 x 106
0.00 ±0.00
9.83 ± 3.94 x 105
0.00 ±0.00
1.16±0.44x 106
6.14 ±0.00
0.80 ±0.16
6.09 ±0.00
0.56 ±0.19
Initial RH 45%; Three Fumigate Cycles Totaling 50 mL Injected Over 3x15 min
Dwell between injections ~45 min
9
60 min
126 min
192 min
Carpet
Carpet
Carpet
2.33 x 106§
2.33xl06§
2.33xl06§
4.35 ± 0.42 x 106
4.35 ± 0.42 x 106
4.35 ± 0.42 x 106
9.74 ±3. 81 x 105
5.00 ± 7.62 xlO4
1.61±3.34xl04
0.68 ±0.17
3. 82 ±2.61
4.92± 2.39
Initial RH 45%; Fumigate 50 mL Injected Over 20 min
9a
75 min
Aluminum
Keyboard
Carpet
Joint tape
8.40 x 106
8.40 x 106
8.40 x 106
8.40 x 106
1.26±0.84xl05
1.81±2.32xl05
4.70 ± 0.55 x 106
1.83 ± 0.76 x 106
0.00 ±0.00
0.00 ±0.00
9.81 ± 7.64 xlO5
0.00 ±0.00
5. 10 ±0.00
5.26 ±0.00
0.81 ±0.41
6.26 ±0.00
* Data are expressed as mean ± standard deviation of five replicates.
T Positive control coupons were spiked but not exposed to the fumigant.
* Test coupons were spiked and exposed to the fumigant for the contact time.
§ Application was lower than the target 7.5 x 106 - 1.25 x 107 CPUs/coupon.
-------
B. suis
BIOQUELL Claras S HP fumigation results for B. suis
are presented in Table 5-22. Efficacy against B. suis
was highly variable, depending on the type of material.
Viable bacteria were recovered from the aluminum,
keyboard, and carpet after a 60-min contact time and a
fumigation cycle of 15 mL injected over 15-min (65%
initial RH). However, under these conditions, no viable
bacteria were recovered from joint tape. Part of the loss
of viable bacteria from joint tape was attributable to
possible bactericidal activity of the paint or joint tape, as
shown by the decrease in viable bacteria recovered from
the positive control coupons after the 60-min drying
time and the additional 60 min time before extraction,
corresponding to the CT. The impact of HP fumigation
was still discernible.
As described in the Introduction, the calculated
log reductions reflect the incremental impact of the
fumigation technology. Compared to the amount of
bacteria spiked onto the coupon (6.57 x 107 CPUs),
recovery of no viable bacteria from the joint tape after
the 60-min treatment represents >7 log reduction in
viable bacteria attributable to the fumigation and the loss
of viability from joint tape arising from other (unknown)
causes.
Table 5-22. BIOQUELL Claras S HP Fumigation Results for B. suis
Trial
Contact
Time
Material
Spike
Amount
(CFU/coupon)
Mean Recovered B. suis (CFU/coupon)*
Positive Controlf
Test CouponJ
Mean Log
Reduction*
Initial RH 45%; Fumigate 15 mL Injected Over 15 min
4
4
15 min
30 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
9.93 x 107
9.93 x 107
1.77xl08§
1.77xl08§
9.93 x 107
9.93 x 107
1.77 x 108§
1.77 x 108§
3.79 ± 0.88 x 107
2.76 ± 0.55 xlO7
2.35 ± 0.45 xlO8
2.48 ± 1.97 xlO6
3.65 ± 1.21 xlO7
2.13 ± 0.40 x 107
2.33 ± 0.24 x 108
1.66 ± 1.47 x 106
5. 54 ± 4.32 x 104
1.96 ± 3.93 xlO5
1.41 ± 0.09 xlO8
1.10± 1.94xl05
1.27 ± 2.31 xlO2
0.00 ±0.00
l.ll±0.26x 108
3.63 ± 3.27 x 105
2.95 ±0.35
3.20 ± 1.25
0.22 ± 0.03
2.55 ± 1.45
6.62 ± 1.32
7.33 ± 0.00
0.33 ±0.10
0.79 ±0.38
Initial RH 65%; Fumigate 15 mL Injected Over 15 min
5
5
5
15 min
30 min
60 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
3.10 x 107
3.10 x 107
6.57 xlO7
6.57 xlO7
3.10xl07
3.10xl07
6.57 x 107
6.57 x 107
3.10 x 107
3.10 x 107
6.57 x 107
6.57 xlO7
2.65 ±0.31 x 107
3. 10 ± 0.59 x 107
1.35 ± 1.32 xlO7
1.98 ± 2.18 xlO5
3.20 ± 0.94 xlO7
3.20 ± 0.60 xlO7
1.01 ± 0.66 x 107
8.25 ± 8.05 x 103
7.94 ± 3.60 x 107
2.11 ± 0.38 x 107
1.32 ± 1.78 x 106
2.79 ± 2.21 xlO3
4.70 ± 1.56 x 106
2.49 ± 0.77 x 106
1.25 ± 0.85 xlO6
7.40 ± 14.3x10°
2.37 ± 0.92 xlO6
1.22 ± 1.26xl06
1.09 ± 1.19 x 103
2.40 ± 5.20 x 104
1.50 ± 1.24 x 106
2.71 ± 3.59 x 105
2.81 ± 6.26 x 102
0.00 ±0.00
0.77 ±0.16
1.11±0.13
1.20 ±0.51
4.99 ± 0.68
1.17±0.21
1.68 ±0.58
4.38 ±0.82
1.24 ± 1.85
1.90 ±0.50
2. 27 ±0.74
5.49 ± 1.41
3.45 ± 0.00
* Data are expressed as mean ± standard deviation of five replicates.
T Positive control coupons were spiked but not exposed to the fumigant.
* Test coupons were spiked and exposed to the fumigant for the contact time.
§ Application exceeded the target 1.0 x 106 - 1.0 x 108 CFU/coupon.
-------
F. tularensis
BIOQUELL Claras S HP fumigation results for F.
tularensis are presented in Table 5-23. The Claras S
HP fumigation was efficacious for all materials at the
shortest time used and independent of the starting RH
values investigated. No F. tularensis was recovered
from carpet or joint tape following fumigation at a
contact time of 15 min and a fumigation cycle of 15 mL
injected over 15 min (65% initial RH). No F. tularensis
was recovered from aluminum or keyboard following
fumigation at a contact time of 30 min (shortest contact
time tested) and a fumigation cycle of 15 mL injected
over 15 min (45% or 65% initial RH). As described in
the Introduction, the calculated log reductions reflect
the incremental impact of the fumigation technology.
Compared to the amount of bacteria spiked onto the
coupon (about 107 CPUs/coupon), after a 15-min
treatment ofF. tularensis on carpet or joint tape or a
30 min treatment of F. tularensis on aluminum and
keyboard a >7 log reduction in viable bacteria was
attributable to the fumigation and the loss of viability
arising from other (unknown) time-dependent causes.
Table 5-23. BIOQUELL Clarus S HP Fumigation Results for F. tularensis
Trial
Contact
Time
Material
Spike
Amount (CFU/
coupon)
Mean Recovered F. tularensis (CFU/coupon)*
Positive
Controlf
Test
CouponJ
Mean Log
Reduction*
Initial RH 45%; Fumigate 15 mL Injected Over 15 min
2
2
30 min
60 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
1.33 xlO7
1.33 xlO7
1.58 xlO7
1.58 xlO7
1.33 x 107
1.33 x 107
1.58 x 107
1.58 x 107
1.61 ± 0.51 xlO5
3.80± 1.55 xlO5
5.09 ± 1.52 xlO6
8.67 ± 12.1 xlO2
6.27 ±4. 16 x 104
9.63 ± 1.29 x 104
8.96 ± 6.44 x 105
2.83 ± 1.23 x 102
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
5.21 ±0.00
5. 58 ±0.00
6.71 ±0.00
2.94 ±0.00
4.80 ±0.00
4.98 ±0.00
5.95 ±0.00
2.45 ±0.00
Initial RH 65%; Fumigate 15 mL Injected Over 15 min
3
3
3
15 min
30 min
60 min
Carpet
Joint tape
Carpet
Joint tape
Carpet
Joint tape
1.77 x 107
1.77 xlO7
1.77 xlO7
1.77 xlO7
1.77 xlO7
1.77 x 107
3.30 ± 2.26 x 106
1.38 ± 1.75x10'
3.00 ± 0.69 xlO6
4.01 ± 4.82 xlO3
1.01 ± 1.92 xlO4
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
6.52 ±0.00
1.14±0.00
6.48 ±0.00
3.60 ±0.00
4.00 ±0.00
Not calculable
Initial RH 65%; Fumigate 15 mL Injected Over 15 min
3
3
3
30 min
60 min
90 min
Aluminum
Keyboard
Aluminum
Keyboard
Aluminum
Keyboard
1.65 x 107
1.65 x 107
1.65 x 107
1.65 x 107
1.65 x 107
1.65 x 107
6.63 ± 2.76 x 104
1.30 ± 1.21 x 106
1.91 ± 1.70 x 104
3. 85 ± 3.50 x 105
6.98 ± 2.34 xlO3
1.04 ± 0.30 xlO5
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
4.82 ±0.00
6.11 ±0.00
4.28 ±0.00
5. 59 ±0.00
3. 84 ±0.00
5.02 ±0.00
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
-------
Y. pestis
BIOQUELL Claras S HP fumigation results for Y. pestis
are presented in Table 5-24. Efficacy against Y. pestis
was highly variable, depending on the type of material.
Viable bacteria were recovered from the aluminum,
keyboard, and carpet after a 60-min contact time and a
fumigation cycle of 15 mL injected over 15 min (65%
initial RH). However, under these conditions, no viable
bacteria were recovered from joint tape. Part of the loss
of viable bacteria from joint tape was attributable to
possible bactericidal activity of the paint or joint tape,
as shown by the decrease in viable bacteria recovered
from the positive control coupons. As described in
the Introduction, the calculated log reductions reflect
the incremental impact of the fumigation technology.
Table 5-24. BIOQUELL Claras S HP Fumigation Results for Y. pestis
Compared to the amount of bacteria spiked onto the
coupon (about 107 CPUs/coupon), after a 60-min
treatment of Y. pestis on the various types of coupons,
efficacy ranging from <1 log reduction to >7 log
reduction in viable bacteria was attributable to the
combined effects of fumigation and the loss of viability
arising from other (unknown) time-dependent causes.
Trial
Contact
Time
Material
Spike
Amount
(CFU/coupon)
Mean Recovered Y. pestis (CFU/coupon)*
Positive
Controlf
Test
CouponJ
Mean Log
Reduction*
Initial RH 45%; Fumigate 15 mL Injected Over 15 min
6
6
15 min
30 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
4.93 x 107
4.93 x 107
6.03 x 107
6.03 x 107
4.93 x 107
4.93 x 107
6.03 x 107
6.03 x 107
1.53±0.87xl05
4.02 ± 2.53 xlO4
6.47 ±5. 86 x 106
1.77 ± 1.86 x 104
3.41 ±2.11 x 104
2.92 ± 1.19x 105
1. 14 ± 0.28 x 10s
8.41 ± 5.20 xlO3
1.40 ±2.91x10'
2.67 ± 3.60 xlO2
1.66 ± 2.58 x 107
3.40 ± 4.67 x 101
0.00 ±0.00
7.36 ± 12.8 x 101
2.03 ± 3.53 xlO6
3.58±5.08xl02
4.82 ±0.81
2.72 ± 1.11
0.11 ±0.84
3.48 ±1.05
4.53 ±0.00
4.36 ± 1.08
3.23 ± 1.88
2.46 ± 1.47
Initial RH 65%; Fumigate 15 mL Injected Over 15 min
7
7
7
15 min
30 min
60 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
5.27 xlO7
5.27 xlO7
5.03 x 107
5.03 x 107
5.27 x 107
5.27 x 107
5.03 x 107
5.03 x 107
5.27 xlO7
5.27 xlO7
5.03 x 107
5.03 x 107
1.02±0.46xl05
9.97 ± 6.79 xlO4
6.43 ± 3.28 x 107
5.80 ±5.91 x 106
1.66 ± 0.89 x 104
1.52 ± 0.28 x 105
3.60 ±2.81 x 107
1.79 ±2.91 x 103
1.13±0.28xl05
2.41 ± 0.44 xlO4
2.49 ± 0.67 xlO7
2.18±2.82xl02
7.46 ± 3.92 xlO2
1.31 ± 2.02 xlO4
3.39 ± 1.35 xlO7
6.01 ±6. 12 x 105
4.07 ±5.01 x 102
6.09 ± 4.95 x 103
3.29 ± 1.40 x 107
2.15 ±4. 78 x 102
1.26 ± 1.59x10'
2.92 ± 1.92 xlO2
1.83±0.58xl07
0.00 ±0.00
2.21 ±0.31
1.18±0.50
0.30 ±0.17
1.26 ±0.61
2.24 ± 1.21
1.52 ±0.37
0.08 ±0.22
2.65 ± 1.35
4. 46 ±0.81
2.02 ±0.38
0.15 ±0.15
2.34 ±0.00
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
-------
Summary Statistics for BIOQUELL Clams S HP
Decontamination
Table 5-25 provides a summary of BIOQUELL Claras
S HP fumigation efficacy results. Because of the loss
of viable Y. pestis from positive control coupons due to
unknown, time-dependent causes, the log reductions
attributable only to the fumigation effect may appear
low or may not be determined. The combined effects
attributable to the 60 min fumigation treatment and the
loss of viability over time from other (unknown) causes
result in >7 log reduction in viable F. tularensis bacteria
applied to the test coupons. For B. suis and Y. pestis,
the efficacies varied depending on the type of coupon
material and the CT. The BIOQUELL Claras S HP
fumigation treatment showed high efficacy (no recovered
spores) against B. anthracis, except the treatment showed
low efficacy against spores on aluminum. A reaction
between the HP and aluminum may explain the results.
Table 5-25. Summary of Efficacy (Calculated as Mean Log Reduction) of BIOQUELL Claras S HP
Fumigation Results
Surface Damage
The physical effect of the BIOQUELL Claras S HP
fumigation on the materials was evaluated qualitatively.
The appearance of the decontaminated coupons was
visually inspected for any obvious changes in the color,
reflectivity, and apparent roughness of the material
surfaces. These comparisons were performed for each
material, before extraction of the decontaminated test
coupons. No differences were observed between control
and fumigated coupons for any material, except that the
coupons were visibly moistened by condensation during
fumigation.
Trials
Agent
Material
Trial 2
Trial 3
Trial 4
Trial 5
F. tularensis
F. tularensis
B. suis
B. suis
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Mean Log Reduction (95% CI) and p-Value*
or Mean Log Reduction (# of treated coupons with zero recovery /# of treated coupons) and
p-Valuef or N/A (# of control coupons with zero recovery /# of control coupons # of treated
coupons with zero recovery /# of treated coupons) and p- Value J
15 min
>6.45 (5/5)
p=0.0079
N/A (3/5 5/5)
p=0.4444
2.94 (2.56, 3.32)
p<0.0001
0.22 (0.12, 0.31)
p=0.0032
3.19 (1.90, 4.49)
p=0.0046
2.47 (0.94, 3.99)
p=0.0180
0.77 (0.60, 0.94)
p=0.0002
0.93 (0.11, 1.75)
p=0.0312
1.10 (0.94, 1.26)
p<0.0001
>4.41 (4/5)
p=0.0079
30 min
>5.19 (5/5)
p=0.0079
>6.69 (5/5)
p=0.0079
>5.55 (5/5)
p=0.0079
>2.63 (5/5)
p=0.0079
>4.78 (5/5)
p=0.0079
>6.47 (5/5)
p=0.0079
>5.95 (5/5)
p=0.0079
N/A (1/5 5/5)
p=0.0476
>6.60 (3/5)
p=0.0079
0.33 (0.21, 0.45)
p=0.0010
>7.32 (5/5)
p=0.0079
0.65 (0.059, 1.24)
p=0.0352
1.15 (0.88, 1.41)
p<0.0001
4.24 (3.28, 5.20)
p<0.0001
1.67 (1.06, 2.28)
p=0.0027
>1.05 (1/5)
p=0.3571
60 min
>4.46 (5/5)
p=0.0079
>5.83 (5/5)
p=0.0079
>4.98 (5/5)
p=0.0079
>2.42 (4/4)
p=0.0286
>4.06 (5/5)
p=0.0079
>3.19 (5/5)
p=0.0079
>5.44 (5/5)
p=0.0079
N/A (5/5 5/5)
p=1.0000
1.87 (1.32, 2.42)
p=0.0005
>5.00 (4/5)
p=0.0079
2.26 (1.50, 3.03)
p=0.0022
>3.33 (5/5)
p=0.0079
75 min
90 min
>3.82 (5/5)
p=0.0079
>5.00 (5/5)
p=0.0079
-------
Trials
Agent
Material
Trial 6
Trial 7
Trial 8
Trial 9a
Y. pestis
Y. pestis
B. anthracis
spores
B. anthracis
spores
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Carpet
Keyboard
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Mean Log Reduction (95% CI) and p-Value*
or Mean Log Reduction (# of treated coupons with zero recovery /# of treated coupons) and
p-Valuef or N/A (# of control coupons with zero recovery /# of control coupons # of treated
coupons with zero recovery/# of treated coupons) and p-Value J
ISmin
>4.72 (4/5)
p=0.0079
-0.49 (-2. 17,
1.18)p=0.5198
>2.66 (1/5)
p=0.0079
>3.32 (3/5)
p=0.0079
2.18 (1.81, 2.54)
p<0.0001
0.25 (-0.082,
0.57)p=0.1294
1.06 (0.41, 1.72)
p=0.0062
0.23 (-1.71, 2.16)
p=0.7959
30min
>4.46 (5/5)
p=0.0079
3.22 (1.27, 5.16)
p=0.0187
>4.33 (2/5)
p=0.0079
>2.39 (2/5)
p=0.0079
>2.17 (1/5)
p=0.0079
-0.056 (-0.54,
0.43) p=0.7987
1.51 (1.12, 1.90)
p=0.0005
N/A (1/5 4/5)
p=0.2857
0.80 (0.63, 0.97)
p=0.0002
>6.12 (5/5)
p=0.0079
611 in in
>4.45 (3/5)
p=0.0079
0.14 (-0.056,
0.34) p=0. 1406
2.02 (1.62, 2.41)
p=0.0002
>2.03 (5/5)
p=0.0079
-0.38 (-2.77,
2.02)p=0.7344
>6.08 (5/5)
p=0.0079
75min
0.81 (0.38, 1.24)
p=0.0112
>4.97 (5/5)
p=0.0079
>6.22 (5/5)
p=0.0079
>4.99 (5/5)
p=0.0079
90min
* Mean log reduction is the mean of the base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm
of recovered agent from the treated coupons. A 95 % CI for the difference is shown in parentheses. A p-value is provided for the probability that
the control and treatment recoveries are the same. The p-value is from the two sample t-test with Satterthwaite's method to allow for potentially
different variances in the two groups. p-Values less than 0.05 denote less than 1 in 20 chance that a difference as large as or larger than observed
would occur by chance if the control and treatment means were truly identical. Comparisons with p-values less than 0.05 (statistically significant
at the 0.05 level) are bolded.
| One or more of the treatment coupons had no recovered agent. The mean log reduction of the form ">X" is calculated as the mean of the base-
10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm of recovered agent from the treated coupons
except that "zero recovery" coupons have a substituted recovered value of "1" (base-10 log is 0). Since the log becomes an increasingly negative
value below 1 and is undefined at 0, this substitution is necessary and results in a lower bound on the mean log difference, as indicated by the
">". The number of "zero recovery" treatment coupons and the total number of treatment coupons is shown in parentheses. The p-value is from
the non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance results as different or more different than
observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with p-values less
than 0.05 (statistically significant at the 0.05 level) are bolded.
J One or more of both the control and the treatment coupons had no recovered agent. In this case, the log reduction is indeterminate and the
mean log reduction is identified as "N/A". The number of "zero recovery" control coupons and the total number of control coupons is shown in
parentheses followed by the number of "zero recovery" treatment coupons and the total number of treatment coupons. The p-value is from the
non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance that results as different as or more different
than observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with p-values
less than 0.05 (statistically significant at the 0.05 level) are bolded.
-------
5.4 HP Fumigation (STERIS VHP8)
5.4.7 Description of STERIS VHP9 HP Technology
The STERIS VHP® Generator Series lOOOED, shown in
Figure 5-16, was used to introduce and control the HP
vapor inside a 1270 L BSC III. Because HP vapor is not
stable as a compressed gas, HP vapor must be produced
on site by vaporization of concentrated aqueous solutions
of HP. Thus, this technology includes the equipment
and chemicals for on-site generation, dispersion, and
neutralization of the HP vapor.
Figure 5-16. STERIS VHP® Generator Series 1000ED.1
The HP fumigation technology operates at ambient
temperature and atmospheric pressure in a closed loop
configuration. As depicted in Figure 5-17 (from STERIS
literature), the testing chamber is subjected to four
phases: dehumidification, condition, sterilization, and
aeration. This technology dehumidifies by re-circulating
the chamber air through a reusable or disposable
desiccant cartridge. Once the desired RH is reached,
HP vapor is injected at a rate set to achieve the desired
concentration of HP inside the chamber. The system then
maintains the set concentration for the desired contact
period for decontamination of the biological agent. Once
the decontamination phase is complete, the enclosure air
is re-circulated through the HP fumigation technology to
reduce the HP vapor concentration to the desired level.
The HP vapor in the test chamber is monitored using an
Analytical Technology HP gas sensor with liquid crystal
display, as described in Section 5.2.1. The HP gas sensor
is oriented in the test chamber at a position that is distant
to the HP vaporizer but in proximity to test coupons. The
display unit and power supply are located outside the test
chamber. The concentration of HP vapor is documented
approximately every 20 min during the gassing portion
of the decontamination cycle while test coupons are in
the test chamber.
Figure 5-17. STERIS VHP® Biodecontamination Cycle.16
Typical Biodecontamination Cycle
More
it
I
B
a.
Less
\
\
N.
S
1
o%
Gas CcneHKraticn
=?*ljtv» Humidity
Condensation Point
Dfihumtsif c^rion
Condition
Sierilizaton
-------
5.4.2 Test Matrix for STERIS VHP8 HP Fumigation
The testing performed with STERIS VHP® HP
fumigation is shown in Table 5-26. The experimental
design tested decontamination efficacy by determining
whether there was a difference between the log
reductions in the viable biological agents after
fumigation compared to controls for various materials.
Table 5-26. Test Matrix for STERIS VHP® HP Fumigation
These tests also assessed whether there was any
difference in efficacy at varying fumigant concentrations
and fumigation contact times. Critical parameters in
this testing included fumigant concentration, fumigation
contact time, and the viability of the biological
agents. An adaptive management approach was used
to incorporate new knowledge into the testing as
decontamination efficacy results became available.
Trial
1
4a
4b
5a
5b
6a
6b
7a
7b
2a
2b
3a
3b
Biological Agent*
B. cmthracis
spores
Y. pestis
Y. pestis
F. tularensis
F. tularensis
B. suis
B. suis
Vaccinia virus
Vaccinia virus
B. anthracis
spores
B. anthracis
spores
B. anthracis
spores
B. anthracis
spores
Material
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Aluminum
Keyboard
Aluminum
Keyboard
Aluminum
Keyboard
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Laminate
Ductwork
Carpet
Concrete
Laminate
Ductwork
Carpet
Concrete
Wood
Glass
Ceiling tile
Wood
Glass
Ceiling tile
STERIS VHP8 Concentration
500 ppmv
200-250 ppmv
500 ppmv
200-250 ppmv
500 ppmv
200-250 ppmv
500 ppmv
200-250 ppmv
500 ppmv
500 ppmv
200-250 ppmv
500 ppmv
200-250 ppmv
Contact Times (Sterilization Phase)
30 min, 60 min, 120 min, 240 min, and full cycle
240 min
90 min, 120 min
30 min
90 min
30 min
90 min, 120 min
30 min, 60 min,
90 min
30 min, 60 min1, 120 mint
30 min, 60 min
30 min, 60 min, 120 min, 240 min, and full cycle
240 min
30 min, 60 min, 120 min, 240 min, and full cycle
240 min
30 min, 60 min, 120 min, 240 min, and full cycle
240 min
30 min, 60 min, 120 min, 240 min, and full cycle
240 min
Biological indicators (G. stearothermophilus) were included with each fumigation test.
At 60 min only aluminum and keyboard were used.
At 120 min only aluminum was used.
-------
5.4.3 STERIS VHP9 HP Fumigation Results
Figure 5-18 shows a graph of typical temperature, RH,
and HP concentration during a nominally 225 ppmv HP
fumigation cycle.
Figure 5-18. Typical Temperature (°C), RH (%), and HP Concentration (ppmv) Dynamics During a Fumigation Cycle
(225 ppmv HP) with the STERIS VHP® Generator Series 1000ED.
225 ppmv, 90 Minutes
400
350
300
250
200
150
100
07:45:40
08:45:40
-H202
-96RH
09:45:40
Temp
10:45:40
Figure 5-19 shows a graph of typical temperature, RH, and HP concentration during a nominally 500 ppmv HP
fumigation cycle.
Figure 5-19. Typical Temperature (°C), RH (%), and HP Concentration (ppmv) Dynamics During a Fumigation Cycle
r500 rmmv HP1 with the STERIS VHP® Generator Series 1000ED.
500 ppm, 4 Hours
500
450
400
•H202
PPM
•%RH
-------
G. stearothermophilus
The results for STERIS VHP® HP fumigation of
biological indicators are shown in Table 5-27. The
biological indicators were G. stearothermophilus
nominally 1 x 106 spores on stainless steel in Tyvek®
packaging. In this qualitative test, no growth was
observed at any contact time of 60 min or longer at an
HP concentration of 500 ppmv; no growth was observed
at any contact time of 240 min at an HP concentration
of 200-250 ppmv. On some materials and with some
biological agents (B. anthracis, B. suis, and vaccinia
virus), the results from the biological indicators did not
correlate with the results from the biological agents'
exposure to the same CT. The high efficacy, suggested
by the completed kill of the biological indicators, did not
correlate with complete kill of B. anthracis spores on
other materials.
Table 5-27. STERIS VHP® HP Fumigation Results for G.
stearothermophilus
Trial
1
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
STERIS VHP8
Concentration
500 ppmv
500 ppmv
200-250 ppmv
500 ppmv
200-250 ppmv
200-250 ppmv
500 ppmv
200-250 ppmv
500 ppmv
200-250 ppmv
500 ppmv
200-250 ppmv
500 ppmv
Contact Times (min)
Growth Positive/Total Biological
Indicators
30
5/5
30
5/5
30
5/5
30
4/5
30
5/5
30
1/5
30
1/5
30
1/5
30
3/5
30
0/5
60
0/5
60
0/5
60
5/5
60
0/5
60
5/5
90
0/5
90
0/5
90
0/5
60
0/5
60
0/5
60
0/5
120
0/5
120
0/5
120
2/5
120
0/5
120
2/5
120
0/5
120
0/5
90
0/5
120
0/5
240
0/5
240
0/5
240
0/5
240
0/5
240
0/5
B. anthracis
STERIS VHP® HP fumigation results for B. anthracis
spores are presented in Table 5-28 and Figures 5-20 and
5-21. No viable B. anthracis spores were recovered from
carpet, concrete, glass, aluminum, keyboard, laminate,
ductwork, and ceiling tile at exposures of 120 min to
the 500 ppmv HP fumigation cycle. Decontamination
of wood was more difficult than other materials: B.
anthracis spores were recovered from wood after 120-
min exposure to the 500 ppmv HP fumigation cycle. No
viable B. anthracis spores were recovered from wood at
an exposure of 240 min to the 500 ppmv HP fumigation
cycle.
With the 200-250 ppmv HP fumigation cycle, B.
anthracis spores were not recovered from laminate,
ductwork, concrete, glass, and ceiling tile after 120 min
of exposure, but B. anthracis spores were recovered from
carpet and wood after the entire 240-min fumigation
cycle. For perspective, Vaprox® HP sterilant, when
used with a STERIS VHP® generator for sterilization
of exposed pre-cleaned dry porous and non-porous
surfaces, specifies a sterilization phase with "a minimum
of 250 ppm[v] of VHP sterilant for 90 min in sealed
enclosures up to 4,000 ft3 [113,000 L]".17
-------
Table 5-28. STEMS VHP® HP Fumigation Results for B. anthracis
Trial
Contact Time
Material
Spike Amount (CFU/
coupon)
Mean Recovered B. anthracis (CFU/coupon)*
Positive Controlf
Test CouponJ
Mean Log
Reduction*
500 ppinv Fumigation Cycle
1
1
1
1
1
30 min
60 min
120 min
240 min
Full cycle
240 mm
Aluminum
Keyboard
Carpet - a
Joint tape
Aluminum
Keyboard
Carpet - a
Joint tape
Aluminum
Keyboard
Carpet - a
Joint tape
Aluminum
Keyboard
Carpet - a
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
4.87 x 10s §
4.87 x 10s §
4.87 x 10s §
4.87 x 10s §
4.87 x 108§
4.87 x 10s §
4.87 x 108§
4.87 x 10s §
4.87 x 10s §
4.87 x 10s §
4.87 x 10s §
4.87 x 10s §
4.87 x 10s §
4.87 x 108§
4.87 x 10s §
4.87 x 108§
4.87 x 10s §
4.87 x 10s §
4.87 x 10s §
4.87 x 10s §
7.30 ± 4.03 xlO81
3.96 ± 3.29 x 10s
7.70 ± 4.24 xlO81
5.42 ± 1.72 xlO8
7.30 ± 4.03 x 1081
3.96 ± 3.29 x 10s
7. 70 ± 4.24 x 1081
5.42 ± 1.72 x 10s
7.30 ± 4.03 x 10s t
3.96 ± 3.29 x 10s
7.70 ± 4.24 xlO81
5.42 ± 1.72 x 10s
7.30 ± 4.03 xlO81
3.96 ± 3.29 x 10s
7. 70 ± 4.24 x 1081
5.42 ± 1.72 x 10s
7.30 ± 4.03 x 1081
3.96 ± 3.29 x 10s
7.70 ± 4.24 xlO81
5.42 ± 1.72 x 10s
0.00 ±0.00
0.00 ±0.00
2.73 ± 4.06 xlO5
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
1.85 ± 2.56 x 104
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
8. 86 ±0.00
8.60 ±0.00
4.31 ± 1.21
8.73 ± 0.00
8. 86 ±0.00
8.60 ±0.00
6.70 ±2.35
8.73 ± 0.00
8. 86 ±0.00
8.60 ±0.00
8. 89 ±0.00
8.73 ± 0.00
8. 86 ±0.00
8.60 ±0.00
8. 89 ±0.00
8.73 ± 0.00
8. 86 ±0.00
8.60 ±0.00
8. 89 ±0.00
8.73 ± 0.00
500 ppinv Fumigation Cycle
2a
2a
2a
2a
2a
30 min
60 min
120 min
240 min
Full cycle
240 mm
Laminate
Ductwork
Carpet - b
Concrete
Laminate
Ductwork
Carpet - b
Concrete
Laminate
Ductwork
Carpet - b
Concrete
Laminate
Ductwork
Carpet - b
Concrete
Laminate
Ductwork
Carpet
Concrete
2.80 x 10s §
2.80 x 10s §
2.80 x 108§
2.80 x 108§
2.80 x 108§
2.80 x 10s §
2.80 x 10s §
2.80 x 10s §
2.80 x 10s §
2.80 x 10s §
2.80 x 10s §
2.80 x 108§
2.80 x 108§
2.80 x 108§
2.80 x 10s §
2.80 x 10s §
2.80 x 10s §
2.80 x 10s §
2.80 x 10s §
2.80 x 10s §
2.61 ±1. 58 x 10s
1.67 ± 1.04 x 10s
2.13 ± 2.39 x 10s
2.26 ± 1.60 x 10s
2.61 ± 1.58 x 10s
1.67 ± 1.04 x 108
2.13 ± 2.39 x 10s
2.26 ± 1.60 xlO8
2.61±1.58xl08
1.67 ± 1.04 x 10s
2.13 ± 2.39 xlO8
2.26 ± 1.60 x 10s
2.61 ± 1.58 x 108
1.67 ± 1.04 x 108
2.13 ± 2.39 x 108
2.26 ± 1.60 x 10s
2.61±1.58xl08
1.67 ± 1.04 x 10s
2.13 ± 2.39 xlO8
2.26 ± 1.60 xlO8
0.00 ±0.00
0.00 ±0.00
1.74 ± 1.41 x IQ5
1.20 ± 2.68 x 102
0.00 ±0.00
0.00 ±0.00
6.67 ± 11. 6 x 103
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
8.42 ± 0.00
8.22 ±0.00
3.44 ±0.85
7.80± 1.24
8.42 ± 0.00
8.22 ±0.00
5.57 ± 1.69
8.35 ±0.00
8.42 ± 0.00
8.22 ±0.00
8.33 ± 0.00
8.35 ±0.00
8.42 ± 0.00
8.22 ±0.00
8.33 ± 0.00
8.35 ±0.00
8.42 ± 0.00
8.22 ±0.00
8.33 ± 0.00
8.35 ±0.00
-------
Trial
Contact Time
Material
Spike Amount (CFU/
coupon)
Mean Recovered B. anthracis (CFU/coupon)*
Positive Controlf
Test CouponJ
Mean Log
Reduction*
200-250 ppmv Fumigation Cycle
2b
2b
2b
2b
2b
30 min
60 min
120 min
240 min
Full cycle
240 min
Laminate
Ductwork
Carpet
Concrete
Laminate
Ductwork
Carpet
Concrete
Laminate
Ductwork
Carpet
Concrete
Laminate
Ductwork
Carpet
Concrete
Laminate
Ductwork
Carpet
Concrete
6.93 x 106*
6.93 xlO6*
6.93 xlO6*
6.93 xlO6*
6.93 xlO6*
6.93 xlO6*
6.93 x 106*
6.93 x 106*
6.93 x 106*
6.93 x 106*
6.93 xlO6*
6.93 xlO6*
6.93 xlO6*
6.93 xlO6*
6.93 xlO6*
6.93 x 106*
6.93 x 106*
6.93 x 106*
6.93 x 106*
6.93 xlO6*
3.22 ± 0.50 x 106
5.25 ± 0.79 xlO6
6.17 ± 0.57 xlO6
6.73 ± 0.70 x 106
3.22 ± 0.50 xlO6
5.25 ± 0.79 xlO6
6.17 ± 0.57 x 106
6.73 ± 0.70 x 106
3.22 ± 0.50 x 106
5.25 ± 0.79 x 106
6.17 ± 0.57 xlO6
6.73 ± 0.70 x 106
3.22 ± 0.50 xlO6
5.25 ± 0.79 xlO6
6.17 ± 0.57 xlO6
6.73 ± 0.70 x 106
3.22 ± 0.50 x 106
5.25 ± 0.79 x 106
6.17 ± 0.57 x 106
6.73 ± 0.70 x 106
1.34 ± 3.00 x 101
0.00 ±0.00
3.35 ± 0.74 xlO5
1.65 ± 3.59 xlO3
0.00 ±0.00
0.00 ±0.00
1.11 ±2.01 x 103
6.60 ± 14.8 x 10°
0.00 ±0.00
0.00 ±0.00
2.00 ±4. 47x10'
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
2.00 ±2.99x10'
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
1.34 ±3.00'
0.00 ±0.00
6.14±0.82
6.72 ±0.00
1.27 ±0.09
5.28 ±1.63
6.51 ±0.00
6.72 ±0.00
5.06 ± 1.67
6.52 ±0.68
6.51 ±0.00
6.72 ±0.00
6.39 ±0.89
6.83 ±0.00
6.51 ±0.00
6.72 ±0.00
6.12 ±0.92
6.83 ±0.00
6.51 ±0.00
6.72 ±0.00
6.42 ±0.82
6.83 ±0.00
500 ppmv Fumigation Cycle
3a
3a
3a
3a
3a
30 min
60 min
120 min
240 min
Full cycle
240 min
Wood
Glass
Ceiling tile
Wood
Glass
Ceiling tile
Wood
Glass
Ceiling tile
Wood
Glass
Ceiling tile
Wood
Glass
Ceiling tile
9.77 xlO6
9.77 xlO6
9.77 xlO6
9.77 xlO6
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 xlO6
9.77 xlO6
9.77 xlO6
9.77 xlO6
9.77 xlO6
9.77 x 106
9.77 x 106
5.47 ± 1.57xl05
8.18 ± 10.5 xlO6
7.49 ± 1.40xl05
5.47 ± 1.57xl05
8.18 ± 10.5 x 106
7.49 ± 1.40x 105
5.47 ± 1.57x 105
8.18 ± 10.5 x 106
7.49 ± 1.40xl05
5.47 ± 1.57xl05
8.18 ± 10.5 xlO6
7.49 ± 1.40xl05
5.47 ± 1.57xl05
8.18 ± 10.5 x 106
7.49 ± 1.40x 105
1.82±3.23xl04
2.73 ± 3.52 xlO2
0.00 ±0.00
4.51 ±4.41 x 103
0.00 ±0.00
0.00 ±0.00
7.19 ± 8.28 x 102
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
1.95 ±0.66
5.40 ±1.42
5. 87 ±0.00
2.97 ±1.69
6.91 ±0.00
5. 87 ±0.00
3.51± 1.31
6.91 ±0.00
5. 87 ±0.00
5.74 ±0.00
6.91 ±0.00
5. 87 ±0.00
5.74 ±0.00
6.91 ±0.00
5. 87 ±0.00
-------
Trial
Contact Time
Material
Spike Amount (CFU/
coupon)
Mean Recovered B. anthracis (CFU/coupon)*
Positive Controlf
Test CouponJ
Mean Log
Reduction*
200-250 ppmv Fumigation Cycle
3b
3b
3b
3b
3b
30 min
60 min
120 min
240 min
Full cycle
240 mm
Wood
Glass
Ceiling tile
Wood
Glass
Ceiling tile
Wood
Glass
Ceiling tile
Wood
Glass
Ceiling tile
Wood
Glass
Ceiling tile
1.02 x 107
1.02 xlO7
1.02 xlO7
1.02 xlO7
1.02 xlO7
1.02 xlO7
1.02 x 107
1.02 x 107
1.02 x 107
1.02 x 107
1.02 xlO7
1.02 xlO7
1.02 xlO7
1.02 xlO7
1.02 xlO7
3. 59 ± 0.63 x 105
3.53±1.68xl06
7.62 ± 2.18 xlO5
3.59±0.63xl05
3.53±1.68xl06
7.62 ± 2.18 xlO5
3. 59 ± 0.63 x 105
3.53± 1.68 x 106
7.62 ± 2.18 x 105
3. 59 ± 0.63 x 105
3.53±1.68xl06
7.62 ± 2.18 xlO5
3.59±0.63xl05
3.53±1.68xl06
7.62 ± 2.18 xlO5
2.06 ± 1.60 x 104
1.34*3.00x10'
3.32*4.08x10'
6.83 ± 10.9 xlO3
2.66 ±5.95x10'
2.00 ±2.99x10'
2.68 ± 0.60 x 103
0.00 ±0.00
0.00 ±0.00
1.61 ± 1.67 x 103
0.00 ±0.00
0.00 ±0.00
2.73±3.84xl04
0.00 ±0.00
0.00 ±0.00
1.35 ±0.35
6. 18 ±0.82
5.18 ±0.98
2. 15 ±0.70
6. 12 ±0.95
5.21 ±0.92
2.14±0.10
6.55 ±0.00
5.88 ±0.00
3.52 ± 1.87
6.55 ±0.00
5.88 ±0.00
3.32 ±2.45
6.55 ±0.00
5.88 ±0.00
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
Time zero was lower than target recovery of >10% of spike amount.
T|Time zero exceeds target recovery of <120% of spike amount.
§ Application was inadvertently about 1 log higher than the target 1 x 10' CPUs/coupon.
# Application was lower than the target 7.5 x 105 CPUs/coupon.
Figure 5-20. STERIS VHP® Fumigation Results for B. anthracis at the 500 ppmv HP Fumigation Cycle, Line Chart.
•500 ppmv: Aluminum
• 5 0 0 ppmv: Keybo ard
•500ppmvr Carpet- a
500 ppmvr Carpet - b
500 ppmv. Joint tap e
5 00 ppmv: Laminate
500 ppmvr Ducuvork
500 ppmv. Concrete
500ppmv:Wood
•500ppmv;Glass
500 ppmv7 Ceiling rile
10
9
S
7
4
500
1000
CT. ppmv-hr
1500
2000
-------
Figure 5-21. STERIS VHP® Fumigation Results for B. anthracis at the 200-250 ppmv HP Fumigation Cycle, Line
Chart.
8
200-2 50 ppmv. Laminate
2 0 0 - 2 5 0 ppmv. Ductwork
-200-250ppmv. Carpet
200-250 ppmv. Concrete
200-250ppmv, \Vood
•200-2 50 ppmv, Glass
200-250 ppmv, C eiling til e
200
400
600
SOO
1000
CT. ppmv-hr
B. suis
STERIS VHP® HP fumigation results for B. suis are
presented in Table 5-29 and Figures 5-22 and 5-23. After
exposure to the 500 ppmv fumigation cycle, B. suis was
For carpet and joint tape shorter exposures (30 min)
resulted in no recoveries of viable B. suis. After a 120-
min exposure to the 200-250 ppmv HP fumigation cycle.
viable B. suis was recovered at low levels (-10° to -101
not recovered from any material after a 90-min exposure. CPUs/coupon) from keyboard and joint tape; no viable B.
suis was recovered from aluminum and carpet.
Table 5-29. STERIS VHP® HP Fumigation Results for B. suis
Trial
Contact
Time
Material
Spike Amount (CFU/
coupon)
Mean Recovered B. suis (CFU/coupon)*
Positive
Controlf
Test
CouponJ
Mean Log
Reduction*
200-250 ppmv Fumigation Cycle
6a
6a
90 min
120 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
6.83 x 107
6.83 x 107
6.83 x 107
6.83 x 107
9.57 x 107
9.57 xlO7
9.57 xlO7
9.57 xlO7
5.11 ± 0.89 x 107
4.99 ± 0.18 x 107
9.41 ± 2.64 x 104
5.06 ± 3.48 x 105
3.00 ± 0.74 x 107
1.93 ± 1.51 xlO7
1.99±0.39xl06
2.25 ± 0.13 xlO6
7.98 ± 8.37 x 101
1.77 ± 1.84 x 103
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
6.66 ± 14.9x10'
0.00 ±0.00
6.60 ± 14.8x10°
6.22 ±0.91
5.03 ± 1.06
4.97 ±0.00
5.70 ±0.00
7.48 ± 0.00
6.78 ± 1.13
6.30 ±0.00
6.04 ±0.70
500 ppmv Fumigation Cycle
6b
6b
6b
30 min
60 min
90 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
5.33 x 107
5.33 x 107
5.33 x 107
5.33 x 107
7.03 x 107
7.03 x 107
7.03 x 107
7.03 x 107
8.80 xlO7
8.80 xlO7
4.07 ± 0.29 x 107
3.79 ± 0.50 x 107
3. 13 ± 0.49 x 106
1.98 ± 1.13x 104
3.76 ± 1.62 x 107
3.32 ±0.51 x 107
7.43 ± 3.65 x 105
4.02 ± 2.01 xlO4
5.06 ± 0.59 xlO7
5.01 ± 1.05 x 107
2.16±0.68xl03
1.75 ± 0.42 x 103
0.00 ±0.00
0.00 ±0.00
2.40 ± 2.73 x 102
8.68 ±9.31 x 101
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
4.29 ±0.13
4.35 ±0.12
6.50 ±0.00
4.30 ±0.00
5.76 ± 1.11
6.25 ± 1.17
5.87 ±0.00
4.60 ± 0.00
7.70 ± 0.00
7.70 ± 0.00
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
-------
Figure 5-22. STERIS VHP® Fumigation Results for B. suis at the 200-250 ppmv and 500 ppmv HP Fumigation cycles,
Line Chart.
'200-250 ppmv. Aluminum
500 ppmv. Aluminum
•200-250 ppmv. Keyboard
500 ppmv. Keyboard
•2 00-2 50 ppmv, Carpet
5 00 ppmv. Carpet
•2 00-2 50 ppmv. Joint tape
500 ppmv. Joint tape
200 400 600
CT, ppmv-hr
soo
Figure 5-23. STERIS VHP® Fumigation Results for B. suis at the 200-250 ppmv and 500 ppmv HP Fumigation
Cycles, Column Chart.
I Aluminum
I Keyboard
Carpet
I Joint tape
750
*Note: Carpet and joint tape were not tested at 750 CT, ppmv-hr.
-------
F. tularensis
STERIS VHP® HP fumigation results for F. tularensis
are presented in Table 5-30. Viable F. tularensis was
not recovered after any of the fumigation trials; the
associated mean log reductions in F. tularensis ranged
from 5.59 to 6.66. Compared to the amount of bacteria
spiked onto the coupon (about 10s CPUs/coupon), after
a 90-min treatment of F. tularensis on aluminum and
keyboard, a >8 log reduction in viable bacteria was
attributable to the fumigation treatment and the loss of
viability arising from other (unknown) time-dependent
causes.
Table 5-30. STERIS VHP® HP Fumigation Results for F. tularensis
Trial
Contact
Time
Material
Spike Amount (CFU/
coupon)
Mean Recovered F. tularensis (CFU/coupon)*
Positive
Control1
Test
Coupon^
Mean Log
Reduction*
200-250 ppinv Fumigation Cycle
5a
90min
Aluminum
Keyboard
1.91 x 108§
1.91 x 108§
2.18 ± 0.91 xlO6
5.55 ± 1.57 xlO5
0.00 ±0.00
0.00 ±0.00
6.34 ±0.00
5.74 ±0.00
500 ppmv Fumigation Cycle
5b
30 min
Aluminum
Keyboard
4.70 x 108§
4.70 x 108§
4.54 ± 1.83 x 106
3.87± l.lOx 105
0.00 ±0.00
0.00 ±0.00
6.66 ±0.00
5. 59 ±0.00
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
§ Application was lower than the target 1.0 x 106 - 1.0 x 108 CFU/coupon.
Vaccinia virus
STERIS VHP® HP fumigation results for vaccinia
virus are presented in Table 5-31 and Figures 5-24 and
5-25. No vaccinia virus was recovered from carpet
or joint tape following exposure to the 200-250 ppmv
HP fumigation cycle for 30 min. Vaccinia virus was
recovered from keyboard after exposure to both the
200-250 ppmv HP and 500 ppmv HP fumigation cycles
for 30 min, but vaccinia virus was not recovered from
either fumigation cycle when the exposures lasted 60
min. Vaccinia virus was recovered from aluminum after
every fumigation trial at levels <5 x 101 PFUs/coupon;
the associated log reductions ranged from 4.60 to 5.26.
At the 200-250 ppmv fumigation cycle, growth was
observed on three of five biological indicators following
exposure to the fumigant after the 30-min contact time.
At the 500 ppmv fumigation cycle, no growth was
observed on any biological indicator replicate following
exposure to the fumigant.
Compared to the amount of vaccinia virus spiked onto
the coupon (about 107 PFUs/coupon), no viable vaccinia
virus was recovered from carpet or joint tape after a
treatment of 200-250 ppmv HP for 30-min contact time,
equating to about a 7 log reduction in viable bacteria
attributable to the fumigation and the loss of viability
arising from other (unknown) time-dependent causes.
Compared to the amount of vaccinia virus spiked onto
the coupon (about 107 PFUs/coupon), no viable vaccinia
virus was recovered from keyboard after a treatment of
200-250 ppmv HP for 60-min contact time, equating
to about a 7 log reduction in viable vaccinia virus
attributable to the combined effects of the fumigation
and the loss of viability arising from other (unknown)
time-dependent causes. Compared to the amount
of vaccinia virus spiked onto the coupon (about 107
PFUs/coupon), no viable vaccinia was recovered from
keyboard after a 200-250 ppmv HP fumigation for 60-
min contact time or after a 500 ppmv HP fumigation
with a 60-min contact time, equating to about a 7 log
reduction in viable bacteria attributable to the combined
effects of the fumigation and the loss of viability arising
from other (unknown) time-dependent causes.
-------
Table 5-31. STEMS VHP® HP Fumigation Results for Vaccinia Vims
Trial
Contact
Time
Material
Spike Amount (PFU/
coupon)
Mean Recovered Vaccinia Virus (PFU/coupon)*
Positive
Controlf
Test
CouponJ
Mean Log
Reduction*
200-250 ppmv Fumigation Cycle
7a
7a
7a
30 min
60 min
120 min
Aluminum
Keyboard
Carpet
Joint tape
Aluminum
Keyboard
Aluminum
9.64 xlO6
9.64 xlO6
9.64 xlO6
9.64 xlO6
5.99 x 106
5.99 x 106
6.51 x 106
2.50 ± 1.59xl06
2.43 ± 0.60 x 105
1.34 ± 1.79 xlO4
1.75±0.65xl04
1.41 ± 0.22 x 106
8.50 ± 1.37 x 104
1.64 ± 0.42 x 106
1.42*0.32x10'
1.49 ± 0.48 x 101
0.00 ±0.00
0.00 ±0.00
1.25 ±0.21 x 101
0.00 ±0.00
3.37 ± 0.67 x 101
5.26 ±0.11
4.23 ±0.16
4.13 ±0.00
4.24 ±0.00
5.06 ±0.08
4.93 ± 0.00
4.70 ± 0.09
500 ppmv Fumigation Cycle
7b
7b
30 min
60 min
Aluminum
Keyboard
Aluminum
Keyboard
4.95 x 106
4.95 x 106
1.12xl07
1.12xl07
1.28 ± 0.68 x 106
2.58 ± 0.66 x 105
2.27 ± 0.17 xlO6
4.78± 1.37 xlO5
3.43 ± 1.28 x 101
1.08 ± 0.28 x 101
2.23 ±0.23x10'
0.00 ±0.00
4.60 ±0.19
4.39 ±0.12
5.01 ±0.05
5.68 ±0.00
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
Figure 5-24. STERIS VHP® Fumigation Results for Vaccinia Virus at the 200-250 ppmv and 500 ppmv HP Fumigation
Cycles, Line Chart.
~200-250ppmv. Aluminum
500 ppmvr Aluminum
-2 00-250 ppmv. Keyboard
500 ppmvr Keyboard
500
600
-------
Figure 5-25. STERIS VHP® Fumigation Results for Vaccinia Virus at the 200-250 ppmv and 500 ppmv HP Fumigation
Cycles, Column Chart.
7
• Aluminum
• Keyboard
Carpet
• Joint tape
u 4
•3
"si 3
-
2
13
225 250
CT. ppmv-hr
500
Y. pestis
STERIS VHP® HP fumigation results for Y. pestis
are presented in Table 5-32. Viable Y. pestis was
not recovered after any of the fumigation trials; the
associated mean log reductions in Y. pestis ranged
from 1.90 to 4.73. Compared to the minimum amount
of bacteria spiked onto the coupon (3.83 x 106 CPUs/
coupon), after a 30-min treatment of Y. pestis on
aluminum and keyboard, a >6.5 log reduction in viable
bacteria was attributable to the fumigation treatment and
the loss of viability arising from other (unknown) time-
dependent causes.
Table 5-32. STERIS VHP® HP Fumigation Results for Y. pestis
Trial
Contact
Time
Material
Spike Amount (CFU/
coupon)
Mean Recovered Y. pestis (CFU/coupon)*
Positive
Controlf
Test
CouponJ
200-250 ppmv Fumigation Cycle
4a
4a
90 min
120 min
Aluminum
Keyboard
Aluminum
Keyboard
3.83 x 106
3.83 x 106
3.57x 106
3.57x 106
3.29 ± 0.52 x 104
4.13± 1.57 x 102
5.31 ± 3.46 x 104
8.00 ± 6.92 x 101
Mean Log
Reduction*
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
4.52 ±0.00
2.62 ±0.00
4.73 ± 0.00
1.90 ±0.00
500 ppmv Fumigation Cycle
4b
30 min
Aluminum
Keyboard
7.33 x 106
7.33 x 106
2.87± 1.70x 104
4.91 ± 0.73 x 104
0.00 ±0.00
0.00 ±0.00
4.46 ± 0.00
4.69 ± 0.00
* Data are expressed as mean ± standard deviation of five replicates.
| Positive control coupons were spiked but not exposed to the fumigant.
J Test coupons were spiked and exposed to the fumigant for the contact time.
-------
Summary Statistics for STERIS VHP® HP
Decontamination
Table 5-33 provides a summary of STERIS VHP® HP
decontamination efficacy, calculated as the difference in
the mean log of viable bacteria recovered from positive
control coupons and the mean log of viable bacteria
recovered from coupons after fumigation for a given
contact time. The 95% CI and p-value are also shown.
Significant log reduction was observed against all
combinations of biological agents and materials at all
contact times tested.
Surface Damage
The physical effect of the STERIS VHP® HP fumigation
on the materials was evaluated qualitatively. The
appearance of the decontaminated coupons was
visually inspected for any obvious changes in the color,
reflectivity, and apparent roughness of the material
surfaces. These comparisons were performed for each
material, before extraction of the decontaminated test
coupons. No physical differences were observed between
control and fumigated coupons for any material.
Table 5-33. Summary of STERIS VHP® HP Fumigation Efficacy (Calculated as Mean Log Reduction)
Mean Log Reduction (95% CI) and P-value*
Or Mean Log Reduction (# of treated coupons with zero recovery /# of treated coupons) and
Trials Agent Material P-valuef
Trial 1
(500
ppmv)
Trial 2a
(500
ppmv)
Trial 2b
(200-250
ppmv)
Trial 3a
(500
ppmv)
Trial 3b
(200-250
ppmv)
Trial 4a
(200-250
ppmv)
B. anthracis
spores
B. anthracis
spores
B. anthracis
spores
B. anthracis
spores
B. anthracis
spores
Y. pestis
Aluminum
Carpet
Keyboard
Joint Tape
Paper
Carpet
Concrete
Galvanized
Metal
Laminate
Carpet
Concrete
Galvanized
Metal
Laminate
Ceiling Tile
Glass
Wood
Ceiling Tile
Glass
Wood
Aluminum
Keyboard
30min
>8.70 (5/5)
p=0.0079
4.17 (2.82, 5.51)
p=0.0007
>8.35 (5/5)
p=0.0079
>8.72 (5/5)
p=0.0079
3.23 (2.23, 4.24)
p=0.0003
>7.72 (4/5)
p=0.0079
>8.17 (5/5)
p=0.0079
>8.35 (5/5)
p=0.0079
1.27 (1.17, 1.37)
p<0.0001
>5.28 (2/5)
p=0.0079
>6.72 (5/5)
p=0.0079
>6.14 (4/5)
p=0.0079
>5.87 (5/5)
p=0.0079
>5.16 (2/5)
p=0.0079
1.93 (1.24, 2.62)
p=0.0023
>4.86 (2/5)
p=0.0079
>6.11 (4/5)
p=0.0079
1.35 (0.98, 1.72)
p=0.0007
611 niin
>8.70 (5/5)
p=0.0079
>6.57 (2/5)
p=0.0079
>8.35 (5/5)
p=0.0079
>8.72 (5/5)
p=0.0079
>5.36 (1/5)
p=0.0079
>8.28 (5/5)
p=0.0079
>8.17 (5/5)
p=0.0079
>8.35 (5/5)
p=0.0079
>5.06 (2/5)
p=0.0079
>6.52 (4/5)
p=0.0079
>6.72 (5/5)
p=0.0079
>6.50 (5/5)
p=0.0079
>5.87 (5/5)
p=0.0079
>6.67 (5/5)
p=0.0079
>2.95 (1/5)
p=0.0079
>5.20 (3/5)
p=0.0079
>6.05 (4/5)
p=0.0079
2.14 (1.42,
2.87)
p=0.0022
90min
>4.51 (5/5)
p=0.0079
>2.59 (5/5)
p=0.0079
120 min
>8.70 (5/5)
p=0.0079
>8.75 (5/5)
p=0.0079
>8.35 (5/5)
p=0.0079
>8.72 (5/5)
p=0.0079
>8.12 (5/5)
p=0.0079
>8.28 (5/5)
p=0.0079
>8.17 (5/5)
p=0.0079
>8.35 (5/5)
p=0.0079
>6.39 (4/5)
p=0.0079
>6.83 (5/5)
p=0.0079
>6.72 (5/5)
p=0.0079
>6.50 (5/5)
p=0.0079
>5.87 (5/5)
p=0.0079
>6.67 (5/5)
p=0.0079
>3.49 (1/5)
p=0.0079
>5.87 (5/5)
p=0.0079
>6.48 (5/5)
p=0.0079
2.13 (2.00,
2.26)
p<0.0001
>4.62 (5/5)
p=0.0079
>1.80 (5/5)
p=0.0079
240 min
>8.70 (5/5)
p=0.0079
>8.75 (5/5)
p=0.0079
>8.35 (5/5)
p=0.0079
>8.72 (5/5)
p=0.0079
>8.12 (5/5)
p=0.0079
>8.28 (5/5)
p=0.0079
>8.17 (5/5)
p=0.0079
>8.35 (5/5)
p=0.0079
>6.12 (3/5)
p=0.0079
>6.83 (5/5)
p=0.0079
>6.72 (5/5)
p=0.0079
>6.50 (5/5)
p=0.0079
>5.87 (5/5)
p=0.0079
>6.67 (5/5)
p=0.0079
>5.72 (5/5)
p=0.0079
>5.87 (5/5)
p=0.0079
>6.48 (5/5)
p=0.0079
>3.51 (2/5)
p=0.0079
Gassing + 4hr
>8.70 (5/5)
p=0.0079
>8.75 (5/5)
p=0.0079
>8.35 (5/5)
p=0.0079
>8.72 (5/5)
p=0.0079
>8.12 (5/5)
p=0.0079
>8.28 (5/5)
p=0.0079
>8.17 (5/5)
p=0.0079
>8.35 (5/5)
p=0.0079
>6.42 (4/5)
p=0.0079
>6.83 (5/5)
p=0.0079
>6.72 (5/5)
p=0.0079
>6.50 (5/5)
p=0.0079
>5.87 (5/5)
p=0.0079
>6.67 (5/5)
p=0.0079
>5.72 (5/5)
p=0.0079
>5.87 (5/5)
p=0.0079
>6.48 (5/5)
p=0.0079
>3.31 (2/5)
p=0.0079
-------
Trials
Trial 4b
(500
ppmv)
Trial 5 a
(200-250
ppmv)
Trial 5b
(500
ppmv)
Trial 6a
(200-250
ppmv)
Trial 6b
(500
ppmv)
Trial 7a
(200-250
ppmv)
Trial 7b
(500
ppmv)
Agent
Y. pestis
F. tularensis
F. tularensis
B. suis
B. suis
Vaccinia
Virus
Vaccinia
Virus
Material
Aluminum
Keyboard
Aluminum
Keyboard
Aluminum
Keyboard
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Carpet
Keyboard
Painted Joint
Tape
Aluminum
Keyboard
Mean Log Reduction (95% CI) and P-value*
Or Mean Log Reduction (# of treated coupons with zero recovery /# of treated coupons) and
P-valuef
30min
>4.22 (5/5)
p=0.0079
>4.69 (5/5)
p=0.0079
>6.63 (5/5)
p=0.0079
>5.58 (5/5)
p=0.0079
4.29 (4.16, 4.42)
p<0.0001
>6.49 (5/5)
p=0.0079
4.34 (4.21, 4.48)
p<0.0001
>4.22 (5/5)
p=0.0079
5.20 (4.92, 5.47)
p<0.0001
>3.09 (5/5)
p=0.0079
4.22 (4.02, 4.43)
p<0.0001
>4.22 (5/5)
p=0.0079
4.54 (4.21, 4.88)
p<0.0001
4.38 (4.21, 4.55)
p<0.0001
611 niin
>5.69 (1/5)
p=0.0079
>5.79 (5/5)
p=0.0079
>6.25 (2/5)
p=0.0079
>4.56 (5/5)
p=0.0079
5.05 (4.95,
5.16)
p<0.0001
>4.92 (5/5)
p=0.0079
5.01 (4.95,
5.07)
p<0.0001
>5.66 (5/5)
p=0.0079
90min
>6.30 (5/5)
p=0.0079
>5.73 (5/5)
p=0.0079
>6.21 (1/5)
p=0.0079
>4.96 (5/5)
p=0.0079
5.03 (3.94,
6.13)
p=0.0004
>5.47 (5/5)
p=0.0079
>7.70 (5/5)
p=0.0079
>7.69 (5/5)
p=0.0079
120 min
>7.46 (5/5)
p=0.0079
>6.29 (5/5)
p=0.0079
>6.51 (4/5)
p=0.0079
>6.05 (4/5)
p=0.0079
4.68 (4.52,
4.84)
p<0.0001
240 min
Gassing + 4hr
* Mean log reduction is the mean of the base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm
of recovered agent from the treated coupons. A 95 % CI for the difference is shown in parentheses. A p-value is provided for the probability that
the control and treatment recoveries are the same. The p-value is from the two sample t-test with Satterthwaite's method to allow for potentially
different variances in the two groups. p-Values less than 0.05 denote less than 1 in 20 chance that a difference as large as or larger than observed
would occur by chance if the control and treatment means were truly identical. Comparisons with p-values less than 0.05 (statistically significant
at the 0.05 level) are bolded.
| One or more of the treatment coupons had no recovered agent. The mean log reduction of the form ">X" is calculated as the mean of the
base-10 logarithm of recovered agent from the control coupons minus the mean of the base-10 logarithm of recovered agent from the treated
coupons except that "zero recovery" coupons have a substituted recovered value of "1" (base-10 log is 0). Since the log becomes an increasingly
negative value below 1 and is undefined at 0, this substitution is necessary and results in a lower bound on the mean log difference, as indicated
by the ">". The number of "zero recovery" treatment coupons and the total number of treatment coupons is shown in parentheses. The p-value
is from the non-parametric Kolmogorov-Smirnov test. p-Values less than 0.05 denote less than 1 in 20 chance that results as different as or more
different than observed would occur by chance if the distribution of the control and treatment recoveries were truly identical. Comparisons with
p-values less than 0.05 (statistically significant at the 0.05 level) are bolded.
-------
6.0
Performance Summary
The persistence of the biological agents (B. suis, F.
tularensis, vaccinia vims, and Y. pestis) on various
building materials varied by organism and material
type. All biological agents persisted at least 7 days
(168 hr) on at least one building material. However, the
null hypothesis that, given an equivalent application of
biological agent the amount of biological agent on the
coupons was constant over time, was rejected. For many
combinations of biological agent and material, significant
loss of viable biological agent occurred within 2 hr.
Significant loss of viable biological agent was observed
for all combinations of biological agent and material
within three days.
At the tested conditions, none of the decontamination
technologies evaluated eliminated the recovery of every
tested biological agent from every tested material. The
results of the fumigation testing are summarized in Tables
6-1, 6-2, 6-3 and 6-4. However, the null hypothesis
that there was no difference in the decontamination
efficacy using the treatment compared to the positive
controls was rejected (except in the cases of high loss of
viable biological agent from positive control coupons).
Significant loss of viable biological agent compared to the
positive controls was observed for all biological agents
using all four decontamination technologies. In some
cases the biological agent on certain materials exhibited
a high loss of recoverable biological agent from the
positive control coupons (e.g., F. tularensis on carpet at
40% RH). In these cases, even though little or no viable
biological agent was recovered from the test coupons, the
loss of viable biological agent from the positive control
coupons resulted in a low base for statistical comparison
and the null hypothesis could not be rejected - there
were no difference in the high level of decontamination
efficacy using the treatment compared to the high level
of loss of biological agent from the positive controls. The
combined effect of loss of viability over time (without
decontamination treatment) and decontamination efficacy
determines the overall effectiveness of a treatment.
Sabre CIO, fumigation was generally more efficacious at
75% RH than at lower RH (e.g., 40%).
Sabre C1O2 fumigation at 3,000 ppmv and 75% RH
resulted in no viable B. anthracis spores being recovered
from keyboard (40-min contact time), carpet (90-min
contact time), or joint tape (90-min contact time). At
40% RH, B. anthracis spores remained viable on carpet
and joint tape after 90 min of exposure to 3,000 ppmv
C1O2. Viable B. anthracis spores were recovered from
aluminum following all tests with 3,000 ppmv C1O2,
although the mean amount of recovered spores was
generally multiple logs lower at 75% RH than 40% RH.
Sabre C1O2 fumigation at 50-100 ppmv and 75% RH
resulted in no viable B. suis being recovered from
aluminum, carpet or joint tape after a 60-min contact
time. However, at 50-100 ppmv C1O2 with a 60 min
contact lime and 40% RH, viable B. suis was recovered
from aluminum, carpet, and joint tape at greater than
103 CPUs/coupon. B. suis was recovered from keyboard
following all tests with 50-100 ppmv C1O2, although the
mean amount of recovered agent was generally lower at
75% RH than 40% RH.
Sabre C1O2 fumigation at 50-100 ppmv and 75% RH
resulted in no viable F. tularensis being recovered from
aluminum or keyboard after a 120-min contact time. At
50-100 ppmv ck)2 and 40% RH, viable F. tularensis
was recovered from aluminum and keyboard at levels
greater than 105 CFUs/coupon. No viable F. tularensis
was recovered from any of the tests with carpet or joint
tape; natural degradation of F. tularensis may have been
an important contributing factor (especially at 40% RH)
as the associated positive controls demonstrated relatively
low recoveries.
Vaccinia vims generally remained viable on keyboard for
all tests conducted with Sabre C1O2. No viable vaccinia
virus was recovered from aluminum, carpet, or joint tape
with 50 - 100 ppmv C1O2 at 75% RH following a 30-min
contact time. Comparable testing at 40% RH resulted in
viable vaccinia virus being detected at greater than 10'1
PFUs/coupon.
Tests with Sabre C1O2 fumigation at 50-100 ppmv and
40% RH or 75% RH resulted in no viable Y. pestis
recovered from any of the tests with aluminum, keyboard,
carpet, or joint tape; natural degradation of Y. pestis
may have been an important contributing factor as the
associated positive controls generally demonstrated low-
recoveries.
BIOQUELL Claras C HP with a fumigation cycle of 10
min at 8 g/min, dwell at 0.8 g/min with a 180-min contact
time resulted in no B. anthracis spores being recovered
from laminate, ductwork, glass, and ceiling tile; viable
spores were recovered from carpet, concrete, and wood
under these conditions.
BIOQUELL Clarus C HP with a fumigation cycle of 10
min at 8 g/min, dwell at 0.8 g/min with a 180-min contact
time resulted in no viable B. suis, vaccinia virus, or Y.
pestis being recovered from any of the materials tested
(aluminum, keyboard, carpet, glass [vaccinia only], and
joint tape).
-------
BIOQUELL Clams S HP with a fumigation cycle of 50
mL HP injected over 20 min (initial RH: 40%-50%) with
a 75-inin contact time resulted in no B. anthracis spores
being recovered from aluminum, keyboard, and joint
tape; viable spores were recovered from carpet under
these conditions.
BIOQUELL Clams S HP with a fumigation cycle of 15
mL injected over 15 min (65% initial RH) with a 15 or
30-min contact time resulted in no viable F. tularensls
being recovered from the materials tested (aluminum,
keyboard, carpet and joint tape).
BIOQUELL Claras S HP with a fumigation cycle of 15
mL injected over 15 min (65% initial RH) with a 60-min
contact time resulted in viable B. suis and Y. pestis being
recovered from the aluminum, keyboard, and carpet, but
not from joint tape.
STERIS VHP® HP fumigation with a nominal
concentration of 500 ppmv and a 120-min contact time
resulted in no B. anthracis spores being recovered
from any material tested (except wood). Materials
tested included aluminum, keyboard, joint tape, carpet,
laminate, ductwork, ceiling tile, glass, concrete. No viable
spores were recovered from wood after a 240-min contact
time under these conditions (-500 ppmv).
STERIS VHP® HP fumigation with the 500 ppmv
fumigation cycle resulted in no viable B. suis from
carpet or joint tape (after a 30-min contact time) or from
aluminum or keyboard (after a 90-min contact time).
After a 120-min contact time in the 200-250 ppmv HP
fumigation cycle, viable B. suis was recovered from
keyboard and joint tape, but not aluminum and carpet.
STERIS VHP® HP fumigation with the 200-250 ppmv
fumigation cycle for 90 min and fumigation with the 500
ppmv fumigation cycle for 30 min resulted in no viable
F. tularensis or Y. pestis recovered from aluminum or
keyboard (carpet and joint tape were not tested).
STERIS VHP® HP fumigation with the 200-250 ppmv
fumigation cycle for 30 min resulted in no viable
vaccinia virus being recovered from carpet or joint
tape (but vaccinia vims was recovered from aluminum
and keyboard). When the contact time was increased
to 60 min, vaccinia vims was no longer recovered
from keyboard. However, vaccinia virus continued to
be recovered from aluminum even when the contact
time was increased to 120 min (at the 200-250 ppmv
fumigation cycle). Following a 60-min exposure to the
500 ppmv fumigation cycle, viable vaccinia virus was
recovered from aluminum but not keyboard.
Biological indicators were used in parallel with
the biological agent decontamination testing. The
biological agents that were used were B. atrophaeus
spores (nominally 10s spores) on steel in Tyvck®
packaging, for the Sabre CIO, fumigation testing and
G. stearothermophilus (nominally 1 x 106 spores) on
stainless steel in Tyvek® packaging for the three HP
technologies. The results from qualitative evaluation of
the biological indicators did not correlate consistently
with the results from quantitative evaluation of viable
biological agent remaining on coupons of various
materials. For example, the B. atrophaeus biological
indicators used for the Sabre CIO, fumigation were all
positive for growth at the 180-min contact time (the
longest time tested), indicative of incomplete kills. The
biological indicators (on steel) are consistent with B.
anthracis on aluminum which retained viable spores
under all treatment conditions. However, no B. anthracis
spores were recovered from keyboard or carpet under (lie
same conditions. In contrast, the G. stearothermophilus
biological indicators were negative for growth,
indicating complete kills, after fumigation treatments
in which viable B. anthracis, B. suis, and vaccinia virus
were recovered from some materials. For these hardy
biological agents, observations of no growth of biological
indicators cannot be assumed to correlate to no viable
biological agent remaining on any material.
-------
Table 6-1. Summary of Sabre CIO Fumigation
Decontamination Method
Sabre C1O2 3,000 ppmv,
40%RH
Sabre C1O2 3,000 ppmv,
75%RH
SSabre C1O2 50-100 ppmv,
40%RH
Sabre C1O2 50-100 ppmv,
60%RH
Sabre C1O2 50-100 ppmv,
75%RH
Sabre C1O2 50-100 ppmv,
40%RH
Sabre C1O2 50-100 ppmv,
75%RH
Sabre C1O2 50-100 ppmv,
40%RH
Sabre C1O2 50-100 ppmv,
60%RH
Sabre C1O2 50-100 ppmv,
75%RH
Sabre C1O2 50-100 ppmv,
40%RH
Sabre C1O2 50-100 ppmv,
75%RH
Biological Agent
B. anthracis
spores
B. anthracis
spores
B. suis
B. suis
B. suis
F. tularensis
F . tularensis
Vaccinia virus
Vaccinia virus
Vaccinia virus
Y. pestis
Y. pestis
Aluminum
Viable spores @ 1 80 min
Viable spores @ 1 80 min
Viable bacteria @ 120
min
Viable bacteria @ 120
min
0 CPUs @ 60 min
Viable bacteria @ 120
min
0 CPUs @ 120 min
Viable virus @ 120 min
Mixed results @ 120 min
0 PFUs @ 30 min
0 CPUs @ 30 min
0 CPUs @ 30 min
Keyboard
0 CPUs @ 20 min
0 CPUs @ 40 min
Viable bacteria @
120 min
Viable bacteria @
120 min
Viable bacteria @
120 min
Viable bacteria @
120 min
0 CPUs @ 120 min
Viable virus @ 120
min
Mixed results @
120 min
Viable virus @ 120
min
0 CPUs @ 30 min
0 CPUs @ 30 min
Carpet
OCFUs@ 180
min
0 CPUs @ 90
min
0 CPUs @ 120
min
0 CPUs @ 120
min
0 CPUs @ 30
min
0 CPUs @ 30
min
0 CPUs @ 30
min
Viable virus @
120 min
0 PFUs @ 120
min
0 PFUs @ 30
min
0 CPUs @ 30
min
0 CPUs @ 30
min
Joint Tape
Viable spores @
180 min
0 CPUs @ 90
min
0 CPUs @ 120
min
0 CPUs @ 30
min
0 CPUs @ 30
min
0 CPUs @ 30
min
0 CPUs @ 30
min
Viable virus @
120 min
0 PFUs @ 120
min
0 PFUs @ 30 min
0 CPUs @ 30
min
0 CPUs @ 30
min
Table 6-2. Summary of BIOQUELL Claras C HP Fumigation
Decontamination Method
BIOQUELL Claras C
HP injection for 10 min at 8 g/
min; dwell at 0.8 g/min
BIOQUELL Claras C
HP injection for 5 min at 8 g/
min; dwell at 0.8 g/min
BIOQUELL Claras C
HP injection for 10 min at 8 g/
min; dwell at 0.8 g/min*
BIOQUELL Claras C
HP injection for 10 min at 8 g/
min; dwell at 0.8 g/min
BIOQUELL Claras C
HP injection for 10 min at 8 g/
min; dwell at 0.8 g/minj1
BIOQUELL Claras C
HP injection for 10 min at 8 g/
min; dwell at 0.8 g/min
Biological Agent
B. anthracis
spores
B. anthracis
spores
B. anthracis
spores
B. suis
Vaccinia virus
Y. pestis
Aluminum
"
"
"
0 CPUs @ 180 min
OPFUs@ 180 min
0 CPUs @ 180 min
Keyboard
"
"
"
OCFUs@ 180 min
OPFUs@ 180 min
OCFUs@ 180 min
Carpet
0 CPUs @ 180
min
Viable bacteria
@ 180 min
Viable bacteria
@ 180 min
0 CPUs @ 180
min
0 PFUs @ 180
min
0 CPUs @ 180
min
Joint Tape
"
"
"
OCFUs@ 180
min
0 PFUs @ 180
min
OCFUs@ 180
min
* No spores were recovered from laminate, ductwork, glass, and ceiling tile under these conditions
(0 CPUs @ 180 min); viable spores were recovered from carpet, concrete, and wood under these conditions (180 min).
| No viable bacteria were recovered from glass under these conditions (0 CPUs @ 180 min).
- Not tested.
-------
Table 6-3. Summary of BIOQUELL Claras S HP Fumigation
Decontamination Method
BIOQUELL Claras S
3 x —15 mL injection,
45%RH
BIOQUELL Claras S
15 mL injection, 45%RH
BIOQUELL Claras S
50 mL injection, 45%RH
BIOQUELL Claras S
15 mL injection, 45%RH
BIOQUELL Claras S
15 mL injection, 65%RH
BIOQUELL Claras S
15 mL injection, 45%RH
BIOQUELL Claras S
15 mL injection, 65%RH
BIOQUELL Claras S
15 mL injection, 45%RH
BIOQUELL Claras S
15 mL injection, 65%RH
Biological Agent
B. anthracis
spores
B. anthracis
spores
B. anthracis
spores
B. suis
B. suis
F. tularensis
F. tularensis
Y. pestis
Y. pestis
Aluminum
"
--
0 CFUs @75 min
Viable bacteria @ 30
min
Viable bacteria @ 60
min
OCFU@ 15 min
0 CFUs @ 30 min
0 CFUs @ 30 min
Viable bacteria @ 60
min
Keyboard
"
0 CFUs @ 30
min
0 CFUs @75
min
0 CFUs @ 30
min
Viable bacteria
@ 60 min
OCFUs® 15
min
0 CFUs @ 30
min
Viable bacteria
@ 30 min
Viable bacteria
@ 60 min
Carpet
Viable bacteria @
192 min
Viable bacteria @
60 min
Viable bacteria @
75 min
Viable bacteria @
30 min
Viable bacteria @
60 min
OCFUs@ 15 min
OCFUs@ 15 min
Viable bacteria @
30 min
Viable bacteria @
60 min
Joint Tape
"
--
0 CFUs @75 min
Viable bacteria @
30 min
0 CFUs @ 60 min
OCFUs® 15 min
OCFUs@ 15 min
Viable bacteria @
30 min
0 CFUs @ 60 min
-- Not tested.
Table 6-4. Summary of STERIS VHP® HP Fumigation
Decontamination Method
STERIS VHP®
200 - 250 ppmv*
STERIS VHP*
500 ppmv|
STERIS VHP*
200 - 250 ppmv
STERIS VHP*
500 ppmv
STERIS VHP*
200 - 250 ppmv
STERIS VHP*
500 ppmv
STERIS VHP*
200 - 250 ppmv
STERIS VHP*
500 ppmv
STERIS VHP*
200 - 250 ppmv
STERIS VHP*
500 ppmv
Biological Agent
B. anthracis
spores
B. anthracis
spores
B. suis
B. suis
F. tularensis
F. tularensis
Vaccinia virus
Vaccinia virus
Y. pestis
Y. pestis
Aluminum
--
0 CFUs @ 30
min
0 CFUs @ 120
min
0 CFUs @ 90
min
0 CFUs @ 90
min
0 CFUs @ 30
min
Viable virus @
120 min
Viable virus @
60 min
0 CFUs @ 90
min
0 CFUs @ 30
min
Keyboard
--
0 CFUs @ 30 min
Viable bacteria @
120 min
0 CFUs @ 90 min
0 CFUs @ 90 min
0 CFUs @ 30 min
0 PFUs @ 60 min
0 PFUs @ 60 min
0 CFUs @ 90 min
0 CFUs @ 30 min
Carpet
Viable bacteria @
full cycle 240 min
0 CFUs @ 120 min
0 CFUs @ 90 min
0 CFUs @ 30 min
--
--
0 PFUs @ 30 min
--
--
Joint Tape
--
0 CFUs @ 30 min
0 CFUs @ 90 min; 2nd
ran, viable bacteria @
120 min
0 CFUs @ 30 min
--
--
0 PFUs @ 30 min
--
--
* No spores were recovered from ductwork after a 30-min contact time, from laminate after a 60-min contact time; concrete, glass or ceiling tile
after a 120-min contact time under these conditions (200 - 250 ppmv); viable spores were recovered from wood after 240-min contact time under
these conditions.
| No spores were recovered from laminate, ductwork, or ceiling tile after a 30-min contact time, from glass or concrete after a 60-min contact
time; or from wood after a 240-min contact time at this condition (500 ppmv).
-- Not tested.
-------
7.0
References
1. Battelle, Technology Testing and Evaluation
Program Test/QA Plan for Systematic Investigation
ofFmnigant Technologies for Decontamination
of Biological Agents from Contaminated Building
Materials. Version 1, Columbus, Ohio, May 2007.
2. Batteiie, BBRC SOP Number: MREF. X-074,
Standard Operating Procedure (SOP) for the
Production of Bacillus anthracis Spores.
3.
4.
6.
7.
8.
9.
Rogers, J.Y, C.L, Sabourin, Y.W. Choi, W.R.
Richter, D.C. Rudnicki, K.B. Riggs, M.L. Taylor,
and J. Chang, Decontamination Assessment of
Bacillus anthracis, Bacillus subtilis, and Geobacillus
stearothermophilus Spores on Indoor Surfaces using
a Hydrogen Peroxide Gas Generator. Journal of
Applied Microbiology, 2005(99): p. 739-748.
Battelle, BBRC Standard Operating Procedure for
the Operation and Maintenance of Primus General
Purpose Steam Sterilizer Model: PSS5-A-MSSD.
2006.
Institute for International Cooperation in Animal
Biologies and The Center for Food Security and
Public Health, Brucellosis. Iowa State University,
College of Veterinary Medicine, Ames, Iowa, 2005.
Centers for Disease Control and Prevention,
American Society for Microbiology, and Association
of Public Health Laboratories, Basic Protocols
for Level A Laboratories for the Presumptive
Identification of'Francisella tularensis. 2001.
American Society for Microbiology, Sentinel
Laboratory Guidelines for Suspected Agents of
Bioterrorism: Yersinia pestis. 2005.
Battelle, Quality Management Plan (QMP) for the
Technology Testing and Evaluation Program (TTEP).
Version 3. Columbus. Ohio. January 2008.
Leslie, S. B., E. Israeli, B.Lighthart, J.H. Crowe, and
L. M. Crowe. Trehalose and sucrose protect both
membranes and proteins in intact bacteria during
drying. Applied and Environmental Microbiology,
61(10), p 3592-3597, 1995.
10. Inglesby, TV, T O'Toole, D.A. Henderson, J.G.
Bartlett. M.S. Ascher, E. Eitzen. A.M. Friedlander,
J. Gerberding, J. Hauer. J. Hughes, J. McDade, M.T
Osterholm, G. Parker, T.M. Perl, P.K. Russell, and
K. Tonat, Anthrax as a Biological Weapon, 2002
Updated Re commendations for Management. JAMA.
2002(287): p. 2236-2252.
11. Battelle, Systematic Evaluation of Developmental
and Commercially Available Methods for Biological
Agent Decontamination, Task 1: Recommendations
for Agents/Scenarios, Decontamination
Technologies, and Scale of Testing. Columbus, Ohio,
December 2004. (For Official Use Only)
12. Battelle, BBRC SOP Number MREF. X-135,
Standard Operating Procedure (SOP) for Operation
of the Sabre Bench Chlorine Dioxide Gas Generator.
13. APHA (American Public Health Association),
American Water Works Association and Water
Environment Federation. Standard Methods for the
Examination of Water and Wastewater. 21st edition.
American Public Health Association, Washington,
DC. 2005.
14. BIOQUELL, The Clams C - Multi Function
Hydrogen Peroxide Vapor Generator. http://www.
bioqucll.com/US/default.asp?id=562&ex526=l&cx2
562=l&pid=526.
15. BIOQUELL, Equipment Bio-Decontamination
Service (EBDS). http://www.bioquell.co.uk/default.as
p?id=656&ex590=l&ex2656=l&pid=590.
16. STERIS, VHP® WOOED Mobile Biodecontamination
System, http://www.steris.com/documents.
cfm?id=M2370EN.
17. STERIS, Package Insert for Vaprox* Hydrogen
Peroxide Sterilant, EPA Registration No. 58779-4.
Available on the EPA Pesticide Product Label System
at: http://oaspub.epa.gov/pestlabl/ppls.srchreslt7Com
pNum=058779&ProdNum=00004 (see approval date
31-Mar-2009).
18. Satterthwaite, F. E. (1941). "Synthesis of Variance,"
Psychometrika, 16. 5, 309-316.
19. Satterthwaite, F. E. (1946). "An Approximate
Distribution of Estimates of Variance Components,"
Biometrics, 2, 11 0- 114.
20. Harnett, Donald L. 1982. Statistical Methods,
Third Edition. Addison-Wesley Publishing Co.
Phillippines. Page 712.
-------
-------
Not
Method Demonstration - Biological Agent Recovery Testing:
B. anthracis spores target application range: 7.50 x 106 -1.25 x 107 CPUs
Actual application: 9.17 x 107 - 9.87 x 107 CPUs
(Inadvertently used an application that was one log too high; results do not impact
test results.)
Persistence Testing with K tidarensis:
Target positive control % recover}' range: > 10% - <120%
Actual recover}' from carpet: 169%
Actual recovery' from keyboard keys: 121%
Persistence Testing with Vaccinia Virus:
Target positive control recover}- criteria: >1 x 105 PFUs and CV <25%
Actual CV associated with joint tape: 25.5%
Persistence Testing with Y. pestis:
Target positive control % recovery range: >10% - <120%
Actual recovery from aluminum: 224%
Actual recover}' from keyboard keys: 136% and 288%
Actual recover}' from carpet: 329%
Actual recover}' from joint tape: 150%
Sabre Fumigation Testing with B. anthracis spores:
Target positive control % recover}' range: > 10% - <120%
Actual recover}' from aluminum at 75% RH: 109.5% -138.2%
Actual recovery- from keyboard keys at 75% RH: 232.5% - 433.3%
Actual recover}' from joint tape at 75% RH: 66.2% - 133.7%
B. anthracis spores target application range: 7.50 x 106 -1.25 x 107 CPUs
Actual application at 75% RH: 3.83 x 10s - 5.73 x 106 CPUs
Sabre Fumigation Testing with B. xuix:
Target positive control % recovery range: >10% - <120%
Actual recovery from joint tape (40% RH, 60% RH, and 75%RH): 0.01% - 0.88%
Actual recover}' from aluminum at 75% RH: 1557%
Sabre Fumigation Testing with F. tidarensis:
F. tidarensis target application range: 1.0 x 10s -1.0 x 10s CPUs
Actual application: 5.17 x 107 -1.15 x 108 CPUs
Target positive control % recovery range: >10% - <120%
Actual recovery from joint tape (40% RH and 75% RH): 0.0% -1.09%
Actual recover}' from aluminum at 40% RH: 4.07%
Sabre Fumigation Testing with vaccinia virus:
Target positive control recovery criteria: >1 x 105 PFUs and CV <25%
Actual CV from aluminum (40% RH, 60% RH, and 75%RH): 43.7% - 79.6%
Actual CV from keyboard keys (40% RH, 60% RH, and 75%RH): 20.4% - 74.5%
Actual CV from carpet (40% RH, 60% RH, and 75%RH): 36.6% -112.2%
Actual CV from joint tape (40% RH, 60% RH, and 75%RH): 25.9% - 53.3%
-------
Sabre Fumigation Testing with Y. pestis:
Target positive control % recover}' range: > 10% - <120%
Actual recover}' from aluminum (40% RH and 75% RH): 0.00% - 0.16%
Actual recovery- from keyboard keys (40% RH and 75% RH): 0.03% - 0.08%
Actual recover}' from joint tape (40% RH and 75% RH): 0.00%
BIOQUELL Clarus C Fumigation Testing with 5. anthracis spores:
Target positive control % recover}' range: >10% - <120%
Actual recover}' from wood: 4.86%
Actual recover}' from ceiling tile: 6.17%
BIOQUELL Clarus C Fumigation Testing with vaccinia virus:
Target positive control recover}1 criteria: >1 x 105 PFUs and CV <25%
Actual recover}' from carpet: 3.45 x 104 PFUs and CV 56.3%
Actual recover}' from keyboard keys: CV74.1%
Actual recovery from joint tape: CV 54.9%
Actual recover}' from aluminum: CV79.5%
Actual recover}' from glass: CV26.1%
BIOQUELL Clarus C Fumigation Testing with Y. pestis:
Target positive control % recover}' range: >10% - <120%
Actual recover}' from keyboard keys: 4.12%
Actual recover}' from joint tape: 1.82%
Actual recovery from aluminum: 0.80%
BIOQUELL Clarus S Fumigation Testing with B. anthracis spores:
B. anthracis spores target application range: 7.50 x 106 - 1.25 x 107 CFUs
Actual application: 2.33 x 106 - 8.40 x 10s CFUs
BIOQUELL Clarus S Fumigation Testing with B. suis:
B. suis target application range: 1.0 x 10s -1.0 x 10s CFUs
Actual application: 3.10 x 107 - 1.77 x 10s CFUs
BIOQUELL Clarus S Fumigation Testing with F. tularensis:
Target positive control % recovery range: >10% - <120%
Actual recovery from keyboard keys: 1.19%
Actual recover}' from joint tape: 0.02%
Actual recover}' from aluminum: 0.06%
BIOQUELL Clarus S Fumigation Testing with Y. pestis:
Target positive control % recover}' range: >10% - <120%
Actual recover}' from keyboard keys: 0.10%
Actual recover}' from joint tape: 0.63%
Actual recovery from aluminum: 0.53%
STERTS VHP® Fumigation Testing with B. anthracis spores:
B. anthracis spores target application range: 7.50 x 106 - 1.25 x 107 CFUs
Actual application: 6.93 x 106 - 4.87 x 10s CFUs
Target positive control % recovery' range: >10% - <120%
Actual recover}' from carpet (Trial 1): 158.1%
Actual recovery from aluminum (Trial 1): 149.9%
Actual recovery from wood (Trials 3a and 3b): 3.52% - 5.60%
Actual recover}' from ceiling tile (Trials 3a and 3b): 7.47% - 7.66%
-------
STERIS VHP® Fumigation Testing with B. suis:
Target positive control % recover}' range: >10% - <120%
Actual recover}' from joint tape: 0.06% - 2.11%
STERIS VHP® Fumigation Testing with F. tularensis:
F. tularensis target application range: 1.0 x 106 -1.0 x 10s CFUs
Actual application: 2.53 x 107 - 4.70 x 108CFUs
Target positive control % recover}' range: >10% - <120%
Actual recover}' from keyboard keys: 0.08% - 0.39%
Actual recover}' from aluminum: 3.18% - 5.31%
STERIS VHP® Fumigation Testing with vaccinia virus:
Target positive control recovery criteria: >1 x 105 PFUs and CV <25%
Actual recover}' from carpet: 6.63 x 104 PFUs and CV 42.31%
Actual recover}' from keyboard: CV 25.5% - 26.8%
Actual recovery from joint tape: 1.94 x 104 PFUs and CV 23.50%
STERTS VHP® Fumigation Testing with Y. pestis:
Target positive control % recover}' range: >10% - <120%
Actual recovery from keyboard keys: 0.01% - 0.95%
Actual recover}' from aluminum: 3.30% - 5.45%
Further, during persistence testing with B. suis, contamination of blanks occurred for a limited
number of trials.
Blank acceptance criteria: no observed CFUs
Actual results: 2.30 x 102 CPUs/coupon on aluminum at 0 hr,
6.70 x 10' CPUs/coupon on aluminum at 2 hr, and 3.00 x 10' CPUs/coupon on
keyboard keys at 3 days.
The biological agent contamination issue was limited in scope and was not expected to affect
the overall results of the associated persistence test results. Once the contamination was
noted, fumigation of the hoods, increased physical separation of the coupons, and discussions
with technical staff to raise awareness of the problem were used to successfully eliminate the
contamination issue.
-------
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
------- |