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Indoor Air. 2018;1–9. wileyonlinelibrary.com/journal/ina
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1
© 2018 John Wiley & Sons A /S.
Publish ed by John W iley & Sons Ltd
1 | INTRODUCTION
Filtering facepiece respirators (FFR) should be discarded after use for
one work sh ift to control infec tion,1 especiall y if they come into conta ct
with airborne pathogens, such as Mycobacterium tuberculosis,2 or in-
fluenza virus.3,4 During the severe acute respiratory syndrome (SARS)
epidemic outbreak, consumers’ demand for N95 respirators increased
owing to their high collection ef ficiency. During the outbre ak of Middle
East res piratory syn drome (MERS) in Korea, p harmacies in Sout h Korea
sold many times more N95 FFRs than usual. However, these FFR s are
sometimes reused, especially during a shortage or when their distribu-
tion is delayed.5 Economic considerations may also apply.6 The price of
certified FFRs, such as National Institute for Occupational Safety and
Health (NIOSH)- approved N95 FFRs, typically exceeds those of non-
certified masks. Affordability considerations favor reuse.
National Institute for Occupational Safety and Health recom-
mends practices for the extended use and limited reuse of NIOSH-
certified N95 FFRs.1 NIOSH defines reuse as the use of the same
N95 respirator for multiple encounters with patients but removing
it (“doffing”) after each encounter. The respirator is stored between
encounters to be put on again (“donned”) before the next encounter
with a patient. To prevent tuberculosis, the CDC recommends that
a disposable respirator can be reused by the same worker as long as
it maintains its physical integrity and its proper use provides pro-
tection (exposure reduction) consistent with the assigned protection
factor for respirator of its class.1 Furthermore, NIOSH requires that,
between uses, used respirators should be hung in a designated stor-
age area or kept in a clean, breathable container such as a paper bag.
To minimize potential cross- contamination, respirators are stored
without touching each other and the use of the respirator is clearly
Received:16August2017
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Accepted :28May2018
DOI : 10.1111 /in a.12475
ORIGINAL ARTICLE
Relative survival of Bacillus subtilis spores loaded on filtering
facepiece respirators after five decontamination methods
T.-H. Lin1 | F.-C. Tang2,3 | P.-C. Hung4 | Z.-C. Hua5 | C.-Y. Lai5,6
1Department of Dental Hygiene, China
Medical University, Taichung, Taiwan
2Department of Occupational
Medicine, Changhua Christian Hospital,
Changhua, Taiwan
3Department of Leisure Services
Management, Chaoyang University of
Technology, Taichung, Taiwan
4Instit ute of Lab or, Occupational Safety and
Health , Minist ry of Labor, New Taipei Cit y,
Taiwan
5Depar tment of O ccupati onal Safe ty and
Health , Chung Shan Medic al Unive rsity,
Taichung, Taiwan
6Department of Occupational
Medicine, Chung Shan Medical University
Hospital, Taichung, Taiwan
Correspondence
C.-Y. Lai, Depar tment of O ccupational Safe ty
and Heal th, Chung Shan Medical Universit y,
Taichung, Taiwan.
Email: cylai511@gmail.com
Funding information
Ministry of Science and Technology, Taiwan;
Instit ute of Lab or, Occupational Safety and
Health, Ministry of Labor, Taiwan
Abstract
This study determines the relative sur vival (RS) of Bacillus subtilis spores loaded on an
N95 filtering facepiece respirator (FFR) after decontamination by five methods under
worst- case conditions. Relative survival was obtained by testing after decontamina-
tion and after storing the FFRs at 37°C and 95% relative humidity for 24 hours. The
decontamination methods involved ethanol, bleach, ultraviolet irradiation (UVA
365 nm, UVC 254 nm), an autoclave, and a traditional electric rice cooker (TERC) that
was made in Taiwan. Without decontamination, 59 ± 8% of the loaded spores sur-
vived for 24 hours. When 70% ethanol was added to the N95 FFR at a packing den-
sity of 0.23, the RS was 73 ± 5% initially and decayed to 22 ± 8% in 24 hours. Relative
survival remained above 20% after 20 minutes of UVA irradiation. The other four
decontamination measures achieved 99%- 100% biocidal efficacy, as measured im-
mediately after the methods were applied to the test FFRs. Relative survival is a use-
ful parameter for measuring sterilization or degree of disinfection. Bleach, UVC, an
autoclave, and a TERC provide better biocidal efficacy than ethanol and UVA. Not
only a higher filter quality but also a lower value of RS produced the most decontami-
nated FFR.
KEY WORDS
bioefficacy, decontamination, N95, respirator, spore, survive
2
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LIN et aL.
identified. Storage containers should be disposed of or cleaned reg-
ularly.1 The FDA defines three kinds of reuse: between patients with
adequate reprocessing, reuse by the same person with adequate re-
processing/decontamination, and repeated use by the same person
over a period with or without reprocessing.7,8
Before FFRs are reused, they may be decontaminated to con-
trol the growth of microorganisms on them. However, whether a
decontaminated N95 FFR can be reused is an issue that requires
detailed consideration. In some cases, the use of chemical disin-
fectants may require that an employer train workers on protect-
ing themselves against chemical hazards and on complying with
OSHA’s Hazard Communication, 29 CFR 1910.1200, and other
standards.9 Contaminated objec ts with porous surfaces that cannot
be disinfec ted may have to be disposed of.9 All personnel, clothing,
equipment, and samples that leave a contaminated area (generally
referred to as the Exclusion Zone) must be decontaminated to re-
move any harmful chemicals or infec tious organisms that may be
attached to them.10 Decontamination methods (i) physically remove
contaminants, (ii) inactivate contaminants by chemical detoxifica-
tion or disinfection/sterilization, or (iii) remove contaminants by a
combination of both physical and chemical methods.10 NIOSH has
published a series of research articles on mask decontamination.11-1 4
In selecting decontamination methods, both decontamination
and protective capability are considered.15 For example, ultraviolet
germicidal irradiation (UVGI) and bleach reportedly do not signifi-
cantly reduce the protective capability (penetration by contami-
nants) of FFRs.13,14 Bergman et al13 tested many methods, involving
UVGI and bleach, and found that FFRs that were treated with these
two decontaminants and control samples exhibited expected filter
aerosol penetration (<5%) and filter airflow resistance. Physical dam-
age varied with treatment method. Further research is needed be-
fore any particular decontamination methods can be recommended.
Other chemical and energetic methods also have potential for de-
contaminating FFRs,11,12,16 but few studies have addressed the elimi-
nation of viable microorganisms from FFRs. UVGI had been reported
effectively to eliminate H5N117 or MS2 coliphages18 from FFRs
and was found not to affect drastically the filtration efficiency of
FFRs. Related studies have not evaluated the efficiency with which
decontamination methods destroy bacteria. Therefore, objective
experimentally obtained information concerning the destruction of
bacteria using various decontamination methods is required to sup-
port the reuse of FFRs.
The FDA has not cleared alcohol as the main active ingredient in
liquid chemical sterilants or high- level disinfectants, because alco-
hol is rapidly bactericidal rather than bacteriostatic against vegeta-
tive forms of bacteria, and it does not destroy bacterial spores.15,19
However, ethanol or isopropanol can eliminate the electrostatic
charges on filters which were used before the test of par ticle pene-
tration through the electret masks.20-2 2
In subtropical areas, such as Taiwan, temperatures and humidity
are high all year round, favoring the growth of bacteria. Therefore,
this study compare s the cultivation of airborne Bacillus subtilis spores
that are loaded on N95 FFRs af ter treatment by a commercially
available decontamination method with that after storage at a con-
stant worst- case temperature and relative humidity (RH) to elucidate
the survival and reproduction of bacteria on N95 FFRs. The poten-
tial for N95 reuse during a shortage of epidemic- preventive supplies
is evaluated, and recommendations concerning decontamination
methods and FFR reuse criteria are made to increase public health.
OSHA has recommended that decontamination methods in-
clude chemical disinfection, irradiation, gas/vapor sterilization or
steam sterilization, and dry heat sterilization.10 To understand the
biological effect of decont amination on FFRs, the following five de-
contamination methods were compared: low- temperature chemical
decontamination using (i) ethanol and (ii) bleach, and physical decon-
tamination using (iii) UVGI, (iv) an autoclave to provide moist heat,
and (v) a traditional electric rice cooker (TERC), which was made in
Taiwan, to provide dry heat. The first four methods are preferred
methods for the disinfection or sterilization of patient- care medical
devices.15 TERC is frequently used in hospitals in Taiwan. 22 The fil-
ter qualit y of FFRs, including particle penetration and pressure drop,
was originally published elsewhere.22
2 | MATERIALS AND METHODS
2.1 | Test system and decontamination methods
The main test variable in this study is the survival of bacteria that
were loaded on N95 FFRs that were decontaminated by various
methods under worst- case temperature and humidity, which prevail
when an FFR is placed in a zipper bag in a healthcare worker’s pocket
with the goal of preventing cross- contamination,21 and touching
of the respirator.1,23 In the experiment, B. subtilis spores were the
tested microbial strain; a six- jet Collison nebulizer (BGI, Waltham,
MA) sprayed the spores into a test system, shown in Figure 1, where
they were loaded on N95 FFRs by suction to simulate the respiratory
Practical Implications
• The survival of bacteria of reclaimed National Institute
for Occupational Safety and Health-cer tified N95 filter-
ing facepiece respirators (FFRs) after decontamination is
important, especially for healthcare workers.
• Safe respirator usage after decontamination using vari-
ous methods improves infection control and protection
against biohazards.
• The optimal dosages of decontamination methods are
important for determining a comprehensive infection
control strategy.
• Our work addresses the potential for cross-contamina-
tion of reused respirators with a view to overcoming
FFR shortages and so to increase capacity for control-
ling future outbreaks.
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LIN et aL .
flow of workers during intensive activity.21 The experimental FFR
was an N95 FFR (8210, 3M, St. Paul, MN), certified by NIOSH. It was
divided into six pieces, to which five decontamination methods were
applied; they involved ethanol, bleach, UV, an autoclave, and a tra-
ditional electric rice cooker (TERC), made in Taiwan, without steam.
The treatment proceeded as follows.
• Ethanol: Ethanol with various concentrations and volumes was
added to the center of the surface of the N95 FFR using a pi-
pette,21 the FFR was then dried in a petri dish that was placed in a
biosafety cabinet (BSC) for 10 minutes.
• Bleach: A 0.4 mL volume of bleach with various concentrations
(5.4% (w/w) as Cl2: original; 2.7%: one part bleach to one part of
deionized water; 0.54%: one part bleach to nine parts of deion-
ized water13) was added to the center of the surface of the N95
FFR using a pipette,21 the FFR was then dried in a petri dish in a
BSC for 10 minutes.
• UV: An N95 FFR was placed 10 cm below a 6 W handheld UV
lamp (model UVGL-58, VUP LLC, Upland, CA) that emitted a
wavelength of 254 nm (UVC, 18.9 mW/cm2) or 365 nm (UVA,
31.2 mW/c m2). Both sides of each N95 FFR were exposed for dif-
ferent times - 1, 2, 5, 10 and 20 minutes - in a BSC. The UV inten-
sity was measured using a handheld laser power and energy meter
(OPHIR NOVAII, model Nova II PD300-UV) and was reported as
a mean of five measurements over a 10 × 10 mm aperture with a
swivel mount and a removable filter.
• Autoclave: The N95 FFR was heated for 15 minutes at 121°C and
103 kPa.
• TERC: The N95 FFR was placed in an electric rice cooker for dry
heating for 3 minutes (149-164°C, without added water).22
2.2 | Sampling procedure
Each N95 FFR was placed into the system (Figure 1) for 30 minutes
of bacterial bioaerosol sampling. The respiratory flow (85 L/min) of
workers during high- intensity activities was used,24 and the face ve-
locity for the whole N95 FFR was calculated as 8.3 cm/s. The N95
FFRs were cut into pieces with a diameter of 45 mm. Each had an
effective diameter of 40 mm and a filtration area of 12.6 cm2. The
sampling flow rate of the pump was 6.3 L/min, which produced the
desired face velocity.17,21,25
Bacillus subtilis prototype strains (CCRC 12145, Taiwan Food
Industry Research and Development Institute) were used to prepare
an endospore suspension liquid for generating bacterial bioaero-
sols.21 The suspension was centrifuged at 1917 g for 5 minutes. The
supernatant was discarded and the pellet was resuspended in ster-
ile distilled water. This washing process was repeated two times,26
and spores were resuspended in approximately 55 mL of sterile dis-
tilled water to yield a uniform mixture which was poured into the
Collison nebulizer.21 The spores were aerosolized at a pressure of
25 psi when the dilution air flow rate was 8 0 L/min, as presented in
Figure 1. The stability of the bioaerosol concentration in the system
was verified using an Andersen single- stage sampler (Andersen Inc.,
Atlanta, GA).21
The aqueous packing density (αaq) of the retained liquid decon-
taminants was modified that in a previous report, in which αaq was
the volume fraction of the filter27; it is calculated using the equation,
where Vaq is the volume of liquid disinfectant that was spiked onto
the test N95 FFR . The volume of the test N95 FFR (Vf) was 1.84 mL,
which was estimated from the volume of water that was displaced
by it. When 0.15, 0.4, 0.8, and 1.6 mL of ethanol were spiked onto
the surface of the test N95, αaq values of 0.082, 0.23, 0.44, and 0.87,
respectively, were obtained.
After spores were loaded onto the FFRs for 30 minutes, the FFRs
were decontaminated using one of the five aforementioned methods,
and then p laced in an incubato r (Model: HONG- YU, HRM- 80, Taichung,
Taiwan) at the worst- case scenario temperature of 37°C (similar to
body temperature) and 95% RH (the maximum feasible RH value), re-
spectively, for 24 hours for another day of usage. Each batch test was
conducted in triplicate. Figure 2 displays the sampling procedure.
After decontamination, elution was performed. It involved plac-
ing the test filter in a 50 mL centrifuge tube and then adding 20 mL
(1)
α
aq =
Vaq
V
f
FIGURE1 Experimental system setup.21,22 A filter holder
contains one piece of N95FFR
Exhaust
Dilution air 80 L/min
Compressed air
Suction pump
Collison nebulizer
Filter holder
HEPA filter
Flow controller
Pressure regulator
Aerosol neutralizer
Andersen 1 stage
Sampling flow rate = 28.3 L/min
4 sets of holders and pumps
6.28 L/min for each pump
Face velocity = 8.3 cm/s
ID: 40 mm, OD: 45 mm
4
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LIN et aL.
of sterilized water to soak the filters completely.21 A centrifuge was
used to recover the loaded spores by desorption from the FFRs
at a centrifugation speed of 3500 rpm for 10 minutes, followed by
1 minutes of vortexing. The centrifuged and vortexed suspension
(0.1 mL) was uniformly applied to the TSA and then placed in an
incubator for 24 hours. Colony forming units (CFUs) were counted
and their relative sur vival (RS) was calculated as follows.21,28,29
where Cf is the number of CFUs after decontamination and Ci is the
number of CFUs before decontamination (Figure 2).
3 | RESULTS
Figure 3 presents the decontaminating effect of 0.4 mL (αaq = 0.23)
ethanol at various concentrations on the RS of B. subtilis spores that
were loaded on the N95 FFR. An RS of 89 ± 6% was obtained after
spiking with 50% ethanol, and 73 ± 5% was obtained after spiking
with 70% ethanol. The lowest RS of 68 ± 3% was obtained when
the concentration of ethanol was 80%. The result that was ob-
tained using 95% ethanol (RS = 73 ± 7%) was close to that obtained
using 70% ethanol although the samples that were spiked with 95%
ethanol sometimes yielded slightly higher values of RS than were
obtained using the 80% ethanol samples. An RS of 59 ± 8% was
obtained in 24 hours without decontamination. The 50%, 70% ,
80%, and 95% ethanol- treated samples had RS values of 33 ± 8%,
22 ± 8%, 20 ± 2% and 26 ± 7% after 24 hours, respectively.
Figure 4 shows the effect of 70% ethanol on the RS of B. subtilis
spores. Just after spiking with ethanol, the RS was found to have
declined from 100% to 68%- 75%. When 0.4 mL (αaq = 0.23) of 70%
ethanol was applied, the RS fell to 22% in 24 hours. The RS fell to
20% when 80% ethanol was used.
In the bleach decontamination test, no colony was recovered
after 5.4%, 2.7% or 0.54% NaOCl was used, constituting no dilution,
twofold, and 10- fold dilution,9 respectively (Figure 5). This study
found that NaOCl, even when diluted 10- fold from standard bleach,
had a strong decontamination effect, with a 100% bactericidal ef fect.
Similar results were achieved using UVC. No colony was re-
covered after exposure to UVC for as little as 5 minutes (Figure 6).
However, RS remained above 20% af ter 20 minutes of irradiation by
UVA, exponentially decaying with increased exposure time (Figure 6).
Figure 7 present s the RS values that were achieved using the five
decontamination methods. Four of the methods - involving 0.54%
NaOCl, UVC, an autoclave and a TERC - ef fectively sterilized almost
100% of the bacteria. Decontamination with 70% ethanol yielded an
initial RS of approximately 75% and an RS that remained above 20%
after 24 hours of storage.
4 | DISCUSSION
Decontamination using ethanol yielded higher RS of spores than
the other four decontamination methods (Figure 7). The other four
(2)
RS
=
C
f
C
i
∗
100%
FIGURE2 Sample treatment flowchart
Airborne B. subtilis sampling and loading onto N95
FFR for 30 min in the test system
Decontamination
0. Clean & untreated
1. Ethanol spiking
2. NaOClspiking
3. Ultraviolet germicidal irradiation
4. Autoclave sterilization
5. Dry-heated in a traditional electric rice cooker
Relative survival (RS) =
Filters stored
at 37oC, 95%
RH for 24 h
CiCf
Filters,
0 h
Elution
Elution
Plates incubated at 37oC for 24 h Plates incubated at 37oC for 24 h
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5
LIN et aL .
methods yielded RS values of close to zero, indicating effective
sterilization. The biosafet y manual that was published by the World
Health Organization (WHO) notes that alcohols are effective against
vegetative bacteria but not spores.30 The US CDC also states that
alcohols can eliminate many or even all pathogenic microorganisms
except for bacterial spores.15 Our findings are consistent with the
recommendations of the WHO and the US CDC.
In this study, 59 ± 8% of the loaded spores sur vived on N95
FFRs for 24 hours without decontamination. This result is consis-
tent with some previous studies of the survival rates of B. subtilis
FIGURE3 Relative survival as a
function of concentration of 0.4 mL
ethanol for Bacillus subtilis spores loaded
on N95 filtering facepiece respirator. Error
bars represent one standard deviation
Ethanol concentration (EC), %
010203040506070809
0100
% ,)SR( lavivrus evitaleR
0
20
40
60
80
100
Storage Condition
Temperature = 37
o
C
Relati ve humidity = 95%
0.4 ml Et hanol
Initial
24 h
RS = y
0
+a(EC)+b(EC)
2
y
0
SD a SD b SD R
2
initial 198.5 11.9 –3.16 0.34 0. 019 0.002 0.995
24 h 121.4 9.7 –2.60 0.28 0.017 0. 001 0.991
Range of USCDC
optimal bactericidal
concentration
FIGURE4 Relative survival as a
function of aqueous packing density
(αaq) of 70% ethanol for Bacillus subtilis
spores loaded on N95 filtering facepiece
respirator. Error bars represent one
standard deviation
Packing density, %
02040608
0100
Relative survival, %
0
20
40
60
80
100
Initial
24 h
Storage Condition
Temperature = 37
o
C
Relative humidity = 95%
6
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LIN et aL.
spores at 37°C and an RH of 85%31 or 95%21 Moreover, the results
of Sagripanti and Bonifacino32 suggest that the nature of the chal-
lenged sur face may af fect the sporicidal activity of some chemical
agents. For example, a study by Li et al revealed that the average
values of RS (%) that were obtained by elution from a Nuclepore
filter for hardy B. subtilis were 64% and 48% at sampling times of 1
and 30 minutes, respectively. The average RS (%) values of B. subtilis
from a gelatin filter were 128% and 108% at sampling times of 1 and
30 minutes, respectively.26 Our results were comparable to those
obtained for Nuclepore filter samples.
The WHO biosafety manual mentions that alcohols should
be used at concentrations of approximately 70% (v/v) in water to
FIGURE5 Relative survival as a
function of concentration of 0.4 mL
NaOCl for Bacillus subtilis spores loaded
on N95 filtering facepiece respirator. Error
bars represent one standard deviation
NaOCl concentration, %
012345
6
Relative survival, %
0
20
40
60
80
100
Initial
24 h
Storage Condition
Temp. = 37
o
C
RH = 95%
0.4 ml NaOCl
FIGURE6 Relative survival as a
function of wavelength and duration of
exposure to ultraviolet for Bacillus subtilis
spores loaded on N95 filtering facepiece
respirator. Error bars represent one
standard deviation
Exposure time (ET), min
0510 15 20
% ,)SR( lavivrus evitaleR
0.001
0.01
0.1
1
10
100
UVA 365 nm
UVC 254 nm
RS = a*ex p(-b*ET)
a SD b SD R2 half-life
UVA 365 nm 107.5 8. 1 0. 076 0.012 0. 941 10.07
UVC 254 nm 100.0 4.4 4.79 0. 48 0.998 0.14
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7
LIN et aL .
maximize their germicidal effectiveness. The WHO and the US CDC
both recommend the use of 70% alcohol.15,30 The US CDC also notes
that the biocidal activity of alcohol diminishes sharply at dilutions of
weaker than 50% (v/v), and the optimal bactericidal concentration is
60%- 90% (v/v).15 From the result of quadratic polynomial regression,
the lowest RS occurred at the 83% and 76% alcohol for the initial and
24 hours samples, respec tively (Figure 3). The RS values of approx-
imately 70%- 95% (v/v) (Figure 3) that were obtained in this study
support the US CDC’s recommendation.
The optimal bactericidal concentrations for various microorgan-
isms may var y.15 Pseudomonas aeruginosa was destroyed by ethanol
at concentrations of 30%- 100%, and Serratia marcescens, E. coli, and
Salmonella typhosa were destroyed by ethanol at concentrations of
40%- 100%. Gram- positive organisms, such as Staphylococcus aureus
and Streptococcus pyogenes, were slightly more resistant to ethanol,
being des troyed by ethanol con centrations of 60% to 95%. When the
effect of ethanol against M. tuberculosis was evaluated, 95% ethanol
was found to kill the tubercle bacilli in water or sputum suspension
within 15 seconds. As the challenge bioaerosol in the current study
is B. subtilis spore, the RS remained at 75 ± 17% (Figure 7) indicating
that the B. subtilis spores were more resistant than those mentioned
above, including tubercle bacilli. Ethanol does not affect the viabilit y
of Bacillus spores in current guidance,1,10,15, 30 and a comprehensive
examination of disinfection against different target microorganisms
should therefore be performed.
The average RS (%) values for hardy B. subtilis that were obtained
by elution from FFRs decayed to 23 ± 8% in 24 hours of treatment
with 70% ethanol but to 59 ± 8% without ethanol (Figure 7). The
decay to 59% was caused mainly by the nature of the FFR surface
and effects of storage. The combined effect of FFR surface, storage,
and ethanol treatment might have been expected to yield an RS of
44% (59% × 75%), but was only 22% in this study. As ethanol should
not have this much of an effect on spores,1,10,15 ,30 the nature of the
FFR surface, treatment with ethanol, and the storage conditions may
have an interaction ef fect. Therefore, the mechanism by which eth-
anol affects the amounts of spores that survive on FFRs should be
further investigated.
Figure 4 presents the ef fect of the αaq of 70% ethanol on the
RS of B. subtilis spores. When αaq exceeded 0.23, the initial RS was
around 70% and that after 24 hours was about 20%. Lin et al21 found
that when 1.5 mL of artificial saliva was dropped onto an N95, its
surface tension caused it to form a sphere- like droplet that was at-
tached to the hydrophobic first layer, before it slowly penetrated
the second and third layers. However, in the present study, which is
based on observation, 70% ethanol penetrated the filter rapidly and
quickly evaporated to the air. Accordingly, increasing αaq had little
effect on the RS of B. subtilis spores and may have been responsible
for the rapid evaporation of 70% ethanol.
About 60%- 70% of the B. subtilis spores that were loaded on N95
FFRs survived after 24 hours of storage without decontamination,
whereas only approximately 20% of spores retained their cultiva-
bility af ter 20 minutes of irradiation by UVA (Figure 6), whose dis-
infection effec t was comparable to that of ethanol (Figure 4). From
the result of exponential decay regression, the half- life (the value of
RS reduces to 50%) was 10 and 0.17 minutes for the UVA and UVC
irradiation, respectively (Figure 6). Although UVA could not decon-
taminate as effectively as UVC, it did have some decontaminating
effect. This finding warrants further study.
The results in our study verif y the biocidal efficacy of bleach
(0.54% NaOCl), UVC, and an autoclave, which are well known means
of sterilization.15,30 Interestingly, the TERC exhibited biocidal effi-
cacy as a sterilizing device. In the WHO biosafety manual,30 heat is
FIGURE7 Relative survival of Bacillus
subtilis spores loaded on N95 filtering
facepiece respirator. Error bars represent
one standard deviation. (TERC: Taiwan
traditional electric rice cooker)
0
20
40
60
80
100
Initial
24 h
Relative survival, %
Storage Condition
Temperature = 37
o
C
Relative humidity = 95%
Pretreatme nt Autoclave
(15 min)
Ethanol
70%
(0.4 mL)
TERC
(3 min)
NaOCl
0.54%
(0.4 mL)
UVC
254 nm
6 Watt
(1 min)
Rela tive Survival initia l (%) 24 h (%)
Methods Me an STD Mean STD
Pr etreatment 100.0 0.0 58.6 7.8
Ethanol 70% 74.6 16.7 22.3 8.1
NaOCl 0.54% 0.0 0.0 0.0 0.0
UV C 254 nm 6 W 0.8 0.4 0.0 0.0
Autoclave 0.0 0.0 0.0 0.0
TERC 0.3 0.0 0.1 0.0
8
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LIN et aL.
regarded as one of the most commonly used physical agent s for de-
contami nation against pa thogens. “Dr y” heat, which i s non- cor rosive,
is applied to many items of laborator y- ware, which can withstand
temperatures of at least 160°C for 2- 4 hours. In this study, the TERC
is used as a dr y heating device and was found to exhibit a biocidal
efficacy that reaches ef fective sterilization in 3 minutes. The results
achieved using the TERC provide useful information regarding effec-
tive means for decontaminating and reusing FFRs.
Notably, when an N95 FFR is reused, the biocidal efficacy of
the decontamination treatment, filter qualit y, fit factor (which is af-
fected by physical damage to the frame or rubber strap), and toxic
residual chemicals on FFR14 must all be considered. For example,
bleach can harm the wearer if not properly used to decontaminate
an N95 FFR before reuse.15 Safe disposal of spent bleach is import-
ant, and users may decide to neutralize the microbicidal activity of
the bleach before disposal. Solutions can be neutralized by reaction
with chemicals such as sodium bisulfite, or glycine.15 Considering the
potential health risks, the method of decontamination using bleach
must be modified such as by the use of chemical methods for neu-
tralizing residuals.12
The RS is a function of decontamination and the biological char-
acteristics of pathogens. The filter quality combines penetration and
pressure drop and is affected by the physical characteristics of the
FFR. However, this study focused on RS because it is a useful metric
for quantifying sterilization or degree of disinfec tion. In summary,
bleach, UVC, the autoclave, and the TERC provide effec tive steril-
ization. However, ethanol and UVA are ineffective and not cleared as
high- level disinfectant by US FDA.19 A better reuse FFR has a higher
filter quality and a lower RS.
ACKNOWLEDGEMENTS
This work was supported by grants from the Institute of Labor,
Occupational Safet y and Health, Ministry of Labor, and from the
Ministr y of Science and Technolog y, Republic of China, Taiwan.
ORCID
C.-Y. La i http://orcid.org/0000-0003-4365-8673
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How to cite this article: Lin T-H, Tang F-C, Hung P-C , Hua
Z-C, Lai C-Y. Relative sur vival of Bacillus subtilis spores loaded
on filtering facepiece respirators after five decontamination
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