2 Facepiece Respirators for the Evaluation of ... · 2 1 Abstract 2 3 A chamber to apply...

1

Development of a Test System to Apply Virus Containing Particles to Filtering 1

Facepiece Respirators for the Evaluation of Decontamination Procedures 2

3

Edward Fishera, Samy Rengasamy

b, Dennis Viscusi

b, Evanly Vo

b, and Ronald Shaffer

bc 4

5

aEG&G Technical Services, Inc, Pittsburgh, PA 15236 6

bNational Institute for Occupational Safety and Health, National Personal Protective 7

Technology Laboratory, Pittsburgh, PA 15236 8

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cCorresponding author. National Institute for Occupational Safety and Health, National 10

Personal Protective Technology Laboratory, 626 Cochrans Mill Rd., P.O. Box 18070, 11

Pittsburgh, PA 15236. Email [email protected] 12

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Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01653-08 AEM Accepts, published online ahead of print on 9 January 2009

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Abstract 1

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A chamber to apply aerosolized virus-containing-particles to air permeable 3

substrates (coupons) was constructed and validated as part of a method to assess the 4

virucidal efficacy of decontamination procedures for filtering facepiece respirators. 5

Coliphage MS2 was used as a surrogate for pathogenic viruses in the confirmation of the 6

Bioaerosol Respirator Test System. The distribution of virus applied onto and within the 7

coupon was characterized and the repeatability of applying a targeted virus load was 8

examined. The average viable virus loaded onto ninety coupons over the course of five 9

days was found to be 5.09 ± 0.19 log10 (relative standard deviation=4%). To determine 10

the ability to differentiate the effectiveness of disinfecting procedures with differing 11

levels of performance, sodium hypochlorite and steam treatments were tested in varying 12

dose and time experiments, respectively. The role of protective factors was assessed by 13

aerosolizing the virus with varying concentrations of the aerosol generating medium. 14

Sodium hypochlorite (bleach) concentrations of 0.6% and steam treatments of 45 seconds 15

and longer demonstrated log reductions (>4 logs) which reached detection limits for both 16

levels of protective factors. Organic matter (ATCC medium 271) as a protective factor 17

afforded some protection to the virus in the sodium hypochlorite experiments but was not 18

a factor in the steam experiments. The evaluation of the Bioaerosol Respirator Test 19

System demonstrated a repeatable method to apply a targeted viral load onto respirator 20

coupons and provided insight into the properties of aerosols that are of importance to the 21

development of disinfection assays for air permeable materials. 22

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INTRODUCTION 1

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The Institute of Medicine reports that a 42-day influenza pandemic outbreak may 3

require over 90 million N95 filtering facepiece respirators (FFRs) to protect workers in 4

the healthcare industry (12). High demand for FFRs may result in local shortages. 5

Decontamination and reuse of FFRs may provide a solution to increased demand in 6

emergency situations (36). Test methods exist to assess the efficacy of biological 7

decontamination methods (BDMs) for viruses in suspension and on hard-surface-8

porous/nonporous carriers (4, 5, 30). However, a test method to evaluate the 9

effectiveness of BDMs applied to air permeable materials (APMs), such as disposable 10

FFRs, is lacking. 11

Because of a lack of a standardized method for an aerosolized application of 12

viruses, current carrier tests are not realistic for aerosols and APMs involving airflow 13

through the test sample. Modifications to current carrier test protocols, specifically to the 14

method of viral challenge application, the use of air permeable substrates, and airflow 15

through the substrate, are needed for testing FFR decontamination under conditions that 16

better simulate a real world scenario. Ideally, the test procedure will mimic the airborne 17

transmission of viruses, allow for penetration into and through APMs, and provide a 18

manner for depositing a reproducible viral challenge on the substrate surface (even for a 19

water resistant surface such as some surgical masks and some types of FFRs). 20

In this work, a Bio-Aerosol Respirator Test System (BARTS) was developed, 21

characterized, and validated to provide a method to load APMs (FFR sample coupons) 22

with a repeatable targeted load of aerosolized virus-containing-particles (VCPs). The 23

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physical properties of the bioaerosol generated from BARTS were examined to better 1

identify the parameters that are important in measuring the virucidal efficacy of BDMs 2

on FFRs contaminated with VCPs. A tryptone based protective factor medium similar in 3

protein concentration to the recommended organic challenge of ASTM method E 1053 4

was examined at two levels (100% and 1%) to determine its effects on the physical 5

properties of the VCPs and on BDMs (5). The particle size distribution of VCPs 6

generated using BARTS and the deposition of the VCPs on the surface of the respirator 7

sample and inside the filter media were determined for each protective factor level. The 8

ability to differentiate the effectiveness among BDMs of various degrees of severity was 9

assessed by performing experiments with a range of sodium hypochlorite concentrations 10

and steam exposure times using both protective factor levels. 11

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MATERIALS AND METHODS 1

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BARTS. A 43L (36.5 cm x 34.5 cm x 34.5cm) aerosol testing chamber was 3

constructed from anti-static acrylic panels. As shown in Fig. 1, the chamber is equipped 4

with an aerosol inlet (2 cm diameter), a 25 mm diameter pressure relief port and six 25 5

mm diameter test specimen holder ports. Coliphage MS2 in suspension was aerosolized 6

using a 6-jet Collison nebulizer (BGI Incorporated, Waltham MA). Air flow regulators 7

(Ashcroft, Costa Mesa, CA), one upstream from the 6-jet nebulizer and one for the dilution 8

air, were used to control the nebulizer air flow at 10 L/min (20 psi) and the dilution air 9

flow at 50 L/min (13 psi). A 100 mm (4 in.) diameter fan inside the chamber was used to 10

mix the bioaerosol. Six test specimen holders (SKC Inc., Eighty Four, PA) (Fig. 2), each 11

containing an FFR coupon, were connected directly to the specimen holder ports for 12

loading with virus. Each specimen holder was connected to a flowmeter (Cole-Parmer, 13

Vernon Hills, IL), which controlled and measured the flow rate between the vacuum inlet 14

and each test specimen holder at a calibrated level of 4.0 L/min for a face velocity of 13 15

cm/s. 16

BARTS was housed inside a Safeaire® chemical fume hood (Fisher Hamilton, 17

Pittsburgh, PA). The chemical fume hood is appropriate when working with Biosafety 18

Level 1 agents. The productions of bioaerosols containing potentially hazardous agents 19

would require the use of a biosafety cabinet. 20

N95 Respirator. The N95 respirator used for this study is a NIOSH-approved 21

filtering facepiece respirator (FFR) commonly utilized by healthcare workers for 22

protection against particulate hazards. NIOSH certified N95 respirators are 95% efficient 23

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or better at filtering particles around 300 nm aerodynamic mass median diameter. This 1

model of FFR is comprised of 3 layers. The exact composition of the various layers in 2

the FFR is often considered proprietary information. However, most FFRs share 3

common traits regarding the types of materials used and the roles of the various layers. 4

The innermost layer, which directly contacts the users face, is designed to be soft and is 5

often fluid resistant to prevent perspiration or saliva from interfering with, or exiting, the 6

facemask (12). The second layer consists of the non-woven filter media designed to 7

capture particulates. Typical filter media used in the FFRs today are made from electret 8

treated spun-bonded polypropylene. The outermost layer is designed from fabric 9

materials to impart stability during handling and contact and to prevent larger particles 10

and debris from reaching the inner layers. 11

Media, virus, and host cell. American Type Culture Collection (ATCC) medium 12

271 (www.atcc.org) was used for the growth of Escherichia coli (E. coli) and the 13

preparation, storage, recovery and assay of MS2. The aerosol-generating medium was 14

comprised of 100% or 1% ATCC medium 271 (deionized water was used as the diluent 15

for 1% ATCC 271 medium). When used as the aerosol generating solution, 100% ATCC 16

medium 271 is referred to as high protective factor (HPF) medium and 1% ATCC 17

medium 271 as low protective factor (LPF) medium. The two levels of the protective 18

factor were chosen to assist in the characterization of the properties of the generated 19

bioaerosol and not necessarily to replicate the exact organic challenge one may expect in 20

a FFR reuse situation. The ATCC 271 medium was amended with 2.5 g of agar (Sigma 21

A7002) for the soft agar medium used in the plaque assays. 22

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Coliphage MS2 (ATCC 15597-B1) were replicated using E. coli (ATCC. 15597) 1

as the host. A 5-hour culture of E.coli (1 ml) was used to inoculate 30 ml of ATCC 2

medium 271. After 2.5 hours, 1.5 ml of MS2 stock was added to the flask and incubated 3

overnight. The sample was centrifuged at 7100 x g for 30 min at 4°C (IEC Multi RF, 4

Thermo Electron Corporation), and the supernatant was filtered through a 0.22 µm-pore-5

size filter (Fisherbrand). The virus suspension (1011

plaque forming units (PFU)/ml) was 6

stored at 4°C in 50 ml conical tubes (Falcon, Becton Dickinson) for use in the 7

experiments. MS2 was selected for the study based on its moderate resistance to 8

disinfectants, survivability, ease of preparation and assay, and nonpathogenicity (18, 38). 9

Virus aerosol generation and loading onto respirator coupons. Circular 10

coupons (5 cm2) were cut from respirators from a single model of a NIOSH-approved 11

N95 FFR. The respirator coupons were not sterilized prior to loading, but were handled 12

with sterile forceps to minimize contamination with biological or chemical matter. Six 13

respirator coupons (excised from at least 2 different respirators) were placed into separate 14

test specimen holders and attached to the six BARTS sample ports. A quantified titer 15

(105-10

8 pfu/ml dependent upon aerosol medium and viral loading target level) was 16

suspended in LPF or HPF. The virus challenge solution (23 ml) was added to the 17

nebulizer glass jar. The compressed air valves were opened to the nebulizer (20 psi, 10 18

L/min) and the dilution air (13 psi, 50 L/min). After allowing 5 min for the aerosol 19

concentration to stabilize within the chamber, the vacuum was initiated and regulated at 4 20

L/min for each sample port. The aerosol was passed through the coupons for 30 min. 21

Virus recovery. Three procedures, vortexing (Vortex Genie 2®, Scientific 22

Industries, Bohemia, NY), sonication (Model 8510, Bransonic®, Danbury, CT) and 23

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shaking (GyromaxTM

SteadyshakeTM

757, Amerex Instruments, Inc., Lafayette, CA), 1

were tested in triplicate to evaluate MS2 recovery efficiency. MS2 in HPF medium (10 2

µl of a 105 PFU/ml solution) was applied to coupons using a micropipette and allowed to 3

dry for 10 min. The coupons were resuspended in 10 ml ATCC medium 271 in 50 ml 4

conical tubes or 50 ml flasks (shaking). Triplicate sets were subjected to 2 min of 5

vortexing at the highest setting, shaking at 200 rpm for 15 min, or placed in a sonicating 6

bath for 10 min. Upon completion of the recovery procedure, the coupons were 7

discarded and the supernatant was assayed for active MS2 as previously described. The 8

virus recovery methods were not tested using the LPF medium as this experiment 9

preceded the inclusion of a low protective factor level in the overall study. Upon review 10

of the literature and consideration of the results from the HPF recovery experiments, the 11

vortex method was chosen for the recovery of virus from coupons for both levels of 12

protective factor. 13

Plaque assay. A single agar layer method was used to enumerate the viruses (1, 14

35). Eight ml of soft agar medium 271 was placed into glass culture tubes and incubated 15

at 47°C in a water bath. Log phase E. coli (0.5 ml) and 1ml of the MS2 suspension were 16

added to the culture tubes. The soft agar containing E. coli and MS2 was poured into an 17

empty Petri plate and mixed by swirling. The plates were allowed to harden at room 18

temperature and placed in an incubator at 37°C and 30% relative humidity overnight. 19

Plates were counted the following day and those containing 30-300 (PFU) were recorded. 20

Effect of charge neutralization of MS2 aerosol. The use of a particle 21

neutralizer (TSI model 3012) was assessed to determine the effect of particle charge on 22

loading coupons with the MS2 challenge. Neutralizers use a radioactive source to ionize 23

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the surrounding atmosphere into positive and negative ions. Particles carrying charges 1

capture ions of opposite polarity. The particles reach charge equilibrium and the aerosol 2

demonstrates a bipolar distribution. Virus was loaded onto the coupons under two 3

conditions, with and without neutralization, in quadruplicate for 10, 20, and 30 min 4

following the loading procedures previously described. After exposure to MS2, the 5

coupons were removed from the filter holders and the virus was recovered and assayed as 6

previously described. 7

Validation studies. (i) Repeatability. Six coupons were loaded simultaneously 8

with MS2 (a targeted load of 5 logs/coupon) during 3 runs per day for 5 days. MS2 was 9

recovered and quantitated for all 90 coupons as described above. 10

(ii) Particle size and desiccation determination. MS2 was aerosolized in water, 11

LPF and HPF suspension medium as previously described using BARTS. Particle size 12

and concentration were measured at the filter sample holder/aerosol chamber interface 13

using an Aerodynamic Particle Sizer (APS, Model 3321, TSI Inc) and a Scanning 14

Mobility Particle Sizer (SMPS, Model 3080, TSI Inc.), including a Condensation Particle 15

Counter (CPC, Model 3025A, TSI Inc.). The APS measured particle sizes ranging from 16

500 to 20,000 nm, and the SPMS measured particle sizes ranging from 20 to 900 nm. 17

The full scale (20-20,000 nm) size distribution, which expresses the concentration of 18

particles as a function of particle diameter, was obtained by combining the data from the 19

SMPS and the APS. Matching the SMPS and APS data was performed by calculating the 20

ratio value from the overlap data between 500 and 900 nm. The APS data was then 21

multiplied by the ratio value to give the best fit to a lognormal distribution of the SMPS 22

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data. The chamber door was opened for 15 min and purged of particles between 1

loadings. 2

To characterize the extent of evaporation of water from particles generated by 3

BARTS, the mass median equilibrium diameter of a completely desiccated particle was 4

calculated for LPF and HPF media using the equation previously described by Nicas et 5

al.: 6

7

deq = (Cnonvolatile/ρnonvolatile)1/3

* d0 (1) 8

9

where deq represents the equilibrium particle diameter, d0 represents the initial diameter, 10

Cnonvolatile is the mass concentration (g/L) of the solutes in the medium (20g/L for HPF 11

and 0.2g/L for LPF) and ρnonvolatile is dry density of the solutes (often estimated to be 12

the density of water or 1000 g/L) (23). The output distribution of the BGI nebulizer in 13

the BARTS has a mass median diameter of 2.5 µm and was used for d0 in the above 14

equation (10). The calculated particle size was compared to the measured values for LPF 15

and HPF mediums. 16

(iii) Virus distribution on/within the respirator coupon. Eight respirator 17

coupons (3.1 cm2 aerosol exposed area) for each suspension medium (LPF and HPF) 18

were exposed to MS2 as previously described. Using a cork borer, 4 coupons for each 19

suspension medium type were separated into two samples; inner circles measuring 1.5 20

cm2 and outer rings measuring 1.6 cm

2. The MS2 deposited on quadruplicate samples of 21

the inner circles and outer rings were enumerated as previously described. The three 22

layers of the other four coupons were separated and enumerated to test viral penetration 23

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through the respirator coupon. 1

(iv) Decontamination treatment of viruses. (a) Sodium hypochlorite. The 2

decontamination efficacy of sodium hypochlorite (Clorox®, EPA Reg. No. 5813-50) in 3

concentrations of 0.0006, 0.006, 0.06, and 0.6% on MS2 viability was tested in triplicate. 4

Respirator coupons were used as air permeable inanimate carriers and exposed to MS2 5

(approximately 6 logs/coupon) as described under BARTS methods. Upon completion of 6

exposure to MS2, one coupon was placed directly into 10 ml of ATCC medium 271 as a 7

loading level control; one was submerged in water (dipping control) and one each in 8

sodium hypochlorite at different concentrations in 50 ml conical tubes. After 10 min of 9

treatment the coupons were carefully removed from the sodium hypochlorite solution and 10

air dried for 2 min. Each coupon was then placed in 10 ml of medium 271. The tubes 11

were capped and vortexed for 45 sec. The active MS2 remaining on the coupon was 12

enumerated by plaque assay as described above. The process was repeated for triplicate 13

sample sets for each suspension medium. 14

(b) Microwave-generated steam. Steam treatment times of 0, 15, 30, 45, 60, 15

and 75 sec were tested in triplicate for efficacy against MS2. Steam sterilization 16

chambers were constructed from plastic pipette tip boxes. Nine holes measuring 17

approximately 4 mm in diameter were drilled into the top of the pipette tip box lid for 18

ventilation. The base of the pipette tip box was filled with 50 ml of room temperature 19

water. Respirator coupons were loaded with MS2 as previously described. After MS2 20

loading, the coupons were placed on a rack inside the steam sterilization chamber 21

approximately 2.5 cm above the water level. The vented pipette tip box lid was placed 22

over the base and the chamber was heated in the microwave on high for the appropriate 23

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treatment time. The commercially available microwave (Model no. R305KS, Sharp 1

Electronics, Mawwah, NJ) was validated previously for power output (rated 1100W 2

measured 750W) by Viscusi et al. (36). Upon treatment, the coupons were carefully 3

removed from the chamber and placed in 10 ml of medium 271 in a conical tube. The 4

tubes were capped and vortexed for 45 sec. The coupons were removed, discarded, and 5

the suspension assayed by plaque assay. 6

(v) Toxicity controls. To determine whether the residual sodium hypochlorite on 7

treated coupons had been neutralized by the ATCC medium 271, three coupons 8

containing no MS2 challenge were treated with 0.6% sodium hypochlorite, allowed to 9

dry, and vortexed in medium 271. Suspensions of MS2 were made by placing 3 ml of the 10

coupon recovery medium and 3 ml of fresh medium 271 into separate 10 ml conical tube. 11

A known titer of MS2 (100 µl) was added to all conical tubes and plaque assays were 12

performed. 13

Data analysis. The MS2 recovered from experimental and control coupons for 14

all experiments unless otherwise specified was quantified by calculating log10 of the 15

viable MS2 titer recovered from each coupon and the average and the standard deviations 16

were calculated. 17

The loading of MS2 onto FFR coupons was further assessed by calculating the 18

relative standard deviation (RSD) for all 90 coupon samples. An analysis of variance 19

(ANOVA) was performed using EXCEL (Microsoft Office 2003) for comparison of the 20

variability among the sample ports. 21

The dispersion of MS2 on the outer surface of the FFR coupon was determined by 22

enumerating the viable MS2 for both the outer ring and inner circle as log10(PFU/cm2). 23

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The three layer deposition of MS2 was evaluated as a percentage of the viable MS2 per 1

layer compared to the total MS2 loaded for the full coupon. 2

Antimicrobial activity of bleach and steam treatments of FFR coupons was 3

determined by calculating log10 (N/X), where N is the titer of viable MS2 recovered from 4

untreated samples and X is the titer of viable MS2 recovered from the treated coupons. 5

A two-tailed t test (Student test) was completed by using EXCEL (Microsoft Office 6

2003) for each decontamination treatment where log reduction (LR) was greater than one 7

(for at least one sample set) and less than the limit of detection (for both sample sets). 8

For statistical analysis where applicable, the p-values were compared at the 95% 9

confidence level.10

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RESULTS 1

2

Preliminary experiments to optimize VCP application onto coupons and recovery 3

from coupons were performed before the validation and characterization of BARTS. The 4

difference in the number of VCPs loaded onto respirator coupons with and without 5

charge neutralization was examined. Figure 3 shows control and charge neutralized VCP 6

loading onto FFR samples at 3 different time points. The active MS2 recovered from the 7

coupons generated from the control and neutralized aerosol was similar for all time points 8

(10, 20 and 30 min). A marginal but statistically significant (p < 0.003) increase in the 9

number of MS2 extracted from coupons loaded from the control aerosol was seen for the 10

20 and 30 min time points. The optimal method of MS2 recovery from coupons was 11

examined by comparing efficiencies of sonicating, vortexing and shaking. Each method 12

demonstrated similar efficiency and repeatability in extraction of MS2 from coupons 13

(Table 1); however, vortexing was chosen as the recovery method for all other tests. 14

BARTS was validated and characterized to assess its applicability for evaluating 15

decontamination efficacies of BDMs for FFRs. Figure 4 shows the repeatability of the 16

application of MS2 onto respirator coupon samples using BARTS. The bars represent the 17

average log 10 (PFU/coupon) for each filter sample port (1-6). The error bars represent 18

the standard deviation of the mean. The difference among the ports was not significant 19

(p=0.68). Moreover, the average viable virus loaded onto the 90 coupons was found to 20

be 5.09 ± 0.19 log10, indicating sufficient repeatability (relative standard deviation = 4%). 21

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The distribution of MS2 among the 3 layers of the respirator coupon and deposited 1

over the entire surface of the outer layer of the coupon was determined for both LPF and 2

HPF aerosol mediums. Figure 5 shows the deposition of MS2 on the three layers of an 3

N95 FFR coupon. Virus aerosolized from LPF medium is predominately deposited on 4

the middle layer, with significant deposition also occurring on the outer layer. Virus in 5

the HPF medium became entrapped on the outer layer, with significant deposition also 6

occurring in the middle layer. Neither medium type demonstrated a significant 7

accumulation of virus on the inner layer in comparison to the middle and outer layer. 8

The average and standard deviation log10(PFU/cm2) loaded onto the outer layer of the 9

coupon for the inner circle and outer ring was 4.4 ± 0.26 and 4.7 ± 0.06 (p=0.11)

10

respectively for LPF and 5.5 ± 0.16 and 5.3

± 0.20 (p=0.03)

respectively for HPF. 11

Similar concentrations of viable virus were recovered from the inner circle and outer ring 12

of the outer layer for each medium type (5.5 vs. 5.3 for HPF and 4.4 vs. 4.7 for LPF in 13

units of log10(PFU/cm2) indicating uniform deposition on the outer layer. 14

The particle mass distributions from 20-20,000 nm for water and each aerosol 15

medium type are shown in Figure 6. The mass median diameter was also calculated for 16

water, LPF, and HPF and found to be 58, 141, and 492 nm, respectively with 17

corresponding mass mean diameters of 72, 187, and 629 nm. In count mode, very few 18

particles larger than 1,000 nm were observed. The count median diameter for water was 19

27 nm compared to 30 nm for LPF and 56 nm for HPF suspension media. The count 20

mean diameter was determined for water, LPF, and HPF and found to be 29, 32, and 62 21

nm, respectively. At constant flow conditions, an increase in particle number and an 22

increase in particle size coincided with the increase in protective factor in the suspension 23

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medium. The addition of MS2 to the suspension medium had no effect on the particle 1

size (data not shown). 2

The efficacy of sodium hypochlorite decontamination on FFR samples loaded 3

with MS2 is shown in Figure 7. The LR of MS2 resuspended in HPF and LPF medium 4

increased with increasing concentrations of sodium hypochlorite. However, the increase 5

in MS2 LR was greater for the LPF set at all sodium hypochlorite concentrations 6

compared to the HPF set (p < 0.02), except for where both sets reached detection limits 7

(0.6% sodium hypochlorite). The recovery buffer presumably neutralized the 8

experimental samples for sodium hypochlorite treatments as indicated by the toxicity 9

tests (LR = 0.03 ± 0.11). Plaque assays enumerating the same sample (0.6%) of recovery 10

medium on consecutive days confirmed that the recovery solution was adequate at 11

neutralizing the active components in bleach as quantities remained constant over an 12

approximate 24 hour period (data not shown). 13

Figure 8 shows the decontamination efficacy of steam treatment at different time 14

exposures. For steam sterilization the sample sets for HPF and LPF had similar LRs for 15

all treatment times. As expected, longer steam treatment time demonstrated higher 16

efficacy. Within the limits of detection, the protective factor had little effect against 17

steam treatment. Statistical significance for the steam treatment was not calculated as 18

only a single time point fell within the parameters outlined in the data analysis section. 19

20

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DISCUSSION 1

Current disinfectant testing methods, the most common of which are suspension 2

tests and carrier tests, are inadequate in assessing the antimicrobial activity of 3

decontamination treatments of APMs exposed to a bioaerosol. Suspension tests are 4

convenient for initial screenings, but inanimate carrier tests are more appropriate for 5

evaluating surface disinfectants. Carrier tests, which are commonly performed through a 6

dipping or spiking of the viral challenge onto the substrate, do not account for the 7

physical characteristics of aerosols, such as particle size, composition, velocity, 8

desiccation and the subsequent substrate penetration (8, 21, 30). It has been shown in the 9

literature that the testing method has an effect on the activities of disinfectants (9, 24, 38). 10

Woolwine and Gerberding showed that the inactivation of MS2 in suspension tests and 11

carrier tests using glass slides, ceramic tile, and pigskin as the carrier substrate displayed 12

different decontamination efficacies for the same chemical treatment under identical 13

conditions (38). Thus, it is not only the testing method (e.g. carrier or suspension test), 14

which alters the activities of the decontaminant; it is also dependant on the carrier 15

substrate (e.g. porous, nonporous, air permeable). The method of application of the viral 16

challenge may have similar effects on activities of BDMs. By passing a bioaerosol 17

through the FFR sample coupon, BARTS provides a more realistic assessment of 18

decontamination efficiency for APMs than other carrier or suspension tests. 19

The complexity of testing methods for the assessment of virucidal activities of 20

decontamination procedures results in little or no standardization, procedural 21

deficiencies, and a lack of guidelines among the established protocols (8, 30, 38). 22

Developing a test method using bioaerosols adds to the complexity of decontamination 23

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studies. The preliminary experiments addressed two issues associated with developing a 1

bioaerosol decontamination test method, particle charge neutralization and virus recovery 2

from the substrate. The use of a neutralizer is common in many aerosol applications such 3

as filtration testing where charge neutralization presents a more challenging set of 4

conditions. However, many of the experimental bioaerosol generating systems developed 5

for other research applications have not included a neutralizer (11, 15, 17, 37). Aerosol 6

charge may affect the culturability and viability of microorganisms and may cause a loss 7

of microbes which may stick to the walls of the test system through electrostatic 8

interactions (20, 33). Although it is possible that the neutralizer may have had a positive 9

impact on viability or wall effect, the total number of viable viruses recovered from the 10

coupon was lower (Fig. 3), thus the neutralizer was deemed unnecessary for this study. 11

Using the BARTS for other applications such as bioaerosol filtration testing of APMs 12

containing integrated antimicrobial technologies may require a modification to include a 13

neutralizer to accommodate filtration testing recommendations. 14

The elution and recovery of virus from a substrate is a problem for all carrier tests 15

and the use of an aerosol and an APM increases the difficulty of the procedure. 16

Although each recovery method was comparable in MS2 recovery from FFR coupons, 17

vortexing proved to be the most time efficient. The recovery methods tested in this study 18

were also previously examined for the elution of bacteria from FFR samples, in which 19

vortexing was not only more time efficient, but provided the most energy and relative 20

motion to separate the bacteria from respirator fibers (37). 21

It is documented in the literature that water from aerosol particles evaporates 22

almost immediately upon generation, at typical levels of room relative humidity and 23

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room temperature to form droplet nuclei (29, 32). A study examining the size 1

distribution of droplet nuclei generated from the cough of human subjects at 35% relative 2

humidity found particle sizes ranging from 0.58 to 5.42 µm with 82% of the particles in 3

the 0.74-2.12 µm range (39). Particles < 3 µm essentially remain airborne because of 4

low settling velocities (32). One would expect that ultrafine and submicron sized VCPs 5

would be readily trapped on FFRs through inhalation in a high risk exposure setting such 6

as a hospital during a pandemic. Also in the case of influenza virus, transmission is 7

greater at lower relative humidity, which would further suggest the importance of 8

studying particles in the ranges used in this study (19). The particle size determination 9

data for both LPF and HPF demonstrates a comparable size distribution to the system 10

developed by Hogan, with the majority of particles in the submicron and ultrafine particle 11

range (17). The mass median diameters of the VCPs at room relative humidity (20-25%), 12

as measured at the sample collection ports of the aerosol chamber, were approximately 13

141 nm and 492 nm for LPF and HPF media, respectively. These values are similar to 14

the calculated desiccated particle size (from eq. 1) for LPF and HPF (146 nm and 15

678nm), indicating the formation of droplet nuclei. 16

The constituents of the aerosol medium provide the protective factor and 17

contribute to the particle size (17). Protective factors, used to mimic the organic and 18

inorganic molecules that are often associated with the biological contaminant in real 19

world decontamination scenarios, may provide protection to the virus in the VCPs by 20

neutralizing the antimicrobial compound or by acting as a physical barrier. The aerosol 21

media, LPF and HPF, which were chosen to provide two distinct levels of protective 22

factor to the decontamination test, provided different particle size distributions (see 23

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Figure 6). The two particle size distributions in this study resulted in different deposition 1

patterns inside and on the surface of the respirator coupons. For example, viruses 2

recovered from experiments using HPF were more likely to be found on the outer layer 3

compared to LPF (see Figure 5). This may be important in cases where the 4

decontamination treatment (for example UV light) may not have access to the viral 5

challenge if the viral particles are deposited on an internal layer of an FFR. It may be 6

possible to expose multiple layers of an APM to a viral challenge using typical 7

application methods of carrier tests such as spiking or dipping, however; other properties 8

of aerosols such as protective factor concentration would be neglected. 9

The FFR decontamination test method using BARTS was able to differentiate 10

between BDMs with varying degrees of expected efficacy. The experiments included 11

both a chemical (sodium hypochlorite) and physical (steam) treatment and two distinct 12

protective factor challenges. For the chemical treatment, the BARTS test method was 13

able to discern the efficacy of decontamination among the various concentrations of 14

sodium hypochlorite as well as the effect of the protective factor. The physical 15

decontamination treatment test detected a difference in efficacy for the different steam 16

treatment times yet demonstrated no effect from the protective factor. As expected, for 17

both the physical and chemical treatment, higher LRs coincided with the potency of the 18

treatment. Yet, within the detection limits, the protective factor only influenced the 19

chemical test. Protective factors in the HPF medium as deposited on the carrier substrate 20

(in this case the FFR) may act to neutralize the chemical disinfectant or inhibit the 21

diffusion of the chemical treatment to the MS2, whereas in the steam treatment, the 22

constituents of the protective factor have little effect on the rate of heat conduction. 23

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The primary purpose of this work was to develop and validate a test system to 1

apply VCPs to APMs for use in disinfection assays. Sodium hypochlorite disinfection is 2

well characterized in the literature (7, 13, 22, 25-28, 38). Steam treatment is also a well 3

known method for inactivating viruses on surfaces (6, 14, 16, 26, 31). However, typical 4

steam sterilization requires the use of an autoclave which utilizes pressurized steam at 5

121°C, which was shown to have a deleterious effect on FFR performance (36). In this 6

work, a commercial microwave oven was used to generate a less rigorous ambient 7

pressure steam treatment. Initial attempts at studying microwave irradiation for FFR 8

decontamination resulted in melted coupons (data not shown). Other studies involving 9

microwave decontamination have found that water was needed for sterilization (26). 10

The use of microwave irradiation / heating to generate steam for decontamination or 11

sterilization is not new and affords benefits. Microwave energy was used to heat water to 12

sterilize surfaces for use with NASA’s Mammalian Cell Bioreactor, where thermal 13

impact on the system was minimized through the rapid and selective properties of 14

microwave heating (6). Microwave–generated steam has also been shown to 15

decontaminate medical waste and respirators (14, 16). 16

One concern with using a commercially available microwave oven to generate 17

steam is repeatability. The data in figure 8 suggest that the microwave oven and steam 18

chamber used in this study were able to produce repeatable results. Steam treatments for 19

45 sec or longer demonstrated LRs which reached detection limits for all 18 of the 20

coupons tested (LPF and HPF). Treatment times at 15 and 30 seconds demonstrated 21

RSD ranging from 9 to 26%, which is similar to other decontamination methods 22

described in the literature as is discussed further below. Although promising, the data 23

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presented in this study using a single microwave oven and one model of FFR do not 1

indicate that the use of a commercial microwave oven to generate steam should be 2

adopted as a means of sterilization without further studies. The use of microwaves for 3

sterilization of medical devices has not been cleared by the Food and Drug 4

Administration (26). Furthermore, additional studies are needed to separate the effects of 5

microwave heating, microwave irradiation, and steam on the efficacy of decontamination. 6

In germicide testing, the assessment of repeatability and reproducibility are of 7

great importance to the validation of a standard method. ASTM International and AOAC 8

International require an evaluation of the precision of the test method within the 9

laboratory (repeatability) and interlaboratory precision (reproducibility) before a test 10

method becomes a standard (2, 3). However, there is no known recommendation of how 11

precise a test method must be until it is accepted as repeatable or reproducible. Tilt and 12

Hamilton performed a literature review to survey the repeatability and reproducibility of 13

germicide tests and found that germicides with a LR > 2 had (RSD) ranging from 3 to 14

36% (34). The precision of BARTS as a means to expose FFR to a consistent viral 15

challenge provided a RSD of 4% (Fig. 4), which corresponds to the lower limit of typical 16

RSDs in germicide testing. The variability of the loading process is the cumulative 17

variation of all the steps in the procedure including the day-to-day production of the virus 18

aerosol solution, the virus recovery step, and the plaque assays. However, loading the 19

viral challenge is only part of the complete test method. A better estimate of the 20

repeatability of the entire method can be found in Figs. 7 and 8. Within the triplicate 21

samples generated for sodium hypochlorite and steam decontamination test methods that 22

demonstrated a LR > 2, RSD was found to range from 2 to 32%. These data were 23

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generated from a series of experiments and do not incorporate other sources of error such 1

as day-to-day differences. However, from three independent experiments performed in 2

triplicate on separate days using 0.006% sodium hypochlorite (the only condition that 3

demonstrated a LR > 2 without complete reduction for all tests), the RSD was 27%, 4

which falls within the range (3% to 36%) described previously. Further work is needed 5

to address inter-laboratory reproducibility including repeatability and reproducibility 6

assessment of experiments demonstrating higher LRs. 7

The limit of detection for the decontamination experiments reported in this study 8

allowed for a demonstration of an approximate four log reduction in viable virus for both 9

the bleach and steam treatments. The ability to demonstrate a higher log reduction using 10

BARTS as the method of virus application may be possible with modifications to the 11

experimental parameters. For example, an increase in the initial titer of the virus in the 12

nebulization medium, virus loading times, or airflow through the coupon, would result in 13

an increase in the number of virus loaded onto the substrate and allow for the reporting of 14

greater log reductions. Using a different nebulization medium, virus enumeration 15

method, or virus may also impact the limits of detection. 16

BARTS proved to be valuable in achieving a targeted viral load on the substrate 17

and in the assessment of properties important in bioaerosol testing methods. Further 18

studies are needed to develop a decontamination test method for APMs exposed to 19

bioaerosols, specifically VCPs. A comparison of the efficacy of decontamination 20

procedures on virus applied using standard techniques (pipetting or dipping) to VCPs 21

applied as droplet nuclei will further characterize the effects of bioaerosols. Many of the 22

standard suspension tests and carrier tests use similar mediums amended with defined 23

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protein challenges such as tryptone or bovine serum albumin. A standardization of 1

aerosol medium may be possible as well. The requirements for the protective factor 2

have to be determined from real world scenarios. Consideration must also be given to 3

other FFR types as well as other APMs. Evaluation of other APMs may require the 4

addition of filters and particle detecting instruments placed downstream of the test 5

substrate to enhance characterization of the APM performance. 6

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ACKNOWLEDGEMENTS 1

The authors wish to express our sincere gratitude to Dr. William P. King, Dr. 2

Raymond Roberge, Dr. Benjamin C. Eimer, Mr. Mike Bergman, and the manuscript 3

reviewers for their suggestions and contributions. 4

5

DISCLAIMER 6

The findings and conclusions in this manuscript are those of the authors and do not 7

necessarily represent the views of the National Institute for Occupational Safety and 8

Health (NIOSH). Mention of company names or products does not constitute 9

endorsement by NIOSH.10

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39. Yang, S., Lee, G. W. M, Chen, C-M., Wu, C-C. and Yu, K-P. 2007. The size 1

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Aerosol Med. 20:484-494 3

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Table 1. Comparison of MS2 Recovery Methods 1

FIG. 1. Scematic of the Bio-Aerosol Respirator Test System (BARTS) 2

FIG. 2. Schematic diagram of Test Specimen Holder Assembly. 3

FIG. 3. The effects of a neutralizer on the efficiency of MS2 application to FFR 4

coupons. Results represent averages and the error bars represent the standard deviation 5

of 4 coupons loaded in two experimental runs. 6

FIG. 4. Repeatability of the application of MS2 using BARTS. MS2 was loaded onto 90 7

FFR coupons over a 5 day period. Six coupons were loaded simultaneously during 3 runs 8

per day. Results represent the averages for each filter sample port (I-VI). 9

FIG. 5. The percentage of MS2 deposited on each coupon layer for Low Protective 10

Factor (LPF) and High Protective Factor (HPF) suspension media. Error bars represent 11

standard deviations of 4 samples. 12

FIG. 6. Size distribution of particles generated from the nebulization of suspension 13

media. Combined SMPS and APS Size distribution of particles as measured at the 14

sample ports for Water, Low Protection Factor, and High Protection Factor. The data 15

was normalized to the peak bin value of the scan for each medium type. 16

FIG. 7. Sodium hypochlorite concentration dependent reduction of MS2 aerosolized in 17

Low Protective Factor (LPF) and High Protective Factor (HPF) media. Error bars 18

represent the standard deviation of 3 samples. Note: Bleach concentrations of 0.006, and 19

0.06, and 0.6 for LPF and 0.6% for HPF demonstrated log reductions which reached 20

detection limits. 21

FIG. 8. Steam treatment time dependent reduction of MS2 aerosolized in Low 22

Protection Factor (LPF) and High Protection Factor (HPF) media. Error bars represent 23

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the standard deviation of 3 samples. Note: Steam treatments of 45 seconds and greater 1

for both LPF and HPF demonstrated log reductions which reached detection limits. 2

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Extraction Method Log Recovery SD

Sonication 5.69 0.04

Vortexing 5.87 0.11

Shaking 5.82 0.15

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use

Air

Ho

use

Vac

uu

m

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21

2

3 4

5

6 7

8

910

6. Fan

7. Exhaust port

8. Sample ports

9. Test specimen holders

10. Rotameters

1. Air flow regulator

2. HEPA filter

3. 6-jet Collison Nebulizer

4. Dilution air inlet

5. Aerosol chamber

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6 7

5

4. Filtering Facepiece Respirator Coupon

5. Stainless Steel Screen

6. Hose Barb

7. Hose (to vacuum)

1. O-Ring of Test Specimen Holder Port

(adhered to the exterior of aerosol chamber)

2. Test Specimen Holder (plugs into O-ring of

the test specimen holder port)

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2 Facepiece Respirators for the Evaluation of ... · 2 1 Abstract 2 3 A chamber to apply...

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