aem accepts, published online ahead of print on 31 august...

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Evaluation of Sample Recovery Efficiency of Bacteriophage P22 on Fomites 1

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Amanda B. Herzog 1, 4, Alok K. Pandey 1, David Reyes-Gastelum 5, Charles P. Gerba 4,6, Joan B. 3

Rose 3,4, and Syed A. Hashsham 1,2• 4

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1 Department of Civil and Environmental Engineering, 2Center for Microbial Ecology, 6

3Department of Fisheries and Wildlife, 4Center for Advancing Microbial Risk Assessment, 7

5Center for Statistical Training and Consulting, Michigan State University, East Lansing MI; 8

6Department of Soil, Water and Environmental Science, University of Arizona, Tucson AZ 9

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• Corresponding author: 12

Syed A. Hashsham 13

Department of Civil and Environmental Engineering 14

A126 Research Complex-Engineering 15

East Lansing, MI 48824 16

Phone: (517) 355 8241 17

Fax: (517) 355 0250 18

Email: [email protected] 19

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01370-12 AEM Accepts, published online ahead of print on 31 August 2012

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ABSTRACT 24

Fomites are known to play a role in the transmission of pathogens. Quantitative analysis of the 25

parameters that affect sample recovery efficiency (SRE) at the limit of detection of viruses on 26

fomites will aid in improving quantitative microbial risk assessment (QMRA) and infection 27

control. The variability in SRE as a function of fomite type, fomite surface area, sampling time, 28

application media, relative humidity (RH), and wetting agent was evaluated. To quantify the 29

SRE, bacteriophage P22 was applied onto the fomites at average surface densities of 0.4 ± 0.2 30

and 4 ± 2 PFU/cm2. Surface areas 100 and 1000 cm2 of nonporous fomites found in indoor 31

environments (acrylic, galvanized steel, and laminate) were evaluated with pre-moistened 32

antistatic wipes. The parameters with the most effect on the SRE were sampling time, fomite 33

surface area, wetting agent, and RH. At time zero (initial application of bacteriophage P22), the 34

SRE for 1000 cm2 was on average 40% lower than 100 cm2. For both fomite surface areas, 35

application media trypticase soy broth (TSB) and/or the laminate fomite predominantly resulted 36

in a higher SRE. After the applied samples dried on the fomites (20 min), the average SRE was 37

less than 3%. A TSB wetting agent applied on the fomite, improved the SRE for all samples at 38

20 min. In addition, RH greater than 28% generally resulted in a higher SRE than RH less than 39

28%. Parameters impacting SRE at the limit of detection have the potential to enhance sampling 40

strategies and data collection for QMRA models. 41

42

INTRODUCTION 43

Nonporous fomites (inanimate or nonliving objects) can be an important vehicle in the 44

transmission of viral disease especially for populated indoor environments such as schools, 45

daycare centers, nursing homes, hospitals, food preparation settings or any civil infrastructure (4-46


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6, 26, 33). Human exposure can be through touching and transfer of pathogens present on the 47

fomite to the hands and then to the mouth, nasopharynx, and eyes (5, 25). Exposure can also be 48

from the inhalation of re-aerosolized organisms from contaminated fomites (5, 26). A 49

challenging task can be to control and remediate an indoor environment from an outbreak 50

resulting from an accidental or intentional release of viruses (1, 26). 51

52

After decontaminating an indoor environment, to declare a site as “clean”, quantification of the 53

loss due to sample recovery which is specific to the method(s) used is essential to verify the 54

efficacy of decontamination (17). Quantitative analyses of the parameters that affect SRE from 55

fomites are vital for implementing efficient sampling and detection methods (17). The use of 56

infection transmission models that include the environmental dynamics (environmental 57

conditions, human behavior, survival characteristics of the agent in the environment, etc.) can be 58

used to make decisions on interventions to prevent viral outbreaks (20). Without a quantitative 59

assessment of the abundance of such agents in the environment, generic intervention 60

recommendations could be ineffective (4, 36). 61

62

Survival and SRE studies with viruses have generally been conducted on fomites at surface 63

densities of 102 PFU/cm2 or higher by applying virus stocks in volumes ranging from 5 to 500-µl 64

on fomite areas ranging from 0.38 to 32 cm2 (Table 1). Use of higher initial titers is known to 65

extend the viral survival rate on fomites (5). Under these optimal conditions, results may 66

represent the upper limits of SRE. Surface densities may also be lower than what has been 67

studied so far and pose significant risk (Table 1). However, parameters affecting survival at very 68

low surface densities are less well studied. To our knowledge, only two survival studies (3, 7) 69


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and two SRE studies (18, 37) have been conducted at surface densities ranging from 0.02 – 50 70

PFU or TCID50/cm2 (Table 1 and Table S1 in the supplemental material). These factors may 71

have a significant effect on quantifying the risk to human health after decontamination. 72

73

The objective of this study was to evaluate the parameters that affect SRE of bacteriophage P22, 74

a surrogate for DNA viruses (21, 31), at concentrations close to the limit of detection. 75

Bacteriophage P22 was chosen because it is a surrogate for DNA viruses such as Adenovirus 76

(13, 31), meets many of the desired characteristics of a surrogate (21, 31, 34), and has been 77

successfully used by our group in environmental release and recovery studies (21, 31). The 78

variability of SRE from the parameters such as fomite type, fomite surface area, sampling time, 79

application media, wetting agent, and RH was evaluated. Results presented here have 80

implications for sampling strategies and subsequent microbial risk assessment at low 81

concentrations. 82

83

MATERIALS AND METHODS 84

Bacteriophage P22: preparation, application and sample recovery 85

Bacteriophage P22 that infects the bacterial host Salmonella typhimurium LT2 (ATCC 19585), 86

was provided by Charles P. Gerba (University of Arizona). Bacteriophage P22 is a dsDNA 87

icosahedral-shaped virus with a short tail, 52 to 60 nm in size, and belongs to the family 88

podoviridae (31). To prepare bacteriophage P22, 1-ml of bacteriophage P22 stock was added to 89

25-ml of the bacterial host, S. typhimurium, at log phase in TSB (Difco, Becton Dickinson and 90

Company, Sparks, MD). After 24 hr incubation at 37 oC, 0.1-ml of lysozyme and 0.75-ml of 91

ethylenediamine tetraacetic acid (EDTA) were added to the solution and centrifuged at 2390 x g 92


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for 10 minutes. The supernatant was filtered through a 0.45 µm filter (Millipore) to remove the 93

bacterial cells and debris (31). Bacteriophage P22 was then diluted in suspensions of phosphate 94

buffered saline tween-80 (Fisher Scientific, NJ) (PBST), TSB or sterile distilled water. 95

96

Fomites, simulating an indoor environment, included acrylic (Optix, Plaskolite Inc, Columbus, 97

OH), galvanized steel (Type 28 GA galvanized, MD Building Products, Oklahoma City, OK) 98

and laminate (Type 350 #60 mate finish, Wilsonart International Inc, Temple, Texas) with 99

surface areas of 100 and 1000 cm2. Fomites and testing area were disinfected with 70% ethanol, 100

rinsed with sterile distilled water and dried. Bacteriophage P22 was applied in PBST, TSB or 101

water on the fomite in a grid formation comprising of fifty 1-µl droplets. The average amount of 102

bacteriophage P22 applied to the fomite was 433.1 ± 194.5 PFU, approximately 8.66 103

PFU/droplet, with average surface densities of 4.3 ± 1.9 PFU/cm2 for 100 cm2 and 0.4 ± 0.2 104

PFU/cm2 for 1000 cm2. The recovery material, pre-moistened Fellowes Screen Cleaning Wipes 105

are generally used to remove dirt, dust and finger prints from office equipment (#99703, 106

Fellowes, Itasca, IL) and are antistatic, non-toxic and alcohol free. The pre-moistened wipes are 107

made of crepe fabric (crepe material is treated as a trade secret by Fellowes) and wetted by the 108

manufacture with water and detergent (propylene glycol ethers). Pre-moistened wipes were cut 109

into 48 cm2 pieces using sterilized scissors and stored in sterile whirl pack bags at room 110

temperature during the experiment lasting no more than 12 hr. Fresh pieces were cut and used 111

each day. Sampling was done by moving the pre-moistened wipes over the entire fomite twice 112

(in perpendicular directions to each other). Two samples were taken; one immediately after the 113

initial application (referred to as 0 min) and another after the samples were visibly dry (which 114

was 20 min). Control experiments conducted with bacteriophage P22 suspensions to determine 115


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if the moistening agent had an effect on the viability of the virus indicated that on average 116

95% (range 80 – 125%) of bacteriophage P22 could be recovered with inoculation directly onto 117

the wipe and dissolution with PBST. Very high recoveries were also seen at time zero on 118

fomites with no drying. 119

120

After sampling, the recovery material was placed into a 50-ml tube containing 5-ml of PBST and 121

vortexed for 30 seconds. Bacteriophage P22 was assayed using a double agar layer method (14). 122

The sample containing bacteriophage P22 (1-ml) was added to 2.5-ml of melted 1% agar overlay 123

(1 g bacto agar/100 ml TSB) (Bacto agar: Difco, Becton Dickinson and Company, Sparks, MD) 124

with 0.3-ml of S. typhimurium in the log phase. The solution was rolled by hand for mixing and 125

immediately dispensed evenly onto 1.5% trypticase soy agar (TSA) (Difco, Becton Dickinson 126

and Company, Sparks, MD) plates. After the overlay agar solidified, the plates were incubated 127

at 37 oC for 24 hr; the number of plaque forming units was then counted. The SRE experiments 128

conducted used a total of 324 plates. These consisted of 3 fomite types, 2 sampling times, 3 129

application media, and 2 fomite surface areas. Each SRE measurement was made in triplicate 130

and repeated on three different days. Because the PFUs recovered were already very low, 131

dilution of samples was not necessary. For each sample recovery experiment, positive controls 132

were conducted in triplicate. Fifty 1-µl droplets of bacteriophage P22 inoculated in 950-μl of 133

PBST (same as extraction solution) were dispensed into a 1.5-ml microcentrifuge tube. The 1-ml 134

bacteriophage P22 control was dispensed as described above. 135

136

Single agar layer method to separate bacteriophage P22 survival from sample recovery 137

When evaluating the survival of bacteriophage P22 on fomites, fifty 5-μl droplets containing an 138


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estimated 3.96 PFU/droplet suspended either in TSB or water were applied on polystyrene Petri 139

dish surface (100 x 15 mm) in a grid formation. An average of 198 ± 65 PFU were applied to 140

each plate with an average surface density of 2.5 ± 0.9 PFU/cm2. For this experiment, the time 141

of first sampling (other than the initial at time zero) was changed to 1 hr instead of 20 min 142

because the 5-μl droplets took longer to visibly dry on the Petri dish. Samples were evaluated at 143

0, 1, 2, 4, 8, 12, and 24 hr by implementing a single agar layer method. This method allowed us 144

to evaluate the PFU remaining but eliminated the need to recover them from a surface because 145

bacteriophage P22 was directly applied on the Petri dish surface. The assay consisted of 146

dispensing 3-ml of melted 1% agar overlay (1 g bacto agar/100 ml TSB) with 0.5-ml of S. 147

typhimurium in the log phase and 2-ml of TSB onto the Petri dish surface where bacteriophage 148

P22 were applied. After the overlay agar solidified, the plates were incubated at 37 oC for 24 hr 149

at which point the number of plaque forming units was then counted. The experiment was 150

conducted twice using six replicates per time point spanning 7 sampling time points and two 151

application media (thus using a total of 168 plates). For each survival experiment a positive 152

control was also included in triplicate which consisted of fifty 5-μl droplets of bacteriophage P22 153

inoculated in 750-μl of PBST in 1.5-ml microcentrifuge tubes. One ml of this positive control 154

was plated as described above. 155

156

Relative humidity and TSB wetting agent 157

The RH and temperature in the laboratory were measured before each experiment with a digital 158

RH and temperature meter (VWR Scientific Products). The average temperature of the 159

laboratory during these experiments was 20.8 ± 0.23 (mean ± standard deviation). The RH 160

ranges of 9 – 23% and 28 – 32% were the natural RHs of the laboratory during the winter and 161


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summer months respectively (Figure 3). For RH range of 55 – 58%, a small laboratory space (14 162

ft x 7 ft x 9 ft) was equipped with a humidifier (Bionaire, Milford, MA). 163

164

Previous studies support that use of a wetting agent applied to the recovery material (wipe or 165

swab) to enhance the SRE (9, 18, 19, 29). In a preliminary experiment, PBST and TSB were 166

compared as wetting agents applied on the fomite surface to evaluate its effect on SRE 167

enhancement at 20 min. There was no statistical differences between the SREs when PBST or 168

TSB as wetting agent was applied on the fomite (p=0.232, n=27, student t-test, data not shown). 169

Hence in further SRE experiments, a TSB wetting agent was used (this step is referred to as TSB 170

wetting). Using a disposable spreader, 200 and 400 µl of TSB was applied and uniformly 171

distributed over 100 and 1000 cm2 fomite surface area, respectively. The recovery material 172

sampled both the disposable spreader and the fomite. The recovery materials were processed as 173

described above. This experiment used a total of 162 plates consisting of 2 wetting conditions, 1 174

fomite surface area, 1 sampling time, 3 fomites, 3 application mediums, and 3 RH. Each 175

measurement was made in triplicate. A positive control was also included in triplicate as 176

described previously for the SRE experiments. 177

178

Percent surface recovery efficiency computations and statistical analysis 179

Percent sample recovery efficiency was calculated as: 180

181

( )100% ×=

control

assay

N

DNSRE (1) 182

183


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where %SRE was the sample recovery efficiency from the fomite, Nassay was the number of 184

plaque forming units counted on the agar plate from sampling the fomite, D was the dilution 185

factor (the total extraction volume divided by the volume of sample assayed), and Ncontrol was the 186

number of units on the agar plate from the control experiment. 187

188

The data (%SRE) had considerable differences in variance, especially between 0 min and 20 189

min. Due to this, the data were transformed by adding 1 (to account for the zero values) and 190

converted to a log scale. After analyzing the residuals, it was determined that the normality 191

assumption of the residual did not fit the equation, therefore the residuals were fitted under the 192

assumption of a gamma distribution. Two equations for the transformed outcome were used to 193

study the relationships between fomite type, application media, RH, and wetting condition. 194

195

Log (%SRE +1) = βo + β1X1 + β2X2 + β3X1X2 + e (2) 196

197

Where for equation 2, Log (%SRE +1) is the log transformed SRE, X1 is an independent variable 198

that denotes the fomite type so X1 is a nominal variable with no numerical value (acrylic, 199

laminate, and galvanized steel as categories) for which laminate was taken as the reference 200

category in the analyses, X2 is an independent variable that denotes the application media so X2 201

is a nominal variable (PBST, TSB, and water as categories) for which water was taken as the 202

reference category in the analyses, X1X2 is the interaction between fomite type and application 203

media, and e is the error term. The intercept βo represents the average value of the reference 204

group, in this case, the average value of the log of the reference categories laminate fomite and 205


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water media. The terms β1, β2, and β3 are the regression coefficients known as the effect for the 206

corresponding independent variable X1, X2, and X1X2, respectively. 207

208

Log (%SRE +1) = βo + β1X1 + β2X2 + β3X3 + β4X4 + β5X1X2 + β6X2X3 + β7X1X3 + e (3) 209

210

In equation 3, Log(%SRE +1) is the log transformed SRE, X1 and X2 are defined as in equation 211

(2), X3 is an independent variable that denotes RH range so X3 is a nominal variable (9 – 23%, 212

28 – 32%, and 55 – 58% as categories) for which 55 – 58% was taken as the reference category 213

in the analyses, X4 is an independent variable that denotes the use of TSB wetting for the sample 214

collection so X4 is a nominal variable (no wetting and TSB wetting as categories) for which TSB 215

wetting was taken as the reference category in the analyses, and e is the error term. As before, 216

the intercept βo represents the average value of the log of the reference categories laminate 217

fomite, water media, 55 – 58% RH and TSB wetting. The interaction terms are X1X2 (fomite 218

type and application media), X2X3 (application media and RH), and X1X3 (fomite type and RH). 219

The regression coefficients (β1-7) are known as the effect for the corresponding independent 220

variable X1-4, X1X2, X2X3, and X1X3, respectively. 221

222

Because the independent variables used were nominal, dummy variables were used to compare 223

the different categories to the corresponding reference categories. The dummy variable described 224

the set of experimental conditions consisting of fomite type, application media, RH, and wetting 225

condition as single entity and evaluate the SRE for each set to the next by treating two such sets 226

as reference (laminate and water). A regression was run using SAS 9.2® with the GLIMMIX 227

procedure to evaluate the equations. Data was analyzed to evaluate the type III test of fixed 228


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effects (emanating from the factors being investigated) to determine the significance of each of 229

the parameters specified in the model statement (27). Analysis of the model was performed in 230

groups wetting condition, fomite surface area and sampling time. Patterns in the experimental 231

data indicated differences to explore certain effects. This limited error rates and avoided 232

canceling of significant effects. 233

234

RESULTS 235

SRE of bacteriophage P22 from various fomites 236

For 100 cm2 fomite surface area and the three application media (PBST, TSB, or water), the 237

average SRE for the experimental data at 0 min was 46 ± 6.9% (SRE ± standard deviation) for 238

acrylic, 70 ± 7.7% for galvanized steel, and 92 ± 6.4% for laminate (Figure 1A). The type III 239

test of fixed effects (equation (2)) for 100 cm2 at 0 min was significant for fomite type 240

(p<0.0001), application media (p<0.0001) and the interaction between fomite type and 241

application media (p=0.0128) (Table S2). Based on equation (2), laminate yielded the highest 242

SRE while acrylic gave the lowest SRE regardless of which application media was used. 243

However, use of TSB did result in a higher SRE than the other media. PBST and TSB 244

performed similarly on acrylic while PBST and water performed similarly on laminate (Table 245

S3). At 20 min, the average SRE for acrylic, galvanized steel and laminate were all less than 1 ± 246

0.9% for all application media (Figure 1A). The type III test of fixed effects for 100 cm2 at 20 247

min was significant for fomite type (p=0.0047) and application media (p<0.0001) but not 248

significant (p=0.3589) for the interaction between fomite type and application media (Table S4). 249

For these conditions, the application media significantly affected the SRE and a higher SRE was 250

observed from application media TSB. Similar to 0 min, laminate resulted with a higher SRE 251


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while acrylic resulted with a lower SRE. Similar results were observed on acrylic and 252

galvanized steel when applied in PBST media (Table S3). 253

254

Considering all application media for 1000 cm2 fomite surface area, the average SRE for the 255

experimental data at 0 min was 21 ± 6.9% for acrylic, 26 ± 3.1% for galvanized steel, and 42 ± 256

19.2% for laminate (Figure 1B). The type III test of fixed effects for 1000 cm2 at 0 min was 257

significant for fomite type (p<0.0001), application media (p=0.0037) and the interaction between 258

fomite type and application media (p=0.0998) (Table S5). The laminate fomite yielded the 259

highest SRE while acrylic fomite gave the lowest SRE irrespective of the application media. The 260

use of TSB resulted in higher SRE while PBST and water had statistically equivalent SREs 261

(Table S6). At 20 min, the average SRE for the 1000 cm2 fomite surface area was 2 ± 1.4% or 262

less for all surfaces and application media (Figure 1B). The type III test of fixed effects for 1000 263

cm2 at 20 min was significant for fomite type (p<0.0001) and application media (p=0.0053) but 264

not significant for the interaction between fomite type and application media (p=0.3720) (Table 265

S7). The laminate fomite had the highest SRE while acrylic and galvanized steel had lower and 266

comparable SREs. The use of TSB and water as application media resulted in a higher SRE than 267

PBST (Table S6). 268

269

SRE versus decay for bacteriophage P22 270

The method employed to determine SRE includes the loss due to decay. To separate this loss 271

from the SRE, bacteriophage P22 was directly applied onto a Petri dish (using TSB and water) 272

and decay was quantified as described in the Methods section using the single agar layer method. 273

The decay rates for bacteriophage P22 were 7.97x10-2 hr-1 when applied in TSB and 6.81x10-2 274


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hr-1when applied in water. After 1 hr, when the 5-μl droplets were visibly dry on the Petri dish; 275

majority of the applied bacteriophage P22 were still infective (89.4 ± 6.7% in TSB and 87 ± 276

7.9% in water). This was substantially higher than the SREs at 20 min employing the double 277

agar layer method which was 0% in water and 0.62 ± 1.3% in TSB for the 100 cm2 acrylic fomite 278

and 0.76 ± 1.6% in water and 0.69 ± 1.5% in TSB for the 1000 cm2 acrylic fomite. Even at 24 hr 279

2 – 5% of bacteriophage P22 was detectable using the single agar method (Figure 2). These 280

results indicate that low or zero SRE may not always indicate absence of the target because SREs 281

also include loss due to sample recovery. 282

283

Impact of wetting agent at varying relative humidity 284

From the above experiment, it was clear that significant portion of the bacteriophage P22 was 285

still active on the fomite at 20 min and the recovery material was unable to recover the dried 286

sample. To enhance recovery, TSB was applied to the fomite as a wetting agent for SREs at 20 287

min (Figure 3). Each point on the distribution represents the experimental data for each fomite 288

type, application media, RH and TSB wetting combination. The type III test of fixed effects 289

using equation (3) was significant for application media (p<0.0001), RH (p<0.0001), the 290

interaction between fomite type and application media (p=0.0001), the interaction between 291

fomite type and RH (p=0.0048) and the interaction between application media and RH 292

(p<0.0001). It was not significant for fomite type (p=0.7634). Use of TSB wetting step was 293

significantly different from not using it (p<0.0001) (Table S8). The TSB wetting step improved 294

the mean SRE for all cases. For both TSB wetting and no TSB wetting, bacteriophage P22 295

applied in TSB media resulted in a higher SRE than the PBST and water. Exception to this was 296

the acrylic and galvanized steel where water gave higher SRE at 55 – 58% RH range (Table S9). 297


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298

Overall, regardless of wetting agent, a higher average SRE result was primarily observed at RH 299

of 28 – 32% and 55 – 58%. The SRE values for both these humidity ranges were not statistically 300

different from each other. Mean predicted SRE was predominantly lower for RH range of 9 –301

23% compared to the other two ranges. When water was used as the application media, the 302

highest average SRE was always obtained for 55 – 58% RH and the lowest for 9 – 23% RH 303

range (Table S9). The effects of RH on SRE for the other application media (TSB and PBST) 304

were less obvious than water. 305

306

DISCUSSION 307

Once decontamination has been conducted on an indoor site due to a viral outbreak or 308

bioterrorism event, environmental monitoring and quantitative microbial risk assessment 309

modeling will help determine the risk to human health and if the indoor site can be declared 310

“clean” (16, 17). When monitoring fomites for viruses near the detection limit, the results from 311

the linear regression equation suggest that the sampling priority should be for 100 cm2 laminate 312

fomite. At both sampling times (0 min and 20 min) and both fomite surface areas evaluated (100 313

cm2 and 1000 cm2), laminate resulted in a higher SRE compared to the other fomites under the 314

same conditions. An increase in the fomite surface area from 100 to 1000 cm2 decreased the 315

average SRE at 0 min by approximately 25% for acrylic, 40% for galvanized steel, and 50% for 316

laminate (Figure 1). A lower SRE for the larger surface area was expected because the surface 317

density was also lower. Previous studies suggest that one method may not fit all scenarios and 318

sampling for larger fomite surface areas, use of alternative recovery material may be more 319

appropriate (12). Wipe methods are generally used for fomite surface areas of 10 – 25 cm2 but it 320


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is unknown what influence fomite surface area may have on the SRE (12). Low surface 321

densities will require sampling of larger surface areas. Given that the SRE at 1000 cm2 was 322

lower than the SRE at 100 cm2 and SRE includes decay, sampling at low surface densities must 323

be carried out with caution. 324

325

In general, the application medium TSB produced higher SREs than PBST and water. TSB is an 326

organic medium used for the growth of bacteria and may have properties that were more 327

stabilizing for the bacteriophage P22 on the fomite than other media. It has been suggested that 328

suspension in more complex media may affect resistance to desiccation (30). Most of the SRE 329

studies reported earlier used organic media to suspend viral particles before applying to the 330

fomites (Table 1). The higher SRE in TSB application media suggest that application media may 331

also influence the SRE, especially at low surface densities. 332

333

The most dramatic reduction in the average SRE of bacteriophage P22 from the fomite was with 334

time (0 min vs. 20 min). Initially, inactivation of bacteriophage P22 could be the main reason for 335

this loss in SRE when the sample was dry on the fomite (20 min). Most of the rapid inactivation 336

occurs during the period of desiccation when bacteriophage P22 becomes less stable on the 337

fomites compared to a liquid medium (21). In addition, the concentration that was applied to the 338

fomite was rather low, close to the limit of detection of the plaque assay. Viral survival 339

increases with increase in concentration which can stabilize the virus against environmental 340

stressors (5). On average, less than 3% of bacteriophage P22 was recoverable after 20 min on 341

the fomite. The SREs reported at 20 min varied widely from 3 – 98.4% (Table 1). Each of the 342

studies had a different experimental approach for determining the SRE from fomites which may 343


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account for the broad range of SREs reported. Keswick et al. (19), using cotton swabs recovered 344

rotavirus, poliovirus and bacteriophage f2 immediately after applying the samples to the fomite. 345

Similarly, cotton swabs were used to recover norovirus and rotavirus dried for 15 min on the 346

fomite by Scherer et al. (29). Taku et al. (37) evaluated three methods, moistened cotton swabs 347

or nylon filter, fomite contact with elution buffer and aspiration, and scraping with aspiration to 348

recover feline calicivirus dried for 15 min on the fomite. Recovery materials antistatic cloth, 349

cotton swab and polyester swab were evaluated by sampling bacteriophage MS2 dried for 45 min 350

on the fomite(18). Sattar et al. (28), analyzed the SRE of human rhinovirus 14 dried on 1 cm 351

diameter disks for 1 hr, then eluted the virus by submerging the disk in 1-ml of tryptose 352

phosphate broth and sonication. It is evident that the number of parameters influencing the SREs 353

is rather large posing a challenge for simple comparison. 354

355

A positive sample result indicates surface contamination and potential risk of exposure. 356

However, a negative result does not entirely assure the absence of infectious agents and the 357

absence of the potential risk of exposure (29). Following the same protocol, Masago et al. (21) 358

found bacteriophage P22 to survive for 36 hr on 10 cm2 fomites (aluminum, ceramic, glass, 359

plastic, stainless steel, and laminate) when applied at a surface density of approximately 107 360

PFU/cm2 (Table 1). The decay rate of bacteriophage P22, reported in Masago et al. (21), for the 361

plastic fomite was 5.2x10-3 hr-1. When eliminating the recovery method by applying the 362

bacteriophage P22 (surface density 2.5 ± 0.9 PFU/cm2) directly onto the Petri dish (plastic 363

fomite), 2 – 5% of bacteriophage P22 could be detected at 24 hr. The decay rate for 364

bacteriophage P22 on Petri dish was estimated to be 7.97x10-2 hr-1 when applied in TSB and 365

6.81x10-2 hr-1 when applied in water. The differences between the decay rates in Masago et al. 366


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(21) and this study were most likely due to the sample concentrations, higher initial titers have 367

shown to extend survival on fomites (5). As seen in Figure 2, at 1 hr (5-μl droplets were visibly 368

dry) an average of 88.2 ± 7.3% of the applied bacteriophage P22 was still active. Majority of the 369

loss (40 – 60%) occurred between hours 1 and 2. Compared to this, the average SRE from the 370

100 cm2 and 1000 cm2 acrylic surface at 20 min (1-μl droplets visibly dry) was less than 1% 371

(Figure 2). Survival of organisms on fomites is known to be agent-specific and ranges from 0.75 372

hr for rotavirus to 90 days for astrovirus (Table 1). Temperature, RH, fomite surface area, and 373

sample concentration are all known to effect survival (5, 33, 40). Knowledge of the organisms’ 374

response to environmental stress on the fomite is important in determining the appropriate 375

detection methods and employing clean up strategies. 376

377

The results of the experiment designed to separate the decay from sample recovery (Figure 2) 378

revealed that for surface densities of 0.4 – 4 PFU/cm2, SRE was low due to poor efficiency of 379

recovery method rather than decay. The TSB wetting step improved the SRE for all cases at 20 380

min (Table S9). SRE results doubled in the majority of the cases, especially when the 381

application media was TSB. However this TSB wetting step, the combination of the scraping 382

from the disposable spreader and the TSB wetting solution applied onto the fomite, may have 383

physically dislodged the viral particles resulting with a higher SRE than without the TSB wetting 384

step (37). It can also be speculated that the bacteriophage P22 may adhere strongly to the fomite 385

surface after drying or attach to an imperfection on the fomite and the sampling material cannot 386

desorb the virus off the fomite. Surface roughness has been shown to influence adhesion and 387

cell retention to fomites, which can affect recovery (32, 39). In this study, surface roughness was 388


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not measured. The addition of the TSB wetting step demonstrates the potential to further desorb 389

viruses from the fomite and improve SRE. 390

391

The RH and temperature are crucial parameters to viral survival on fomites (Table 1) (4, 5, 33). 392

A higher SRE was observed for bacteriophage P22 at RH ranges of 28 – 32% and 55 – 58% 393

regardless of the use of a wetting agent (Figure 3). At RH range of 9 – 23%, lowest SREs were 394

obtained (Table S9). The combination of application media and RH may also play a significant 395

role in SRE. Bacteriophage P22 applied in water consistently had the highest SRE at 55 – 58% 396

and the lowest SRE at 9 – 23%. However, for the RH ranges evaluated, its effect was not as 397

obvious for the other application media. The interaction between RH and application media may 398

be a useful parameter in implementing sampling strategies. 399

400

CONCLUSIONS 401

Efficient sample recovery and detection methods are essential in determining the exposure of 402

humans to viruses and the resulting risk in a contaminated indoor environment. The SRE of 403

bacteriophage P22 from fomites at concentrations near the limit of detection was most influenced 404

by time of sampling, fomite surface area, use of a wetting agent and RH. The observations made 405

here using bacteriophage P22 as a surrogate highlight some of the factors that must be 406

considered when sampling for very low surface densities of threat agents. Understanding the 407

contributions of decay and recovery in the overall measured SREs under various conditions and 408

the parameters affecting them will assist in implementing appropriate sampling methods and 409

decontamination strategies. 410


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411

ACKNOWLEDGMENTS 412

This study was supported by the Center for Advancing Microbial Risk Assessment funded by the 413

U.S. Environmental Protection Agency and Department of Homeland Security grant number 414

R83236201. We thank Rebecca Ives, Yoshifumi Masago, and Tomoyuki Shibata at Michigan 415

State University for their technical support. 416

417

LIST OF TABLES 418

Table 1 - Parameters from survival and SRE studies evaluating viruses applied to fomites. 419

420

LIST OF FIGURES 421

Figure 1- Experimental SRE of bacteriophage P22 from fomites acrylic, galvanized steel, and 422

laminate. Bacteriophage P22 was applied in media PBST, TSB and water to fomites surface 423

areas of (a) 100 cm2 and (b) 1000 cm2. The pre-moistened wipe recovered bacteriophage P22 at 424

the initial application time (0 min) and after drying (20 min). Each bar represents the average of 425

nine plates and the error bars represent the standard deviation of those nine plates. 426

427

Figure 2– Loss of bacteriophage P22 due to decay versus the loss due to sample recovery and 428

decay. Survival of bacteriophage P22 is signified by circles, ● applied in TSB and ○ applied in 429

water, and each point represents the mean and standard deviation of twelve plates. SRE from 100 430

cm2 is signified with triangles,▼applied in TSB and ∆ applied in water. SRE from 1000 cm2 is 431

signified by squares, ■ applied in TSB and □ applied in water. Each point of the SRE data 432

represents the mean and standard deviation of nine plates. 433


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434

Figure 3 - The experimental impact of RH and TSB wetting agent on the SRE of bacteriophage 435

P22 after drying (20min) on 100 cm2 fomite surface area. Bacteriophage P22 was sampled with 436

pre-moistened wipes at RH ranges of (a) 9 – 23%, (b) 28 – 32%, and (c) 55 – 58%. Each dot on 437

the distribution represents the SRE from a single fomite (acrylic, galvanized steel, and laminate) 438

and application media (PBST, TSB, and water) combination. Those with the highest SRE were 439

labeled. The solid line of the box plot represents the median SRE and the dashed line represents 440

the mean SRE. The box plot whiskers above and below the box indicates the 90th and 10th 441

percentiles, respectively. 442

443


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Table 1 4

Organism Sample

Concentration

Fomite Area

(cm2)

Application

Volume

(μl)

Surface

Density

Application

Medium

Relative

Humidity

( % )

Temperature

(oC)

Survival or

SRE

Ref.

Survival studies

Alphaviruses

Ebola virus

Lassa virus

1.5x107–4.5x1010 PFU/ml 0.25 NRa 6.4x106 PFU/cm2 (A), 7.6x107 PFU/cm2 (E), 5.6x107 PFU/cm2

NR 30–40 20, 25 6–14 days (26)

Astrovirus 1x105–5x105 PFU 1,3 20, 50 3.3x104-1.6x105PFU/cm2 Phosphate buffered saline (PBS) or 20% fecal suspension (FS)

90±5 4, 20 10–90 days (2)

Avian metapneumovirus

Avian influenza virus

3.1x106–6.3x106 TCID50/mlb

1 10 3.4x104–6.3x104 TCID50/cm2 NR NR NR 1–6 days (39)

Bacteriophage P22 1x109 PFU/ml 10 10 107 PFU/cm2 NR 50 25 859 hours (21)

Calicivirus 107 TCID50/ml 1 20 2.0x105 TCID50/cm2 NR NR NR 4–72 hours (10)

Coronavirus 104–105 MPNc 1 10 104–105 MPN/cm2 Cell culture medium 20±3, 50 ±3, 80 ±3 4, 20, 40 0.25–28 days (8)

Coronavirus 107 PFU/ml 0.79 10 1.3x105 PFU/cm2 PBS 55, 70 21 3–6 hours (35)

Feline calicivirus

Norwalk virus

109 PFU/ml (FC),

106 RT-PCRU/ml (N)

25 NR 4x107PFU/cm2 (FC), 4x102 RT-PCRU/cm2

20% FS in PBS 75–88 22±2 7 days (11)

Hepatitis A virus

Polio virus

10–fold dilution 0.79 10 N/A 10% FS in saline 25±5, 55±5, 80±5, 95±5

5, 20, 35 4–96 hours HAV,

4–12 hours PV

(22)

Hepatitis A virus

Human rotavirus

Enteric adenovirus

Poliovirus

NR 1 20, 50, 100 N/A PBS or FS 50±5, 85±5, 90±5 4, 20 30–60 days HAV,

30–60 days HRV,

5–60 days ADV,

5–60 days PV

(1)

Rotavirus

Poliovirus

Bacteriophage f2

103–105 PFU/ml 250 Misted N/A Distilled water, distilled water with 10% FS

NR NR 0.75–1.5 hours (19)

Influenza A virus 2x108 PFU/ml NR 20 N/A NR 50–60 22±2 6–24 hours (23)

Influenza A virus 106 TCID50/ml 1 10 104 TCID50/cm2 Eagle minimal essential medium with 25mM HEPES and Earle’s salts

30–50 21–28 2 hours–17 days (38)

Influenza A virus 1.5x108 TCID50/ml 2 10 7.5x105 TCID50/cm2 1% BSA 23–24 17–21 4–9 hours (15)

Influenza A and B virus 103–104 TCID50/0.1 ml 7.07–19.63 100 50–1.4x103 TCID50/cm2 NR 35–40(A),

55–56(B)

27.8–28.3(A), 26.7–28.9(B)

24–48 hours (3)

Parainfluenza 1.5x100, 1.5x103,

1.5x104 TCID50/ml

32 500 0.02–2.0x102 TCID50/cm2 Minimun essential medium with Earle’s salts

NR 22 6–10 hours (7)


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a. NR-not reported 5 b. TCID50-median tissue culture infective dose 6 c. MPN-most probable number 7 d. N/A – not applicable, not able to calculate surface density from information reported 8 9

Rhinovirus 107 PFU/ml 0.79 10 1.3x105 PFU/cm2 Tryptose phosphate broth, bovine mucin, human nasal discharge

20±5, 50±5,

80±5

20±1 2–25 hours (28)

Zaire ebolavirus

Lake Victoria marbugvirus

1x106 TCID50/ml 0.38 20 5.2x104 TCID50/cm2 Guinea pig sera, tissue culture media

55±5 4, 22 14–50 days (24)

SRE studies

Bacteriophage MS2 1x106 PFU/ml 25 5 3.7 PFU/cm2 50% solution of TSB and dilution buffer (5 mM NaH2PO4 and 10 mM NaCl)

45–60 20–22 7–40% (18)

Feline calicivirus 7.0x105–1.3x106 TCID50/100µl

25.8, 929, 5,290

20 26–104 TCID50/cm2 10% FS in PBS NR NR 3–71% (37)

Rotavirus

Poliovirus

Bacteriophage f2

103–105 PFU/ml 250 Misted N/A Distilled water, distilled water with 10% FS

NR NR 16.8±6% (R), 42.3±1.9% (P), 10.6±5.7%(B)

(19)

Norovirus

Rotavirus

2.0x107 RT-PCRU/ml(N)

2.0x105 RT-PCRU/ml(R)

10 100 2.0x103 , 2.0x104 RT-PCRU/cm2 (N)

2.0x101 , 2.0x102 RT-PCRU/cm2 (R)

10% PBS NR NR 10.3±13.0–51.9±38.5% (N)

5.4±1.5–57.7±25.9% (R)

(29)

Rhinovirus 107 PFU/ml 0.79 10 1.3x105 PFU/cm2 Tryptose phosphate broth, bovine mucin, human nasal discharge

50±5 22 40.3–98.4% (28)


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Figure 1 450 451 452 453 a. 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 b. 475 476 477 478 479 480

481 482 483 484 485 486 487 488 489 490 491 492 493 494 495

0

20

40

60

80

100

120

Acr

ylic

Gal

vani

zed

Ste

el

Lam

inat

e

PBST PBSTTSB TSBWater Water

SR

E (

%)

100 cm2

0 min 20 min

Application Medium

0 min

0

20

40

60

80

100

120

PBST TSB Water

20 min

PBST TSBWater

Acr

ylic

G

alva

nize

d S

teel La

min

ate

SR

E (

%)

1000 cm2

Application Medium


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496 497 498 499 Figure 2 500 501 502 503

Log Time (hr)

0.1 1 10 100

Su

rviv

al o

r S

RE

(%

)

0

20

40

60

80

100No Loss

Loss due to decay (single agar layer method)

Loss due to sample recovery and decay (double agar layer method)


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Figure 3 504 505

(a) 9-23% rH

SR

E (

%)

0

10

20

30

40

No Wetting

TSB Wetting

(b) 28-32% RH

0

10

20

30

40

No Wetting

TSB Wetting

(c) 55-58% RH

0

10

20

30

40

No Wetting

TSB Wetting

Acrylic + TSB

Laminate + TSB Laminate + TSB

Acrylic + TSB

Laminate + TSB

Acrylic + TSB


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