key: cord-0807822-gbmnlzym
authors: Anderson, Claire E.; Boehm, Alexandria B.
title: Transfer rate of enveloped and non-enveloped viruses between fingerpads and surfaces
date: 2021-06-23
journal: bioRxiv
DOI: 10.1101/2021.06.22.449538
sha: 3d945c9f851afb4ca3267c88acec3102e09a7ae7
doc_id: 807822
cord_uid: gbmnlzym

Fomites can represent a reservoir for pathogens, which may be subsequently transferred from surfaces to skin. In this study we aim to understand how different factors (including virus type, surface type, time since last handwash, and direction of transfer) affect virus transfer rates, defined as the fraction of virus transferred, between fingerpads and fomites. To determine this, 360 transfer events were performed with 20 volunteers using Phi6 (a surrogate for enveloped viruses) and MS2 (a surrogate for non-enveloped viruses), and three clean surfaces (stainless steel, painted wood, and plastic). Considering all transfer events (all surfaces and both transfer directions combined), the mean transfer rates of Phi6 and MS2 were 0.17 and 0.26, respectively. Transfer of MS2 was significantly higher than Phi6 (P<0.05). Surface type was a significant factor that affected the transfer rate of Phi6: Phi6 is more easily transferred to and from stainless steel and plastic than to and from painted wood. Direction of transfer was a significant factor affecting MS2 transfer rates: MS2 is more easily transferred from surfaces to fingerpads than from fingerpads to surfaces. Data from these virus transfer events, and subsequent transfer rate distributions, provide information which can be used to refine quantitative microbial risk assessments. This study is the first to provide a large-scale data set of transfer events with a surrogate for enveloped viruses, which extends the reach of the study to the role of fomites in the transmission of human enveloped viruses like influenza and SARS-CoV-2. Importance This study created the first large-scale data set for the transfer of enveloped viruses between skin and surfaces. The data set produced by this study provides information on modelling the distribution of enveloped and non-enveloped virus transfer rates, which can aid in the implementation of risk assessment models in the future. Additionally, enveloped and non enveloped viruses were applied to experimental surfaces in an equivalent matrix to avoid matrix effects, so results between different viral species can be directly compared without confounding effects of different matrices. Our results indicating how virus type, surface type, time since last handwash, and direction of transfer affect virus transfer rates can be used in decision-making processes to lower the risk of viral infection from transmission through fomites.

Relative humidity during the study ranged from 13% to 74%, with a median value of 58%. Full 116 temperature and humidity data are available in the SI. 117 Transfer Rate Distributions. All negative controls had 0 PFU and all viral stock 118 concentrations had an expected number of PFU/mL. 119 The fraction of virus transferred (f) was determined for 360 transfer events for the two 120 viruses. Out of the 360 transfer events for Phi6, all three dilutions plated were TNTC 8 times. All 121 three dilutions were lower than the detection limit 38 times. As a result, 46 transfer events were 122 removed from the data set for Phi6, leaving 314. Out of the 360 transfer events for MS2, there 123 were no instances where all dilutions exceeded the limit of detection. The three dilutions were 124 lower than the detection limit 4 times for MS2. As a result, 4 transfer events were removed from 125 the data set for MS2, leaving 356. The instances where the transfer rate was irrecoverable for 126 Phi6 and MS2 are not limited to a single surface, time since last handwash, or direction of 127 transfer. The instances also make up less than 7% of the total data, and therefore are not 128 anticipated to affect the overall distribution of the data. More information about these instances 129 of irrecoverable transfer rates can be found in Table 2 . 130 The mean transfer rate for Phi6 was 0.17, while the median was 0.12 and the standard Overlayed on the histogram in Figure 4 is the distribution that best fit the data of the distributions 139 tested, along with the distribution parameters. In the case of both virus type, beta distributions fit 140 the data best. For each virus, the beta distribution had the highest log-likelihood estimate, the 141 lowest AIC, and a p-value greater than 0.05. Although the normal distribution fit the data well (a these data as the Kolmogov-Smirnoff tested suggested the data could be reasonable 148 approximated as normal. The 'time since last handwash' factor was not significant in the model 149 (P=0.87). In terms of interactions between variables, significant two-way interactions were found 150 between the 'virus type' and 'surface type', the 'virus type' and 'time since last handwash', and 151 'surface-type' and 'time since last handwash'. The remaining unlisted interactions were not 152 statistically significant. To parse through these interaction terms, two three-way ANOVAs were 153 performed with Phi6 and MS2 as the dependent variables, separately.

A three-way ANOVA performed with Phi6 transfer rate as the dependent variable 155 indicates that surface type is significant (P<0.001). The post-hoc test shows that there are 156 differences between wood and plastic (mean difference between wood and plastic = -0.13, 157 P<0.001) and wood and stainless steel (mean difference between wood and stainless steel = -158 0.12, P<0.001), but no difference between stainless steel and plastic (P=0.97). Direction of 159 transfer (P=0.16) and time since last handwash (P=0.24) are not significant factors in the model.

There is no statistically significant three-way interaction between 'surface type', 'direction of transfer', or 'time since last handwash' (P=0.14). In terms of possible two-way interactions, the 162 only significant interaction occurs between 'surface type' and 'direction of transfer' (P=0.014); 163 the direction of transfer was found to only significantly impact the transfer rate between 164 fingerpads and plastic (mean difference between finger to plastic transfer and plastic to finger 165 transfer = -0.09).

A separate three-way ANOVA performed for all MS2 data indicates that direction of 167 transfer is the only significant variable (P<0.001). The post-hoc test shows that the mean 168 difference between fingerpad to surface transfer and surface to fingerpad transfer is -0.18.

Surface type (P=0.71) and time since last handwash (P=0.23) were not found to be significant.

Similarly to Phi6, there is no statistically significant three-way interaction between surface type, 171 direction of transfer, or time since last handwash (P=0.73). The only significant two-way 172 interaction occurs between the surface type and direction of transfer (P=0.003). The direction of 173 transfer significantly effects the transfer from all three surfaces, with a higher fraction transferred 174 from surfaces to fingerpads for all surface types (a mean difference of 0.23 for plastic, 0.21 for 175 stainless steel, and 0.10 for wood). 

The transfer rates reported in this study for MS2 and Phi6 are similar to virus transfers 186 reported by others (1, 11, 12, 21) . Specifically, the MS2 mean transfer rate of 0.26 is comparable 187 to the MS2 mean transfer rate of 0.22 between fingertips and glass reported by Julian et al. (10) 188 who used similar methods as those used herein. Previous work reported viral transfer rates 189 between skin and fomites to range between 0.16 and 0.65 for non-porous surfaces (1, (10) (11) (12) 21) .

The higher values in this range were obtained using greater contact pressure and a shorter 191 desiccation time for viral suspensions (1, 10, 12) . According to a physical-chemical model of 192 skin-surface microbial transfer (14), greater contact pressure will likely lead to higher transfers.

Future work should explore the influence of this variable on viruses, and specifically non- Non-enveloped viruses are more readily transferred from surfaces to fingerpads than 220 from fingerpads to surfaces; the mean difference between surface to fingerpad and finger pad to 221 surface transfer rate was found to be 0.23 for plastic, 0.21 for stainless steel, and 0.10 for wood.

In previous studies that report that direction of transfer is important in controlling virus transfer, 223 conclusions regarding the direction in which virus was more readily transferred differed based on 224 virus type (5, 10, 12). This agrees with what was found in this study, where only MS2 showed a 225 greater transfer from surfaces to fingerpads than from fingerpads to surfaces. A greater transfer 226 from surfaces to fingerpads than from fingerpads to surfaces suggests individuals are able to pick 227 up viral particles from a surface and may not be able to spread them to additional surfaces as 228 easily. As a result, viruses may remain on the skin rather than be transferred off. Presence of viruses on the hands and subsequent interaction with the nose, eyes, or mouth, may lead to self-230 inoculation and subsequent infection. A previous study found that the transfer rate for a non-231 enveloped virus (PRD-1) from fingertip to lip is roughly 34% (21). Additional work 232 investigating skin-to-skin transfer rate, in combination with previous results of surface-to-skin 233 transfer rate, can help develop a complete model of the disease transmission pathway. 234 We did not find that 'time since last handwash' affected transfer of virus between 

There are several limitations to this study which have not already been mentioned. First, 246 this study controlled contact pressure even though it is understood that this may affect transfer 247 (12, 14) . Additional work to include contact pressure as a variable may be useful. Second, this 248 study worked with clean surfaces and relatively clean fingerpads. In reality, surfaces and 249 fingerpads may be coated with dirt or oil and this could affect transfer rates by changing physio-250 chemical interactions between viruses and surfaces (14). Further work should consider the use of 251 realistically soiled surfaces and hands, which may provide protection to pathogens when the contact event occurs (26). Third, this study was restricted to two viruses and three surfaces. It 253 would be interesting to expand on these in future studies to investigate whether the trends 254 observed here for enveloped viruses can be confirmed with other surrogate, non-pathogenic 255 enveloped viruses. Finally, our surface sampling technique may not recover all viruses from the 256 surfaces swabbed. An inherent assumption in this work is that the recovery efficiency of virus 257 from fingerpads and tested surfaces was not distinct, so that the transfer rate could be calculated Phi6. Virus stock consisted of TSB with ~10 5 PFU MS2 /mL and between 10 8 PFU Phi6/mL and 324 10 10 PFU Phi6/mL. The higher Phi6 titer stock was used for fingerpad and painted wood donor 325 surfaces while the lower Phi6 stock was used for stainless steel and plastic surfaces. The 326 different Phi6 titers were required to obtain countable plaques from the recipient surfaces.

Temperature and relative humidity of the room during the experiment were recorded using a 328 ThermoPro TP49 Digital Hygrometer.

An hour prior to Experiment A, volunteers were asked by the technician to wash their 330 hands with antibacterial liquid hand soap (Colgate-Palmolive, New York, NY, USA) for 15 331 seconds, rinse them in tap water, and dry them with a Kleenex scientific cleaning wipe 332 (Kimberly-Clark, Irving, TX, USA). They were asked to refrain from using the restroom, eating 333 food, and wearing latex gloves until the start of the experiment. For each volunteer, one surface 334 to be tested was chosen through a random number generator from 1-3 (1=Stainless steel, 335 2=Plastic, and 3=Painted wood). An optional second surface to be tested the same day was also 336 randomly chosen from the remaining 2 surfaces. Next, the finger corresponding to each direction 337 of transfer and the finger used as a control were chosen through a random number generator from After the inoculum on the donor surface was visibly dry, the contact event took place.

The volunteer contacted the surface for 10 s at a pressure of 25 kPa. The appropriate pressure 353 was administered using a triple-balance beam set to 500 g. This pressure is comparable to a child 354 gripping an object, the pressure of adult fingerpads exerted locally on a hand tool, and studies 355 examining transfer of soil from surfaces to skin (29-31). Upon completion of the contact event, a 356 cotton swab wetted with TSB was used to remove the virus from both the donor and recipient 357 surfaces. The swab was swiped firmly across the surface for 10 s using a sweeping motion. The 358 swab was then placed in 1000 µL of TSB and vortexed for 10 s.

After Experiment A was complete, the volunteer was asked to use alcohol-based hand 360 sanitizer (ABHS) and then wash their hands using the same method they used at the start of the 361 experiments. Immediately after washing, Experiment B was initiated using the same surface(s), 

Three dilutions of each sample were assayed, including undiluted, 1:10 dilution, and 375 1:100 dilution samples. In addition, a negative control for each hand and surface was included 376 for each volunteer. The negative control consisted of performing the contact event with a surface 377 and fingerpad that were not inoculated with the virus, swabbing the recipient surface, and 378 processing the swab sample using the plaque assay described. The viral stock concentration was 379 enumerated in each experiment, confirming the plaque assay was working correctly even if no 380 plaque were observed in the surface transfer results. The Phi6 and MS2 hard agar plates were 381 incubated at 30°C and 37°C, respectively, for 18 hours before plaques were counted as PFUs.

The number of PFUs were counted if the number was between 1 and 500. If there were more 383 than 500 PFU, TNTC (too numerous to count) was recorded. If there were no PFU, then a 0 was 384 recorded. 

Transfer 430 Efficiency of Bacteria and Viruses from Porous and Nonporous Fomites to Fingers under 32. EPA. 2001. Method 1602: Male-specific (F+) and Somatic Coliphage in Water by Single 511

Agar Layer (SAL) Procedure 38