recommended by the CDC as the ideal method of protection from COVID-19. Although 24 N95 respirators are traditionally single-use, the shortages have necessitated the need 25 for re-use. Effective methods of N95 decontamination that do not affect the fit or 26 filtration ability of N95 respirators are essential. Numerous methods of N95 27 decontamination exist; however, none are universally accessible. In this study we 28 describe an effective, standardized, and reproducible means of decontaminating N95 29 respirators using widely available materials. The N95 decontamination method 30 described in this work will provide a valuable resource for hospitals, healthcare centers, 31 and outpatients practices that are experiencing increasing shortages of N95 respirators 32 due to the COVID-19 pandemic. 33 INTRODUCTION 35 Since the initial cases in Wuhan, China in late December 2019, the COVID-19 36 pandemic, caused by the novel SARS-CoV-2 virus, has resulted in over 2.3 million 37 infections and 160,000 deaths worldwide (2). Throughout this outbreak, the infection 38 and resultant incapacitation of healthcare providers has been of significant concern. 39 Each sick provider contributes to further nosocomial transmission and reduces the 40 healthcare system's capacity to handle incoming patient volume. One of the greatest 41 threats to healthcare workers' well-being is the critical shortage of personal protection 42 equipment. Of particular concern are shortages in specialized N95 respirators. 43 The National Institute for Occupational Safety and Health (NIOSH) recommends 44 N95 respirators for protection from particles <100nm in size, including viruses (3). 45 Across the U.S. and worldwide, N95 prices have skyrocketed and supplies have 46 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 25, 2020 . . https://doi.org/10.1101 Here, as a quality assurance initiative at our institution, we set out to identify an 67 N95 decontamination method that would allow repeated use of respirators. To assess 68 decontamination, we utilized the Escherichia coli MS2 bacteriophage as a highly 69 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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To address the shortage of N95 respirators that are essential to keep healthcare 76 workers protected from SARS-CoV-2, we set out to identify an effective method of N95 77 decontamination. Our goal was to find a decontamination method accessible to all 78 practitioners in our distributed healthcare network, using MS2 phage as a model for 79 SARS-CoV-2. Like SARS-CoV-2, the MS2 phage is a positive-sense, single-stranded 80 RNA virus. Unlike SARS-CoV-2, the MS2 phage lacks a lipid envelope, making it more 81 resistant to disinfection. Due to its stability, MS2 has been used to model disinfection of 82 viruses such as norovirus and Ebola (9, 10). 83 Microwave-generated steam has proven to be an effective method of 84 decontamination (4-8). In seeking a platform with widespread availability, we initially 85 tested two protocols using common household items. Both protocols involved the use of 86 a 10cm diameter ceramic mug filled with 60 mL water and covered with the mesh from a 87 produce bag secured with a rubber band, on which the respirator was suspended 88 directly above the generated steam ( Figure 1A ). In one assay, we placed the mug inside 89 a ventilated gallon Ziploc bag. In the other assay, we place the mug directly in the 90 microwave without containment. We then examined the ability of both methods to 91 sterilize N95 coupons (excised 1 cm 2 N95 fabric squares) inoculated with 10 7 plaque 92 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 25, 2020 . . https://doi.org/10.1101 forming units (PFU) of MS2 phage. The inoculation of 10 7 PFU represents a higher viral 93 load than any viral droplet a healthcare provider is likely to encounter in the clinical 94 setting (11). After 1 minute of microwave steam decontamination, we saw no significant 95 difference in MS2 phage reduction between the two methods ( Figure 1B ). It is important 96 to note that both methods resulted in greater than a 4-log 10 reduction in MS2 titer after 97 only 1 minute of microwave treatment. The Ziploc bag, however, melted under this 98 treatment and posed the risk of steam burns during retrieval of the N95 respirator. 99 Noting equal efficacy, we proceeded with the open container method of steam 100 decontamination for all further work. 101 To identify the optimal length of microwave time required for MS2 phage 102 decontamination, we performed a dose-response test using 1 minute increments where 103 we examined the decontamination of 10 7 PFU of MS2 on 1cm 2 N95 coupons placed 104 over an open mug (as described above). Following three minutes of microwave steam 105 treatment there were no detectable MS2 phage remaining on the coupons ( Figure 2A ). 106 To accurately assess the ability of this method to decontaminate all areas of an N95, we 107 inoculated 10 7 PFU of MS2 phage onto ten discrete sections of an N95 respirator 108 ( Figure 2C ). Following three minutes of treatment, we observed a greater than 4-log 10 109 reduction in PFU on all N95 respirator segments, except the elastic straps which only 110 showed a 1-3 log 10 reduction in PFU ( Figure 2D ). Due to the limited diameter of the 111 mug, the elastic straps draped over the edges, and presumably were minimally exposed 112 to microwave-generated steam ( Figure 2B ). Consequently, we hypothesized that direct 113 exposure to steam is essential for effective decontamination, and sought to identify a 114 commercial container of sufficient diameter to treat an entire respirator.
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The copyright holder for this preprint this version posted April 25, 2020. . https://doi.org/10. 1101 Ultimately we selected a generic glass container sized at 17 x 17 x 7.5 cm 116 (LxWxH) that had an opening large enough to expose the entire N95 respirator to the 117 vertical column of generated steam. As with the ceramic mug, we secured mesh from a 118 produce bag over the top of the container with a rubber band and added 60ml of water 119 to the basin ( Figure 3A -B). We repeated a sterilization time course against 1cm 2 N95 120 respirator coupons in 1-minute increments. After 2 minutes of microwave steam 121 treatment we were unable to detect residual viable phage on the coupons ( Figure 3C ). 122 This represents a 1 minute reduction in sterilization time compared to the ceramic mug 123 decontamination assay, indicating that the glass container is a more efficient 124 decontamination system. 125 We next examined the ability of the glass container to sterilize a whole N95 126 respirator. As described above, we inoculated ten sections of an N95 respirator with 127 MS2 phage and treated the respirator for 3 minutes over the container system ( Since it is essential that any decontamination method not reduce the filtration or 136 integrity of N95 respirators, we examined N95 respirator fit and function after sequential 137 treatments. After 1, 5 or 20 three-minute treatment cycles, no damage was evident in 138 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 25, 2020. open vessel was not associated with significant water retention (6). This is likely due to 153 the fact that the N95 respirator is suspended above the steam and is not in any 154 sustained, direct contact with water. The lack of water retention means little to no drying 155 time is required post-treatment prior to N95 respirator use. 156 In summary, we identified an effective method of N95 decontamination by 157 microwave generated steam utilizing universally accessible materials. Our method 158 resulted in almost complete sterilization after only three minutes of treatment and did 159 not appear to affect the integrity of N95 filtration or fit with repeated treatment. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 25, 2020. but is also a known carcinogen and teratogen, and exposure has been correlated with 184 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 25, 2020. 190 In order to identify a generally accessible N95 respirator decontamination 191 method, we focused our efforts on microwave generated steam decontamination. (6). Although Ziploc-enclosed decontamination was effective, our results 203 indicated that it is a more cumbersome system and containment could be dispensed 204 with entirely. Furthermore, Ziploc bags began to melt when exposed to more than one 205 minute of microwave generated steam and posed the risk of thermal burns from 206 contained steam making the enclosed method less compelling.
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The copyright holder for this preprint this version posted April 25, 2020. . https://doi.org/10.1101/2020. 04.22.20076117 doi: medRxiv preprint With additional study, we found that use of a generic glass container measuring 208 17x17x7.5cm (LxWxH) resulted in the most efficient and practical N95 respirator 209 decontamination system. Using this method, we observed almost complete sterilization 210 of the N95 respirator after a single 3-minute treatment. On average, we found a 6-log 10 211 reduction in viable MS2 phage with a minimum of a 5-log 10 reduction. During 212 decontamination treatments, we positioned the N95 respirator with its convex surface 213 pointed downward, onto the mesh-covered container, maximizing steam exposure. 214 Placement was otherwise made without regard to specific orientation of the respirator, 215 simulating real world application. Post-treatment water retention by the N95 was 216 undetectable, eliminating a need for drying time before reuse. Importantly, this method 217 was validated for use of 20 times on a single respirator without detrimental effect on 218 respirator integrity or fit. In contrast, a recent preprint demonstrated that fit and seal 219 integrity was compromised in UV-and heat-treated N95 respirators after 3 treatment 220 cycles, and in ethanol-treated respirators after 2 treatment cycles (17). Given these 221 findings, decontamination by microwave-generated steam may provide an ideal solution 222 for broad N95 respirator re-use, with minimal treatment duration, minimal post-treatment 223 processing, and maximal re-use potential. 224 The MS2 bacteriophage was used as a model of SARS-CoV-2 in this study. It 225 provided a facile system for rapid quantitative evaluation of respirator disinfection. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 25, 2020. . https://doi.org/10.1101/2020. 04.22.20076117 doi: medRxiv preprint has been used as a surrogate for protein capsid-protected noroviruses and also for 231 enveloped Ebola virus, and has been reported to be significantly more resistant to 232 disinfectant than both (9, 18). These differences were highlighted in our present study 233 by observation of complete resistance of MS2 to dry heat inactivation at 105°C for 60 234 minutes ( Figure 1B ) compared with previously reported complete inactivation of SARS-235 CoV-2 by 70°C dry heat treatment for the same duration (17). Therefore, the substantial 236 reduction in viable MS2 by microwave steam decontamination gives confidence in an 237 appropriate safety margin for SARS-CoV-2 decontamination and suggests the 238 additional benefit of disinfection of other viruses that may also contaminate respirators 239 during re-use. 240 Our N95 decontamination system uses only commonly available materials: a 241 glass container, mesh from a produce bag that can be found at any grocery store, and a 242 rubber band, as well as a common household 1100W or 1150W microwave. All 243 microwaves used in this study had a turntable to enable rotation while heating ( Figure   244 1B-C, 3A), a feature that likely promotes uniform heating and steam production. It is 245 important to note that the microwave treatment did not result in sparks even when there 246 was metal present on the N95, which is consistent with previous reports (5). Our 247 decontamination protocol was validated with a glass container, and it is possible that 248 steam generation and results would be affected using containers made of other 249 materials. 250 We began our study using a ceramic coffee mug to generate steam. However, 251 there were several limitations to this method. Notably, sizes of ceramic mugs vary 252 widely, making this method hard to standardize. Although we observed complete 253 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 25, 2020 . . https://doi.org/10.1101 sterilization of the 1 cm 2 coupons on the ceramic coffee mug, we did not observe a 254 similar result using the entire N95 respirator (Figure 2 ) where significant quantities of 255 viable virus remained post-treatment (10 2 -10 6 PFU). In contrast, the larger surface area 256 provided by the glass container led to almost complete sterilization at all locations. 257 Furthermore, the 10 cm diameter mug opening was still too small to suspend the elastic 258 straps of the respiratory above the mug orifice. This led to inefficient decontamination of 259 the elastic straps, in contrast to findings using the larger glass container. Our study 260 highlights the need to examine the whole respirator for disinfection rather than just small 261 coupons isolated from the filtration material as has been generally performed in N95 262 respirator disinfection studies. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 25, 2020. Particulate Respirator and Surgical Mask). Excised 1cm 2 coupons or whole N95 282 respirators were treated with 10 µL of 10 9 PFU/mL MS2 resulting in a ~10 7 PFU 283 inoculation. When a whole respirator was treated, ten sections were demarcated and 284 inoculated, including two spots on the elastic straps. The inoculated N95 coupons and 285 whole respirators were then allowed to dry inside a biosafety cabinet for 2 hours. Once 286 dried, pre-cut triplicate N95 respirator coupons were removed to quantify viral load prior 287 to intervention. 288 Mesh from produce bags (multiple variants were utilized in this study) were 289 secured across the top of either a ceramic mug or glass container with rubber bands. 290 The mug utilized in this study was 10cm in diameter. The glass container (Snapware 4-291 cup Food Storage Container made with Pyrex Glass) is roughly 17cm x17cm x7.5cm 292 (6.5x6.5x3in). Both the mug and glass container were filled with 60ml (¼ cup) of water 293 for steam generation. N95 coupons and respirators were placed outward facing side 294 down, onto the mesh, for direct suspension above the steam ( Figure 1C, 3B) . When a 295 Ziploc bag was utilized, a 2 cm slit was cut in the upper-right side of the bag to vent 296 excess steam. The N95 respirator coupons and whole respirators were then treated for 297 the indicated time in either a 1150W or a 1100W microwave. Both microwaves utilized 298 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 25, 2020 . . https://doi.org/10.1101 in this study contain a turntable for uniform heating. It is noteworthy that results were 299 consistent between both microwaves used in this study. 300 For dry heat treatment, 1cm 2 N95 respirator coupons were exposed to 105°C dry 301 heat for 60 minutes in a hybridization oven.
303 Viral load on pre-and post-intervention N95 respirators and coupons was 304 measured using established plaque assay protocols (19, 20) . For whole respirators, 305 each treated area was cut and removed from the N95 and processed individually. Each 306 piece was submerged in 1 mL LB broth and vortexed for one minute to elute MS2 307 phage. The LB suspension was then serially diluted, following which 100 µL from each 308 of the phage dilutions was mixed with 100 µL of exponential phase host E. coli W1485 309 cells. This mixture was then vortexed with 3 mL of LB top agar (0.6% agar w/v) and 310 spread on LB agar plates (1.5% agar w/v). All plates were incubated at 37°C overnight 311 to allow for bacterial lawns and viral plaques to grow. Plaques were quantified and the 312 total PFU burden was calculated. The limit of detection of this assay was 10 PFU. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 25, 2020 . . https://doi.org/10.1101 .04.22.20076117 doi: medRxiv preprint regs/regulations/standardnumber/1910 /1910 We quantified water absorption into N95 respirators during the microwave steam 339 decontamination treatment as described previously (6). We measured the mass of N95 340 respirators pre-treatment and immediately post-treatment using an analytical balance. 341 We subtracted the pretreatment mass from the post-treatment to quantify the amount of 342 water absorption. In all assays, there was <1mg difference between the two 343 measurements, which remained constant even with multiple treatment cycles.
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The copyright holder for this preprint this version posted April 25, 2020 . . https://doi.org/10.1101 