key: cord-0920595-i5iz5csd
authors: Daeschler, S. C.; Manson, N.; Joachim, K.; Chin, A. W. H.; Chan, K.; Chen, P. Z.; Tajdaran, K.; Mirmoeini, K.; Zhang, J. J.; Maynes, J. T.; Science, M.; Darbandi, A.; Stephens, D.; Poon, L. L. M.; Gu, F.; Borschel, G. H.
title: Thermal Disinfection Inactivates SARS-CoV-2 in N95 Respirators while Maintaining Their Protective Function
date: 2020-05-27
journal: nan
DOI: 10.1101/2020.05.25.20112615
sha: 3fe95d7c42149f9d7b4cb0155232458478c1fcc2
doc_id: 920595
cord_uid: i5iz5csd

Background: The unprecedented demand and consequent global shortage of N95 respirators during the COVID-19 pandemic have left frontline workers vulnerable to infection. To expand the supply, we validated a rapidly applicable low-cost decontamination protocol in compliance with US-regulatory guidelines to enable the safe reuse of personalized, disposable N95-respirators. Methods: Four common models of N95-respirators were disinfected for 60 minutes at {degrees}C either at 0% or 50% relative humidity (RH). Effective inactivation of SARS-CoV-2 and E. coli was evaluated in inoculated masks. The N95 filter integrity was examined with scanning electron microscopy. The protective function of disinfected N95 respirators was tested against US NIOSH standards for particle filtration efficiency, breathing resistance and respirator fit. Results: A single heat treatment inactivated SARS-CoV-2 (undetectable, detection limit: 100 TCID50/ml) and E. coli (0 colonies at 50%RH) in all four respirator models. Even N95-respirators that underwent ten dry heat, or up to five 50%RH-controlled decontamination cycles maintained their integrity and met US-governmental criteria for approval regarding fit (10x dry heat: 194.48 {+/-} 15.5; 99%CI: 185.5 to 203.5; 5x at 50%RH: 198.2 {+/-} 6.3; 99%CI: 189.4 to 207; n=23 each, pass value [≥] 100,), breathing resistance (10x dry heat: 68.0 {+/-} 8.4 Pa, 99%CI: 61.8 to 74.1, n=16; 5x at 50%RH: 78.5 {+/-} 19.7 Pa, 99%CI: 21 to 135.9, n=4, pass value [≤] 343.23Pa), and filtration levels (10x dry heat: 97.6 {+/-} 1.1%, 99%CI: 96.8 to 98.4; n=16; 5x at 50%RH: 97.18 {+/-} 0.49, 99%CI: 95.7 to 98.6, n=4, pass value [≥] 95 %). Interpretation: This rapid process enables large-scale decontamination of existing N95-respirators using commonly sourced equipment during the COVID-19 pandemic.

As the COVID-19 pandemic has overwhelmed many health care systems world-wide, the unprecedented demand for personal protective equipment exhausted stockpiles and interrupted global supply chains for N95 respirators. Currently, the proportion of frontline healthcare workers among SARS-CoV-2 infected individuals exceed 10% in some regions and is expected to increase as supplies of protective equipment further diminish (1, 2) . As a result, protecting frontline workers from SARS-CoV-2 infection is now an immediate global concern (3).

Disposable N95 respirators protect users against infectious airborne particles and are therefore indispensable for frontline workers during the COVID-19 pandemic (3) . However, the present global shortage of personal protective equipment forced regulating institutions to adjust infection control measures: prior to the pandemic, guidelines recommended disposal of N95 respirators after each patient encounter. Now, evolving guidelines instruct staff to re-use one mask over their whole shift or even longer (4) . This policy of re-using disposable masks in areas of high airborne pathogen exposure, such as COVID-19 patient care, may result in accumulation of contagious material on the mask surface, risking the health and safety of personnel and patients (5, 6) . Inactivating accumulated pathogens in disposable respirators without affecting their protective properties may enable safe reuse and thus help to alleviate the current global shortage temporarily. However, the sterilization methods regularly used in health care institutions potentially deteriorate components of disposable respirators and thereby affect fit or filtration efficiency (7) .

Thermal disinfection may overcome this issue and potentially provides a widely available and cost-effective decontamination strategy for disposable respirators. Recent reports demonstrate a high sensitivity to heat for SARS-CoV-2 as five minutes of heating at 70°C inactivates the All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 May 27, 2020. . virus (5, 8) . Beyond that, the polypropylene microfibers in commercially available N95 respirators have a thermal degradation point above 130°C, suggesting that the filter may withstand repetitive exposure to 70°C (9, 10) . However, the viricidal efficacy of thermal disinfection for SARS-CoV-2-contaminated N95 respirators, and the protective performance of heat-treated respirators has not been validated to a level meeting US-regulatory standards.

We therefore asked whether thermal disinfection at 70°C for 60 min inactivates pathogens, including SARS-CoV-2, while maintaining critical N95 respirator protective properties for multiple cycles of disinfection and re-use in a real-world setting.

We used thermal disinfection in cycles of 60 min at 70°C, either at 0% or 50% relative humidity (RH), to treat four common models of commercially available N95 respirators (8110s, 9105s, 8210, 1860s; all 3M, Minnesota, USA) while preserving their integrity and function. The respirators were wrapped in sterilization pouches (Steril-peel, GS Medical Packaging, Ontario, Canada) prior to disinfection. To control for temperature and RH, we used the BevLes Heated Holding Cabinet with humidity (BevLes Inc, Erie, PA, USA) set to 70°C and either 0% or 50% RH. A digital thermo-and hygrometer (Hadgen Group Inc, Montreal, Quebec, Canada) was used as an added quality control measure. Additionally, we accounted for potential real-world temperature fluctuations by cooling the masks to room temperature for five minutes mid-cycle.

We assessed SARS-CoV-2 inactivation in all four N95 respirator models. We cut untreated and 10x heat-treated N95 respirators into 1 cm 2 pieces and inoculated the outer surface of the respirators with 5 µl of SARS-CoV-2 (~7.8 log TCID50/ml) in a biosafety level 3 laboratory in triplicates (n=3 per respirator type). The virus-inoculated respirators underwent thermal All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 May 27, 2020. . disinfection at 70°C / 0% RH for 60 min, with and without a 5 min cool down midcycle, followed by soaking in 300 µl of viral transport medium for 30 min for virus elution. We then titrated the recovered infectious virus particles by standard TCID50 assay using Vero E6 cells as described (5) . Virus-inoculated respirator surfaces without the heat inactivation step were used as controls.

To test for bacterial inactivation, we cut untreated N95 respirators (1860S, 3M, Minnesota, USA) into 1 cm 2 pieces. The outer surface was inoculated with 100 µl of Escherichia coli (4 × 10 8 CFU/ml, optical density 0.612 at 600 nm) and a negative control was inoculated with pure Luria-Bertani (LB) medium. The inoculated respirators underwent 60 min heat treatment at 70°C either at 0% RH or at 25% RH, 40% RH or 50% RH (n=4 per condition). Controls were and treated at 90°C / 70% RH or left at room temperature for 1 hour. Then, we washed N95 fragments individually in 1ml of LB medium and inoculated 100 µl washing media on LB-agar plates. Colonies were counted after 24 h incubation at 37°C (Figure 1 ). To the 900µl of remaining washing media and N95 fragments, 9.1ml LB media were added and incubated at 37°C in a shaking incubator. Optical density at 600nm was read after 24 hours of incubation to estimate bacteria concentration.

To assess whether exposing the polymer microfibers of the N95 filter media to high temperatures caused fiber degradation, we analyzed a 1 cm 2 filter sample from respirators that underwent 1, 2 and 10 (0% RH and 50% RH) thermal disinfection cycles at 70°C. We coated each sample with 10 nm of carbon and imaged with a scanning electron microscope (XL30, FEI company, Oregon, USA) at magnifications of 150x, 200x, 650x, and 1200x at 5 keV. We analyzed fiber morphology with a blinded observer using ImageJ (https://imagej.nih.gov/ij/) in All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 May 27, 2020. . https://doi.org/10.1101/2020.05.25.20112615 doi: medRxiv preprint ten randomly selected individual fibers from all quadrants of a representative image of each sample. We measured the fiber diameters in 40 individual fibers per condition (10 fibers per mask type) to calculate the mean fiber diameter after each disinfection cycle.

For reliable respiratory protection, respirators need to tightly seal the face against leakage of unfiltered air. Exposure to high temperatures may affect the mechanical properties of the respirator components, such as elasticity of the headbands or adjustability of the nose clip, potentially allowing leakage of particles. To test the respirator fit, we applied a standardized, quantitative fit testing procedure in a total of 46 respirators (n=12 per type, except n=10 for (11) (12) (13) . Particles greater than 0.02 µm in size were detected in a concentration range of 0.01 to 2.5

x 10 5 particles/cm 3 . Average ambient and in-mask particle concentration was measured during standardized exercises and their ratio was calculated as the respirator fit factor, with a fit factor of 100 being defined by the OSHA as the minimum pass value (11) . Fitted respirators were personalized to two test subjects (one male, one female) and underwent thermal disinfection at 0% RH or 50% RH respectively (n=23 each) as outlined above. After 10 and 15 disinfection cycles the quantitative fit testing was repeated for each respirator, with the same test subject.

Additionally, the test subject rated the subjective fit, adjustability and comfort of each decontaminated respirator compared to the unprocessed reference masks on the CSA Comfort Assessment Score (0 -no issues; 1 -discomfort can be ignored; 2 -some discomfort but still able to function; 4 -unacceptable discomfort) (13) .

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We determined the breathing resistance and particulate filter efficiency in a total of n=46 N95 respirators that underwent either five or ten cycles of thermal disinfection at 0% RH or 50% RH, using the abbreviated National Institute for Occupational Safety and Health (NIOSH) Standard Test Procedure per Code of Regulation 42 CFR §84.180 and §84.181(14-16). To measure breathing resistance (airflow resistance), the respirators were mounted on a test fixture with air flowing at continuous rate of 85 ±2 l/min. In accordance with the NIOSH standard, a breathing resistance below 343.23 Pa is considered tolerable (16). For NIOSH filtration efficiency protocols, the respirators were pre-conditioned at 85 ± 5% relative humidity and 38 ± 2.5°C for 25 ± 1 hours and then mounted on a certified condensation particle counter (3772, TSI Incorporated, Minnesota, USA). The respirators were tested against a near monodispersed polystyrene latex bead at a flow rate of 85±2 l/min, at 21 -26°C and a relative humidity of 30.4 -43.2%. Particle filter efficiency was calculated as the percentage of all counted particles (median diameter 0.075 ± 0.020 µm) removed by the respirator. For N95 masks, particle filter efficiency needs to be equal to or greater than 95 % (15).

We conducted statistical analyses using JMP (version 15.1.0, SAS Institute, North Carolina, USA). No sample size calculation was performed. Descriptive statistics were calculated as overall values among all tested respirators for each condition. All means are expressed with standard deviation (±SD). Given the limited sample size due to the global shortage of N95 respirators, one sample t-tests were used to compare the group means of the disinfected masks to the respective pass value for each assessment in compliance with governmental guidelines.

To increase the stringency and adjust for multiple comparisons, an alpha level of 0.01 for onesided p-values was chosen. Additionally, non-inferiority of the disinfected masks was assumed All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 May 27, 2020. . https://doi.org/10.1101/2020.05.25.20112615 doi: medRxiv preprint only when the lower bound of the 99% confidence intervals (99% CI) was greater than the minimum required pass value.

After inoculation with virus, no infectious SARS-CoV-2 could be detected in any dry heattreated respirators (70°C for 60 min) whereas high levels of SARS-CoV-2 could still be detected in respirators that did not undergo heat treatment (Table 1) . Shown are quantifications of the infective doses of SARS-CoV-2 after a single cycle of thermal disinfection (70°C / 0% RH) of new N95 respirators, and respirators that were pre-treated with 10 disinfections. Infectious SARS-CoV-2 could not be recovered from the disinfected masks, indicating effective decontamination.

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No E. coli could be detected in inoculated N95-respirators when heat treated for 60 min at 70°C at 50% RH and at 90°C at 70% RH. As shown in Figure 1 , in samples exposed to dry heat (70°C / 0% RH), > 1000 bacterial colonies were still detectable, while exposure to 70°C with humidity (25% RH and 40% RH) dramatically reduced colony formation. Consequently, thermal disinfection for 60 min at 70°C and 50% RH eliminated E. coli contamination on N95 respirators ( Table 2 ). All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 May 27, 2020. .

We analyzed the N95 filter media in new, unprocessed (control) respirators and observed an overall mean fiber diameter of 3.88±2 µm. Even after ten cycles of thermal disinfection of 60 min at 70°C and either 0 or 50% RH, the mean overall fiber diameter remained within the range for unprocessed N95 filters as specified in the US patent ( Figure 2 ) (9). diameter of unprocessed and ten times disinfected N95 filters (0% and 50%RH), with the typical fiber diameter range of 3M N95 filters highlighted in green (9) . Shown are the mean fiber diameters with 99% confidence intervals as error bars. Graphs are labelled with mean±SD.

Quantitative fit testing was conducted with four common types of commercially available N95 respirators that underwent 10 and then 15 cycles of thermal disinfection at 0% and 50% RH respectively (n=23 for each condition; Figure 3 ). All tested groups of thermally disinfected respirators significantly exceeded the fit factor of 100, as the OSHA-defined pass value for sufficient respiratory protection (p<0.001 for all groups) and so did the lower bound of their All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 May 27, 2020. . https://doi.org/10.1101/2020.05.25.20112615 doi: medRxiv preprint 99% confidence intervals (Figure 3) . In a total of 92 performed quantitative fit tests with disinfected respirators (0% and 50% RH), none failed the test. Also, the subjective fit and wearing comfort of the decontaminated respirators did not differ from new masks and were rated with zero, or no issues, on the CSA Comfort Assessment Score.

Further, we tested the particle filter efficiency and breathing resistance in the same four types of commercially available N95 respirators that underwent five cycles or ten cycles of thermal disinfection at 0% and 50% RH respectively (Figure 3 ). Dry heat disinfected respirators significantly exceeded the required pass value even after ten cycles of disinfection (p<0.001).

Respirators that underwent disinfection at 50% humidity significantly exceeded 95% filtration efficiency after five disinfection cycles (p<0.001) but not after ten (p=0.024). In addition, the breathing resistance of the same set of disinfected respirators was significantly lower than the maximum tolerable resistance of 343.23 Pa for all tested groups (p<0.001). Filtration efficiency and breathing resistance of the disposable N95 respirators are maintained after up to ten cycles of thermal disinfection at either 0% RH or 50% RH. particle filtration efficiency (centre) and breathing resistance (right) of thermally disinfected N95 respirators (0% and 50% RH). The US-governmental pass values for each test are indicated by a dashed line (11, 15, 16) . Graphs are displayed as means with 99% confidence intervals and labelled with the mean ± SD. Groups that significantly exceeded the pass value are labelled with a double asterisk (** p < 0.01; * p=0.024).

The unprecedented demand for personal protective equipment during the COVID-19 pandemic forced regulatory institutions to adjust infection control measures, now instructing frontline workers to re-use disposable protective equipment (4, 17) . Strategies to disinfect and redistribute personalized, disposable N95 respirators would increase the safety of health care workers and expand the supply. However, at present, a safe and universally available largescale decontamination protocol for N95 respirators is unavailable.

Based on recent reports demonstrating the heat sensitivity of SARS-CoV-2, we applied thermal disinfection protocols for four common, disposable N95 respirator models (5, 8) . To test for effective virus inactivation, we inoculated new and ten times pre-treated respirators with SARS-CoV-2. After a single disinfection cycle for 60 min at 70°C and 0% relative humidity, no active virus could be recovered from any of the tested N95 respirators, indicating effective SARS-CoV-2 inactivation. In addition, although airborne bacterial contamination of the N95 respirators to a level representing a respiratory risk for the user may be less concerning in COVID-19 patient care, we also tested for bacterial inactivation of E. coli. Thermal disinfection thoroughly eliminated E. coli in N95 respirators when the relative humidity is kept at 50%, but not below. Thus, we recommend applying 50% humidity during thermal disinfection.

To further validate the safety of the procedure, we determined the effect of the thermal disinfection process on the respirator's integrity and function. The filters in N95 respirators consist of an electrostatically charged polypropylene microfiber mesh (electret) (9) . Effective filtration of airborne particles relies on both, physical filter pore size and electrostatic attraction (18) . The process of disinfection potentially affects these filter properties (19) . Thus, we examined the decontaminated masks for N95 filter deterioration using electron microscopy and software-based image analysis, performed a quantitative fit testing according to OSHA All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 May 27, 2020. . https://doi.org/10.1101/2020.05.25.20112615 doi: medRxiv preprint guidelines and determined particle filter efficiency and breathing resistance in compliance with official NIOSH test standards (11, 15, 16) .

After ten cycles of disinfection, using dry heat or heat at 50% relative humidity, the polypropylene microfibers of the N95 filter layer maintained a mean diameter within the range of new N95 filters. These data suggest that the physical structure of the electret N95 filter media is upheld after repetitive thermal disinfection. Further, the subjective fit and wearing comfort was maintained was maintained for all four types of disposable N95 respirators, following repetitive disinfections at 0% and 50% relative humidity. Additionally, dry or 50% RH heat disinfected respirators maintained their required level of respiratory protection in compliance with US standards for N95 respirators and maintained a low breathing resistance with particulate filtration efficiencies greater than 95% for at least five cycles of thermal disinfection.

In conclusion, disposable N95 respirators can undergo repetitive thermal disinfections without losing their protective performance.

To effectively cope with the current global supply shortage, strategies for disinfection and reuse require widespread scale-up. The U.S. Food and Drug Administration (FDA) recently issued emergency use authorizations for the vaporized hydrogen peroxide gas sterilization of disposable N95 respirators (20, 21) . However, this technology is limited to non-cellulose based respirators, therefore making a large proportion of N95s ineligible for reprocessing, and is also unavailable in most hospitals and other facilities (22). Thermal disinfection can be performed at low cost in conventional mechanical convection ovens, which are widely available in commercial kitchens, laboratories or sterilization facilities. Their large capacity enables the simultaneous disinfection of thousands of masks per oven per day, allowing a potential to scale the decontamination to a level sufficient to expand the supply of protective equipment globally. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 May 27, 2020. . https://doi.org/10.1101/2020.05. 25.20112615 doi: medRxiv preprint Other alternative decontamination procedures have been proposed as well (23) (24) (25) . In conjunction with those strategies, thermal disinfection may be used as a rapidly applicable emergency measure to alleviate the shortage of N95 respirators during the current COVID-19 pandemic.

Thermal disinfection for 60 min at 70°C uses widely available equipment to enable the safe reuse of disposable N95 respirators without affecting their protective performance. Thus, it provides a feasible, effective and rapidly scalable low-cost method to temporarily expand the supply of N95 respirators during the COVID-19 pandemic.

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