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Year : 2022  |  Volume : 20  |  Issue : 3  |  Page : 172-176

Effect of a hydrogen peroxide gas-plasma sterilization on physical integrity and quality of N95 respirators: An experimental study during the COVID-19 pandemic

1 Department of Clinical Microbiology, Hospital Infection Control Committee, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Infectious Diseases, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Infectious Diseases, Christian Medical College, Vellore, Tamil Nadu, India; Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool; Department of Tropical and Infectious Diseases, Royal Liverpool University Hospital, Liverpool, UK
4 Department of Bioengineering, Christian Medical College, Vellore, Tamil Nadu, India
5 Department of Clinical Microbiology, Hospital Infection Control Committee, Christian Medical College; Department of Infectious Diseases Christian Medical College, Vellore, Tamil Nadu, India

Date of Submission20-Jan-2022
Date of Decision22-Mar-2022
Date of Acceptance23-Mar-2022
Date of Web Publication01-Aug-2022

Correspondence Address:
Dr. Priscilla Rupali
Department of Infectious Diseases, Christian Medical College Hospital, Vellore - 632 004, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cmi.cmi_9_22

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Background: N95 respirators have prevented transmission among health-care workers during the COVID-19 pandemic. During times of intense shortage of respirators and border closures during the pandemic, re-use strategies with available decontamination methods were necessitated. This in-house experimental study evaluated the effect of hydrogen peroxide gas-plasma sterilization on respirators and helped establish an evidence-based protocol for their re-use in a resource-poor setting. Materials and Methods: A three-dimensional experimental model using saline nebulization as the aerosol exposure and a particle counter to measure the filtration of particles through the mask pre- and post-sterilization was used. Multiple cycles of plasma sterilization were done till the physical integrity/fit was lost. Total filtration volume was used as a surrogate marker to assess the filtration efficiency (FE). Results: The total volume of particles filtered on a 3M respirator was 99.9%. Unused Halyard and Venus respirators were compared against 3M and found to have FE of 99.9% and 60.5%, respectively. After repeated sterilization cycles, the total volume of particles filtered was 59.3% for Halyard in the seventh cycle and 36.2% for Venus in the fifth cycle. When the physical integrity and fit was tested, the appropriate fit was lost after eight cycles of sterilization for Venus and was not lost for Halyard even after the tenth cycle. Conclusion: This low-cost experimental study helped implement an effective and safe decontamination strategy for safe re-use of N95 respirators in an emergent situation with no access to commercial testing in a resource poor health-care setting during the pandemic.

Keywords: COVID-19, N95 respirators, re-use, sterilization

How to cite this article:
Murugesan M, Radha S, Singh B, Mathews P, Devasahayam S, Rupali P. Effect of a hydrogen peroxide gas-plasma sterilization on physical integrity and quality of N95 respirators: An experimental study during the COVID-19 pandemic. Curr Med Issues 2022;20:172-6

How to cite this URL:
Murugesan M, Radha S, Singh B, Mathews P, Devasahayam S, Rupali P. Effect of a hydrogen peroxide gas-plasma sterilization on physical integrity and quality of N95 respirators: An experimental study during the COVID-19 pandemic. Curr Med Issues [serial online] 2022 [cited 2022 Aug 10];20:172-6. Available from: https://www.cmijournal.org/text.asp?2022/20/3/172/352982

  Introduction Top

COVID-19 disease is transmitted mainly by contact, droplet, and through aerosols during aerosol-generating procedures.[1] Universal respiratory protection plays a crucial role in preventing cross-transmission of infections from health-care workers to patients and vice versa.[2] In an ideal situation, N95 respirators are intended for single use. In order to meet the increasing demand, Occupational Safety and Health Administration (OSHA) and Food and Drug Administration provided initial guidance on re-using respirators with appropriate decontamination methods,[3] the early stages of the pandemic. With the surge in demand overwhelming the usual supplies of certified N95 masks, a rapid uncontrolled increase in the production of respirators by hitherto unknown companies bypassing the usual certification norms and processes was noted. As a result, many different types and brands of respirators flooded the markets which lead to administrative and health-care worker anxiety regarding their filtration efficiency (FE) and safety when re-used after decontamination.

Globally, India had the second-highest cumulative COVID-19 cases during the first wave of the pandemic in July 2020. With uncertainty regarding the route of transmission droplet versus airborne of COVID 19 infection, there was widespread panic among health-care workers seeing patients with COVID 19 infection. An initial policy of single use and discarding N95 respirators led to a massive increase in consumption, thereby increasing the costs and a break in the supply chain during a period of border closures. Hence, circumstances dictated the deployment of a re-use policy for respirators with an extended usage of 8 h/day, followed by a period of drying for 48 h and then re-use. However, this created anxiety with unsatisfactory feedback from health-care workers. Queries were also raised by the clinicians about the quality and protection of respirators available in the market during this crisis time. The supply of fit test kits, respirators, and evaluation of FE became impossible because of a national lockdown during this pandemic phase. Hence, an alternate low-cost, low-technology method to assess the quality of respirators to safeguard our health-care workers was the need of the hour. In consultation with the bioengineering department in our institution, we created a cost-effective three-dimensional (3D) model which simulated a cough from an infected person. This study aimed to document the effect of hydrogen peroxide gas-plasma sterilization on the physical integrity and FE of respirators, thus allowing the establishment of an evidence-based protocol for their safe re-use during a crisis in a resource-poor setting.

  Materials and Methods Top

Study setting

This study was conducted by the Hospital Infection Control Committee and Department of Bioengineering, Christian Medical College, Vellore, during the first wave of the COVID-19 pandemic crisis.

  • Study design: Experimental study
  • Study period: May to June 2020.


An experimental method using a 3D-molded manikin head with a tubular oronasal passage and a commercial nebulizer for the production of aerosolized particles were used [Figure 1]. The particle counter's (HACH ULTRA Met One 3413®) long inlet hose was fitted within the manikin's mouth, with the nozzle around 5 cm from the nebulizer outlet. The particle counter's inward flow rate was fixed at 28.3 l/min, which corresponds to the mean inspiratory flow rate observed in healthy people engaged in light work. The particle counter normally quantifies particles in six size channels: 0.3, 0.5, 1.0, 3.0, 5.0, and 10.0 μm, and counts the number of particles over a 30-s period, with a coincidence loss of 5% at 14,126,000 particles/m3. Two NIOSH-approved N95 respirator models (Halyard Health, Inc N95 62126 NIOSH TC-84A-7525 and Venus® V-4400N95 NIOSH TC-84A-8126) were available at our institution at the time of the study. We had a very limited supply of 3M (model 1870+) respirators, which we used as a reference standard for initial validation. After successful validation, new unused Halyard and Venus respirators were tested. The respirators were subjected to a user seal check fit test with a volunteer and two observers, following the OSHA procedure.[4] A three-layer surgical mask was also tested using the experimental method to compare the efficiency with N95 respirators.
Figure 1: Experimental model design to test the quality of respirators.

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Particle counts at each step (baseline, aerosol generated by the nebulizer without a respirator and aerosol generated with respirators fitted onto the 3D human manikin face) were measured three times in succession and the mean was taken as the particle count for that step. After an initial purge of particles left in the channel using a handheld HEPA filter, a background count of particles inside the cabinet was recorded, to ensure that this was similar between experiments. Then, 0.9% normal saline was aerosolized using the nebulizer, initially without applying the respirator followed by applying the respirator fitted on to the manikin face, with the box placed over it ensuring an appropriate seal with no dispersal of aerosol. It was observed that the number of 0.3μm particles increased several fold following the application of the respirators and masks consistently, with reductions in the larger particle sizes. We attributed this on consultation with the engineer to be due to a relative increase as the respirator prevented the entry of larger sizes and also the collision of larger particles led to the creation of smaller particles. Hence, total filtration volume, i. e.(the percentage reduction of total particle volume through the respirators, using the total volume after vs. before application of the respirator to the manikin) was used as a surrogate marker of FE. This experiment was repeated after respirators were subjected to sequential cycles of decontamination using the validated Sterrad® 100S sterilization system.[5] After each decontamination cycle, the respirator was subjected to fit testing to ensure physical integrity by the same volunteer and then tested on our experimental model. Decontamination cycles were repeated until on visual inspection the physical integrity was lost or failed a fit test.

Statistical analysis

The total particle volume was defined as the sum of the combined volume of particles in each size range, using the following formula: (mid-point of the particle size channel range in um) x (P/6) x (mean particle count). Sensitivity bounds were calculated for the FE estimates, using 95% confidence bounds for the mean total particle volumes: the upper sensitivity bound used the lower 95% confidence bound for the prerespirator value and upper 95% confidence bound for the post-respirator value, and vice versa for the lower sensitivity bound.

Ethical statement

The above experimental method was approved by the institutional review board (IRB Min No. 12848 dated May 05, 2020).

  Results Top

An initial assessment of fit was satisfactory for the 3M, Halyard, and Venus respirators. After decontamination, during fit test, a metallic odor was noticeable in some of the respirators, which increased with the number of cycles of decontamination. Based on this finding observed by the volunteers, it was decided to wait 48 h after the decontamination procedure before assessing the fit of the respirator. The respirators were stored in a brown paper bag during the 48 h waiting period. Fit testing in decontaminated Venus respirators revealed a compromised fit at the eighth cycle of decontamination, due to the nose bridge piece becoming progressively less pliable. Halyard respirators retained physical integrity even after the tenth and final cycles of decontamination.

FE on a new 3M respirator showed 99.9% FE as measured by total particle volume. For the new Halyard and Venus respirators, the corresponding figures were 99.9% and 60.5%, respectively. We also evaluated a three-layered surgical mask, when applied singly resulted in 40.6% efficiency and when two were applied, one over the other, there was no increase, with 41.3% efficiency.

After successive cycles of decontamination, the respirators showed a reduction in FE with some fluctuations [Figure 2]. Compared to predecontamination FE of 99.9% (sensitivity bounds 99.4-100%) and 99.2% (98.6%–99.7%) after one cycle, the Halyard respirator dropped to 70.5% (63.2%–77.7%) efficiency after two cycles. The Venus respirator started at a lower filtration of 60.5% (56.7%–63.8%); after four cycles, this was 46% (39.6%–51.6%).
Figure 2: Percentage reduction of total particle volume during testing of respirators and surgical masks.

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To evaluate the effect of potential variation between respirators of the same model and batch and validate our results, we tested four different Halyard respirators independently from each other predecontamination, and then again after two cycles of decontamination. Similar results were obtained in our experiment and showed a consistent drop in efficiency between baseline (mean: 99.7%, 95% confidence interval [CI] 99.4% to 99.99%) and postdecontamination (mean 70.4%, 95% CI 63.2% to 77.7%).

We used these results to subsequently modify our hospital policy of re-use and extended use of N95 respirators in COVID and non-COVID areas.

  Discussion Top

Many tertiary care institutions had embarked upon different methods to continue using and reusing N95 respirators safely over extended periods of time.[6] However, our health-care workers were anxious, as extended use over 8 h followed by a period of 48 h of drying did not ensure adequate decontamination of SARS CoV2. There were no studies which documented the absence of SARS CoV2 on the surface of the respirators after 48 h of drying leading to a considerable unwillingness among our Health care worker (HCW) re-using the respirators. Hence, it became the responsibility of the Hospital Infection Control Committee (HICC) to come up with an innovative solution to address this. Therefore, this study describes a low cost in-house experimental methodology to assess the quality of N95 respirators before and after plasma sterilization using a 3D model during a period of supply chain uncertainties and border closures. Commercially, this is performed by an automated filter tester or an equivalent instrument which was not accessible or practical for us to pursue during COVID-19-initiated border closures. Hence, we used this experimental model with the total filtration volume as a surrogate marker of FE to guide the implementation of a re-use/extended use of N95 respirators in our hospital. The total filtration volume rather than stratification according to particle size to judge FE was used, as larger fluid drops during nebulization were noted to break down into minute droplets upon collision with the mask. We found that decontamination using a hydrogen peroxide gas-plasma sterilizer is an effective method to allow the safe re-use of respirators, though the number of cycles after which these respirators can be re-used varies between manufacturers. Based on the study results, we inferred that a specific model of Halyard and Venus respirators could be re-used up to two cycles of decontamination, beyond which the FE declines and would be unsafe for further use. The above study findings helped us formulate a safe re-use policy for N95 respirators during an emergent crisis situation with no access to commercial techniques for sterilization [Table 1]. This evidence-based policy boosted the staff morale and brought confidence among all categories of health-care workers when re-use of N95 respirators after plasma sterilization was implemented. In addition, this considerably eased the financial burden and restored the supply chain of N95 respirators, and helped maintain stock in a sustained cost-effective manner.
Table 1: Policy for re-use of N95 respirators based on the experiment

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We used total particle volume to assess FE which limited the comparability of our results with the published literature. We also noted that there were wide fluctuations in the FE after repeated cycles of decontamination. The sudden increase in the filtering capacity could be due to a change in porosity caused by an alteration in conformation of fibers within the layers of the respirator.[5]

  Conclusion Top

There was a huge demand for N95 respirators during the COVID-19 pandemic. A break in supply chains all over the country warranted cost-effective and safe re-use strategies. Innovating during a pandemic crisis and developing cost-effective, low technology methods to ascertain the safety of decontamination of respirators helped us optimize safe re-use strategies, in a low-resource setting.

Research quality and ethics statement

All authors of this manuscript declare that this scientific study is in compliance with standard reporting guidelines set forth by the EQUATOR Network. The authors ratify that this study required Institutional Review Board/Ethics Committee review, and hence, prior approval was obtained, IRB Min. No 12848 dated May 5, 2020. We also declare that we did not plagiarize the contents of this manuscript and have performed a plagiarism check.


We thank Mr. Kavin, Mr. Aravind, and Mr. Sathish from the bioengineering department who helped in the 3D printing of our simulation model. We thank Dr Alok Srivastava, Mr Augustine Thambaiah, Dr Sathya Subramani, and Dr Soosai Manickam for graciously allowing us to use the particle counter and biosafety cabinet in the Center for Stem Cell Research and Physiology Department. We also thank Ms. Florence Ponnie who helped us in the sterilization of N95 respirators in our CSSD department.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

CDC. Coronavirus Disease 2019 (COVID-19) – Transmission. Centers for Disease Control and Prevention; June 16, 2020. Available from: https://www.cdc.gov/coronavirus/2019-ncov/prevent-getting-sick/how-covid-spreads.html. [Last accessed on 2020 Jun 24].  Back to cited text no. 1
MacIntyre CR, Wang Q, Cauchemez S, Seale H, Dwyer DE, Yang P, et al. A cluster randomized clinical trial comparing fit-tested and non-fit-tested N95 respirators to medical masks to prevent respiratory virus infection in health care workers. Influenza Other Respir Viruses 2011;5:170-9.  Back to cited text no. 2
Final Report for the Bioquell Hydrogen Peroxide Vapor (HPV) Decontamination for Reuse of N95 Respirators. Final Rep; 2016. p. 46.  Back to cited text no. 3
Fit Testing Procedures (Mandatory). 1910.134 App A | Occupational Safety and Health Administration. Available from: https://www.osha.gov/pls/oshaweb/owadisp.show_document?p_id=9780&p_table=STANDARDS. [Last accessed on 2020 Oct 14].  Back to cited text no. 4
Liao L, Xiao W, Zhao M, Yu X, Wang H, Wang Q, et al. Can N95 respirators be reused after disinfection? How many times? ACS Nano 2020;14:6348-56.  Back to cited text no. 5
Standard Operating Procedure (SOP) for Extended Use of N95 Mask for Personal Safety of Health Care Workers (HCW) at AIIMS; April 08, 2020. Available from: https://www.aiims.edu/images/pdf/notice/SOP_N95_09_04_20.pdf. [Last accessed on 2020 Jun 24].  Back to cited text no. 6


  [Figure 1], [Figure 2]

  [Table 1]


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