key: cord-1024692-7azerrez
authors: Ibáñez-Cervantes, Gabriela; Bravata-Alcántara, Juan Carlos; Nájera-Cortés, Alan Steve; Meneses-Cruz, Sergio; Delgado-Balbuena, Laura; Cruz-Cruz, Clemente; Durán-Manuel, Emilio Mariano; Cureño-Díaz, Monica Alethia; Gómez-Zamora, Erika; Chávez-Juárez, Sonia; Sosa-Hernández, Oscar; Aguilar-Rojas, Antonio; Bello-López, Juan Manuel
title: Disinfection of N95 masks artificially contaminated with SARS-CoV-2 and ESKAPE bacteria using hydrogen peroxide plasma: impact on the reutilization of disposable devices
date: 2020-07-06
journal: Am J Infect Control
DOI: 10.1016/j.ajic.2020.06.216
sha: 73095848e538023d30102365d9a1e3d2fe12f473
doc_id: 1024692
cord_uid: 7azerrez

INTRODUCTION: One of the serious consequences of the SARS-CoV-2 pandemic is the shortage of protective equipment for health personnel. N95 masks are considered one of the essential protective equipment in the management of patients with COVID-19. The shortage of N95 masks implies potential health risks for health personnel and significant economic losses for the health institution. The objective of this work was to investigate the disinfection of N95 masks artificially contaminated with SARS-CoV-2 and ESKAPE bacteria by using hydrogen peroxide plasma. MATERIAL AND METHODS: We examined the disinfection capacity of hydrogen peroxide plasma against the SARS-CoV-2 and two members of the ESKAPE bacteria (Acinetobacter baumannii and Staphylococcus aureus) through a study of artificial contamination in situ of N95 masks. Amplification of specific genes by RT-PCR of SARS-CoV-2 and microbiological culture of ESKAPE bacteria was performed before and after the disinfection process. RESULTS: SARS-CoV-2 was not detected in all assays using five different concentrations of the virus, and A. baumannii and S. aureus were not cultivable with inoculums of 10(2) to 10(6) CFU after disinfection tests of N95 masks with hydrogen peroxide plasma. CONCLUSION: Disinfection of N95 masks by using the hydrogen peroxide plasma technology can be an alternative for their reuse in a shortage situation. Implications for the use of disinfection technologies of N95 masks and the safety of health personnel are discussed.

One of the serious consequences of the SARS-CoV-2 pandemic is the shortage of protective equipment for health personnel. N95 masks are considered one of the essential protective equipment in the management of patients with COVID-19. The shortage of N95 masks implies potential health risks for health personnel and significant economic losses for the health institution. The objective of this work was to investigate the disinfection of N95 masks artificially contaminated with SARS-CoV-2 and ESKAPE bacteria by using hydrogen peroxide plasma. Material and methods: We examined the disinfection capacity of hydrogen peroxide plasma against the SARS-CoV-2 and two members of the ESKAPE bacteria (Acinetobacter baumannii and Staphylococcus aureus) through a study of artificial contamination in situ of N95 masks. Amplification of specific genes by RT-PCR of SARS-CoV-2 and microbiological culture of ESKAPE bacteria was performed before and after the disinfection process. Results: SARS-CoV-2 was not detected in all assays using five different concentrations of the virus, and A. baumannii and S. aureus were not cultivable with inoculums of 10 2 to 10 6 CFU after disinfection tests of N95 masks with hydrogen peroxide plasma. Conclusion: Disinfection of N95 masks by using the hydrogen peroxide plasma technology can be an alternative for their reuse in a shortage situation. Implications for the use of disinfection technologies of N95 masks and the safety of health personnel are discussed.

One of the main difficulties worldwide is the insufficient supply of N95 masks for the care of cases arising in hospital units, for example, United States calculated that will need 3.5 billion N95 respirators for health workers during this pandemic, and currently it calculates that it has 1% of the necessary volume. 1 The shortage of protective equipment for the management of patients carrying the COVID-19 infection caused by the SARS-CoV-2 pandemic has generated the need to implement alternatives that allow its reuse. [2] [3] [4] . The respiratory tract, being the main route of entry for SARS-CoV-2, is considered one of the main critical points that need protection. 5, 6 To avoid health personnel of being infected, N95 masks are essential in the management of suspected and confirmed patients of SARS-CoV-2 due to their high level of protection as they prevent the virus from spreading through the bioaerosols generated by the patients' exhalation [7] [8] [9] . Therefore, their shortage implies potential risks of contagion between health personnel and patients. In USA, an alarming number of health workers infected with SARS-CoV-2 has been reported, and more than 100 deaths have been reported worldwide, including doctors and nurses. 10, 11 This marks the importance of providing safety and sufficient supply of protective equipment to health workers. The protection of the respiratory tract of health personnel is a challenge, therefore, to provide safety to health personnel through the use of N95 masks is crucial during the management of COVID-19 patients. In previous works, the effectiveness of disinfection of N95 masks has already been investigated by using diverse methods, including the use of ultraviolet light and vaporized hydrogen peroxide. 4 causing Ventilator-Associated Pneumonia (VAP) are also a potential risk for health personnel, since the Intensive Care Unit (ICU) is the main hospital area considered a reservoir for this type of bacteria 14 . In a previous study by our working group, it was reported that two members of the ESKAPE group (Acinetobacter baumannii and Staphylococcus aureus) are the main causative agents of VAP in the "Hospital Juárez de México" (HJM). 14 Therefore, the study of the effect of disinfection on this type of bacteria also provides valuable information on microbiological safety for health personnel.

The objective of this work was to investigate the disinfection of N95 masks artificially contaminated with SARS-CoV-2 and ESKAPE bacteria by using hydrogen peroxide plasma.

Implications for the use of disinfection technologies of N95 masks and the safety of health personnel are discussed. Strains were previously isolated from patients admitted to the ICU of the HJM and were genetically identified by 16S rRNA gene sequence analysis ( Table 1) .

Serial dilutions (1:10, 1:100, 1:1000, and 1:10000) of SARS-CoV-2 virus were performed in viral preservation media. The undiluted clinical sample containing SARS-CoV-2 was also included in the study. In all dilutions, the presence of SARS-CoV-2 was verified by RT-PCR assays as aforementioned.

Acinetobacter baumannii and S. aureus were streaked on LB-agar and incubated at 37 o C overnight. Strains were inoculated in LB-broth under agitation at 200 rpm at 37 o C for 24 h.

The cultures were adjusted to 10 6 CFU/50 l by using a Spectrophotometer 3000

SmartSpec™ flow (BIORAD) at 600 nm and diluted to 10 2 , 10 3 , 10 4 , and 10 5 CFU/50 l. In order to confirm the bacterial concentration, cultures were spread onto LB-agar and incubated for the CFU/ml counting (per triplicate). Bacterial suspensions were stored on an ice bed before use.

All contamination assays were carried out under aseptic conditions in a laminar flow cabinet.

Ten N95 3M ® masks (Model 8210) were divided in two groups A (problem) and B (control).

Fifty microliters of each dilution of the SARS-CoV-2 and ESKAPE bacteria were inoculated into 1 cm 2 area by the outer surface of the N95 masks as shown in Figure 1 . Subsequently, inoculums were incubated at room temperature for 20 min for their total absorption in the N95 mask material. Over time, "group A" N95 masks (problems) were individually placed in 20 cm X 20 cm sterilization bags "Tyvek ® " (with STERRAD ® chemical indicator and STERRAD VELOCITY TM biological indicator) and were hermetically sealed and subjected to disinfection by using hydrogen peroxide plasma as indicated below. Disinfection experiments were carried in a STERRAD ® 100NX ® sterilization system (ASP TM , USA), by using the "standard STERRAD ® NX" sterilization cycle under 47 min to exposure to peroxide hydrogen plasma and according to the manufacturers' protocols. "Group B" N95 masks (controls) were subjected to recovery of SARS-CoV-2 and ESKAPE bacteria by RT-PCR and bacterial culture, respectively, as follows.

After disinfection of N95 masks, inoculation areas of N95 masks with SARS-CoV-2 and ESKAPE bacteria were cut out. The virus and ESKAPE bacteria were separately eluted from the N95 masks in viral preservation medium and saline solution, respectively. The first five aliquots were subjected to RNA extraction and specific amplification of E, RdRp, and RNAse P genes, and the last five aliquots were appropriately diluted, viable count was made by plating in LB-agar. After incubation, colonies (only if there was growth) were counted and reported as CFU, and the average of CFU was compared "before and after" disinfection process. Death bacterial percentage after disinfection at different bacterial concentrations was calculated. Each experiment was conducted by triplicate.

To examine the disinfectant activity of hydrogen peroxide plasma, five different dilutions of SARS-CoV-2 (including not diluted clinical sample) were inoculated on N95 masks in order to simulate the "real" contamination of the N95 masks in health personnel ( Figure 1 ). All dilutions of SARS-CoV-2 were detected by RT-PCR after inoculation in N95 masks, therefore, the possible not detection of SARS-CoV-2 was due to treatment with hydrogen peroxide plasma. We found that hydrogen peroxide plasma inhibited the detection of the SARS-CoV-2 in all virus dilutions by RT-PCR (compared to control) as soon as it was exposed to treatment ( Table 2) .

Acinetobacter baumannii and S. aureus strains were completely killed on N95 masks after treatment with hydrogen peroxide plasma at the five bacterial concentrations tested.

Calculations of viability before and after treatment reflected as 100% bacterial death. This observation was confirmed by absence of bacterial growth in all assays of bacterial recovery by bacterial culture on TSA plates (Table 2 ).

According to the recommendations on the mandatory Personal Protective Equipment (PPE)

for the care of patients with suspected and/or confirmed SARS-CoV-2, this must include gloves, a gown, protective glasses, and face masks; however, N95 respirators allow to have a hermetic seal to the skin, conferring greater protection than surgical masks, being relevant for the processes that can generate aerosols. In compliance with the recommendations and protocols regarding infection prevention and control, these types of respirators have to be discarded, since they are considered single-use disposable devices. 1 With these results we speculate that due to the mechanism of action of the hydrogen peroxide plasma, genetic material of SARS-CoV-2 virus was significantly damaged, since it could not be detected by RT-PCR. Hydrogen peroxide gas plasma generated in a closed chamber under vacuum are known to produce charged particles, many of which are highly reactive free radicals. These radicals have the ability to disinfect materials due to their interaction with essential components of bacteria, fungi, and viruses, such as essential enzymes, nucleic acids (DNA and RNA) and therefore, avoid their replicative and infectious capacity. [16] [17] [18] The strategy of conducting artificial contamination tests with a specimen of clinical origin of SARS-CoV-2, and not with a viral culture, is fully justified. The tests were aimed to achieve, as much as possible, a real situation of contamination by means of bioaerosols. Health personnel are known to be exposed to SARS-CoV-2 contamination during the exhalation of patients with COVID-19. Bioaerosols generated by infected people have been described as the main cause of contagion of SARS-CoV-2, with saliva particles of breathable size from 1 to 10 μm being the main concern 8 . In contrast, the co-infections reported by ESKAPE bacteria causing VAP in patients with COVID-19 in other parts of the world, are an important factor that could promote the acquisition of bacterial infections 19, 20 and where health personnel could also be affected. Bacterial strains used in this study have genetic characteristics (virulence and antimicrobial resistance factors) that allow them to form mature biofilms on inert surfaces, such as N95 masks ( Table 1 ). The complete elimination of the strains tested in this study, reinforces the mentioning evidence of the direct damage of hydrogen peroxide plasma in vital macromolecules for the replication of pathogens. With the evidence showed in this work, we believe that the use of hydrogen peroxide plasma under standard conditions of treatment of N95 masks, may be an alternative for disinfection and possibly for other protective devices in shortage situations.

Additionally, we contribute to the efforts to generate solid research evidence that supports the viability to disinfect used N95 masks, by using a method that preserves the physical characteristics of protection and that avoids the degradation of the materials with which they are made.

Hydrogen peroxide plasma could be considered an option in the disinfection of N95 masks due to its activity. 

This study is part of the project "CONACyT 313771":Análisis del efecto del ozono sobre SARS-CoV2 como alternativa de producto desinfectante en equipos de protección del personal de salud de alta demanda" 

There was no financial support 5 9.7X10 4 NA 100 10 6 9.8X10 5 NA 100 *By RT-PCR method **Plating on TSA agar method ***Not applicable

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