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. 2022 Oct 26;7(5):e0030322.
doi: 10.1128/msphere.00303-22. Epub 2022 Aug 30.

Aerosolized Hydrogen Peroxide Decontamination of N95 Respirators, with Fit-Testing and Viral Inactivation, Demonstrates Feasibility for Reuse during the COVID-19 Pandemic

Affiliations

Aerosolized Hydrogen Peroxide Decontamination of N95 Respirators, with Fit-Testing and Viral Inactivation, Demonstrates Feasibility for Reuse during the COVID-19 Pandemic

T Hans Derr et al. mSphere. .

Abstract

In response to the demand for N95 respirators by health care workers during the COVID-19 pandemic, we evaluated decontamination of N95 respirators using an aerosolized hydrogen peroxide (aHP) system. This system is designed to dispense a consistent atomized spray of aerosolized, 7% hydrogen peroxide (H2O2) solution over a treatment cycle. Multiple N95 respirator models were subjected to 10 or more cycles of respirator decontamination, with a select number periodically assessed for qualitative and quantitative fit testing. In parallel, we assessed the ability of aHP treatment to inactivate multiple viruses absorbed onto respirators, including phi6 bacteriophage, herpes simplex virus 1 (HSV-1), coxsackievirus B3 (CVB3), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For pathogens transmitted via respiratory droplets and aerosols, it is critical to address respirator safety for reuse. This study provided experimental validation of an aHP treatment process that decontaminates the respirators while maintaining N95 function. External National Institute for Occupational Safety & Health (NIOSH) certification verified respirator structural integrity and filtration efficiency after 10 rounds of aHP treatment. Virus inactivation by aHP was comparable to the decontamination of commercial spore-based biological indicators. These data demonstrate that the aHP process is effective, with successful fit-testing of respirators after multiple aHP cycles, effective decontamination of multiple virus species, including SARS-CoV-2, successful decontamination of bacterial spores, and filtration efficiency maintained at or greater than 95%. While this study did not include extended or clinical use of N95 respirators between aHP cycles, these data provide proof of concept for aHP decontamination of N95 respirators before reuse in a crisis-capacity scenario. IMPORTANCE The COVID-19 pandemic led to unprecedented pressure on health care and research facilities to provide personal protective equipment. The respiratory nature of the SARS-CoV2 pathogen makes respirator facepieces a critical protective measure to limit inhalation of this virus. While respirator facepieces were designed for single use and disposal, the pandemic increased overall demand for N95 respirators, and corresponding manufacturing and supply chain limitations necessitated the safe reuse of respirators when necessary. In this study, we repurposed an aerosolized hydrogen peroxide (aHP) system that is regularly utilized to decontaminate materials in a biosafety level 3 (BSL3) facility, to develop a method for decontamination of N95 respirators. Results from viral inactivation, biological indicators, respirator fit testing, and filtration efficiency testing all indicated that the process was effective at rendering N95 respirators safe for reuse. This proof-of-concept study establishes baseline data for future testing of aHP in crisis-capacity respirator-reuse scenarios.

Keywords: COVID-19; CURIS; N95 respirators; SARS-CoV2; aerosolized hydrogen peroxide; decontamination; disinfection; filtering facepiece (FFP) respirators (FFR); fit-testing; sterilization; virologic testing; virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Sequential fit testing (QLFT and QNFT) of N95 respirators subjected to repeated aHP decontamination cycles. Results demonstrate that all 3M model 8511 respirators successfully pass QLFT and QNFT after 1, 5, and 10 cycles. In particular, 8 of 9 QNFT results for the 3M model 8511 respirator surpassed a fit factor of 200, the maximum reportable by the test method, providing qualitative, yet objective, evidence of the safety margin related to fit integrity after 10 aHP decontamination cycles. All respirator models except Alpha Pro Tech (which had inconsistent fit-test results; see Materials and Methods for details) passed all QLFT and QNFT to which they were subjected. Model Alpha Pro Tech (aHP cycle 1) was only included for a single cycle and thus is not shown here.
FIG 2
FIG 2
Infectious titer of phi6 bacteriophage inoculated on N95 respirator facepieces was eliminated after aerosolized H2O2 (aHP) decontamination. (A) Data are plotted for each aHP cycle in which viral testing was done (see Table 4). Multiple models of N95 respirator (see Table 1) were inoculated and either treated as drying-only controls (red) or subjected to aHP treatment (blue). The respirator surface and model are indicated by the symbol shape and fill. The median of all points within a given aHP cycle and treatment is indicated by a solid horizontal line. The dashed horizontal line indicates the limit of detection (LOD) at 1 viral PFU in the resuspended but undiluted volume from the site of viral inoculation. (B) Petri dish plating of bacterial lawns exposed to phi6 from drying-only (left side) or aHP-treated (right side) respirator inoculation sites. These were applied to the bacterial lawn either as an undiluted resuspension (top row) or 1:106 dilution applied to focal points (bottom row). For the purposes of illustrating the decontaminated sites where zero plaques were detected, these numbers were replaced with fractional values (0.5), to allow their visualization on this log-scale plot. See Table S1 for all data values.
FIG 3
FIG 3
The infectious titer of HSV-1 inoculated on N95 respirator facepieces was reduced by drying and eliminated after aerosolized H2O2 (aHP) decontamination. Data are plotted for each aHP cycle in which viral testing was done (see Table 4). For HSV-1, the sole positive plaque after aHP treatment occurred in aHP cycle no. 3, when the modification 1 parameters were in use (Tables 3 and 4). This failure, in concert with a spore-based biological indicator and 2 CVB3 plaques (Fig. 4), motivated the addition of a dwell time in the final aHP parameters. As in Fig. 2, multiple models of N95 respirator (see Table 1) were inoculated and either treated as drying-only controls (red) or subjected to aHP treatment (blue). The respirator surface and model are indicated by the symbol shape and fill. The median of all points within a given aHP cycle and treatment is indicated by a solid horizontal line. The dashed horizontal line indicates the limit of detection (LOD) at 1 viral PFU in the resuspended but undiluted volume from the site of viral inoculation. For the purposes of illustrating the decontaminated sites where zero plaques were detected, these numbers were replaced with fractional values (0.5) to allow their visualization on this log-scale plot. See Table S1 for all data values.
FIG 4
FIG 4
The infectious titer of CVB3 inoculated on N95 respirator facepieces was reduced by drying and eliminated after aerosolized H2O2 (aHP) decontamination. Data are plotted for each aHP cycle in which viral testing was done (see Table 4). For CVB3, two positive plaques after aHP treatment occurred in aHP cycle no. 3, when the modification 1 parameters were in use (Tables 3 and 4). This failure, in concert with a spore-based biological indicator and 1 HSV-1 plaque (Fig. 3), motivated the addition of a dwell time in the final aHP parameter. The only other positive CVB3 plaque after aHP treatment occurred in aHP cycle 6, and no plaques were detected in the replicate or in parallel samples. As in Fig. 2, multiple models of N95 respirator (see Table 1) were inoculated and either treated as drying-only controls (red) or subjected to aHP treatment (blue). The respirator surface and model are indicated by the symbol shape and fill. The median of all points within a given aHP cycle and treatment is indicated by a solid horizontal line. The dashed horizontal line indicates the limit of detection (LOD) at 1 viral PFU in the resuspended but undiluted volume from the site of viral inoculation. For the purposes of illustrating the decontaminated sites where zero plaques were detected, these numbers were replaced with fractional values (0.5) to allow their visualization on this log-scale plot. See Table S1 for all data values.
FIG 5
FIG 5
The infectious titer of SARS-CoV-2 inoculated on N95 respirator facepieces was reduced by drying and eliminated after aerosolized H2O2 (aHP) decontamination. (A and B) Data are plotted separately for each aHP cycle in which viral testing was done (see Table 4). For SARS-CoV-2, no infectious virus was detected by TCID50 assay after aHP treatment. As in Fig. 2, multiple models of N95 respirator (see Table 1) were inoculated and either treated as drying-only controls (red) or subjected to aHP treatment (blue). The respirator surface and model are indicated by the symbol shape and fill. The median of all points within a given aHP cycle and treatment is indicated by a solid horizontal line. Viral titer was determined by 50% tissue culture infectious dose (TCID50) assay in 96-well plates, with a limit of detection (LOD) of 1.2 (see Materials and Methods for details). For the purposes of illustrating the decontaminated samples where no virus was detected, these numbers were plotted as a value of 1. See Table S1 for all data values.

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