Addressing personal protective equipment (PPE) decontamination: Methylene blue and light inactivates severe acute respiratory coronavirus virus 2 (SARS-CoV-2) on N95 respirators and medical masks with maintenance of integrity and fit

Abstract

Objective: The coronavirus disease 2019 (COVID-19) pandemic has resulted in shortages of personal protective equipment (PPE), underscoring the urgent need for simple, efficient, and inexpensive methods to decontaminate masks and respirators exposed to severe acute respiratory coronavirus virus 2 (SARS-CoV-2). We hypothesized that methylene blue (MB) photochemical treatment, which has various clinical applications, could decontaminate PPE contaminated with coronavirus.

Design: The 2 arms of the study included (1) PPE inoculation with coronaviruses followed by MB with light (MBL) decontamination treatment and (2) PPE treatment with MBL for 5 cycles of decontamination to determine maintenance of PPE performance.

Methods: MBL treatment was used to inactivate coronaviruses on 3 N95 filtering facepiece respirator (FFR) and 2 medical mask models. We inoculated FFR and medical mask materials with 3 coronaviruses, including SARS-CoV-2, and we treated them with 10 µM MB and exposed them to 50,000 lux of white light or 12,500 lux of red light for 30 minutes. In parallel, integrity was assessed after 5 cycles of decontamination using multiple US and international test methods, and the process was compared with the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O₃) decontamination method.

Results: Overall, MBL robustly and consistently inactivated all 3 coronaviruses with 99.8% to >99.9% virus inactivation across all FFRs and medical masks tested. FFR and medical mask integrity was maintained after 5 cycles of MBL treatment, whereas 1 FFR model failed after 5 cycles of VHP+O₃.

Conclusions: MBL treatment decontaminated respirators and masks by inactivating 3 tested coronaviruses without compromising integrity through 5 cycles of decontamination. MBL decontamination is effective, is low cost, and does not require specialized equipment, making it applicable in low- to high-resource settings.

Fig. 1.

Graphical representation of the DeMaND study methodology. (A) Overview of the coronaviruses, respirators, masks, and decontamination methods used. (B) FFRs and medical masks were inoculated with virus and treated with MBL. The remaining infectious virus was quantified using TCID₅₀ or plaque assay. (C) In parallel with the virucidal testing of MBL, intact FFRs and medical masks were subjected to 5 cycles of decontamination before mask integrity was tested using the indicated methods. Note. FFR, filtering facepiece respirator; PRCV, porcine respiratory coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; MHV, murine hepatitis virus; MBL, methylene blue + light; VHP+O₃, vaporized hydrogen peroxide plus ozone. See Supplemental Table S1 (online) for the respirator and mask decontamination and testing matrix.

Fig. 2.

Inactivation of PRCV and SARS-CoV-2 using MBL. (A) To determine the efficacy of different MB concentrations, we added serial dilutions of MB to wells of a 48-well plate containing 10 µL PRCV (2×10⁷ TCID₅₀/ml). Plates were either exposed to red light (12,500 lux) for 30 minutes or were protected from light (<100 lux). The dotted line indicates the limit of detection. (B) We added serial dilutions of MB to wells of a 12-well plate containing ~50 PFU SARS-CoV-2 in MEM plus 15% FCS. Plates were either exposed to white light (50,000 lux) for 45 minutes or protected from light (<100 lux). We determined viral titers using 2–3 replicate samples. Note. FCS, fetal calf serum; MEM, minimum essential media, ND, not detected.

Fig. 3.

MBL inactivates MHV and SARS-CoV-2 on FFR and medical mask material. (A) Effect of MBL treatment on MHV and SARS-CoV-2 titers. We applied a 10-µL aliquot of MHV or SARS-CoV-2 to coupons derived from an FFR (R3) or medical mask (FW) and they were left to dry for 20 minutes. Subsequently, we added 10 or 30 µL MB to each coupon at the indicated concentrations. We exposed the samples to white light (50,000 lux) for the indicated periods or left them in the biosafety cabinet with the lights off. We measured each virus titer using 2–6 replicate samples by TCID₅₀ or plaque assay. Data are represented as mean ± SD. Note. PFU, plaque forming units; R3, 3M panel respirator (1870+) FW, Type II EN 14683 generic face mask. The dotted line indicates the limit of detection.

Fig. 4.

MBL inactivates MHV, SARS-CoV-2, and PRCV on multiple FFR and medical mask types. (A–C) We applied a 10-µL aliquot of SARS-CoV-2 or MHV to coupons of the indicated masks and dried them for 20 minutes. Depending on coupon size, we added 10–30 µL of 10 µM MB to each coupon and then treated the samples with light (50,000 lux) or protected them from light. (D) We injected 100 µL PRCV under the outer layer of intact FFRs or medical masks and allowed them to dry for 30 minutes. Subsequently, we sprayed the FFRs and medical masks with 10 µM MB and dried them for 30 minutes in the dark before exposure to red light (12,500 lux). We determined each virus titer using 2–6 replicate samples by TCID₅₀ or plaque assay. Data are represented as mean ± SD. Note. PRCV, porcine respiratory coronavirus; MBL, methylene blue, and light; MHV, murine hepatitis virus; FFR, filtering facepiece respirator; MB, methylene blue; TCID₅₀, median tissue culture infectious dose; ND, not detected; RH, Halyard duckbill respirator (Fluidshield-46727); RM, 3M half-sphere respirator (1860); R3, 3M panel respirator (1870+); FW, Type II EN 14683 generic face mask; FH, Type IIR ASTM F2100 Level 2 Halyard face mask. The dotted line indicates the limit of detection.

Fig. 5.

Potential applications of MBL in a clinical setting. (A) Effect of low light levels on SARS-CoV-2 inactivation using MB. We applied a 10-µL aliquot of SARS-CoV-2 to R3 coupons and dried them for 20 minutes. We added 10 µL of 10 µM MB to each coupon before treatment with 700 lux (the light level produced by the biosafety hood lights) or <100 lux of light. (B) Effect of MB pretreatment on SARS-CoV-2 inactivation. We cut coupons from R3 masks and soaked them for 1 hour in 10 µM MB. We then dried the coupons in the dark for 2 days before adding 10 µL virus to either the inner or outer layers. We exposed the samples to white light (50,000 lux) for 30 minutes and determined the virus titer by plaque assay. (C) Inactivation of a SARS-CoV-2 clinical specimen by MBL. We obtained a saliva specimen from a COVID-19 patient with a titer of 1.1 × 10⁵ PFU/mL for SARS-CoV-2. We applied 10 µL aliquots to coupons cut from an R3 mask, treated with 10 µM MB and exposed to white light (50,000 lux) for 30 minutes. We determined virus titer by plaque assay. (D) Effect of MB pretreatment on MHV inactivation using intact masks. We pretreated intact RM and FH masks with 10 µM MB by spraying the front and back with a total of 7–8 mL MB and allowed them to dry overnight in the dark. We inoculated the dried masks with MHV and exposed them to white light (50,000 lux) for 30 minutes. We then excised inoculated areas before elution and titration. We determined the virus titer by TCID₅₀ assay. Note. ND, not detected; R3, 3M panel respirator (1870+); RM, 3M 1860 half-sphere respirator; FH, Type IIR Halyard face mask. The dotted line indicates the limit of detection.

Fig. 6.

Effect of MBL and VHP+O₃ treatments on NaCl submicron filtration efficiencies and breathability before and after 5 cycles of decontamination. (A) NaCl submicron filtration efficiency is a measure of the ability of an FFR or medical mask to capture aerosolized particles <1 µm, expressed as a percentage of particles that do not pass the material at a given velocity or flow rate. (B) Inhalation and (C) exhalation breathing resistances before and after 5 cycles of decontamination. The resistance to airflow during inhalation and exhalation is an indication of the difficulty in breathing through the respirators and masks. *Results from decontaminated FFRs and medical masks are significantly different from untreated masks (Student t-test or Mann-Whitney U test, P < .01). **Horizontal solid line in (A) represents the N95 filtration efficiency requirement of ≥95% particle filtration efficiency according to 42 CFR Part 84. Horizontal lines in (B) and (C) represent the following breathing resistance standards: inhalation: ≤35 mmH2O; exhalation: ≤25 mmH₂O for respirators according to 42 CFR Part 84. EN 149 maximum inhalation resistance at 95 L/minute is 2.4 mbar, or ˜24 mmH₂O. At a higher flow rate according to EN 149, the equivalent breathing resistance may increase slightly but can be similar to the 42 CFR Part 84 maximum inhalation resistance at 85 L/minute. Note. RH, Halyard duckbill respirator (Fluidshield-46727). RM, 3M half-sphere respirator (1860). R3, 3M panel respirator (1870+). FW, EN 14683 Type II generic face mask. FH, ASTM F2100 Level 2 Halyard face mask.

Fig. 7.

Effect of MBL and VHP+O₃ treatments on human and manikin fit factor of FFRs and medical masks. (A) We performed human fit testing with volunteer participants who adjusted the FFRs and medical masks to achieve the highest fit factor or seal and subsequently performed head movements and remeasured fit or seal. (B) Manikin fit factors using advanced, realistic manikin headforms is a reproducible method to test fit without volunteer participants. We used the PortaCount PRO+ 8038 machine (TSI, Inc, Shoreview, MN) to determine the overall fit for both human participants and manikins headforms. *Indicates significantly different values between treated and untreated FFR or medical mask at P < .05, Student t-test or Mann-Whitney U test, as appropriate. **Horizontal line represents the following standard: Per OSHA 1910.134(f), if the overall fit factor as determined through an OSHA-accepted quantitative fit-testing protocol is ≥100 for tight-fitting half facepieces, then the fittest has been passed for that respirator. Percentages on or above each bar represent % of respirators or masks tested that surpassed this standard. Although the standard does not apply to face masks, we present the % to note the strong difference between respirator and face mask test results. Note. RH, Halyard duckbill respirator (Fluidshield-46727); RM, 3M half-sphere respirator (1860); R3, 3M panel respirator (1870+); FW, EN 14683 Type II generic face mask; FH, ASTM F2100 Level 2 Halyard face mask.