Addressing Personal Protective Equipment (PPE) Decontamination: Methylene Blue and Light Inactivates SARS-CoV-2 on N95 Respirators and Masks with Maintenance of Integrity and Fit

Abstract

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has resulted in severe shortages of personal protective equipment (PPE) necessary to protect front-line healthcare personnel. These shortages underscore the urgent need for simple, efficient, and inexpensive methods to decontaminate SARS-CoV-2-exposed PPE enabling safe reuse of masks and respirators. Efficient decontamination must be available not only in low-resourced settings, but also in well-resourced settings affected by PPE shortages. Methylene blue (MB) photochemical treatment, hitherto with many clinical applications including those used to inactivate virus in plasma, presents a novel approach for widely applicable PPE decontamination. Dry heat (DH) treatment is another potential low-cost decontamination method.MethodsMB and light (MBL) and DH treatments were used to inactivate coronavirus on respirator and mask material. We tested three N95 filtering facepiece respirators (FFRs), two medical masks (MMs), and one cloth community mask (CM). FFR/MM/CM materials were inoculated with SARS-CoV-2 (a Betacoronavirus), murine hepatitis virus (MHV) (a Betacoronavirus), or porcine respiratory coronavirus (PRCV) (an Alphacoronavirus), and treated with 10 µM MB followed by 50,000 lux of broad-spectrum light or 12,500 lux of red light for 30 minutes, or with 75°C DH for 60 minutes. In parallel, we tested respirator and mask integrity using several standard methods and compared to the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method. Intact FFRs/MMs/CM were subjected to five cycles of decontamination (5CD) to assess integrity using International Standardization Organization (ISO), American Society for Testing and Materials (ASTM) International, National Institute for Occupational Safety and Health (NIOSH), and Occupational Safety and Health Administration (OSHA) test methods.FindingsOverall, MBL robustly and consistently inactivated all three coronaviruses with at least a 4-log reduction. DH yielded similar results, with the exception of MHV, which was only reduced by 2-log after treatment. FFR/MM integrity was maintained for 5 cycles of MBL or DH treatment, whereas one FFR failed after 5 cycles of VHP+O3. Baseline performance for the CM was variable, but reduction of integrity was minimal.InterpretationMethylene blue with light and DH treatment decontaminated masks and respirators by inactivating three tested coronaviruses without compromising integrity through 5CD. MBL decontamination of masks is effective, low-cost and does not require specialized equipment, making it applicable in all-resource settings. These attractive features support the utilization and continued development of this novel PPE decontamination method.

Of masks and methylene blue - the use of methylene blue photochemical treatment to decontaminate surgical masks contaminated with a tenacious small non-enveloped norovirus

“…To establish optimal MNV treatment conditions, a preliminary microplate-based assay examined concentration- and LB exposure time-dependent virucidal kinetics of MB photochemical treatment. Again, treatment with 10 µM MB concentrations and 30 minutes of LB exposure (treatment conditions previously utilised to successfully inactivate SARS-CoV-2 and its surrogates by more than three orders of magnitude [23]) reduced viral titres by less than one order of magnitude (0.73 (±0.53) log 10 TCID 50 /mL). Protocols combining the same LB exposure time with 100 µM and 1000 µM MB concentrations both reduced MNV titres by 0.93 (± 0.35) log 10 TCID 50 /mL.…”

“…The light box designed by the Terra Research Centre and previously used for MB activation and PRCV decontamination in the context of the DeMaND study [15] contains six 180 W horticultural LED lamps (Roleadro Culture Indoor IP65 LED Horticultural T5 Grow), which together emit 12,500 LUX (or 10.474 W/m²) at 660 nm wavelength (luminescence verified using a light metre (DeltaOHM, Model HD2102.2)). The box interior is cooled by a ventilator equilibrating temperatures within the box (thus eliminating possible temperature effects on viral titres).…”

“…The same protocol was then tested for decontamination of MNV-inoculated SMs. The general workflow followed previously described protocols for PRCV or MNV [12–14] inoculation, MB photochemical treatment [15], and virus elution from SMs [12–14] and is also described in more detail below. Briefly, following injection of 100 µl undiluted PRCV or MNV suspension (7.80 and 7.06 log 10 TCID 50 /mL, respectively; verified via back-titration) under the first outer layer of designated SM coupons, inoculated SMs were comprehensively sprayed with 7 – 8 mL of a 10 µM MB solution (Sigma-Aldrich (M9140); solution prepared in deionised water; total dose of 0.024 mg MB per SM), allowed to dry, and were then either kept in the dark or submitted to LB exposure for a duration of 30 minutes.…”

“…To address this issue, the Development and Methods for N95 Respirators and Mask Decontamination (DeMaND) study recently established a low-cost methylene blue (MB) photochemical treatment for the efficient decontamination of SMs and FFRs contaminated with infectious SARS-CoV-2 or surrogate animal coronaviruses [15]. Photosensitive MB dye, FDA-approved as an oxidation-reduction agent for the treatment of acquired methaemoglobinemia, has a long-standing history of use in pathogen inactivation both in the context of plasma treatment [16–18] and maxillary sinusitis therapy [19].…”

“…For its application to photochemical PPE decontamination, contaminated materials were coated with MB and subsequently exposed to a visible light source triggering the generation of virucidal singlet oxygen. A 10 µM MB concentration and a 30-minute exposure to 12,500 lux (10.474 W/m 2 ) of red light or 50,000 lux (39 W/m 2 ) of white light reduced titres of SARS-CoV-2 and two surrogate viruses by more than three orders of magnitude on all tested materials [15].…”