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. 2022 Aug;50(8):871-877.
doi: 10.1016/j.ajic.2022.01.024.

Of masks and methylene blue-The use of methylene blue photochemical treatment to decontaminate surgical masks contaminated with a tenacious small nonenveloped norovirus

Constance Wielick  1 , Allyson Fries  1 , Lorène Dams  1 , Ravo M Razafimahefa  1 , Belinda Heyne  2 , Brian H Harcourt  3 , Thomas S Lendvay  4 , Jean-François Willaert  5 , Simon de Jaeger  5 , Eric Haubruge  5 , Etienne Thiry  6 , Louisa F Ludwig-Begall  1
Affiliations

Of masks and methylene blue-The use of methylene blue photochemical treatment to decontaminate surgical masks contaminated with a tenacious small nonenveloped norovirus

Constance Wielick et al. Am J Infect Control. 2022 Aug.

Abstract

Background: In the context of the SARS-CoV-2 pandemic, reuse of personal protective equipment, specifically that of medical face coverings, has been recommended. The reuse of these typically single-use only items necessitates procedures to inactivate contaminating human respiratory and gastrointestinal pathogens. We previously demonstrated decontamination of surgical masks and respirators contaminated with infectious SARS-CoV-2 and various animal coronaviruses via low concentration- and short exposure methylene blue photochemical treatment (10 µM methylene blue, 30 minutes of 12,500-lux red light or 50,000 lux white light exposure).

Methods: Here, we describe the adaptation of this protocol to the decontamination of a more resistant, non-enveloped gastrointestinal virus and demonstrate efficient photodynamic inactivation of murine norovirus, a human norovirus surrogate.

Results: Methylene blue photochemical treatment (100 µM methylene blue, 30 minutes of 12,500-lux red light exposure) of murine norovirus-contaminated masks reduced infectious viral titers by over four orders of magnitude on surgical mask surfaces.

Discussion and conclusions: Inactivation of a norovirus, the most difficult to inactivate of the respiratory and gastrointestinal human viruses, can predict the inactivation of any less resistant viral mask contaminant. The protocol developed here thus solidifies the position of methylene blue photochemical decontamination as an important tool in the package of practical pandemic preparedness.

Keywords: Equitable decontamination; Large enveloped virus; Light activation; Personal protective equipment; Photosensitiser; Singlet oxygen; Small nonenveloped virus.

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Figures

Fig 1
Fig 1
Validation of the previously established methylene blue (MB) photochemical treatment protocol using an enveloped animal coronavirus (left panel) and evaluation of its efficacy in inactivating a small non-enveloped norovirus (right panel). Porcine respiratory coronavirus (PRCV) - or murine norovirus (MNV) - inoculated surgical mask coupons remained untreated (c+) or were treated with a 10 µM MB solution and exposed to a 12,500-lux red light source (light box) for 30 min (MBL). The infectivity of PRCV recovered from surgical mask coupons (n = 3) was analyzed in swine testicular cells. The infectivity of MNV recovered from mask coupons (n = 9) was analyzed in RAW 264.7 cells. Values for positive controls (MB-untreated, but light box-exposed) ranged between 5.80 and 6.63 log10 TCID50/mL (PRCV) 5.05 and 6.05 log10TCID50/mL (MNV). The cell culture limit of detection (LOD) was 0.80 log10 TCID50/mL for all analyses. Means log10 TCID50/mL and standard deviations are represented. P-values were computed by using a two-sided independent sample t-test (where ****P < 0.0001).
Fig 2
Fig 2
Evaluation of concentration- and time-dependent virucidal kinetics of methylene blue (MB) photochemical treatment on murine norovirus (MNV) in vitro. Murine norovirus was mixed with different MB concentrations in microplates and was exposed to a 12,500-lux red light source (light box) for varying amounts of time (0, 30, 60, 90, 120 minutes). The infectivity of surviving MNV recovered from microplate wells was then analysed in RAW 264.7 cells. The cell culture limit of detection (LOD) was 0.80, 1.8, and 2.8 log10 TCID50/mL for 10 µM MB, 100 µM MB, and 1,000 µM MB concentrations, respectively. All assays were performed as technical duplicates (n = 2). Means log10 TCID50/mL and standard deviations are represented. Values for positive controls (MB-untreated, but light box-exposed MNV) ranged between 4.80 and 5.30 log10 TCID50/mL.
Fig 3
Fig 3
Evaluation of concentration- and longer exposure time-dependent virucidal kinetics of methylene blue (MB) photochemical treatment on murine norovirus (MNV) in vitro. Murine norovirus was mixed with different MB concentrations in microplates and was exposed to a 12,500-lux red light source (light box) for varying amounts of time (0, 1, 2, 3, 4 hours). The infectivity of surviving MNV recovered from microplate wells was then analysed in RAW 264.7 cells. The cell culture limit of detection (LOD) was 1.8, and 2.8 log10 TCID50/mL for 100 µM MB and 1,000 µM MB concentrations, respectively. All assays were performed as biological triplicates and technical duplicates (n = 6). Means log10 TCID50/mL and standard deviations are represented. Values for positive controls (MB-untreated, but light box-exposed MNV) ranged between 5.69 and 6.19 log10 TCID50/mL.
Fig 4
Fig 4
Evaluation of concentration- and time-dependent virucidal kinetics of methylene blue (MB) photochemical treatment on murine norovirus (MNV) – inoculated surgical masks. Murine norovirus-inoculated masks were treated with 100 µM or ,1000 µM MB concentrations and subsequently exposed to a 12,500-lux red light source (light box) for varying amounts of time (0, 2, 3, 4 hrs). The infectivity of surviving MNV recovered from surgical mask coupons was analyzed in RAW 264.7 cells. The cell culture limits of detection (LOD) were 0.8 and 1.8 log10 TCID50/mL for 100 µM and 1,000 µM MB concentrations, respectively. All assays were performed as biological triplicates (n = 3). Means log10 TCID50/mL and standard deviations are represented. Values for positive controls (MB-untreated, but light box-exposed MNV) ranged between 4.90 and 5.71 log10 TCID50/mL.
Fig 5
Fig 5
Evaluation of methylene blue (MB) photochemical treatment on murine norovirus (MNV) – inoculated surgical masks. The infectivity of MNV recovered from mask coupons treated with 100 µM MB and subsequently exposed to a 12,500-lux red light source (light box) for 30 minutes was analysed in RAW 264.7 cells. The cell culture limit of detection (LOD) was 0.8 log10 TCID50/mL. All assays were performed as biological quadruplicates and technical triplicates (n = 12). Means log10 TCID50/mL and standard deviations are represented. Values for positive controls (MB-untreated, but light box-exposed MNV) ranged between 5.80 and 6.05 TCID50/mL. P-values were computed by using a two-sided independent sample t-test (where ****P < 0.0001).
Fig 6
Fig 6
Evaluation of methylene blue (MB) photochemical treatment on murine norovirus (MNV) – inoculated surgical masks. The infectivity of MNV recovered from mask coupons treated with 100 µM MB and subsequently exposed for 15 min to a 12,500-lux red light source (light box) was analysed in RAW 264.7 cells. The cell culture limit of detection (LOD) was 0.8 log10 TCID50/mL. All assays were performed as biological and technical triplicates (n = 9). The left panel represents a summary analysis of all obtained values. The right panel shows results for individual masks (n = 3) and illustrates the varying decontamination efficacies of this protocol. Means log10 TCID50/mL and standard deviations are represented. Values for positive controls (MB-untreated, but light box-exposed MNV) ranged between 5.05 and 6.55 TCID50/mL. P-values were computed by using a 2-sided independent sample t-test (where ****P < 0.0001).
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