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. 2022 Jan 10;11(1):83.
doi: 10.3390/pathogens11010083.

Ultraviolet-C Irradiation, Heat, and Storage as Potential Methods of Inactivating SARS-CoV-2 and Bacterial Pathogens on Filtering Facepiece Respirators

Rhodri Harfoot  1 , Deborah B Y Yung  1 , William A Anderson  2 , Cervantée E K Wild  3 , Nicolene Coetzee  3 , Leonor C Hernández  1 , Blair Lawley  1 , Daniel Pletzer  1 , José G B Derraik  3 , Yvonne C Anderson  3 , Miguel E Quiñones-Mateu  1   4
Affiliations

Ultraviolet-C Irradiation, Heat, and Storage as Potential Methods of Inactivating SARS-CoV-2 and Bacterial Pathogens on Filtering Facepiece Respirators

Rhodri Harfoot et al. Pathogens. .

Abstract

The arrival of SARS-CoV-2 to Aotearoa/New Zealand in February 2020 triggered a massive response at multiple levels. Procurement and sustainability of medical supplies to hospitals and clinics during the then upcoming COVID-19 pandemic was one of the top priorities. Continuing access to new personal protective equipment (PPE) was not guaranteed; thus, disinfecting and reusing PPE was considered as a potential alternative. Here, we describe part of a local program intended to test and implement a system to disinfect PPE for potential reuse in New Zealand. We used filtering facepiece respirator (FFR) coupons inoculated with SARS-CoV-2 or clinically relevant multidrug-resistant pathogens (Acinetobacter baumannii Ab5075, methicillin-resistant Staphylococcus aureus USA300 LAC and cystic-fibrosis isolate Pseudomonas aeruginosa LESB58), to evaluate the potential use of ultraviolet-C germicidal irradiation (UV-C) or dry heat treatment to disinfect PPE. An applied UV-C dose of 1000 mJ/cm2 was sufficient to completely inactivate high doses of SARS-CoV-2; however, irregularities in the FFR coupons hindered the efficacy of UV-C to fully inactivate the virus, even at higher UV-C doses (2000 mJ/cm2). Conversely, incubating contaminated FFR coupons at 65 °C for 30 min or 70 °C for 15 min, was sufficient to block SARS-CoV-2 replication, even in the presence of mucin or a soil load (mimicking salivary or respiratory secretions, respectively). Dry heat (90 min at 75 °C to 80 °C) effectively killed 106 planktonic bacteria; however, even extending the incubation time up to two hours at 80 °C did not completely kill bacteria when grown in colony biofilms. Importantly, we also showed that FFR material can harbor replication-competent SARS-CoV-2 for up to 35 days at room temperature in the presence of a soil load. We are currently using these findings to optimize and establish a robust process for decontaminating, reusing, and reducing wastage of PPE in New Zealand.

Keywords: COVID-19; New Zealand; PPE; SARS-CoV-2; UV-C; bacteria; disinfection; personal protective equipment.

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Conflict of interest statement

The authors declare no conflict of interests, including no further patents, products in development or marketed products. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Use of UV-C irradiation to inactivate SARS-CoV-2 replication in stitched filtering facepiece respirator (FFR) coupons. (A) Stitched FFR coupons, four replicates per condition, were inoculated with 5 µL SARS-CoV-2 (107 TCID50/mL) in a solution of cell-culture medium, mucin (simulating saliva), or soil load (mimicking respiratory secretions), then exposed to different applied doses of UV-C irradiation (250 to 2000 mJ/cm2). Following the treatment with UV-C, the FFR coupons were inverted so that the inoculated surface was in contact with the cell-culture medium in a 24-well plate containing 50,000 VeroE6/TMPRSS2 cells/well. Cells were incubated at 37 °C, 5% CO2 for 72 h and SARS-CoV-2 replication quantified by measuring cytopathic effect (CPE). Cells only were used as negative control, including untreated FFR coupons, with no SARS-CoV-2. (B) Quantification of the distribution of UV-C radiation across the UV-C chamber using chemical actinometry. The mean of the UV-C doses absorbed (mJ/cm2) from triplicate experiments for each position in a 24-well plate are indicated.
Figure 2
Figure 2
Use of UV-C irradiation to inactivate SARS-CoV-2 replication in unstitched filtering facepiece respirators (FFR) coupons. (A) Unstitched FFR coupons, four replicates per condition, were inoculated with 5 µL SARS-CoV-2 (107 TCID50/mL) in a solution of cell-culture medium, mucin (simulating saliva), or soil load (mimicking respiratory secretions), then exposed or not to two applied doses of UV-C irradiation (1000 to 2000 mJ/cm2). FFR coupons were processed and analyzed as described in Figure 1A. (B) Head-to-head comparison of stitched vs. unstitched FFR coupons inoculated with SARS-CoV-2 and exposed to a single UV-C applied dose of 1000 mJ/cm2. Negative (cells only) and positive (cells plus SARS-CoV-2) controls correspond to FFR coupons not treated with UV-C irradiation. Cytopathic effect (CPE) score: 0, no CPE; 1, <25% CPE; 2, 25–49% CPE; 3, 50–74% CPE; and 4, 75–100% CPE.
Figure 3
Figure 3
Use of dry heat to inactivate SARS-CoV-2 replication in stitched filtering facepiece respirators (FFR). Stitched FFR coupons, six replicates per condition, were inoculated with 5 µL SARS-CoV-2 (107 TCID50/mL) in a solution of cell-culture medium, mucin (simulating saliva), or soil load (mimicking respiratory secretions), then incubated for 15 to 90 min at room temperature (approximately 22 °C), 60 °C, 65 °C, or 70 °C. Following the dry heat treatment, FFR coupons were processed and analyzed as described in Figure 1A. CPE, cytopathic effect.
Figure 4
Figure 4
Inactivation of Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa on filtering facepiece respirator (FFR) coupons exposed to dry heat treatment. FFR coupons were inoculated with 1 × 106 colony-forming units (CFU) bacteria in PBS, mucin (simulating saliva), or a colony biofilm with ~3.26 × 107 to 4.4 × 108 CFU. Dry heat treatment was performed at 75 °C and 80 °C for 30, 60, 90, or 120 min. Bacterial survival was determined by growth or no growth from the coupons and reported as the number of coupons (out of six replicates) with positive CFU. nd, not determined.
Figure 5
Figure 5
Stability of SARS-CoV-2 on filtering facepiece respirator (FFR) coupons over time. (A) The relative humidity in the PC3 laboratory was monitored during the first nine days of the viral stability experiment to establish the normal range of % humidity in the environment where the SARS-CoV-2-contaminated FFR coupons were stored. S, Sunday; M, Monday; T, Tuesday; W, Wednesday; T, Thursday; F, Friday; and S, Saturday. (B) Stitched FFR coupons, six replicates per condition, were inoculated with 5 µL SARS-CoV-2 (107 TCID50/mL) in a solution of cell-culture medium, mucin (simulating saliva), or soil load (mimicking respiratory secretions), then stored for up to 35 days inside a Biosafety Cabinet in the PC3 laboratory, as described in Materials and Methods. FFR coupons were processed and analyzed as described in Figure 1A 2, 3, 6, 7, 9, 14, 21, 28, and 35 days post-inoculation with SARS-CoV-2. CPE, cytopathic effect.
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