Approach of using a household device in decontaminating respirators with ultraviolet C during the scarcity in the COVID-19 pandemic

Abstract


Introduction
The COVID-19 pandemic has led to an unexpectedly expanded number of infected cases as well as a shortage of standard protective equipment for health care workers.The strain on resources in the healthcare setting is an extremely challenging problem.Amid the COVID 19 crisis and the shortage of protection, reusing the masks, particularly N95 respirators, a core component of the personal protective equipment among the healthcare workers, seems to be the reasonable practice.The ideal reuse strategy must disinfect pathogenic viruses and bacteria, while conserving the effectiveness of N95 respirators.Another challenge is that the method should be feasible, quick, easy, and practical for all hospitals.
A prior study had shown that SARS-CoV was mostly inactivated by dry heat of 65ºC for 20 min or greater, and inactivated by ultraviolet C (UV-C) by 4016 µW/cm 2 at a distance of 3 cm for 15 min [1].Another study also showed an effectiveness of viral inactivation by heat using temperature at 56ºC and 60ºC [2].
We investigated the possibility of reusing N95 respirators after decontamination by the UV-C sterilizer using household devices.A baby bottle UV-C sterilizer box was tested to ensure the safety for implementation in the hospitals (Fig. 1).

Quantitation of viral titers for the UV-C disinfection method
A Porcine Epidemic Diarrhea virus (PEDV), a swine coronavirus, harmless to humans and, previously shown to possibly transmit via an aerosol route [3] was used for evaluating the inactivation of virus on the N95 respirators.This virus has been successfully used to investigate several aspects of coronavirus replication [4].For the assessment of viral infection, recombinant PEDV bearing a uorescent mCherry gene in its genome (PEDVmCherry) was used [5].
To investigate the effect of UV-C radiation on the infectivity of PEDV bearing the mCherry gene (PEDVmCherry), droplets of Opti-MEM containing PEDVmCherry on the N95 respirator were allowed to be exposed to UV-C for 20 mins before being adsorbed onto Vero E6 cells.The 200 µL (10 4 tissue culture infectious dose [TCID 50 ] per mL) of the recombinant PEDVmCherry was sprayed on the outer layer of N95 respirators and placed at a distance of 5 cm from the UV-C bulb.The N95 respirators were divided into 4 groups; 1. no UV-C exposure, undisturbed at room temperature for 1 hour, 2. only heat mode of the sterilizer box for 50 mins, 3. only UV-C mode of the sterilizer box for 20 mins, 4. combination mode of heat 50 mins and UV-C 10 mins.
Samples of N95 respirator were incubated in 2 mL of Opti-MEM to release the virus at room temperature for 1 hour, then overnight at 4°C.The 500 µL of Opti-MEM containing released virus was adsorbed onto Vero E6 cells for 2 hours, washed with phosphate buffer saline, maintained in Opti-MEMâ (Gibcoâ, Thermo Fischer Scienti c Inc., Waltham, MA , USA) with trypsin (2 µg/mL) and incubated for 48 hours at 37°C.Hoechst dye was used to stain the nuclei of the infected cells.A uorescence microscope was used to observe the cytopathic effect and visualize the mCherry expression in infected cells.

Bacterial culture for the UV-C disinfection method
A used N95 respirator was swabbed before and after decontamination with UV-C.The respirator was hung in the sterilizing box at around 5-8.5 cm directly below the UV-C bulb.The colonies were cultured on chocolate, blood and MacConkey agars.Colonies of bacteria were counted and identi ed by MALDI-TOF mass spectrometry ( MALDI-Biotyperâ, Bruker Cooperation, Germany) after 24-48-hour incubation.
Scanning Electron Microscope (SEM) before/after heat and UV-C exposure The samples of N95 respirator were cut and placed on SEM sample holder before sputter coated with thin gold layer.Their structures as well as the contained bers were examined using scanning electron microscope with a 1000-time magni cation (SEM, Hitachi S-3400N).

Measurement of radiation in the UV-C sterilizer box
The emitted UV-C radiation at the middle and corner of the box, at distances 5 cm to 15 cm below the bulb was measured by a UV radiometer (LT Lutron YK-37UVSD SD Card Data logger UV light meter).The radiated power was calculated using Stefan-Boltzmann law.

Qualitative t testing for N95 respirators
The t test procedure was done on N95 respirators (3M models 1870) as previously described [6].Three participants who had no prior experience with respirator-t testing underwent the initial t test.The masks were reused after 10 mins decontamination with UV-C each time.The follow-up t test was assessed after decontaminating and reusing the N95 respirators after four and six times.

Effect of UV-C radiation on the infectivity of a pig coronavirus
While unexposed PEDV mCherry gave rise to robust mCherry expression, undetectable level of mCherry were observed in the UV-treated group.When the sample was incubated in the heat mode alone, viral infectivity was hardly affected (Fig. 2).The temperature measured during that time reached above 56ºC only 9 mins (6-15 mins after the heat mode started) while it was 54-55ºC for the rest of the time.

Effect of UV-C radiation on bacterial growth
All the bacteria that grew were grown from swabs taken from the facial side of the respirator.They were normal skin and oral ora.All but one were destroyed by 10 mins of UV-C exposure in the sterilizer box.
However, a few colonies of Bacillus beringensis continued to be cultured from swabs taken from the folds of the respirator after 30 mins of UV-C exposure.

Effect of UV-C on SEM characterization
The contour of N95 respirator ber, structure and spaces between the bers remained unaffected after a cumulative of 240 mins UV-C exposure (Fig. 3).

Radiometric analysis
A signi cant variation in the UV-C level at different points in the box was detected, depending on the closeness and angle from the bulb.At the position of testing the recombinant PEDVmCherry coronavirus, 5 cm below the bulb, the level was 0.930-0.932mW/cm 2 .The lowest level was found at the top corner of the box at 0.055 mW/cm 2 .The minimum and maximum level measured at the oor of the box, measured at the corner and the centre respectively, were 0.26 mW/cm 2 and 0.30 mW/cm 2 .
Qualitative t testing for N95 respirators after reuse All 3 subjects passed the t test at the rst time, and the fourth time after reuse.Two subjects passed and one subject failed the t test after the sixth time of reuse.

Discussion
The strategy of reusing respirators might be a very reasonable practice to conserve available supplies for further use during this unprecedented threat of COVID-19.Nevertheless, ineffective sterilization of respirators can result in contamination, transmission and self-inoculation of mucous membranes resulting in COVID-19 infection among the reusers.To our knowledge, there has only been limited data that describe experimental models of coronavirus inactivation on the N95 respirator.SARS-CoV-2 is primarily transmitted through respiratory droplets and contact routes [7][8][9] but airborne transmision is possible even in the absence of aerosols-generating procedures [10].Therefore, the safety of healthcare workers caring for the COVID-19 cases during the current SARS-CoV-2 pandemic has been a major concern.Previous studies have shown that heat effectively inactivated SARS-CoV and MERS-CoV at the lowest temperature range 56-65 °C, after an average time of 30 min [2,11,12].
Prior to the present study, published literature showed that viruses could be inactivated by UV-C but the reported effective dose of UV-C varied between studies.The UV-C exposure dose required for 90% inactivation (D90) for coronaviruses including SARS-CoV varied between studies from 3-3046 J/m 2 in air or liquid media [1,[13][14][15][16][17].However, the relatively high UV-C dose of 1,000-18,000 J/m 2 was used for inactivating the majority of in uenza viruses and coliphage on contaminated N95 facepieces [18][19][20].
Our study found that the emitted radiation in different parts of the UV-C sterilizer box ranged between 0.05-0.93mW/cm 2 .The effective time of viral inactivation by UV-C in our study was observed at 20 min.
The calculation of UV dose in the sterilizer box, excluding the corners of the box, for 20 min and 10 min were 3,600 − 11,160 J/m 2 , and 1,800-5,580 J/m 2 , respectively.
Although heat alone had no effect on virus, the combination of heat 54-55ºC and UV-C 10 min showed no detectable viruses.The uctuation in temperature may have been the reason for the poor viral inactivation in the heat mode.Alternatively, this could be interpreted as the effectiveness of 10-mins UV-C exposure or the synergistic effect of heat combined with UV-C.However, the MERS-CoV was still detectable after heat at 56ºC after 30 min in a previous study [11].Therefore, it is likely that 10-min UV-C is su cient for the inactivation of the virus.
UV radiation degrades polymers.This may result in the loss of protective function of N95 respirators.
However, a study using relatively high UV irradiation at 120 J/cm 2 (1,200,000 J/m 2 ) found minimal increase in particle penetration (1.25%) and had little effect on the ow resistance.This high UV dose decreased the strength of the layers of respirator material by 5-42% depending on the models and layer of respirators, but it had very small effect on the strength of the straps [21].Although we did not test particle penetration or ow resistance in our study, we used the physical appearance of the bers, as determined by SEM, and t testing as secondary measures to imply any changes in respirator function that might occur after UV-C decontamination.The SEM showed no change in the ber arrangement and spaces between bers after 240 min of UV-C exposure, compared to the structure of unexposed UV-C samples.
Inactivation of organisms by UV-C depends on the type of organisms, energy of the light source, distance between light source and object surface, and the time of exposure.All these factors must be integrated in order to determine the optimal disinfection strategy.Our approach using a UV-C sterilizer box, with a 4 watt bulb, at the distance of 5-8.5 cm, for at least 10 min should be enough to inactivate the coronaviruses, according to our test result, and previous studies.There were several limitations in our study.We did not perform the viral and SEM test on the strap of N95 respirator.Viral inactivation requires direct UV-C exposure of the contaminated surface.It is important to ensure that all contaminated surfaces are exposed to the UV-C radiation.Previous study suggested that UV-C had minimal effect on the respirator straps.A UV-C dose of 590 J/cm 2 (5,900,000 J/m 2 ) decreased the strap strength by 10-21% [21].The ltration e ciency after reuse was not conducted in our study.However, no structural integrity change was observed using SEM in this study, and the t testing has been shown to correlate well with the particle-size-selective protection factors [22].Following the recommended guidelines, a seal check must always be performed prior to each reuse.
Our disinfection strategy of using a home device has several advantages.UV-C sterilization devices are already available in the markets.The process takes only 10-20 min.Users take responsibility for the disinfection process and keep their own N95 respirators.The disinfection process is easy, as no new invention or additional workers are required for the process.

Figure 2 The
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