Construction and validation of an ultraviolet germicidal irradiation system using locally available components.

Coronavirus disease (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is responsible for a global pandemic characterized by high transmissibility and morbidity. Healthcare workers (HCWs) are at risk of contracting COVID-19, but this risk has been mitigated through the use of personal protective equipment such as N95 Filtering Facepiece Respirators (FFRs). At times the high demand for FFRs has exceeded supply, placing HCWs at increased exposure risk. Effective FFR decontamination of many FFR models using ultraviolet-C germicidal irradiation (UVGI) has been well-described, and could maintain respiratory protection for HCWs in the face of supply line shortages. Here, we detail the construction of an ultraviolet-C germicidal irradiation (UVGI) device using previously existing components available at our institution. We provide data on UV-C dosage delivered with our version of this device, provide information on how users can validate the UV-C dose delivered in similarly constructed systems, and describe a simple, novel methodology to test its germicidal effectiveness using in-house reagents and equipment. As similar components are readily available in many hospitals and industrial facilities, we provide recommendations on the local construction of these systems, as well as guidance and strategies towards successful institutional implementation of FFR decontamination.

Introduction

One emergent challenge during the current COVID-19 pandemic has been that the SARS-CoV-2 virus frequently infects health care workers, threatening to deplete the pool of people available to care for sick patients at a time when they are most needed. Respiratory protection is an important component of interrupting the chain of SARS-CoV-2 transmission. The Centers for Disease Control and Prevention (CDC) recommends N95 Filtering Facepiece Respirators (FFRs) or the equivalent for those engaged in the care of SARS-CoV-2 positive patients [1]. However, N95 FFRs have been in critically short supply due to the intersection of dramatically increased global demand and fractured supply chains, presenting an urgent need for the implementation of decontamination strategies for used N95 FFRs during this crisis.

N95 FFRs can be decontaminated and re-used several times without loss of protective efficacy [2-9]. Although a wide variety of modalities effectively decontaminate N95 FFRs, some of these modalities (such as spraying with alcohol) degrade the mask material’s aerosol filtration abilities or alter subsequent FFR fit [4]. Other modalities, such as vaporized hydrogen peroxide, are highly effective and preserve FFR function [4], but require equipment that could be difficult to obtain and operationalize during a pandemic. Ultraviolet-C germicidal irradiation (UVGI) is a mode of N-95 FFR decontamination that is effective and more easily set-up. After the 2003 severe acute respiratory syndrome (SARS) epidemic, numerous studies demonstrated that coronaviruses similar to SARS-CoV-2, are inactivated by light in the UV-C spectrum (200-280nm) [6]. Inactivation occurs by photochemical degradation of the coronavirus genetic material which is composed of single-stranded RNA (ssRNA) [10, 11]. Institutions have developed protocols to employ commercial light sources to perform UVGI-based N95 FFR decontamination [10]. However, commercial UVGI systems are costly and in short supply during the current COVID-19 pandemic.

Here, we outline how we developed and tested an inexpensive system for UVGI-based N95 FFR decontamination and confirmed its germicidal efficacy. Furthermore, we discuss technical considerations, practical guidance, and alternative approaches to outline how to rapidly construct a system to permit decontamination of N95 FFRs and thus attenuate the impact of critical PPE shortages.

Methods

UVGI chamber design and construction

Light sources capable of producing UV-C light are normally widely available. During the COVID-19 pandemic, many “implementation ready”, commercial UVGI systems became unavailable, so we designed and constructed an in-house UVGI system, using equipment already available on site. Apart from biological safety hoods, UV-C bulbs are used in HVAC and water decontamination applications, and are frequently available from a hospital’s physical plant. A variety of bulbs exist for this purpose, which can vary both in power as well as in terms of far UV-C wavelength production (175–210 nm). Far UV-C light can generate hazardous ozone gas [11], and additional design considerations would be necessary if these wavelengths were produced.

We designed and built a chamber specifically for UVGI decontamination of N95 FFRs using a local supply of low-pressure UV-C bulbs (Philips TUV G30T8 30W / 35 inch bulbs; dominant emission at 254 nm, minimal emission at 175–210 nm). Our design consisted of two planar arrays of UV-C bulbs facing each other to produce an adequate field of UV illumination to both sides of each mask and allow for simultaneous decontamination of multiple masks (). Our electrical shop assembled the arrays using standard low pressure light mounts (‘tombstones’) and regulated power supplies (ballasts; see for materials). Each array was 4 bulbs wide (12 cm between centers of adjacent bulbs) and 3 bulbs long end-to-end (12 bulbs per array), and the distance between arrays was 70 cm. FFRs are suspended from stretches of nylon line (Black and Decker), strung between two hooks with a thick rubber band at one end to maintain tension. Although not performed at our facility, this system could be scaled up by increasing the number of paired 4-bulb arrays, in a linear or side-by-side arrangement, with consideration to facilitate staff access for FFR loading/unloading.

Light fixtures were mounted onto flat aluminum stock bases, with mounting bars bracketed across an 8 x 10 ft room and bolted to walls (). An additional safety rail was constructed on the operator side of the array to protect bulbs and staff from inadvertent contact. UV-C exposure is harmful to skin and eyes, so a UV-C-blocking barrier (glass) was used to protect staff from UV-C exposure during decontamination, while still allowing UVGI technicians to observe the light meter readings. UV-C light did not penetrate the glass, as verified by our UV-C dosimeter.

UVGI intensity calibration and measurement

To measure UV-C dose, we used the ILT2400-UVGI meter (International Light Technologies, Peabody, MA), which is selectively sensitive to the wavelengths in the UV-C spectrum and reports UV-C light irradiance in W/cm2. This dosimeter can also integrate light exposure in real time, and report total UV-C dose delivered in J/cm2. We mounted this detector inside of our UVGI chamber to verify effective light delivery with each UVGI decontamination cycle, and positioned it to measure light intensity in a direction/location corresponding to the least amount of UV-C energy that would be received by any FFR component (see below), thus ensuring that all sections of the mask receive adequate irradiation. We strongly recommend irradiance/energy measurement for newly assembled systems and during each cycle as a quality control parameter, as UV-C bulb output can vary between bulbs, within the same bulbs as they “burn-in” and age, and even during use (in our hands, irradiance increased substantially over the first minute of being turned on). An optimal UV-C dosimeter must be calibrated, which can be verified through ISO17025 accreditation (quality assurance) and NIST traceability (calibrated to a known source), and ideally indicate both irradiance and total energy delivered for ease-of-use. However, although UV-C dosimeters are frequently available in larger-scale industrial hygiene programs, their availability might also be subject to resource availability and cost constraints. This has led to the evaluation of UV-C sensitive photochromic paper as a low-cost alternative methodology for the quantitative evaluation of UVGI processes [12].

Locally-produced bactericidal assay

Glass coverslips (Fisherbrand 18CIR-1.5) were sterilized in 3% bleach and rinsed in 70% ethanol in a biosafety cabinet with laminar flow. DH5α Escherichia coli (E. coli) frozen at -80C in 10% glycerol was grown in sterile Luria broth (LB) until bacterial concentration following 10-fold dilution reached an optical density 600 (OD600) of 0.25–0.4 (Amersham Biosciences Ultrospec 10 spectrophotometer). OD600 was recorded for each run to limit variation between experiments. Biological indicators (BIs) were made by plating 4 μL of E. coli–containing suspension onto sterilized coverslips in a biosafety cabinet and allowing them to dry for 30 minutes. Bacteria-free glass coverslips were employed as negative controls.

These BIs were placed inside sterile petri dishes in a cell culture hood, transported to our UVGI device, and then exposed to UV-C light in our custom built UV-C light apparatus using Philips G30T8 bulbs (see above). The BI-containing dishes were positioned on a platform positioned directly in the midpoint of the array (where FFRs would be positioned), directly facing the top UV-C array, with petri dish lids removed. Cumulative UV-C doses ranged from 6 to 1000 mJ/cm2, as measured by a calibrated UV-C specific light meter (ILT2400-UVGI; International Light Technologies, Inc., Peabody, MA). After UV-C irradiation, petri dishes were covered, and the BIs were transferred to a biosafety cabinet where they were placed in conical tubes (50 mL) with 5 mL of LB. The loosely-capped tubes were placed in an orbital shaker at 37 C and 250 RPM. After 16 hours of growth, the OD600 of each sample was measured (Amersham Biosciences Ultrospec 10 spectrophotometer). E.coli amounts were obtained by comparing the OD600 for each sample with the OD600 from similarly and simultaneously prepared standards derived from a range of non-irradiated control coverslips that had been plated with serially diluted stock E. coli. Hydrogen peroxide vapor was used as a decontamination positive control with which to compare UV-C. These BIs were similarly prepared on glass coverslips as above, and then wrapped in sterile Tyvek and placed in a Biosafety Level 3 laboratory at the VA Portland HealthCare System. A negative control was a placed in the room. One positive and one negative control were placed in the hallway and were never exposed to HPV. In addition, hydrogen peroxide chemical indicators (Steris VERIFY HPU Chemical Indicator) were placed throughout the room. HPV was generated using a Bioquell Clarus C and 30% hydrogen peroxide (Fisher). The HPV cycle was 30 minutes of conditioning, pre-gassing, 45 minutes of gassing at 8 gram/minute, 60 minutes of gassing-dwell at 8 gram/minute, followed by an aeration phase. An additional aeration unit was also used. Peak levels of hydrogen peroxide were >400–600 PPM. We used a PortaSens II handheld detector to verify the concentration of hydrogen peroxide was < 1ppm before entering the room. After treatment with HPV, BIs were processed as described above and compared with the OD600 from samples that had not been exposed to HPV.

Results

UVGI dose measurements

Our UVGI chamber design consisted of two planar arrays of UV-C bulbs facing each other, producing a strong and uniform field of UV illumination, and designed to allow for simultaneous decontamination of multiple masks from both sides. Irradiance diminishes as one moves away from a light source, as described by the inverse square law. This relationship means that shorter inter-array distances provide stronger irradiance (and possibly greater rates of decontamination and throughput), but result in a greater difference in irradiance between the proximal and distal aspects of each FFR facing each array. Our intent was to provide a strong irradiance with minimal non-uniformity between the arrays over the region occupied by the N95s. Between 28–42 cm from each array (the space occupied by the hanging masks directly between the arrays), our arrangement produces UV-C irradiance of 0.47–0.78 mW/cm2 (). This range corresponds to the closest and furthest FFR components facing each identical array (proximal and distal regions), as shown in . This irradiance provides a dosage of 28–47 mJ/cm2 in one minute, and 1.0–1.6 J/cm2 in approximately 30 minutes. Irradiance was observed to drop off markedly in the outermost 10 cm of the bulb array in the longitudinal direction and beyond 20 cm from midline in the transverse direction ( p < 0.01 for outermost locations compared with any other location; repeated measures ANOVA with post-hoc Tukey’s test), and these regions are not used for decontamination. Cycle time was adjusted such that the minimum dosage within the FFR zone would reach 1.0 J/cm2 (which corresponded to irradiance at outermost edges of the region), which our facility chose as a minimum UV-C dose for mask decontamination, and which is consistent with evidence and recently released guidance from governmental and non-governmental agencies [13, 14].

UV-C light intensity measured within the array.

A. Schematic demonstrating array dimensions and axes. B. Light intensity variation along longitudinal stretch of each array beginning at the terminal tombstone (= 0 cm) moving inwards along the x axis between the center bulbs (mean mW/cm2 ± standard deviation, n = 3 at each location). The slight decrease in irradiance at 85–90 cm corresponds to the region between end-to-end bulbs. C. Light intensity variation moving outwards from the center of the linear array (along the y axis), measured at x = 45 cm. Measurements were recorded with dosimeter facing the array at the proximal and distal vertical extents of each hanging FFR (in this case, z = 28 and 42 cm from array), demonstrating decreased light irradiance at both levels when moving away from the middle of the exposure zone (n = 4 measurements per location ± standard deviation).

Germicidal activity verified using locally produced biological indicators

Although we verified the UV-C dosage produced by our array by a specific UV-C detector, additional evidence of functional efficacy of novel decontamination processes is typically obtained through the use of surrogate biological indicators. Biological indicators utilize microorganisms with a defined resistance to a sterilization process [15]. Various organism classes may be ranked in terms of resistance to decontamination, with bacterial spores ranked amongst the most difficult to kill organisms, while enveloped viruses such as SARS-CoV-2 are ranked amongst the most susceptible to standard disinfection processes [16, 17]. Due to supply disruptions reducing the availability of commercially produced BIs, we developed our own BIs using E. coli. E. coli was selected because it is much more resistant to decontamination than enveloped single-stranded RNA viruses [17], and provides a faster readout than G. stearothermophilus spores, which require days of incubation post-decontamination.

The sensitivity to of E.coli to UV decontamination was measured via the optical density of broth after incubation with irradiated, or control non-irradiated, E.coli-coated glass discs (Methods; ). The E.coli-coated glass discs were exposed to discrete doses of UV-C, and subsequently incubated overnight in LB. The optical density of the LB after incubation, which is proportional to bacterial number [18], was plotted against the UV-C irradiance level (). These data demonstrate that doses as little as 6 mJoule/cm2 reduced the optical density by ~50%. To characterize the lower limit of detection, we prepared E. coli-coated glass discs under identical conditions, but with concentrations of E. coli that had been serially 10-fold diluted. We found that even after 5 serial dilutions (1/100,000) the optical density of non-irradiated samples was higher than we observed following an irradiance of 30 mJ/cm2 (Fig 3D). These data indicate that 30 mJoule/cm2 irradiance provided ≥ 5-log reduction (99.999%) of viable E.coli, which is an organism more resistant to UV-C than single-stranded RNA viruses such as SARS-CoV-2. Our positive controls utilized hydrogen peroxide vapor (HPV) as a gold standard for decontamination, and similarly demonstrated ≥ 5-log reduction of E.coli.

Fig 3

UV-C light is germicidal to homemade E. coli biological indicators.

A. A homemade Escherichia coli (E. coli) Biological Indicator (BI) shown a on glass coverslip. B. Homemade E. coli BIs were incubated for 16 hrs in an orbital shaker. Conical on the left is a negative control (no E. coli on disk) and conical on the right is a positive control (E. coli on disk). C. Serial dilutions of E. coli on glass coverslips. The coverslips were incubated as described above and the Optical Density at 600 nm (OD600) was measured. The mean and SEM are shown. Data are pooled from 3 separate experiments. D. Homemade BIs were exposed to discrete doses of UV-C (mJ/cm2) and then incubated as described above. OD600 was measured and plotted. The mean and SEM are shown. Data are pooled from separate experiments (0 mJ/cm2: n = 4; 500 mJ/cm2: n = 6; all other doses: n = 7).

Alternative chamber designs

Prior to construction of our UVGI device, we investigated alternative chamber designs, and will briefly describe them here. First, we re-purposed standard cell culture biosafety hoods, which are widely available in facilities with biomedical research labs and which have been previously evaluated for N95 FFR decontamination by others [19]. These unmodified biosafety hoods (SterilGARD, The Baker Company, Sanford, ME) contain a single UV-C bulb in its stock configuration, as well as a UV-C impermeable sliding glass door to protect staff. The bulb in our hood was a GE G64T5 65W bulb, with an expected light intensity of 0.26 mW/cm2 at 1 m distance. However, as irradiance decreases with bulb age, we performed several measurements of irradiance in our hood. We measured an irradiance of 0.18 mW/cm2 of UV-C intensity at the working surface 60 cm directly below the bulb and 0.13 mW/cm2 at the working surface at the front of the hood (75 cm from the bulb). A raised platform 30 cm from the center of the bulb received 0.34 mW/cm2. While readily available, these units did not permit both surfaces of an N95 FFRs to be irradiated simultaneously, complicating the decontamination process, and the low intensity of the UV-C also necessitated prolonged irradiance (> 4 hours to reach the necessary 1 J/cm2 dose for each surface), which we deemed impractical for high throughput [19].

In a subsequent refinement, additional bulbs (Philips TUV G30T8 30W; see above) were placed in the upper and lower parts of the cabinet, to allow simultaneous UVGI decontamination of both surfaces of each N95 mask, improving the performance of the device (. The intensity of UV-C at the vertical midpoint of the chamber was 0.66 mW/cm2 in either direction, but was uniform moving laterally until the last 25 cm of each bulb run. Thus, depending on the particular bulbs available at any individual institution, biosafety hoods could be safely modified to accommodate additional light sources to provide bidirectional irradiation. In addition, the use of UV-C reflective material such as household aluminum foil [20] to surround the decontamination chamber could not only reduce the processing time, but also improve the incident angles on FFRs, perhaps increasing efficacy. Although this was not formally evaluated in any of our designs, it has been incorporated in other UVGI systems [21].

UVGI decontamination chamber constructed within a modified cell culture biosafety hood.

Two identical four-bulb UV-C arrays were placed facing each other within the hood, and were used to decontaminate masks prior to construction of the larger array (Fig 1).

Fig 1

UVGI arrays.

A. Each array consists of 12 UV-C bulbs in a 3x4 array, facing each other, with masks suspended between arrays. B. Longitudinal end of one array showing lampholder mounts on aluminum stock. C. Light dosimeter probe on left (white arrow) mounted to measure light at iso-irradiant site relative to lowest exposure of each mask. Light meter console (arrowhead) in foreground for monitoring total dose delivered (as seen from clean workroom). D. “Dirty” workroom entry to UVGI Suite. E. “Clean” workroom for packaging decontaminated masks.

Discussion

The burden on healthcare systems caused by the COVID pandemic has necessitated the adaption of new approaches to ensure availability of PPE for healthcare workers. However, even decontamination strategies typically require resources and supplies that can be difficult to obtain during global supply line disruption. Our description of simple and cost-effective methods to develop and test a decontamination chamber using locally available components may be of use to other institutions to attenuate the impact of shortages of PPE during the pandemic.

Limitations and risks of UV-C decontamination

Although UV-C-based N95 FFR decontamination is effective and can be achieved with a variety of either commercially available or custom-built chamber designs [2–6, 10, 22, 23], it does have some limitations. First, these systems can be labor intensive, as they require careful FFR positioning within the UVGI array, such that all surfaces of the FFR are exposed to UV-C irradiation. Second, certain N95 FFR models include the potential requirement for a secondary decontamination method for the straps [5, 24], as this component of each FFR is more variably decontaminated by UVGI. Additionally, although UVGI has been carefully validated for a variety of FFR models, many newer FFR models, including non-surgical N95s or internationally-sourced KN95 FFRs, have not been formally evaluated in terms of their penetration to UV-C light, and thus might be variably decontaminated, as UV-C transmission through N95 layers is material-dependent [25]. Although effective UV-C decontamination doses have been established for many widely available FFR models [2, 5], for FFRs that have not been formally tested, prior work has proposed methodologies for evaluating dose requirements for FFR decontamination [25, 26]. Additionally, although manufacturers and the CDC continue to recommend new FFRs whenever feasible, some have evaluated the effects of various decontamination methods on FFR integrity [27], and could potentially be contacted for reprocessing guidance in crisis situations with low FFR availability. Finally, although prior work specifically evaluated FFR decontamination in regards to enveloped viruses, including SARS-CoV2, more resistant pathogens, such as bacterial spores, might not be inactivated with a 1 J/cm2 dose [13, 24], and thus FFRs intended for decontamination are user-specific, and are often labeled so that they can be returned to the original wearer, to prevent cross-contamination between users. Other decontamination methodologies such as moist heat [7, 28, 29], or a combination of moist heat and UV-C, could be considered as alternative approaches to FFR decontamination [30], and may still retain implementation advantages over vaporized hydrogen peroxide [4], which is very effective but also requires expensive equipment and dedicated facilities that might be difficult to obtain in resource-constrained settings.

Of note, we noticed a nutty/smoky odor when masks were donned shortly after UV-C decontamination. This did not affect fit-testing and was noticed to “off gas” spontaneously after several hours. Although this odor has not been fully characterized, a limited analysis performed by Applied Research Associates demonstrated that levels of 62 volatile organic compounds were either undetectable or several orders of magnitude below permissible exposure limits [24]. We recommend that off-gassing time be considered in UVGI FFR decontamination protocols when feasible. Finally, some UV-C bulbs also produce ozone, which is hazardous and should be appropriately vented.

Finally, some of the limitations of our design relate specifically to its relatively low throughput (as designed, 30 minutes for each batch of ~20 FFRs), and current design requiring in-room changes of FFRs (removal of decontaminated FFRs and hanging of contaminated FFRs). Throughput could be increased with a larger array (to allow more FFRs to hang simultaneously) and reflective material (to increase irradiance and decrease cycle time). We also considered a rolling cantilevered rack that could be loaded and unloaded outside of the chamber, to minimize inter-batch delay, but to facilitate that we would recommend separate doors into the chamber so that decontaminated FFRs could leave via the “clean” exit door, while contaminated FFRs could enter via a separate door, which would help maintain an appropriate processing workflow and decrease the potential for cross-contamination. Finally, we would recommend that any system be implemented with auto-shutoff features, to turn off the UV-C arrays when staff enter the chamber, to prevent accidental hazardous exposures.

Benefits of E. coli as a BI

To validate decontamination, we explored the use of homemade Bis, which offer several advantages. First, the E. coli based BIs described in this report are relatively easy to make and assay with basic laboratory equipment. Second, these BIs serve as a useful surrogate for viral killing because E. coli is more difficult to kill than viruses such as SARS-CoV-2 the E.coli readout provides results more rapidly than growing viruses or G. stearothermophilus. Third, they are suitable for validating both UV and HPV decontamination methods.

Using homemade BIs, we demonstrated at least 5-log killing efficiency with UV-C doses of 1 Joule/cm2 or an HPV cycle reaching >400 PPM. Since viruses are less resistant to decontamination than E. coli [17], this indicates that true viral killing efficiency in our experiments is likely greater than 5-logs. Although a 3-log reduction in coronavirus is considered an acceptable level of decontamination of hard surfaces in healthcare settings [31, 32], N95 FFRs are only rated to block >95% of all infective particles. It is not known whether the infectious risk of FFRs after clinical use derives from the risk of touching a contaminated FFR, in which case it might be treated as a surface, or from potential inhalation of viral particles liberated from FFR fibers during breathing. However, in a crisis situation, an FFR which has been decontaminated and retains its filtering capacity is clearly preferable to either providing clinical care without an FFR or with a non-decontaminated one, so even in the absence of clinical data it remains prudent to pursue decontamination strategies.

One limitation of E. coli-based BIs on glass coverslips relates specifically to the alternative medium (glass vs. synthetic N95 fibers) as well as application method, since contaminated FFRs could have been contaminated within deeper layers of the FFR by aerosolized virus-containing particles. Although UVGI-mediated decontamination of FFRs exposed to aerosolized viral particles has been previously validated [2, 5, 7], the ability to test our system using aerosolized virions is beyond our capacity (and beyond the capacity of most hospital systems), and thus our UVGI process design relies on these prior data in combination with validated surrogate measures. Similarly, as each individual FFR cannot be tested without destroying it, the use of these surrogate measures (UVGI irradiance, biological indicators, and prior studies) is critical in implementing these processes. As has been previously emphasized, FFR decontamination is only to be recommended when new FFRs are unavailable, as FFRs begin to degrade in performance after multiple don/doff cycles [33].

To determine the feasibility of making these BIs in resource limited settings, we performed a cost analysis. We excluded fixed costs for a spectrophotometer, shaking incubator and an autoclave. Overall, we found the cost of our process was 1.89 dollars per BI. However, if reusable conical tubes, coverslips and cuvettes are used, the cost decreases to 2.6 cents per BI.

Conclusions

When supply lines may adversely impact PPE availability, a simple, cost-effective approach can be utilized to construct a chamber using locally-available components for N95 respirator decontamination. These maneuvers can help protect healthcare workers and hence sustain healthcare delivery services during periods when new respirators are unavailable. In addition to the development of a chamber for UV-C decontamination, successful implementation requires collaboration within the institution to ensure the smooth collection, processing, and storage of decontaminated respirators. These other steps are outlined in the S1 Appendix.

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PONE-D-21-08724

Construction and Validation of an Ultraviolet Germicidal Irradiation System using Locally Available Components

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Reviewer #1: The article makes up for an interesting read in context of its goal of locally developing an in-house UV-C based decontamination system for reprocessing of FFRs. However, it fails to address the most important issues while considering a UV-C based system for reprocessing of FFRs i.e. Decontamination Efficacy & available dose of UV-C to the electret media (filtering layer). Biggest critique of UV-C based FFR reprocessing system is that model specific doses need to be established before it can be recommended for widespread use, an issue which this study doesn’t address at all.

The authors have checked the microbicidal efficacy using Escherichia coli as a surrogate micro-organisms which though not ideal but is acceptable. However, this knowledge about UV-C being germicidal is well known and already established by multiple studies. What is novel about the study is how they have integrated this indigenous system into their workflow which has been provided in the Appendix.

Hence, I would suggest the authors that they should reformat the manuscript by putting the Appendix in the main manuscript and the Germicidal efficacy part can be given as Appendix.

Minor Corrections:

1. “The widely used Spaulding Classification indicates true killing efficiency is greater that 5-logs”: Spaulding classification categorizes items into critical, semicritical and non-critical. It doesn’t tell what should be the log efficiency for killing of micro-organisms. In any case, authors should provide proper reference

2. In Figure 3, authors should provide description of abbreviations.

3. In figure 3, the following is not clear: “Data are pooled from separate experiments (0 mJ/cm2: 2 experiments, n=4; 500 mJ/cm2: 2 experiments, n=6; all other doses: 3 experiments, n=7)” What do they mean by Experiments?.

4. The authors should include a separate paragraph for limitations of the system as well as the study in the Discussion section

5. 5. In title “validation” word should not be used as effect of various variables associated with the delivery of UV-C dose on FFRs is not evaluated. A suitable title can be “Construction & Implementation of an Indigenous UVGI System for Reprocessing of FFRs”.

Reviewer #2: I applaud the authors on a well done manuscript. The references and figures are appropriate and everything is well explained. The constructed UV cabinet is a good low cost alternative for decontaminating FFR in a crisis situation. I do not have any criticisms to offer. Perhaps one thing that can be added is a sentence to check with the FFR manufacturer to see if any additional decontamination guidance is available from them.

Reviewer #3: Dr. Schnell et al. seem to be outlining the relatively simple steps that can be taken to develop and operate an effective UV disinfection system for filtering facepiece respirators during initial stages of a diseases pandemic. This information needs to be understood by more people. While there are multiple ways to develop effective and safe UV disinfection systems for this purpose, information like that presented in the manuscript can help readers understand that it can be done when it seems like everything is going wrong in the world. If we use early in the COVID-19 pandemic as an example, whether we want to admit it or not, healthcare workers were disinfecting respirators for their reuse in multiple ways. Many of the ways employed by individual workers and entire hospitals were likely less effective than the UV disinfection system described here. Personally, I would have designed it differently. In particular, I would have enclosed it with UV-reflective surfaces as much as possible. As it is, there is a lot of wasted UV energy that could have been used to enhance disinfection, lessen disinfection time, or both. An enclosed system also would be safer for those workers responsible for operating the system. Still, the important thing is that the system as described worked. In the throes of a pandemic, things don’t need to be perfect ore even pretty, they just need to do the job. The authors should be commended for their efforts.

The authors have been very responsive to comments from previous reviewers. While I agree with those reviewers, that the work would be strengthened if respirators had been exposed to real coronaviruses (or even good surrogates) as part of the work, the results presented here are still valuable. As one reviewer pointed out, there is a large variability in the UV dose it takes to disinfect different respirator models exposed to the same organism. Similarly, if multiple laboratories did their own experiments to determine what that dose actually is, there is no doubt there would be multiple right answers. Much of the work in the UV field is done with surrogates and/or results from one study are applied to other studies. The authors are not doing anything different in this manuscript. The key is to use a very conservative UV dose so there is no doubt. That is what the authors proposed (and what the FDA has stated).

Ultimately, I think this article is worth publication as is. With that said, there are a few issues where small changes to the manuscript would help:

1.) Were the UV lamps burned-in for at least 100 hours prior to any reported test results? UV lamp output is quite variable in that first 100 hours, so that should have been done.

2.) It is not clear exactly how the coverslip bioindicators were positioned in the UV array during testing. It seems like there were maintained in the petri dishes without the lids, which meant they must have been kept flat. More detail on that step would help.

3.) As part of the discussion, the authors do well to describe limitations of the work. Are there any lessons learned from the work that readers should know about? What changes to the UV disinfection chamber would you make knowing what you know now?

**********

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Reviewer #1: Yes: Ayush Gupta

Reviewer #2: No

Reviewer #3: No

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23 Jun 2021

Response to Reviewers

Overall, we are pleased that our reviewers were broadly supportive of our manuscript, referring to our work as commendable, “well done”, and an interesting read, with solid appreciation of the goals and importance of this work in crisis situations with limited resource availability. Of note, two of our three reviewers stated that the paper should be published in its current form, although they did make minor suggestions that we have incorporated into our revision. The other reviewer was also generally supportive of our manuscript, although their primary critique was that we did not address the specific doses of UV-C required for specific Filtering Facepiece Respirator (FFR) models. However, as noted in the text, our manuscript explicitly does not attempt to validate FFR decontamination per se, and we agree (and state) that UV-C doses are model specific and in many cases have already been described in previously published literature, and we have provided some additional information in that regard. We still feel, however, that our description of how to deliver UV-C irradiation using locally available equipment, with the data provided on dose and validation, remain as an important contribution worthy of publication.

We have made additional revisions to clarify this issue, as well as several additional minor corrections as suggested by the reviewers, which we believe have further improved the manuscript. Replies to reviewer comments are in blue text, with changes to the manuscript described in bold. We also would like to amend our Funding Statement as suggested in Dr. Donlan’s letter, and this text is given below in red font.

Major point.

Reviewer #1: The article makes up for an interesting read in context of its goal of locally developing an in-house UV-C based decontamination system for reprocessing of FFRs. However, it fails to address the most important issues while considering a UV-C based system for reprocessing of FFRs i.e. Decontamination Efficacy & available dose of UV-C to the electret media (filtering layer). Biggest critique of UV-C based FFR reprocessing system is that model specific doses need to be established before it can be recommended for widespread use, an issue which this study doesn’t address at all.

Our contribution is to detail the construction and dose validation of a device to deliver UV-C light (to FFRs or other items) using widely available components, and simple instructions on how to potentially implement a decontamination process. We have attempted to avoid implication that our device has been validated for FFRs per se by leaving this out of the title, and have instead focused on demonstrating that the device delivers germicidal irradiation, at quantifiable doses, in a manner that could be used to effectively decontaminate FFRs using doses evaluated in many previous studies.

We already address the issue with FFR-specific doses with this phrase already in discussion: “Additionally, although UVGI has been carefully validated for a variety of FFR models, many newer FFR models, including non-surgical N95s or internationally-sourced KN95 FFRs, have not been formally evaluated in terms of their penetration to UV-C light, and thus might be variably decontaminated, as UV-C transmission through N95 layers is material-dependent [21].” However, to expand on this issue, we have now provided additional references and suggestions which could potentially assist in helping determine specific dose ranges for newer FFR models in the discussion. Importantly, we also emphasize that FFR decontamination are only intended for crisis situations in which standard supply lines and resource availability have become prohibitive (and life threatening).

Minor points

Reviewer #1:

The authors have checked the microbicidal efficacy using Escherichia coli as a surrogate micro-organisms which though not ideal but is acceptable. However, this knowledge about UV-C being germicidal is well known and already established by multiple studies. What is novel about the study is how they have integrated this indigenous system into their workflow which has been provided in the Appendix. Hence, I would suggest the authors that they should reformat the manuscript by putting the Appendix in the main manuscript and the Germicidal efficacy part can be given as Appendix.

We appreciate our reviewer’s suggestion on the organization of this manuscript, but in our prior review process, demonstration of germicidal efficacy of our specific system was requested, along with a recommendation that the non-data-supported systems workflow recommendation might be best omitted (or placed into an appendix). Additional testing of our indigenous UVGI decontamination system with surrogate biological indicators adds both an additional level of validation (beyond simple UV-C irradiance/dose measurement), and also provides an additional method through which resource-constrained systems might be able to locally test their system efficacy. Thus, we would prefer to keep the manuscript organization as it currently stands. We agree with the reviewer that the gold standard validation (actual SARS-CoV2 on FFRs) would be ideal, but this assay is rarely feasible outside of BSL-2 laboratory conditions, and thus the use surrogate organisms/measures to evaluate relative efficacy is widely employed and accepted.

Minor Corrections:

1. “The widely used Spaulding Classification indicates true killing efficiency is greater that 5-logs”: Spaulding classification categorizes items into critical, semicritical and non-critical. It doesn’t tell what should be the log efficiency for killing of micro-organisms. In any case, authors should provide proper reference

Thank you for pointing this out. We have provided a more appropriate reference in regards to the relative susceptibility of different microorganisms to decontamination in the Methods. Additionally, the question of “how much decontamination is enough” addressed by Spaulding relates to different medical devices, in large part related to how these devices are used (e.g. critical devices that touch internal/sterile human tissues need to be completely sterile, vs. devices that only touch intact skin are notably less critical). We have also have added additional comments relating to this point in the Discussion.

2. In Figure 3, authors should provide description of abbreviations.

Thank you for catching this omission; abbreviations are now defined in the legend.

3. In figure 3, the following is not clear: “Data are pooled from separate experiments (0 mJ/cm2: 2 experiments, n=4; 500 mJ/cm2: 2 experiments, n=6; all other doses: 3 experiments, n=7)” What do they mean by Experiments?.

The sample size (n) refers to the number of coverslips exposed to each dose of UV-C, and each coverslip was subsequently cultured in its own tube of LB. “Experiments” refers to different batches of coverslips processed in parallel (we ran three separate batches on different dates to increase sample size). Two of the UV-C dose groups were only run twice. The legend was revised to just cite “n” to avoid confusion.

4. The authors should include a separate paragraph for limitations of the system as well as the study in the Discussion section

The limitations inherent to UVGI and reprocessed (vs. new) single-use FFRs is extensively reviewed in the discussion and the references contained therein; but we have expanded our discussion section with an additional paragraph relating to limitations of our specific system and suggestions for improvement.

5. 5. In title “validation” word should not be used as effect of various variables associated with the delivery of UV-C dose on FFRs is not evaluated. A suitable title can be “Construction & Implementation of an Indigenous UVGI System for Reprocessing of FFRs”.

We appreciate our reviewer’s constructive advice on our title, but we actually removed reference to N95s/FFRs and added the word ‘validation’ to the title based on feedback from the prior review of this manuscript at PLOS ONE (which originally had a title quite close to the reviewer’s suggestion). As we believe that it would be inappropriate to imply that our system was specifically validated for FFRs, we would respectfully prefer to keep the title as it stands.

Reviewer #2: I applaud the authors on a well done manuscript. The references and figures are appropriate and everything is well explained. The constructed UV cabinet is a good low cost alternative for decontaminating FFR in a crisis situation. I do not have any criticisms to offer. Perhaps one thing that can be added is a sentence to check with the FFR manufacturer to see if any additional decontamination guidance is available from them.

We appreciate the reviewer’s enthusiasm. Regarding manufacturer guidance, most FFRs are intentionally designed for single-use, and thus most manufacturers have not evaluated or recommended specific decontamination methodologies. We have, however, included this suggestion in the 2nd paragraph of the discussion, as some manufacturers (e.g. 3M) have actually evaluated various decontamination methods (please see reference 27, also added). We also note that although UV-C decontamination is acceptable in maintaining FFR function, 3M continues to not recommend decontamination unless in a crisis situation.

Reviewer #3: Dr. Schnell et al. seem to be outlining the relatively simple steps that can be taken to develop and operate an effective UV disinfection system for filtering facepiece respirators during initial stages of a diseases pandemic. This information needs to be understood by more people. While there are multiple ways to develop effective and safe UV disinfection systems for this purpose, information like that presented in the manuscript can help readers understand that it can be done when it seems like everything is going wrong in the world. If we use early in the COVID-19 pandemic as an example, whether we want to admit it or not, healthcare workers were disinfecting respirators for their reuse in multiple ways. Many of the ways employed by individual workers and entire hospitals were likely less effective than the UV disinfection system described here. Personally, I would have designed it differently. In particular, I would have enclosed it with UV-reflective surfaces as much as possible. As it is, there is a lot of wasted UV energy that could have been used to enhance disinfection, lessen disinfection time, or both. An enclosed system also would be safer for those workers responsible for operating the system. Still, the important thing is that the system as described worked. In the throes of a pandemic, things don’t need to be perfect ore even pretty, they just need to do the job. The authors should be commended for their efforts.

Thank you for the positive feedback. Although we designed our light array for simplicity and minimal resource utilization, we agree that UV-reflective surfaces would likely not only reduce the processing time, but also improve the incident angles on FFRs, perhaps increasing efficacy. Household aluminum foil is actually quite reflective (~75% for UV-C), often widely available, and has been incorporated in other designs to date, and we have added this suggestion (and references) to our ‘Alternative chamber designs’ section.

The authors have been very responsive to comments from previous reviewers. While I agree with those reviewers, that the work would be strengthened if respirators had been exposed to real coronaviruses (or even good surrogates) as part of the work, the results presented here are still valuable. As one reviewer pointed out, there is a large variability in the UV dose it takes to disinfect different respirator models exposed to the same organism. Similarly, if multiple laboratories did their own experiments to determine what that dose actually is, there is no doubt there would be multiple right answers. Much of the work in the UV field is done with surrogates and/or results from one study are applied to other studies. The authors are not doing anything different in this manuscript. The key is to use a very conservative UV dose so there is no doubt. That is what the authors proposed (and what the FDA has stated).

Ultimately, I think this article is worth publication as is. With that said, there are a few issues where small changes to the manuscript would help:

1.) Were the UV lamps burned-in for at least 100 hours prior to any reported test results? UV lamp output is quite variable in that first 100 hours, so that should have been done.

The UV bulbs were not burned in. We agree that UV-C bulb output can vary between bulbs, within the same bulbs as they age/burn-in, and even during use (in our hands, irradiance increased substantially over the first minute of being turned on). For these reasons, we (and others) have strongly recommended that a UV-C irradiance meter is used to measure total energy delivered for every UVGI run as a quality control parameter. We added a comment regarding the variable output of UV-C bulbs during burn-in as an additional justification for this recommendation.

2.) It is not clear exactly how the coverslip bioindicators were positioned in the UV array during testing. It seems like there were maintained in the petri dishes without the lids, which meant they must have been kept flat. More detail on that step would help.

The coverslip bioindicators were positioned on a platform positioned directly in the midpoint of the array (where FFRs would be positioned), directly facing the top UV-C array, with petri dish lids removed. These details were clarified to the Methods.

3.) As part of the discussion, the authors do well to describe limitations of the work. Are there any lessons learned from the work that readers should know about? What changes to the UV disinfection chamber would you make knowing what you know now?

Thank you. During evaluation and implementation of several different FFR decontamination processes in parallel, we eventually settled on vaporized H2O2 as our primary methodology due to is higher throughput. However, it was also clear that this method is not only very expensive (~$100K set up, at a minimum), time consuming (weeks to construct an H2O2-safe room/facility) requires incredibly high level training/safety protocols/infrastructure, and requires devices that would be almost impossible to source during a crisis. The resource-intensiveness of vaporized H2O2 is already mentioned in the introduction.

However, during our ongoing work with the UVGI-system, we did learn several lessons relating to throughput and design improvements (including the suggestion relating to the addition of low-cost reflective material). Although several of these insights were originally in the appendix, we have now included some of the lessons learned in a new paragraph in the discussion.

Housekeeping:

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Our statement regarding funding information was removed from our Acknowledgements section, but even though this funding was not explicitly intended for this work, we feel that it would be appropriate to mention these sources in our “Funding Statement”. Thank you for this opportunity. If possible, please change the Funding Statement on our online submission form to include the following text:

The authors would like to acknowledge grant support by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development Merit Review Awards I01-BX004938(ES), I01 CX001562 (MH), I01-BX002547 (SMS), a Department of Defense CDMRP Award W81XWH-18-1-0598 (ES), an NIH NINDS 1R21NS102948 (ES), an NIH R01 AI129976 (MH) and an 1R21AI151079-01 (EK).

12 Jul 2021

Construction and Validation of an Ultraviolet Germicidal Irradiation System using Locally Available Components

PONE-D-21-08724R1

Dear Dr. Schnell,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: I accept the revision based on the author's replies to the original review. This is a valuable paper for communities with low budgets to perform decontamination.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Ayush Gupta

Reviewer #2: No

16 Jul 2021

PONE-D-21-08724R1

Construction and Validation of an Ultraviolet Germicidal Irradiation System using Locally Available Components

Dear Dr. Schnell:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Table 1

Construction materials for UV-C bulb arrays.

Bulb arrays:
24x Philips TUV G30T8 30W / 35 inch germicidal bulbs (or equivalent).
48x Fluorescent Lampholder, T8, Bi-Pin (“tombstone”).
6x Electronic Ballast, Fluorescent, Input Voltage: 120-277V, Instant Start, Centium Series.
#18 copper conductor (red, yellow, blue, black, white) to wire lampholders to ballast.
Flat aluminum stock (for lampholder mounting)
SO cord #12–30’
1 male cord cap
1–4” junction box and cover
4-Cord grips for SO cord
3 port wago connectors
1-20A switch and single gang box
Mounting Hardware:
Unistrut, 3-deep channel, 40’, and 4–90 degree 4 hole brackets
20’-Aluminum angle, 8-Flat aluminum stock 4”X21”
Black duct tape to cover wires in the Unistrut
6–32 screws to mount lampholders to flat aluminum stock
¼” toggle anchors to mount deep strut to walls
¼” strut hardware to mount fixtures to the strut