Decontamination of Geobacillus Stearothermophilus using the Arca Aerosolized Hydrogen Peroxide decontamination system.

INTRODUCTION: In response to the limited supply of personal protective equipment during the pandemic caused by SARS-CoV-2, recent studies demonstrate that gaseous H2O2 is an effective decontaminant of N95 filtering facepiece respirators to enable reuse of these items in a clinical setting. This paper evaluates the efficacy of the Arca Aerosolized Hydrogen Peroxide Decontamination System (Arca), a novel aerosolized H2O2 decontamination system, using biologic indicator testing.
MATERIALS AND METHODS: The Arca produces and circulates H2O2 aerosol inside of a sealed stainless steel chamber. The Arca's decontamination efficacy was evaluated in 8 decontamination trials with 2 H2O2 concentrations (3% and 12%) and 4 decontamination cycle durations (45, 60, 90, and 120 minutes). Efficacy was evaluated by testing: 1) the concentration in parts per million (ppm) of H2O2 produced inside the chamber and the concentration in ppm of H2O2 vented from the chamber, and 2) the decontamination of Mesa Biologic Indicator filter strips (BI) inoculated with Geobacillus Stearothermophilus. Control tests were conducted by submerging BI strips in 3mL of 3% and 12% H2O2 for 120 minutes (negative controls) and by not exposing one BI strip to H2O2 (positive control).
RESULTS: Greater than 5000 ppm of H2O2 was detected on the concentration strips inside the chamber for each of the eight decontamination trials. No vented H2O2 was detected on the external concentration strips after any decontamination trial. No growth was observed for any of the negative controls after seven days. The positive control was positive for growth.
CONCLUSION: The Arca Aerosolized Hydrogen Peroxide Decontamination System is effective at decontaminating bacterial G. Stearothermophilus at a cycle time of 45 minutes utilizing 6mL of 3% H2O2 solution.

Introduction

The SARS-CoV-2 coronavirus (COVID-19) pandemic has caused a worldwide shortage in personal protective equipment (PPE) including N95 filtering facepiece respirator (FFR) and surgical masks. Both mask types have been shown to dramatically reduce the rate of infection by airborne viruses such as influenza, but the masks are designed for single use before disposal [1]. Due to a shortage of FFRs and the critical role FFRs play in protecting health care workers at the outset of the COVID-19 pandemic, the Center for Disease Control recommended limited reuse of decontaminated FFRs to extend supplies of PPE [1,2]. The Occupational Safety and Health Administration has similarly produced guidelines for the limited reuse of FFRs [3].

Aerosolized or vaporized hydrogen peroxide (H2O2) is an established reactant for decontaminating surfaces inoculated with resistant bacterial and viral pathogens [4]. Recent studies demonstrate that H2O2 delivered in an aerosol or vapor as low as 500 ppm has been shown to achieve 1000 times reductions in viral organism activity on inoculated FFRs while maintaining adequate mask fitment through multiple decontamination cycles [5-8]. Previous work has demonstrated that scalable, proof-of-concept H2O2 decontamination systems could achieve adequate minimum concentrations reported in the literature to eradicate Coronavirus and other pathogens [9,10].

To mitigate infection risk with reuse of FFRs, the Food and Drug Administration granted emergency use authorizations (EUAs) to several commercially available hydrogen peroxide FFR decontamination procedures [11]. However, the commercially available methods of FFR decontamination are costly and limited in availability [12-14]. The H2O2 decontamination methods which received an EUA include those listed in Table 1. The number of maximum decontamination cycles per FFR varies by method with a range of two to 20 cycles [15]. The price per institution for these systems can exceed $50,000 (Table 1). Low-resource institutions are often unable to access these services and purchase adequate supplies of PPE [16]. Therefore, the development of a smaller-scale, less-costly decontamination devices is warranted.

Table 1

Value Comparison of H2O2 decontamination systems with EUAs.

Decontamination System	Estimated Annual Cost to Institution	Maximum Decontamination Cycles per FFR
Bioquell Technology System	$53,000 [17]	4
Battelle Critical Care Decontamination System	$0a	20
STERRAD Sterilization System	$149,000 [18]	2
Sterilucent HC 80TT Vaporized Hydrogen Peroxide Sterilizer	Not Reported	10
Stryker STERIZONE VP4 Sterilizer for N95 Respirator Decontamination	Not Reported	2
Stryker Sustainability Solutions VHP N95 Respirator Decontamination System	Not Reported	3
Duke Decontamination System	Not Reported	10
Technical Safety Services (TSS) 20-CS Decontamination System	Not Reported	20
Michigan State University Decontamination System	Not Reported	3
Roxby Development Zoe-Ann Decontamination System	Not Reported	4

aBattelle was awarded a federal contract for subsidized N95 decontamination with costs upwards of $1 million per system [19].

This study seeks to evaluate the efficacy of a low-cost design engineered for use in low-resource settings to expand the supply of decontaminated PPE for safe re-use by frontline workers. Our objectives were to test the device’s efficacy at different H2O2 concentrations and Arca device cycle durations by evaluating: 1) the concentration in parts per million (ppm) of H2O2 produced inside the chamber and the concentration in ppm of H2O2 vented from the chamber, and 2) the decontamination of Mesa Biologic Indicator filter strips (BI) inoculated with 106 Geobacillus Stearothermophilus.

Materials and methods

The Arca Aerosolized Hydrogen Peroxide Decontamination System (Arca) produces H2O2 inside of a sealed stainless steel chamber and circulates the aerosol with four CG IP67 personal computing fans. The Arca uses a Venturi tube design to generate an aerosol: an air compressor forces high velocity air through a tube where the cross-sectional area is reduced with the result of lowering the air pressure (Fig 1). The design lowers the pressure to below 2300 Pa, the vaporization pressure of 10% H2O2 in water solution [20]. The H2O2 is fed to the low pressure opening of the tube, where the low pressure induces partially vaporization, forming small droplets of solution (i.e. an aerosol). A catch is included at the low pressure point to trap large droplets and recirculate them back to the low-pressure area. FFRs are placed on a wire mesh rack inside the chamber and exposed to the H2O2 for decontamination (Fig 2). Circulated H2O2 aerosol is removed from the chamber interior using a Speedair 4ZL07 condenser unit to avoid operator exposure when removing decontaminated FFRs (Fig 3). The device’s manufacturing and component costs totaled less than $2,000.

Fig 1

Nebulizer Venturi tube design [21].

Fig 2

Arca interior demonstrating mesh rack, circulating fans, and Venturi tube nozzle.

Fig 3

Arca exterior showing nebulizer (top), condenser unit (bottom), and electronics housing (left).

The Arca’s decontamination efficacy was evaluated by a testing protocol exposing MesaStrip BI filter strips inoculated with 106 G. Stearothermophilus [. We performed a total of 8 trials varying the H2O2 concentration (3% and 12%) and decontamination cycle duration (45, 60, 90, and 120 minutes). Decontamination efficacy was evaluated by H2O2 concentration and biologic testing within the device chamber and venting system.

Concentration testing was performed using Bartovartion Very High Level Peroxide Test Strips, which change color to indicate exposure to H2O2 at concentrations from 0 to 5000 ppm [23]. For each trial, one BI was secured with Scotch MagicTM Tape to an FFR located centrally inside the chamber. An additional concentration strip was placed on the exterior of the door and evaluated for vented H2O2 from the chamber after each cycle was completed and the door to the chamber opened. All cycles were performed in a well-ventilated area.

Additionally, one BI was submerged in 3 mL of 3% and one in 3 mL of 12% H2O2 for 120 minutes each to serve as negative controls. One BI was not exposed to H2O2 to serve as a positive control.

The BIs were handled using sterile technique and after testing were shipped in sealed plastic bags to STERIS Laboratories. Sterility testing was performed according to STERIS Laboratories protocol ST/01, in which the BIs were incubated in 10 mL of Tryptic Soy Broth at 55–60 degrees Celsius for seven days. The cultures were evaluated daily and interpreted as “No growth” only if the medium remained clear without color change or turbidity after seven days [24].

Results

Concentration testing

The concentration strips for all decontamination cycles showed internal concentrations of H2O2 exceeding 5000 ppm. The exterior concentration strips monitoring for vented H2O2 all showed undetectable levels of H2O2 (Tables 2 and 3).

Table 2

Concentration strip results for 3% H2O2 solution testing.

	45 Minutes Cycle	60 Minutes Cycle	90 Minutes Cycle	120 Minutes Cycle
Internal Strip	>5000 ppm	>5000 ppm	>5000 ppm	>5000 ppm
Exterior Strip	No color change	No color change	No color change	No color change

Table 3

Concentration strip results for 12% H2O2 solution testing.

	45 Minutes Cycle	60 Minutes Cycle	90 Minutes Cycle	120 Minutes Cycle
Internal Strip	>5000 ppm	>5000 ppm	>5000 ppm	>5000 ppm
Exterior Strip	No color change	No color change	No color change	No color change

Decontamination testing

All BIs exposed to H2O2 had no growth at seven days of incubation (Tables 4 and 5). The positive control that was not exposed to H2O2 demonstrated bacterial growth.

Table 4

BI growth at 7 days following exposure to 3% H2O2 solution.

Trial	120 Minutes Submersion	45 Minutes Cycle	60 Minutes Cycle	90 Minutes Cycle	120 Minutes Cycle
Growth Result	No growth	No growth	No growth	No growth	No growth

Table 5

BI growth at 7 days following exposure to 12% H2O2 solution.

Trial	120 Minutes Submersion	45 Minutes Cycle	60 Minutes Cycle	90 Minutes Cycle	120 Minutes Cycle
Growth Result	No growth	No growth	No growth	No growth	No growth

Discussion

The limited supply of FFRs during the SARS-Cov-2 pandemic endangers health care and other frontline workers. While the FDA and CDC have encouraged the decontamination of FFRs for reuse, existing decontamination systems are inaccessible in low-resource settings. Development of systems designed for deployment in low-resource settings will increase the supply of PPE for at-risk personnel.

This study attempted to evaluate a novel device’s ability to eradicate bacterial pathogens from the surface of an FFR without exposing users to unsafe levels of H2O2. Our testing demonstrates that the novel Arca device can decontaminate bacterial pathogens without exposing users to high concentrations of H2O2. These results are consistent with the literature on existing gaseous H2O2 technologies. The eradication of bacteria achieved at every trial suggests that the Arca could achieve adequate decontamination with a lower concentration H2O2 solution, a shorter cycle duration, or a smaller volume of H2O2. Based on previously reported H2O2 testing, the bactericidal properties of the device suggest virucidal efficacy as well [9,25].

The Arca has a throughput of 16 FFRs per cycle, with a daily output of up to 512 FFRs. The marginal cost to clean each additional FFR is significantly less than $1, as operating expenses include small volumes of widely available concentrations of H2O2 and the variable cost of electricity to power the device. The Arca’s production cost at less than $2,000 per unit is significantly lower than the price of existing commercially available solutions. At scale, this manufacturing cost should decrease. These devices are intended to be donated at no cost to recipient institutions, therefore the operating expenses are the only costs passed on to end users. Future iterations of the device could reduce costs further by using more off-the-shelf components in its manufacturing process. The decreased cost of the Arca improves its accessibility in low-resource settings compared with existing FFR decontamination systems.

Limitations

The sterility testing performed on the BI strips resulted in a binary “No growth” or “Growth” outcome, and did not demonstrate log kill rates. Therefore, it is possible that small populations of bacteria survived decontamination cycles but did not change the TSB medium’s turbidity. However, the same testing methodology is used as part of quality assurance testing to assess sterility achieved by FDA-approved devices, with an industry standard sterility assurance level (SAL) of 10−6. The FDA requires only an SAL of 10−3 for products that contact intact skin [26].

Testing was performed under optimal conditions using a single FFR. Real world use may lead to decreased efficacy due to variations in power supply to the Arca, increased pathogen burden, or greater surface area of multiple FFRs. Additionally, while bacteria are considered more resistant to H2O2 than viruses, the eradication of this study’s BIs may not be generalizable to all mutations of SARS-CoV-2.

Future directions

Experimental derivation of the absolute minimum cycle duration, volume of H2O2, and percent H2O2 solution to achieve decontamination will allow for greater throughput of the Arca device. Testing in a BSL-3 lab will evaluate the Arca’s efficacy at decontamination of SARS-CoV-2 and other pathogens directly inoculated on PPE.

Fitment testing of FFRs after decontamination will allow for experimental derivation of the maximum decontamination cycles per FFR. Additional testing of other PPE and various equipment may expand the use cases for the Arca.

Additionally, “real world” field testing will help to determine any potential causes of diminished aerosol concentration, inadequate decontamination, or non-optimal user experience.

Conclusion

The Arca Aerosolized Hydrogen Peroxide Decontamination System can safely decontaminate FFRs inoculated with bacteria using a minimum cycle time of 45 minutes with 6 mL of H2O2 solution.

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

16 Jun 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Peter Setlow

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

2. Thank you for stating the following in the Competing Interests section:

[The authors of this publication are members of Abaton's Board of Directors, which could be perceived as a competing interest. However, the authors do not receive a salary or any gifts-in-kind for their service on the Board, and Abaton is a 501(c)3 non profit. The Arca device design has not, nor ever will be submitted for patent. The intention of the authors is to disseminate the design as an open source document for replication in locations where it might be of assistance. The authors further declare that they in no way financially or otherwise benefitted from the grant funding and personal donations made to Abaton.]

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

3. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Dear Dr. Loren,

Your manuscript has been reviewed by two experts in the field, and while both reviewers are positive about the manuscript, they make a few minor comments that should be attended to, as they will improve the manuscript.

Sincerely,

Peter Setlow

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: N/A

**********

3. 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: Yes

Reviewer #2: Yes

**********

4. 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

**********

5. 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: The manuscript by Mead et al "Decontamination of Geobacillus Stearothermophilus using the Arca Aerosolized Hydrogen Peroxide decontamination system" is a well written description of a device that uses vaporized hydrogen peroxide to disinfect N95 respirators. The methods are straightforward and are consistent with previous literature that vaporized H2O2 is an effective disinfectant.

The main issue to consider is that the authors repeatedly discuss this as an option for low resource areas at a cost of 2000. It is not clear from figure 2 how many masks fit in the device and what the throughput is. In table 1 many of those were high throughput set ups such that it is not an equitable comparison. Perhaps including the masks in Figure 2 would be helpful. A cost per N-95 might be more applicable as if one can only repurpose 20 per day even at 2000 it may not be cost effective especially as manufacturing and stockpiles have increased. It would be helpful in the discussion to discuss throughput (how many masks per day for a single unit) as well as cost in this context.

Minor point

The authors state sterility was demonstrated by clear TSB and lack of color change at 7 days. Unfortunately I was unable to access the cited reference (error code) for the steris procedure and the manufacturer of the BI strip is not provided. TSB turbidity requires significant growth and lack of turbidity is not equivalent to no growth. The media will not necessarily change color with small amounts of growth (perhaps this was a reference to a color indicator on the strip) Filtering the media and plating the filter to catch any organisms is one mechanism to capture small amount of organisms in a volume too large to plate. Some clarification of this procedure as well as the limit of detection for colony forming units would be helpful to the reader. It would also be helpful to note how much growth was seen with the positive control as well to confirm the log killing and disinfection achieved if we assume the TSB was in fact sterile.

Reviewer #2: A very straightforward paper. Only a few minor comments.

1) l 42 - In light of ramping up a variety of responses to the pandemic that drove up supplies of PPE and drove down costs, might be helpful to give an estimate of the times/costs to process 1, 10, 100 etc M-95s would be helpful.

2) l-90 . Presumably these are G. stearothermophilus spores? Are these BIs the ones used for H2O2 sterilizers, as I believe these spores are not generally the ones most resistant to H2O2, although are for wet heat.

3) Since the TSB incubated with H2O2-treated BIs gave no obvious turbidity/color change, most spores have been killed. But what are the limits of this assessment? I could not access reference 23 to get any information about this, and thus it is not clear what the confidence in "sterility is.

**********

6. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.