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A do-it-yourself test protocol using commercial Bacillus atrophaeus spores to evaluate the effectiveness of ultraviolet-C light room-decontamination devices

Published online by Cambridge University Press:  14 June 2023

Jennifer L. Cadnum and
Curtis J. Donskey
Jennifer L. Cadnum
Affiliation:
Research Service, Louis Stokes Cleveland Veterans’ Affairs (VA) Medical Center, Cleveland, Ohio
Curtis J. Donskey*
Affiliation:
Case Western Reserve University School of Medicine, Cleveland, OhioGeriatric Research, Education, and Clinical Center, Louis Stokes Cleveland VA Medical Center, Cleveland, Ohio
*
Corresponding author: Curtis J. Donskey; Email: Curtis.Donskey@va.gov
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Abstract

We developed a do-it-yourself test protocol using commercial Bacillus atrophaeus spores to assess the efficacy of ultraviolet-C (UV-C) light room-decontamination devices. Overall, 4 UV-C devices reduced B. atrophaeus by ≥3 log10 colony-forming units in 10 minutes, whereas a smaller device required 60 minutes. Of 10 in-use devices, only 1 was ineffective.

Information

Type
Concise Communication
Information
Infection Control & Hospital Epidemiology , Volume 44 , Issue 10 , October 2023, pp. 1676 - 1679
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Creative Commons
This is a work of the US Government and is not subject to copyright protection within the United States. Published by Cambridge University Press on behalf of The Society for Healthcare Epidemiology of America.
Copyright
© Department of Veterans Affairs, 2023.

Ultraviolet-C (UV-C) light room-decontamination devices are not regulated by the US Food and Drug Administration (FDA), and there are no standard test protocols to demonstrate efficacy. Reference Donskey1–Reference Kreitenberg and Martinello3 This is an important concern because variations in test methods can markedly impact reductions of pathogens achieved by UV-C, Reference Donskey1,Reference Cadnum, Tomas, Sankar, Jencson, Mathew, Kundrapu and Donskey4 and devices may vary substantially in efficacy. Reference Pearlmutter, Haq, Cadnum, Jencson, Carlisle and Donskey5 Standardized test protocols could improve consumer confidence when assessing the claims of manufacturers. Reference Kreitenberg and Martinello3,Reference Kreitenberg and Polacci6,Reference Poster, Miller and Martinello7 Such testing requires technical expertise and has typically been performed by commercial laboratories or research groups.

For several reasons, it would be advantageous for healthcare facilities to have access to do-it-yourself test protocols to evaluate UV-C efficacy. First, in-house testing could be used to compare devices being considered for purchase. Second, in-use devices could be tested intermittently to ensure appropriate performance. Third, there can be substantial discrepancies in results obtained by commercial laboratories and research groups using similar methods (authors’ unpublished data). Finally, some manufacturers’ service agreements prohibit testing of devices by outside parties. Here, we developed a do-it-yourself test protocol for UV-C room decontamination devices that would require limited or no onsite microbiological expertise.

Methods

Test organisms

Bacillus atrophaeus (ATCC 9372) and Geobacillus stearothermophilus (ATCC 12980) spores were purchased from Mesa Laboratories (Lakewood, CO) as spore suspensions or on preprepared 8×12-mm steel disks inoculated with 103 or 106 colony-forming units (CFU) of spores. For comparison, a clinical methicillin-resistant Staphylococcus aureus (MRSA) isolate and Clostridioides difficile American type culture collection (ATCC) strain 43598 were tested. Reference Cadnum, Tomas, Sankar, Jencson, Mathew, Kundrapu and Donskey4 MRSA and C. difficile carriers were prepared by spreading 10 µL containing 6 log10 CFU onto 20-mm steel disks. C. difficile spores were prepared as previously described. Reference Cadnum, Tomas, Sankar, Jencson, Mathew, Kundrapu and Donskey4

Devices tested

We included 5 low-pressure mercury UV-C room decontamination devices in laboratory testing: Tru-D (Professional Disposables International, Woodcliff Lake, NJ), Rapid Disinfector (Steriliz LLC, Rochester, NY), UVDI-360 Room Sanitizer (UltraViolet Devices, Santa Clarita, CA), Guardian (Camillus LLC, North Canton, OH), and VORTEX-UV Portable UVC Room Sanitizer (MRSA-UV LLC, West Palm Beach, FL). The VORTEX-UV device is a small device intended for decontamination of small rooms up to ∼6×6 m (∼20×20) feet in 15 minutes. For larger rooms, concurrent use of multiple devices is recommended. We tested 10 additional UV-C room decontamination devices used in hospitals against B. atrophaeus.

Test protocol

A 3-log10 or greater reduction in test organisms in comparison to untreated controls was considered an indication of effective decontamination. Reference Cadnum, Tomas, Sankar, Jencson, Mathew, Kundrapu and Donskey4 In initial experiments, 10 minutes of UV-C exposure from the UVDI-360 Room Sanitizer consistently reduced B. atrophaeus, G. stearothermophilus, and C. difficile spores by >3 log10 CFU. Therefore, the test protocol was defined as 10 minutes of exposure to steel disk carriers aseptically adhered to petri-dish bottoms positioned parallel to lamps 0.914 m from the device at the midpoint of the lamps. B. atrophaeus was selected as the indicator organism because cultures were easier to process than G. stearothermophilus cultures. A detailed description of the proposed do-it-yourself test protocol is provided as Supplementary Material (online).

After identification of standard test conditions, the devices were tested against 6-log10 CFU disks. Additional testing was conducted with longer cycle times of up to 60 minutes if a 3-log10 reduction in spores was not achieved in 10 minutes. Each test was repeated in triplicate.

For B. atrophaeus, we assessed whether 10-minute UV-C cycles would consistently reduce 3-log10 CFU disks to undetectable levels. For each device, 10 disks exposed to UV-C were aseptically transferred to tubes containing 2 mL trypticase soy broth that were incubated for up to 7 days. The percentages of disks with positive cultures were calculated.

Testing of devices used in hospitals

In addition to the 5 devices included in laboratory testing, 10 UV-C room decontamination devices used in hospitals were tested against B. atrophaeus using the standard test protocol. For devices not achieving a ≥3-log10 reduction, the manufacturer was contacted to ensure that maintenance was up to date.

Microbiology

Disks were processed to quantify MRSA and C. difficile. Reference Pearlmutter, Haq, Cadnum, Jencson, Carlisle and Donskey5 For B. atrophaeus and G. stearothermophilus, disks were processed as described in the Supplementary Materials (online). Log10 CFU reductions were calculated by subtracting viable organisms recovered from treated versus untreated carriers. Reference Pearlmutter, Haq, Cadnum, Jencson, Carlisle and Donskey5

After UV-C exposure, the 3-log10 CFU B. atrophaeus disks were transferred to tubes containing 2 mL trypticase soy broth. The tubes were incubated at 30°C for up to 7 days. Aliquots from all tubes were plated on trypticase soy agar and were assessed for growth of B. atrophaeus.

Results

As shown in Figure 1, the Tru-D, UVDI-360, Rapid Disinfector, and Guardian devices reduced MRSA and the spore-forming organisms by >6 log10 and >3 log10 CFU, respectively. The VORTEX device did not reduce the spore-forming organisms by ≥3 log10 when operated for 10 or 30 minutes but did when operated for 60 minutes.

Figure 1.

Log10 reductions in recovery of the test organisms on steel disk carriers after exposure to ultraviolet-C light from room decontamination devices. The carriers were placed 0.91 m from the device and oriented parallel to the bulbs. MRSA, methicillin-resistant Staphylococcus aureus. Note. CFU, colony-forming unit. The dashed line indicates a 3-log10 reduction, which was considered an indication of effective decontamination.

Figure 2 shows the percentage of the 3-log10 B. atrophaeus disks with negative cultures after exposure to a 10-minute cycle of UV-C. For the Tru-D, UVDI-360, Rapid Disinfector, and Guardian devices, all 10 treated disks had negative cultures, whereas only 1 of 10 disks treated with a 10-minute cycle with the VORTEX device had a negative culture.

Figure 2.

Percentage of the steel disks inoculated with 103 colony-forming units of Bacillus atrophaeus spores with negative cultures after exposure to a 10-minute cycle of ultraviolet-C light from room-decontamination devices.

Of the additional 10 devices used in hospitals, 1 (10%) did not achieve a 3-log10 reduction in B. atrophaeus using the proposed protocol or the manufacturer’s recommended protocol. For this device, 10 of 10 disks with 3 log10 CFU of B. atrophaeus had positive cultures after UV-C exposure. The manufacturer stated that the bulb was not due for replacement.

Discussion

Infection prevention personnel often face uncertainty when assessing claims regarding the efficacy of UV-C light devices. Reference Kreitenberg and Polacci6 To address this uncertainty, we propose a do-it-yourself test protocol analogous to the use of biological indicators to assess sterilization technologies. Our findings provide proof of concept that this protocol could be useful for onsite evaluation of UV-C light room-decontamination devices.

The proposed test method requires limited or no onsite microbiological expertise. The use of B. atrophaeus spores from commercial vendors specializing in production of biological indicator spores provides standardization of the test organism. After UV-C exposure, the disks can be placed in sterile containers and mailed to the vendor for nonquantitative (103 CFU disks) or quantitative (106 CFU disks) cultures. Alternatively, cultures can be processed on site by microbiology laboratories. The process for nonquantitative cultures is particularly straightforward, and B. atrophaeus is nonpathogenic with no safety concerns. 8

The inexpensive VORTEX device was less effective than the standard room devices. However, with a 60-minute cycle the device achieved a ≥3 log10 CFU reduction in B. atrophaeus. Thus, for devices that have relatively low UV-C output, similar results may be achieved with longer cycle times and/or by placing multiple devices in the room.

Our study had several limitations. The proposed test protocol was not intended to address all variables that impact UV-C performance. Reference Cadnum, Tomas, Sankar, Jencson, Mathew, Kundrapu and Donskey4 Rather, it was well suited for direct comparison of devices and assessment of in-use devices. Others have proposed test protocols in simulated patient rooms. Reference Kreitenberg and Martinello3 Colorimetric indicators and irradiance measurements using a radiometer could be useful as adjunctive methods to assess UV-C dose delivery. Reference Cadnum, Pearlmutter, Redmond, Jencson, Benner and Donskey9,Reference Masse, Hartley, Edmond and Diekema10 Therefore, we have included information and results for these methods as Supplementary Material (online). Finally, additional studies are needed to validate the proposed protocol and to determine whether mailing specimens has an impact on culture results.

Supplementary material

The supplementary material for this article can be found at https://doi.org/10.1017/ice.2023.24

Acknowledgments

We thank Camillus, LLC (Cleveland, OH), for providing the Guardian device used for testing and David Dudik from Current Lighting (Cleveland, OH) for assistance obtaining irradiance readings. The UV-C device manufacturers did not provide input on study design and did not contribute to data analysis or interpretation or writing or editing the manuscript.

Financial support

This work was supported by the Department of Veterans’ Affairs.

Competing interests

C.J.D has received research grants from Clorox, Pfizer, and Ecolab. All other authors report no conflicts of interest relevant to this article.

References

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Safety Data Sheet: Mesa disc and spore disc biological indicator disc. Mesalabs website. https://info.mesalabs.com/hubfs/1.%20Sterilization%20and%20Disinfection%20Control/Website/Safety%20Data%20Sheets/20-Literature-SDC-Safety-Data-Sheet-TS-216-R01.pdf. Accessed September 18, 2022.Google Scholar
Cadnum, JL, Pearlmutter, BS, Redmond, SN, Jencson, AL, Benner, KJ, Donskey, CJ. Ultraviolet-C (UV-C) monitoring made simple: colorimetric indicators to assess delivery of UV-C light by room decontamination devices. Infect Control Hosp Epidemiol 2022;43:306–311.10.1017/ice.2021.113CrossRefGoogle ScholarPubMed
Masse, V, Hartley, MJ, Edmond, MB, Diekema, DJ. Comparing and optimizing ultraviolet germicidal irradiation systems use for patient room terminal disinfection: an exploratory study using radiometry and commercial test cards. Antimicrob Resist Infect Control 2018;7:29.CrossRefGoogle ScholarPubMed
Figure 0

Figure 1.Log10 reductions in recovery of the test organisms on steel disk carriers after exposure to ultraviolet-C light from room decontamination devices. The carriers were placed 0.91 m from the device and oriented parallel to the bulbs. MRSA, methicillin-resistant Staphylococcus aureus. Note. CFU, colony-forming unit. The dashed line indicates a 3-log10 reduction, which was considered an indication of effective decontamination.

Figure 1

Figure 2.Percentage of the steel disks inoculated with 103 colony-forming units of Bacillus atrophaeus spores with negative cultures after exposure to a 10-minute cycle of ultraviolet-C light from room-decontamination devices.

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