Ozone disinfection for viruses with applications in healthcare environments: a scoping review

IRIE, Milena Suemi; DIETRICH, Lia; SOUZA, Gabriela Leite de; SOARES, Priscilla Barbosa Ferreira; MOURA, Camilla Christian Gomes; SILVA, Gisele Rodrigues da; PARANHOS, Luiz Renato

doi:10.1590/1807-3107bor-2022.vol36.0006

Milena Suemi IRIE

Universidade Federal de Uberlândia – UFU, School of Dentistry, Department of Periodontology and Implantology, Uberlândia, MG, Brazil.

http://orcid.org/0000-0002-2919-2910

Lia DIETRICH

Universidade Federal dos Vales do Jequitinhonha e Mucuri - UFVJM, School of Dentistry, Department of Dentistry, Dental Prosthesis, Diamantina, MG, Brazil

http://orcid.org/0000-0001-7887-8591

Gabriela Leite de SOUZA

Universidade Federal de Uberlândia – UFU, School of Dentistry, Department of Endodontics, Uberlândia, MG, Brazil.

http://orcid.org/0000-0003-3505-5258

Priscilla Barbosa Ferreira SOARES

Universidade Federal de Uberlândia – UFU, School of Dentistry, Department of Periodontology and Implantology, Uberlândia, MG, Brazil.

http://orcid.org/0000-0002-4492-8957

Camilla Christian Gomes MOURA

Universidade Federal de Uberlândia – UFU, School of Dentistry, Department of Endodontics, Uberlândia, MG, Brazil.

http://orcid.org/0000-0002-5653-8403

Gisele Rodrigues da SILVA

Universidade Federal de Uberlândia – UFU, Department of Operative Dentistry and Dental Materials, Uberlândia, MG, Brazil.

http://orcid.org/0000-0002-9358-1339

Luiz Renato PARANHOS

Universidade Federal de Uberlândia – UFU, School of Dentistry, Department of Community and Preventive Dentistry, Uberlândia, MG, Brazil.

http://orcid.org/0000-0002-7599-0120

Corresponding Author: Luiz Renato Paranhos paranhos.lrp@gmail.com

Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.

Database	Search strategy (July 2020)
Embase (http://www.embase.com)	(‘ozone’ OR ‘ozonotherapy’ OR ‘ozone therapy’ OR ‘o3’ OR ‘ozonized’) AND (‘disinfection’ OR ‘infection control’ OR ‘prevention and control’ OR ‘biological control agents’ OR ‘disease transmission’ OR ‘safety management’ OR ‘personal management’ OR ‘disinfectant’ OR ‘decontamination’ OR ‘ppe’ OR ‘virus inactivation’ OR ‘airborne transmission’ OR ‘healthcare workers’) NOT (‘food’ OR ‘agriculture’ OR ‘drinking water’)
LILACS (http://lilacs.bvsalud.org/)	((“Ozone” OR “Ozonotherapy” OR “Ozone Therapy” OR “O3” OR “Ozonized”) AND (“Disinfection” OR “Infection Control” OR “Prevention and Control” OR “Biological Control Agents” OR “Disease Transmission” OR “Safety Management” OR “Personal Management” OR “Disinfectant” OR “Decontamination” OR “PPE” OR “Virus Inactivation” OR “Airborne Transmission” OR “Healthcare Workers”))
LIVIVO (http://livivo.de)	((“Ozone” OR “Ozonotherapy” OR “Ozone Therapy” OR “O3” OR “Ozonized”) AND (“Disinfection” OR “Infection Control” OR “Prevention and Control” OR “Biological Control Agents” OR “Disease Transmission” OR “Safety Management” OR “Personal Management” OR “Disinfectant” OR “Decontamination” OR “PPE” OR “Virus Inactivation” OR “Airborne Transmission” OR “Healthcare Workers”) NOT (“Food” OR “Agriculture” OR “Drinking Water”))
PubMed (http://www.ncbi.nlm.nih.gov/pubmed)	((“Ozone” OR “Ozonotherapy” OR “Ozone Therapy” OR “O3” OR “Ozonized”) AND (“Disinfection” OR “Infection Control” OR “Prevention and Control” OR “Biological Control Agents” OR “Disease Transmission” OR “Safety Management” OR “Personal Management” OR “Disinfectant” OR “Decontamination” OR “PPE” OR “Virus Inactivation” OR “Airborne Transmission” OR “Healthcare Workers”) NOT (“Food” OR “Agriculture” OR “Drinking Water”))
SciELO (http://www.scielo.org/)	((“Ozone” OR “Ozonotherapy” OR “Ozone Therapy” OR “O3” OR “Ozonized”) AND (“Disinfection” OR “Infection Control” OR “Prevention and Control” OR “Biological Control Agents” OR “Disease Transmission” OR “Safety Management” OR “Personal Management” OR “Disinfectant” OR “Decontamination” OR “PPE” OR “Virus Inactivation” OR “Airborne Transmission” OR “Healthcare Workers”))
Scopus (http://www.scopus.com/)	((“Ozone” OR “Ozonotherapy” OR “Ozone Therapy” OR “O3” OR “Ozonized”) AND (“Disinfection” OR “Infection Control” OR “Prevention and Control” OR “Biological Control Agents” OR “Disease Transmission” OR “Safety Management”))
Scopus (http://www.scopus.com/)	((“Ozone” OR “Ozonotherapy” OR “Ozone Therapy” OR “O3” OR “Ozonized”) AND (“Personal Management” OR “Disinfectant” OR “Decontamination” OR “PPE” OR “Virus Inactivation” OR “Airborne Transmission” OR “Healthcare Workers”))
Web of Science (http://apps.webofknowledge.com/)	((“Ozone” OR “Ozonotherapy” OR “Ozone Therapy” OR “O3” OR “Ozonized”) AND (“Disinfection” OR “Infection Control” OR “Prevention and Control” OR “Biological Control Agents” OR “Disease Transmission” OR “Safety Management” OR “Personal Management” OR “Disinfectant” OR “Decontamination” OR “PPE” OR “Virus Inactivation” OR “Airborne Transmission” OR “Healthcare Workers”) NOT (“Food” OR “Agriculture” OR “Drinking Water”))
OpenGrey (http://www.opengrey.eu/)	((“Ozone” OR “Ozonotherapy” OR “Ozone Therapy” OR “O3” OR “Ozonized”) AND (“Disinfection” OR “Infection Control” OR “Prevention and Control” OR “Biological Control Agents” OR “Disease Transmission” OR “Safety Management” OR “Personal Management” OR “Disinfectant” OR “Decontamination” OR “PPE” OR “Virus Inactivation” OR “Airborne Transmission” OR “Healthcare Workers”))
OpenThesis (http://www.openthesis.org/)	((“Ozone” OR “Ozonotherapy” OR “Ozone Therapy” OR “O3” OR “Ozonized”) AND (“Disinfection” OR “Infection Control” OR “Prevention and Control” OR “Biological Control Agents” OR “Disease Transmission” OR “Safety Management” OR “Personal Management” OR “Disinfectant” OR “Decontamination”))

Authors (year)	Virus type	Interventions	Virus carrier/contact surface	Method of assessing viral inactivation	Main results
Sato, Wananabe, Miyata, 1990²⁴	TMEV	Ozone	Dry phase: Lyophilized virus samples in glass vials were exposed to ozone	Plaque assay	Effective: More than 100 ppm ozone and 80% of RH was strongly virucidal
	HVJ	Control (no treatment)	Liquid phase: A portion of a hundredfold virus sample in 35-mm dishes
	RV
	MHV
Tseng, Li, 2006³²	MS2	Ozone exposed	Virus stock solutions were diluted in sterile, deionized water for nebulization; 3% gelatin plates were used to collect virus-containing aerosols before and after ozone treatment.	Plaque assay	Effective: The survival fraction of all four viruses decreased exponentially with increasing ozone dose
	φ6	Ozone unexposed
	φX174
	T7
Hudson et al., 2007²⁵	Norovirus and its animal surrogate feline calicivirus	Ozone exposed	Virus samples were dried on sterile plastic or other surfaces (fabrics and carpet, cotton tips, plastic)	Plaque assays	Effective: Substantial inactivation of FCV and NV samples was achieved, with a comparable reduction in RT-PCR values, indicating that infectivity of both viruses would be similarly affected if it were possible to assay for NV infectivity.
Hudson et al., 2007²⁵	Norovirus and its animal surrogate feline calicivirus	Ozone unexposed		RT-qPCR assay
Lin et al., 2007²³	Enterovirus 71	Ozone	Virus stock in glass dishes	Plaque assay	Ineffective: No statistically significant differences in cell viability were noted among the control group and 0.5 or 1 ppm ozone exposed infected cells (Fig. 3B). The 1.5 or 2 ppm-exposed cells had 45–40% viability.
Lin et al., 2007²³	Enterovirus 71	Control (no treatment)	Virus stock in glass dishes	Plaque assay
Tseng, Li, 2008²¹	MS2	Ozone exposed	A diluted culture of virus stock solution was spread on the surface of gelatin-based medium.	Plaque assay	Effective: The survival fraction of all four viruses decreased exponentially with increasing ozone dose
	φ6	Ozone unexposed
	φX174
	T7
Hudson et al., 2009⁷	Influenza	Ozone exposed	Aliquots of virus, diluted when necessary in PBS, were spotted onto glass slides, stainless steel circular disks, and pieces of fabric and cotton.	Plaque assays	Effective: All viruses tested, showed similar kinetics of virus inactivation on three hard surfaces, plastic, glass and stainless steel. The combination of ozone gas plus high RH consistently yielded substantial inactivation.
	HSV	Ozone unexposed
	Rhinovirus
	Adenovirus
	Mouse coronavirus
	Sindbis virus
	Yellow fever virus
	Vesicular stomatitis virus
	Poliovirus
	Vaccinia virus
Cannon, Kotwal, Wang, 2013²⁰	Murine norovirus (MNV-1)	Ozone	FCV or MNV-1 stocks were spread uniformly onto glass Petri dishes using a cell scraper. One uninoculated Petri dish was also included in each experimental replicate and served as a negative control.	Plaque assays	Effective: exposure of two norovirus surrogates to 20 ppm atmospheric ozone for 18 min and 80% of RH significantly reduced virus infectivity on smooth glass surfaces.
Cannon, Kotwal, Wang, 2013²⁰	Feline calicivirus (FCV)	Ozone		Plaque assays
Petry et al., 2014²²	Herpes Simplex Virus 1 (HSV-1)	Ozone	Aliquots of HSV-1 and BoHV-1 propagated in MDBK cells were added to 35-mm Petri dishes.	Plaque assay	Effective: Ozone promoted a significant reduction of more than 90% of viral replication for both viruses tested after 3 h of exposure.
Petry et al., 2014²²	Bovine Herpes Virus 1 (BoHV-1)	Control (no treatment)		Plaque assay
Guo et al., 2015²⁶	Hepatitis B	Formaldehyde oxidization fumigation	Serum was collected from HBV-infected people with a HBV DNA copy number of 10-7 copies/ml. The serum was diluted 10-fold with sterile distilled water and added to cloth. Negative control groups were composed of sterile distilled water samples.	RT-qPCR	Ineffective: Application of ozone to disinfect HBV-contaminated hospital linen was ineffective.
Guo et al., 2015²⁶	Hepatitis B	Ozone		RT-qPCR
Nayak et al., 2018²⁹	FCV	Ozone	Gas-phase: Sterile stainless steel discs were placed in wells of a 24-well microtiter plates. The surface of each disc was spiked with 15 μl of FCV.	Plaque assay	Effective: Gas-phase FCV inactivation: Complete inactivation was achieved within 3 min of treatment for the humidified biosamples at 1 cm distance from the discharge; 1 cm is similar to 40 cm. Liquid-phase FCV inactivation was also effective: Significant reduction in FCV titer was achieved by treating sterile water for 5 min at 1 cm in dry air.
Nayak et al., 2018²⁹	FCV	Control (no treatment)	Liquid-phase: Aliquots of FCV were added to 96-well plates.	Plaque assay
Cía et al., 2020 (preprint)³³	Lentiviral vector	UV-C light	Open Petri dishes containing pSIN-GFP lentivector stock in DMEM and containing dried bacterial cultures inside an ambulance.	Fluorescence microscopy	Ineffective: Ozone treatments currently applied in emergency vehicles in this study did not significantly affect virus or bacteria viability.
		Ozone		Flow cytometry
				Plaque assays
Blanchard et al., 2020 (preprint)²⁸	Influenza A	Ozone	Virus solutions in growth medium were deposited by pipette onto pieces of each candidate material: cloth face masks, Tyvek (spun high-density polyethylene) fabric used in disposable gowns and PAPR (powered air purifying respirator) hoods, and N95 respirators. Uninoculated samples of each material served as a negative control.	RT-qPCR	Effective: Ozone treatment at 20 ppm or greater, 70% or greater RH at room temperature, and for at least 40 minutes should reliably inactivate enveloped viruses on a variety of materials used for medical PPE.
	Human respiratory syncytial	70% ethanol		Plaque assay
				NanoLuc
Dubuis et al., 2020³¹	φ6	Ozone	The virus buffer was placed in an aerosol generator and nebulized for 10 minutes.	Plaque assays	Effective: 40 minutes and 55% of RH of exposure was required for φX174 and MS2 inactivation.
	φX174			qPCR	10 minutes and 85% RH for φX174, PR772 and MS2. φ6 and MNV-1 viruses showed inactivation levels of at least two orders of magnitude after 40 minutes.
	PR772
	MS2
	MNV-1
Lee et al., 2021²⁷	Human coronavirus HCoV-229E	Ozone	HCoV-229E culture was added to face masks.	Plaque assays	Effective: When face masks experimentally contaminated with a human coronavirus (HCoV-229E) as a surrogate were exposed to ozone gas (approximately 120 ppm) produced by the plasma generator for either 1 or 5 min, the virus lost infectivity.
Lee et al., 2021²⁷	Human coronavirus HCoV-229E	Ozone	HCoV-229E culture was added to face masks.	RT-qPCR
Westover et al., 2020 (preprint)³⁰	Twist Synthetic SARS-CoV-2 RNA Control 2	Ozone	Aliquots of Twist Synthetic SARS-CoV-2 RNA were added to open tubes in the Sani Sport Supreme.	qPCR r	Effective: Synthetic SARS-CoV-2 RNA was shown to undergo significant degradation for 1 hr under ozone treatment. Ozone-treated samples exhibited 65.13%, 25.82%, 11.24%, 12.46% and 6.16% of RNA remaining for 30 mins, 1 hr, 2 hr, 3 hr, and 4 hrs. RNAse-treated samples showed complete degradation.
Westover et al., 2020 (preprint)³⁰	Twist Synthetic SARS-CoV-2 RNA Control 2	Control (no treatment)		qPCR r

Authors (year)	Ozonation method	Ozone concentration (ppm)	Temperature (°C)	Humidity (%)	Time evaluated (min)
Sato, Watanabe, Miyata, 1990²⁴	Ozone Generator (Elios Ozonizer, Shinryo. Reinetsu. Co. Tokyo. Japan)	HJV samples: 200	22–25	50–90	30 mi
Sato, Watanabe, Miyata, 1990²⁴		TMEV samples: 100 and 200	22–25	50–90	30 mi
Tseng; Li, 2006³²	Ozone generator (OZ1PCS-V/SW, Ozotech Inc., Yreka, CA)	0.1–10	25–28	55–85	13.8 sec
Tseng; Li, 2006³²	Ozone generator (OZ1PCS-V/SW, Ozotech Inc., Yreka, CA)	0.1–10	25–28	55–85	18.4 sec
Hudson et al., 2007²⁵	Multiple corona discharge units (Viroforce 1000; Viroforce Systems, Kelowna, BC, Canada)	20–25	23	70 in excess	20
Lin et al., 2007²³	Ozone Generator (Tenco, XV1043CA, Taiwan)	0.5, 1, 1.5; 2	25		120
Tseng; Li, 2008²¹	Ozone generator (OZ1PCS-V/SW, Ozotech Inc., Yreka, CA)	0.6; 0.9; 1.2	25–28	55–85	5
					10
					15
					20
					40
					60
					90
					120
Hudson et al., 2009⁷	Ozone generators (Treated Air Systems) for the initial field trials	25	20	40	10 (RH tests > 90%)
Hudson et al., 2009⁷	Multiple corona discharge units (Viroforce 1000; Viroforce Systems, Kelowna, BC, Canada)	25	20	> 90	60
Cannon, Kotwal, Wang, 2013²⁰	Ozone generator (ZONOsanitech, Alpharetta, GA).	20	Room temperature	80	18
Petry et al., 2014²²	Ozone generator (Brizzamar, Ronda Alta, RS, Brazil)	0.02– 0.05	26.2– 29.2	30–37	60
					120
					180
Guo et al., 2015²⁶	Computer-controlled bed unit ozone sterilizer (Kz-x-dL1, Guangdong Kangzhen Medical Equipment Co. Ltd., Guang-dong province, China)	150*			15
					30
					60
					80
Nayak et al., 2018²⁹	Dieletric barrier discharge	20			5
Nayak et al., 2018²⁹	Dieletric barrier discharge	20			20
Cía et al., 2020³³	Ozone generator (Industrial Global Supply S.L.)	Higher than 10		37–48	10
Cía et al., 2020³³	Ozone generator (Industrial Global Supply S.L.)	Higher than 10		37–48	20
Blanchard et al., 2020²⁸	Global Ozone Decon-Zone 4201A Cabinet//Global Ozone	Approximately 20	Room temperature	40	160
	OT-100 Trailer//Zono SC 1 Cabinet//VirtuCLEAN			50–70	320
	2.0 Waterless CPAP Cleaning Pouch			80
Lee et al., 2020²⁷	Dieletric barrier discharge	Approximately 120	25	--	1
Lee et al., 2020²⁷	Dieletric barrier discharge	Approximately 120	25	--	5
Dubuis et al., 2020³¹	Ozone generator (model EMO3-VTTL, EMO3, Quebec City, CANADA)	Phages: 1.13 ± 0.26		20%	10
		MNV-1: 0.23 ± 0.03		55%	40
				85%	70
Westover et al., 2020³⁰	Ozone generator (Sani Sport Supreme)	20			30
					60
					120
					180
					240

Authors (year)	Sponsorship status

	Sponsored	Nonsponsored	Unclear
Sato, Wananabe, Miyata, 1990²⁴			x
Tseng, Li, 2006³²			x
Hudson et al., 2007²⁵			x
Lin et al., 2007²³			x
Tseng, Li, 2008²¹			x
Hudson et al., 2009⁷			x
Cannon, Kotwal, Wang, 2013²⁰			x
Petry et al., 2014²²			x
Guo et al., 2015²⁶		x
Nayak et al., 2018²⁹		x
Cía et al., 2020³³		x
Blanchard et al., 2020²⁸		x
Dubuis et al., 2020³¹		x
Lee et al., 2021²⁷		x
Westover et al., 2020³⁰		x

[1] Corresponding Author: Luiz Renato Paranhos paranhos.lrp@gmail.com

Brasil

Brasil

Ozone disinfection for viruses with applications in healthcare environments: a scoping review

Abstract