Clinical effectiveness of medical ozone therapy in COVID-19: the evidence and gaps map

Abstract

Ozone therapy (OT), a medical procedure, has been showing good results during the coronavirus disease (COVID-19). We aimed to build an evidence and gaps map (EGM) of OT in the COVID-19 ranking the articles found according to levels of evidence and outcomes. The EGM brings bubbles of different sizes and different colors according to the articles. The OT intervention used was major or minor autohemotherapy, rectal insufflation and ozonized saline solution. EGM was based on 13 clinical studies using OT for COVID-19 involving a total of 271 patients. We found 30 outcomes related to OT in COVID-19. Our EGM divided the outcomes into six groups: 1-clinical improvement; 2-hospitalization; 3-inflammatory, thromboembolic, infectious, or metabolic markers; 4-radiological aspects, 5-viral infection and 6-adverse events. Major autohemotherapy was present in 19 outcomes, followed by rectal insufflation. Improvement in clinical symptoms of COVID-19, improvement of respiratory function, improvement of oxygen saturation, reduction in hospital internment, decrease in C-reactive protein, decrease in ferritin, decrease in lactate dehydrogenase, decrease in interleukin 6, decrease in D-dimer, radiological improvement of lung lesions and absence of reported adverse events were related in the papers. The most commonly used concentrations of OT in major autohemotherapy and in rectal insufflation were 40 μg/mL and 35 μg/mL, respectively. Here, we bring the first EGM showing the efficacy and safety of OT in the treatment of COVID-19. OT can be used as integrative medical therapy in COVID-19 at a low cost and improve the health conditions of the patients.

INTRODUCTION

The novel coronavirus disease 2019 (COVID-19) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the global pandemic. The scientific community has been looking for effective and safe treatments for COVID-19.

Ozone is triatomic oxygen with high oxidizing power. Ozone has been used for a variety of purposes, including ozone therapy (OT). OT is the therapeutic administration of medicinal ozone to treat some diseases. OT is available and recognized as a medical procedure in Germany, Italy, Russia, Portugal, Spain, Turkey, Switzerland, Austria, Greece, Egypt, China, Japan, Cuba, Mexico, Honduras and several Eastern European countries.[1]

OT can be administered by different routes according to the desired therapeutic purpose, considering safety and efficacy concentrations that ranged from 1 μg/mL to 100 μg/mL.[2] OT has been used as an alternative for the treatment of dentistry,[3] osteoarthritis,[4] wound healing,[5,6,7] inactivation of viruses and bacteria8 and backache.[9,10] The ozone has also effectiveness in general viral infections,[11,12,13,14,15,16] including cornovarius.[17] Evidence and gap maps (EGMs) are an innovative rapid review method that involves a systematic search and characterization of a topic of interest, identifying scientific evidence, trends, knowledge gaps and future research needs.[18] The EGMs apply a reproducible and clear method, in addition to an explicit bibliographic search process and inclusion and exclusion criteria.[18] EGMs allow descriptive and/or visual analysis of the database, such as bubble charts, to identify gaps in clinical research and support the formulation of evidence-based policies to provide accessible resources in health decision-making. Here, we present, for the first time, an overview of existing evidence and gaps on the effects of OT in COVID-19, based on case reports, clinical trials, case-control studies, prospective case-control studies, quasi-experimental prospective cohort studies, randomized clinical trials and non-randomized clinical trials to promote evidence-based practice. We also sought to identify the possible mechanisms of action of OT in the treatment of SARS-CoV-2.

DATA AND METHODS

The map was built with the articles found between December 2020 and March 2022. The systematic evidence search process was adapted from the EGM of the International Initiative for Impact Evaluation (3iE) and included: 1-definition of limits and map contexts; 2-research and selection of relevant scientific studies; 3-characterization of the studies.

The results are presented in the form of an evidence map, highlighting positive points related to the potential gains from the clinical application of OT in COVID-19.

Research methods to identify studies

The electronic databases MEDLINE (PubMed), Virtual Health Library (VHL) and WHO-COVID19 (Table 1) were used. The main terms included in the search were: ozonotherapy, ozonetherapy, ozone therapy and medical ozone, COVID-19 and Coronavirus.

Table 1:

Search strategies applied to identify OT studies at COVID-19

Database	URL	Search strategy
VHL	http://www.bvsalud.org	("ozone therapy" OR "ozonioterapia" OR (("oxygen-ozone" OR ozono* OR ozone* OR ozonio* OR "ionized medical oxygen" OR ozone*) (treat* OR tratam* OR therap* OR terap* OR medic* OR clinic*))) AND (((("2019-2020" OR 2019 OR da:202*) ("New Coronavirus" OR "Novel Coronavirus" OR "Nuevo Coronavirus" OR "Novo Coronavirus" OR "Coronavirus disease" OR "Enfermedad por Coronavirus" OR "severe acute respiratory syndrome coronavirus 2")) OR ((2019-ncov) OR (ncov 2019) OR 2019ncov OR covid19 OR (covid-19) OR covid2019 OR (covid-2019) OR (covid 2019)) OR ((srag-cov-2 OR sars-cov-2 OR sars2 OR (sars 2) OR (sars cov 2) OR cov19 OR cov2019 OR Coronavirus* OR "Severe Acute Respiratory Infections" OR "Severe Acute Respiratory Infection" OR "Coronavirus 2" OR "acute respiratory disease" OR mh:Betacoronavirus OR mh:"Coronavirus infections" OR mh:"sars virus") AND (tw:2019 OR da:202*) AND NOT da:201*) OR (Wuhan market virus) OR (virus mercado Wuhan) OR "Wuhan Coronavirus" OR "Coronavirus de Wuhan"))
MEDLINE (PubMed)	http://pubmed.gov	(("ozone therapy" OR "Ozonated Autohemotherapy" OR (("oxygen-ozone" OR ozone* OR "ionized medical oxygen") (treat* OR therap* OR medic* OR clinic*))) AND (Severe Acute Respiratory Syndrome Coronavirus 2[Supplementary Concept] OR COVID-19[Supplementary Concept] OR coronavirus[MeSH Terms] OR coronavirus infections[MeSH Terms] OR betacoronavirus[MeSH Terms] OR Betacoronavirus*[TIAB] OR Coronavirus Infection[TIAB] OR Severe Acute Respiratory Syndrome Coronavirus 2[TIAB] OR Coronavirus Disease 2019[TIAB] OR 2019 Novel Coronavirus[TIAB] OR Wuhan Coronavirus[TIAB] OR COVID- 19[TIAB] OR SARS-CoV-2[TIAB] OR 2019-nCoV[TIAB] OR 2019 Novel Coronavirus[TIAB] OR Coronavirus Infection Disease 2019[TIAB] OR 2019 nCoV Infection[TLAB] OR Novel Coronavirus Pneumonia[TIAB] OR nCoV[TIAB]))
WHO-COVID19	https://search.bvsalud.org/global-literature-on-novel-coronavirus-2019-ncov/	("ozone therapy" OR "Ozonated Autohemotherapy" OR (("oxygen-ozone" OR ozone* OR "ionized medical oxygen") (treat* OR therap* OR medic* OR clinic*)))

Note: COVID-19: Coronavirus disease 2019; OT: ozone therapy.

Unpublished articles from other sources were also identified and included for final evaluation.

Study inclusion criteria

Case reports, clinical trials, case-control studies, prospective case-control studies, quasi-experimental prospective cohort studies, randomized clinical trials and non-randomized clinical trials, in any language or publication date, reporting cases of clinical application of OT in patients affected by the SARS-CoV2 virus were also included, according to Additional Table 1.

Additional Table 1.

List of included studies

Title	Author	Source
A preliminary evaluation on the efficacy of ozone therapy in the treatment of COVID-19.	Zheng, Zhishui; Dong, Minglin; Hu, Ke	J Med Virol; 92(11): 2348-2350, 2020 11.
Blood ozonization in patients with mild to moderate COVID-19 pneumonia: a single centre experience.	Tascini, Carlo; Sermann, Giovanni; Pagotto, Alberto; Sozio, Emanuela; De Carlo, Chiara; Giacinta, Alessandro; Sbrana, Francesco; Ripoli, Andrea; Castaldo, Nadia; Merelli, Maria; Cadeo, Barbara; Macor, Cristiana; De Monte, Amato	Intern Emerg Med; 2021 Feb 03.
Case report: recovery of one ICU-acquired COVID-19 patient via ozonated autohemotherapy.	Junping Wu; Cherie S. Tan; Hongzhi Yu, M.M; Youwei Wang; Yutao Tian; Wenwei Shao; Yifei Zhang; Kuo Zhang; Hongxia Shao; Guangjian Ni; Jun Shen; Qi Wu, M.M; Dong Ming.	The Innovation; 2020
Complementary application of the ozonized saline solution in mild and severe patients with pneumonia COVID-19: A non-randomized pilot study	Schwartz, A.; Martínez-Sánchez, G.; de Lucía, A. M.; Viana, S. M.; Constanta, A. M.	J Pharm Pharmacognosy Res; 2021 2(9):126-46
Effect of Rectal Ozone (O3) in Severe COVID-19 Pneumonia: Preliminary Results.	Fernández-Cuadros, Marcos Edgar; Albaladejo-Florín, María Jesús; Álava-Rabasa, Sandra; Usandizaga-Elio, Isabel; Martinez-Quintanilla Jimenez, Dolores; Peña-Lora, Daiana; Neira-Borrajo, Inmaculada; López-Muñoz, María Jesús; Rodríguez-de-Cía, Javier; Pérez-Moro, Olga Susana	SN Compr Clin Med: 1-9, 2020 Aug 03.
Oxygen-ozone (O(2)-O(3)) immunoceutical therapy for patients with COVID-19. Preliminary evidence reported.	Franzini, Marianno; Valdenassi, Luigi; Ricevuti, Giovanni; Chirumbolo, Salvatore; Depfenhart, Markus; Bertossi, Dario; Tirelli, Umberto	Int Immunopharmacol; 88: 106879, 2020 Nov.
Ozone therapy for patients with COVID-19 pneumonia: Preliminary report of a prospective case-control study.	Hernández, Alberto; Viñals, Montserrat; Pablos, Asunción; Vilás, Francisco; Papadakos, Peter J.; Wijeysundera, Duminda N.; Bergese, Sergio D.; Vives, Marc	Int Immunopharmacol; 88: 106879, 2020 Nov.
Potential Role of Oxygen-Ozone Therapy in Treatment of COVID-19 Pneumonia.	Hernández, Alberto; Viñals, Montserrat; Isidoro, Tomas; Vilás, Francisco	Am J Case Rep; 21: e925849, 2020 Aug 17.
Safety and efficacy of ozone therapy in mild to moderate COVID-19 patients: A phase 1/11 randomized control trial (SEOT study).	Shah, Mili; Captain, Jignasha; Vaidya, Vidyadhar; Kulkarni, Arvind; Valsangkar, Kedar; Nair, Pradeep M. K.; Ganu, Gayatri	Int Immunopharmacol; 91: 107301, 2021 Feb.
Uso de ozonoterapia rectal en paciente anciana con neumonía grave por COVID-19	Peña-Lora, Daiana Yulissa; Albaladejo-Florín, María Jesús; Fernández-Cuadros, Marcos Edgar	Rev Esp Geriatr Gerontol; 55(6): 362-364, 2020.
Utilization of Ozone as a Complementary Therapy for COVID-19 Patients.	Husham A. Razzaq, Mohammad S. Hasan, Muthanna F. Al-Dhalemy, Wurood M. Al-Silaykhee, Hekmat B. Alhmadi, Zaid A. Majeed	Int J Psychosoc Rehab; 24(7):10577-88, 2020.
Old Treatment for a New Disease: Can Rectal Ozone Insufflation Be Used for COVID-19 Management? A Case Report.	Hendawy, H.A.; Mosallam, W.; Abuelnaga, M.E.; Sabry, A.M.	SN Compr Clin Med. 3(6):1424-1427, 2021.
A pilot study for treatment of COVID-19 patients in moderate stage using intravenous administration of ozonized saline as an adjuvant treatment-registered clinical trial.	Sharma A, Shah M, Lakshmi S, Sane H, Captain J, Gokulchandran N, Khubchandani P, Pradeep MK, Gote P, Tuppekar B, Kulkarni P, Paranjape A, Pradhan R, Varghese R, Kasekar S, Nair V, Khanbande U.	Int Immunopharmacol. 96:107743, 2021.

Exclusion criteria from the study

Bibliographic and editorial reviews, comments and opinions papers were excluded, as well as articles that used OT associated with other therapies and in pre- or post-COVID-19 patients, according to Additional Table 2.

Additional Table 2.

List of exclusion studies

Reason for exclusion	Title	Author	Source
Ozone associated with other therapies	A novel approach to treating COVID-19 using nutritional and oxidative therapies	David Brownstein; Richard Ng; Robert Rowen; Jennie-Dare Drummond; Taylor Eason; Hailey Brownstein; Jessica Brownstein	Science, Public Health Policy, and The Law, 2020, 2:4-22
Ozone associated with other therapies	Ozone as adjuvant support in the treatment of COVID-19: A preliminary report of probiozovid trial	Araimo, Fabio; Imperiale, Carmela; Tordiglione, Paolo; Ceccarelli, Giancarlo; Borrazzo, Cristian; Alessandri, Francesco; Santinelli, Letizia; Innocenti, Giuseppe Pietro; Pinacchio, Claudia; Mauro, Vera; Recchia, Gregorio Egidio; Zancla, Serena; Calò, Andrea; Poscia, Roberto; Ruberto, Franco; d'Ettorre, Gabriella; Bilotta, Federico; Mastroianni, Claudio; Pugliese, Francesco	Journal of medical virology, 2020, 4(93):2210-20
Ozone associated with other therapies	Ozone gas applied through nebulization as adjuvant treatment for lung respiratory diseases due to COVID-19 infections: a prospective randomized trial	Dengiz, E.; Özcan, Ç.; Güven, Y.İ.; Uçar, S.; Ener, B.K.; Sözen, S.; Yağcı, B.; Güzel, İ.A.; Yiğit, B.; Andaç, A.; Güneş, B.; Bor, E.; Karabudak, U.; Kaya, A.	Med Gas Res. 2022 Apr-Jun;12(2):55-59
Ozone associated with other therapies	Effectiveness of ozone therapy in addition to conventional treatment on mortality in patients with COVID-19	Çolak, S.; Genç, Y B.; Yavuz, M.; Özçelik, B.; Öner, M.; Özgültekin, A.; Senbayrak, S.	Int J Clin Pract; 75(8): e14321, 2021 Aug.
Ozone associated with other therapies	CORonavirus-19 mild to moderate pneumonia Management with blood Ozonization in patients with Respiratory failure (CORMOR) multicentric prospective randomized clinical trial	Sozio, E.; De Monte, A.; Sermann, G.; Bassi, F.; Sacchet, D.; Sbrana, F.; Ripoli, A.; Curcio, F.; Fabris, M.; Marengo, S.; Italiani, D.; Luciana, Boccalatte-Rosa, D.; Tascini, C.	Int Immunopharmacol. 2021 Sep;98:107874.
Ozone associated with other therapies	Compassionate Use of Rectal Ozone (O 3) in Severe COVID-19 Pneumonia: a Case-Control Study	Fernández-Cuadros, M.E.; Albaladejo-Florín, M.J.; Álava-Rabasa, S.; Gallego-Galiana, J.; Pérez-Cruz, G.F.; Usandizaga-Elio, I.; Pacios, E.; Torres-García, D.E.; Peña-Lora, D.; Casique-Bocanegra, L.; López-Muñoz, M.J.; Rodríguez-de-Cía, J.; Pérez-Moro, O. S.	SN Compr Clin Med. 2021 Mar 22;1-15.
Pre-covid study	Intravenous ozonized saline therapy as prophylaxis for healthcare workers (HCWs) in a dedicated COVID-19 hospital in India – A retrospective study	Sharma, A.; Shah, M.; Sane, H.; Gokulchandran, N.; Paranjape, A.; Khubchandani, P.; Captain, J.; Shirke, S.; Kulkarni, P.	Eur Rev Med Pharmacol Sci, 2021; 25 (9): 3632-3639
Pre-covid study	Could the minor autohemotherapy be a complementary therapy for healthcare professionals to prevent COVID-19 infection?	Orscelik, A.; Karaaslan, B.; Agiragac, B.;Solmaz, I.; Parpucu, M.	Annals of Medical of Research, 28(10):1863-1869, 2021
Post-covid study	Amelioration of symptoms and oxidative stress in hospitalized convalescent post SARS-COV-2 patients treated with rectal ozonetherapy and nutritional supplementation	Lizette Gil-del-Valle; Olga Elena López-Fernández; Joniel Arnoldo Sánchez- Márquez; Zullyt ZamoraRodríguez; Ana Librada Carballo- Reyes; Lidia Asela Fernández-García; Mario Manuel Delgado-Guerra; Faustina Fonseca-Betancourt; Ernesto Miyares-Díaz; Yasmina Piloto, Orraca.; Yusimit, B.A.; Maria Carla Hdez Glez-Abreu; Sarahi Mendoza-Castaño; Yamila Ramona de Armas-Aguila.	International Journal of Modern Pharmaceutical Research, 2020, 4(6):94-107
Post-covid study	Fatigue in post-acute sequelae of SARS-CoV2 (PASC) treated with oxygen-ozone autohemotherapy - preliminary results on 100 patients	Tirelli, U.; Franzini, M.; Valdenassi, L.; Pisconti, S.; Taibi, R.; Torrisi, C.; Pandolfi, S.; Chirumbolo, S.	Eur Rev Med Pharmacol Sci, 2021 Sep;25(18):5871-5875
Editorials	Homeopathic use of Ozonum for some post-Covid dyspnea and/or asthenia. Study based on a cases serie	Renoux, H.	La Revue d'Homéopathie, 2021, 12 (4), 222-227
Editorials	Oxygen–ozone treatment and COVID-19: antioxidants targeting endothelia lead the scenery	Varesi, A.; Chirumbolo, S.; Ricevuti, G.	Intern Emerg Med, 2022, 17(2):593-596
Editorials	COVID-19 profilaxis with ozonetherapy	Falzoni, W.; Senvaitis, M. I.; Iwasa, S.	Acupuncture and Electro-Therapeutics Research, 2021, 46(1):35-36
Editorials	Persistent anosmia after COVID-19 Infection: Treatment options according to BDORT	Dotta Barros, F. C.; Iwasa, S.	Acupuncture and Electro-Therapeutics Research, 2021, 46(1):33-34
Editorials	Ozone therapy may be an option for COVID-19 patients	Şahin, M.; Eryilmaz, F.; Keser Şahin, H.H.	Eur Rev Med Pharmacol Sci, 2021; 25 (6): 2470-2472
Editorials	Oxygen-ozone autohemotherapy against COVID-19 needs to fit highly experienced, customized, and standardized protocols to succeed	Chirumbolo, S.; Franzini, M.; Simonetti, V.; Valdenassi, L.; Ricevuti, G.; Bertossi, D.; Pandolfi, S.	J Med Virol, 2021, 93(5):2580-2582
Editorials	Estado vegetativo persistente por ozonoterapia contra Covid-19	Zavala Jonguitud, L.F.; Solís, J.G.; Noyola García, M.	Salud Publica Mex, 2021, 63:155
Editorials	A Plausible "Penny" Costing Effective Treatment for Corona Virus - Ozone Therapy	Robert Jay Rowen; Howard Robins	Infectious Diseases and Epidemiology, 2020, 6(2):1-5
Editorials	Inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by gaseous ozone treatment.	Yano, H.; Nakano, R.; Suzuki, Y; Nakano, A.; Kasahara, K.; Hosoi, H.	The Journal of hospital infection, 2020, 4(106)837-8
Editorials	Oxygen-ozone immunoceutical therapy in COVID-19 outbreak: facts and figures	Giovanni Ricevuti; Marianno Franzini; Luigi Valdenassi	Ozone Therapy, 2020, 1(5)
Editorials	Oxygen-ozone therapy as adjuvant in the current emergency in SARS-COV-2 infection: a clinical study.	Marini, S.; Maggiorotti, M.; Dardes, N.; Bonetti, M.; Martinelli, M.; Re, L.; Carinci, F.; Tavera, C.	Journal of biological regulators and homeostatic agents, 2020, 3(34):757-66
Editorials	Ozone Therapy as a Possible Option in COVID-19 Management.	Gavazza, Alessandra; Marchegiani, Andrea; Rossi, Giacomo; Franzini, Marianno; Spaterna, Andrea; Mangiaterra, Sara; Cerquetella, Matteo	Frontiers in Public Health, 2020, 8:417
Editorials	Ozonioterapia como adjuvante no tratamento da COVID-19 / Ozone as a complement therapy in the treatment of COVID-19	Jorge Bomfim Fróes de Farias; Antonio Pedro Fróes de Farias; Anelize Gimenez de Souza	Revista Brasileira de Fisiologia do Exercicio, 2020, 19(2):5-8
Editorials	Potential mechanisms by which the oxygen-ozone (O2-O3) therapy could contribute to the treatment against the coronavirus COVID-19.	Valdenassi, L.; Franzini, M.; Ricevuti, G.; Rinaldi, L.; Galoforo, A. C.; Tirelli, U.	European review for medical and pharmacological sciences, 2020, 8(24):4059-61
Editorials	Recovery of Four COVID-19 Patients via Ozonated Autohemotherapy.	Wu, Junping; Tan, Cherie S.; Yu, Hongzhi; Wang, Youwei; Tian, Yutao; Shao, Wenwei; Zhang, Yifei; Zhang, Kuo; Shao, Hongxia; Ni, Guangjian; Shen, Jun; Galoforo, Antonio Carlo; Wu, Qi; Ming, Dong	Innovation (New York, N.Y.), 2020, 3(1)100060
Editorials	The need for a correct oxygen-ozone autohemotherapy (O(3)-AHT) in patients with mild to moderate COVID -19 pneumo nia	Chirumbolo, Salvatore; Pandolfi, Sergio; Valdenassi, Luigi; Bertossi, Dario; Franzini, Marianno	Internal and Emergency Medicine, 2021, P.1-2
Editorials	Utilização do gás ozônio e da ozonioterapia no combate à disseminação da COVID-19 submetidas pela Sociedade Brasileira de Ozonioterapia Médica (SOBOM)	Brasil. Ministério da Saúde. Secretaria de Ciencia, Tecnologia, Inovação e Insumos Estratégicos em Saúde.	Brasília; Brasil. Ministério da Saúde; abr. 2020.
Literature review	Dos terapias conocidas podrían ser efectivas como adyuvantes en el paciente crítico infectado por COVID-19	Hernández, A.; Papadakos, P. J.; Torres, A.; González, D. A.; Vives, M.; Ferrando, C.; Baeza, J.	Rev. esp. anestesiol. reanim, 2020, 5(67):245-242
Literature review	Implicações sobre o uso do ozônio (O3) no tratamento coadjuvante do COVID-19	Jorge Bomfim Fróes de Farias; Antonio Pedro Fróes de Farias; Anelize Gimenez de Souza	Revista Brasileira de Fisiologia do Exercício, 2020, 4(106):5-8
Literature review	Ozone (O3) and SARS-CoV-2: Physiological Bases and Their Therapeutic Possibilities According to COVID-19 Evolutionary Stage.	Fernández-Cuadros, Marcos Edgar; Albaladejo-Florín, María Jesús; Peña-Lora, Daiana; Álava-Rabasa, Sandra; Pérez-Moro, Olga Susana	SN comprehensive clinical medicine, 2020, p.1-9
Literature review	Ozone potential to fight against SAR-COV-2 pandemic: facts and research needs.	Blanco, Angeles; Ojembarrena, Francisco de Borja; Clavo, Bernardino; Negro, Carlos	Environmental science and pollution research international, 2021, p.1-15
Literature review	Ozone therapy for the treatment of COVID-19 pneumonia: A scoping review.	Izadi, Morteza; Cegolon, Luca; Javanbakht, Mohammad; Sarafzadeh, Ali; Abolghasemi, Hassan; Alishiri, Gholamhossein; Zhao, Shi; Einollahi, Behzad; Kashaki, Mandana; Jonaidi-Jafari, Nematollah; Asadi, Mosa; Jafari, Ramezan; Fathi, Saeid; Nikoueinejad, Hassan; Ebrahimi, Mehrdad; Imanizadeh, Sina; Ghazale, Amir Hosein	Int Immunopharmacol, 2021, 92:107307
Literature review	Ozone therapy in COVID-19: A narrative review.	Francesco Cattel; Susanna Giordano; Cecilia Bertiond; Tommaso Lupia; Silvia Corcione; Matilde Scaldaferri; Lorenzo Angelone; Francesco Giuseppe De Rosa	Virus Research, 2021, 291:198207
Literature review	Ozone: A Potential Oxidant for COVID-19 Virus (SARS-CoV-2)	Chedly Tizaoui	Ozone: Science & Engineering, 2020, 42(5):378-85
Literature review	Ozonetherapy: a multirole weapon, topical pathway against SARS-COV-2	Giovanni Ranaldi; Emanuele Rocco; VillaniLaura Franza; Giulia Motola	Frenxiv, 2020, p.1-11
Literature review	Ozonoterapia enteral: una posible opción segura y económica para pacientes con COVID-19	Vivian Borroto Rodríguez; Antonio Jiménez Tapia; María Esther Dragustinovis Ruiz	Revista Cubana de Medicina Física y Rehabilitación, 2020, 12(3):1-16
Literature review	Ozonoterapia para el tratamiento de pacientes adultos con COVID-19	Peru. EsSalud Instituto de Evaluación de Tecnologías en Salud e Investigación	Peru. EsSalud. Instituto de Evaluación de Tecnologías en Salud e Investigación. s.l; IETSI; 1 jun. 2020.
Literature review	Potential Cytoprotective Activity of Ozone Therapy in SARS-CoV-2/COVID-19.	Martínez-Sánchez, Gregorio; Schwartz, Adriana; Donna, Vincenzo Di	Antioxidants (Basel, Switzerland), 2020, 9(5)1-12
Literature review	Potential use of ozone in SARS-CoV-2 / COVID-19	Therapy, International Scientific Committee of Ozone	International Scientific Committee of Ozone Therapy. Madrid; s.n; mar. 2020. p.2-15
Literature review	Rationale for ozone-therapy as an adjuvant therapy in COVID-19: a narrative review.	Ranaldi, Giovanni Tommaso; Villani, Emanuele Rocco; Franza, Laura	Medical Gas Research, 2020, 3(10):134-8
Literature review	Recent case studies on the use of ozone to combat coronavirus: Problems and perspectives.	Manjunath, Soumya Nagashri; Sakar, M.; Katapadi, Manmohan; Geetha Balakrishna, R.	Environmental Technology & Innovation, 2021, 21:101313
Literature review	Safety, pitfalls, and misunderstandings about the use of ozone therapy as a regenerative medicine tool. A narrative review.	Re, L.; Noci, J. B.; Gadelha Serra, M. E.; Mollica, P.; Bonetti, M.; Travagli, V.	Journal of biological regulators and homeostatic agents, 2020, 4(34):1-3
Literature review	SARS-COV-2 and Ozonetherapyin microcirculation: Devils and Angels~lll	Giovanni T. Ranaldi; Emanuele Rocco Villani; Laura Franza; Giulia Motola.	Frenxiv, 2020
Literature review	Therapeutic Effects of Ozone Therapy that Justifies Its Use for the Treatment of COVID-19	Silvia Menendez-Cepero; José Antonio Marques-Magallanes-Regojo; Alberto Hernandez-Martinez; Francisco Javier Hidalgo Tallón; JoséBaeza-Noci	Journal of Neurology and Neurocritical Care, 2020, 3(1):1-6
Literature review	Two known therapies could be useful as adjuvant therapy in critical patients infected by COVID-19.	Hernández, A.; Papadakos, P. J.; Torres, A.; González, D. A.; Vives, M.; Ferrando, C.; Baeza, J.	Revista espanola de anestesiologia y reanimacion, 2020, 5(67):245-52
Literature review	Observancia de reacciones adversas y análisis de cumplimiento de bioseguridad en la aplicación rectal de ozonoterapia en pacientes cubanos con infección aguda o convalecientes de COVID-19.	Lizette Gil del Valle, Mario M. Delgado Guerra, Ana L. Carballo- Reyes, Joniel A. Sánchez Márquez, Olga E. López Fernández, Faustina Fonseca Betancourt, Zullyt Zamora-Rodríguez, Lidia A. Fernández García, Rodolfo Suárez Iznaga, Raiza Martinez Casanueva, Lisandra Castro de la Fe, Niorkidis Gonzalez Carvajal, Silvia V. Castellanos Veitia, Yamila R. de Armas-Aguila	J Pharm Pharmacogn Res, 2021, 9(4): 465-473
Literature review	Promising gas therapies for severe COVID-19	Wang, T.; Xiang, Q.; Bian, Jinjun.	Journal of Intensive Medicine; 2021.
Literature review	The possibilities of using the effects of ozone therapy in neurology	Masan, J.; Sramka, M.; Rabarova, D.	Neuro Endocrinol Lett. 2021 Mar;42(1):13-21.
Literature review	COVID-19 and ozone	Yilmaz, N.; Eren, E.; Oz, C.	Cyprus Journal of Medical Sciences, 2020, 5(4):365-372
Literature review	Ozonum—The Global Impact	Gajdos, P. S.	Homoeopathic Links, 2020, 33(4):283-296
Literature review	Potential therapeutic effect of oxygen-ozone in controlling of COVID-19 disease.	Yousefi, B.; Banihashemian, S.Z.; Feyzabadi, Z.K.; Hasanpour, S.; Kokhaei, P.; Abdolshahi, A.; Emadi, A.; Eslami, M.	Med Gas Res, 2022, 12(2):33-40
Literature review	Insights on the mechanisms of action of ozone in the medical therapy against COVID-19	Chirumbolo, S.; Valdenassi, L.; Simonetti, V; Bertossi, D.; Ricevuti, G.; Franzini, M.; Pandolfi, S.	Int Immunopharmacol, 2021, 96:107777
Literature review	Using Ozone Therapy as an Option for Treatment of COVID-19 Patients: A Scoping Review	Radvar, S.; Karkon-Shayan, S.; Motamed-Sanaye, A.; Majidi, M.; Hajebrahimi, S.; Taleschian-Tabrizi, N.; Pashazadeh, F.; Sahebkar, A.	Adv Exp Med Biol, 2021, 1327:151-160
Literature review	Drug Repurposing in the COVID-19 Era: Insights from Case Studies Showing Pharmaceutical Peculiarities	Gatti, M.; De Ponti, F.	Pharmaceutics, 2021, 25;13(3):302
Literature review	Alternative therapies for Covid-19	Sundararajan, G.; Isaac, PJ.; Andal, V.; Lakshmipathy, R.	Mater Today Proc, 2021, 5;55:327-9
Literature review	Ozonoterapia: una propuesta terapéutica para la prevención y el tratamiento de la infección por coronavirus SARS-COV-2 "COVID 19	Longas Vélez, B.; Muñoz Mejía, C. A.; Ramírez Amaya, C. A.; Gaviria Morales, C.; Sarasti Vanegas, D. A.; Ramírez Zuluaga, E.; Trujillo Escobar, G.; Ríos Arenas, J. A.; Gaitán Sánchez, J. F.; Londoño Murillo, M. C.; Martínez Cadavid, M. M.; Soto Velásquez, M. L.; Álvarez Upegui, N.; Pérez Garcíí, C. L.; Mejía Alfaro, J. H.	Medellin – Colombia 2020
Clinical trials registers	Ozone Plasma on Lung Function and Inflammatory Parameters in Pulmonary Sequelae Associated With Coronavirus 19 Infection	Fernando Grover Paez, Centro Universitario de Ciencias de la Salud, Mexico	https://clinicaltrials.gov/show/NCT05089305
Clinical trials registers	Rectal ozone therapy in high-risk symptomatic SARS-CoV-2 positive patients	National Center for Scientific Research (CNIC)	https://rpcec.sld.cu/en/trials/RPCEC00000383-En
Clinical trials registers	Evaluation of Post-covid 19 Patients Who Receive Ozonetheraphy With Thorax CT	Gaziosmanpasa Research and Education Hospital	https://clinicaltrials.gov/show/NCT04789395
Clinical trials registers	Study on ozonated blood in COVID-19 severe pneumonia	Políclinica Nuestra Señora del Rosario	https://www.clinicaltrialsregister.eu/ctr-search/search?query=eudract_number:2020-005020-11
Full paper in Russian	Ozone Therapy In Patients With The New Coronavirus Infection Covid-19	Hammad, E.V.; Nikitin, I.G.; Fedorova, K.V	Bulletin of Rehabilitation Medicine, 2020, 5 (99): 94-100
Full paper in Russian	Ozone therapy as a component of a comprehensive rehabilitation program for patients after polysegmental pneumonia associated with SARS-CoV2 infection	Baranova, I. V; Gumeniuk, A. F.; Semenenko, A. I.; Iliuk, I. A.; Osypenko, I. P.	Zaporozhye Medical Journal, 2021, 23(6):752-758

Study analysis

The titles and abstracts of the 606 studies identified were analyzed. 341 duplicates were excluded, and 190 studies did not use OT for medical purposes. Subsequently, the full text of 75 pre-selected studies was read, of which only 13 met the inclusion criteria, as shown in Figure 1. Additional Tables 1 and 2 detail the included and excluded studies, respectively.

Figure 1:

Flow of OT literatures according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2009 flow diagram.

Note: OT: Ozone therapy.

Characterization of the studies

The characterization of the studies depends on the identification of the data: type of study (methodology), countries where the study was conducted, type of intervention (routes of application of OT) and the results obtained.

Size of points on the bubble chart

According to the number of studies found, the “Result” versus “Routes of administration” items received different point sizes. The larger points were related to three or more studies, the medium points were related to two studies and the small points were only related to one study.

Bubble chart color

Circles located at the intersections between “interventions” and “outcomes” items represent the identified studies and quantity. The color of the circle represents the study design: green for randomized clinical trials, yellow for non-randomized clinical trials and quasi-experimental clinical studies, and red for case reports and case studies.

Administration routes

The routes of administration were identified in the 13 studies (Table 2).

Table 2:

Routes of administration of the ozone therapy

Routes	Method
Intravenous systemic (venipuncture for collection of the blood, followed by reinfusion of ozonated blood with oxygen-ozone gas mixture using the same venous access)	Major autohemotherapy
Systemic by intramuscular administration (venipuncture for collection of the blood, followed by injection of ozonated blood with oxygen-ozone gas mixture intramuscularly)	Minor autohemotherapy
Intestinal systemic (rectal oxygen-ozone gas mixture insufflation)	Rectal insufflation
Parenteral ozonated saline solution	Intravenous

Outcomes mentioned in scientific studies

The main topics observed in the original articles were analyzed following:

A) Clinical improvement: (1) General improvement of clinical symptoms of COVID-19; (2) Improvement of cough; (3) Reduction of body temperature; (4) Improvement of shortness of breath; (5) Improvement of respiratory function; (6) Improvement of oxygen saturation; (7) Early weaning from oxygen support; (8) Reduction in the need for invasive mechanical ventilation; (9) Faster recovery from acute respiratory distress syndrome; (10) Prevention of clinical worsening; (11) Reduction of mortality.

B) Hospitalization: (1) Reduction of hospital stay; (2) Reduction in the need for admission to the intensive care unit.

C) Inflammatory/thromboembolic/infectious/metabolic markers: (1) Decrease in C-reactive protein (CRP);(2) Reduction of ferritin; (3) Decreased lactate dehydrogenase (LDH); (4) Decreased fibrinogen; (5) Decrease in interleukin (IL) 6; (6) D-dimer decrease; (7) Leukocyte normalization; (8) Normalization of lymphocytes; (9) Decrease in procalcitonin; (10) Decreased neutrophil-lymphocyte ratio; (11) Normalization of alanine aminotransferase or glutamate-pyruvate transaminase.

D) Radiological aspects: Radiological improvement of lung lesions.

E) Viral infection: (1) Reduction of viral load; (2) Faster reverse transcriptase polymerase chain reaction negative.

F) Adverse events: (1) Discrete meteorism (gas sensation); (2) Epistaxis associated with the concomitant use of heparin; (3) Improvement of refractory hypoxemia; (4) Mild pain at the injection site and headache.

RESULTS

The results of existing evidence and gaps in the effects of OT for the treatment of COVID-19 are shown in the colored bubble graph (Figure 2). The colored bubble chart summarizes the results of the 13 selected studies (date of survey: March 15, 2022).

Figure 2:

Medical evidence of OT and EGM in the COVID-19.

Note: COVID-19: Coronavirus disease 2019; EGM: evidence and gaps map; OT: ozone therapy.

All included studies were primary studies, being case reports (n = 6), randomized clinical trials (n = 2), case and control studies (n = 2), quasi-experimental clinical study (n = 1), observational study (n = 1) and non-randomized clinical trial (n = 1). Publications occurred in 2022. The countries that carried out the studies were: Spain (n = 5), China (n = 2), Italy (n = 2), India (n = 2), Iraq (n = 1) and not identified (n = 1).

The 13 selected clinical studies, involving a total of 271 patients with COVID-19 that received OT showed positive results, both in mild to severe cases, being mild to moderate in 3 studies, moderate to severe in 1 study, mild to severe in 1 study, moderate in 1 study, severe in 7 studies. The outcomes found were: general improvement of clinical symptoms of COVID-19 (13 studies); improvement of cough (2 studies); reduction of body temperature (3 studies); improvement of shortness of breath (2 studies); improvement of respiratory function (6 studies); improvement of oxygen saturation (6 studies); early weaning from oxygen support (2 studies); reduction in the need for invasive mechanical ventilation (3 studies); faster recovery from acute respiratory distress syndrome (2 studies); prevention of clinical worsening (4 studies); reduction of mortality (2 studies); reduction of hospital stay (6 studies); reduction in the need for admission to the intensive care unit (2 studies); decrease in CRP (10 studies); reduction of ferritin (7 studies); decreased LDH (8 studies); decreased fibrinogen (2 studies); decrease in IL-6 (6 studies); D-dimer decrease (8 studies); leukocyte normalization (1 study); normalization of lymphocytes (2 studies); decrease in procalcitonin (1 study); decreased neutrophil-lymphocyte ratio (1 study); normalization of alanine aminotransferase or glutamate-pyruvate transaminase (1 study); radiological improvement of lung lesions (7 studies); reduction of viral load (1 study) and faster reverse transcriptase polymerase chain reaction negative (1 study). The information compilation of the 13 articles is shown in Additional Table 3.

Clinical improvement

Hospitalization

Inflammatory/thromboembolic/infectious/metabolic markers

Radiological aspects

Viral infection

Adverse events

General improvement in clinicalsymptoms of COVID-19

Improvement of cough

Reduction of body temperature

Improvement of shortness of breath

Improvement of respiratory function

Improvement of oxygen saturation

Early weaning from oxygen support

Reduction in the need for invasivemechanical ventilation

Faster recovery from acute respiratorydistress syndrome (ARDS)

Prevention of clinical worsening

Reduction of mortality

Reduction of hospital stay

Reduction in the need for admission tothe intensive care unit (ICU)

Decrease in C-reactive protein

Reduction of ferritin

Decreased lactate dehydrogenase

Decreased fibrinogen

Decrease in interleukin 6

D-dimer decrease

Leukocyte normalization

Normalization of lymphocytes

Decrease in procalcitonin

Decreased Neutrophil-LymphocyteRatio

Normalization of alanineaminotransferase

Radiological improvement of lunglesions

Reduction of viral load

Faster RT-PCR negative

Presence

Absence

Unmentioned

Study

Name

Country

Therapy

Type of study

Number of hospitalized patients who received Ozone Therapy

Pneumonia severity level

A

Wu et al. (2020)*

China

GAHT, 40 mcg / ml, 100 ml, 1: 1, once a day on the first day and twice a day on the second to the fifth day, for 5 days, totaling 9 sessions

Report of a case

1

Severe

X

X

X¶

X

X

X

X

X

X

X

X

B

Zheng et al. (2020)*

China

GAHT, 20 mcg / ml, 100 ml, 1: 1, once a day for 7 days, totaling 7 sessions

Report of a case

2

Severe

X

X

X

X

X

X

X

X

C

Franzini et al. (2020)

Italy

Clinical trial

50

Moderate

X

X

X

X

X

X

X

X

X

X

X

D

Hernández et al. (2020)*

Spain

GAHT, 40 mcg / ml, 200 ml, 1: 1, 2 timer a day, for 3 days, totaling 6 sessions

Report of a case

3

Severe

X

X

X

X

X

X

X

X

X

X

X

E

Tascini et al. (2020)

Italy

GAHT, 40 mcg / ml, 200 ml, 1: 1, once a day, for 3 days, totaling 3 sessions

Cases and cortrols study

30

Mild to moderate

X

X

X

X

X

F

Hernández et al. (2021)

Spain

GAHT, 40 mcg / ml, 200 ml, 1: 1, 2 times a day for 5 days, totaling 10 sessions

Prospective case-control study

Severe

X

X

X

X

X

X

X

G

Fernández-Cuadros et al.(2020)*

Spain

IR, 35 mcg / ml, 100 ml, once a day for 5 to 10days, for a total of 5 to 10 sessions

Quasi-experimental prospective cohort study

Severe

X

X

X

X

X

X

X

X

XΔ

H

Pena-Lora et al. (2020)*

Spain

IR, 35 mcg / ml, 100 ml, once a day for 5 days, totaling 5 sessions

Report of a case

1

Severe

X

X

X

X

X

X

X

X

X

X

X

I

Shah et al. (2020)

India

1st) IR, 40 mcg / mL, 150mL, 2 times a day for9 days, totaling 18 sessions 2nd) PAHT 25mcg /mL, 5mL of O3 and 2 to 3 ml of blood, once aday for 9 days, totaling 9 sessions

Randomized clinical trial

30

Mild to moderate

X

X

X

X

X

X

X¤

X¤

X¤

X

X

J

Razzaq et al. (2020)*

Iraq

SFO3, 35-45mcg / mL, 500mL, 3 to 5 minutes of preparation in continuous bubbling, infusion in15 minutes, 1 to 2 times a day, for 5-10 days.

Clinical trial

104

Moderate to severe

X

X

X

X

K

Schwartz et al. (2020)

Spain

SFO3, 3-5mcg / mL, 200mL, 10 minutes of preparation in continuous bubbling, infusion of15 to 30 minutes, once a day, for 10 days.

Non-randomized clinical trial

35

Severe

X

X

X

X

X

X

X

X

X

X

X

X

X

J

Sharma et al. (2021)

India

SFO3. 5mcg / mL, 200mL, 20 minutes of preparation in continuous bubbling, infusion of 1hour, once a day, for 8 days.

Clinical trial

10

Moderate

X

X

X

X

X

X

X

X

X

X

X

X

X

XΦ

L

Hendawy et al. (2021)

Egypt

1) IR, 12.6 mcg / mL, 2 liters, two sessions2) IR, 12.6mcg/mL, 2 liters, one session

Report of a case

3

Mild and severe

X

X

X

X

X

X

X

Total Results

13

2

3

2

6

6

2

3

2

4

2

6

2

10

7

8

2

6

8

1

2

1

1

1

7

1

1

3

6

4

Note: * Studies without statistics; Δ Discreet weather, gas sensation; . Epistaxis associated with the use of heparin; ¤ Falling of inflammatory markers, without significant statistics; ¶ Improvement of refractory hypoxemia; Φ Mild pain at the injection site and headache.

The most reported type of intervention was major autohemotherapy (MAH), followed by rectal insufflation (RI). The reported outcomes were: general improvement in clinical symptoms of COVID-19 (n = 13), improvement of respiratory function (n = 6), improvement of oxygen saturation (n = 6), reduction in hospital stay (n = 6), decrease in CRP (n = 10), decrease in ferritin (n = 7), decrease in LDH (n = 8), decrease in IL-6 (n = 6), decreased D-dimer (n = 8), radiological improvement of lung lesions (n = 7) and absence of reported adverse events (n = 10). The concentration of medicinal ozone in the MAH ranged from 20 to 45 μg/mL, with 40 mcg/mL being the most used concentration, and the number of sessions was 3–10. For the RI route, a concentration of 12.6 to 40 μg/mL was used, with the concentration of 35 μg/mL being the most used, and the average of sessions was only one session.

DISCUSSION

The best findings of this work are high evidence (randomized clinical trials) for MAH in decreasing ferritin, D-dimer, IL-6, LDH, procalcitonic, normalization of alanine aminotransferase, improved respiratory function, faster acute respiratory distress syndrome recovery, improved oxygen saturation and improves symptoms of COVID-19. OT can be beneficial in reducing the length of hospital stay and decreasing inflammatory markers. RI results in negative reverse transcriptase polymerase chain reaction, breathlessness relief, cough relief, prevention of clinical worsening and decrease in clinical symptoms of COVID-19.

The advantages of OT in COVID-19 are:

• General improvement in oxygen metabolism;

• Increasing oxygenation and gas exchange in the lungs and peripheral tissues;

• Restoring the balance of the cellular redox state;

• Increasing the synthesis of intracellular antioxidant enzymes;

• Promoting interferon-gamma induction;

• Reducing pro-inflammatory cytokines;

• Increasing the effectiveness of antigen-antibody dynamics;

• Having antiviral action, vasodilation, increase of tissue perfusion, modification and attenuation of the viruses’ structural conformation, favoring the immune response;

• Increasing hemorheological, reducing the aggregation of red blood cells, decreasing blood viscosity and optimizing blood flow;

• Favoring the regeneration of injured tissues, due to the release of growth factors and obtaining a feeling of well-being, as reported by most patients, through the activation of the neuroendocrine system.

OT can improve hemorheological properties,[19] favoring blood flow,[20] reducing erythrocyte aggregation and systemic and erythrocyte oxidative stress[21] in patients with COVID-19.

COVID-19 patients are also likely to have mild nonspecific hepatitis[22,23] and pulmonary fibrosis.[23] OT stabilizes hepatic metabolism and plasma fibrinogen and prothrombin levels tend to normalize in infected patients, suggesting an improvement in hepatic protein synthesis.[24]

There is also a demonstration that OT protects against oxidative damage to the lung,[25] kidney,[26] heart,[27] intestine,[28] muscles[29] and brain.[30]

OT causes an increase in the glycolysis rate of red blood cells, which produce more ATP. This leads to the stimulation of 2,3-diphosphoglycerate, which can shift the oxyhemoglobin dissociation curve to the right, increase the partial pressure of arterial oxygen and decrease the partial pressure of venous oxygen called Bohr effect, which generates an increase in the amount of oxygen released to the tissues. This effect improves the oxygen supply to ischemic tissues. OT also improves oxygenation and gas exchange in the lungs and peripheral tissues due to vasodilation provided by nitrosothiols, activation of nitric oxide synthase (with increased production of nitric oxide, a potent vasodilator) and produces more red blood cells because of better functioning of the Na⁺/K⁺ ATPase pump.[31] Ozone can improve the circulation and perfusion of the lung and all organs in a state of hypoxia, as it improves oxygen metabolism.[32]

In summary, the mechanisms of action of OT applicable to the main complications of COVID-19 are based on: (1) General improvement in oxygen metabolism, increasing oxygenation and gas exchange in the lungs and peripheral tissues. (2) Restoring the balance of the cellular redox state. (3) Increased synthesis of intracellular antioxidant enzymes. (4) Promotion of interferon-gamma induction (main endogenous humoral mechanism of antiviral control). (5) Reduction of pro-inflammatory cytokines. (6) Increasing the effectiveness of antigen-antibody dynamics. (7) Antiviral action. (8) Vasodilation and increased tissue perfusion. (9) Modification and attenuation of the structural conformation of virions, favoring the antiviral immune response (“autovaccine”). (10) Hemorheological improvement, reducing the aggregation of red blood cells, decreasing blood viscosity, and optimizing blood flow. (11) Regeneration of injured tissues, due to the release of growth factors. (12) Obtaining a feeling of well-being, as reported by most patients, through the activation of the neuroendocrine system.

The most widely used administration routes for OT are MAH, minor autohemotherapy (MiAH) and RI, described in the clinical studies for COVID-19.[33,34,35,36,37,38,39,40,41] In MAH, approximately 250 mL of blood is collected from the participant and gently homogenized with the same amount of oxygen-ozone mixture, and then injected again into the participant intravenously. In MiAH, a smaller volume of blood (5 mL) is taken from the participant, homogenized with the same amount of the oxygen-ozone mixture, and the application is made intramuscularly.[31] In the pathophysiological context of thrombogenesis of COVID-19, in which there may be diffuse occlusive arterial disease, we find another potential applicability of OT. Clinical practice has already consolidated in the treatment of peripheral arterial occlusive disease. MAH is useful in improving both the rheological properties of blood and oxygen release in ischemic tissues,[19] in addition to the dose-dependent vasodilator mechanism related to the production of prostacyclin.

The OT used by RI has local and systemic action, since the gas is quickly dissolved in the contents of the intestinal lumen, where there are mucoproteins and other secretion products with a strong antioxidant activity, so that it reacts quickly and produces reactive oxygen species and lipid peroxidation products, which, once generated, are absorbed and pass into the systemic circulation, being able to promote therapeutic effects. To avoid damage to the rectal mucosa, it is not convenient to reach a concentration above 40 μg/mL in the RI. The volume of the gas mixture to be applied is variable, depending on the disease and the different institutions of OT, which varies between 100 to 500 mL per session for adults. RI is able to achieve similar results to MAH, but it needs a high number of sessions and a higher dose.[42] RI was already used in the treatment of patients infected with Ebola, obtaining a significant improvement after 5 sessions, without deaths.[43] It was believed that this benefit could be extrapolated to patients with COVID-19.[44] In a case-control study evaluating 14 patients with COVID-19 severe bilateral accompanied by pneumonia, the patients were treated with standard of care plus OT via RI, for 10 days. The OT protocol consisted of 8 sessions (1 session per day), at a volume of 150 mL and a concentration of 35 μg/mL. The standard of care protocol included oxygen delivery, antivirals (remdesivir), corticosteroids (dexamethasone/methylprednisolone), monoclonal antibodies (anakinra/tocilizumab), antibiotics (azithromycin) and anticoagulants (enoxaparin). The lymphocyte count improved statistically in the OT group (P < 0.05). Inflammation biomarkers (fibrinogen, D-dimer, urea, LDH, CRP and IL-6) were decreased in both groups, but only significantly in the OT group (P < 0.05). Ferritin showed a significant decrease in the OT group and an increase in the standard of care group. Pneumonia decreased in both groups, but it was significant only in the OT group (improved Taylor’s radiological score) (P < 0.0001). Mortality and length of stay, although not significant, were lower in the OT group. Therefore, OT via RI improved oxygen saturation, decreased inflammation biomarkers and improved Taylor’s radiological scale in the treatment of COVID-19.[45]

The precise mechanism of COVID-19 disease is not completely defined; however the link between mitochondria and viruses is known. The virus SARS-CoV-2 silences the body’s innate inflammatory response and does so by diverting mitochondrial genes from their normal function. SARS-CoV-2 has been shown to reduce levels of a group of mitochondrial proteins, known as Complex One, which are encoded by nuclear DNA. This effect can "calm" the cell's metabolic production and the generation of reactive oxygen species, which, when functioning properly, produce an inflammatory response that can kill the virus.[46] It has also been observed that the SARS-CoV-2 virus can be even more complex, suggesting that in the peripheral blood mononuclear cells of patients with COVID-19, the virus deliberately manipulates the metabolic functions of mitochondria to its own benefit. Ajaz et al.[47] found increases in the fibroblast growth factor 21 mitokine and in glycolysis. Since mitochondria play a key role in the initiation and development of the cytokine storm, specific mitochondrial pathways in immune cells can be targeted clinically.

Mitochondria have a high sensitivity to oxidative damage, caused by free radicals that can compromise their structure and function, reducing their activity.[48] Systemic OT can activate the action of mitochondria.[49]

The pathogenesis of COVID-19 is also associated with a hyperinflammatory response. The protein spike (S) potently induces inflammatory cytokines and chemokines, including IL-6, IL-1β and tumor necrosis factor α. Protein S triggers inflammation by activating the nuclear factor κB pathway in a MyD88-dependent manner.[50] OT is able to inhibit the NF-κβ gene,[51] a pathway that would play an important role in stimulating hyperinflammation or cytokine storm.[52] These findings would explain why inflammatory variables such as ferritin, IL-6 and CRP decrease in patients with COVID-19 treated with OT via RI.[37]

The detection and control of the pro-inflammatory response are crucial in the early stages of COVID-19 and IL-6 is one of the main pro-inflammatory cytokines. The control of systemic levels of IL-6 in patients infected with SARS-CoV-2 may signal an improvement in the disease.[53] Systemic OT can decrease IL-6 and other pro-inflammatory cytokines.[54] It has also been measured in vivo that the release of numerous cytokines from ozonated blood, at a concentration between 30 and 55 μg/mL, causes an increase in interferon production and less production of tumor necrosis factor α[55] and IL-2.[56]

OT is also associated with the production of interferon alpha, beta and gamma, as well as ILs, granulopoietin, and in the transformation of growth factor transforming growth factor β and possibly other proteins are also stimulated.[31] These changes can be observed for days after ozonation, suggesting that when leukocytes are activated by OT, they migrate to lymphoid environments where the release of cytokines triggers the activation of other immune cells, reactivating the depressed immune system.[57]

The activation of nuclear factor erythroid 2-related factor 2 can also be a strategy against COVID-19, since its action promotes the resolution of inflammation and, in parallel, restores cellular redox and protein homeostasis, and favors tissue repair. The possible benefits of pharmacological activation of nuclear factor erythroid 2-related factor 2 in the context of SARS-CoV-2 infection are: to increase fitness and provide protection to the host cell; promote the anti-inflammatory phenotype during the activation of macrophages, avoiding the uncontrolled production of pro-inflammatory cytokines and pyroptosis; and inhibit viral spread.[58] They may, as a result, be important in mitigating the severity of COVID-19, acting through endoplasmic reticulum stress or angiotensinconverting enzyme-angiotensin-II-ATVia of axis 1 receptor.[59]

OT also modulates the nuclear factor erythroid 2-related factor 2 system[60] producing three effects. First, it normalizes the redox balance by increasing antioxidants in the cytoplasm, in the mitochondria and finally in the plasma, mainly glutathione peroxidase, but also glutathione reductase, NADPH and superoxide dismutase. Second, it induces the production of heme oxygenase-1, a protective enzyme, along with heat shock proteins (HSPs) like HSP-60, HSP-70 and HSP-90.[61] Third, it activates the nuclear factor κB system, which modulates the production of pro-inflammatory ILs in damaged tissues.[62] All effects contribute to restoring the normal functioning of inflamed tissues and decreasing the plasma ILs.

HSPs also play a role at different levels in viral infections. They can regulate viral infections through interaction with the virus, including cell entry and nuclear import, viral replication and gene expression, viral protein folding/assembly, regulation of apoptosis, and host immunity. They are also effective carrier molecules for cross-presentation to antigen-presenting cells. Thus, HSPs and especially HSP70 can increase the development of innate and adaptive immune responses against infectious agents.[63] Mortality in a rat model with sepsis-induced acute respiratory distress syndrome) has been reduced by overexpression of HSP70 chaperone adenovirus. A natural increase in body temperature during low fever can naturally accumulate high cellular levels of HSP70 that can stop apoptosis and protect alveolar lung cells from inflammatory damage. However, in addition to 1–2 hours of fever, no HSP70 is being produced and a decrease in body temperature is needed to restore the cell’s ability to produce more HSP70 in a subsequent fever cycle. Therefore, it is suggested that antipyretics may be beneficial in patients with COVID-19, allowing lung cells to accumulate protective HSP70 against damage from the inflammatory response to the SARS-CoV-2 virus.[64]

OT can also induce HSP70.[32] The confluence of human vascular endothelial cells was challenged with different concentrations of these inducers and the production of nitric oxide (NO); and heme oxygenase-1 was measured by nitrite or bilirubin formation, or/and protein immune reactivity by Western blot using a rabbit anti-human heme oxygenase-1 and HSP70. The production of NO and heme oxygenase-1 was quite dose-dependent and it was particularly high using human plasma after exposure to ozone. The HSP70 induction was also observed. Therefore, the results clarify that OT can also induce HSP70. OT also increased the length of the mitochondrial ridges and the content of the mitochondrial HSP70.[65]

An important source of cytokines and chemokines are mast cells, which are ubiquitous in the body, especially in the lungs, and are essential for allergic and lung diseases.[66] Activated mast cells were recently detected in the lungs of deceased patients with COVID-19 and were associated with pulmonary edema, inflammation and thrombosis.[67] Hyperinflammation was also observed in patients with COVID-19 and post-COVID-19, suggesting that this hyperinflammation is linked to mast cell activation syndrome.[68]

Mast cells are typically activated by allergic triggers, but they can also be triggered by pathogen-associated molecular patterns via the activation of Toll-like receptors. In addition, mast cells express the renin-angiotensin system, the ectoprotease angiotensin-converting enzyme 2 required for SARS-CoV-2 binding, and serine proteases, including transmembrane protease serine 2, required for priming of the corona spike protein.[65] Such triggers could lead to the secretion of multiple proinflammatory mediators selectively, without the release of histamine or tryptase.[69]

In addition to the proinflammatory cytokines and chemokines, activated mast cells could release matrix metalloproteinases and transforming growth factor beta, which could contribute to lung fibrosis, including thromboxanes and platelet-activating factor, leading to the recently reported microthromboses in the lungs of deceased patients with COVID-19.[67] Moreover, mast cells communicate with endothelial cells, fibroblasts, and macrophages, further stimulating the release of proinflammatory, fibrotic, thrombogenic, and vasoactive mediators.

Peden and Dailey[70] showed the immunoregulatory action of OT on mast cells in vitro and verified that during OT, the plasma histaminopexic and serotoninopexic activity increased. These findings can be explained by the elimination of histamine and serotonin from the body.

For the prescription of a medication, by a qualified professional, but for an unapproved indication, and therefore, that is not included in the package leaflet, this practice is called off-label use. The definition for off-label by the Brazilian National Health Surveillance Agency (ANVISA) is a use in situations that differ from the package insert for a drug registered. It may include differences in indication, age/weight, dose, frequency, presentation, or route of administration. In Brazil, off-label use is not illegal, and since it is supported by robust scientific evidence and the absence of a therapeutic alternative, it is considered legitimate or even necessary when considering the clinical situation that demanded it.

In view of the seriousness of the medical-political-social and economic situation and in the absence of known treatments for COVID-19, some treatments are being researched and used as off-label (hydroxychloroquine, tocilizumab, invermectin, colchicine, remdesivir, etanercept, corticosteroids, antibiotics, convalescent heterologous plasma, immunoglobulins, antivirals and immunomodulators), however, bring with them, to a greater or lesser degree, a high cost, be it financial or a potential adverse effect. OT is a feasible health resource, if judiciously practiced, it can achieve excellent therapeutic results associated with a very low rate of complications and risks.[71]

In our study, we verified the presence of very few adverse events, which were: mild meteorism (sensation of gas) and epistaxis that was associated with the use of heparin. These results agree with the Jacobs’s study[72] which tabulated data from nearly 5.6 million OT treatments and found only 0.0007% risk of complications and 0.0001% risk of death.

OT can also be used in patients after COVID-19,[73] evaluated the effects of nutritional supplementation and RI with ozone. The 40 patients received nutritional supplementation capsule (400 mg) daily or 2 capsule nutritional supplementation plus RI with ozone daily during 1 month. Twenty healthy patients were enrolled as controls. Improvements in symptoms of COVID-19 were observed in both groups, resulting in 85% of patients for nutritional supplementation plus RI with ozone and 37% for nutritional supplementation (P < 0.05). The symptoms that persist in COVID-19 were reduced and no side effects were observed. These results corroborate that substantial oxidative stress and symptoms persist in COVID-19 powders can be improved by the treatments of RI with ozone plus nutritional supplementation.

Our EGM has some limitations such as the fact that we only used published studies to provide an overview of the research and that no further evidence was included. We did not calculate effect sizes in a Meta-analysis, nor provide risk of bias assessments, but tried to overcome these limitations by relying on the authors’ skills in conducting and evaluating the study quality, choice of outcomes, analysis of effects and susceptibility to publication and outcome reporting bias. We suggest that future studies should adopt health and economic impact assessments for health services, as well as robust methodologies for evaluating clinical trials.

The EGM showed that all routes of administration (MAH, MiAH, RI) have high evidence levels in some outcomes. The MAH presented high evidence levels for the improvement of clinical symptoms of COVID-19, of oxygen saturation, in the recovery from acute respiratory distress syndrome, in the respiratory function, and decrease D-dimer, CRP, IL-6, procalcitonin, LDH. MAH also normalized ALT levels. The high evidence levels for MiAH were found for the decrease of LDH, CRP and ferritin. And finally, RI improves clinical symptoms of COVID-19, including cough, prevents clinical worsening and shortness of breath, and faster reverse transcriptase polymerase chain reaction negative.

In this way, the main high evidence levels were found with the use of MAH route. MAH was able to improve the clinical signs of the patients and inflammatory markets. The EGM demonstrates in a preliminary way (13 articles) that OT can be beneficial in reducing the length of hospital stay and decreasing inflammatory markers with few adverse events. These benefits are seen in patients at a mild and/or moderate stage of the disease and may accelerate recovery to avoid the entry into severe stages of COVID-19.

Author contributions

Conceptualization: MEGS, MML, APA; methodology, software, formal analysis: CVMA; validation: MEGS, APA; investigation: MEGS, JBN, CDB, APA; resources: MEGS, CDB, APA; data curation: MEGS, CVMA, CDB, APA; writing – original draft: MEGS, MML, CDB, APA; writing – review & editing: MEGS, JBN, CDB, APA; visualization: MEGS, JBN, MML, CVMA, DFS, CDB, APA; supervision: CDB, APA; project administration, and funding acquisition: MEGS. All authors approved the final manuscript for publication.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Open access statement

This is an open access journal, and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Additional files

Additional Table 1: List of included studies.

Additional Table 2: List of exclusion studies.

Additional Table 3: Methodology and outcomes observed in the 13 final article.