Preprints (www.preprints.

org) | NOT PEER-REVIEWED | Posted: 3 July 2020

Research Article
Complementary Application of the Ozonized Saline Solution in
Mild and Severe Patients with Pneumonia Covid-19
Adriana Schwartz. 1 Gregorio Martí
nez-Sánchez. 2,* Alejandra Menassa de Lucí
a.3 Sergio Mejí
a
4
Viana. Alina Mita Constanta. 5

1. Director Fiorela Clinic, Pain Management Unit, Madrid (28035), Spain; adriana@clinicafiorela.com
2. Scientific advisor, Freelance, Ancona (60126), Italy. gregorcuba@yahoo.it
3. Integrative Medicine Clinic, Madrid (28002), Spain; alejandra.menassa@medicinaintegrativa.com
4. Cardiologist at Cardio Care of Hospital High Care Marbella and Hospital CHIP of Malaga (29010), Spain;
info@drsergiomejiaviana.com
5. Emergency physician at Hospital Virgen De La Paloma, Madrid (28003) Spain; constanta_alina_m@hotmail.com
* Correspondence: gregorcuba@yahoo.it
Received: date; Accepted: date; Published: date

Abstract
Currently, there is no effective antiviral therapy recommended for the new coronavirus
disease 2019 pneumonia (COVID-19). The purpose of this pilot study was to evaluate the
safety of Ozonized Saline Solution (O3SS) used as a complementary therapy in adult patients
COVID-19. Twenty-five adult patients who were hospitalized with mild to severe COVID-
19 symptoms, who met the inclusion criteria and were being treated from April 3rd to April
26th, 2020, at the Viamed Virgen De La Paloma Hospital, Madrid, Spain were included in
this study. Patients were allocated to receive standard care (SC) that included 200-400 mg
hydroxychloroquine twice daily for 5-7 days plus Tocilizumab 400 mg twice daily for 5 days,
low molecular weight heparin (LMWH) and 40 mg-60 mg metil-prednisone plus O3SS, 200
mL, 3-5 µg/mL daily for 10 days. Non control group was included, data were compared to
clinical trials in this subject. Primary outcomes of treatment with O3SS were an improvement
of clinical symptoms and a reduction in mortality. Secondary end points evaluated included
participant clinical status, laboratory examinations, and duration of viral shedding. None of
the patients treated with SC + O3SS died. Improvements in symptoms such as dyspnea,
weakness, and reduction in body temperature were observed and corresponded with an
improvement of laboratory finding including D-dimer, fibrinogen, LDH, and CRP. No side
effects from the O3SS treatment were observed. Conclusions: COVID-19 patients with mild
to severe symptoms who received intravenous O3SS as a complementary therapy
demonstrated no side effects. This preliminary data will be served as base for a future study
of the efficacy of this therapy.

Keywords: ozone therapy; ozonized saline solution; SARS-CoV-2; COVID-19; pneumonia.

1. Introduction
A coronavirus named Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2)
was isolated as the causative agent of severe pneumonia. This infections has been termed
Coronavirus Disease 2019 (COVID-19) by the World Health Organization (WHO) who
officially declared COVID-19 a pandemic on March 11, 2020 [1]. COVID-19 rapidly spread
into at least 213 countries and killed more than 500,000 people by June 30, 2020. There are
not specific therapies available to treat the Covid-19 infection.

© 2020 by the author(s). Distributed under a Creative Commons CC BY license.

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Hydroxychloroquine (HCQ) and chloroquine (CQ) have garnered unprecedented

attention as potential therapeutic agents against COVID-19. Apart from their antimalarial
use, they have also shown an in vitro activity against COVID -19 [2]. However, there is a
growing body of scientific data of their side effects particularly in QTc prolongation and
cardiac arrhythmias [3].
There are multiple physiological pathway dysregulations that appear to be disrupted by
the SARS-CoV-2 and related viruses. Angiotensin-converting enzyme (ACE2) has recently
been identified as the SARS-CoV-2 receptor. The ACE2 system is a critical protective
pathway from inflammatory injuries due to excess oxidative stress. An unregulated ACE2
dysfunction worsens COVID-19 and could initiate multi-organ failure. The imbalance in the
action of ACE1 and ACE2-derived peptides [Angiotensin II (Ang II) and Angiotensin 1-7
(Ang 1-7), respectively] may explain most of the pathological consequences of SARS-CoV-
2 infections [4].
Health risk factors that predispose COVID-19 patients for a progression of the disease
to more advanced stage include proinflammatory conditions such as hypertension, diabetes
and cardiovascular disease [5]. Earlier studies of the related SARS-CoV virus infections of
primates suggest that the severe lung injury was due to an exacerbated inflammatory
response mediated through an activation of the innate immune system and an upregulation
of the NF-κB pathway [6]. A recent human study demonstrated that in critically ill patients,
the SARS-CoV-2 virus cause an exacerbated inflammatory response which is not self-
limiting, it is uncontrolled generating an inflammatory cytosine storm. This is happening
because, in theory the lymphocytes NK and T cytotoxic (CD8 and CD4+) should inhibit the
macrophage activity, but by down regulation this inhibition does not occur because the
infected cells lack Major Histocompatibility Complex, who should inhibit the action of the
macrophage. This is caused a suppression of functional lymphocytes (lymphopenia) resulting
in decreased immune function and increased susceptibility to infection [7]. Additional animal
studies have demonstrated that the pulmonary fibrosis that is a hallmark of the SARS disease
process is mediated through an induction of TGF-beta1 by way of an upregulation of the
ROS/ p38 MAPK/ STAT3/Egr-1 pathway both in vitro and in vivo [8]. Host genetics may
also play a role in pathogenesis since studies on knock-out mice have demonstrated that a
genetic deficiency of ACE2 receptors resulted in a reduction of Ang 1,7 thus increasing
oxidative stress and susceptibility to advanced disease progression [9].

Medical ozone (O2/O3) at a low dose which is produced by a mixture of oxygen (carrier)
and ozone (active component) in a carrier of 99.9% pure oxygen. Medical ozone therapies
(O3X) have been demonstrated to be effective in treating a range of human pathologies that
have a physiological basis of inflammatory dysregulation (oxidative stress). Properly dosed
and timed treatments have the ability to induce endogenous oxidative preconditioning [9].
Potentially, O3X may improve the symptoms of COVID-19 acting as an inductor of
adaptation to OS, a modulator of pro-inflammatory cytokines and improving tissues
oxygenation [10]. A preliminary case report shown the benefit of the treatment with ozone in
two COVID-19 patients in China [11] also in 18 patients treated in Ibiza (Spain) as part of a
single-center prospective cohort study [12]. These findings allowed us to evaluate the safety
and efficacy of ozonized saline solution (O3SS) in patients with mild to severe COVID-19
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as a complementary therapy. Here, we report data from 25 patients who received standard
care (SC) plus O3SS. The results suggest that O3SS as a complementary therapy accelerates
the recovery of the patients, stabilizes their biochemical index, reduces the need for oxygen
support and shows no side effects.

2. Materials and Methods

The complementary application of O3SS was done in accordance with the principles of the
Declaration of Helsinki [13] and the Good Clinical Practice Guidelines of the International
Conference on Harmonization [14]. All patients and/or legal representatives were informed
about the objectives and risks of participation. They were given time to carefully read and
sign the informed consent form. Random online clinical monitoring and quality control were
performed. A virtual independent data safety and monitoring board (DSMB), composed of
O3x experts, clinicians, and experts in infectious diseases from AEPROMO (Spanish
Association of Medical Professionals in Ozone Therapy) and ISCO3 (International Scientific
Committee of Ozone Therapy), was selected to review the protocol and hold daily meetings
to follow the daily results of the application of O3SS. The trial was reported according to the
Consolidated Standards of Reporting Trials (CONSORT) reporting guideline [15]. The
protocol was approved by the Regional Ethics Committee of Madrid (Number 05/20). This
manuscript is a partial report (pilot study) of the full study involved the comparison between
two parallel groups SC alone and SC plus O3SS. Full study will be performed after
administrative approved, using the pilot study experience. Results will be reported in a future
manuscript.
2.1. Design and Site

2.1.1. Site
The complementary application of O3SS was done following the criteria of a pilot, open
label, phase III clinical trial, between April 3th to April 26th 2020, aiming to first treat
hospitalized patients with mild to severe respiratory syndrome secondary to SARS-CoV-2
infection COVID-19; and as a second aim, to assess the safety and efficacy of O3SS. These
patients were hospitalized at the Viamed Virgen de la Paloma Hospital, Madrid (declared
COVID-19 center during the epidemic). The hospital has all source documents registered in
an electronic medical recording system. Clinical analyses, laboratory examinations, and
routine Chest radiographs are also available locally.

2.1.2. Participants
Hospitalized patients with clinical suspicion of COVID-19 (i.e., history of fever and any
respiratory symptom, e.g., cough or rhinorrhea); male or female aged 18-98 years old at the
time of inclusion; within 1 week of onset; who did not participate in other clinical studies
within the last three months; willing and able to sign the informed consent for participation
in the application of O3SS; Chest radiographs confirmed pulmonary lesions (for moderate
cases); were included. Patients were enrolled before laboratory confirmation of COVID-19
by reverse transcription–polymerase chain reaction (RT-PCR Covid-19), considering that
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this procedure could delay inclusion. The flow chart (Fig. 1) presents clinical-epidemiologic
suspected cases as well as cases already confirmed by RT-PCR Covid-19.
The exclusion criteria included: Female participants who were pregnant, lactating or
planning pregnancy during the course of the trial. Patients with significant renal or hepatic
impairment or with scheduled elective surgery or other procedures requiring general
anesthesia during the application of O3SS. Participants who had participated in any clinical
trial involving an investigational product within the past 12 weeks prior to the study. Patients
with G-6PD defect (Favism). Patients who continually used immunosuppressant, or were
organ transplant recipients within the last 6 months. Patients with history of not controlled
hyperthyroidism, unstable period of severe cardiovascular disease, copper or iron
supplementation via IV or any situation that did not allow the patients safety during the study.
The patient had to be transferred to a non-participating hospital within 72 h. Patients
receiving copper or iron supplementation i.v.

39 Hospitalized patients (≥18 years) assessed for eligibility

9 Excluded
4 Did not meet
inclusion criteria
1 Declined to
25 Allocated to SC + O3SS O3SS participate

Figure 1. Study flow chart. Eligible participants were allocated to receive standard care (SC), basically: (40 mg-
60 mg methyl prednisone daily for 5-7 days plus Tocilizumab 400 mg twice daily for 5 days, low molecular weight
heparin (LMWH) and hydrocortisone) plus O3SS, 200 mL, 3-5 µg/mL daily for 10 days) plus ozonized saline
solution (O3SS), 3-5 µg/mL daily for 10 days.

Participants were allocated at the time of inclusion and were subsequently identified
throughout the application of O3SS only by their allocated number, always assigned in
chronological order. This was an open label application of O3SS.

2.3. Sample Size Calculation

The sample size calculation for this pilot complementary application of O3SS was
estimated according to the suggestion of Whitehead A.L. et al [16]. Medium sample (25
subjects) was selected for a future main trial designed with 90% power and two-sided 5%
significance.

2.4. Procedures
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According to hospital protocol, all patients meeting the same criteria of the study (i.e.,
acute respiratory distress syndrome) received intravenous ceftriaxone (250 mg – 2 g twice
daily for 7 days) plus azithromycin (500 mg once daily for 5 days), Enoxaparina (Clexane®)
40 – 60 mg daily, HCQ 200 mg, methyl prednisone 40 mg or prednisone 5 mg systematically,
starting on day 0. Tocilizumab (Actemra®), 0,4 mg twice daily for 5 days, was also prescribed
when influenza infection was suspected.
O3SS consists of bubbling and saturating 200 mL of sterile physiological solution (0.9%)
with O2/O3 mixture during 10 min, at concentrations ranging 3-5 µg/NmL. Keeping
bubbling, using the infusion set, the solution was administered i.v. (thought the basilic
brachial or cephalic veins) during (15 to 30) min. Ozonation (bubbling) was stopped when
about 50 mL of liquid remaining in the bottle [17]. Patients received SC plus O3SS. The first
5 days the bubbling concentration used was 5 µg/NmL (total dose per day 250 µg of O3),
administered daily. In the following 5 sessions, the bubbling concentration was lowered to 3
µg/NmL (total dose per day 150 µg of O3), and administered daily. Patients received 10
sessions of O3SS in total. The concentration of ozone was measured by a build in
spectrophotometer in the ozone generator (254 nm). The concentration of ozone in saline
solution during the continuous bubbling flow was calculated as ¼ of the bubbling
concentration [18]. Under this ozonisation condition it has been demonstrated that no H2O2
or HOCl appeared in relevant concentration (H2O2 not exceeding 0.0004 % [19] HOCL
concentration are less than 0.001 g/mL [20]. The ozone decomposition processes in NaCl 0.9
% aqueous solutions is not accompanied by formation of products other than oxygen [21].
Ozone was generated by a medical class IIb CE device (Ozonobaric P®, SEDECAL®,
Spain). The container that administered the solution was disposable, made of medical-grade
materials, free of phthalates and fully compatible with ozone. It had a classification as
medical devise class IIb obtained from Bexozone® (Bexen medical®, Spain). Physiological
Saline Solution (NaCl 0.9 %) from (Lab. ERN, Spain) was used.
Clinical parameters were measured daily by the routine clinical staff from day 0 to
discharge or death, and then at day 28 for discharged patients, to assess efficacy (day 7 and
14) and safety outcomes. Laboratory parameters and electrocardiograms were performed at
the clinician’s discretion. Data were recorded on the case report form and then transferred
into an electronic database (Excel®, Microsoft®), which were further validated by external
trial monitoring staff.
Dyspnea was scaled as: Grade 0, no dyspnea; grade 1, slight dyspnea; grade 2, moderate
dyspnea; grade 3, severe dyspnea; grade 4 very severe dyspnea [22]. Weakness was scaled
as: 0, paralysis; 1, severe weakness; 2, slight weakness; 3, normal strength [23].

2.1. Outcomes
Safety outcomes included adverse events that occurred during treatment, serious adverse
events, and premature or temporary discontinuation of treatment. Adverse events were
classified according to the National Cancer Institute Common Terminology Criteria for
Adverse Events. The null hypothesis was that the complementary application of O3SS in the
experimental group would have a mortality rate that was 50% lower than the mortality
reported for only SC by day 14. Thus, the primary end point was mortality by day 14.
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Secondary end points included participant clinical status, laboratory examinations, chest
radiographs on days 7 and 14, daily clinical status during hospitalization, duration of
mechanical ventilation (if applicable) and supplementary oxygen (if applicable), and the time
(in days) from treatment initiation to death. Here we present analyses until day 14, with
lethality as the primary outcome. Virologic measures included viral RNA detection was
performed daily until 2 consecutive negative values was obtained.
To evaluate the efficacy outcome the seven-category ordinal scale was used, and
consisted of the following categories: 1, not hospitalized with resumption of normal
activities; 2, not hospitalized, but unable to resume normal activities; 3, hospitalized, not
requiring supplemental oxygen; 4, hospitalized, requiring supplemental oxygen; 5,
hospitalized, requiring nasal high-flow oxygen therapy, noninvasive mechanical ventilation,
or both; 6, hospitalized, requiring extracorporeal membrane oxygenation, invasive
mechanical ventilation, or both; and 7, death [24]. Disease severity was defined as: Mild, No
signs of pneumonia on imaging; Moderate, Fever and respiratory symptoms with
radiological findings of pneumonia; Severe Dyspnea, respiratory frequency ≥ 30/min, blood
oxygen saturation ≤ 93%, PaO2/FiO2 ratio < 300, and/or lung infiltrates > 50% within 24–
48 h; Critical Respiratory failure, septic shock, and/or multiple organ dysfunction/failure
[25].

2.2. Laboratory Analysis

Hematology and biochemistry analyses were performed in automatized machines.
Samples (2 nasopharyngeal or 1 oropharyngeal swabs) were submitted to Novel Coronavirus
(2019-nCoV) Real Time RT-PCR test, using a kit from Biopath-Unilabs (France) by Cobas
z480 qPCR (Roche), with the use of LightMix Modular SARS-CoV-2 (COVID19).
Sampling did not stop when a swab at a given time point was negative. Baseline throat swabs
were tested for detection of E gene, RdRp gene, and N gene, and samples on the subsequent
visits were qualitatively detected for E gene.

2.3. Statistical Analysis

Descriptive statistics were used for demographic, laboratory, and clinical data. To assess
the safety of the SC + O3SS compared to SC, the proportion (and 95% CI) of deaths in SC +
O3SS group was compared with the historical proportion (and 95%CI) of deaths in patients
who did not use O3SS in Spain and Europe in the same period [26-28]. For qualitative
variables, χ2 tests and Fisher exact tests were performed. We used the t test or Mann-Whitney
test to compare means and medians. The Wilcoxon signed-rank test and Hodges–Lehmann
estimate was used to compare inter quantile ranges (IQR). Statistical analyses were
performed in IBM SPSS statistic version 17, and a 2-tailed P < 0.05 was considered
significant.

3. Results
3.1 Demografic and clinical characteristics
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A total of 25 patients that were allocated to the SC + O3SS group completed the study.
(Fig. 1). Some patients (4 of 25 [16%]) had COVID-19 confirmed a posteriori by reverse
transcription–polymerase chain reaction testing. The patients with initial unconfirmed
disease who had clinical and epidemiological presentation compatible with COVID-19 were
analyzed together. Overall baseline characteristics are presented in Tab. 1.
Table 1: Demographic and clinical findings of patients at baseline.1
Variable Total Men Women
n 25 11 14
Age, Median (Min-Max) years 55 (30-95) 55(30-90) 55(45-95)
Current Smoker n (%)2 2 (8) 2 (18) 0
History of drug abuse n (%) 1 (4) 1 (9) 0
Comorbidities n (%)
Hypertension 4 (16) 4 (36)* 0
Asthma 3 (12) 2 (18) 1 (7)
Hypothyroidism 3 (12) 1 (9) 2 (14)
Obesity 2 (8) 2 (18) 0
Alcohol use disorder 1 (4) 1 (9) 0
COPD 1 (4) 1 (9) 0
Rheumatic diseases 1 (4) 1 (9) 0
Raynaud's syndrome 1 (4) 0 1 (7)
Tuberculosis 1 (4) 1 (9) 0
Chronic kidney disease 1 (4) 1 (9) 0
Diabetes 1 (4) 0 1 (7)
Heart disease 1 (4) 1 (9) 0
Peripheral arterial disease 1 (4) 0 1 (7)
Oxygen therapy on admission 14 (56) 10 (90)* 4 (28)
Body temperature, °C
<37.5 12 (48) 3 (27)* 9 (64)
37.5-38.0 1 (4) 1 (9) 0
38.1-39.0 12 (48) 7 (63)* 5 (35)
Blood pressure (mm Hg)
Systolic, Mean (Min-Max) 120 (110-151) 120 (110-151) 120 (110-125)
Diastolic, Mean (Min-Max) 80 (70-90) 80 (70-90) 80 (70-80)
O2 saturation, Median (Min-Max) % 93 (80-98) 90 (80-93) 93 (83-98)
Clinical symptoms3
Weakness n (%) 21 (84) 9 (82) 12 (86)
Dyspnea n (%) 19 (76) 10 (91) 9 (64)
Dry cough n (%) 14 (56) 5 (45) 9 (64)
Anosmia n (%) 12 (48) 5 (45) 7 (50)
Polymyalgia n (%) 9 (36) 3 (27)* 6 (43)
Headache n (%) 8 (32) 2 (18)* 6 (43)
Diarrhea n (%) 6 (24) 3 (27) 3 (21)
Legend: 1. In all cases the race was white; 2. No former smoker was found; 3. Symptoms with frequency
lower that 20 % n (%): cough with phlegm 4(16); central chest pain 4(16); pharyngodynia 3(12); abdominal
distension 3 (12); abdominal colic 3 (12); flatulence 2(12); lateral chest pain 3(12); lower limb edema 1(4)
and oliguria 1(4). COPD, chronic obstructive pulmonary disease. *, significant difference (p<0.05), χ2 tests
for proportion between gender.

Baseline characteristics show an overall median (min -max) age of 44 (30 -95) years and
a predominance of women (14 [65%]). The most frequent comorbidities were hypertension
(4 of 25 [16%]), asthma (3 of 25 [12%]), hypothyroidism (3 of 25 [12%]), smoking (2 of 25
[8%]), and obesity (2 of 25 [8%]). Hypertension was more frequent in male vs female (4 of
11 [36%] vs 0). On admission, oxygen support was required in 14 of the 25 patients (56%),
which was more frequent requirement in males vs females (10 of 11 [90%] vs 4 of 14 [28%]).
Baseline body temperature was greater than 37.5 °C in 13 of 25 patients (52%); with a greater
frequency in males vs females (8 of 11 [72%] vs 5 of 14 [35%] respectively). Main presenting
clinical symptoms were weakness (21 of 25 [84%]), dyspnea (19 of 25 [76%]), dry cough
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(14 of 25 [56%]) and anosmia (12 of 25 [48%]). Polymyalgia and headache were present
more frequently in females (both 43%) than in males (27% and 18 % respectively).
Laboratory findings (Tab. 2) show borderline low levels of hemoglobin in male patients.
Increased levels of serum ferritin, fibrinogen, D-dimer, LDH, CPR, ALT and AST was found
in all patients. Serum ferritin values were significantly (p<0.05) higher in women as
compared to men and CRP was significantly (p<0.05) higher in men as compared to women.
All patients were positive for qualitative SARS-CoV-2 PCR at baseline.
Table 2: Laboratory and radiographic findings of patients at baseline.
Variable NR Total Men Women
n 25 11 14
Leucocytes count, mean ±SD (4.5 – 11) x 109 cells /L 7.00±3.68 5.97±1.86 8.30±4.97
Lymphocytes count, mean ±SD (1.0 – 4.8) x 109 cells /L) 1.49±1.64 1.35±0.52 1.67±2.46
Platelets count, mean ±SD (150-450) x 109 cells /L 287±100 270±99 309±101
Eosinophils, mean ±SD (0-0.4) x 109 cells /L 0.04±0.05 0.03±0.04 0.06±0.06
Male 138-172 g/L
Hemoglobin, mean ±SD 135±16 131±15 ↓ 140±16
Female 120-156 g/L
Male 18-350 µg/L
Serum Ferritin, mean ±SD 561±567 ↑ 335±256 ↑* 829±718 ↑
Female 18-204 µg/L
Fibrinogen, mean ±SD 2 – 4 g /L 7.6±3.2 ↑ 6.7±3.2 ↑ 8.7±3.0 ↑
D-Dimer, mean ±SD < 250 µg/L 905±769 ↑ 807±695 ↑ 1030±872 ↑
LDH, mean ±SD < 270 U/L 423±182 ↑ 333±111 ↑ 538±194 ↑
ALT, mean ±SD < 48 U/L 68±58 ↑ 50±21 ↑ 91±80 ↑
AST, mean ±SD < 42 U/L 49±22 ↑ 39±16 61±24 ↑
CRP, mean ±SD < 10 mg/L 33.7±71.0 ↑ 46.9±86.1 ↑* 9.2±9.5
Radiologic findings
GGOI Unilateral n (%) 4 (16) 1 (9) 3 (21)
Bilateral n (%) 6 (24) 3 (27) 3 (21)
Pleural effusion n (%) 5 (20) 4 (16) 1 (7)
Pulmonary auscultation
Rales Unilateral n (%) 2 (8) 1 (9) 1 (7)
Bilateral n (%) 8 (32) 5 (45) 3 (21)
Rales / Rhonchi Bilateral n (%) 3 (12) 2 (18) 1 (7)
Wheezing Unilateral n (%) 1 (4) 1 (9) 0
Disease severity
Mild disease (4)2 n (%) 6 (24) 1 (9) 5 (36)
Severe disease (5) 2 n (%) 19 (76) 10 (91) 9 (64)
Legend: 1. Viral RNA SARS-CoV-2 load in throat swabs sample; 2. Value according to seven-category
ordinal scale; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; GGOI, Ground-glass
opacity infiltration; LDH, Lactate dehydrogenase; NR, Normal Range; CRP, C-reactive protein; ↑ above the
reference range; ↓ below the reference range; disease severity was done according the criteria of Chinese
Center for Disease Control and Prevention [25]. No significant difference (p>0.05), χ2 tests was found
between data expressed as proportion; *, significant difference (p>0.05) between gender within the same
series.

The most common radiographic finding on a chest radiograph was ground-glass opacity
infiltration (Tab. 2), in 40 % of patients and pleural effusion in 20 %. Pulmonary auscultation
found rales, rales/rhonchi and wheezing sound in 56 % of the patients. A majority of patients
fit the severe disease status (76%) and 6 (24%) meet the criterium of mild disease.

3.2 Clinical outcomes

Overall mortality rate in our group patients was zero. Safety outcomes were evaluated
at 7 and 14 days. Haematological and laboratory findings did not undergo notable
modification with the application of O3SS therapy. No decrease in haemoglobin levels, or
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increased in LDH, ALT or AST compared to baseline were found. No side effects associated
with the investigational drug (O3SS) were detected. Occurrence of epistaxis was detected in
3 patients between days 3-4 of treatment with a suspension of heparin reversing the
symptoms. Progressive reduction of body temperature was observed in patient with >37.5
°C at baseline (Fig. 2a). From the day 3 a significant (p<0.05) reduction was found, at day 8,
all patients return to values of body temperature lover than 37.5 °C.

120
Patients with > 37.5 °C (%)

100

80 *

60

40 *
*
20 *

0
0 1 2 3 4 5 6 7
Time (days)
(a)

4
Range according to scale

* * *
3

2
* *
1
* * * *

0
Dyspnea Weakness
0 1 2 3 4 Time 5(Days) 6 7 8 9 10 (b)
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40.0

35.0
(CRP mg/L) 30.0

25.0

20.0

15.0
* *
10.0 *
*
5.0 * *
* * * *
0.0
0 1 2 3 4 5 6 7 8 9 10
Time (days)
(c)
Figure 2: Change from baseline in: (a) body temperature (percent from baseline and - - trendline), error bars
indicate 95% confidence intervals. * significant difference (p<0.05), χ2 tests was found between data
expressed as proportion, compared to baseline value; (b) Dyspnea and weakness. For Dyspnea [22] and
Weakness [23] score, see Material and Method; * significant difference (p<0.05) compared to baseline value
within the same series. (c) Change from baseline in C-reactive protein (CRP) * significant difference (p<0.05)
compared to baseline, values within the normal range (<10 mg/L). Error bars indicate 95% confidence
intervals.

Dyspnea and weakness was gradually reduced (Fig. 2b). From day 7 dyspnea prevalence
was reduced by 40 % (slight dyspnea) on day 14, only 1 patient (4 %) remained with this
symptom. Weakness was improved on day 7 when 86 % of the patients passed from severe
weakness to slight weakness. On day 14, 91 % of patients shifted from severe weakness to
slight weakness. The CRP values (Fig. 2c) entered into the normal range within 24 h of the
first application of the O3SS.
Serum ferritin, fibrinogen, D-dimer and LDH were progressively decreasing during the
treatment (Fig. 3a). By day 10, fibrinogen and LDH values entered the normal ranges in all
patients. ALT and AST were also decreased during this time and by the day 10 remained
above the normal range in 7 of 25 patients (28%) and 9 of 25 patients (36%), respectively.
The rate of decline in activity of ALT and AST by day 10 were 82±117 U/L and 71±65 U/L,
respectively.
Average duration of viral shedding was 8 days (IQR 6.0-11.5). None of the 25 patients
withdrew throughout the application of O3SS. The average duration of hospitalization from
inclusion to discharge was 14 days (IQR 9.5-15) (Tab. 3). Efficacy outcome based on the
seven-category ordinal scale shows at day 7 an improvement in 19 out of 25 patients (76%).
Out of these 19 patients, 17 patients (68%) shifted from 5 to 3 and 2 patients (8%) shifted
from 4 to 2 on the ordinal scale.
By the end of the treatment with O3SS (day 14) most of the patients (18 out of 25 [72%])
were in score 2 (discharge), (6 out of 25 [24%]) in score 3 (hospitalized, not requiring
supplemental oxygen), and (1 out of 25 [4%]) was admitted to the intensive care unit (ICU).
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1,500.0 15.00

Fibrinogen g/L
Serum Ferritin µ g/L
1,000.0 * 10.00 *
** **
500.0 5.00

0.0 0.00
1 6 10 1 6 10
Time (Days) Time (Days)

(a) (b)

1500.00 600.00
D-Dimer µ g/L

LDH U/L
1000.00 * 400.00 *
**
500.00 200.00
0.00 0.00
1 6 10 1 6 10
Time (Days) Time (Days)

(c) (d)

Figure 3: Follow-up of biochemical findings. (a) Serum ferritin; (b) Fibrinogen; (c) D-Dimer;
(c) LDH. Values represented a mean ± S.E.M. The baseline and follow-up values correspond
to patients with biochemical index out of normal range that complete the three-point test:
for serum ferritin, n=18; fibrinogen, n=10; D-dimer, n=10; LDH, n=20. *, significant
differences (p < 0.05) compared to day 1; **, significant difference (p< 0.05) compared to day
1 and 6.

Table 3: Efficacy outcomes.

SC + O3SS
Characteristic
n=25
Hospital stay — median (IQR) no. of days 14 (11-18)
Time from inclusion to average discharge (IQR) no. of days 14 (9.5-15)
Oxygen support — days (IQR) 9 (6 - 14.5)
Score on seven-category scale at day 7 — no. of patients (%)
2. Not hospitalized, but unable to resume normal activities 2 (8)
3. Hospitalization, not requiring supplemental oxygen 17 (68)
4. Hospitalization, requiring supplemental oxygen 4 (16)
5. Hospitalization, requiring HFNC or noninvasive mechanical
1 (4)
ventilation
6. Hospitalization, requiring ECMO, invasive
1 (4)
mechanical ventilation, or both
7. Death 0
Score on seven-category scale at day 14 — no. of patients (%)
2. Not hospitalized, but unable to resume normal activities 18 (72)
3. Hospitalization, not requiring supplemental oxygen 6 (24)
4. Hospitalization, requiring supplemental oxygen 0
5. Hospitalization, requiring HFNC or noninvasive mechanical
0
ventilation
6. Hospitalization, requiring ECMO, invasive
1 (4)
mechanical ventilation, or both
7. Death 0
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Legend: ECMO, extracorporeal membrane oxygenation; HFNC, high-flow nasal cannula for oxygen therapy;
IQR, interquartile range; SC + O3SS, standard care (SC) plus ozonized saline solution (O 3SS).

4. Discussion
The COVID-19 pandemic represents a global public health crisis. Given the severity
with which this disease has unfolded, empirical treatment recommendations for COVID-19
are being made based on unpowered studies. Because of the mortality and morbidity
associated with the disease untested drugs with a questionable safety profile at higher doses
are being prescribed on a compassionate basis [29]. For facing this pandemic, the repurposing
of existing therapeutic agents happens to be the only pragmatical approach as urgent
response, as most of these drugs have already been tested for their safety [30]. These agents
can be classified into two categories: 1) Agents that directly target the virus replication cycle,
and 2) Agents based on immunotherapy approaches. “The development of vaccine represents
a more long-term strategy to prevent COVID-19 outbreaks in the future” [30]. O3x has been
used to treat different pathologies including viral diseases [17]. There are well known
different mechanisms that presuppose the utility of O3x in COVID-19 infection [10]. In this
indication, O3x can be classified as immunomodulator, either boosting innate antiviral
immune responses or alleviating damage induced by dysregulated inflammatory responses.
The development of vaccine represents a more long-term strategy to prevent COVID-19
outbreaks in the future [30]. O3x has been used to treat different pathologies including viral
diseases [17]. There are well known different mechanisms that presuppose the utility of O3x
in COVID-19 infection [10]. In this indication, O3x can be classified as immunomodulator,
either boosting innate antiviral immune responses or alleviating damage induced by
dysregulated inflammatory responses.
The population distribution by age in this complementary application of O3SS (30-50
years [28%], 50-70 years [52%] and >70 years [20%]) was in line with the international
epidemiologic data reported for this infection [31] and infected Spain population [26,27]. This
finding emphasises that subjects of any age can acquire COVID-19 infection. However,
adults of middle age and older are most commonly affected. The average age ranged in Spain
was 18 – 102 years [26] similar with the average age of our patients 55 (30-95). In others
studies of hospitalized patients with confirmed COVID-19, the average ranged from 49 to
56 years [32,33]. Comorbidities have been associated with severe illnesses and mortality,
however the results of application of O3SS indicates that only (9 of 25 [36%]) patients did
not show comorbidities. The most frequently comorbidities were hypertension, asthma,
hypothyroidism, smoking and obesity, in line with the data available from infected patients
in Spain [26,27]. Except hypothyroidism, all other conditions are considered risk factors for
SARS-CoV-2 infection [34]. All patients were white, therefore an analysis of differences
between races was not performed. In general, more males were affected by the disease as
reported in cohorts studies from China, Italy, and the United States [35-37]. However, we
enrolled more women (14), than men (11), but an analysis of the ratio of gender incidence in
this case is not valid, because the small number of subjects. In addition, in a study performed
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in Madrid in 2226 case, was found e relative more proportion of affected female patients
(51.8 %) [27].
Fever (defined as an axillary temperature over 37.5°C) is not a universal finding on
presentation of COVID-19. In our sample (13 of 25 [52%]) had fever at baseline (Tab. 1). In
a study of over 5000 patients who were hospitalized with COVID-19 in New York, only 31%
had a temperature >38°C at presentation [35]. In another study of 1099 patients from Wuhan
and other areas in China, fever was present in only 44% on admission but was ultimately
noted in 89% during the hospitalization [38]. In an epidemiological study in 18 European
hospitals in 1420 patients’ fever was appeared in 45.5 % of the subjects [28]. Nevertheless,
our patients treated with O3SS (Fig. 2a) had their febrility reduced gradually in line with their
favorable recovery. Nevertheless, our patients treated with O3SS (Fig. 2a) fever reduced
gradually in line with their favorable recovery.
All clinical manifestations found in patients (Tab. 1) were similar to the clinical features
of the disease onset [39]. The main clinical manifestations of disease (dyspnea and weakness)
had a favorable course of resolution in O3SS treated patients. On days 7 and 8 respectively
(Fig. 2b) these symptoms were found to be significantly (p<0.05) improved. Acute
respiratory distress syndrome (ARDS) is the major complication of patients with severe
disease. ARDS was shown in 20%, on average of 8 days after the onset of symptoms in a
study involved 138 COVID-19 patients; in this study mechanical ventilation was
implemented in 12.3% [39]. In addition, some patients with initially no severe symptoms
may progress over the course of a time (in a week). In another study, the median time to
dyspnea was 8 days [33]. However, without exceptions, our 25 patients after the treatment
with O3SS, had a resolution of the dyspnea. Elevation of inflammatory markers (e.g., ferritin,
D-dimer, CPR), were observed in our COVID-19 patients, in line with the results of other
recent reports [40] and epidemiological report form Madrid in the same period [26]. Elevated
ferritin has also emerged as poor prognostic factors. Higher serum ferritin was associated
with ARDS development [41], in addition ferritin was raised in 72.4 % of patients in a study
involved 6 424 subjects in Madrid [26]. In addition, higher D-dimer was detected in 36% of
patients in a descriptive study of 99 COVID-19 cases in Wuhan, China [32] but lower form
the values reported in Madrid in the same period (61.5%) [26]. The higher levels of D-dimer
were significantly associated with increased risk of ARDS [42]. Increased disease severity
and ARDS development were associated with elevated CRP [40]. According to different
studies from China and Singapore, mean values of CPR in patients that did not require
supplemental O2 were 11.1 (IQR: 0.9-19.1 mg/L); in patients that required O2, 65.6 (IQR:
47.5-97.5 mg/L) [43] and in the mortality group, 109.25, (IQR 35.00-170.28 mg/L) [44]. In
our patients, in correspondence with their clinic improvement, the average baseline values
of CPR were 12.5 (IQR: 2.5-19.3 mg/L) after the first 24-48 h (Fig. 2c).
Fibrinogen was also higher in our sample, confirming that the hypercoagulation in
patients with SARS-CoV-2 represent an important complication. Higher level of D-dimer,
and fibrinogen was found in a clinical study, in COVID-19 patients, as compared with
healthy controls (p<0.001) [45]. In addition, high LDH level was significantly associated
with severe COVID-19 on admission [46]. In our findings administration of O3SS reduced
the levels of inflammatory markers (e.g., ferritin, D-dimer, CPR, LDH) and fibrinogen as
marker of coagulation function (Fig. 3).
Radiographic findings and auscultation revealed signs of pneumonia in 60 and 56 % of
patient respectively (Tab. 2) similar to the results reported from COVID-19 patients in
Madrid in the same period, in which rales were present in 52.4% [26]. Chest radiographs
may be normal in early or mild disease. In a retrospective study of 64 patients in Hong Kong
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with documented COVID-19, 20 % did not have any abnormalities on chest radiograph at
any point during the illness [47]. Main abnormal radiograph findings were consolidation and
ground glass opacities, with bilateral, peripheral, and lower lung zone distributions [48]. The
25 patients treated with O3SS, bilateral signs of pneumonia were present only in 24-32% of
them, according to the radiographic findings or auscultation, respectively (Tab. 2). Normally
lung involvement increased over the course of illness, with a peak in severity at 10 to 12 days
after symptom onset [49]. However, in our patients, chest radiographs and auscultation
drastically changed after the third to fifth session of O3SS, with both showing an
improvement of their status.
Time from inclusion to discharge in patients treated with O3SS and SC was not
significantly different (>0.05) as compared to other report of patients treated only with SC
in Madrid in the same period (mean 10 days, range 1-62 d) [26]. However, the inclusion of
O3SS as a complementary treatment, accelerated the improvement of patients in terms of
clinical symptoms (Tab. 3) and laboratory biomarkers (Fig. 2). This improvement avoided
the patient transit to critical status. In addition, the time to median duration of viral shedding
[8 days (IQR 6.0-11.5)] and longest duration of viral shedding (22 days) were reduced
compared to other reports of 20.0 days (IQR 17.0-24.0) and 37 days, respectively [50]. Non
death was recorded during the study at time 7 or 14 d. However, mortality rate in hospitalized
COVIS-19 patients in Madrid in the same period was 20.7-21.1% [26,27].
The most probable mechanism associated to the low doses of ozone, using physiological
saline solution as a carrier and applied as a complementary therapy in COVID-19 patients,
will be thought the modulation of the “cytokines storm” through the balanced regulation of
the Nrf2/NF-κB pathway[10]. The potential benefit of ozone in these clinical condition’s
merits further research. Clinical study with this rational are already propose [51].

5. Conclusions
Results of this pilot study suggest that patients with mild to severe symptoms due to COVID-
19 disease by the inclusion of O3SS treatment as a complementary therapy to standard care
was safe. No side effects were observed during the O3SS treatment. The use of O3x as
adjuvant treatment for the management of the infection by SARS-CoV-2 patients has
molecular and preclinical scientific evidence and clinical justification in term of
cryoprotection and control of the inflammatory response. Based on the results of this clinical
trial, it would be reasonable to conduct further clinical studies with this therapy on other viral
diseases with a similar clinical and pathophysiological profile. Without a sufficiently
powered randomized controlled trial, application of O3SS cannot be recommended for
COVID-19 or other viral infections.

Data Availability
The clinical data used to support the findings of this study are included within the article.
Individual patient data can obtain contacting the corresponding author by e-mail.
Pre-print article is available in https://www.preprints.org/manuscript/202006.0233/v1.
Conflicts of Interest
The funders had no role in the design of the protocol applied to the patients; in the collection,
analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results. Gregorio Martínez-Sánchez is the President of ISCO3 (International
Scientific Committee of Ozone Therapy), Adriana Schwartz is the secretary of the ISCO3
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 3 July 2020

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and the president of AEPROMO (Spanish Association of Medical Professionals in Ozone

Therapy). This study was partially funded by AEPROMO.
Funding Statement
This complementary application of O3SS was funded by AEPROMO and partially by SESMI
(Spanish Society of Health and Integrative Medicine); SEDECAL S.A. (Spanish Society of
Electromedicine and Quality) lent the ozone devices and donated consumables.
Acknowledgments
To all members of AEPROMO for their contribution. For their assistance in data collection:
Clara Barrachina and Fabricio Quintero. For corrections to the English style and wording:
Roberto Quintero, Ana Gutiérrez Gossweiler and Michel Gossweiler. For the technical
support: JoséRamos.

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