Cespedes-Suarez J, Martin-Serrano Y,
Carballosa-Peña MR, Dager-Carballosa
DR. The immune response behavior in
HIV-AIDS patients treated with Ozone
therapy for two years. J Ozone Ther.
doi: 10.7203/jo3t.2.3.2018.11458

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Jose Baeza-Noci,
School of Medicine, Valencia
University, SPAIN

World Federation of Ozone Therapy,
Bologna, ITALY

February 1, 2018

March 24, 2018

December 15, 2018

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Cespedes-Suarez J.

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The immune response behavior in HIV-AIDS patients treated with Ozone therapy for two years.

Javier Cespedes-Suarez, Yanisley Martin-Serrano, Maria Rosa Carballosa-Peña, Diana Rosa Dager-Carballosa

Cardiozono Medical Center, Luanda, Angola

HIV continues to be one of the biggest problems for the global public health.
The African region is one of the most affected, accounting for almost two
thirds of the new infections. Many of them are retroviral medications and
multi-therapies aiming to stop the viral replication and maintaining
immunological stability, which fail to guarantee the quality of life or enhance
the patient’s immunity
Ozone has biological properties, among which the antimicrobial and
modulatory effect on the immune response is highlighted, which makes it
possible to use it in a complementary way for the treatment of these patients.
We conducted the present study in which the effect of the ozone therapy
administered systemically (blood route) is analyzed. We have studied 32
patients to whom we applied Major Autohemotherapy with a protocol of 15
sessions and maintenance every 15 days at 50 μg/mL of concentration; the
initial dose was 4,000 μg/mL up to 12,000 μg/mL.
We indicated viral load, CD4 and CD8 counts before and at the end of
therapy. The results showed a significant decrease in viral load to
undetectable values and an increase of CD4 and CD8 at the end of the 15
sessions, being maintained at 2 years of treatment, thus achieving a
permanent activation of the immune system and improving the quality of life
of these patients.

Keywords: Ozone therapy, HIV-AIDS, Immune Response, Major Autohemotherapy

The Human Immunodeficiency Virus (HIV) is a retrovirus. It means that the
virus genetic material consists of RNA and, once it managed to infect a cell,
has the ability to convert RNA to DNA, so as to be able to incorporate their
genetic material into the cells (DNA) in this case directly affecting CD4
helper T cells.
Currently, HIV continues to be one of the biggest problems for global public
health, it has already taken over 35 million lives. In 2016, one million people
died for causes related to this virus. At the end of that year there were
approximately 36.7 million people infected with HIV worldwide, and 1.8
million new infections occurred. According to data from 2016, in the WHO
African Region there were 25.6 million people infected. This Region is the most affected and accounts for almost two thirds of new HIV infections in the
world [1].
In Angola, the adult population incidence rate was 2.7% in 2015 [2].
The HIV / AIDS treatment consists in three main parts: the disease
preventive treatment, opportunistic diseases treatment and drug treatment.
Of all of them the preventive treatment is the ideal right now, since there is
no cure for the disease. The treatment of opportunistic diseases is based on
the CD4 T cell count, is aimed at treating and preventing them, as well as
prolonging the patient’s life.
Drug treatment, which encompasses a large number of drugs, mainly the
use of antiviral drugs such as Reverse Transcriptase (RT) inhibitors,
analogues as: Zidovudine (AZT), Didanosine (Dd), Zalcitabine, Lamivudine
(TC), Stavudine (DT), Combivir (Glaxo Operations UK Limited. Priory Street,
Ware, Herts SG12 0DJ,United Kingdom), Abacavir and no-analogues as:
Nevirapine, Efavirenz, Delavirdine.
Antiviral agents of the Protease Inhibitors (PI) type. In recent years important
results have been obtained with this class of antivirals; which are
characterized by high antiviral activity translating into a marked decrease in
viral load and an increase in CD4 T lymphocytes. They are used in
combination with analog reverse transcriptase inhibitors. In this group we
found: Saquinavir, Ritonavir, Indanavir, Nelfinavir, Atazanavir, Lopinavir /
ritonavir kaletra. In addition, integrase inhibitors such as Raltegravir, Stribild
and Dolutegravir are also known.
Similarly, treatments based on Chemokine Coreceptor Antagonists (CCR5)
have been developed which are targeted against these coreceptors by
selectively inhibiting the interaction between the human CCR5 receptor and
the gp120 viral glycoprotein, preventing HIV from entering the cell
Also known are fusion or entry inhibitors. These inhibit the fusion process of
the glycoprotein complex gp 120 / gp 41 from the viral envelope to the cell
membrane, preventing virus penetration (Enfuvirtide). In the case of
treatments based on fixed combinations (combination of several active
ingredients in a single tablet or capsule), several drugs have been developed
as Combivir, Trizivir and Kivexa (Glaxo Operations UK Limited. Priory Street,
Ware, Herts SG12 0DJ,United Kingdom), Kaletra (AbbVie Inc., 1 North
Waukegan Rd, North Chicago, IL 60064, USA), Truvada, Atripla, Complera,
Eviplera and Stribild (Gilead Sciences Inc., 333 Lakeside Drive, Foster City,
CA 94404,).

The conditions for treatment initiation are:

Evaluation of the CD4 count for initiation of antiretroviral therapy (ARVT):
• Less than 200 cells / mm3: treat regardless of clinical stage.
• From 200-350 cells / mm3: is recommended, because it is considered that
at this level there is a functional immune deficiency.
• >350-500 cells / mm3: no treatment is recommended; except for some
special conditions [3].
Despite these advances, there are still factors that limit the achievement of
good long-term results. In a research protocol (termed ACTG 320 clinical
trial) the combination of Zidovudine, Lamivudine and Indinavir did not
suppress plasma detectable viral load in 60% of patients with advanced disease. The same occurred in 50% of patients treated with a regimen based
on Indinavir or Ritonavir. Because the cross-resistance frequency to all three
types of antiretroviral drugs (nucleoside and non-nucleoside reverse
transcriptase inhibitors and protease inhibitors), failure with one regimen
limits future treatment options. This implies the need to develop new drugs, a
slowly progressing process [4,5].
Faithful adherence to HIV treatment is not always easy and, in some cases,
impossible. Side effects and interaction with other medications are common.
These effects call into question the innocuousness of the newest treatments
when used for prolonged periods [6].
On the other hand, there are also other natural treatments that perfectly
complement the pharmacological ones. Multiple references point to ozone
therapy as an encouraging alternative for HIV-infected patients. Recent
studies show that, in addition to potentiating the effect of antiretrovirals, it is
useful in the prevention and treatment of many complications of this disease
Ozone therapy alone has very significant effects as a modulator of the
immune system, and oxidative stress. When oxidative stress is small,
transient and controlled, it restores the antioxidant defense systems needed
in these seropositive patients who are in chronic oxidative stress [9,10,11].
Ozone increases the production of interferon’s that have the ability to
interfere in the replication of viruses in host cells. Gamma interferon levels
can be raised from 400% to 900% with their antiviral activity. They prevent
viral replication and activate CD4 + T lymphocytes (helper),
immunomodulatory functions and natural killer cells (cytotoxic). Also induce
the cytotoxicity of CD8 + T lymphocytes, promotes the proliferation and B
lymphocytes activation, which is why these cells are considered a
fundamental pillar in the mediated-cell immune response. The CD4 + T
lymphocytes activation trigger an immune reactions cascade [12,13]. CD8 +
T lymphocytes, macrophages, neutrophils, eosinophils, NK cells and the
activation of antibody-dependent cellular cytotoxicity (ADCC) constitute
immune effectors mechanisms to destroy virus-infected cells [14]. Previous
studies have shown different types of ozone treatments against HIV infection
[15]. Considering this background, the aim of this study is to demonstrate the
immunological response in HIV-AIDS patients treated with ozone by major
autohemotherapy during 2 years.

Materials and methods
A retrospective longitudinal study was performed at the Cardiozono Medical
Center located in Luanda, Angola. Thirty-two patients with no gender
distinction were included. They began treatment at the Medical Center in
August 2015 until August 2017, submitted to our Clinic from an Infectology
Consultation and other Hospitals, with 1-3 years of seropositive diagnosis.
9 patients were male (28.1%) and 23 (71.9%) were female. The age ranged
from 32 up to 48 years old. The groups with the highest incidence of disease
were the ranges 36-45 and 46-55 years.

Inclusion criteria: HIV positive patients with high viral load and low CD4
and CD8 counts, under retroviral treatment (Lamivudine 150 mg and
Zidovudine 300 mg one tablet daily) suffering the adverse effects of

Exclusion criteria: HIV positive patients who did not comply with the
treatment time and serological results expressed in the inclusion criteria.
Pregnant women and patients under 18 years of age.

Treatment Protocol: The patients received 15 sessions of major
autohemotherapy (one daily session). In each session we used a Germanmade
Promedic Humazon equipment (Humares GmbH, Bruchsal,
Germany), 3-way stopcock, epicranial needle 19 G, Sangiset (Humares
GmbH, Bruchsal, Germany) with capacity of 350 mL where we put 10 mL of
3.13% sodium citrate to avoid blood clot during the procedure and other
disposable material.
The major autohemotherapy (Table 1) was performed at a concentration of
50 μg / mL in 100 mL of blood; the initial dose of the therapy was 2000 μg
(40 mL of ozone gas at 50 μg / mL concentration); we increased the dose by
2000 μg every 2 days until reaching a dose of 12000 μg (240 mL of ozone
gas at 50 μg / mL concentration), keeping this dose as a maintenance dose
every 15 days throughout the year.

Evaluation criteria
(before and after treatment): Results of the viral load,
CD4 and CD8 counts per month and every 6 months, until 2 years of
The real-time PCR reference values were taken into account as a
standardized method to determine Viral Load with an analytical range of
50-300,000 copies/mL. This technique was developed in a clinic authorized
for the diagnosis of this virus
CD4 values with a range of 500-1600 cells/mm3 and CD8 of 375-1100 cells/mm3 were taken into account according to the reference of the clinical laboratory in which these tests were performed.

Statistical analysis: We used SPSS 19.0 statistic software and we
categorized the values using contingency tables utility and chi-square
analysis for the significance on the changes before and after the treatment.
Values of chi-square under 0,05 were considered significant.

Side effects evaluation: As a marginal target, the increase in the quality of
life referred by most of the patients and the decrease in the rate of side
effects made us compile them in an open list way and calculate incidence
rate before and after the treatment.

Before starting treatment, most patients had a viral load between
15,000-20,000 IU / ml for 46.9%, and after one month the viral load was
undetectable in 100% of the sample during the 2 years under treatment
(Table 2).

The results on the kinetics cell populations of TCD4 and TCD8 are shown in
Tables 3 and 4. Before starting the treatment, the highest number of patients
had a CD4 count between 250-300 cells / mm3 for 71.9%. At one month,
53.1% presented values between 550-600 cells / mm3, reaching the ranges
between 600-700 and 700-800 cells / mm3 at 6 months, being maintained at
one and at two years in 100% of patients (Table 3).
In the case of CD8 cells, the highest number of patients had a CD8 count
between 240-300 cells / mm3 for 81.3%, before starting treatment. At one
month, 59.4% presented values ranging from 400-450 cells / mm3, to
500-1000 cells / mm3 at 6 months, and remained within 2 years of treatment
in 100% of patients (Table 4).

The patients presented with side effects of antiretroviral therapy are as
• Decay
• Lack of appetite,
• Insomnia,
• Nausea, Headache,
• Gastric discomfort.
We can see in Table 5 the evaluation after the treatment. In 7 patients, all
side effects disappeared.

The HIV infection evolution is closely related to the immune response, that
the patient can achieve the effectiveness and control of viral replication.
There have been different studies that suggest that a cell response mediated
by helper T cells (CD4), cytotoxic T lymphocytes (CD8) together with NK
cells predict a favorable prognosis in the control of HIV infection, where there
is a gradual loss of CD4 lymphocytes because these are the target cells of
this retrovirus. Likewise, in the immune response to HIV, NK cells play an
important defensive role due to the CD4 rapid decrease, these NK cells are
of vital importance because they do not have CD4 receptors and some of
them are resistant receptors to HIV infection virus. Considering the
implication of the immune system in the pathogenesis of HIV infection, as
well as the immunomodulatory effect of ozone therapy on TCD4, TCD8 and
NK cells, we have evaluated the efficacy of a treatment based on increased
autohemotherapy against HIV in infected patients [16,17].
Taking into account the values obtained after counting CD4 and CD8 T cell
populations at 6 months of treatment, as well as the impossibility of detecting
viral RNA during the first month of treatment and during the two years of this
study, our results show that the treatment with major autohemotherapy acted
as a modulator of the immune system and as an enhancer of current
antiretroviral therapy. It is important to note that the treatment employed was
able to maintain undetectable viral load during the two years of treatment,
resulting in a potentiating effect on the number of T cells (CD4 and CD8)
during this time. Our results are similar to those previously obtained by
Gonzalez et al., 2003 [18], and Méndez et al., 2005 [15], although in Mendez
study, ozone treatment was administered rectally. This can be an option
when major autohemotherapy can`t be performed and the results are the
same20 adjusting the dosification. Due to cultural reasons in Angola, rectal
insufflation is usually refused.
We must also point out an important aspect, HIV infection causes oxidative
stress in these patients, this is because in their pathogenesis and evolution,
reactive oxygen species (ROS) may be involved. The oxidative stress acts
on the immune system, producing a decrease in the intracellular antioxidant
systems and therefore an increase in the apoptosis of the cells of the immune system (for example TCD4 cells)21. The induction of an oxidative
stress with medical ozone, through controlled treatments (for example, main
autohemotherapy) stimulates the endogenous antioxidant systems that
control the excessive production of ROS, by Diaz L. et al., 2018 [22], thus
supporting the activation of the system through the decrease as a
consequence of the apoptosis of the TCD4 cells, which in turn are attacked
by the virus.

Our results support previous studies that demonstrate the immunomodulatory
potential of ozone, as well as the beneficial effect of ozone therapy at the level
of the immune system, in the control of oxidative stress and its relationship
with the immune response that improves HIV infection. Considering that there
is not much information in the scientific literature on this type of strategies, this work provides information on the immunomodulatory capacity of ozone against one of the most relevant infections in human health that does not yet have an effective treatment.
No side effects were presented because of the ozone treatment. The quality of
life improved not only because the improvement of the immunological status
but also thanks to the decrease of antiretroviral therapy side effects.
We recommend carrying out studies with a greater number of patients,
including other parameters to be studied, so that the introduction of ozone
therapy in HIV-AIDS treatment protocols could be considered in the future.

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