Effect of thermal helium-oxygen mixture on viral load in COVID-19

In this paper, we will discuss the use of t-Не/О 2 in the treatment of patients with the viral disease COVID-19. The aim of the study is to evaluate the effect of thermal helium-oxygen therapy on viral load, inflammatory markers, and antibody synthesis. Methods. A single-center, randomized, prospective study included 60 patients with COVID-19. Patients were divided into two groups: 1 ( n = 30; 17 male, 13 female) – t-Не/О 2 therapy was included in the standard COVID-19 treatment Protocol; 2 ( n = 30; 16 male, 14 female) – standard therapy in accordance with the clinical recom-mendations of Healthcare Ministry of Russia for patients with COVID-19. Of the 60 patients included in the study, 28 (46.7%) were medical pro- fessionals. The median age of patients in the study was 56.7 (45 – 61) years old. In the group 1 – 58 (45 – 59.5) years old, in the group 2 – 55 (46 – 66) years old. All patients had a positive test of SARS-CoV-2 coronavirus RNA, CT signs of “ground-glass opacity” type lung damage, and areas of air space consolidation. Patients were comparable by gender, age, body mass index (BMI), area of lesion of the pulmonary parenchyma, laboratory data. Results . As a result of the use of t-Не/О 2 , the elimination of the SARS-CoV-2 virus occurred within 48 – 72 hours from the start of inhalation and was confirmed by PCR test. The following changes were found in all patients: synthesis of IgM and IgG antibodies, increase in lymphocytes level, decrease of C-reactive protein, restoration of alanine aminotransferase and aspartate aminotransferase levels, D-dimer, and ferritin. These signs became more pronounced in the 1 st group within 72 – 168 hours, compared with the 2 nd group, where these results were achieved on the 10 th day of therapy. Conclusion . Тhe inclusion of thermal inhalation a gas mixture of helium and oxygen (t-Не/О 2 ) in the standard therapy of patients carrying infectious disease caused by SARS-CоV-2 with CT signs of COVID-19 pneumonia (СT1, CT2 grades) reduces the viral load by stimulating antibody synthesis, as the type of immunoglobulin G, and immunoglobulin M causing the effect of “termovaccination”; increases the effectiveness of treatment, reducing the markers of inflammation.

The first outbreak of the new coronavirus disease 2019 (COVID-19) began in December 2019 in Wuhan, China and has evolved into a major pandemic [1,2]. A severe acute respiratory syndrome coronavirus (SARS-CoV-2) was later identified as the causative agent. This virus is enveloped in a positive-stranded RNA [3,4]. About 28,506,254 cases of COVID-2019 and 915,920 deaths (as of) were registered globally by September 11, 2020 1 . The fight against the coronavirus infection is an extremely acute issue that requires the development of new methods to expand the therapeutic and preventative options. WHO have not recommended any medicinal product for the treatment of COVID-19 yet. The currently recommended and used therapeutics are supportive. Some repurposed anti-HIV and antiviral drugs are currently in use, including hydroxychloroquine, remdesivir, lopinavir/ritonavir, interleukin 6 (IL-6) receptor inhibitors, and plasma therapy [5][6][7][8].
Great hopes are set on the development of synthetic vaccines that affect the antibody synthesis and specifically interact with certain proteins of the virus. The issue of finding new methods for treatment of COVID-19 is pressing.
We have developed a new method based on the use of thermoheliox (inhalation of a high-temperature mixture of helium and oxygen -t-He/O 2 ).
Helium is an inert gas. It was discovered by P.Janssen and N.Lockyer, and both reported the discovery independently to the French Academy of Sciences in 1868. Academician P.L.Kapitsa played an important role in the study of the physicochemical properties of helium. He began his research during his internship in Rutherford laboratory in London (1938), and continued it in Moscow together with Academician D.L.Landau [9][10][11][12][13]. Both scientists were awarded the Nobel Prize for their research on the physical properties of helium.
The authors of this article have more than 20 years of clinical experience with heliox. Initially, we used a thermal helium-oxygen mixture in patients with hypoxemic respiratory failure, and then in patients with hypercapnic respiratory failure 2, 3 .
In the recent years, this experience has been used in a neurological clinic for the treatment of patients with ischemic stroke [14,15] and in obstetrics to correct the oxygen status of pregnant women in the III trimester 4  Our clinical study was preceded by a theoretical analysis of the development of acute viral infection with an assessment of the potential therapeutic effects of t-He/O 2 inhalation. The kinetic model included a description of growth and reproduction of the virus in the human body, the viral damage to the recipient cells, the effects of thermal destruction of viruses, and the antibody response. Theoretical analysis predicted the potential effects of inhalation of t-He/O 2 , i.e. the production of antibodies against the proteins of the destroyed viral particles [16]. The analysis of the protein composition of the patient's exhaled air condensate confirmed the safety of thermoheliox [17].
This article discusses the use of t-He/O 2 in the treatment of COVID-19.

Materials and methods
Study group. A single-center, randomized, prospective study included 60 patients with COVID-19. Patients were divided into two groups. Group 1 (n = 30) received the standard COVID-19 treatment protocol together with the t-Не/О 2 . Group 2 (n = 30) received the standard treatment in accordance with the clinical recommendations of the Ministry of Health of the Russian Federation "Prevention, diagnostics, and treatment of the novel coronavirus infection COVID-19" [version 5 (approved by the Ministry of Health of the Russian Federation on March 08, 2020), version 6 (approved by the Ministry of Health of the Russian Federation on April 28, 2020), version 7 (approved by the Ministry of Health of the Russian Federation on June 3, 2020)] [18]. Of the 60 patients included in the study, 28 (46.7%) were medical professionals. The male/female ratio was 17/13 in Group 1, and 16/14 in Group 2. The groups were matched by the sex ratio, p = 0.403. The median age of the patients was 56.7 years (45 to 61 years). The median age was 58 years (45 years; 59.5 years) in Group 1 and 55 years (46 years; 66 years) in Group 2. The groups were matched by age, p = 0.537. The general characteristics of the patients at the enrollment are shown in Table 1.
The clinical symptoms in Groups 1 and 2 are characterized in Table 2. The symptoms included: a loss of smell and taste, runny nose, shortness of breath, dyspnea, weakness, fever, headache, muscle pain, sore throat, and dry cough.
All patients who were enrolled in the study according to the protocol No.11 -20 dated April 20, 2020, approved by the Ethics Committee on Biomedical Ethics of the N.V.Sklifosovsky Research Institute for Emergency Medicine of Moscow Department of Health received treatment for pneumonia caused by the SARS-CoV-2 virus from April 21 to June 2020 both inclusive.
The diagnostic procedures included specific molecular tests of the respiratory samples (throat and nasopharyngeal swab) (detecting amplifier CFX-96 REAL TIME, Bio-Rad, USA), lung CT scan, express analysis of the gas composition of arterial blood on an automatic analyzer ABL-500 (Radiometer Copenhagen, Denmark), measurement of serum IgM and IgG on an immunoche-miluminescent analyzer (Mindray 6000, USA), complete blood count, analyses of serum markers (D-dimer, C-reactive protein [CRP], ferritin, lymphocytes, aspartate aminotransferase, AST, and alanine aminotransferase, ALT).
All patients had a positive test of SARS-CoV-2 coronavirus RNA, the ground-glass opacities in the lung CT, and the areas of air space consolidation.

Study design
All patients underwent swabs from the mucous membrane of the nasal cavity and oropharynx to detect SARS-CoV-2 coronavirus RNA; sampling of venous blood for IgG (semiquantitative) and IgM (semiquantitative) antibodies of SARS-CoV-2 coronavirus by the standard enzyme immunoassay method, for CPB, D-dimer, ferritin, lymphocytes, AST, and ALT levels. Schedule of the examinations is presented in Table 3. The study design is shown in Figure 1.

Treatment with gas mixtures
The   The patients underwent 4 daily inhalation procedures for 15 minutes with 15-minute intervals. The concentration of He and O 2 was selected individually for each patient in the range from 79 to 50% (He) and from 21 to 50% (O 2 ) to maintain SpO 2 within 97 -99% at temperatures from 75 to 96 °C, depending on the saturation index, tidal volume, and the patient comfort.
At SpO 2 ≥ 93%, the inhalation of t-He/O 2 started with 79% He and 21% O 2 at a temperature of 85 -96 °C with a gradual increase in O 2 fraction by 2% every minute until the target SpO 2 97 -99%.
At 85 ≤ SpO 2 ≤ 92%, the inhalation of t-He/O 2 started with 70 He and 30% O 2 at a temperature of 85 -96 °С with a gradual increase in O 2 fraction by 2% every minute until the target SpO 2 97 -99%.
At SpO 2 < 85%, the inhalation of t-He/O 2 started with 65% He and 35% O 2 at the temperature of 75 -84°C with a gradual increase in O 2 fraction by 2% every minute. The O 2 fraction did not exceed 50%, i.e. the ratio of helium and oxygen not more than 50 : 50% while maintaining SрO 2 97 -99%.
The maximal allowed tidal volume (TV) was 1,000 mL. If the TV was more than 1000 ml, we interrupted the breathing cycle from the circuit of the apparatus and the patients made one or two breaths of an air mixture with FiO 2 21%. Then, the face mask was put back on, and breathing with t-He/O 2 was continued with the same concentration of He and O 2 . SрO 2 was monitored using an OxyShuttle pulse oximeter (Sensor Medics, USA). The TV was monitored with a monitor built into the Heliox-Extreme apparatus.

Statistical analysis
The statistical processing of the data was carried out using the SPSS 17.0 software package (SPSS Inc., USA). The quantitative parameters are presented as median (Me) and quartiles (lower and upper quartiles). The nonparametric statistic methods with Mann-Whitney U-test were used to compare the variables between the groups. Friedman rank analysis of variance followed by paired comparison with the Wilcoxon test was used to assess the changes over time in each group. The differences were considered statistically significant at p < 0.05.

Results
No patients had any objective procedure-related side effects during the inhalation therapy with t-He/O 2 . One patient refused inhalation therapy on Day 2 because he did not tolerate the fever well. From that moment on, 29 patients in Group 1 and 30 patients in Group 2 continued the therapy. None of the patients were transferred to mechanical ventilation. There were no lethal outcomes in both groups. All patients were discharged. The median hospital stay was 15 (13.35) [12,7; 34,6] days.
Changes of the PCR results over time. Group 1 showed statistically significant decrease in positive RNA tests for the SARS-CoV-2 coronavirus. According to our observations, most COVID-19 patients from Group 1 who received inhalation of t-He/O 2 had a negative PCR on Day 3 and some patients had a negative result as early as 1 day after the start of therapy. The patients in the standard therapy group has a positive test for the viral antigen from 7 days to 4 weeks from the onset of the disease, in some cases even longer (Figure 2).
Changes in the D-dimer test results over time. Group 1 showed a statistically significant decrease in the D-dimer level on Day 3 as compared to Day 7 in the Group 2. The change of D-dimer level within 10 days was significantly higher in the Group 1 (Figure 3).
Changes in the ferritin level over time. The ferritin level stayed high in both groups. The statistically significant decrease in the ferritin level was observed on Day 7 and Day 10 ( Figure 4).
Changes in the CRP level over time. The standard therapy group kept a statistically significant increase in CRP on Day 3 and the statistically significant decrease on    in the Group 1 on Day 3 and to achieve full recovery on Day 7. Group 2 showed a statistically significant decrease in lymphocyte level on Day 3 as compared to the baseline. The lymphocyte level increased significantly on Day 3 and Day 7, but the growth was significantly lower than in Group 1 ( Figure 6).

Changes in ASTs over time.
High AST levels were reported in Groups 1 and 2 at baseline. Group 1 showed a statistically significant decrease in AST level on Day 3 and a continued decrease on Days 7 and 10. Group 2 showed an increase in AST on Day 3, and the difference from the baseline was statistically significant. The AST level dropped on Days 7 and 10, but a lesser extent than in Group 1 (Figure 7). Changes in ALT levels over time. High ALT levels were reported in Groups 1 and 2 at baseline. Group 1 showed a statistically significant decrease in ALT level on Day 3 and a continued decrease on Days 7 and 10. Group 2 showed a continued growth of the ALT level on Day 3. The ALT level decreased on Day 3 and 7 but it was still significantly higher than the baseline. The ALT level decreased on Days 3 and 7 but to a lesser extent than in Group 1 (Figure 8).
Changes in IgM levels over time. The immune response was confirmed in both groups at baseline. The immune response showed a statistically significant peak on Day 7 and decreased by Day 10 in Group 1. Meanwhile, Group 2 showed the peak immune response on Day 10 ( Figure 9). Changes in IgG levels over time. Group 1 had a statistically significant increase in IgG levels on Day 3 of the t-Не/О 2 therapy and a continued significant growth on Days 7 and 10. The Group 2 had a statistically insignificant increase in IgG levels on Days 3 and 7 of the standard therapy. The statistically significant increase in the IgG levels was reported on Day 10 as compared to the baseline. The increase in the IgG levels on Days 3, 7, and 10 in Group 1 was significantly higher than in Group 2 ( Figure 10).

Discussion
The findings demonstrate that the full-size viral particles detected by PCR are eliminated by days 2 -3 of t-He/O 2 . The antibody production is continued, apparently, as a response to the protein products of the thermal destruction of the virus. The use of t-He/O 2 stimulates the synthesis of IgM and IgG from the first procedure. 60% of patients in Group 2 that did not receive the t-He/O 2 inhalations, had almost no IgM and IgG antibodies during the first three days. Antibody synthesis begins on the second or third day after the induction period.
The changes in the IgG and IgM levels over time clearly demonstrate that t-He/O 2 leads to activation of the immune system and stimulates production of specific antibodies in patients with the coronavirus infection.
The immune response is complex and involves various biochemical systems. In particular, CRP is considered one of the components of a complex chain of biochemical processes and one of the first respondents to bacterial and viral infections. We compared the changes in the accumulation and reduction of CRP in the course of standard treatment and treatment with t-He/O 2 inhalations. A significant difference in the response was found.
A relatively slow accumulation of CRP occurs in the typical course of the disease. In most cases, the CRP level reaches maximum on Days 2 -4 of treatment. The CRP level decreases as the treatment is continued. In our study, inhalations with t-He/O 2 stimulate the rapid accumulation of CRP with the subsequent exponential decrease in the level of CRP.
Stimulation of the immune response with t-He/O 2 can be defined as "thermal vaccination". Our study showed that the full-size viral particles detected by PCR are eliminated by Days 2 -3 of t-He/O 2 . Apparently, the antibody production is continued as a response to the protein products of the thermal destruction of the virus. A "classical" vaccination with a weakened or destroyed antigen takes place. The fundamental positive difference is that the process takes place in vivo with the participation of natural viral proteins, and the "thermal vaccination" can have a wide-range specificity.
The mechanism of the observed effect of stimulation of the immune response with t-He/O 2 requires further research. The kinetic model that was developed and evaluated by us 2 [13] explains the observed effects by an increase in the antigen concentration during thermal destruction of the virus.

Conclusion
The addition of thermal inhalation of a gas mixture of helium and oxygen (t-Не/О 2 ) to the standard therapy of patients with the infectious disease caused by SARS-CоV-2 and with CT signs of COVID-19 pneumonia (СT1, CT2 grades) reduces the viral load by stimulating both IgG and IgM antibody production. This effect of "thermal vaccination" increases the effectiveness of treatment and reduces the markers of inflammation. We are currently studying the response of patients with the CT3 signs of pneumonia to t-He/O 2 . The intermediate results demonstrate a positive effect in the critically ill patients. The results require further research and statistical analysis.