Abstract
Introduction:
An exaggerated immune response is considered the most important aspect of COVID-19 pathogenesis. Hypertonic saline (HS) has shown promise in combating inflammation in several respiratory diseases. We investigated the effects of nebulized HS on clinical symptoms and inflammatory status in patients with severe novel coronavirus infection (COVID-19) pneumonia.
Materials and Methods:
We randomly assigned 60 adults admitted to the intensive care unit (ICU) due to severe COVID-19 pneumonia to the experimental (received nebulized 5% saline) and control (received nebulized distilled water) groups. All interventions were applied 4 times daily for 5 days. The levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and other clinical factors from venous blood were evaluated before and after intervention application. Mortality rate, intubation rate, and durations of ICU and hospital stay were also compared between groups.
Results:
The levels of TNF-α (MD: −21.35 [−32.29, −10.40], P = 0.000) and IL-6 (−9.94 [−18.86, −1.02], P = 0.003) were lower in the experimental group compared to the control group after applying the interventions. The levels of white blood cell count, PO2, and serum sodium were also statistically significant differences between groups. However, we did not observe significant differences in terms of hospitalization durations and mortality rates.
Conclusion:
Nebulization of HS in patients with severe COVID-19 pneumonia appears to be effective in reducing inflammation, but does not appear to affect intubation rates, mortality, hospitalization, or length of stay in ICU.
Introduction
In late 2019, SARS-CoV-2, an enveloped, positive-sense single-stranded RNA virus of the coronavirus family, caused a pandemic, with over 500 million infected and over 6 million infected worldwide by 2022. 1 Symptoms include fever, cough, fatigue, hemoptysis, diarrhea, shortness of breath, lymphopenia, and chest computed tomography showing evidence of pneumonia. 2 A variety of symptoms result from an overactive immune response called a “cytokine storm.” 3 This is not unique to COVID-19, and previous studies have reported it in other situations as well as systemic lupus erythematosus, hemophagocytic lymphocytic leukemia, certain infections, etc. Impaired immune responses can lead to fever, cytopenia, splenomegaly, incidence of hepatitis, coagulopathy, multiorgan dysfunction, and death. 4 Previous outbreaks of coronaviruses known as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) caused similar illnesses. SARS causes a cytokine storm driven primarily by interleukin 6 (IL-6), IL-12, and tumor necrosis factor-α (TNF-α), whereas IL-1β, IL-8, and IL-6 were higher in MERS. 5 Studies of the pathogenesis of novel coronavirus disease (COVID-19) have shown that IL-6 and granulocyte-macrophage colony-stimulating factor play a major role in the development of its cytokine storm, resulting in high levels of IL-12, IL-17, and TNF-α. 6 Some reports have associated elevated plasma IL-6 levels with prolonged duration of systemic inflammatory response syndrome. 7 Many studies have highlighted the inhibitory effects of hypertonic saline (HS) on inflammation and related mechanisms, including pro-inflammatory cytokines. Injection of HS into pigs has been shown to reduce airway inflammation. 8 Previous reviews on airway inflammatory diseases have reviewed and discussed the benefits of HS in improving inflammatory markers, clinical status, and patient quality of life.9,10 This study evaluated the effects of nebulized 5% saline on clinical status, inflammatory markers, intensive care unit (ICU) stay, hospitalization, and mortality in patients with severe COVID-19 pneumonia.
Materials and methods
Study design
The study was a single-center, double-blind, prospective, two parallel-armed randomized-controlled trial comparing the effect of 5% HS with distilled water nebulization on inflammatory mediators in patients admitted to ICU with severe COVID-19 pneumonia. Between December 2021 and February 2022, participants were recruited at the ICUs of Imam Khomeini Hospital Complex (IKHC), Tehran, Iran.
Participants
The inclusion criteria were individuals with (1) the age of 18 to 70 years old; (2) confirmed COVID-19 by positive real-time polymerase chain reaction test; (3) severe pneumonia identified with respiratory rate > 30, SpO2 < 93% in free air breathing condition and, 100 ≤ PaO2/FiO2 ≤ 200 mmHg 1 ; (4) fully conscious verbal consent of them or their guardian (if the patient is not conscious); (5) no history of positive human immunodeficiency virus infection or other immunodeficiency disorders; and (6) no history of any kind of cancer, malignancy, chronic lung diseases include pulmonary fibrosis, bronchiectasis, asthma smoking, or previous thoracic and lung surgery.
The exclusion criteria were: (1) receiving interleukin production inhibitors such as Actemra; (2) patient with active bacterial pneumonia; (3) patients with hypernatremia; and (4) intubated patients.
Randomization and masking
After obtaining the consent, eligible individuals were assigned (1:1) to one of the groups using the randomization method by the random numbers: (1) the experimental group (received 10 mL of 5% NaCl every 6 hours for 5 days) or (2) the control group (received 10 mL of distilled water every 6 hours for 5 days as the sham intervention). To randomize participants into two groups, one of the researchers used the random list numbers (https://www.sealedenvelope.com). Afterward, he followed the numbers from top to bottom consecutively and gave them to each participant. Even numbers and odd ones were allocated to the experimental and control groups, respectively. The solutions were pre-prepared and coded in sterile, identical syringes. To double-blind the study, the syringe of each patient was delivered to the nurse based on the allocation. The nurse who nebulized the solutions was not aware of their type of them. Next, the patient's blood sample was prepared and sent to the laboratory without being known the type of nebulized solution. The lab experimenter was not aware of the allocation of patients and did not know which patient the blood sample belonged to. Finally, the researcher examined the laboratory results of each patient based on the set codes. Other researchers who were responsible for randomization collected data from the type of applied intervention and results from the lab.
Procedures
Researchers enrolled and recruited participants on the same day. They were also undergoing clinical evaluation including laboratory analyses of the venous blood and sequential organ failure assessment (SOFA). For venous blood analysis, blood samples were taken from the upper extremity veins and immediately were transferred to the central lab of IKHC in an anticoagulant-containing test tube for complete blood count analysis and an upright tube for erythrocyte sedimentation rate (ESR) and, clotted tube for other analyses. The resulting serum for measuring TNF-α and IL-6 markers was stored by the researcher at minus 80°C. IL-6 and TNF-α levels were measured by an enzyme-linked immunosorbent assay (ELISA) method using ELISA kits (Bioassay Technology Laboratory company, China; TNF-α ELISA kit: sensitivity = 1.52 ng/L, detection range = 3–900 ng/L; human IL-6 ELISA kit: sensitivity = 1.03 ng/L, detection range = 2–600 ng/L) by the manufacturer's instructions. Analysis of total bilirubin (BT), direct bilirubin (BD), C-reactive protein (CRP), blood urea nitrogen (BUN), creatinine (Cr), sodium (Na), aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALK), D-dimer, albumin, and procalcitonin of the venous blood was done by Turbidometric method (Biosystem Company Kit, Spain). The interventions were applied during 5 days of participants’ stay in the ICU. All participants in both groups received usual medical care including nursing and medication based on the national guideline of COVID-19. 2 Patients underwent a second evaluation at the end of day 5. Their ICU and hospital stay days and 1-month mortality were also collected.
Control group
Allocated patients to the control group received 10 mL of distilled water through nebulization 4 times per day for 5 days during their ICU stay. This intervention was applied by a nurse who was not aware of the randomization and allocation process. As the shape of sodium chloride (NaCl) and distilled water are similar, the patient was not able to distinguish them.
Intervention group
Allocated participants to the intervention group received 10 mL of 5% HS through nebulization 4 times per day for 5 days during the ICU stay.
Outcome measures
The primary outcomes included IL-6 and serum TNF-α. The secondary outcomes were levels of white blood cell (WBC), neutrophils, lymphocytes, platelets, hemoglobin, ESR, BT, BD, CRP, BUN, Cr, Na, oxygen pressure (PO2), carbon dioxide pressure (PCO), pH, bicarbonate (HCO3), AST, ALT, ALK, D-dimer, albumin, and procalcitonin of the venous blood sample before and after the 5-day intervention. SOFA was analyzed by the intensivist using the lab data and patients’ condition. ICU and hospital stay and the mortality rate were also evaluated.
Statistical analysis
The sample size of this study was calculated based on the results of the study by McElvaney et al. Considering means of 348.2 and 217.7 pg/mL, SD of 264 and 168.7 pg/mL, two steps of measurements (before and after), the correlation between measurements of .7, type I error of .05, and 80% power, a sample size of 28 participants per group were calculated. After considering the risk of loss to follow up, 30 patients per group was considered as the final sample size. 3
Stata version 13 (Stata, College, Statin, Texas, USA) was used to analyze the data. We reported means ± SD and frequency counts (%) for continuous and categorical data, respectively. Data normality was checked for all continuous variables using the P-P plot, Q-Q plot, and Shapiro-Wilk test. As the data of IL-6 had no normal distribution in the intervention group, data transformation was utilized using ladder and gladder commands (formula: log (IL-6)) to perform an independent sample t-test and calculate the standardized mean difference (SDM). Data were then retransformed to analyze the mean difference (MD). 11 An independent sample t-test was used to determine the differences between all continuous data. 12 The point estimates of effects were reported as MD with a 95% confidence interval (CI), SMD with 95% CI analyzed by Cohen's d test. We considered .2 to .49, .5 to .79, .8 to 1.19, and >1.2 valuables as small, moderate, large, and very large SMD effects, respectively.
Logistic analysis with odds ratio (OR) effect size was utilized to compare the categorical data between groups.
Results
From December 2021 to February 2022, 137 patients were screened and 60 of them (age: 50.9 ± 10.7 years) met the criteria. They were then randomly allocated to the HS group (n = 30) or sterile water group (n = 30). All patients received the interventions for 5 days and participated in the final assessment session. No attrition was observed during the assessment, intervention, and analysis processes. The trial profile is presented in Figure 1.
Trial profile.
The demographic and clinical characteristics of all participants are presented in Table 1.
Demographics and clinical characteristics of participants.
Defined as a prescribed ventilation type for the patient by a physician which should be used most of the time.
*All of the participant's characteristics are statistically similar between groups based on the t-test and chi-square test.
n: number; NIV: noninvasive ventilation; SD: standard deviation.
The result of analysis of variance showed that the mean of post-intervention TNF-α (MD: −21.35 [−32.29, −10.40], SMD: −1.01 [−1.54, −0.46]) and IL-6 (MD: −9.94 [−18.86, −1.02], −0.58 [−1.09, −0.06]) were statistically different between two groups (Table 2 and Figure 2).
The result of TNF-α (a) and IL-6 (b) before and after interventions. IL-6: interleukin 6; TNF: tumor necrosis factor.
Distribution of primary outcomes according to two arms in addition to related effect sizes.
*Expressed as mean ± standard deviation. The baseline and after values present the result of participants’ assessment before and after 5 days, respectively.
**Analyzed using independent samples t-test.
CI: confidence interval; IL-6: interleukin 6; MD: mean difference; SMD: standardized mean difference (based on Cohen's d test); TNF: tumor necrosis factor.
The results of the independent sample t-test showed that the difference of WBC, ESR, Na, and PO2 was a statistical difference between groups (P < 0.05). Details of the results of the statistical analysis of secondary outcome measurements are presented in Table 3.
Distribution of continuous secondary outcomes according to two arms in addition to related effect sizes.
*Expressed as mean ± standard deviation. The baseline and after values present the result of participants’ assessment before and after 5 days, respectively.
Analyzed using independent samples t-test.
Analyzed using the Mann–Whitney U test.
ALK: alkaline phosphatase; ALT: alanine transaminase; AST: aspartate transaminase; BUN: blood urea nitrogen; CI: confidence interval; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; HCO3: bicarbonate; ICU: intensive care unit; MD: mean difference; mm: cells per cubic millimeter; Na: sodium level; PCO2: partial pressure of carbon dioxide; SMD: standardized mean difference (based on Cohen's d test); SOFA: sequential organ failure assessment; WBC: white blood cell.
Out of 30 participants allocated to the experimental group, 11 were intubated and died until 1 month. In the control group, 9 patients were intubated and then died until 1 month (OR: 0.74 [.25, 2.17], P: 0.58).
Discussion
We aimed to investigate the effects of nebulized HS (5%) on inflammatory markers, ICU and hospitalization, and mortality in adults with severe novel coronavirus disease (COVID-19) pneumonia. TNF-α, IL-6, Na, ESR levels, leukocyte count, and PO2 were significantly different in the intervention group. They were all important. Mortality was slightly higher within the intervention group (11; section 9 within the control group). There were also no significant differences between the study groups in terms of intubation rate, length of hospital stays, or length of stay in the ICU.
Several studies have reported the anti-inflammatory effects of HS. Although not significant, pre-operative intravenous (IV) administration of HS reduced IL-6 and TNF-α levels and increased the anti-inflammatory cytokine IL-10 in patients undergoing coronary artery bypass grafting. 4 Treatment of a patient with pancreatitis with IV HS is associated with a significant reduction in her IL-6 and TNF-α levels within pancreatic tissue. 5 Several animal studies have shown that IV administration of HS may also reduce airway inflammation. 6 For a long, the inhalation of HS has been a helpful and recommendable option in approaching patients with respiratory conditions such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), 4 initially due to its rheological characteristics. Treating patients with CF with inhaled HS reduces the frequency of their respiratory exacerbations and improves lung function.5,6 Several studies have associated inhaled HS with a reduced number of respiratory tract polymorphonuclear cells; neutrophil (PMNs), decreased sputum viscosity and easier sputum expectoration, amelioration of clinical condition, shorter durations of hospitalization, and improved quality of life in patients with bronchiectasis and CF.7–10
HS delivers its anti-inflammatory effects through various mechanisms: IL-10 inhibits the biosynthesis of TNF-α and IL-1β as well as several pathways that involve leukocyte chemotaxis, adhesion, and activation.13–15 Nebulized HS decreases the biosyntheses and expression of several pro-inflammatory agents, namely IL-1α, IL-6, IL-8, IL-33, IL-36, and TNF-α.16–23 It also regulates pathways involved in inflammation and immune responses, like inhibition of T helper 17 (Th-17), lymphocytes, and mitogen-activated protein kinase. 24 Carpagnano et al. 25 reported that inhalation of HS decreases IL-6 and TNF-α in patients with COPD and asthma. Likewise, we found lower levels of TNF-α, IL-6, ESR, and WBC after treating patients with nebulized HS. Also, higher PO2 levels in our intervention group can echo the improvement in lung function which was reported in earlier studies.
Inflammatory markers were significantly reduced after treatment with aerosolized HS, but mortality in the treatment group was not. This may be due to underlying medical conditions in the study participants unknown to us. The novel coronavirus disease (COVID-19) affects multiple systems in the human body, causing a phenomenon known as a “cytokine storm” including a series of severe and intense inflammatory reactions throughout the body. Pneumonia is not the only cause of death in patients who die from COVID-19, so it is important to study the causes of death in patients who die as a result of COVID-19 pneumonia. These include hypotension and organ hypoperfusion, hypercoagulability, altered lipid metabolism, and heart attack. Our limitation is due to the large mortality impact in such studies. So, greater power and larger samples may need to be confirmed. Therefore, we propose to design a similar multicenter study with a larger sample size to answer this question.
Given the attenuating effect of HS on inflammatory conditions, future studies may help determine whether aerosolized HS has a prophylactic role in preventing disease exacerbation in patients with COVID-19.
Conclusion
According to the results, the administration of nebulized HS to patients with COVID-19 pneumonia decreases inflammation and improves respiratory function. Our results are consistence with that of previous studies in terms of how inhaled HS affects the inflammatory state in the inflammatory conditions of the respiratory system. However, we did not observe any difference in mortality and intubation rates and duration of hospital and ICU stays.
Footnotes
Acknowledgements
The researchers sincerely thank all physicians and nurses of the intermediate care wards of IKHC. The authors would like to appreciate the methodologist's support and constructive comments research development office, IKHC, Tehran, Iran.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Ethics approval
This trial was approved by the Ethical Committee of the Tehran University of Medical Sciences (TR.TUMS.IKHC.REC.1400.016).
Research registration number
Author biographies
Mohammad-Taghi Beigmohammadi is a professor of anesthesiology and intensive care and is a fellowship of critical care medicine. He is head of the ICU at Imam Khomeini Complex Hospital.
Laya Amoozadeh has a Ph.D. in physiotherapy and works and research in the school of rehabilitation at Tehran University of Medical Sciences.
Nikoosadat Naghibi is an assistant professor of anesthesiology at Abadan University of Medical Sciences and works in the ICU.
Babak Eslami is an assistant professor of anesthesiology and research on pulmonary diseases.
Samrand Fattah Ghazi is an assistant professor of anesthesiology and research on viral pulmonary infections and treatment.
Mohammad Javaherian has a Ph.D. in physiotherapy and works and research in the school of rehabilitation at Tehran University of Medical Sciences.
Mohammad-Amin Khajeh-Azad is a general practitioner and does statistical analysis as a consultant.
Bahram Tabatabaei has a Ph.D. in physiotherapy and works and research in the school of rehabilitation at Tehran University of Medical Sciences about supplementary treatments.
Alireza Abdollahi is a professor of clinical and anatomical pathology. He is the head of the clinical laboratory and cancer institute pathology laboratory in Imam Khomeini Hospital Complex.
Elham Nazar is an assistant professor of clinical and anatomical pathology and is a fellowship of cytopathology. She researches oncology and viral diseases.