Effects of Nasal-spraying LiveSpo Navax in Treatment of Acute Respiratory Infections in Children

Study Overview
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Brief Summary
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Rationals: Infection with the Respiratory Syncytial Virus (RSV) is one of the most common causes of respiratory tract diseases. However, treatment for pediatric RSV infection remains supportive to prevent co-infection bacteria and respiratory failure. In recent years, preventive and supportive probiotic therapies for respiratory tract infections (RTIs) have been increasingly strengthened, however, the use of oral administrative probiotics as functional foods is effective only for mild symptoms and not applicable for Acute RTIs (ARTIs). Here, we propose that direct spraying of probiotics into the nose can be a fast and effective symptomatic treatment for ARTIs. Objectives: Investigate symptomatic treatment effects of probiotic product LiveSpo Navax, as liquid-suspension form containing Bacillus spores of safe B. subtilis ANA4 and B. clausii ANA39 strains, in children having acute respiratory diseases caused by RSV: Primary Objective: Evaluation of improved efficacy and reduced treatment time of LiveSpo Navax in children infected with RSV. Secondary Objectives: Measurement of changes in RSV viral load, co-infectious bacterial concentrations, and major cytokine indicators in the nasopharyngeal mucosa before and after 3 days using LiveSpo Navax. Endpoints: Primary endpoint: LiveSpo Navax alleviates RSV-infection symptoms about 25% more effectively, as indicated by 90% of patients using LiveSpo Navax (Navax group) are symptom-free at day 3-6 of intervention depending on symptoms, compared to 65% of patients in Control group. Secondary endpoint: Patients in Navax group had more significant reductions in RSV load (>10 fold) than patients in Control group at day 3 of intervention. Study Population: Sample size is 100. Description of Sites: The study is carried out at Vietnam National Children's Hospital. Description of Study Intervention: Totally 100 eligible patients are divided randomly into 2 groups (n = 50/group each): Patients in Control group received the routine treatment and three times per day 0.9% NaCl physiological saline while the and patients in Navax group received three times per day LiveSpo Navax in addition to the same standard of care treatment. The standard treatment regimen is 3-6 days but can be extended further depending on the severity of the patients' respiratory failure. Study Duration: 12 months

Detailed Description
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Respiratory syncytial virus (RSV) is the most common virus that causes Acute Respiratory Tract Infections (ARTIs) in young children, with a high risk of serious bronchiolitis. RSV infection symptoms range from mild fever, cough, runny nose, and wheezing to severe symptoms such as difficulty breathing and respiratory failure. Cytokines in the airways of children with bronchiolitis, such as tumor necrosis factor (TNF-alpha), Interleukin-6 (IL-6) and IL-8, have been shown to increase at a very high level in primary RSV infection, and extreme elevation of IL-6 is associated with sudden death in children with RSV infection. The World Health Organization (WHO) estimates that 160,000-600,000 children under the age of five die or hospitalize each year as a result of RSV infection. There is currently no vaccine or specific treatment for RSV-infected children because monoclonal antibody palivizumab therapy and antiviral nucleotide drug ribavirin are either too expensive or too dangerous for children and are only recommended for high-risk patients. In recent years, preventive and supportive therapies for respiratory tract infection have grown in popularity, with probiotics emerging as promising safe candidates for therapeutic support and antibiotic reduction. It is suggested that probiotics can capture viruses through direct interactions, or produce secondary growths that inhibit virus growth or stimulate the immune system to capture virus intrusion. However, the efficacy of oral digested probiotics on children's respiratory tracts has been slow to develop (normally it takes about 3-12 months) and is primarily used for prevention rather than supportive treatment of ARITs. As a result, alternative delivery routes for probiotics in the treatment of ARTIs are required. The aim of the study about to evaluate the effectiveness of nasal-spraying probiotics containing two bacterial strains, Bacillus subtilis and Bacillus clausii in preventing and in supporting the treatment of children having acute respiratory symptoms due to RSV infection. Methods: A randomized, blind, and controlled clinical trial are conducted. The patient's parents are required to provide the following information of their children: full name, sex, age, obstetric history, vaccination history, antibiotic use history… After informed consent, 100 patients with ARTIs due to RSV will be randomized into 2 groups (n = 50/group): the control group (named Control group) use 0.9% NaCl physiological saline and an experimental group (named the Navax group) use the probiotics LiveSpo Navax. The patient is given a coded spray in the form of a blind sample to ensure the objectivity of the study. The clinical follow-up will be 6 days, nasopharyngeal samples will be collected at day 0 and day 3 to evaluate potential reductions in viral load and co-infection bacteria, as well as modulation of overreacted cytokine release and the presence of probiotic spores in the patient's nasal mucosa. Real-time PCR for detection of microorganism in nasopharyngeal samples: semi-quantitative assays for measuring changes in RSV load and co-infection bacterial concentrations is conducted by the real-time RT-PCR/PCR routine protocol which has been standardized under ISO 15189:2012 criteria and used in Vietnam National Children's Hospital. Detection of B. subtilis ANA4 and B. clausii ANA39 are also conducted by real-time PCR SYBR Green that has been standardized under ISO 17025: 2017 standard and routinely in the Key Laboratory of Enzyme and Protein Technology, VNU University of Science. ELISA assays for cytokine levels: pro-inflammatory cytokines levels (pg/mL) including interleukin (IL-6, IL-8) and TNF-alpha are quantified using an enzyme-linked immunosorbent assay kit (ELISA) according to the manufacturer's instructions. During treatment, patients are monitored daily for typical clinical symptoms of RSV-induced respiratory tract infections, including runny nose, chest depression, difficulty breathing, dry rales, moist rales, body temperature (oC), oxymetry (SpO2) (%), pulse (beats/min), and breath (beats/min) until discharged. The patients' health conditions are observed by doctors and nurses, and their pieces of information are filled in medical records. During this study, parents' patients are asked to abstain from consumption for their children of other probiotics, either via nasal spray or oral administration and refrain from cleaning nose for their children with other 0.9% NaCl physiological saline sprayers. Data collection and statistical analysis: individual medical records are collected, and the patient's information is then gathered and systematized in a data set. The efficacy of LiveSpo Navax is evaluated and compared to 0.9% NaCl physiological saline based on the following clinical and sub-clinical criteria obtained in Navax and Control groups: (i) the symptomatic-relieving day; (ii) the reduction levels (2^△Ct) of RSV load and co-infection bacteria concentrations. △Ct for target genes is calculated as Ct (threshold cycle) at day 3 - Ct at day 0 while Ct of internal control is adjusted to be equal among all samples; (iii) the reduction levels of IL-6, IL-8, and TNF-alpha cytokines. The tabular analysis is performed on dichotomous variables using the χ2 test or Fisher's exact test when the expected value of any cell is below five. Continuous variables are compared using either the Wilcoxon test, t-test, or the Mann-Whitney test when data are not normally distributed. The correlations among the variables are assessed by Spearman's correlation analysis. Statistical and graphical analyses are performed on GraphPad Prism v8.4.3 software (GraphPad Software, CA, USA). The significance level of all analyzes is set at p < 0.05. P-values. Expected outcomes: (i) LiveSpo Navax alleviates RSV-infection symptoms about 25% more effectively, as indicated by 90% of patients using LiveSpo Navax (Navax group) are symptom-free at day 3-6 of intervention depending on symptoms, compared to 65% of patients in Control group; (ii) Patients in Navax group has more significant reductions in RSV load (>10 fold) than patients in Control group at day 3 of intervention.

Official Title
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Application of LiveSpo Navax in Treatment of Acute Respiratory Disease in Children Infected With Respiratory Syncytial Virus

Conditions
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Acute Respiratory Tract Infections

Intervention / Treatment
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* Combination Product: LiveSpo Navax
* Drug: 0.9% NaCl physiological saline


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Children (male/female) aged from 4 to 60 months. Admitted hospital due to lower respiratory infection. RSV is positive by rapid test. Parents of the pediatric patient agree to participate in the study, explain and sign the research consent form. Exclusion Criteria: Newborn babies. Have a history of drug allergy. Need oxygen therapy. Discharged before day 3. Lost to follow-up. Withdrawn from the trial. Continuing in the trial but missing data. Meeting the criteria for psychiatric disorders other than depression and/or anxiety.

Ages Eligible for Study
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Minimum Age: 4 Months
Maximum Age: 60 Months

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: Blind, randomized, and controlled clinical trial
Masking: Triple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Placebo Comparator: Control<br>Control group receives the routine treatment and uses 0.9% NaCl physiological saline: Routine treatment is as follows: Oral administrative drugs: antipyretic paracetamol (Efferegant®️); anti-inflammatory corticosteroid methylprednisolon; antibiotics e.g. ampicillin and sulbactam complex (Ama-power®️), tobramycin (Medphatobra®️), or cefotaxim (Goldcefo®️), based on the results of antibiotic susceptibility test. Aerosol therapy: bronchodilator e.g. salbutamol (Ventolin ®️inhaler) or budesonide (Pulmicort ®️Respules). | Drug: 0.9% NaCl physiological saline<br>* Nasal-spraying 0.9% NaCl physiological saline is prepared by extracting 5 mL from 0.9% NaCl intravenous infusion 500 mL PP bottle (B.Braun, Germany, product declaration No. VD-32732-19), and then pouring it into the same opaque plastic spraying 10 mL-bottle that is used for LiveSpo Navax.<br>* Other names: Registration number: VD-32723-19;|
| Experimental: Navax<br>Navax group receives the routine treatment and uses NaCl 0.9% plus B. subtilis and B. clausii at 5 billions CFU/5 mL (LiveSpo®️ Navax): Routine treatment is as follows: Oral administrative drugs: antipyretic paracetamol (Efferegant®️); anti-inflammatory corticosteroid methylprednisolon; antibiotics e.g. ampicillin and sulbactam complex (Ama-power®️), tobramycin (Medphatobra®️), or cefotaxim (Goldcefo®️), based on the results of antibiotic susceptibility test. Aerosol therapy: bronchodilator e.g. salbutamol (Ventolin ®️inhaler) or budesonide (Pulmicort ®️Respules). | Combination Product: LiveSpo Navax<br>* In Vietnam, LiveSpo Navax is manufactured as a Class-A medical device product (Product declaration No.210001337/PCBA-HN) under manufacturing standards approved by Hanoi Health Department, Ministry of Health, Vietnam (Certificate No YT117-19) and ISO 13485:2016.<br>* Other names: Registration number: No.210001337/PCBA-HN;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Percentage of patients with free respiratory symptoms | Percentage (%) of RSV-infected patients with free respiratory symptoms including runny nose, chest depression, difficulty breathing, dry rales, and moist rales | Day 0 to day 6 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Patient's breath | RSV-infected patient's breath (beats/min) before and after nasal-spraying | Day 0 to day 6 | 
| Patient's pulse | RSV-infected patient's pulse (beats/min) before and after nasal-spraying | Day 0 to day 6 | 
| Patient's temperature | RSV-infected patient's temperature (oC) before and after nasal-spraying | Day 0 to day 6 | 
| Patient's pulse oxygen (SpO2) | RSV-infected patient's pulse oxygen-SpO2 (%) before and after nasal-spraying | Day 0 to day 6 | 
| RSV concentration | Concentration of respiratory syncytial virus in nasopharyngeal samples, as indicated by real time PCR threshold cycle (Ct) value | Day 0 and day 3 | 
| Co-infection bacterial concentrations | Co-infection bacterial concentrations in nasopharyngeal samples, as indicated by real time PCR threshold cycle (Ct) values | Day 0 and day 3 | 
| Cytokines levels | Levels (pg/mL) of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) in nasopharyngeal samples | Day 0 and day 3 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Respiratory Syncytial Virus (RSV), Acute Respiratory Tract Infections (ARTIs), Children, Nasal-spraying probiotics, Viral load, Co-infection bacteria, Cytokines, Bacillus spores