Effects of Nasal-spraying LiveSpo Navax in Treatment of Influenza Virus in Children

Study Overview
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Brief Summary
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Acute Respiratory Tract Infections (ARTIs) in children are common diseases, with influenza virus types A and B is one of the main causes of serious symptoms in young children. Although an influenza vaccine is available, influenza vaccination requires annual injections, which made it difficult for young children to get effectively immunized. Moreover, antiviral nucleotide drugs including Tamiflu (oseltamivir) are unsafe for young children and are recommended only for high-risk patients. Probiotics have emerged as promising safe candidates for supportive treatment of ARTIs and reduction of antibiotic dependence in recent years. Here, investigators propose that direct spraying of probiotics into the nose can be a fast and effective symptomatic treatment for ARTIs due to influenza virus. The aim of the study about to evaluate the effectiveness of nasal-spraying probiotics containing spores of two bacterial strains, Bacillus subtilis and Bacillus clausii in preventing and supporting the treatment of children having acute respiratory symptoms due to influenza infection. Study Population: sample size is 70. Description of Sites: the study is carried out at Vietnam National Children's Hospital. Description of Study Intervention: totally 70 eligible patients are divided randomly into 2 groups (n = 35/group each): Patients in Control group received the routine treatment and three times per day 0.9% NaCl physiological saline while the patients in the Navax group received three times per day LiveSpo Navax in addition to the same standard of care treatment. The standard treatment regimen is 2-5 days but can be extended further depending on the severity of the patient's respiratory failure. Study duration: 15 months

Detailed Description
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Influenza virus types A and B is the common virus that causes Acute Respiratory Tract Infections (ARTIs) with a high risk of bronchiolitis for young children and infants. Influenza-infection symptoms range from mild fever, cough, runny nose, and wheezing to severe symptoms such as fast pulse, fast breathing, and respiratory failure. In a systematic review and meta-analysis of the global burden of seasonal influenza respiratory infections in young children, they estimated that 28.000 - 111.500 children under the age of 5 die as a result of influenza infections in 2008, of which 99% of these deaths occurred in developing countries. Influenza vaccination requires annual injections, which made it difficult for young children to get effectively immunized. Moreover, antiviral nucleotide drugs including Tamiflu (oseltamivir) are unsafe for young children and are recommended only for high-risk patients. Probiotics have emerged as promising safe candidates for supportive treatment of ARTIs and reduction of antibiotic dependence in recent years. However, the use of oral administrative probiotics as functional foods is effective only for mild symptoms and not applicable for Acute RTIs (ARTIs). Here, investigators propose that direct spraying of probiotics into the nose can be a fast and effective symptomatic treatment for ARTIs. The objective was to investigate the symptomatic treatment effects of probiotic product LiveSpo Navax, as a liquid-suspension form containing Bacillus spores of safe B. subtilis ANA4 and B. clausii ANA39 strains, in children having acute respiratory diseases caused by influenza virus, investigators evaluation of improved efficacy and reduced treatment time of LiveSpo Navax in influenza-infected children; and measurement of changes in influenza viral load, co-infectious bacterial concentrations, and major cytokine indicators in the nasopharyngeal samples before and after 2 days using LiveSpo Navax. Methods: A randomized, blind, and controlled clinical trial is conducted. The patient's parents are required to provide the following information about their children: full name, sex, age, obstetric history, vaccination history, and antibiotic use history… After informed consent, 70 patients with ARTIs due to influenza virus will be randomized into 2 groups (n = 35/group): the control group (named Control group) uses 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 5 days, nasopharyngeal samples will be collected at day 0 and day 2 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 influenza viral 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 Flu-induced respiratory tract infections, including runny nose, fever, 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 into 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 influenza viral load and co-infection bacteria concentrations. △Ct for target genes is calculated as Ct (threshold cycle) at day 2 - 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 influenza-infection symptoms about 30% more effectively, as indicated by 90% of patients using LiveSpo Navax (Navax group) are symptom-free at day 2-5 of intervention depending on symptoms, compared to 60% of patients in Control group; (ii) Patients in Navax group has more significant reductions in influenza viral load (>10 fold) than patients in Control group at day 2 of intervention.

Official Title
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Application of LiveSpo Navax in Treatment of Acute Respiratory Disease in Children Infected With Influenza 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 months to 7 years Admitted to hospital due to upper respiratory infection Influenza A and B 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 Having a history of drug allergies Need oxygen therapy Discharged before day 2 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: 7 Years

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®); expectorant Carbocysteine (Carbothiol®); antiviral Oseltamivir phosphate (Tamiflu®); antibiotics e.g. cefotaxim (Goldcefo®), Amoxicillin / clavulanic acid (Augmentin®) 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 billion CFU/5 mL (LiveSpo®️ Navax): Routine treatment is as follows: Oral administrative drugs: antipyretic paracetamol (Efferegant®); expectorant Carbocysteine (Carbothiol®); antiviral Oseltamivir phosphate (Tamiflu®); antibiotics e.g. cefotaxime (Goldcefo®), Amoxicillin / clavulanic acid (Augmentin®) 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 influenza-infected patients with free respiratory symptoms including runny nose, fever, dry rales, moist rales, fast breath, and fast pulse | Day 0 to day 5 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Patient's breath | Influenza-infected patient's breath (beats/min) before and after nasal-spraying | Day 0 to day 5 | 
| Patient's pulse | Influenza-infected patient's pulse (beats/min) before and after nasal-spraying | Day 0 and day 5 | 
| Patient's temperature | Influenza-infected patient's temperature (oC) before and after nasal-spraying | Day 0 and day 5 | 
| Patient's pulse oxygen (SpO2) | Influenza-infected patient's pulse oxygen-SpO2 (%) before and after nasal-spraying | Day 0 to day 5 | 
| Influenza virus concentration | Concentration of influenza virus in nasopharyngeal samples, as indicated by real-time RT-PCR threshold cycle (Ct) value | Day 0 and day 2 | 
| 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 2 | 
| 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 2 | 

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