Efficacy of LiveSpo Navax in Supportive Treatment of Pneumonia in Children With RSV and Bacterial Co-infections

Study Overview
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Brief Summary
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Respiratory syncytial virus (RSV) infection and bacterial co-infection are the most common causes of pneumonia. Currently, there is no vaccine available for RSV prevention, and the use of the antiviral medication ribavirin is not widely recommended for children. Therefore, the primary treatment approach follows the general protocol for pneumonia, and oxygen therapy is recommended for all cases of pneumonia with respiratory failure. However, in children, the treatment of RSV and bacterial pneumonia remains supportive to prevent bacterial co-infection and respiratory failure. Probiotics have emerged as promising and safe options for supporting the treatment of acute respiratory tract infections (ARTIs) and reducing dependence on antibiotics in recent years. In this study, investigators propose that the direct administration of probiotics through a nasal spray can offer rapid and effective symptomatic treatment for children with pneumonia who require oxygen therapy due to RSV and bacterial co-infections. The aim of the study is to evaluate the effectiveness of nasal-spraying probiotics containing spores of two bacterial strains, Bacillus subtilis and Bacillus clausii (LiveSpo Navax), in preventing and supporting the treatment of severe pneumonia in children (who require oxygen therapy) caused by RSV infection and bacterial co-infection. Study population: The sample size was 100, and the study was conducted at the Vietnam National Children's Hospital. Description of Study Intervention: All 100 eligible patients were randomly divided into two groups (n = 50/each): Patients in the Control group received routine treatment and were administered 0.9% NaCl physiological saline 3 times/day, while the patients in the Navax group received LiveSpo Navax 3 times/day in addition to the same standard of care treatment. The standard treatment regimen typically lasts for 5-7 days, but its duration can be extended based on the severity of the patient's respiratory failure. Study duration: 12 months.

Detailed Description
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Pneumonia is a common disease in children and one of the leading causes of death in young children, especially those under 1 year old, infants, and malnourished children. According to the World Health Organization (WHO), an estimated 12.9 million children die each year, with 4.3 million (33.4%) of those deaths attributed to pneumonia. In Vietnam, the mortality rate from pneumonia is highest among respiratory diseases (75%), compared to the overall mortality rate of 30-35%. Statistics show that on average, a child can experience 3 to 5 episodes of acute respiratory infections per year, including 1 to 2 episodes of pneumonia. Respiratory syncytial virus (RSV) infection and bacterial co-infection are the leading causes of severe pneumonia, and the rate of co-infection can range from 26.3% to 43.6%. Commonly associated bacteria include Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis. These bacteria can invade the lower respiratory tract and cause secondary infections, taking advantage of respiratory damage caused by RSV. Currently, there is no vaccine or specific treatment available for children with RSV infection, and oxygen therapy is generally recommended for children experiencing respiratory failure. The use of the monoclonal antibody palivizumab and the antiviral nucleotide drug ribavirin is considered too expensive or risky for children and is only recommended for high-risk patients. Bacterial co-infections often require antibiotic treatment based on suspected or known pathogens, but the use of antibiotics has significant side effects and raises concerns about the development of resistance. In recent years, probiotics have gained popularity as promising and safe candidates for preventive and supportive therapies in respiratory infections, aiming to aid in the treatment and reduction of respiratory tract infections. Probiotics, which are live microorganisms providing health benefits when consumed in adequate quantities, have traditionally been used to promote gut health. However, their potential role in respiratory infections, specifically RSV pneumonia, has not been extensively explored. Some studies suggest that certain strains of probiotics can directly interact with viruses, capturing them, inducing secondary growth to inhibit virus entry and growth, or modulating the immune response to reduce the risk of respiratory infections. Nevertheless, the impact of oral probiotics on the respiratory tract of infants is typically delayed (usually around 3-12 months) and primarily used for prophylaxis rather than as an adjunct to ARTI treatment. Hence, there is a need for alternative delivery routes of probiotics in ARTI treatment. In our recent study, we have proven that the nasal-spraying Bacillus spore liquid-form probiotics (LiveSpo Navax containing > 5 billion B. subtilis ANA4 and B. clausii ANA39/ 5 mL ampoule) can rapidly and effectively relieve symptoms of ARTIs due to respiratory syncytial virus (RSV) infection while exhibiting strong impacts in reducing the viral load and inflammation. This finding is the first demonstration that spraying probiotics directly into the nose could be a quick and effective symptomatic treatment for ARTIs. In this following study, we conducted the double-blind, randomized, and controlled clinical trial to further examine the efficacy of LiveSpo Navax in supporting the treatment of children with severe pneumonia who require oxygen therapy due to RSV and bacterial co-infections. Methods: A randomized, blind, and controlled clinical trial will be conducted. The parents of the patients will be required to provide various information about their children, including their full name, sex, age, obstetric history, vaccination history, and antibiotic use history. After obtaining informed consent, 100 patients with severe pneumonia due to RSV and bacterial co-infection will be randomly assigned to two groups (n = 50/group): the control group (referred to as the Control group) will receive 0.9% NaCl physiological saline, and the experimental group (referred to as the Navax group) will receive the probiotics LiveSpo Navax. The patients will be given a coded spray in the form of a blind sample to ensure the objectivity of the study. Clinical follow-up will be conducted for 7 days or more, and nasopharyngeal samples will be collected on day 0 and day 3 to evaluate potential reductions in viral load, co-infection bacteria, modulation of overreacted cytokine release, and the presence of probiotic spores in the patient's nasal mucosa. Real-time PCR will be used to detect microorganisms in the nasopharyngeal samples. Semi-quantitative assays will be performed to measure changes in RSV load and co-infection bacterial concentrations using the real-time PCR routine protocol, which has been standardized under ISO 15189:2012 criteria and is routinely used at the Vietnam National Children's Hospital. Detection of Bacillus strains belonging to B. subtilis and B. clausii will be conducted using real-time PCR SYBR Green assay, which is routinely performed at the Spobiotic Research Center in Hanoi, Vietnam. ELISA assays will be used to quantify pro-inflammatory cytokine levels (e.g., IL-6, IL-8, TNF-alpha...) and Immunoglobulin A (IgA) levels. The assays will be performed using an ELISA kit according to the manufacturer's instructions. Nasal microbiome analysis will be conducted using next-generation sequencing (NGS) of the 16S rRNA gene at Macrogen in Seoul, Korea, on the Illumina MiSeq platform with a 2 x 250 bp run configuration. During the treatment, patients will be monitored daily for typical clinical symptoms of severe pneumonia due to RSV and bacterial co-infection, including runny nose, chest depression, dry rales, moist rales, oxymetry (SpO2) (%), pulse (beats/min), and breath (beats/min), as well as the number of days requiring oxygen therapy... until discharge. The patients' health conditions will be observed by doctors and nurses, and their information will be recorded in medical records. Throughout the study, parents will be asked to refrain from administering other probiotics, either via nasal spray or oral administration, and from using other 0.9% NaCl physiological saline sprays for nasal cleaning. Data collection and statistical analysis will involve collecting individual medical records and systematically organizing the patient's information into a dataset. The efficacy of LiveSpo Navax will be evaluated and compared to 0.9% NaCl physiological saline based on various clinical and sub-clinical criteria obtained from the Navax and Control groups. These criteria include the number of days until symptomatic relief, the reduction levels (2^△Ct) of RSV load, and co-infection bacterial concentrations. The △Ct for target genes will be calculated as Ct (threshold cycle) at day 3 - Ct at day 0, while the Ct of the internal control will be adjusted to be equal among all samples. Additionally, the reduced levels of cytokines (e.g. IL-6, IL-8, TNF-alpha...), and IgA will be assessed. The tabular analysis will be performed using the χ2 test or Fisher's exact test for dichotomous variables with expected cell values below five. Continuous variables will be compared using the Wilcoxon test, t-test, or Mann-Whitney test when data are not normally distributed. The correlations among variables will be assessed using Spearman's correlation analysis. Statistical and graphical analyses will be performed using GraphPad Prism v8.4.3 software (GraphPad Software, CA, USA). The significance level for all analyses will be set at p < 0.05. Expected outcomes: (i) LiveSpo Navax is expected to alleviate RSV infection symptoms about 25% more effectively, with 90% of patients in the Navax group being symptom-free at day 3-7 of intervention (depending on symptoms), compared to 65% of patients in the Control group; (ii) Patients in the Navax group are expected to experience more significant reductions (>10 fold) in RSV load compared to patients in the Control group on day 3 of intervention.

Official Title
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Efficacy of Nasal-sprayed Probiotics Containing Bacillus Subtilis and Bacillus Clausii (LiveSpo Navax) in the Supportive Treatment of Pneumonia in Children Infected With Viral Respiratory Syncytial Virus (RSV) and Bacterial Co-infections

Conditions
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Acute Respiratory Tract Infections, Pneumonia, Respiratory Syncytial Virus (RSV)

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


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Children (male/female) aged from 1 to 24 months. Hospitalization due to pneumonia. RSV is positive by rapid test. Bacterial co-infection (Yes or No). Oxygen therapy (Yes or No). Parents of the pediatric patient agree to participate in the study, explain and sign the research consent form. Exclusion Criteria: Children with underlying medical conditions (congenital heart disease, airway malformation). Hospital-acquired pneumonia. Newborn babies. Have a history of drug allergy. 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: 1 Month
Maximum Age: 24 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: Treatment medications: antipyretic paracetamol, antibiotics following the treatment protocol for community-acquired pneumonia in children by the Ministry of Health, and antibiotics with susceptibility results, such as Amoxicillin, Augmentin, or Benzylpenicillin. In cases of severe pneumonia, the following antibiotics may be used: Benzylpenicillin + Gentamicin; Cephalosporins (Cefotaxime, Ceftriaxone) + Amikacin; Oxacillin, Bristopen, Vancomycin if Staphylococcal pneumonia is suspected. Oxygen therapy: Indicated for all cases of severe pneumonia when SpO2 <92%. Use an oxygen mask or nasal cannula. | 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: Treatment medications: antipyretic paracetamol, antibiotics following the treatment protocol for community-acquired pneumonia in children by the Ministry of Health, and antibiotics with susceptibility results, such as Amoxicillin, Augmentin, or Benzylpenicillin. In cases of severe pneumonia, the following antibiotics may be used: Benzylpenicillin + Gentamicin; Cephalosporins (Cefotaxime, Ceftriaxone) + Amikacin; Oxacillin, Bristopen, Vancomycin if Staphylococcal pneumonia is suspected. Oxygen therapy: Indicated for all cases of severe pneumonia when SpO2 <92%. Use an oxygen mask or nasal cannula. | 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 7 | 
| Number of days requiring oxygen therapy | Number of days the patient requires oxygen therapy intervention. | Day 0 to day 7 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Patient's breath | Monitoring the patient's breath (beat/min) on a daily basis during treatment | Day 0 to day 7 | 
| Patient's pulse | Monitoring the patient's pulse (beat/min) on a daily basis during treatment | Day 0 to day 7 | 
| Patient's pulse oxygen (SpO2) | Monitoring the patient's pulse oxygen - SpO2 (%) on a daily basis during treatment | Day 0 to day 7 | 
| Change RSV concentration | Change concentration of respiratory syncytial virus in nasopharyngeal samples, as indicated by real-time PCR threshold cycle (Ct) value at day 3 (after treatment) compared with day 0 (before treatment) | Day 0 and day 3 | 
| Change co-infection bacterial concentrations | Change concentration of Co-infection bacterial in nasopharyngeal samples, as indicated by real-time PCR threshold cycle (Ct) value at day 3 (after treatment) compared with day 0 (before treatment) | Day 0 and day 3 | 
| Change cytokines levels | Change cytokines levels (pg/mL) (e.g., tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8)...) in nasopharyngeal samples at day 3 (after treatment) compared with day 0 (before treatment) | Day 0 and day 3 | 
| Change Immunoglobulin A (IgA) level | Change IgA level (mg/mL) in nasopharyngeal samples at day 3 (after treatment) compared with day 0 (before treatment) | Day 0 and day 3 | 
| Change the nasal microbiota (If any) | Alter the nasal microbiota, as indicated by changes in the diversity of nasal microbial species based on the data analysis of next-generation sequencing (NGS) on day 3 (after treatment) compared to day 0 (before treatment). | 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, Oxy therapy, Nasal-spraying probiotics, Co-infection bacteria, Viral load, Cytokines, Bacillus spores