Materials

Nasal-spraying probiotics LiveSpo Navax (LiveSpo Pharma, Hanoi, Vietnam) was formulated as a 0.9% NaCl physiological saline suspension containing Bacillus subtilis ANA4 (accession no. MT123906.1 in NCBI) and B. clausii ANA39 (accession no. MT275656.1 in NCBI) spores at ≥ 5 × 109 CFU/ 5 mL26. The product was manufactured as a Class-A medical device product (No: 210001337/PCBA-HN) under ISO 13,485:2016 standard. Taste and smell were indistinguishable between LiveSpo Navax and 0.9% NaCl physiological saline (B.Braun, Germany). Because of the opaque plastic container, the color and turbidity of LiveSpo Navax suspension is unrecognizable.

Ethical issues, study design, and patient collection

This study received ethics approval by the Ethics Committee in Medical Research of the Vietnam National Children’s Hospital under Decision No. 441/BVNTW-VNCSKTE, and was conducted with the ethical principles in accordance with the Helsinki statement and the ICH GCP guidelines, the Health Department's current ethical regulations and standards of research using subject’s human. All parents of pediatric patients who volunteered to participate in the study were given information about the study and signed an informed consent form. The study was registered with ClinicalTrials.gov of US. National Library of Medicine with Identifier No: NCT05378022 on 17/05/2022.

This was a double-blind, randomized, controlled intervention pilot study. Patients were randomly assigned to either the control group (named “Control” group) received 0.9% NaCl or the experimental group (named “Navax” group) received the probiotics LiveSpo Navax. The study was implemented at the International Center, Vietnam National Children’s Hospital from December 2020 to April 2022. The size of the patient cohorts (n = 43 per group) was calculated based on a hypothesis that LiveSpo Navax treatment effectively alleviates influenza-infection symptoms by 25% more than standard of care with α = 0.05 and the power level of 0.826. A total of 218 participants were screened for eligibility and 100 eligible participants (n = 50 per group) were randomly assigned by lottery to Control and Navax group to reduce the risk of about 20% patient’s drop out during follow-up treatment. After parents of children signed the informed consent form, the chief nurse randomly selected paper sheets with the coding numbers 1 or 2 from a carton box and immediately assigned the coding number to each participant. The Control and Navax groups were assigned the numbers 1 and 2, respectively, and this information was also confidential to parents of children, nurses, and investigators, with the exception of the principal investigator and the data analyst26. The flowchart of the study design is shown in Fig. 1.

The inclusion criteria for this study included children of both genders, aged between 4 months and 7 years, who were admitted to the hospital due to upper respiratory infections and tested positive for both flu A and flu B through rapid testing and had consents from parents. Exclusion criteria included newborn babies, having a history of drug allergies, the need for oxygen therapy, being discharged before day 2, being lost to follow-up, being withdrawn from the trial, continuing in the trial but missing data, receiving flu vaccination within one year, and meeting the criteria for psychiatric disorders other than depression and/or anxiety.

Questionnaires, treatment procedures, and clinical observation

The patient's parents were required to provide information of their children. Nurses were given coded sprayers in the form of blind samples and were educated to use the sprayers with dosages of about 50 µl 0.9% NaCl physiological saline (with/without 2.5 × 108 Bacillus spores) per each nasal cavity/ time × 3 times/ day directly into the nasal cavity continuously for maximal follow-up 5 days of treatment. As shown in Table 2, the nasal spray products were applied in parallel with routine treatment drugs at hospital. Antibiotics were assigned for all patients who tested positive for co-infection pathogens. For primary outcomes, patients were monitored daily during treatment for typical clinical symptoms of influenza infections, including runny nose, dry rales, moist rales, body temperature (oC), pulse oxymetry (SpO2) (%), pulse (beats/min), and breath (beats/min) until discharged.

Routine diagnostics at hospital

Screening of influenza-infected cases from nasopharyngeal samples at day 0 was firstly conducted by using “BD Veritor System for Detection of Flu A + B” kit (Bection Dickison, NJ, US). Serum C-reactive protein (CRP) concentrations and white blood cell counts were measured to access the level of infection. Cardiopulmonary X-ray was appointed for visualization of lung hyperinflation, osler’s nodes, atelectasis… Due to ethical concerns, all these tests were conducted only at day 0 (Table 2).

Real-time PCR for detection of microorganism in nasopharyngeal samples

DNA/RNA from 200 µl nasopharyngeal specimens (repeated twice) was extracted by MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics, IA, US), and 100 µl of the purified DNA/RNA was aliquoted into three PCR tubes (approximately 30 µl/tube) for storage at – 80 °C.

As secondary outcomes, semi-quantitative assays for measuring changes in viral load of influenza virus (types A and B) and bacterial co-infection concentrations (Streptococcus pneumoniae and Haemophilus influenzae) in nasal tract between days 0 and 2 was conducted by the real-time RT-PCR/PCR, following a similar methodology as described previously26,39,40,41,. The primers and probes used for specific amplification of influenza virus types A, B, and Ribonuclease P as internal control39,40,41 by real-time RT-PCR TaqMan probe was presented in Table S1. Reactions were set up to include an initial reverse transcription at 50 °C for 30 min, 95 °C for 2 min, followed by 45 amplification and detection cycles at 95 °C for 15 s, 55 °C for 30 s. To simultaneously detect multiple bacterial co-infections, the Allplex Respiratory Panel 4 kit (Seegene, Seoul, Korea) was utilized. This commercial kit allows for the detection of seven pathogenic bacterial species, including Bordetella parapertussis (BPP), Bordetella pertussis (BP), Chlamydophila pneumoniae (CP), Haemophilus influenzae (HI), Legionella pneumophila (LP), Mycoplasma pneumoniae (MP), Streptococcus pneumoniae (SP). Reactions following company’s protocol used an initial denaturation at 95 °C for 15 min, followed by 45 amplification and detection cycles at 95 °C for 10 s, 60 °C for 1 min, 72 °C for 10 s. The read-out standardization for the analysis of influenza virus and bacterial co-infection needed to be adjusted to a Ct of < 40 to confirm whether they are a true positive or not. The protocols for influenza virus and the bacterial co-infection detection had been standardized according to ISO 15,189:2012 criteria and routinely employed for testing of clinical samples in Department of Molecular Biology for Infectious Diseases, Vietnam National Children’s Hospital.

Detection of B. subtilis ANA4 and B. clausii ANA39 in nasopharyngeal samples was also conducted at day 0 and day 2 by real-time PCR SYBR Green using primers specific for detection of B. subtilis42 and B. clausii43 (Table S1) at the following condition: 95 °C for 10 min, amplification for 40 cycles at 95 °C for 15 s, 60 °C for 20 s, 72 °C for 30 s. The read-out standardization for B. subtilis and B. clausii analysis was set at Ct < 35 to confirm true positive. The protocol for B. subtilis and B. clausii detection had been developed following the ISO 17,025:2017 guideline and applied for research purpose only at Department of Molecular Biology for Infectious Diseases, Vietnam National Children’s Hospital.

ELISA assays for cytokine and IgA levels

Other secondary outcomes including (i) pro-inflammatory cytokine levels (pg/mL) of interleukin (IL-6, IL-8) and TNF-α, and (ii) IgA levels (mg/mL) in nasopharyngeal samples at days 0 and 2 were quantified using an enzyme-linked immunosorbent assay kit (ELISA). IL-6 and TNF-α were quantified from 100 µL samples by the Human IL-6 DuoSet ELISA and the Human TNF-α ELISA kit, respectively (R&D Systems, MN, US). IL-8 was quantified by 50 µL samples by IL-8 Human ELISA kit (Invitrogen/Thermo Fisher Scientific, MA, US). IgA was quantified by 10 µL samples by Human IgA ELISA kit (Invitrogen/Thermo Fisher Scientific, MA, US).

Data collection and statistical analysis

Individual medical records were collected, and the patient's information was then gathered in a data set. The reduction levels (2Ct) of influenza viral load and bacterial co-infection concentrations. Ct for target genes was calculated as Ct (threshold cycle) at day 2–Ct at day 0 while Ct of internal control was adjusted to be equal among all samples. Tabular analysis was conducted on dichotomous variables using either χ2 test, Fisher’s exact test. The Wilcoxon test or Mann–Whitney test was utilized to compare the medians of quantitative variables within the same group or between two groups. GraphPad Prism v8.4.3 software was used for statistical and graphical analyses (GraphPad Software, CA, USA). All analyses were conducted with a significance level of p < 0.05.

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