Dipartimento Area Materno Infantile S.C. Pronto Soccorso Pediatrico - Direttore: Prof. Carlo V. Agostoni Tel. 02 5503.2349 E-mail: [email protected]

The original version of the full study protocol was in Italian (attached below). We provide a translated version in English.

Effect of Supplementation with a Mixture of Probiotic Strains (Bifidobacterium breve M16V, Bifidobacterium lactis HN019, Lactobacillus rhamnosus HN001) in Febrile Children with Upper Respiratory Tract Infections

Acronym: PROBIOS2021 Sponsor: Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Via Sforza 28, 20122 Milano, Italy Coordinating Center: Pediatric Emergency Room Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Via della Commenda 9, 20122 Milano, Italy Principal Investigator: Carlo Agostoni Document Type: Non-Pharmacological Interventional Study Protocol Version Number: v.1.0 Date: 12/05/2021

CONFIDENTIALITY STATEMENT All information presented in this document will be considered confidential and will remain the exclusive property of Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. The use of such confidential information should be limited to the recipient for the agreed purpose and must not be disclosed, published, or otherwise communicated to unauthorized persons for any reason in any form without prior written consent from Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.

FLOWCHART

INDEX

1. 1. INTRODUCTION

1. 2.2 Secondary Objective(s)
2. 3.1 Study Design

1. 3.2 Inclusion Criteria

1. 3.3 Exclusion Criteria

1. 4. STUDY PROCEDURES
2. 5. ENDPOINTS
3. 6. STUDY DURATION/TIMELINE
4. 7. STATISTICAL ANALYSIS
5. 8. ADVERSE EVENTS
6. 9. RISK/BENEFIT ASSESSMENT
7. 10. STUDY MANAGEMENT

1. 10.1 Data Collection and Management
2. 10.2 Regulatory and Ethical Considerations
3. 10.3 Investigator Duties
4. 10.4 Study Monitoring

1. 10.5 Quality Assurance
2. 10.6 Study Closure
3. 10.7 Document Archiving
4. 10.8 Information Disclosure
5. 10.9 Intellectual Property Rights

1. 11. Compensation for Damages
2. 12. Protocol Amendments
3. 13. Financial Agreements
4. 14. Conflict of Interest Disclosure
5. 15. References
6. 16. Appendix

 Carlo Agostoni: Principal Investigator

1. 1.1 Background and Rationale

The human body is the habitat of billions of microorganisms that together form an ecosystem called the microbiota. The gastrointestinal tract is the compartment with the highest density of microbial cells and is the most studied portion to date (1). The gut microbiota is increasingly considered an "invisible organ" of the human body essential for host health; a symbiosis is created between bacteria and the host where the body provides nutrients necessary for bacterial survival and development, and microorganisms produce substances necessary for the development and well-being of the individual (2). At birth, the newborn’s gastrointestinal tract is virtually sterile but is quickly exposed to colonization by opportunistic bacteria from the maternal vaginal, fecal, or skin microbiota; indeed, there is a significant similarity between the newborn's microflora and the mother’s, consistent with previous evidence of vertical transmission of microorganisms (3). Thus, the mode of delivery influences the composition of the newborn’s microbiota. During the first 24 hours of extrauterine life, the gastrointestinal tract is mainly colonized by facultative anaerobes such as Streptococci, Enterococci, and Escherichia coli; later, obligate anaerobes such as Bifidobacteria and Lactobacilli settle in (4). The development of the bacterial flora is also influenced by the infant’s diet, i.e., breastfeeding or formula feeding. Numerous studies have demonstrated the different composition of the intestinal microbiota of breastfed versus formulafed individuals. Breast milk is rich in monosaccharides such as lactose, glycolipids, glycoproteins, sialic acid, fucose, oligosaccharides, and N-acetylglucosamine (3). These components, called prebiotics, reach the intestine undigested and serve a defensive function by creating a shield

against pathogenic bacteria. Moreover, since bacteria can ferment milk compounds, there is an increase in fermentative microorganisms with a parallel decrease in putrefactive ones and a reduction in pH. Under these conditions, there is an improvement in digestive and absorption functions, a relative stimulation of the immune system, and a reduction in the risk of allergies and vitamin deficiencies (5).

The introduction of solid foods complementary to milk has been correlated with a reduction in variability of the gut microbiota among individuals (6). However, the maturation of the intestinal microbial community is generally reached around 2 years of age (7). The gut microbiota tends to maintain a certain homeostatic state throughout life; that of children, unlike that of adults, which is considered relatively stable, is highly dynamic and prone to alterations. Numerous factors can modulate this maturation, including the family environment in which the child is placed and other influences related to culture, traditions, and geographic area. Another external factor that can alter the composition of the microbiota is inflammation. Inflammation is a biological response to the activation of the immune system after contact with infectious external agents.

Probiotics, according to the WHO (World Health Organization), are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (8). It is known that probiotics can strengthen the immune response by restoring symbiosis within the gut microbiota, thereby aiding immune protection against various pathogens, including those responsible for respiratory infections (9).

Finally, exposure to antibiotics can also alter the composition of the gut microbiota. Antibiotics (substances produced by bacteria, fungi, or synthetic origin capable of inhibiting the growth of other microorganisms) are the most commonly used drugs in neonatal and pediatric populations in Western countries. Antibiotic therapies can alter the initial microbial composition and cause loss of colonization resistance (3). Consequently, the host may become susceptible to colonization by one or more pathogens. Since the composition of the infant gut microbiota is very important for maintaining an optimal state of health, it is equally important to accurately identify the bacterial species residing in the gastrointestinal tract at various times of life, both in good health and in

pathological conditions. The first years of life are thus the most critical and fundamentally important period for the stabilization of the microbiota (4).

Evaluation of the Effects of the Probiotic Formulation Ofmom Synteract Bimbi on Children’s Health Status. The hypothesis posits that children receiving the probiotic mixture will experience a shorter duration and lower intensity of fever compared to those in the placebo group. Furthermore, the probiotic formulation may contribute to the prevention of gastrointestinal disturbances commonly associated with antibiotic therapy.

1. 2.1 Primary Objective
2. 3.1 Study Design This is a prospective, biological, randomized, controlled, non-pharmacological interventional study with a 1:1 allocation ratio in triple-blind conditions. The study duration for each patient will be 14 days (administration of Ofmom Synteract Bimbi, one stick per day or five drops per day). Probiotics will be administered after the first stool sample is collected. The study will be proposed to parents during their visit to the Pediatric Emergency Department, where they

will complete informed consent. After 12 months, the parents of the recruited patients will be recontacted and offered the third stool collection (T2). Parents will complete informed consent at this time as well.

1. 3.2 Inclusion Criteria Patients who meet the following criteria will be included and asked to participate voluntarily:
2. 4.1 Intervention

Parents of patients meeting the inclusion criteria will be asked to enroll their child/ward in the study. Upon arrival at the emergency department (ED), patients will undergo a medical evaluation to determine whether antibiotic therapy is required. Patients will then be randomly assigned to either the intervention group or the placebo group. The intervention group will be asked to collect a stool sample within 24 hours of ED admission (T0) and, after collection, to begin a 14-day

regimen of the Ofmom Synteract Bimbi probiotic mixture (one sachet or five drops per day). At the end of the intervention period (T1), a second stool sample will be collected. The placebo group will also be required to provide two stool samples (T0 and T1) and will receive a placebo formulation consisting of Medium Chain Triglycerides (MCT) (one sachet or five drops per day). After 12 months, parents of participants who completed the 14-day regimen (either probiotic or placebo), provided both stool samples (T0 and T1), and submitted them for analysis will be contacted for a follow-up study. They will be asked to collect a third stool sample (T2) at the 12month mark. The sample collection kit will either be provided at the time of recruitment in the ED or delivered to the participant’s home. Parents will collect the stool sample at home, and they may either deliver the refrigerated sample to the ED during a scheduled follow-up visit or arrange for courier shipment of the sample.

1. 4.2 Randomization Enrolled subjects will be progressively assigned to a treatment group based on a computergenerated numerical sequence, with a 1:1 allocation ratio.

1. - 48 subjects will receive supplementation with Ofmom Synteract Bimbi (one sachet per day or five drops per day) (technical data sheet attached).
2. - 48 subjects will receive the placebo (technical data sheet attached).

Neither the investigator, the patient, the data manager, nor the personnel responsible for transportation will be aware of the treatment assignment. Both the probiotics and the placebo will be packaged in identical, neutral containers labeled only with the letters “A” or “B.” The random allocation sequence will be generated by AAT, which will be responsible for safeguarding the randomization codes and conducting the decoding process at the end of the study.

1. 4.3 Blinding Participants, all healthcare professionals providing care, researchers, and data managers will remain blinded to the group assignment (placebo or intervention) throughout the duration of the trial.

1. 5.1 Primary Endpoint

Duration and degree of fever.

1. 5.2 Secondary Endpoints
2. 6. STUDY DURATION / TIMELINE
3. 7. STATISTICAL ANALYSIS

1. 7.1 Sample Size The sample size calculation is based on the primary endpoint, specifically the duration of fever. The estimates are based on an average fever duration in children with upper respiratory tract infection in ED departments of 4 days (SD 2.6) (10). Data will be analyzed using intention-to-treat analysis. Assuming a reduction in fever duration in the intervention group by 37% (SD 1.5) and considering a Type I error of 0.05 and Type II error of 0.20, the calculated number of children to recruit is at least 48 per group.
2. 7.2 Data Analysis Categorical or ordinal variables will be expressed as frequency (percentage, %); continuous variables as mean and standard deviation if normally distributed, and as median and interquartile range if not. Within-group and between-group comparisons will be performed using parametric or non-parametric statistical tests, as appropriate: for between-group comparisons, the Student's t-test (2 groups) or ANOVA (>2 groups) will be used for normally distributed continuous variables, the Mann-Whitney test (2 groups) or Kruskal-Wallis test (>2 groups) for asymmetrically distributed continuous variables, and the Chi-square test or Fisher's exact test (as appropriate) for categorical variables. The level of statistical significance is set at a p-value < 0.05, and where appropriate, 95% CIs will be calculated. Statistical software used will be SPSS (Statistical Package for Social Science version 20) and R Project for Statistical Computing.

Interim analyses will be performed to decide whether it is appropriate to terminate early or continue recruitment beyond the defined end of the trial. If there are deviations from the original statistical plan, the interim analyses will be described and justified in the final report, in the most appropriate manner.

The project does not involve the administration of drugs or other substances nor invasive clinical practices. Therefore, no adverse events are expected. Notification of any adverse events will follow current regulations in phytovigilance for products authorized for market release.

The probiotic bacterial strains contained in the Ofmom Synteract Bimbi product (Lactobacillus rhamnosus HN0019, Bifidobacterium lactis HN001, and Bifidobacterium breve M16V) have been widely used for years in the Italian market as probiotic supplements by various companies. No risks have emerged related to the use of these strains in humans. The product's contribution to the patient is based on rebalancing their intestinal microbiota through a significant intake of bifidobacteria, which are a primary component of a child’s intestinal microbiota, providing essential support for the digestion of breast milk and formula milk and for the proper development of the immune system. Therefore, it is believed that the intake of the probiotic mixture can help the child's immune system, both mucosal and systemic, to react to bacterial and viral infections.

1. 10.1 Data Collection and Management All data will be collected by designated evaluators using specifically developed data collection forms (Case Report Form, CRF). All collected data will be processed, analyzed, and stored confidentially. Each participant will be assigned a unique code at the time of enrollment. The file associating the participant's code with their identifying data will be stored separately on a password-protected computer accessible only to study personnel designated by the principal investigator. The data will be de-identified so that individuals accessing the database cannot trace the identity of the subjects. Only local investigators will be able to trace the identity of enrolled subjects.
2. 10.2 Regulatory Aspects and Ethical Considerations

1. 10.2.1 Competent Authority Approval In accordance with current regulations, the principal investigator must obtain approval from the appropriate Competent Authority before starting the clinical study. This study will be conducted in accordance with ICH/GCP (International Conference of Harmonization/Good Clinical Practice) guidelines and all applicable laws, including the Declaration of Helsinki of June 1964, as amended by the most recent World Medical Association General Assembly in Seoul, 2008.

1. 10.2.2 Ethics Committee Approval The investigator must ensure that the protocol has been reviewed and approved by the local independent Ethics Committee (EC) before starting the study. The EC must also review and approve the informed consent form (IC) and all written information provided to the patient before enrollment in the study. If it is necessary to amend the protocol and/or IC during the study, the investigator will ensure the review and approval of such modified documents as requested by the EC. The content of such amendments will be implemented only after EC approval. Until then, the previously approved document version must be referred to.
2. 10.2.3 Informed Consent (IC) The investigator or designated personnel is responsible for informing individuals about all aspects and procedures of the study. The investigator will seek the willingness to participate or not in the study and obtain informed consent. The process of obtaining informed consent must comply with current regulatory procedures. The investigator (or a designated collaborator) and the subject must date and sign the informed consent form before the patient initiates any study-related procedures. The subject will receive a copy of the IC dated and signed by both parties; the original copy will be stored in the study's designated archives. Neither the investigator nor designated personnel should in any way exercise any coercion or influence on a subject to induce them to participate or continue to participate in the study. A subject's decision to participate in the study must be completely voluntary. The investigator and designated personnel must emphasize to the subject that they can withdraw their consent at any time without any penalty or loss of any benefits they may be entitled to. Written or oral information about the study, including the written consent form, must not contain any language that coerces the subject to waive (even apparently) their legal rights or that exonerates the investigator, entity, or sponsor from liability for negligence. Patients involved in the study for a total period of 14 days will be asked to provide three fecal samples (T0, T1, and T2).

1. 10.3 Investigator Responsibilities In accordance with applicable local regulations, the investigator must submit periodic reports on the progress of the study at their center to the EC and notify them of the study's closure. Periodic reports and closure notification are part of the investigator's responsibilities.

1. 10.4 Study Monitoring In accordance with applicable regulations and good clinical practice (GCP), the monitor must visit or contact the center periodically. The duration, nature, and frequency of such visits/contacts depend on recruitment frequency, the quality of documents held by the center, and their adherence to the protocol.
2. 10.5 Study Quality Assurance

As the sponsor, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico may, at its discretion, conduct a quality control of the study. In this case, the investigator must allow the

monitor direct access to all pertinent documentation and dedicate part of their time and staff to the reviewer to discuss monitoring results and any other aspects of the study. Additionally, regulatory authorities may conduct inspections. In this case, the investigator must authorize the inspector direct access to all pertinent documentation and dedicate part of their time and staff to the inspector to discuss monitoring results and any other aspects of the study.

1. 10.6 Study Closure Upon study closure, the monitor and investigator must activate a series of procedures:
2. 10.7 Document Archiving In accordance with current national regulations, the investigator must retain a copy of all documentation and store it in a dry and safe place after the study's closure.
3. 10.8 Disclosure of Information Regarding Scientific Findings

1. 10.8.1 Confidentiality The investigator and other personnel involved in the study must treat all study-related information (including the protocol, obtained data, and all documentation produced during the study) confidentially and must not use such information, data, or reports for purposes other than those described in the protocol. These restrictions do not apply to:
2. 10.8.2 Study Publications

Any publication of the study results (such as a manuscript or oral presentation) generated by the investigator must be submitted for review to the sponsor at least 60 days before submission for publication. The sponsor may require deletions and/or modifications if the publication contains confidential information or patentable material, which must be removed before publication. If the sponsor does not notify the investigator within the above 60 days of its objections, the publication can be submitted. If the sponsor provides comments within this period, the investigator must consider these comments in good faith. However, the investigator retains the right to submit the publication without the sponsor's consent. The study results will be published regardless of the study outcome.