Comparison of two-month consumption of multi-strain probiotics with placebo for general health and patients with multiple sclerosis

The aim of this study was to compare the effects of two months of taking a multi-strain probiotic with a placebo on the general health and depression of patients with multiple sclerosis. The practical aim of this study is to improve the quality of life of patients with MS by reducing depressive symptoms and increasing their psychological well-being.

A controlled, double-blind, randomized, phase 3 clinical trial with parallel groups on 50 patients. Block Randomization method was used for randomization.

This clinical trial is being conducted at Kashani Hospital. After screening and obtaining informed consent, 50 eligible patients will be enrolled in the study and will be allocated to two groups of 25 (probiotic and placebo) by an independent person using Block Randomization. The study is double-blind; participants, healthcare providers, and data analysts are unaware of the type of intervention. Probiotic and placebo capsules are identical in appearance and packaging and are coded.

inclusion ; Patients with relapsing-remitting multiple sclerosis (RRMS) with a definitive diagnosis by a specialist Patients aged 18 to 65 years. Patients willing and able to participate in the study Patients who report anxiety, depression, and other general health problems as a complication of the underlying multiple sclerosis exclusion; Patients taking probiotics or similar supplements in the past 3 months Patients with depression and anxiety as their chief clinical complaint History of allergy to any component of probiotics or similar drugs

Comparison of two-month consumption of multi-strain probiotics with placebo for general health and patients with multiple sclerosis

General health based on the GHQ-28 questionnaire ;Depression severity based on the BDI-II questionnaire

After screening patients based on inclusion and exclusion criteria and obtaining informed consent, eligible participants are enrolled in the study. The recruitment process continues until a minimum of 50 patients are reached. To allocate participants to the two intervention (multi-drug probiotic) and control (placebo) groups, a block randomization method with four blocks is used to maintain a balance in the number of subjects in the two groups during the recruitment process. The basic idea of ​​block randomization is to divide patients into M blocks of size 2N, such that in each block N patients are assigned to A and N patients to B. The block is then randomly selected. This method ensures equal treatment allocation in each block provided that the block is fully used. . The block size depends on the number of treatments, it should be short enough to avoid imbalance, and large enough to avoid guessing the treatment assignment in each group during the study. The block size should be at least 2 times the number of treatment nodes. The block size was not stated in the study so that the investigators were blinded to it. If the blocks were stated, the treatment series in each block could be guessed. For example, in a 2N=4 block, A A B should be B and in A A it can be inferred as B B. This can lead to selection bias. The solution to avoid this error is to: 1) not disclose the blocking mechanism 2) use random block sizes. The randomization sequence is generated by an independent person who is not involved in the recruitment and evaluation of patients, using reliable randomization software (such as Random Allocation Software or the website randomization.com). The final sequence is stored in the form of a coded list. Allocation Concealment: To prevent bias, the randomization sequence is placed in opaque, light-tight, numbered, and sealed envelopes. Each envelope is opened only at the time of each patient's entry into the study and in numerical order. The person responsible for opening the envelope is a person who is not involved in the outcome assessment. Allocation method: After opening the envelope, the patient is allocated to one of two groups based on the code inside the envelope: - Intervention group: at least 25 people receiving multi-faceted probiotics - Control group: at least 25 people receiving placebo Intervention standardization: The probiotic and placebo are completely similar in shape, color, size, and packaging and are coded to maintain blinding of participants and clinical caregivers.

This study will be double-blind. Participants, clinical caregivers, outcome assessors, and data analysts will be unaware of the type of intervention (probiotic or placebo). In the intervention group, patients will be treated with FamiLact capsules, and in the control group, patients will be treated with placebo capsules. The probiotic and placebo capsules are completely similar and indistinguishable in appearance, size, color, odor, packaging, and method of administration. The probiotic capsule is marketed by Zist Takhim Pharmaceutical Company [Iran] under the brand name FamiLact Capsule®, which was also used in this study. FamiLact® is a synbiotic formula (probiotic + prebiotic). This product contains high levels of safe and beneficial bacterial strains (Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus casei, Bifidobacterium bruchid, Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus plantarum, Bifidobacterium bifidum, Streptococcus thermophilus) along with fructooligosaccharide (FOS) as a prebiotic. In the intervention group, probiotic capsules were administered at a rate of one daily. In the control group, placebo capsules were used instead of Femi Lact capsules, which were completely identical in appearance to the original capsules and were in the form of a hard, two-part, white capsule. The ingredients in the Femi Lact capsules used are as follows: probiotic + prebiotic, and an inactive ingredient that included glycerin, distilled water, and titanium dioxide. Only similar inactive ingredients were used in the placebo capsules. The packaging and dosage form of the probiotic and placebo capsules were identical and indistinguishable, thus maintaining blinding. Both products are packaged with identical codes, the only difference being the specific code used for blinding. The codes for each package (A and B) are assigned by an independent individual who is not involved in the data collection, evaluation, and analysis process and are stored in sealed envelopes. Researchers and patients will have no idea which code is for the probiotic and which is for the placebo.In the data analysis phase, the analysis is performed by a statistical consultant who is aware of the contents of the drug packages; therefore, the study was double-blind and the contents of the two drug groups are not known from the patient's entry into the study to the study and data collection. Maintaining blinding throughout the study: - The intervention and placebo packages are identical in appearance. - The person responsible for distributing the packages is not involved in the outcome assessment. - Participants will not be informed of their intervention type until the end of the study. - Data analysis is also performed based on A and B codes and decoding is done only after the statistical analysis is completed. In the event of a severe complication or a situation where treatment decision-making requires information about the type of intervention, the principal investigator may open the envelope related to that participant in coordination with the ethics committee. These cases will be recorded and reported.

Description of Shared Documents and Data Files In this trial, only de‑identified datasets will be eligible for sharing. Individual Participant Data (IPD), including baseline characteristics, primary outcome scores (GHQ‑28 and BDI‑II), and variables required to reproduce the main analyses, will be shared after complete removal of all personal identifiers. If full de‑identification of the entire dataset is feasible, the complete de‑identified dataset may be shared; otherwise, only the subset of data related to primary outcomes and essential analytical variables will be made available. The following documents may be shared: - De‑identified Individual Participant Data (IPD) - Final study protocol - Statistical Analysis Plan (SAP) - Generic version of the Informed Consent Form (ICF) - Statistical analysis code - Data Dictionary (variable definitions and coding structure) All documents will be shared only upon reasonable written request from qualified researchers, subject to Ethics Committee approval and a signed Data Use Agreement ensuring confidentiality and appropriate use of the data.

Timing of Data and Document Release The release of study documents and datasets will follow a predefined schedule. The access period will begin 6 months after publication of the main trial results and will remain open for at least 3 years after the final manuscript is published. Detailed timeline: - De‑identified Individual Participant Data (IPD): Available 6 months after publication of the primary results. - Final study protocol: Released upon completion of trial registration (IRCT) or at the time of protocol paper publication. - Statistical Analysis Plan (SAP): Available at the time of publication of the main results, typically as a supplementary file. - Generic Informed Consent Form (ICF): Accessible upon publication of the protocol or main article. - Statistical analysis code: Available 6 months after publication of the main results. - Data Dictionary: Released concurrently with the IPD dataset.

Eligibility for Requesting Study Data and Documents Access to the study data and documents will be limited to qualified researchers with a legitimate scientific purpose. Applicants must have an established institutional affiliation and provide a clear research rationale. Eligible groups include: - University faculty members and academic researchers - Researchers affiliated with recognized research institutes - Graduate students, with supervision and approval from their academic advisor - Industry researchers, only if the intended use is non-commercial, scientific, and accompanied by a signed confidentiality agreement Access will not be granted to individuals without research affiliation, to requests lacking a valid analysis plan, or to purposes unrelated to scientific inquiry. All requests require approval from the Ethics Committee and the principal investigators, and data will be shared only after signing a Data Use Agreement (DUA).

Additional Conditions for Using Shared Data and Documents In addition to eligibility criteria for requesting access, the use of de‑identified data and study documents is subject to several scientific and ethical requirements. The following conditions must be met for submitting a request and for using the shared materials: Requirements for submitting a data access request - Submission of a formal written request specifying the research purpose, analysis plan, and required data elements - Demonstration of a valid institutional affiliation (academic, research institute, or non‑commercial industry research) - Provision of a clear and methodologically sound secondary analysis plan - Description of data confidentiality and security measures - Signing a Data Use Agreement (DUA) - Approval by the Ethics Committee and the principal investigators Requirements for using the data and documents - Data may only be used for scientific, academic, and non‑commercial purposes - Analyses must strictly follow the approved analysis plan submitted with the request - Any new or modified analysis requires a new request and ethical approval - Attempts to re‑identify participants are strictly prohibited - Secondary publications must include proper citation of the original trial - Data may not be shared with third parties without explicit written permission - Data must be stored in secure, encrypted environments - Use of documents (protocol, SAP, ICF, analysis code) is limited to reproducing analyses or conducting scientifically valid secondary analyses

Applicant Guide to Requesting Data and Documentation Applicants seeking data or documentation for a study should submit their formal request through the following channels. These channels are listed in order of priority for response: 1. Primary and Priority Channel – Corresponding Investigator Email Name: Dr. Sasan Zandi, Isfahan Email: sasanzandiesfahan@yahoo.vom Description: All requests should be submitted first via email. The request should include the researcher’s introduction, institutional affiliation, scientific objective, and proposed analysis plan. 2. Secondary Channel – Faculty of Medicine Research Office Postal Address: Faculty of Medicine, Islamic Azad University, Najafabad Branch, Daneshgah Boulevard, Najafabad, Iran Email: . 299752@iau.ir Telephone: 00983142292929 Description: Requests that require administrative review or institutional approval will be processed through this channel. 3. Third Path – Research Ethics Committee Postal Address: Research Ethics Committee, Islamic Azad University, Najafabad Branch, Iran Description: Requests that involve access to more sensitive data or require ethical approval are referred to the Ethics Committee after initial review. Important Notes for Applicants - The request must be in writing and include a clear scientific objective. - Submission of a secondary analysis plan is mandatory. - Access is only possible after signing a Data Use Agreement. - Initial response is usually sent within 2-4 weeks.

English Version – Detailed Process for Requesting and Receiving Study Data/Documents Applicants must follow a clear, multi‑step process to obtain access to de‑identified datasets or study documents. The steps below ensure scientific integrity and protection of participant confidentiality. Steps in the Data Access Process 1. Submission of a formal request (1–2 weeks) The applicant must submit a written request including: - Scientific purpose and justification - Proposed secondary analysis plan - Specific data elements required - Institutional affiliation Initial screening typically takes 1–2 weeks. 2. Scientific and ethical review (2–4 weeks) The request will be reviewed by: - The principal investigators - The Research Ethics Committee (if required) This review phase usually requires 2–4 weeks. 3. Signing the Data Use Agreement (1 week) If approved, the applicant must sign a Data Use Agreement (DUA) outlining: - Scientific and non‑commercial use only - Prohibition of re‑identification - Prohibition of sharing data with third parties - Requirements for secure data storage This step typically takes about 1 week. 4. Preparation and release of data (1–2 weeks) After the DUA is signed: - Data are fully de‑identified - Only approved variables are extracted - Files are encrypted and transferred securely This step requires 1–2 weeks. Estimated total time for the full process Approximately 4 to 9 weeks (depending on request complexity, dataset size, and ethical review requirements)