Adjunctive Probiotic Therapy in Major Depressive Disorder (ProMOOD)

Overview

A double-blind, randomized controlled trial (RCT) aimed at evaluating the efficacy of probiotic supplementation in patients undergoing concurrent antidepressant therapy. The study will also assess gut microbiome composition and function, and correlate microbiome changes with clinical and psychological outcomes. Participants will be evaluated by a psychiatrist/clinician, alongside standardized psychological assessments at: * Baseline (inclusion) * Week 4 * Week 12 The following validated instruments will be used: * HAM-D - Hamilton Depression Rating Scale (clinician-rated depression severity) * BDI - Beck Depression Inventory (self-reported severity of depression) * PSS - Perceived Stress Scale (self-reported stress perception) Inclusion Criteria * Age 18-65 years * Confirmed diagnosis of depression according to ICD 11 (International Classification of Diseases) criteria * Written informed consent * Stable antidepressant therapy (same medication for at least 6 weeks to ensure stable clinical response) Exclusion Criteria * Acute suicidality * Severe cardiovascular disease * Pregnancy or breastfeeding * Substance/ alcohol abuse * Severe neurological or systemic disease * Recent antibiotic use * Probiotic use in past year * Treatment resistant depression * Psychotic depression Sample Size * Initial recruitment target: 150-200 participants * Expected dropout rate: approximately 50%, based on similar studies * Final expected sample size: \~100 participants * Sample size calculation based on Nikolova et al., 2023. Primary outcome Change in Hamilton Depression Rating Scale (HAM-D-17) score from baseline to Week 12. Secondary outcomes Change in Beck Depression Inventory-II (BDI-II) score from baseline to Week 12. Change in Perceived Stress Scale (PSS-10) score from baseline to Week 12. Changes in gut microbiome composition and diversity (α-diversity, β-diversity and relative abundance of bacterial taxa) between baseline and Week 12. Safety and tolerability of probiotic supplementation, assessed by adverse events and treatment discontinuation. Intervention: OmniBiotic Stress Repair Microbiome Analysis Subgroup Stool samples will be collected at: * Baseline * 1 month * 3 months Analysis will follow methodologies similar to Mörkl S et al. (2025). Remark: Additional sampling at 1 week (as done in the referenced study) is considered unnecessary, as it falls outside routine clinical monitoring. Antidepressant Strategy * Include patients receiving SSRI (Selective Serotonin Reuptake Inhibitors) and possibly SNRI (Serotonin-Norepinephrine Reuptake Inhibitors) * Advantage: broader applicability * Limitation: increased variability

A prospective, randomized, placebo-controlled, double-blind clinical trial will be conducted. Participants will be randomly allocated to the intervention or control group in a 1:1 ratio. According to the proposed study design, block randomization with a block size of four will be performed using a computer-generated randomization schedule. Allocation concealment will be maintained. Randomization will be performed by the supplier, who will not otherwise be involved in the study as an investigator. The randomization schedule will be unblinded only after the final participant has completed follow-up. The probiotic and placebo products will be comparable in appearance, taste, nutritional composition, and packaging, thereby ensuring the blinding of participants, investigators, and outcome assessors. 4.2 Study population The study will include adult outpatients aged 18-65 years with a confirmed diagnosis of a depressive disorder who are receiving stable antidepressant treatment. Potential participants will be invited to participate during a routine outpatient appointment. The diagnosis will be confirmed by a psychiatrist through a clinical interview and a review of the available medical documentation. Before enrolment, participants will receive both oral and written information about the study and will provide written informed consent. Proposed inclusion and exclusion criteria have already been submitted in the Brief Summary. 4.3 Sample size The sample size calculation will be based on published data from a comparable study published in 2025 (Mörkl S et al). The statistical parameters will be set as follows: a two-sided alpha level of 5%, statistical power of 80%, and a 1:1 allocation ratio. Assuming an attrition rate of 50%, a total of 200 participants are expected to be enrolled, with approximately 100 participants expected to complete the study and be available for the final assessment. The anticipated duration of the study is two years or until the required sample size has been reached. 4.4 Intervention and follow-up In addition to unchanged antidepressant treatment, participants in the intervention group will receive the multi-strain probiotic preparation OMNi-BiOTiC® STRESS Repair as a lyophilised powder, with 7.5 × 10⁹ CFU per sachet (administered twice daily) of the following live probiotic strains: * Lactobacillus casei W56 * Lactobacillus acidophilus W22 * Lactobacillus paracasei W20 * Bifidobacterium lactis W51 * Lactobacillus salivarius W24 * Lactococcus lactis W19 * Bifidobacterium lactis W52 * Lactobacillus plantarum W62 * Bifidobacterium bifidum W23 The additional ingredients include: corn starch, maltodextrin, inulin, potassium chloride, rice proteins, magnesium sulfate, fructooligosaccharides, enzymes (amylases), and manganese sulfate. Participants in the control group will receive a corresponding placebo formulation. The intervention will last 12 weeks. Clinical and psychological assessments will be conducted at baseline and after 4 and 12 weeks. Participants will undergo a clinical assessment at study enrollment, conducted by the treating psychiatrist. Depression severity and perceived stress will subsequently be assessed using the 17-item Hamilton Depression Rating Scale (HAM-D-17), the Beck Depression Inventory-II (BDI-II), and the 10-item Perceived Stress Scale (PSS-10). The clinical assessment and administration of these instruments will be repeated at the scheduled follow-up visits at weeks 4 and 12. Stool samples will be collected at baseline, week 4, and week 12. The samples will be temporarily stored for 1-2 weeks at most in a laboratory freezer (between -20 and -25 degrees C) until they are transported in batches to the AllergoSan facilities in Graz, Austria, where microbiome analyses will be performed. 4.5 Clinical and psychological outcomes The primary outcome will be the change in depression severity, as measured using the HAM-D-17 total score, from baseline to week 12. Secondary clinical outcomes will include the duration of sickness absence as measured in days as stated by the official recors ( on the decision of ZZZS - Slovenian health insurance department); change in self-reported depressive symptom severity, as measured using the BDI-II; change in self-reported perceived stress, as measured using the PSS-10, from baseline to week 12; and the safety and tolerability of the intervention. 4.6 Gut microbiome analysis Stool samples will be analyzed in a randomly selected subgroup of 20 participants. Samples will be collected at baseline and after 4 and 12 weeks. The planned analyses will follow an approach similar to that described by Mörkl et al. (2025). Changes in α-diversity, β-diversity, and the relative abundance of bacterial taxa will be assessed. 4.7 Statistical analysis The primary statistical analyses will be conducted according to the intention-to-treat principle. Accordingly, all randomized participants will be analyzed in the groups to which they were originally allocated, irrespective of their adherence to the intervention or whether they completed the study. For continuous variables, descriptive statistics will be reported as means and standard deviations or as medians and interquartile ranges, depending on the distribution of the data. Categorical variables will be presented as frequencies and percentages. The primary outcome will be analyzed using a statistical method that accounts for repeated measurements and group allocation and permits estimation of between-group differences in changes in the outcome over time. The same analytical approach will be applied to the BDI-II and PSS-10 scores. Microbiome analyses will include measures of α- and β-diversity and differential-abundance analyses, with appropriate correction for multiple testing. Associations between microbiome changes and clinical response will be examined using correlation or regression models, as appropriate. Statistical analyses will be performed using IBM Statistical Package for Social Sciences (IBM SPSS 28.0, SPSS Inc., Chicago, IL, USA).. Statistical significance will be defined as a two-sided p value \< 0.05. Microbiome analyses will be conducted using validated R packages appropriate to the sequencing and bioinformatics pipeline. 4.8 Ethical considerations Participation will be voluntary and based on written, informed, and freely given consent. Participants may withdraw from the study at any time without any consequences for their subsequent medical care. The study has been approved by the National Medical Ethics Committee of the Republic of Slovenia (reference no. 0120-315/2026-2711-3). It will be conducted in accordance with the Declaration of Helsinki, the Oviedo Convention, applicable personal-data-protection legislation, and relevant professional ethical standards. Study data will be anonymized, and the results will be published in a manner that prevents the identification of individual participants.