Recent research links gut microbiota alterations to Autism Spectrum Disorders (ASD), a neurobiological condition with multifactorial bases. In some ASD patients, altered gut flora and increased intestinal permeability are observed, influencing the central nervous system's development and function. Chronic gastrointestinal (GI) symptoms are commonly associated with ASD and correlate with its severity. This non-pharmacological interventional clinical study aims to investigate the role of gut microbiota on ASD and the effectiveness of postbiotic-based dietary supplements in children aged 3-8 years old. Gastrointestinal symptoms, behavioral profile and analysis of intestinal metagenomic and metabolomic profiles will be assessed before and after one-month treatment. The results of the study could enhance understanding of non-pharmacological therapeutic approaches in ASD and improve clinical management strategies and the behavioural functioning for children with ASD.
Modulating the microbiota could be an effective way to improve gastrointestinal issues and related behavioral symptoms in children with ASD, offering new treatment avenues. There is particular interest in alternatives to fecal microbiota transplantation, such as postbiotic supplements, which contain molecules naturally released during microbiota metabolism. These molecules can regulate intestinal homeostasis and microbe-host interactions generating health benefits, possess anti-inflammatory and immunomodulatory activities, and contribute to the barrier activity of the intestinal epithelium. In line with findings for other nutraceutical therapies, dietary supplementation with postbiotics could resolve bowel problems in children with ASD and have positive consequences on behavioral regulation; however, the data collected in the literature is still scarce and limited to studies on mouse models of ASD. This non-pharmacological clinical study, part of a collaboration between Fondazione IRCCS Istituto Neurologico Carlo Besta (FINCB) and Istituto Clinico Humanitas (ICH), evaluates postbiotic-based supplements' impact on gut microbiota's role in ASD. Stool samples will be collected from patients at FINCB and FDG, and transported to ICH for analysis of the intestinal bacterial community and intestinal permeability. Participants will then receive a postbiotic-based dietary supplement. After treatment, changes in bowel habits, intestinal bacteria characteristics, intestinal permeability, and any behavioral changes will be evaluated. Existing literature highlights the protective effects of postbiotics derived from Lactobacillus paracasei CNCM I-5220 on the intestine, specifically contributing to the maintenance of intestinal barrier integrity and protection against leaky gut syndrome. Unlike probiotics, it provides immediate benefits without live bacteria risks, offering a safe alternative for fragile populations. PostbiotiX Comfort®, which includes the same fermented FOS by L. paracasei found in various infant food supplements (such as Smart D3 Matrix, Polivit Immuno Matrix, and Idra Matrix), will be utilized in this study. These products have been safely used in other formulations without any recorded safety concerns and have demonstrated an excellent toxicological profile.
Study Type
INTERVENTIONAL
Allocation
NON_RANDOMIZED
Purpose
TREATMENT
Masking
NONE
Enrollment
90
Treatment with a postbiotic dietary supplement, PostbiotiX Comfort®, administered for 1 month in participants from Group 1 and Group 2.
Participants in Group 3 do not receive any treatment.
Fondazione IRCCS Istituto Neurologico Carlo Besta
Milan, MI, Italy
RECRUITINGIRCCS Humanitas Reasearch Hospital (ICH) Laboratory of Microbiota and Mucosal Immunology
Milan, Italy
RECRUITINGModulation of Gastrointestinal Symptoms in ASD with Postbiotic Supplement
The presence of gastrointestinal symptoms will be assessed at recruitment (T0) using the GI Severity Index. Subjects recruited will be treated with the postbiotic-based dietary supplement, PostbiotiX Comfort®, once daily for 1 month. The GI Severity Index will be repeated within one week after the end of the treatment (T1) to quantify changes in bowel regulation and one month after the end of the treatment (T2) to assess the stability of the observed changes.
Time frame: 24 months
Effects of postbiotics on sleep regulation
Standardized behavioral assessments will be conducted through parents/caregivers reported questionnaires to evaluate the effects of postbiotic supplementation on sleep regulation. Sleep disturbances will be assessed through validated instruments designed to investigate difficulties related to sleep onset and maintenance, respiratory sleep disturbances, disorder of arousal, wake-sleep transition, excessive daytime sleepiness, and nocturnal hyperhidrosis. Sleep regulation will be assessed using the italian version of the Sleep Disturbance Scale for Children. The scale consists of 26 items rated on a 5-point Likert scale (1 = never; 2 = occasionally; 3 = sometimes; 4 = often; 5 = always). The total raw score ranges from 26 to 130, with higher scores indicating greater severity of sleep disturbances. Assessments will be conducted at three time points: baseline (T0), at enrollment in the interventional study; post-treatment (T1), within one week after the end of treatment; and follow-up (T
Time frame: 24 months
Effects of postbiotics on behavioral regulation
Standardized behavioral assessments will be conducted through parents/caregivers reported questionnaires to evaluate the effects of postbiotic supplementation on behavioral regulation. Emotional and behavioral regulation will be assessed through validated instruments designed to investigate the presence and severity of emotional and behavioral problems, including internalizing and externalizing symptoms. Behavioral and emotional problems will be assessed using the Italian versions of the Child Behavior Checklist (CBCL) according to age. The instrument consists of items rated on a 3-point Likert scale (0 = not true; 1 = somewhat or sometimes true; 2 = very true or often true). Higher scores indicate greater severity of emotional and behavioral problems. Raw scores will be converted into standardized T-scores (mean = 50, standard deviation = 10). Assessments will be performed at three time points: baseline (T0), at enrollment in the interventional study; post-treatment (T1)
Time frame: 24 months
Effects of postbiotics on sensory profile
Standardized behavioral assessments will be conducted through parents/caregivers reported questionnaires to evaluate the effects of postbiotic supplementation on sensory profile. The sensory profile will be assessed using the Sensory Profile-2 (Italian version), completed by parents/caregivers for children aged 3-8 years. The questionnaire evaluates sensory functioning, self-regulation behavioral responses and the frequency of behavioral responses to environmental sensory stimuli. Raw scores are calculated for each sensory domain; higher scores indicate greater deviation from typical sensory processing patterns. Assessments will be performed at three time points: baseline (T0), at enrollment in the interventional study; post-treatment (T1), within one week after the end of treatment; and follow-up (T2), within one month after the end of treatment to assess the stability of the observed changes. All questionnaires completed by the parents/caregivers will subsequently be discussed with
Time frame: 24 months
Metagenomic analyses in typically developing children and in ASD before and after postbiotic treatment
Fecal samples obtained from control subjects and children with ASD with and without gastrointestinal symptoms. For the latter group, samples will be collected both prior to the initiation of supplement therapy (T0) and after treatment (T1). Taxonomic and functional profiling of the fecal microbiota will be performed using shallow shotgun metagenomic sequencing. Total bacterial DNA will be extracted from fecal samples and sequenced using Illumina platforms. Taxonomic analyses will be performed at Species-level Genome Bin (SGB) resolution (8th taxonomic rank). Functional analyses will be based on destratified gene family and metabolic pathway abundances, with pathway annotation performed against the MetaCyc database. Differential abundance analyses (DAAs) will be performed on taxonomic profiles expressed as relative abundances and on gene family and pathway profiles normalized to copies per million (CPM) Unit of measure: Relative abundance of bacterial taxa (%)
Time frame: 24 months
Metabolomic analyses in typically developing children and in ASD before and after postbiotic treatment
The following laboratory tests will be performed on fecal samples obtained from control subjects and children with ASD with and without gastrointestinal symptoms. For the latter group, samples will be collected both prior to the initiation of supplement therapy (T0) and after treatment (T1). Untargeted metabolomic analysis will be performed on fecal samples to identify metabolites of bacterial origin. Metabolites will be detected and quantified using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), followed by bioinformatic analysis. Unit of measure: • Relative or absolute metabolite abundance (normalized peak intensity/concentration)
Time frame: 24 months
Intestinal permeability analyses in typically developing children and in ASD before and after postbiotic treatment
The following laboratory tests will be performed on fecal samples obtained from control subjects and children with ASD with and without gastrointestinal symptoms. For the latter group, samples will be collected both prior to the initiation of supplement therapy (T0) and after treatment (T1). Intestinal permeability will be assessed by quantifying fecal levels of zonulin and albumin using commercially available enzyme-linked immunosorbent assays (ELISA). Increased fecal zonulin levels will be interpreted as indicative of tight junction disassembly and increased epithelial permeability. Elevated fecal albumin concentrations will be interpreted as a marker of intestinal barrier dysfunction associated with inflammatory leakage across the intestinal mucosa . Both biomarkers will be analyzed as indicators of altered intestinal permeability and gut barrier integrity. Unit of measure: * Zonulin family peptides: concentration (e.g., ng/mL or ng/g of feces) * Albumin: concentration (e.g., ng/mL
Time frame: 24 months
Investigation of the relationships between metagenomic/metabolomic profiles and behavioral/GI outcomes in children with ASD
Correlation analyses will be performed to investigate the relationships between metagenomic and metabolomic profiles and behavioral and gastrointestinal outcomes in children with ASD. This initial analysis will enable the identification of significant associations between microbial diversity, metabolite abundance, and clinical measures potentially modulated by postbiotic treatment. Metagenomic, metabolomic, and intestinal permeability data will then be integrated with behavioral outcomes using advanced computational multi-omics approaches. Following data normalization and harmonization through standardized pipelines, metagenomic and metabolomic datasets will be combined using network-based integration strategies and multivariate methods, including Canonical Correlation Analysis (CCA), sparse Partial Least Squares (sPLS), and Multi-Omics Factor Analysis (MOFA), to identify shared functional signatures, key microbial-metabolic interactions, and convergent pathways across datasets. Cross
Time frame: 24 months
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