Autism Spectrum disorder (ASD), is a group of neurodevelopmental abnormalities that begin in early childhood, frequently characterized by problems in communication and social behavior. Metabolites produced by the gut microbiome may affect gastrointestinal (Gl) tract and central nervous system (CNS) functions supporting the presence of the gut- brain-axis. Previous studies have demonstrated changes in the intestinal microbiome in children with ASD compared with neurotypical controls. It is possible that dietary modifications, including the use of probiotics, in children with ASD could positively modify Gl and CNS functions. Few clinical studies have been carried out to assess the effects of probiotic consumption by children with ASD on their microbiota composition, intestinal function and behavior in developing countries. Consequently, the general objective of the study is to determine the effects of probiotics supplementation in gut microbiota composition, nutritional status and immune status in children with ASD.

A randomized crossover study will be conducted including children diagnosed with ASD, living within the Metropolitan District of Quito. After verifying the inclusion and exclusion criteria, 66 participants will be selected. Initially, children diagnosed with ASD will be randomly organized into 2 groups. Group A (n=33) will receive Sb supplements at a dose of 250 mg 3 times a day for a period of 4 months. While Group B (n=33) will not receive supplementation for 4 months. This will be followed by a 30-day "washout" period, during which time neither group consumes probiotics. After this time, the children's groups will change treatment for another four months. The investigators will analyze behavior, gut microbiome, metabolome, nutritional status and gastrointestinal symptoms to determine the changes caused by Sb consumption. Participants will be followed up at months 1, 4, 5, 9 10 of the study. At each follow-up, stool, blood and urine samples will be collected for the different analyzes.

A sociodemographic survey will be applied to determine the epidemiological parameters of the participants. The behavioral parameters of each child will be evaluated by means of psychological assessments. Tests that will be applied are: Autism Diagnostic Observation Schedule, Second Edition (ADOS- 2); Autism Diagnostic Interview-Revised (ADI-R); Wechsler Intelligence Scale for Children (WISC); Adaptive Behavior Assessment System (ABAS); Wechsler Preschool and Primary Scale of Intelligence (WPPSI); Battelle Developmental Inventory (BATELLE). For the evaluation of nutritional status, anthropometric measurements (weight, height, BMI) will be taken and percentiles and Z-scores will be calculated. In addition, a 24-hour recall questionnaire will be applied. Roma IV test will be applied to evaluate gastrointestinal symptoms. To determine the immunological status, the investigators will measure interleukins and growth factors in blood.

Regarding the analysis of the gut microbiota, the investigators will start with DNA extraction from stool samples. All raw sequences obtained from the Illumina sequencer will be analyzed to determine: taxonomic composition, alpha and beta diversity, functional annotation and differential microbial relative abundance between the two groups. Functional annotation will be performed using Human Microbiome Project Unified Metabolic Analysis Network (HumanN) software. Taxonomic assignment will be done using Metagenomic Phylogenetic Analysis (Metaphlan) software. Diversity metrics will be calculated using the Vegan diversity R package. To compare alpha diversity analysis, Chao 1, Shannon and Simpson's index will be calculated. For Beta diversity comparison, the Analysis of Ecological Data (ade4) function in the R package will be used to generate principal component analysis using Bray-Curtis, Jaccard, unweighted Unifrac and weighted Unifrac distance metrics. Linear discriminant analysis will be used in conjunction with the sample size effect measurement test (LEfSe) to perform differential microbial relative abundance analysis between the two groups.

The investigators will perform metabolome analysis on blood, fecal samples and urine. Metabolome assignment will be by 2D experiments based on signal in Proton nuclear magnetic resonance (H-NMR), using the human metabolome database and the biological magnetic resonance metabolome database.