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.