Exploring the Gut-Brain-Behavior Axis With Biomarkers, Probiotic Efficacy, and Artificial Intelligence Algorithms

We will enroll 110 ASD patients (age 4-15 years) in probiotic clinical trials with Clostridium butyricum GKB7 (S.I), 120 ASD children (2-6 years old) in treatment biomarker of NDBIs study (S.II), and 24 autistic adolescents and 15 young adults in treatment biomarker of PEERS® study (S.III). Assessments include diagnostic evaluations and dimensional measures regarding ASD and other diagnoses (ADI-R, ADOS, K-SADS-E, CGI), digestive symptoms and eating (GSQ, FFQ), autistic symptoms (SRS, CAST, GSQ, SP, EQ, SCQ), psychopathology (CBCL, SNAP-IV), social functions/quality of life (SAICA), neuropsychological functions (CPT, CANTAB, BRIEF), images (fNIRS, T1, T2, rfMRI, DTI, MRS), metabolome (blood) and microbiome (stool). We will conduct multidimensional data analysis to identify the mutual regulation pathways of the gut-brain-behavior axis and key biochemical molecules using machine/deep learning algorithms to establish a multimodal auxiliary platform for ASD classification and prediction (S.IV). We will also use the case-control and longitudinal data to identify microbiota and metabolomics biomarkers and endophenotypes of ASD (S.V).

S.I.1 Pilot Study-Open-Label in 20 autistic children： This pilot clinical trial will employ an open-label study design to explore the effects of a 12-week treatment with the probiotic Clostridium butyricum GKB7 on clinical symptoms as primary outcome, and cognitive function, brain activity, and gut microbiota as secondary outcome measures in 20 autistic children, aged 4-15 years. Participants and their parents will undergo clinical and neuropsychological assessments at baseline, Month 1, Month 2, and Month 3 or the last observation day. MRI scans, and stool and blood samples will be collected at baseline and at Month 3 or the last observation day SI.2 Double-Blind Randomized Placebo-Controlled Trial in 90 autistic children： The objectives of this double-blind randomized placebo-controlled trial are to evaluate the tolerability, safety, and efficacy of the probiotic intervention with Clostridium butyricum GKB7 among 90 4-15-year-old children and adolescents with a clinical diagnosis of ASD according to the DSM-5. They will be randomized to two treatment arms [GKB7, n = 60; the placebo comparison group, n = 30] for three months followed by another 3-month open-label clinical trial as follows. At the end of 3rd month, all the 90 participants will receive continuous treatment with GKB7 for 3 months. In summary, among the 90 ASD participants, 60 will receive GKB7 for 6 months, 30 will have routine follow-up assessments for three months, followed by GKB7 for 3 months. The participants and their parents will receive clinical and neuropsychological assessments at baseline (before treatment, V0), month 1 (V1), month 2 (V2), month 3 (V3, transition to open-label study), month 4 (V4), and month 6 (V5). Besides, the participants will undergo MRI scanning, blood and stool sampling at baseline (before treatment, V0), month 3 (V3), and month 6 (V5).

Inclusion criteria

• Children and adolescents aged 4 to 15 who are clinically diagnosed with ASD according to DSM-5 criteria and confirmed by the ADI-R/ADOS.
• Caregivers cooperate with all the assessments and stool and blood collection.

Exclusion criteria

• A history of major psychiatric disorders (e.g., schizophrenia, bipolar disorder, major depression), neurological disorders, and substances use disorders.
• Difficulty following instructions.
• Consumption of antibiotics and yogurt or probiotic products two weeks prior to enrollment.