With an increased prevalence of autism spectrum disorder (ASD) worldwide and in Taiwan, ASD demands urgent attention due to its lasting impact, unclear causes, and limited diagnostic and treatment options. Recent studies highlight disruptions in the gut-brain axis, particularly in ASD. Building on PI Gau's prior research on altered gut microbiota in autism spectrum disorder (ASD), this proposed 4-year study aims to develop evidence-based probiotic intervention (GKB7) and treatment biomarkers for behavioral interventions (Naturalistic Developmental Behavioral Interventions, NDBI, and Program for the Education and Enrichment of Relational Skills, PEERS®) through comprehensive assessments. The ultimate goal is to establish probiotic treatments and diagnostic/treatment biomarkers for ASD utilizing a deep-learning multimodal auxiliary platform.

Specific Aims:

S.I To conduct a randomized, double-blind, placebo-controlled trial to assess the treatment effects of Clostridium butyricum GKB7 on ASD.

S.II To investigate the effect of NDBI intervention trials using metagenomics as the outcome measure.

S.III To evaluate the effect of PEERS® interventional trials with metagenomics as the outcome measure.

S.IV To develop an artificial intelligence (machine/deep learning) multimodal auxiliary prediction platform that integrates environmental, behavioral/clinical, neurocognitive/imaging, and metagenomic data for ASD diagnosis, subtyping, and impairment prediction.

S.V To identify microbiome and metabolomic biomarkers and endophenotypes of ASD, and to establish Gut-Brain-Behavioral Axes in ASD.

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.

This study consists of two sequential stages with different masking procedures. Stage 1 is an open-label pilot study in which all participants receive GKB7, and both investigators and participants are aware of the assigned intervention. Stage 2 begins with a 3-month randomized, double-blind, placebo-controlled period during which participants are randomized in a 2:1 to receive either GKB7 (n = 60) or placebo (n = 30). Participants, care providers, investigators, and outcome assessors remain blinded to treatment allocation throughout this period. At the end of Month 3, all 90 participants enter a 3-month open-label extension and receive continuous treatment with GKB7. During the open-label extension, treatment assignment is no longer masked.