This paper describes the protocol for a multicentre prospective longitudinal study (CARE/MiCrobiota-gut-brain Axis in Resistant Epilepsy) investigating how gut microbial features and associated host epigenetic signatures affect clinical outcomes in people with epilepsy undergoing diverse treatment strategies.
Key Findings
Background
Approximately one-third of people with epilepsy experience resistance to treatment across multiple therapeutic modalities.
Treatment modalities affected by resistance include pharmacological therapies, epilepsy surgery, vagus nerve stimulation (VNS), and dietary interventions such as the ketogenic diet (KD).
This treatment resistance rate is cited as the motivation for investigating alternative mechanistic explanations such as the microbiota-gut-brain axis.
The study background frames treatment resistance as an unresolved clinical problem necessitating new biomarker identification.
Methods
The study will enroll participants aged 3–50 years stratified into four distinct intervention cohorts based on treatment type.
Cohort 3: DRE receiving vagus nerve stimulation (VNS).
Cohort 4: DRE initiating the ketogenic diet (KD).
The study involves four clinical centres in Italy and one self-financing partner.
Methods
Biological and clinical data will be collected at baseline and at two or three longitudinal timepoints over a 12-month observation period.
Biological samples collected include stool and blood.
Clinical assessments include BMI calculation, self-reported monthly seizure count, dietary evaluation, quality of life scale, and gastrointestinal symptoms scale.
Neurological assessments include electroencephalography (EEG) and MRI.
The study design is described as multicentre, prospective, and longitudinal.
Methods
Gut microbiota will be characterized using metagenomics via 16S ribosomal RNA sequencing and metaproteomics.
16S ribosomal RNA sequencing is specified as the metagenomics methodology.
Metaproteomics will be used alongside metagenomics to assess gut microbiota changes over time.
Host DNA methylation profiles will be obtained from blood using Illumina EPIC arrays.
Multi-omics integration approaches will be used to combine omics data with corresponding metadata.
Methods
The primary endpoints are identification of microbial or host methylation changes predictive of therapeutic response, defined as reduction from baseline in monthly seizure count.
Therapeutic response is operationalized as 'reduction from baseline in monthly seizure count' for each intervention cohort.
Data will be analysed using multivariate models and mixed-effect regression.
The study aims to identify 'molecular signatures biomarkers predictive of treatment response and prognosis in people with epilepsy.'
Both microbial features and host epigenetic (DNA methylation) signatures are included as candidate predictive biomarkers.
Methods
The study received ethical approval from the Research Ethics Board Comitato Etico Territoriale Lombardia 3 and is registered on ClinicalTrials.gov.
Ethics approval ID: 4896 - parere numero 4896_17.07.2024_N_bis.
ClinicalTrials.gov Identifier: NCT07010445, registered on 2 May 2025.
All participants or their legal guardians will provide written informed consent.
Results will be disseminated through peer-reviewed publications, conference presentations, and lay summaries targeting patient organisations.
What This Means
This paper describes the design of a research study called CARE (MiCrobiota-gut-brain Axis in Resistant Epilepsy), which will investigate whether the community of microorganisms living in the gut — the gut microbiota — plays a role in why some people with epilepsy do not respond well to treatments. About one in three people with epilepsy continues to have seizures despite trying medications, surgery, nerve stimulation, or special diets like the ketogenic diet, and scientists do not fully understand why. This study proposes that differences in gut bacteria and related changes in how genes are switched on or off in the body (epigenetics) might help explain treatment resistance.
The study will follow participants between 3 and 50 years old across five sites in Italy, grouped by the type of treatment they are receiving: new medications, epilepsy surgery, vagus nerve stimulation, or the ketogenic diet. Researchers will collect stool and blood samples, perform brain scans and electrical recordings, and track seizure frequency over 12 months. Advanced laboratory techniques will be used to identify the types and functions of gut microbes present, and to measure chemical tags on DNA that can change gene activity. The goal is to find biological patterns — 'signatures' — that can predict whether a treatment will work for a given patient.
This research suggests that analyzing gut bacteria and epigenetic markers at the start of treatment could eventually help doctors identify which epilepsy patients are likely to respond to which therapies, potentially enabling more personalized treatment planning. It also opens the possibility that the gut microbiome could become a target for future therapies aimed at reducing treatment resistance in epilepsy. As this is a study protocol paper, no results have yet been reported.
Borghi E, Tassi L, d'Orsi G, Uzzau S, Pivari F, Ricci E, et al.. (2026). Microbiota-gut-brain axis and treatment resistance in epilepsy: a multicentre prospective study protocol (CARE).. BMJ open. https://doi.org/10.1136/bmjopen-2025-111607