*2.6. Study Design*

This prospective, observational, multicenter study will include patients with CD who require anti-TNF therapy to control IBD. Anti-TNF (infliximab or adalimumab) treatment will be initiated under medical prescription according to the drug data sheet and the MAISE (drugs with a high health or economic impact) of the Valencian Health Agency. Administration of treatment will be performed in routine clinical practice and will not be promoted by this study. Fecal samples will be collected for gu<sup>t</sup> microbiota analysis prior to exposure to the anti-TNF drug and six months after commencement of treatment. The patients will be monitored prospectively by their responsible physician during routine clinical practice during the first six months of anti-TNF treatment, recording the presence of inflammatory activity and whether there was no response, partial response or clinical remission at three months and at the end of the study follow-up.

Data to be recorded will include the presence of inflammatory activity (through calculation of the HBI), laboratory analytical data (complete blood count, general biochemistry, CRP) and the FC value prior to and three and six months after anti-TNF drug exposure, as well as treatments at the time of inclusion and any treatment changes throughout the duration of the study. The patient will be instructed to avoid taking probiotics and antibiotics during the study period. However, if antibiotics have to be taken, the patient must contact the study coordinator. In all cases, the second stool sample for mass sequencing will be performed after four weeks without treatment with probiotics/antibiotics, maintaining the same dietary conditions to avoid variability.

Whenever available, radiological and endoscopic activity data used in clinical practice to assess disease activity will be collected when performed, up to 12 weeks prior to patient inclusion and during the study.

Patients who undergo surgical resection within the study period will remain included but will be classified as non-responders to anti-TNF. Prior to surgery, clinical evaluation will be performed and blood and stool samples collected for fecal microbiota analysis, identical to the last determination at week 24 performed per protocol.

The patients will be provided with a 72-h dietary survey designed to record their dietary intake three days prior to stool collection at inclusion, repeated at six months to minimize bias concerning diet. Similarly, patients must complete a validated survey of adherence to the Mediterranean diet at inclusion and at six months to assess significant changes in their eating habits. An outline of the different data collection phases is shown in Figure 1.
