*2.7. Inclusion Criteria*

All the patients must be adults (aged ≥ 18 years) diagnosed with CD with a clinical course of moderate-severe disease requiring anti-TNF treatment, in whom the indication for such treatment was inflammatory bowel activity.

## *2.8. Exclusion Criteria*

Excluded from the study will be all patients who had taken antibiotics, probiotics, or proton pump inhibitors four weeks prior to the start of the study and stool collection; patients with chronic hepatitis C virus and chronic HIV infection; indication for anti-TNF treatment for reasons other than control of their luminal disease (e.g., enteropathic arthropathy, perianal disease, or prevention of recurrence); patients with previous ileum or colon surgery; and patients who will have received previous anti-TNF treatment in the 24 weeks prior to the start of the study.

#### *2.9. Sample Size Considerations*

Few studies have evaluated the variability in gu<sup>t</sup> microbiota after anti-TNF therapy in adult patients with IBD [14,21–23,26–30]. However, from the results obtained it is not possible to determine the exact percentage of expected variability. Accepting an alpha risk of 0.05 and a beta risk of 0.2 in a bilateral contrast, 44 subjects will be required assuming that the initial proportion of events is 0.95 (degree of dysbiosis in the population at the start of the study) and 0.70 at the end. A loss to follow-up rate of 10% has been estimated.

#### *2.10. Planning of the Sample Collection*

The participating centers will be responsible for taking samples and determining conventional analytical and fecal data (complete blood count, stool samples, parasitology, FC, etc.) within routine clinical practice. The specific samples for the analysis of microbiota to be conducted externally will be collected by the patients themselves. The samples must be frozen after collection (by the patient in his or her freezer) until they are delivered to the laboratory of the hospital of origin, where they will be stored at −20 ◦C. The samples will subsequently be centralized.

#### *2.11. Sequencing and Bioinformatics*

The samples will be coded and the bacterial microbiota present will be analyzed using capture of the v3-v4 region of the 16S rRNA subunit [8], ultra-sequencing with Library Illumina 15044223 B protocol (ILLUMINA) comparative bioinformatics analysis. From 200 mg of stool, DNA will be obtained through a combination of mechanical and enzymatic lysis, and purified using the PowerFecal Pro DNA isolation (Qiagen) protocol with modifications. The DNA will be processed and prepared for sequencing. The sequences obtained will be filtered by parameters of quality (threshold value Q20) and length (sequences greater than 250 nucleotides). This strategy avoids erroneous ascriptions that generate an incorrect distribution of taxa. The minimum number of readings per sample will be 5000 and the mean length greater than 400 nt. A rarefaction analysis will be performed on the sample sequences to assess saturation for microbial biodiversity. Subsequently, the analysis of taxonomic identification at di fferent taxonomic levels will be performed using the Microbiome bioinformatics with QIIME 2 2019.4 protocol [31] (Figure 2).

**Figure 2.** Sequencing and bioinformatics scheme.

#### *2.12. Identification and Evaluation of Potential Biomarkers*

To identify potential non-invasive biomarkers from the characterization of the intestinal microbiome in various stages of CD, we will use LEfSe (Linear Discriminant Analysis E ffect Size), an algorithm designed for the discovery of metagenomic biomarkers through class comparison, biological consistency testing and e ffect size estimation [32].

Due to the need to develop a rapid method of analysis such as the ratios of *Faecalibacterium prausnitzii*/*Escherichia coli* and *Faecalibacterium prausnitzii*/*Clostridium coccoides* group, a triplex digital droplet PCR will be implemented to allow the absolute quantification of the *Faecalibacterium prausnitzii*, *Escherichia coli* and *Clostridium coccoides* species, as well as the total number of bacteria present in a given sample through the 16S rRNA gene, all with a minimum volume and in a single reaction. Therefore, the appropriate primers and probes have been selected to perform this digital droplet PCR (Table 3), as well as to optimize the different concentrations and hybridization temperatures, based on numerous relevant studies [20,33–36].



#### *2.13. Anti-TNF Dosage and Safety Evaluation*

Anti-TNFs are monoclonal antibodies that neutralize pro-inflammatory cytokine tumor necrosis factor (TNF) involved in the inflammatory cascade of CD and other immune-mediated diseases. The anti-TNFs used are infliximab and adalimumab by indication for induction of remission in patients with moderate-severe CD. The administered doses of anti-TNFs are those listed in the data sheet, both at induction and maintenance doses. Infliximab dose regimen for adult patients with CD 5 mg/kg is given as an IV induction regimen at 0, 2, and 6 weeks followed by a maintenance regimen of 5 mg/kg IV every 8 weeks thereafter; treatment with 10 mg/kg IV may be considered for patients who respond and then lose their response. Adalimumab dose regimen for adult patients with CD is 160 mg initially on Day 1 (given in one day or split over two consecutive days), followed by 80 mg two weeks later (Day 15). Two weeks later (Day 29) they begin a maintenance dose of 40 mg every other week. During the prospective follow-up, according to usual clinical practice, the gastroenterologist responsible will modify or maintain a maintenance pattern according to the response to the drug by means of clinical (HBI) and analytical (fecal C-reactive protein and calprotectin) variables, which will be recorded in each patient's data collection notebook.

To identify any adverse effects associated with the administration of the anti-TNFα drug, a combination of physical examination, recording of vital signs (BP, pulse, temperature), and questionnaires are evaluated after administration (30 min later) and at follow-up visits at weeks 12 and 24. These questionnaires inquire about possible adverse effects related to adverse reactions to infliximab and adalimumab drugs (nausea, headache, dizziness, fever, hives, reactions at the infusion site, nervous system disorders, cardiac arrhythmia or myocardial ischemia, hypertensive or hypotensive events, skin reactions, gastrointestinal disorders, infections, hypersensitivity or anaphylaxis). In addition, patients are asked open-ended questions about their general well-being to request notification of any other adverse effects not listed in the data sheet. In order to assess the causality of adverse effects related to infliximab and adalimumab, the ADR (Adverse Drug Reaction) Naranjo probability scale is applied [37].

Laboratory parameters, including white blood cell count, liver transaminase levels, phosphate levels, and kidney function are studied prior to anti-TNF treatment and at the 12-week and 24-week follow-up (post-anti-TNF).
