*ProjectReport***Evaluation of Changes in Gut Microbiota in Patients with Crohn's Disease after Anti-Tnfα Treatment: Prospective Multicenter Observational Study**

**Laura Sanchis-Artero 1 , Juan Francisco Martínez-Blanch 2,\*, Sergio Manresa-Vera 2 , Ernesto Cortés-Castell 3 , Josefa Rodriguez-Morales 1 and Xavier Cortés-Rizo 1**


Received: 4 June 2020; Accepted: 10 July 2020; Published: 15 July 2020

**Abstract:** *Background:* Crohn's disease is believed to result from the interaction between genetic susceptibility, environmental factors and gu<sup>t</sup> microbiota, leading to an aberrant immune response. The objectives of this study are to evaluate the qualitative and quantitative changes in the microbiota of patients with Crohn's disease after six months of anti-tumor-necrosis factor (anti-TNF α) (infliximab or adalimumab) treatment and to determine whether these changes lead to the recovery of normal microbiota when compared to a control group of healthy subjects. In addition, we will evaluate the potential role of the *Faecalibacterium prausnitzii*/*Escherichia coli* and *Faecalibacterium prausnitzii*/*Clostridium coccoides* ratios as indicators of therapeutic response to anti-TNF α drugs. *Methods*/*Design:* This prospective multicenter observational study will comprise a total of 88 subjects: 44 patients with Crohn's disease scheduled to start anti-TNF α treatment as described in the drug specifications to control the disease and 44 healthy individuals who share the same lifestyle and eating habits. The presence of inflammatory activity will be determined by the Harvey-Bradshaw index, analytical parameters in blood, including C-reactive protein, and fecal calprotectin levels at commencement of the study, at three months and at six months, allowing the classification of patients into responders and non-responders. Microbiota composition and the quantitative relationship between *Faecalibacterium prausnitzii* and *Escherichia coli* and between *Faecalibacterium prausnitzii* and *Clostridium coccoides group* as indicators of dysbiosis will be studied at inclusion and six months after initiation of treatment using ultra sequencing with Illumina technology and comparative bioinformatics analysis for the former relationship, and digital droplet PCR using stool samples for the latter. Upon inclusion, patients will complete a survey of dietary intake for the three days prior to stool collection, which will be repeated six months later in a second collection to minimize dietary bias. *Discussion:* In this study, massive sequencing, a reliable new tool, will be applied to identify early biomarkers of response to anti-TNF treatment in patients with Crohn's disease to improve clinical managemen<sup>t</sup> of these patients, reduce morbidity rates and improve e fficiency.

**Keywords:** gu<sup>t</sup> microbiota; anti-TNF α; Crohn's disease; *Faecalibacterium prausnitzii*; *Escherichia coli* and *Clostridium coccoides* group
