Microbiota profiling in pediatric inflammatory bowel disease by means of IS-pro

Inflammatory bowel disease (IBD) compromises a group of chronic, relapsing
inflammatory disorders of the gastrointestinal tract, consisting of two major
entities; Crohn*s disease (CD) and ulcerative colitis (UC). During the last few
years significant advance has been achieved in the understanding of the
pathogenesis of IBD. It is commonly accepted that both genetic as well as
environmental factors play an important role in its etiology. Furthermore, an
imbalance in the intestinal microflora is considered to be characteristic for
IBD. Analysis of the enteric bacterial flora has revealed major differences in
the composition of this flora between adult patients with active IBD and
healthy controls. Until now no specific pathogenic micro-organism has been
associated directly with the pathogenesis of IBD.

In children with newly diagnosed CD primary induction therapy consists of
exclusive enteral nutrition (EEN) with polymeric liquid formula during 6 weeks.
Change of the intestinal microflora during this period has been described and
this suggests a crucial role of enteral bacteria in CD. Data concerning
microflora in the pathogenesis of pediatric IBD and the role in achieving
induction and remission are far from complete yet. As a consequence, the role
of bacteria in the development of IBD remains to be clarified.

Recent development of culture-independent techniques, based on bacterial DNA
analysis, has resulted in an increased knowledge of the intestinal microbiota.
These new molecular techniques include fluorescent in situ hybridization
(FISH), denaturing gel electrophoresis studies (DGE), real-time quantitative
PCR (qPCR), and cloning and sequencing. All these methods have limitations,
including low reproducibility, high labour intensiveness or restriction to
analysis of user-defined species. Deeper insight into the intestinal flora can
be of great importance as this may lead to improved diagnostic, prognostic and
therapeutic options. A highly reproducible, broad-range molecular techniques
has recently been developed and validated to investigate intestinal microbiota:
IS-pro, based on the 16S-23S interspacer (IS) region (Budding 2010). In this
study the microflora of children with newly diagnosed IBD are analysed
throughout time by means of this new IS-pro method.

Primary objectives:
to describe the composition of intestinal microbiota in the course of pediatric
IBD-patients by IS-pro
to describe the composition of intestinal microbiota of apparently healthy
children, aged 4-17 years, by IS-pro

Secondary objectives:
1- to compare composition of faecal microbiota in active IBD (CU and CD) with
controls
2- compare composition of faecal microbiota in active IBD and inactive IBD
3- to correlate microbiota in mucosal biopsies (mucosa associated microbiota)
and faecal samples at time of diagnosis IBD.

First visit
Children suspected of IBD and meeting the inclusion criteria, and their parents
are asked to participate in this study.

Subsequent visits (interval according routine procedure IBD)
Written informed consent is obtained at first subsequent visit (see informed
consent letter)
- During routine diagnostic colonoscopy mucosal biopsy is taken from ileum,
ascending colon and sigmoid colon (10 cm above the rectal verge). So in total 3
extra biopsy samples are taken.
- Patient is asked to collect a faecal sample in a provided container prior to
bowel cleansing in suspected IBD and at week 1, 3, 6, 18, 26 and 52, after the
diagnosis has been established. An addiotional faecal sample will be collected
in case of a relapse of IBD. The patient is asked to cool faecal samples at
home preferably at -20ºC, within 2 hours of collection, and bring these samples
along at subsequent regular outpatient ward visits. In the hospital faecal
samples are stored at -20ºC before further handling. DNA isolation of all
samples will be carried out according to above described procedure.

Routine laboratory tests are performed at subsequent outpatient visits,
including C-reactive protein, leucocytes, platelets, albumin. (hence no
additional laboratory investigations)
Routine faeces test on calprotectin test is performed at diagnosis and at 3
months after start induction therapy.
No extra blood samples are taken and no extra hospital visits are required for
this study

harvesting of 3 mucosal biopsies (Ileum, colon and sigmoid)

Collection of 3 extra mucosal samples during the first routine colonoscopy
participation will lead to only minimal increased risk and burden to the
patient.