Recurrent abdominal pain has long been acknowledged to be the most common chronic pain entities in children. The purpose of this study is to describe the microbiome in children with FD and to explore relationships between the microbiome and postprandial distress syndrome, anxiety scores, and mucosal biomarkers or anxiety. The specific goals of this study are to: 1) Determine the frequencies and relative proportions for specific bacteria or bacteria phyla in children with FD in both duodenal mucosal specimens and stool samples. 2) Determine if the frequencies or proportions of specific bacteria or bacteria phyla differ between children with and without PDS. 3) Determine bi-variate correlations between bacteria/phyla frequency, bacteria/phyla proportions, anxiety scores, and mucosal biomarkers, respectively.
The purpose of the current study is to describe the microbiome in children with FD and to explore relationships between the microbiome and postprandial distress syndrome, anxiety scores, and mucosal biomarkers or anxiety. SPECIFIC AIMS 1. To determine the frequencies and relative proportions for specific bacteria or bacteria phyla in children with FD in both duodenal mucosal specimens and stool samples. 2. To determine if the frequencies or proportions of specific bacteria or bacteria phyla differ between children with and without PDS. 3. To determine bi-variate correlations between bacteria/phyla frequency, bacteria/ phyla proportion, anxiety scores, and mucosal biomarkers, respectively.
Study Type
OBSERVATIONAL
Enrollment
40
In this study patients are being scoped(EGD) as standard of care and being asked to allow .5 aggregate of tissue biopsy to be taken for research purposes.
We will be collecting stool samples from each participant for research purposes. Within two weeks after EGD tissue collection.
The Children's Mercy Hospital
Kansas City, Missouri, United States
Microbiome Analysis
Fecal samples and 8 mucosal biopsies (descending duodenum) will be obtained from each subject. Samples will be processed and DNA isolated per standard lab techniques.16S rDNA sequence analysis of microbial communities within patient samples will be analyzed utilizing our HiSeq 1500 rapid run technologies. We will perform 2 X 150 bp paired end sequencing which will give exceptional coverage (reads \> 30,000). We will sequence the V4 region of 16s RNA (E. coli 515-806), which is the method optimized for Illumina HiSeq 1500 currently. We will also sequence simultaneously the V3 region of 16s RNA to provide a dual-control analysis of samples. Confirmatory PCR will be employed when appropriate. Data will be reported as total counts for each identified bacterial species.
Time frame: within 12 months from collection
Inflammatory Cell Density
Routine histology slides previously stained with hematoxylin and eosin will be utilized for determination of eosinophil density. IHC techniques will be utilized for determination of mast cell density. Serial 3-μm paraffin sections will be air dried and heat fixed on slides. The sections will be deparaffinized with xylene and iodine and rehydrated in a graded series of alcohol. Utilizing tryptase monoclonal mouse antihuman mast cell tryptase antibody (clone AA1, Dako, Carpinteria, CA), sections will be stained on an automated Dako Autostainer 3400 using Dako's LSAB+ kit with streptavidin conjugated to horseradish peroxidase. To determine density for each cell type, the entire specimen will be scanned to determine the subjective area of greatest involvement. Density will be determined by counting cells within 5 consecutive high-power fields (400X magnification). Peak and mean cell densities expressed as cells/hpf will be determined. This will be completed before study end.
Time frame: within 12 months from collection
Immunohistochemistry (IHC)
IHC staining will be performed and may include DCLK1, BDNF, TRPV1, and/or CRH-R1, respectively, depending on the results of an on-going pilot study. Staining intensity will be evaluated in a blinded fashion by a pathologist to assign scores for average IHC signal intensity (i.e. 0= none, 1= mild, 2= moderate, and 3= strong) as well as the percentage of tissue cells or fibers showing positive immunoreactivity. A sem-quantitative scoring system will be applied in which the final immunoreactive score will equal the product of the percentage of positive cells times the average staining intensity. Percentage of positive cells or fibers will be graded as follows: 0= negative-10%, 1= 11-33%, 2= 33-66%, and 3= \> 67%. Immunoreactivity will be considered positive when the combined score ranges between 2-6 and negative with a score of 0-1. Other IHC stains may be performed should they become relevant as indicated by the literature. This will be performed before study end.
Time frame: within 12 months from collection
T-scores for the BASC will be collected as part of this study on the Data Collection Form.
This information is used to assess psychosocial information from the patient.This will be collected and recorded before study end.
Time frame: within 6 months from completion
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