Identification of Novel Bio-markers for Environmental Enteropathy in Children Using an Evidence Based Approach
Condition: Malnutrition; Enteropathy
MeSH terms: Intestinal Diseases; Malnutrition
Study type: Observational

Summary: 
      EE is increasingly recognized as a key factor underlying malnutrition, weakened immune
      response and impaired cognitive development in children in developing countries. Absence of a
      distinct biomarker of EE in the blood, urine or stool makes it difficult to study the impact
      of interventions against it. Biomarkers for EE have been challenging to find, partly because
      of our inadequate understanding of its pathophysiology. Investigators aim to identify novel
      biomarkers for EE, based on our hypothesis that EE is a result of two processes: 1) repeated
      exposure to enteric pathogens and environmental toxins leading to gut inflammation and 2)
      weaning on diets high in carbohydrates but low in proteins and fat, leading to atrophy of the
      intestinal mucosa. This leads to gut dysfunction, including leaky gut, small bowel stasis,
      bacterial overgrowth, decreased immune response to infections, and frequent diarrhea. The
      candidate biomarkers investigators have selected for our study (CRP, GLP- 2, Claudin 3,
      Reg-1, plasma amino acids profile, serum cytokine profile, Neopterin and Myeloperoxidase) are
      markers of inflammation, hormonal dysfunction and tight junction malfunction of the small
      intestines. The 'gold standard test' for EE will be direct histopathologic analysis of the
      duodenal mucosa, which will be available in a subset of study children undergoing upper GI
      endoscopy. For other study subjects, clinical surrogates for EE will be used to calculate the
      sensitivity and specificity of biomarkers being tested. These clinical surrogates of EE
      include HAZ and WAZ score < 2 SD at 12 months and 15 months of age, and the worsening in HAZ
      and WAZ scores between 6, 9, 12 and 15 months of age. Investigators plan to study and compare
      duodenal biopsies from children with and without EE using cutting edge technologies including
      electron microscopy, immunofluorescence, and mRNA sequencing. This will allow direct
      correlation of the biomarkers in the blood, urine and stools with the histopathologic
      features of the gut mucosa. The mRNA sequencing of the gut tissue will allow us to identify
      new evidence-based biomarkers for EE, which could be further tested in the future.

      This is a strong, multidisciplinary collaboration between investigators in Pakistan and the
      United States with expertise in complementary areas including chemokines, inflammation, gut
      architecture, infectious diseases, field studies, and technology development.
    

Detailed description: 
      Title of protocol:

      Identification of Novel Biomarkers for Environmental Enteropathy in Children using an
      Evidence Based Approach

      Background

      Childhood malnutrition is a major problem in third world countries. One of the primary
      mechanisms behind childhood malnutrition is that consumption of low quality food and frequent
      gastrointestinal (GI) infections damage the intestinal mucosa of children. The intestines are
      thus not able to absorb the available nutrients and also fail to function as an effective
      barrier against the translocation of GI flora into the systemic circulation. The limited
      energy resources available to the child are diverted to overcome these inflammatory
      challenges at the expense of normal growth. This disorder of intestines (enteropathy) is
      called Environmental Enteropathy (EE). Since EE is thought to be a precursor of chronic
      malnutrition, detecting EE early with the use of biomarkers in the blood, urine or stools
      will allow us to predict chronic malnutrition in children early. The purpose of this research
      is to identify biomarkers for EE.

      Objectives

        1. To test novel candidate biomarkers of EE in a cohort of children 6-15 months of age in a
           rural district in Pakistan.

        2. To study the histo-pathologic characteristics of small bowel mucosa of moderate to
           severely malnourished children of the study cohort A, and correlate findings with the
           biomarkers studied in aim 1.

        3. To study the histo-pathologic characteristics of small bowel mucosa of children
           (well-nourished or malnourished) who are undergoing UGI endoscopy for any clinical
           indication e.g. retrieval of foreign body, evaluation of UGI bleeding etc., (cohort B)
           and correlate the findings with the blood, urine and stool biomarkers studied in aim 1.

        4. To identify new candidate biomarkers for EE through studying mRNA expression in the
           small bowel mucosa of children with and without EE for wide range of proteins involved
           in inflammation, hormonal dysfunction, immune system and tight junction structure and
           function.

      Methods

      In a cohort of children already being followed for growth and infections since birth for the
      RESPAK study in Matiari, Sindh (Cohort A), investigators will collect blood, urine and stools
      at 6 and 9 months of age and test for biomarkers including GLP-2, Claudin 3, CRP, Reg 1,
      serum cytokines profile, plasma amino acids profile, Neopterin and Myeloperoxidase. These
      biomarkers will be correlated with clinical surrogates of EE, including Height for Age Z
      score (HAZ) and Weight for Age Z score (WAZ) of children at 6, 9, 12 and 15 months of age,
      and the change in HAZ and WAZ scores between 6, 9, 12 and 15 months of age Investigators will
      identify 20-30 children within cohort A who are moderately or severely malnourished and have
      history of frequent or prolonged diarrhea at 10 -11 months of age, whose diagnosis remains
      unknown despite a comprehensive non-invasive workup and who fail to respond to first line of
      nutritional interventions. These children will undergo upper GI endoscopy at the Aga Khan
      University, in line with the American Society for Gastroenterologlists (ASGE) guidelines for
      the evaluation of children who are FTT and have associated frequent or prolonged diarrhea(1).
      The duodenal biopsies obtained through the endoscopies will be used for H&E staining
      (standard of care to diagnose celiac disease, other inflammatory and autoimmune causes of
      enteropathy) as well as EE research, including study of inflammation, hormone dysfunction and
      tight junction malfunction at AKU, University of Chicago and University of Virginia.

      In a separate cohort of children (Cohort B) 6-24 months old who present to our collaborating
      pediatric gastroenterologists at the Aga Khan University or at Children's Hospital Lahore for
      UGI endoscopy for any appropriate indication, additional duodenal biopsies and
      blood/urine/stool samples will be collected after informed consent. The workup done on these
      children will be as described above.

      The RNA will be extracted from the entire biopsy specimen obtained, and the mRNA targets
      which are differentially expressed in children with and without EE will be identified, so
      their downstream proteins could be identified and tested as candidate biomarkers for EE in
      the future.

      Adverse effects

      Obtaining blood, urine and stools from the child at 6 and 9 months of age will cause
      discomfort to the child though any serious adverse event resulting from these procedures is
      unlikely. For subjects not responding to nutritional rehabilitation, and in whom the first
      line of diagnostic workup is unrevealing, advanced workup will be offered per the ASGE
      guidelines, which include UGI endoscopy in selected cases. Although rare, risks of endoscopy
      include general anaesthesia related complications, perforation, bleeding and infection.

      Ethical issues

      Besides the issue of privacy invasion and blood, urine and stool collection, the major
      ethical challenge in cohort A is the conduct of UGI endoscopies in some children in whom
      cause of malnutrition is unclear despite first line of investigations and interventions.
      Investigators think that these endoscopies are in the interest of these children, since the
      ASGE recommends them as part of the workup in children who are failing to thrive and have
      gastrointestinal symptoms like chronic or recurrent diarrhoea. The endoscopies may help
      identify treatable inflammatory, infectious or autoimmune causes of failure to thrive in
      these children.

      Investigators are collecting additional biopsies for research during the endoscopies. The
      risks associated with endoscopy are primarily with anaesthesia and procedure. Taking
      additional biopsies is relatively safe since GI mucosa is one of the fastest healing organs
      of the body and essentially no residual sign of the biopsy is left after 24 hours of the
      procedure. Endoscopies at AKU and Children's hospital will be conducted by expert paediatric
      gastroenterologists. Nonetheless, investigators are well equipped to handle any complication
      that may arise.

      In cohort B, investigators are taking additional biopsies from children who are already
      undergoing UGI endoscopies for an appropriate indication not related to this study. The only
      additional risk to the subjects in cohort B is with taking additional biopsies, which should
      be minimal as explained above.
    

Intervention name(s): Upper Gastrointestinal Endoscopy
Intervention type(s): Procedure
Intervention description(s): Procedure Upper gastro-intestinal endoscopic procedure involves collection of intestinal samples and it can be helpful in identifying the causes of malnutrition. Upon parental approval, the child along with two relatives will be provided transportation to Karachi, accommodation and the compensation for their time and expenses.
For this procedure the child will be admitted to the hospital a day before the procedure till one day after the procedure. This procedure takes 30 minutes, requires general anesthesia and involves insertion of a flexible tube with a small TV camera and a light on one end and an eyepiece on the other. The endoscope will allow the doctor to examine the inside of certain tube-like structures in the body. Some tissue samples for laboratory testing will be taken.
Other intervention name(s): UGI endoscopy
Observational model: Cohort
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        children 6- 24 months
      
Biospecimen description: 
      collect blood, urine and stools at 6 and 9 months of age and test for biomarkers including
      GLP-2, Claudin 3, CRP, Reg 1, serum cytokines profile, plasma amino acids profile, Neopterin
      and Myeloperoxidase
    
Keywords: Environmental Enteropathy; Childhood malnutrition; biomarkers