The Environment and Childhood Stunting Dr Tahmeed Ahmed Executive Director icddr,b Permission taken for display for educational purpose only Stunting Height for age less than –2SD Date of Birth : 28 February, 2010 January 1, 2012 December 5, 2012 September 2, 2013 September 15, 2014 Permission taken for display for educational purpose only Global Target for 2025 2016 43% of linear growth faltering in children under-2 can be explained by small gut enteropathy • Sub-acute inflammation of small intestinal mucosa with abnormal small intestinal biopsy findings • Reduced capacity in tests of intestinal absorption and evidence of increased permeability of the intestinal mucosa • Prevalent in areas of poverty with poor sanitation • Tropical enteropathy, environmental enteropathy and environmental enteric dysfunction Keusch GT 2014; Prendergast A 2012; Kelly P 2004 Lindenbaum J 1966, 1971; Lunn P1991 Environmental Enteric Dysfunction Normal intestinal villi Total villous atrophy Environmental Enteropathy Healthy Bangladesh Environmental Enteric Dysfunction (BEED) Study Islam, Ahmed Eur J Clin Nutr 2012 • 40% of all CF contaminated with E. coli • Attributable to faulty food preparing practices • Associated with higher rates of diarrhea and malnutrition • 15,000 metric tons of human waste produced daily in Dhaka city • 70% of all human waste dumped in the rivers without any treatment 20% Central sewerage 30% drainage networks & open channels 3% Small bore holes 25% Improved onsite sanitation 22% Unhygienic onsite Sanitation coverage in Dhaka City Area, 2012 Dhaka WASA Masterplan 2012 Most studies of EED have relied on non-validated fecal or plasma biomarkers Esophagogastroduodenoscopy (EGD) is rarely performed Conflicting data about the incidence of EED and its relationship to stunting Evidence that perturbed development of the microbiota contributes to undernutrition Recent data suggest a potential role for bacteria in the upper gastrointestinal tract Pathophysiological features of EED, and its contribution to impaired linear growth hampered by the difficulty in directly sampling the small intestinal mucosa and microbial community Known to the Unknown To investigate the role of EED in malnutrition, particularly stunting To validate currently available non-invasive biomarker candidates To examine the biology of EED to identify common biological pathways for potential interventions To validate a system for histological scoring of EED To test the effectiveness of nutritional interventions in improving the growth parameters in children with stunting and / or EED EGD Biopsy Duodenal aspirates Histopathology Community Survey Stunted LAZ <-2 At risk of stunting LAZ -1 to -2 Children 12-18m Nutrition Intervention 1 egg 150 mL milk MNP 3 Months Graduated Nutritional status Not-improved No secondary causes of malnutrition Screening Eligible for EGD Consent Blood Stool Urine Inclusion Exclusion Consent Enrollment Community meeting Community meeting Steps Towards Endoscopy and Biopsy Community survey Measure Z score/BMI If failing to thrive Bi-weekly anthropometry Treatment, if required Nutrition intervention starts Baseline sample collection Baseline anthropometry Recruitment Consent taking Applying eligibility criteria Screening If agrees Counsel for endoscopy If FTT confirmed 2 weeks before the end of intervention Screening for secondary malnutrition Positive Stool for TAC & microscopy Pre-anesthetic checkup Start counseling Nutrition intervention ends End line sample collection End line anthropometry Negative Treat Screening for endoscopy Treat or discharge from study Follow-up for next 14 days Endoscopy Microbiota Proteomics Metabolome Flocytometry Transcriptomics IHC Methods Methods Not benefited from nutritional Intervention and underwent EGD July 2016 to July 2018 Enrolled 525 Eligible 110 Remained stunted (LAZ <-2) Remained at-risk-for-stunting (-2 ≤ LAZ <-1) Biopsy-confirmed EED (n=104) Plasma proteomic (n=104) Duodenal proteomic (n=80) Duodenal microbiota and enteropathogen (n=80) Fecal microbiota (n=48) and enteropathogen (n=21) Biopsy negative (n= 6 ) Age-matched heathy controls: LAZ and WLZ ≥ 1 27 plasma, 21 fecal samples Proteomic assays in plasma and duodenal biopsy samples Analysis of bacterial taxa present in duodenal aspirates and fecal samples Association between the presence of EED and Duodenal protein Levels of plasma proteins Small intestinal microbiota The histopathological severity score Length-for-age z score Translational study in mouse model to study causal relationship by transfer of enteropathy to germ-free mouse colonized with cultured duodenal strains obtained from children with EED Histopathology definition of EED EED: 3 features in H&E stain 1. Infiltration of inflammatory cells in the lamina propria 2. Blunting or atrophy of small intestinal villi 3. Hyperplasia or elongation of crypts Histopathology definition of EED EED: 3 features in H&E stain 1. Infiltration of inflammatory cells in the lamina propria 2. Blunting or atrophy of small intestinal villi 3. Hyperplasia or elongation of crypts No correlation observed between histopathological score and the length-for-age z score Methods: Proteomic Assays Proteomic assays (SOMAScan, SomaLogic) to quantify several thousand proteins spanning a broad range of biologic functions Comparisons made between the plasma proteomes of anthropometrically healthy children and children with EED We analyzed 4077 plasma proteins and 2619 duodenal proteins that passed quality-control filtering