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The Environment and Childhood Stunting Dr Tahmeed Ahmed Executive Director icddr,b Stunting Height for age less than –2SD Permission taken for display for educational purpose only 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 Environmental Enteric Dysfunction • 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 Total villous atrophy Normal intestinal villi Healthy Environmental Enteropathy Bangladesh Environmental Enteric Dysfunction (BEED) Study • 40% of all CF contaminated with E. coli • Attributable to faulty food preparing practices • Associated with higher rates of diarrhea and malnutrition Islam, Ahmed Eur J Clin Nutr 2012 • 15,000 metric tons of human waste produced daily in Dhaka city • 70% of all human waste dumped in the rivers without any treatment Sanitation coverage in Dhaka City Area, 2012 22% 20% Central Unhygienic sewerage onsite 30% 25% drainage Improved networks & onsite open sanitation channels 3% Small bore holes Dhaka WASA Masterplan 2012 Known to the Unknown  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 To validate currently available non-invasive biomarker candidates To investigate the role of EED in malnutrition, particularly stunting 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 Community Survey Children 12-18m EGD Biopsy Stunted At risk of stunting Duodenal aspirates LAZ <-2 LAZ -1 to -2 Histopathology Inclusion Exclusion Consent Consent Screening Enrollment No secondary 150 mL milk Not-improved causes of Eligible for EGD Nutritional status malnutrition 1 egg MNP Nutrition Intervention 3 Months Graduated Blood Stool Urine Steps Towards Endoscopy and Biopsy Community Community Applying eligibility Screening Consent taking meeting survey criteria Positive Treat Recruitment Screening for secondary malnutrition Counsel for Baseline Negative If agrees anthropometry endoscopy If FTT confirmed Baseline sample Treat or discharge from Follow-up for Stool for TAC & collection End line study next 14 days microscopy anthropometry Microbiota Screening for Nutrition End line sample Proteomics endoscopy intervention starts collection Metabolome Endoscopy Pre-anesthetic Flocytometry checkup Bi-weekly Nutrition intervention ends Transcriptomics anthropometry IHC If failing to Measure Z 2 weeks before the end Treatment, if Start counseling of intervention required thrive score/BMI Methods Methods July 2016 to July 2018  Proteomic assays in plasma and duodenal Enrolled 525 biopsy samples  Analysis of bacterial taxa present in Not benefited from nutritional Intervention and underwent duodenal aspirates and fecal samples Eligible 110  Association between the presence of EED EGD  Remained stunted (LAZ <-2)  Remained at-risk-for-stunting (-2 ≤ and LAZ <-1)  Duodenal protein Biopsy negative (n= 6)  Levels of plasma proteins Biopsy-confirmed EED (n=104)  Small intestinal microbiota  The histopathological severity score  Length-for-age z score Plasma proteomic (n=104) Age-matched  Translational study in mouse model to Duodenal proteomic (n=80) heathy study causal relationship by transfer of controls: LAZ and WLZ ≥ 1 Duodenal microbiota and enteropathy to germ-free mouse colonized enteropathogen (n=80) with cultured duodenal strains obtained 27 plasma, 21 from children with EED fecal samples Fecal microbiota (n=48) and enteropathogen (n=21) 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 No correlation observed between small intestinal villi histopathological score and the length-for-age 3. Hyperplasia or elongation of crypts 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 Methods: DNA Sequencing and Enteropathogen Detection  We analyzed bacteria present in duodenal aspirates and fecal samples by sequencing amplicons generated from variable region 4 of bacterial 16S ribosomal DNA (rDNA) genes  We grouped V4-16S rDNA reads into amplicon sequence variants (ASVs) and then normalized their levels  ASVs utilized to classify groups of species based on DNA sequences Methods: Analysis in Gnotobiotic Mice 5.5-week-old, male, germ-free C57BL/6J Diet representative of that consumed by 18-month-old children living in Mirpur Oral gavage: 39 bacterial strains cultured from Cecal microbiota of a duodenal samples conventionally raised from study aged-matched mouse children with EED Euthanized DNA extracted from intestine -3 0 1 2 3 4 5 6 7 8 9 Fecal sample Determine Colonization bacterial strains Sacrifice Assays done from mice samples Community profiling by The distribution of bacterial Illumina NextSeq sequencing strains along the length of the gastrointestinal tracts Duodenal RNA-Seq Gene counts Illumina NextSeq 500 Differential expression analysis DESeq2 Flow cytometry Intestinal tissue and FACSCanto II mesenteric lymph nodes Data analyzed using FlowJo Assaying spleens for Cultured isolates were initially Vitek MALDI-TOF mass viable bacteria classified by their spectrometry morphotypes, followed by mass spectrometry and sequencing full-length 16S rDNA amplicons Data analyses plan Proteomic assays in plasma and duodenal biopsy samples Independent Identifying modules of co-expressed duodenal proteins components analysis (ICA) Analysis of bacterial taxa present in duodenal aspirates and fecal samples by culture-independent methods Determined the strength of the association between the Pearson correlation presence of EED and coefficient  duodenal protein modules  levels of plasma proteins  levels of duodenal ASVs  the histopathological severity score  length-for-age z score Identify relationships between the LAZ score and the Spearman’s rank enteropathogen burden correlation Relationships between the duodenal proteome and absolute Singular value levels of duodenal ASVs decomposition of a cross-covariance matrix Clinical Characteristics of the Study Children Duodenal Microbiota Analysis  We characterized the composition of the duodenal microbiota in the 36 children with EED who had an available aspirate sample  Shotgun sequencing of aspirate DNA revealed that bacteria dominated the microbial community  Sequencing analysis of V4-16S rDNA revealed 165 distinct ASVs (taxa) with relative levels of at least 0.01% in 1 or more of the 36 samples  A core group of 14 bacterial taxa were present in at least 80% of the aspirates obtained from children with EED Duodenal Microbiota 36 children with EED 14 core duodenal bacterial taxa present in 80% of children with EED  Streptococcus sp. Duodenal aspirates  Gemella sp.  Granulicatella elegans DNA  Haemophilus sp.  Neisseria subflava  Leptotrichia sp. Shotgun sequencing  Rothia mucilaginosa  Veillonella sp. Bacteria dominated microbial community  Fusobacterium sp.  Prevotella melaninogenica  Actinomyces sp. Sequencing V4-16S rDNA  Leptotrichia sp.  Corynebacterium sp. 165 distinct ASVs  Johnsonella sp. Relationship between Plasma Proteins and LAZ Plasma proteins most strongly correlated with LAZ Proteins Function Pearson r 1 Insulin-like growth factor 1 (IGF-1) Growth promotion 0.53 2 IGF acid-labile subunit (IGFALS), and Stabilize IGF-1 and increases its half- 0.51 life and 3 IGF binding protein 3 (IGFBP-3 bioavailability 0.48 4 Procollagen C endopeptidase enhancer Facilitate bone 2 (PCOLCE2) formation 0.40 5 IGF binding protein 2 (IGFBP-2) Inhibits functions of IGF-1 -0.39 Total bacterial load as well as many core-group taxa negatively correlated with LAZ 14 core duodenal bacterial taxa present in 80% of children with EED Streptococcus sp. Gemella sp. Granulicatella elegans Haemophilus sp. Neisseria subflava Leptotrichia sp. Rothia mucilaginosa Veillonella sp. Fusobacterium sp. Prevotella melaninogenica Actinomyces sp. Leptotrichia sp. Corynebacterium sp. Johnsonella sp. CC-SVD analysis of duodenal proteins and duodenal bacterial taxa Cross-correlation singular value decomposition (CC-SVD) Identifies groups of duodenal bacteria whose abundances are most strongly cross- correlated to the levels of expressed duodenal proteins The 50 most positively and 50 most negatively correlated proteins with the absolute abundances of 14 core duodenal bacterial taxa identified by CC-SVD Abundances of duodenal core group taxa are correlated with mucosal inflammation Cross-correlation singular value decomposition (CC-SVD) Identifies groups of duodenal bacteria whose abundances are most strongly cross-correlated to the levels of expressed duodenal proteins Result: dramatically Symbol Name Function reduced set of taxa CAMP Cathelicidin antimicrobial peptide Innate immune defense against bacteria and proteins whose CHI3L1 LCN2 Chitinase-3-like protein 1 Lipocalin-2 Biomarker of inflammation in multiple disease contexts Proinflammatory cytokine upregulated during intestinal infection/inflammation abundances are ICOSLG Inducible costimulator ligand Immune ligand involved in wound healing PGLYRP1 Peptidoglycan recognition protein 1 Bactericidal proinflammatory cytokine tightly coupled RETN Resistin Proinflammatory cytokine; induces insulin resistance MMP8 Matrix metalloproteinase 8 Neutrophil collagenase upregulated in intestinal inflammation Core group duodenal taxa are enriched in the fecal microbiota of stunted children with EED Test of Causality CONV-D: cecal contents from a conventionally raised mouse BEED: 184 bacterial isolates mapping to 39 bacterial taxa Mirpur-18: diet fashioned after dietary habits of 18-month-old children living in Bangladesh Characterize: - Histopathology - Duodenal inflammation - Barrier dysfunction/permeability Summary  Using aptamer-based proteomics, quantified levels of 4,077 plasma proteins and 2,619 duodenal proteins in 80 children with histopathologic evidence of EED  Absolute abundances of 14 duodenal bacterial taxa, present in >80% of subjects and not typically classified as enteropathogens, negatively correlated with LAZ and positively correlated with duodenal proteins involved in immunoinflammatory Responses  Plasma proteins (e.g., REG3A and LCN2) with levels that correlate with features of the duodenal proteome (e.g., MMP8) together with the levels of these 14 duodenal taxa in feces represent candidate biomarkers of EED  Representation of these 14 duodenal taxa distinct in fecal samples from EED versus healthy children What is the solution then? • Awareness about WASH among people, among policymakers, development partners • Population control • Need to go beyond usual indicators of WASH • We need to review our work plan on WASH, are we doing enough? icddr,b thanks its core donors for their on-going support