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Environmental enteric dysfunction pathways and child stunting : a systematic review Harper, Kaitlyn Mae 2018

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ENVIRONMENTAL ENTERIC DYSFUNCTION PATHWAYS AND CHILD STUNTING:  A SYSTEMATIC REVIEW   by  KAITLYN MAE HARPER  B.Sc., Pacific University, 2013 M.A., Loyola Marymount University, 2015      A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE   in   THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Population and Public Health)      THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)       April 2018   © Kaitlyn Mae Harper, 2018   i Abstract Background Environmental enteric dysfunction (EED) is commonly defined as an acquired subclinical disorder of the small intestine, characterized by villous atrophy and crypt hyperplasia. EED has been proposed to underlie stunted growth among children in developing countries. A collection of biomarkers, organized into distinct domains, has been used to measure different aspects of EED. Here, we examine whether these hypothesized relationships, among EED domains and between each domain and stunting, are supported by data from recent studies.  Methodology A systematic literature search was conducted using PubMed, MEDLINE, EMBASE, Web of Science, and CINAHL between January 1, 2010 and April 20, 2017. Information on study objective, design, population, location, biomarkers, and results were recorded, as well as qualitative and quantitative definitions of EED. Biomarkers were organized into five EED domains, and the number of studies that support or do not support relationships among domains and between each domain with stunting were summarized.  Results There was little evidence to support the pathway from intestinal permeability to microbial translocation and from microbial translocation to stunting, but stronger support existed for the link between intestinal inflammation and systemic inflammation and for intestinal inflammation and stunting. There was conflicting evidence for the pathways from intestinal damage to intestinal permeability and intestinal damage to stunting.  ii  Conclusions These results suggest that certain EED biomarkers may require reconsideration, particularly those most difficult to measure, such as microbial translocation and intestinal permeability. We discuss several issues with currently used biomarkers and recommend further analysis of pathogen-induced changes to the intestinal microbiota as a pathway leading to stunting.    iii  Lay summary Globally, one-quarter of children under the age of five are affected by poor linear growth, known as stunting. Stunting leads to life-long cognitive deficits and lower adult economic productivity. Environmental enteric dysfunction (EED) is a disease characterized by damage to the lining of the small intestine and is thought to contribute to stunting, though the exact mechanism is unclear. EED is assessed by numerous tests, including intestinal wall damage and function, inflammation, and the movement of pathogenic bacteria from the gut into the bloodstream. We conducted a systematic review to evaluate the evidence of relationships between each aspect of EED and stunting. We found the most evidence for the relationship between inflammation and stunting, but less evidence for the relationship between stunting and the presence of pathogenic bacteria outside the gut. Our results suggest that EED may be more complex than previously thought and that some frequently used EED tests may need to be reconsidered.  iv  Preface  This thesis is original work by the author, K. Harper. A version of Chapters 1-5 has been published [Harper KM, Mutasa M, Prendergast AJ, Humphrey J, Manges AR (2018) Environmental enteric dysfunction pathways and child stunting: A systematic review. PLOS Neglected Tropical Diseases 12(1): e0006205.]. I was the lead author, responsible for data collection and analysis, as well as manuscript composition. Mutasa M was involved in data collection. Prendergast AJ was involved in the early stages of concept formation and contributed to manuscript edits. Humphrey J contributed to manuscript edits. Manges A was the supervisory author on this project and was involved throughout the project in concept formation, manuscript composition and manuscript edits.   v  Table of Contents   Abstract .......................................................................................................................................... ii  Lay Summary ................................................................................................................................ iv  Preface ….........................................................................................................................................v   Table of Contents ...........................................................................................................................vi   List of Tables ...............................................................................................................................viii   List of Figures ............................................................................................................................... ix  List of Abbreviations ......................................................................................................................x  Acknowledgements ........................................................................................................................xi   Chapter One: Introduction ..............................................................................................................1   Literature Review ...............................................................................................................1 Research Question ..............................................................................................................8  Chapter Two: Methodology ............................................................................................................9   Data collection and abstraction ...........................................................................................9 Definition of EED domains ..............................................................................................10 Study inclusion and synthesis of results ...........................................................................14  Chapter Three: Results ..................................................................................................................19   Study inclusion and population characteristics .................................................................19 Markers of intestinal damage and repair ...........................................................................20 Markers of permeability and absorption ...........................................................................22 Markers of microbial translocation ...................................................................................24 Markers of intestinal inflammation ...................................................................................25 Markers of systemic inflammation ...................................................................................25  Chapter Four: Conclusions ...........................................................................................................27 Overview ...........................................................................................................................27 Challenges of EED research .............................................................................................29 Significance and future directions ....................................................................................33 Study Limitations ..............................................................................................................34 Summary ...........................................................................................................................35   vi  Bibliography .................................................................................................................................36  Appendices ....................................................................................................................................58    A: Summary data table ......................................................................................................59    B: PRISMA Checklist .....................................................................................................103  vii  List of Tables  Table 1. Evidence of association between EED domains .............................................................15  Table 2. Evidence of association between EED domains and stunting ........................................17   viii  List of Figures  Figure 1. Stunting prevalence rates among children under five years of age..................................2    Figure 2. Height and cognitive achievement among Indian children by level of reading  achievement ....................................................................................................................................4  Figure 3. Histologic sections from distal duodenal biopsy specimens from Zambian patients  with EED .........................................................................................................................................7  Figure 4. Flow diagram of literature search, review, and selection according to PRISMA .........20  Figure 5. Evidence of association in EED pathways ....................................................................32   ix  List of Abbreviations  AAT, alpha-1 antitrypsin AGP, alpha-1-acid glycoprotein CRP, C-reactive protein EndoCAb, endotoxin-core antibody GLP-2, glucagon-like peptide-2 I-FABP, intestinal fatty acid-binding protein IFN-γ, interferon gamma IGF, insulin-like growth factor IgG, immunoglobulin G  IgM, immunoglobulin M KTR, kynurenine-tryptophan ratio L:M, lactulose-mannitol ratio LPS, lipopolysaccharide L:R, lactulose-rhamnose ratio MPO, myeloperoxidase NEO, neopterin REG, regenerating islet derived protein sCD14, soluble CD14 TNF, tissue necrosis factor %L, percent lactulose permeability %M, percent mannitol absorption x Acknowledgements I am very grateful to the many people who have supported me and helped this project come together. A big thanks to my committee members—Andrew Prendergast and David Patrick—as well as Jean Humphrey, for their advice on content and structure throughout various steps in the process. I would like to thank Maxine Mutasa for sifting through hundreds of articles with me and for her enthusiasm about the project from the get-go. I am particularly appreciative of my amazing supervisor, Amee Manges, who enriched my education and personal growth throughout this project. Without her guidance, dedication, and willingness to answer my constant bombardment of questions, I never would have understood the intricate details of epidemiological research. I also want to express gratitude for my friends and family, including my incredible lab mates, Liz and Chad, whose prowess for finding free food (and making me laugh) kept me going on the toughest days. Thanks to the SPPHomies—I’m truly not sure I would’ve made it through these past two years without their words of encouragement! Special shout out to my parents for supporting me emotionally and financially, for letting me raid their fridge every time I come home, and for pushing me to be an overall better human. Last but not least, I’m so grateful for main man, Tanner, for spending countless hours listening to rants, always giving the best advice, and providing me with my daily dose of caffeine. Cheers! 1 1 Introduction  One-quarter of children under the age of five years are stunted, defined as a length-for-age (LAZ) two standard deviations below the median as defined by the World Health Organization (WHO) growth standards [1].Moreover, one-third of stunted children are severely stunted, falling three standard deviations below the global growth standards. Linear growth is an indicator of child health, development and well-being. This literature review discusses the global burden of stunting on children under five years old, including prevalence, consequences, and hypothesized pathophysiological pathways. Nutrition, diarrhea, and environmental enteric dysfunction (EED) are examined as possible risk factors of stunting.    Literature Review Stunting: A global problem Millions of children worldwide are affected by poor linear growth, a result of inadequate nutrition and repeated infections during the first two years of life. Stunting prevalence varies between countries but is highest in low-income regions (Fig 1). In 2010, stunting prevalence of pre-school children was estimated at ~36% in Africa and Oceania, ~27% in Asia, and ~13% in Latin and South America [2,3]. While fewer stunted children are found in the Americas, countries such as Guatemala, Bolivia, and Ecuador have prevalence rates as high as those found in Africa and Asia. Stunting prevalence also varies within countries; large disparities exist between levels of economic earnings, formal education, and household setting (urban versus rural) [4].     2   Figure 1. Stunting prevalence rates among children under five years of age   Adapted from Onis et al, 2016 [4]  Long-term trends in stunting follow different patterns depending on socioeconomic status; stunting prevalence in high-income countries has been stable at 6% since 1990 and is expected to remain at this level, while low-income countries have experienced a decrease from 44% to 29% during the twenty-year period. Despite gradual decreases in overall prevalence in the past 25 years, stunting is considered a major global health priority. In 2012, the WHO set resolutions to reduce the number of children from 162 million to 100 million by 2025 [5]. Stunting reduction is also a main target of the Sustainable Development Goals, created by the World Health Development Programme in 2016, and several initiatives like Scaling Up Nutrition [6] and First One Thousand Days [7] have taken steps to reduce stunting by improving nutrition for mothers and infants.    3 Consequences of linear growth Linear growth is currently the best indicator of a child’s overall health and well-being. It is hypothesized that stunting begins in utero and continues until age two. The first one thousand days of life is considered the critical period for infant growth and development. Growth failure in the first one thousand days has been associated with short stature later in life [8,9]. While short stature itself is not problematic, poor linear growth is indicative of a series of physiological changes that may hinder brain development in young children [10]. In the short-term, stunted children have high morbidity and mortality from infections, including diarrhea, fever, pneumonia, and complicated cough [11,12]. Severely stunted individuals (< 3 SD) exhibit the highest levels of morbidity and mortality [10].   Multiple studies have shown that stunted children have increased risk of poor cognitive development and educational performance [13–17], and children whose linear growth is impaired during the first two years of life are associated with lost productivity and lower adult earnings [18,19]. In 2006, the World Bank estimated that a 1% loss in adult height due to childhood stunting was associated with a 1.4% loss in economic productivity [7]. Short stature has also been associated with reduced late-life cognitive functioning and increased prevalence of Alzheimer’s disease [20].  Childhood stunting has also been associated with the increased risk of lifetime chronic diseases, such as elevated blood pressure, renal dysfunction and altered glucose metabolism [21,22]. Paradoxically, overweight and obesity is also more common in stunted individuals. It is hypothesized that nutrient deprivation in the first two years of life invokes the diversion of  4 nutrients away from growth to preserve vital functions. This results in epigenetic changes in metabolism and organ functioning that may increase the risk of coronary heart disease, diabetes, and other chronic diseases later in life. This is particularly evident in countries undergoing economic transitions, where energy-dense, nutrient-poor foods are more readily accessible [10].  While significant evidence linking stunting with poor health outcomes exists, it is important to note that linear growth is a gradient and cannot necessarily be classified into distinct groups. Thus, some individuals whose growth is considered “normal” in terms of growth standards may experience similar consequences to those considered stunted, and vice versa. For example, a study of Indian children observed a consistent positive association between height and reading level in both stunted and non-stunted children (Figure 3) [23]. Thus, developmental consequences exist for all children and not just those that fall below the global cut-off for stunting. Figure 2. Height and cognitive achievement among Indian children by level of reading achievement  Adapted from Spears, 2012 [23]   5 Stunting pathogenesis Stunting pathogenesis is poorly understood, and consequently no single pathway for intervention exists. The INTERGROWTH-21st Project, a population-based study in eight diverse geographic locations, found that infants born to well-nourished, educated, affluent women had no significant differences in length from birth until age two. Both fetal skeletal growth and newborn linear size were similar among these widespread geographic populations, suggesting that differences in LAZ in the first one thousand days are likely due to environmental and socioeconomic differences, rather than genetic variation [24].  These results suggest that societal and community factors—access to healthcare, education, and healthy food systems, as well as political stability, urbanization, population density and social support networks—may underlie individual factors, such as suboptimal feeding practices, micronutrient deficiencies, and presence of infection that lead to poor growth [25].   For many years, initiatives have focused predominantly on nutrition-specific interventions for stunting; many studies have focused on improving maternal and infant nutrition during the first one thousand days after conception [26–35]. However, previous systematic reviews highlight that neither food quantity nor quality fully explains impaired linear growth in children [36,37]. The reason behind nutritional intervention failures is not yet understood, though it is hypothesized that stunted children may have nutrient malabsorption or dysregulated nutrient partition [38]. Additionally, diarrhea has been proposed as a major contributor to growth failure in young children, though results are inconsistent [39–42]. While diarrheal episodes in the first few months after birth lead to increased prevalence of stunting at 24 months [43], catch-up growth between diarrheal episodes can be sufficient for linear growth recovery in some children  6 [44]. It is possible that the combination of diarrhea, nutrition, and presence of other unknown pathogens induce an inflammatory response in the gut that contributes to poor growth.  Environmental enteric dysfunction Environmental enteric dysfunction (EED) is commonly defined as an acquired subclinical disorder of the small intestine, characterized by villous atrophy and crypt hyperplasia (Fig 2). Previous reviews have described the history and epidemiology of environmental enteric dysfunction in detail [45–50] and have refocused attention on EED as a potential cause of stunting in developing countries. Exposure to bacteria through fecal contamination is postulated to induce morphological changes, leading to intestinal epithelial damage, increased permeability, and microbial translocation into the lamina propria. This invasion prompts an influx of inflammatory cells to the intestine and leads to local and systematic inflammation, resulting in the redirection of resources away from child growth and development, and the disruption of hormonal pathways that regulate growth plate activity in long bones. Chronic inflammation and reduced intestinal nutrient absorption are also hypothesized to affect brain development, inducing lasting negative effects on cognition, educational achievement, and linear growth [51].    7 Figure 3. Histologic sections from distal duodenal biopsy specimens from Zambian patients with EED.  (A) Biopsy sample of normal gut mucosa. Villi are long with short crypts in between. Villus height:crypt depth ratio is 3:1 (B) Biopsy sample of gut mucosa from child diagnosed with EED. Villus height:crypt depth ratio is approximately 1:1. Adapted from Owino et al, 2016 [38]   There are currently no clear diagnostic criteria for EED, which presents a major problem in investigating the role of EED in stunting, and in evaluating treatment and prevention strategies. Intestinal biopsy is used to diagnose diseases with similar pathological changes, such as celiac disease [52]; however, collection of small bowel biopsy samples is technically and ethically infeasible in young children. Over recent years, studies have evaluated a range of potential biomarkers of EED, with a general agreement that these should be organized into distinct domains to measure different aspects of the pathogenic pathway that characterizes EED. Studies  8 have included noninvasive biomarkers of intestinal damage and repair, epithelial permeability and absorption, digestion, epithelial morphology, intestinal inflammation, microbial drivers, systemic immune activation, and non-small intestine organ function.   Research Question Multiple research groups have used this domain-based approach, focusing on longstanding physiologic relationships to study the complex mechanisms that may underlie EED. Here, we examine whether these relationships are supported by recent and rich new data from studies conducted between 2010-2017, building on the review conducted by Denno et al for the time period 2000-2010 [53]. We define five contributing domains of EED to provide supporting evidence for our two aims: (i) to evaluate the relationships between individual EED domains; and (ii) to evaluate the relationships between each EED domain and stunting. We focus on stunting as the primary outcome in this review, as it is the most common outcome of the included studies and is objectively measured. Although our ultimate interest is in cognition and child development, these have not been as commonly measured, and the mechanistic pathways between stunting and neurodevelopment remain unclear [10].   9 2 Methodology  Data collection and abstraction Search strategy and selection criteria  Our search strategy followed PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines for the reporting of systematic reviews [54]. A search for articles in any language between January 1, 2010, and April 20, 2017, was conducted using PubMed, MEDLINE, EMBASE, Web of Science, and CINAHL. Abstracts were independently screened by two reviewers (K.M.H. and M.M.) and full-text articles that were related in any way to “environmental enteric dysfunction”, “environmental enteropathy”, or “tropical enteropathy” were selected for review. The reference lists of all review articles and original publications were also screened for any relevant studies. Published study abstracts were also included in this review. Disagreements regarding study inclusion were resolved by consensus.    Characteristics of study populations Our population of interest included individuals of all ages for whom two or more EED domains, or at least one domain and stunting, were measured. Only human studies were included. While most studies focused on children under 5 years, no age restriction was imposed in the search criteria as some adult studies provide valuable histopathological data that is less frequently collected in young children. Studies in both developed and developing countries were included in the search, though only one study in the final selection included individuals from a developed country [55].     10 Data Abstraction  Studies selected for inclusion can be categorized into three groups: (i) observational studies in which EED was defined as either an exposure or as an outcome; (ii) studies investigating potential EED biomarkers and/or identification/diagnosis of EED; and (iii) intervention studies designed to treat or prevent EED. A standardized data abstraction form was used to extract information from each study. Abstracted data included: study objective, study design, location, subject eligibility and description of study population, inclusion or exclusion of subjects with diarrhea or human immunodeficiency virus (HIV), results and final conclusions. Biomarkers and diagnostic tests were recorded, as well as any qualitative and quantitative definitions of EED provided by the authors (Appendix 1).   Definition of EED domains To review the evidence supporting the complex mechanisms that may contribute to child stunting we have elected to organize results according to these five EED domains: (1) intestinal damage and repair, (2) permeability and absorption, (3) microbial translocation, (4) intestinal inflammation, and (5) systemic inflammation. These domains were determined by consensus from reviewing previous conceptual frameworks and descriptions [46,47,53,56–66].  Each domain and its respective non-invasive biomarkers are described below.  Markers of intestinal damage and repair. Citrulline is a non-essential amino acid mainly produced by enterocytes de novo. Circulating concentrations of citrulline therefore reflect total enterocyte mass and low concentrations indicate reduced surface area [67]. Intestinal fatty acid binding protein (I-FABP) is an intracellular epithelial protein located primarily at the tips of  11 small intestinal villi that is rapidly released into the circulation after injury to the epithelia. It has been used to indicate the severity of intestinal damage in both adults [68] and children [69] and has a very short half-life, reflecting recent intestinal injury. Regenerating (REG) family proteins are involved in tissue regeneration and cell proliferation [70,71], and increased concentrations of fecal REG proteins indicate epithelial injury [72,73]. Glucagon-like peptide 2 (GLP-2) is a gut trophic factor released by enteroendocrine L-cells of the ileum. GLP-2 aids in mucosal regeneration, leading to increased villus length, improved gut barrier and absorptive functions, and anti-inflammatory mucosal activity [74].    Markers of permeability and absorption. The most widely accepted biomarker for epithelial integrity and permeability is the dual-sugar absorption test, although it has several limitations [75]. Intestinal inflammation is thought to create small pores between epithelial cells allowing for paracellular permeation of lactulose, while villous atrophy reduces epithelial surface area and mannitol (or rhamnose) absorption. A higher lactulose-mannitol (L:M) or lactulose-rhamnose (L:R) ratio has been used as an indication of EED, although recovery of the two sugars is sometimes reported separately to disaggregate absorption and permeability. However, because many studies report these measurements only as a ratio, we chose to combine these measurements into one single domain.   Alpha-1-Antitrypsin (AAT) is a protein released during inflammation to protect cells against proteolytic enzymes released by neutrophils during infection [76]. However, AAT is not synthesized in the gut, and the presence of AAT in stool reflects protein loss and increased permeability from the blood into the gut lumen. Claudins and zonulin are proteins that modulate  12 tight junctions, which form paracellular barriers between intestinal epithelial cells, thereby governing permeability and selectivity. High concentrations of zonulin suggest increased permeability [77]. Increased concentrations of claudin-2 and -15 indicate decreased intestinal absorption; increased concentration of claudin-4 indicates increased cell shedding [78].  Markers of microbial translocation. Microbial translocation is defined as the passage of microbes or microbial products, which are strongly immunogenic, through the epithelial barrier into the lamina propria and local mesenteric lymph nodes [79]. Lipopolysaccharide (LPS) and flagellin, two outer components of bacterial structure, are frequently used to indicate microbial translocation. Elevated plasma endotoxin core antibody (EndoCAb) titers and anti-LPS immunoglobulin G (IgG) and A (IgA) are used to identify an immune response to systemic LPS [80–82].  Markers of intestinal inflammation. Translocated LPS and other microbial products engage the mucosal immune system, prompting the activation of inflammatory cells such as neutrophils, macrophages and dendritic cells. Myeloperoxidase (MPO), an enzyme stored inside neutrophils, is involved in the process of killing bacteria [83]. Neopterin (NEO) is produced by macrophages or dendritic cells upon stimulation by interferon-gamma [84], which is released during pro-inflammatory responses by Th1 lymphocytes [85]. Additionally, it is hypothesized that a combination of biomarkers may explain linear growth deficits better than any single biomarker[86], and some studies calculate an “EED composite score” comprised of three fecal biomarkers—AAT, MPO, and NEO [66,86–88]. While AAT is primarily a measure of intestinal  13 permeability, we include the measures of the composite score in the intestinal inflammation domain as each biomarker may be used to measure aspects of gut inflammation.  Calprotectin is a calcium- and zinc-binding protein released by neutrophils as a result of cell stress or damage and is excreted in feces [89]. Accordingly, fecal calprotectin has been used as an indicator of gut damage [90].  However, in young healthy breastfed infants, fecal calprotectin concentrations are high [91] and decline with increasing infant age [92].  Therefore, high fecal calprotectin levels in young infants appears to be physiologic rather than an indicator of gut inflammation.    Markers of systemic inflammation. Interferon gamma (IFN-γ), tumour necrosis factor (TNF), and interleukins (e.g., IL-6, IL-10) are cell-signalling cytokines that activate and drive differentiation of immune cells upon infection [93]. Alpha-1-acid glycoprotein, C-reactive protein, and ferritin are upregulated acute-phase proteins involved in the inhibition of microbial growth. Soluble CD14 (sCD14) is a circulating co-receptor for LPS secreted by monocytes and macrophages [94]. Other systemic markers include total IgG and IgM, which are released by antibody-secreting B-lymphocytes, termed plasma cells. The kynurenine-tryptophan ratio (KTR) also indicates immune activation [95]: kynurenine is formed from the essential amino acid tryptophan by the enzyme indolamine 2,3-dioxygenase, which is upregulated by pro-inflammatory cytokines such as IFN-γ. Immune activation therefore leads to formation of kynurenine and depletion of tryptophan, and a higher KTR indicates a systemic immune response.    14 Study inclusion and synthesis of results Studies were included if they reported results for two or more EED domains, or at least one domain and stunting. In the situation where multiple studies included the same EED measurements on the same study subjects, only the most complete report was included in the review. For each of the 5 EED domains, we first present the number of studies that support (or not) the relationships of each domain to stunting. Second, we present the number of studies that report data in support of the relationship between each EED domain and the other domains, where data are available. These associations are summarized in Table 1 and 2.    15 Table 1. Evidence of association between EED domains. Evidence that supports pathway Evidence that does not support pathway Intestinal Damage and Repair—Permeability and Absorption 2017 Semba: L:M associated with low citrulline and high homocitrulline 2013 Agapova: REG4 gene best differentiated children with increased L:M from children with normal L:M (p = 0.01) 2016 Ordiz: REG1A gene was part of a model that predicted severe EED (LM > 0.45)  2016 Guerrant: REG1B associated with AAT 2016 Kosek: %L negatively associated with citrulline 2010 Papadia: Citrulline associated with xylose absorption in HIV-positive individuals after Benjamini-Hochberg correction 2013 Agapova: REG1B was not significantly different between children with high and low L:M 2016 Kosek: Citrulline not associated with %M 2015 Gosselin: No association between L:M and citrulline  2013 Wessells: Citrulline not associated with L:M recovery ratio  2010 Papadia: Citrulline not associated with rhamnose:glucose ratio or lactulose:rhamnose ratio after Benjamini-Hochberg correction  Intestinal Damage and Repair—Systemic Inflammation 2016 Kosek: Citrulline inversely correlated with CRP, AGP, IL-6 at 7,15,24 months 2016 Guerrant: Citrulline negatively correlated with KTR  Intestinal Damage and Repair—Intestinal Inflammation 2016 Guerrant: REG1B associated with MPO and NEO  Intestinal Damage and Repair—Microbial Translocation 2016 Guerrant: REG1B associated with anti-LPS antibodies 2016 Kelly: Log-transformed LPS associated with cell shedding; LPS associated with epithelial perimeter and villous surface area-volume ratio; LPS inversely associated with GLP2  2016 Uddin: I-FABP positively associated with anti-LPS IgG  2016 Campbell: GLP-2 inversely associated with EndoCAb  Permeability and Absorption—Intestinal inflammation 2017 Campbell: AAT was modestly correlated with MPO (r=0.33, p<0.01) 2017 Kosek: MPO associated with L:M z-score at age 1 2016 Guerrant: MPO associated with both L:M and %L; zonulin negatively associated with MPO and NEO; Claudin-15 negatively associated with L:M and AAT 2015 George: Calprotectin associated with MPO; AAT associated with MPO 2017 Kosek: No biomarkers of intestinal inflammation associated with L:M z-score at age 2 2017 Campbell: No significant correlation of L:M with any biomarkers of intestinal inflammation 2013 Agapova: Transcripts for MPO and calprotectin were not significantly different between children with high and low L:M   16 Evidence that supports pathway Evidence that does not support pathway Permeability and Absorption—Systemic Inflammation 2016 Ordiz: TNF, CD53 associated with L:M (sensitivity = 84%, specificity = 83%) 2016 Yu: 51 fecal transcripts involved in systemic inflammation associated with %L	2016 Semba: KTR significantly correlated with gut permeability; serotonin:tryptophan ratio also correlated with gut permeability 2013 Agapova: Transcripts of TNF, IFN-γ, and other markers of systemic inflammation were not significantly different between children with high and low L:M 2013 Wessells: AGP and CRP not associated with L:M Permeability and absorption—Microbial translocation 2016 Guerrant: Zonulin associated with anti-LPS IgG and anti-FliC IgG; L:M weakly associated with anti-LPS IgG 2010 Kelly: Log-transformed xylose recovery negatively correlated with log-transformed anti-LPS IgG (p = 0.006)  2017 Campbell: No significant correlation of L:M with other biomarkers 2016 Guerrant: L:M not significantly associated with LPS, anti-LPS IgA, anti-FliC IgG/IgA; %L not significantly associated with LPS, anti-LPS IgA/ IgG, anti-FliC IgG/IgA 2015 Benzoni: No significant association between log-EndoCAb and log-%L or log-L:M 2010 Kelly: No associations between anti-LPS antibodies or LPS and %R, %L, or glucose Microbial translocation—Systemic inflammation 2017 Campbell: EndoCAb and total IgG/IgM were consistently intercorrelated 2016 Uddin: anti-LPS antibodies positively associated with sCD14 (p=0.07) 2010 Kelly: Correlation between TNF receptor p55 and anti-LPS IgG and IgM 2016 Kelly: Plasma LPS was not associated with CRP, sCD14, CD163, or LPS-binding protein Microbial translocation—Intestinal inflammation 2016 Uddin: MPO positively associated with anti-LPS antibodies  Intestinal inflammation—Systemic inflammation 2017 Kosek: At age 2, MPO and AGP were positively correlated; NEO negatively correlated with AGP 2016 Guerrant: KTR and CRP associated with MPO; KTR associated with CRP  2015 Naylor: Inclusion of both intestinal and systemic inflammatory markers in clusters 1 and 2: (i) systemic inflammatory markers plus diarrheal burden; (ii) intestinal inflammatory markers plus CRP and sCD14 
 2017 Campbell: Systematic inflammation loaded on their own components when added to the PCA model rather than combining with gut markers   Evidence supporting or not supporting the pathways between five domains of EED. Empty cells indicate that no included studies contained evidence to support or not support the pathway between domains.    17 Table 2. Evidence of association between EED domains and stunting. Evidence that supports pathway Evidence that does not support pathway Intestinal Damage and Repair—Growth 2016 Guerrant: HAZ associated with lower citrulline at baseline; Higher citrulline predicted less stunting in girls only; Lower HAZ associated with higher I-FABP at baseline; Higher I-FABP associated with ∆HAZ 2016 Kosek: In Peru, lower levels of citrulline at 3 mo were associated with higher stunting over subsequent 6 mo 2013 Peterson: Higher REG1B concentrations at 3 mo significantly associated with future HAZ in cohorts of Peruvian and Bengali children 2016 Kosek: Tanzania site only: No association between citrulline and HAZ  2015 Gosselin: Malawi, no significant difference in mean citrulline levels between stunted and non-stunted children; Tanzania, citrulline did not predict subsequent stunting 2014 Prendergast: I-FABP not associated with stunting; I-FABP increased between 3-12 mo and decreased between 12-18 mo 2017 Campbell: Higher GLP-2 associated with lower HAZ in children 6 mo of age Permeability and Absorption—Growth 2017 Campbell: Gut permeability score associated with growth at 6 mo and between 6-18 mo 2016 Ordiz: L:M associated with ∆HAZ at 3 mo 2016 Guerrant: ∆HAZ associated with higher L:M; Zonulin associated with stunting in children > 12 mo 2016 Faubion: %L significantly predicted HAZ  2016 Yu: %L was associated with reduced ∆HAZ 2015 Lima: Higher AAT associated with impaired catch-up growth 2013 Kosek: AAT predicted declines in HAZ in subsequent 6 mo 2013 Lin: L:M strongly associated with HAZ 2012 Weisz: %L associated with ∆HAZ  2017 Brown: Children with higher HAZ (i.e. taller children) had higher L:C ratio 2017 Kosek: HAZ not associated with L:M z-score 2017 Semba: HAZ not associated with L:M 2017 Faubion: HAZ not associated with %R nor L:R 2017 Ordiz: Baseline HAZ not associated with L:M 2016 Arndt: AAT was not associated with subsequent 3-month linear growth 2013 Wessells: HAZ not associated with L:M recovery ratio at baseline 2012 Weisz: HAZ not associated with L:M Microbial translocation—Growth 2016 Guerrant: Lower HAZ associated with IgA anti-flagellin and anti-LPS antibodies at baseline; Higher LPS associated with smaller ∆HAZ  2016 Syed: Higher anti-LPS IgA levels were associated with a decrease in ∆HAZ from 6-18 months  2014 Jones: EndoCAb at day 28 negatively correlated with growth at day 56; EndoCAb positively correlated with IGF-1 at day 56 2017 Campbell: Higher EndoCAb associated with greater baseline HAZ  2016 McDonald: Microbial translocation associated with underweight but not stunting; None of the biomarkers were significantly associated with risk of stunting in either the unadjusted or adjusted models 2016 Syed: anti-LPS IgG not associated with ∆HAZ 2015 Benzoni: No significant association between log-EndoCab and HAZ or ∆HAZ 2014 Prendergast: No association between EndoCAb and stunting 2013 Lin: HAZ not strongly associated with IgG EndoCAb   18 Evidence that supports pathway Evidence that does not support pathway Intestinal inflammation—Growth 2017 Kosek: Negative association between MPO and ∆HAZ at age 1 2016 Arndt: High MPO levels were associated with decreases in 3-mo growth at age 2; Kosek Score barely associated with HAZ in mo 1-3 and mo 12-21 2016 Guerrant: MPO associated with ∆HAZ 2016 Naylor: Calprotectin at 12 wks negatively correlated with HAZ; MPO associated with ∆HAZ from enrolment to age 1 2015 Lima: MPO associated with impaired catch-up growth 2013 Kosek: Kosek Score associated with decreased HAZ in subsequent 6 mo in Brazil, Nepal, and South Africa; NEO predicted declines in HAZ in subsequent 6 mo 2014 Jones: IGF-1 weakly associated with calprotectin at day 56 2017 Campbell: Gut inflammation score (MPO, NEO, AAT) was not associated with growth 2016 Arndt: NEO was not associated with subsequent growth; MPO not associated with 6-mo linear growth at any time; Kosek Score not associated w/LAZ in mo 3-9 nor any other time when Benjamini-Hochberg adjustments were applied 2015 George: Baseline markers of intestinal inflammation (calprotectin and EED activity score) were not associated with stunting Systemic inflammation—Growth 
2017 Kosek: Higher AGP concentrations were associated with decreased ∆HAZ at both ages 2016 Kosek: KTR significantly inversely related to ∆HAZ in Tanzania 2016 Naylor: CRP, ferritin, sCD14 strongly negatively correlated with ∆HAZ 2014 Prendergast: Higher log10 levels of CRP and AGP between 6 wk–12 mo were associated with increased odds of stunting; At birth, IGF-1 was strongly associated with AGP, CRP, and sCD14 2014 Jones: CRP, IL-1, IL-10 significantly negatively associated with IGF-1 at baseline but not day 28 or day 56 2016 Guerrant: CRP not associated with stunting 2016 Kosek: KTR not significantly associated with ∆HAZ in Peru 2014 Prendergast: No associations between levels of IL-6 or sCD14 and stunting 2013 Lin: Total IgG not strongly associated with HAZ 2014 Jones: No association between IGF-1 and sCD14 nor TNF; IL-7, IL-8, IL-15, IL-17a, IL-22, IFN-γ not associated with IGF-1  Evidence supporting or not supporting the pathways between each of the five EED domains and stunting.    19 3 Results  Study inclusion and population characteristics The electronic search identified 598 potentially relevant abstracts and articles. Two additional records were identified from checking reference lists. A total of 190 records remained after duplicates were removed. 126 articles were excluded based on a review of the title and abstract (Fig 3). Of the 64 reports selected for full-text review, 24 were excluded: 7 studies reported results for one domain but did not include measurements of linear growth [96–102], 6 studies reported measurements of linear growth but did not report domain measurements [103–108], and 1 study abstract did not report results for domains or stunting [109]. Five abstracts [110–114] and five full-text articles [115–119] included overlapping EED measurements on the same study subjects and were excluded from our review. The final review therefore includes 40 reports (5 conference abstracts, 35 full articles).    Of the 40 reports included in this analysis, 10 (25%) of the studies were conducted in Central or South America, 16 (40%) were conducted in Asia, and 29 (73%) were conducted in Africa. Half of the studies were conducted in rural locations, and 1 study was conducted in both urban and rural populations, while 9 (23%) studies did not specify the setting. Thirty-six (90%) reports included participants under the age of 5 years, and 19 (48%) studies were restricted to children less than 2 years. Three studies were conducted exclusively in adults. Diarrhea was used as an exclusion criterion in 16 (40%) studies; 16 (40%) studies included participants regardless of diarrheal status and 8 (20%) studies did not mention diarrhea in their analyses. Twenty-nine (73%) reports did not specify the HIV status of participants. Seven (17%) studies used HIV as an exclusion criterion and 4 (10%) studies included individuals regardless of HIV status.  20  Fig 4. Flow diagram of literature search, review, and selection according to PRISMA.    Markers of intestinal damage and repair  Histopathology. Histopathological analysis of small bowel biopsies is used to assess intestinal mucosal damage. Four studies [78,100,102,120] included in this review evaluated severity of EED in Zambian adults, and therefore could not measure associations between intestinal morphology and child stunting. One study observed an association between histology of small  21 intestinal biopsies and noninvasive markers of intestinal damage and repair [120] and a second study reported associations with microbial translocation [78].   Biomarkers of intestinal damage and repair. I-FABP, REG1, GLP-2, and citrulline were used to investigate the possible associations between noninvasive markers of intestinal damage and repair with other domains and with stunting. Two studies observed significant associations between biomarkers of intestinal damage and repair and linear growth outcomes [121,122]. Two studies reported no association [123,124] and 1 study found conflicting results by study location [95]. One study reported an inverse relationship between GLP-2, an intestinotrophic factor, and linear growth [88]. GLP-2 has been associated with increased nutrient absorption in previous studies [125]; these results suggest that EED growth effects may not be improved by increased nutrient absorption alone.  Markers of intestinal damage and repair were not associated with the L:M [123,126] or L:R ratio [120] in 3 studies, and 2 studies found an inverse relationship between markers of intestinal damage and repair and the L:M test [127] or percent lactulose permeability (%L) [95]. A weak association with absorption was observed in HIV-positive adult individuals [120] but not in young children [95]. REG genes were significantly differentiated in children with increased L:M versus children with normal L:M in one study [128] and were associated with makers of intestinal permeability in two studies [121,129]. Markers of intestinal damage and repair were associated with markers of systemic inflammation in 2 studies [95,121] and with intestinal inflammation in a single study [121]. Intestinal damage and repair was associated with microbial translocation in 4 studies [78,88,121,130], but was not reported in 1 study [131]. In summary, the  22 association between biomarkers of intestinal damage and repair with other domains and with stunting were largely conflicting, but evidence consistently supported the pathway between intestinal damage and repair and microbial translocation.  Markers of permeability and absorption  The dual-sugar test, often expressed as a ratio of lactulose and mannitol (L:M) or lactulose and rhamnose (L:R), is a combined measure of permeability and absorption expected to be inversely associated with linear growth. AAT, zonulin, claudin-4, and claudin-15 are other biomarkers used to measure permeability and absorption. Four studies observed a significant association between permeability/absorption and linear growth [88,121,129,132] and three additional studies found associations between permeability only (%L) and linear growth [55,133,134]. Higher levels of AAT were associated with impaired catch-up growth in one study [135] and predicted declines in HAZ for six months following the test [86].  Five studies observed no association between the permeability/absorption ratio and linear growth [126,133,136–138]. Moreover, AAT was not associated with subsequent 3-month linear growth [66]. One study abstract reported an unexpected positive relationship between linear growth and dual-sugar absorption [139]. However, this study used lactose and creatinine as tracers, which may not accurately measure permeability and absorption. Three studies did not report the relationship of linear growth with permeability/absorption [140–142]. In summary, evidence supporting the pathway between permeability/absorption and linear growth was split almost evenly across recent studies.   23 Increased permeability is hypothesized to be associated with microbial translocation and intestinal inflammation. Permeability may enable translocation into the lamina propria and cause intestinal inflammation; conversely, intestinal inflammation may open up tight junctions to increase permeability and microbial translocation [45,143,144]. One study observed a weak association of anti-LPS IgG with the dual-sugar test; however, measurements of LPS, anti-LPS IgA, anti-FliC IgG, and anti-FliC IgA were not associated with the dual-sugar test or with %L alone in the same study [121]. No association between microbial translocation and permeability/absorption was observed in 2 studies [80,88], and 1 study observed conflicting results between the two domains [145]. Two studies did not report direct associations between permeability/absorption and microbial translocation [58,132].  Intestinal inflammation was associated with permeability/absorption in three studies [87,121,138]. In one study, MPO—a marker of intestinal inflammation—was modestly correlated with AAT, but there were no significant correlation between any markers of intestinal inflammation and the dual-sugar test [88]. In a large multi-site study, MPO was associated with L:M z-score (standardized by age and sex) at age 1, but no intestinal inflammation biomarkers were associated with L:M z-score at age 2 [138].    Systemic inflammation was associated with permeability in a study of children in rural Malawi [127] but not in Burkina Faso [126]. Fecal transcriptomics analysis identified eighteen host mRNA transcripts associated with permeability/absorption, 7 of which were identified as predictors of EED with 84% sensitivity and 73-83% specificity, in a population of rural Malawian children [129]. A study of the same population found 51 transcripts correlated with  24 permeability alone, almost all of which coded for proteins that regulate the systemic immune system [134]. However, a third study in the same geographic location found no association of the dual-sugar test with markers of systemic inflammation or intestinal inflammation [128]. In summary, the dual-sugar test was inconsistently associated with intestinal inflammation, systemic inflammation, and microbial translocation.  Markers of microbial translocation  The presence of anti-lipopolysaccharide (LPS) and anti-flagellin antibodies is used to indicate the host response to microbial translocation. Two studies observed an association of microbial translocation with linear growth [121,146]. In contrast, 4 studies found no association between microbial translocation and linear growth [80,124,132,147], and one study reported an unexpected positive association between microbial translocation and linear growth [88]. One study observed conflicting results between microbial translocation and linear growth [148], and 3 studies did not report associations [58,78,130].   Of the 7 studies that examined microbial translocation and inflammation, only 4 reported relationships; one study found positive associations of anti-LPS antibodies with markers of both systemic inflammation and intestinal inflammation [130]. Two studies observed associations of microbial translocation with markers of systemic inflammation [88,145], while another study found no association [78]. The remaining 3 studies did not report associations between microbial translocation and inflammation [58,130,146]. In summary, the pathway between microbial translocation and linear growth was not well supported, and there was inconsistent support for the pathways between microbial translocation and other domains.  25  Markers of intestinal inflammation Intestinal inflammation is a key feature of EED, and several fecal markers—MPO, NEO, and calprotectin—indicate intestinal inflammation. Individual biomarkers of intestinal inflammation were associated with or significantly predicted linear growth in 4 studies [86,121,138,149] and were associated with catch-up linear growth in 1 study [135]. Moreover, one study found a weak association between a marker of intestinal inflammation and insulin-like growth factor 1, a mediator of growth hormone that is important in childhood growth and development [146]. One study found no association of linear growth with baseline markers of intestinal inflammation [87], and one found conflicting results between growth and different intestinal biomarkers [66]. Overall, there was strong evidence supporting the pathway between intestinal inflammation and linear growth.  The EED composite score—comprised of MPO, NEO, and AAT—accurately predicted change in linear growth in a study of infants [86]. However, the baseline composite score was not associated with stunting [87] or HAZ at 18 months [88]. In one study, the unadjusted composite score was mildly associated with HAZ in months 1-3 and 12-21, but associations were not observed after adjustments were applied [66].  Markers of systemic inflammation Chronic systemic inflammation, which may partly arise from intestinal microbial translocation, has been proposed to contribute to stunting [51]. Biomarkers of both innate and adaptive immune activation have been used, including cytokines (TNF, IFN-γ, IL-6, IL-10), acute-phase proteins (alpha-1-acid glycoprotein, CRP, ferritin), amino acids (kynurenine, tryptophan) and  26 sCD14, a circulating marker of monocyte activation. Two studies observed associations between linear growth and systemic inflammation [138,149], 2 studies observed conflicting results [95,124], and 2 studies did not observe associations [121,132]. Additionally, two studies found negative associations between systemic inflammatory markers and insulin-like growth factor 1 [124,146]. However, 1 of these 2 studies observed an association in children only at baseline and not at later ages in the study [146]. Four studies included systemic inflammatory markers and linear growth measurements, but did not report associations [78,130,146].  Results by study design Characteristics of included studies Of the 40 studies in this review, 6 (15%) were randomized controlled trials (RCTs), 15 (35%) were longitudinal cohort, 11 (27%) were case-control, and 9 (23%) were cross-sectional. It is expected that high-quality RCTs and longitudinal cohort studies are more likely to yield consistent domain-domain and domain-stunting associations, while case-control and cross-sectional studies could be more susceptible to error and bias, leading to more inconsistent associations. In this review, case-control and cross-sectional studies provide equally strong evidence for domain-domain or domain-stunting associations compared to longitudinal studies and RCTs. Therefore, the results in this review do not appear to depend on or be explained by study design, although study quality was not formally assessed. Over half the observational studies included in this review discussed adjustment methods and loss to follow-up. The remaining studies did not directly state whether or not they addressed confounding and individuals lost to follow-up so it is difficult to assess the quality of their results. Additionally, heterogeneity of the studies in this thesis was not formally assessed. Given the description of  27 studies in Chapter 3, we acknowledge that study populations are likely to be very heterogeneous, despite the application of strict inclusion criteria.  Randomized controlled trials Six RCTs were included in this review, which looked at zinc [126,140,141], fatty acid [142,150], or micronutrient supplementation [140,142], antibiotics [140,141], and anti-inflammatory drugs [146] as interventions. Each study in this review used randomization, allocation concealment, and blinding techniques to ensure data quality and validity. In theory, these studies provide the strongest evidence for domain-domain or domain-stunting relationships. However, the few RCTs in this review did not assess the same domain-domain or domain-stunting relationships, therefore providing support for many separate associations but no strong support for any single relationship. Of the RCTs, Wessells et al was one of the strongest RCTs methodologically [126]. Although only 63% of the participants were successfully followed up, there was no significant difference in individual characteristics between treatment groups. This study found no association of permeability and absorption (L:M test) with intestinal damage and repair, systemic inflammation, or stunting, which supports the suggestion that the L:M test may not be the most appropriate assessment of permeability and absorption.  Longitudinal studies Since there are no known treatments to ameliorate EED or stunting, it is difficult to justify the use of expensive and resource-intensive RCTs to target any one specific intervention. Fifteen longitudinal studies were included in this review [66,86,122,123,129,131,132,138,147–149,151–154]. Many studies examined the relationship between individual EED domains and stunting  28 over time, while others assessed the relationship of certain risk factors (e.g. fecal environment contamination) with EED domains and stunting. However, no consistent relationships were identified among longitudinal studies included in this review. The MAL-ED cohort study, conducted by Kosek et al, is highlighted due to its strong methodology [138]. This study followed eight multinational birth cohorts for 24 months and measured biomarkers from multiple EED domains as well as stunting and cognitive development. Prior to enrolment, a pilot study was conducted in each study site to determine individual and community characteristics, including household demographics, socioeconomic status, food access insecurity, and general child health status [86]. This study used linear mixed modelling statistical analyses, with child nested in study site as a random intercept, accounting for non-independence between measurements over the entirety of the study. This study observed a strong support for the association between intestinal inflammation and stunting, and systemic inflammation and stunting. This study also observed no association of permeability and absorption with stunting, and observed conflicting results between permeability and absorption and intestinal inflammation. The results from this high-quality study support the overall conclusions drawn from all of the studies included in this review.   Other study designs  Nineteen studies included case-control or cross-sectional designs. These study designs are less ideal as they lack temporality and present the possibility of reverse causality (i.e. stunting occurs before EED characteristics appear). However, ten of the case-control studies included in this review were case-cohort studies using subjects from previous longitudinal studies or RCTs whose original objectives were not specifically focused on investigating the relationships  29 between EED biomarkers [55,58,120,121,124,128,130,133,135,145]. Case-cohort studies have a distinct advantage over other types of case-control studies, as they combine the advantages of a cohort study while saving time and money by analyzing previously collected data. This is particularly important for EED studies, as collecting biomarker data can be time- and resource-intensive. Prendergast et al provide an excellent example of a case-control study using data collected from the ZVITAMBO Vitamin A supplementation trial [124]. Cases and controls were selected based on mother’s HIV-exposure status. Cases were stunted children, defined by a strict cut point of HAZ < -2 SD. Odds ratios for stunting were calculated using multiple logistic regression, adjusting for covariates of both infant and maternal characteristics. This study provides strong evidence to support the finding in this review that microbial translocation is not associated with stunting. However, this study also found no association of intestinal damage and repair with stunting, which is not supported by the overall trend found in this review.   Six of the nine cross-sectional studies in this review also used previously collected data from one time-point in longitudinal studies or RCTs [80,88,127,137,155,156]. Of these, the study by Campbell et al provided the strongest methodology [88]. This study, nested in a large RCT in Bangladesh, assessed the association between EED biomarkers and stunting in 539 children at 18 months. While the study does not assess temporality, the large sample size and the enrolment of participants from a well-described source population provide strong support for the observed relationships. Notably, this study observed no association between microbial translocation and growth, supporting the overall finding of this review. However, this study also found no association between intestinal inflammation and growth, which does not support the overall conclusions reached in this review.  30  Of the three remaining cross-sectional studies that did not use previous cohort data, two assessed the relationship between biopsy samples and various EED domains, including intestinal damage and repair, microbial translocation, intestinal inflammation, and systemic inflammation [78,127]. Evidence from this study may be used to determine the presence of biomarkers when villous blunting is truly known, though temporality of the relationship was not assessed. The final study, a published abstract, reported an unexpected positive relationship between linear growth and dual-sugar absorption. However, as discussed in Chapter 3, this study used lactose and creatinine as tracers, which may not accurately measure permeability. Moreover, using information from a published abstract alone did not allow for the evaluation of selection or measurement biases or the impact of confounding. Therefore, the results of this study should be interpreted with caution. Although most of the studies included in this review were observation studies, many were embedded in larger cohort or RCTs. However, variations in biomarker assessment and heterogeneity in study populations, make summarizing the relationship between EED domains and between EED domains and stunting challenging.  Overall, even the best designed and executed studies concurred with the overall conclusions of this review. 31 4 Conclusions Overview EED has traditionally been defined as a primary gut disorder that initiates a chain of events from intestinal permeability and microbial translocation, via their impact on immune activation, to stunting. In this review, we found that there was fairly little evidence to support the pathway from intestinal permeability to microbial translocation and from microbial translocation to stunting. There was stronger support for the link between intestinal inflammation and systemic inflammation, and between intestinal inflammation and stunting. Other relationships presented conflicting pictures. For example, evidence for or against the role of intestinal damage in intestinal permeability and intestinal permeability/absorption in stunting was evenly split across studies (Fig 4).   These results suggest (i) that the relationships between EED domains are inconsistent; (ii) that the associations between EED domains and stunting are variable, with the best evidence for intestinal inflammation and stunting; (iii) that some domains are harder to measure than others, which may have led to a bias in study findings; (iv) there remains no consistent definition of EED; and (v) the number of studies comparing histopathological analysis of small bowel biopsies to non-invasive biomarkers is lacking. As EED is characterized by small bowel morphological changes, more studies are needed to investigate associations between biopsy samples and a range of non-invasive biomarkers from each domain.    32 Figure 5. Evidence of association in EED pathways 	Permeability/Absorption (L:M, L:R, L:C, %L, %M,  claudins, zonulin) Intestinal damage and repair (Biopsy, I-FABP, REG, citrulline) Microbial Translocation (LPS, EndoCAb, flagellin) Intestinal Inflammation (MPO, NEO, AAT, composite score, GLP-2, calprotectin) Systemic Inflammation (IFN-γ, AGP, CRP, total IgG/M, interleukins, ferritin, KTR, TNF, sCD14)  Growth Axis (IGF-1)  Stunting 6  5 3   4  2   4    1 3   1 1 3   6 4   4  2   3  3    1 6    76   2  3   1  2      1  2  4 	 Conceptual framework for five domains of EED and supporting evidence for the individual pathways between domains within EED and the pathways between each domain and stunting. Green, supports previous hypothesized relationship; red, does not support previous hypothesized relationship.  33 Challenges of EED research The L:M test has been measured in numerous populations, but procedural details (fasting prior to ingestion, sugar dosage, time of urine collection, assay method) vary, which hinders comparisons between studies; moreover, reference cut-points for the diagnosis of children with EED using the  sugar absorption test have not been established [75]. Twelve studies used L:M ratio cut-points of 0.06-0.07 [58,88,126,140,142,150] or 0.10-0.15 [80,128,133,136,137,155] to define EED. Three studies categorized EED as moderate or severe, where L:M values of 0.28-0.45 were used for severe cases [126,129,137]. Seven studies did not report normal L:M cut-point values [55,95,101,121,123,132,139]. The assignment of normal values is challenging as other non-EED related factors can influence the movement of sugars across the epithelium, including age, gastrointestinal motility, variations in gastric emptying, recent diarrhea, mucosal blood flow, and renal clearance [157], resulting in individual and population variability in L:M values. One large multi-site study by Kosek et al compared L:M values to L:M z-values, standardized by age and sex [138]. While L:M values were significantly associated with HAZ up to 15 months, the L:M z-scores were not associated with HAZ at any time points. This study emphasizes the importance of addressing age-specific norms in dual-sugar clearance and illustrates that the assignment of L:M cut-point values may lead to greater risk of EED misclassification in some populations.  Some performance measures, such as the L:M test, may be under- or overestimated in a single population based on its overall characteristics (e.g. higher incidence of diarrhea, prevalence of stunting). For example, two studies of children in rural Malawi found no association between stunting and L:M values [133,136], but each cohort exhibited low mean HAZ values (-2.4 ± 1.3 and -2.8 ± 1.1, respectively) and used high L:M cut-points to classify EED (0.15 and 0.10,  34 respectively). Thus, the values of L:M were likely elevated even in those not classified with EED. Conversely, a third study in Burkina Faso found no association between L:M and stunting, but the cohort was less stunted (mean HAZ = -1.5 ± 1.1) and had a low mean L:M value (0.042; 95% confidence interval: 0.030, 0.072) [126]. The proportionally greater permeability in all subjects with higher L:M values, and the proportionally lower stunting in subjects with lower L:M values, provide evidence in favor of assessing permeability, particularly in cohorts with high variability in the underlying prevalence of stunting. Moreover, EED may not be a binary (present/absent) condition but a population shift in intestinal structure, wherein some individuals with EED-like characteristics (e.g. increased permeability, malabsorption) may not exhibit gut morphological changes. The variability in underlying characteristics presents challenges in interpreting results from heterogeneous study populations.   Finally, lactulose permeability may not accurately reflect the ability of microbes, toxins or larger molecules to pass through the epithelium. The molecular weight of lactulose (342 Da) is significantly smaller than that of antigenic molecules such as LPS (10-20 kDa) [157]. Holes in the tight junction complex that allow paracellular lactulose permeation may not be large enough to allow the movement of microbes or their byproducts across the epithelium. These factors may partly explain why permeability and absorption were not consistently associated with other EED domains, especially inflammation, or stunting.   The association between microbial translocation and stunting was observed in a study of infants in The Gambia [158] over a decade ago, but few studies included in this review support this relationship. Assuming that stunting is a primary outcome of EED, we propose that current tests  35 for microbial translocation may not be appropriate for EED identification. However, strong evidence supports the relationship between intestinal inflammation and stunting, and it is also possible that factors other than microbial translocation may be responsible for the intestinal inflammation observed in individuals with EED. Several investigators [57,138,159] have suggested that pathogen colonization may promote chronic inflammation and induce changes to the intestinal microbiota, contributing to EED and stunting, though the exact mechanism is unknown. The intestinal microbiota contains over hundreds of different species of bacteria, as well as assorted viruses, archaea, fungi and yeasts; the composition and function of the microbiota is largely determined by a combination of dietary, host genetic, inflammatory, clinical (e.g., antimicrobial exposure), and other environmental factors [160]. To our knowledge, only one human study has investigated microbiota dysbiosis in individuals with EED, which was categorised based on L:M ratios [137]. Three of the 6 differentially abundant bacterial genera—Megasphaera, Mitsuokella, and Sutterella—were enriched in children with EED compared to children without EED; the other 3—Succinivibrio, Klebsiella, and Clostridium cluster XI (Peptostreptococcaceae)—were depleted.     Small intestinal bacterial overgrowth (SIBO) may also contribute to intestinal inflammation [161] and EED. A study of urban Bengali children investigated the relationship between the glucose hydrogen breath test, an indicator of SIBO, with EED domains and outcomes [156]. SIBO was not associated with the L:M ratio nor systemic inflammatory markers. However, children with SIBO had significantly worse linear growth and higher concentrations of calprotectin, an intestinal inflammatory marker, compared to those without SIBO. A study of Burmese children observed a similar association between SIBO and linear growth faltering [162]  36 but this association was not present in slum-dwelling children in Brazil [163]. Additional evidence for the relationship between stunting and SIBO is needed.   The inconsistencies highlighted in this review indicate that EED may be more complex than previously conceived. It is possible that EED is not a single entity, but instead a set of phenotypes dependent on unique environmental exposures that vary geographically. The small intestine has a limited repertoire of responses to insult, and enteropathy resulting from multiple potential exposures may have a similar appearance. Some elements of EED may even be adaptive, rather than pathologic, for children living in conditions of poor water, sanitation and hygiene [75]. It is certainly not firmly established that EED is always consequential to linear growth, or indeed that it is definitively associated with stunting. Study subject selection (e.g., including subjects with diarrhea, HIV, or severe acute malnutrition) may influence EED biomarker results. Other potential confounders, including malnutrition, diarrhea, food insecurity, the intestinal microbiota/microbial factors, concomitant treatments (e.g., antimicrobial therapy) that were measured in some studies, but not all, may influence individual study results and our conclusions.   Recent human intervention studies suggest that micronutrient supplementation is not sufficient for EED recovery [140,145,164]; only one study of Zambian adults observed significant improvement in intestinal morphology after micronutrient supplementation [102]. Morphological studies are more feasible in animals than humans and have the potential to reveal key associations that are otherwise difficult to assess in human studies. An experimental study to evaluate the effects of malnutrition and exposure to Bacteroidales and E. coli in mice observed  37 that malnourished mice with bacterial exposure experienced increased permeability and tail length (a surrogate for length of mouse), as well as blunted intestinal villi characteristic of EED [165]. Mice with a malnourished diet—regardless of bacterial exposure—also had greater microbiota dysbiosis and intestinal permeability, but without blunted intestinal villi. Mice with bacterial exposure but normal diets experienced normal tail growth and no change in permeability. These results suggest an interaction between malnutrition and the presence of specific bacterial species, where EED features are present only in individuals with both.   Significance and future directions This review provides important information for the field of EED research. In particular, the results of the review will be used in guiding the definition of EED in two large randomized controlled trials investigating the causal relationship between EED and childhood stunting. The Water, Sanitation, and Hygiene (WASH) Benefits Study is comprised of two cluster-randomized controlled trials that measured the impact of interventions on infant health outcomes in rural villages in Bangladesh and Kenya [166,167]. While this study found modest improvements in growth outcomes with nutritional supplementation, no association was observed between growth outcomes and water quality, sanitation, or handwashing interventions. The Sanitation, Hygiene, Infant Nutrition Efficacy (SHINE) Project, a cluster-randomized trial conducted in rural Zimbabwe, aims to investigate the impacts of WASH and nutritional interventions on childhood health outcomes, including stunting, anemia, diarrhea, and EED [168]. Both studies plan to test the relationships between the interventions and EED biomarkers, as well as the association between EED biomarkers and childhood growth outcomes. This review provides guidance on biomarkers that best characterize EED. Forthcoming conclusions from these well-conducted  38 studies will provide strong evidence for the true pathophysiological pathways involved in EED, and whether EED related intestinal effects cause child stunting.   Study Limitations This review summarizes evidence for each pathway in a consensus framework model of EED (and its hypothesized component/contributing domains) and stunting. Information from a wide range of published articles representing very different biomarker measurement methods, heterogeneous study populations, and different analysis and reporting methods, was reviewed. We did not assess study quality in this review given the heterogeneity, nor did this review include studies of in vitro and in vivo models of EED. Many studies did not report associations between domains; these missing or non-reported data may have influenced our conclusions.  This is a consistent feature of EED-related studies, as already highlighted by Denno et al in their review of biomarkers from 2000-2010 [53,75]. The studies included in that review, which categorized biomarkers into eight EED domains, provided conflicting evidence for the links between permeability and linear growth [144,169–174], permeability and intestinal inflammation [169,175], as well as systemic inflammation and linear growth [170,172]. Microbial translocation was not included as a domain in their review, but one study reported a positive association between IgG endotoxin-core antibody and permeability [169]. No conclusive set of biomarkers for EED diagnosis were identified in either the 2000-2010 or in our review of the literature from 2010-2017.   The domains and specific pathways defined and investigated in our review are open to debate; however, we focused on pathogenic processes that have been long been hypothesized to be part  39 of EED. Other potential domains (e.g. digestion, microbial drivers) have been identified within EED pathways [57,60–62], but these domains were minimally reported or missing from selected studies and were not included in our results.   Summary This review evaluated individual pathways between domains within EED and between each domain and stunting using studies published between 2010-2017. I found evidence to support the link between intestinal and systemic inflammation and stunting, but little support for the link between microbial translocation and stunting within the limits of current tests. There was conflicting evidence for the associations between intestinal damage and intestinal permeability, as well as intestinal damage and stunting. 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Pediatr Res. 2003;54: 306–311.   58 Appendices  A: Summary data table    B: PRISMA Checklist  Appendix	A	–	Summary	data	table			 59	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2017   Brown G et al  Linking growth and gut function: Environmental enteropathy in children under 2 years in rural Nicaragua  To explore the relationship between intestinal function and growth outcomes in children under 2 years  Rural Nicaragua  Children (mean age = 12.04 mo) Cross-sectional  n=53 Anthropometric  HAZ, WAZ, BAZ  Permeability L:C    Anthropometric 11% were stunted (HAZ < −2) 2% were wasted (BAZ < −2).   Permeability Median L:C ratio = 0.10 (IQR = 0.16) L:C ratio was not significantly correlated with WAZ or BAZ Negative correlation between HAZ and L:C ratio  Children with higher HAZ (HAZ > 0) had worse gut function (p = 0.0492) Abstract only  No definition of EED provided   2017  Campbell RK et al  Biomarkers of environmental enteric dysfunction among children in rural Bangladesh  To evaluate convergence among biomarkers and describe risk factors for EED. Rural Bangaldesh  18 mo children Cross-sectional nested in a community-based RCT of complementary food supplements  n = 566 eligible  n = 539 final  (27 children refused EED assessment) Anthropometric LAZ, WAZ  Permeability  2-h sugar absorption test, dosed by body weight (2-20mL/kg with 255 mg/mL L and 50 mg/mL M)  Intestinal damage and repair GLP-2  Intestinal inflammation MPO, AAT, NEO combined to make gut inflammation Anthropometric - LAZ and WLZ at 6 mo and their change from 6 to 18 mo were associated only with the gut permeability score (L:M ratio) - Lower GLP-2 was associated with higher ∆LAZ  - Higher EndoCAb positively associated with ∆LAZ   Permeability  - Mean L:M: 0.06 [0.05, 0.06] - Elevated in 39% of children - Correlation of L:M with other biomarkers were not significant - A multivariate model including all biomarkers only explained 10.8% of the observed variability in L:M Serum and stool biomarkers demonstrated low agreement internally and with L:M test, and principal component analysis (PCA) and partial least squares (PLS) regression did not identify a subset of markers that closely approximated L:M.  EndoCAb – LAZ results were opposite of what is expected Definition: subclinical condition of partial villous atrophy, crypt hyperplasia, leaky tight junctions and enteric immune cell proliferation  Cutoffs L:M > 0.07 MPO > 2000 ng/mL AAT > 270 μg/mL NEO > 70 nmol/L AGP > 1 g/L CRP > 5 mg/L  	 60	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments (GI) score  Systemic inflammation AGP CRP Total IgG, IgM, IgA   Microbial translocation EndoCAb IgG, IgM, IgA   - MPO was modestly correlated with AAT (r=0.33, p<0.01)  Intestinal inflammation - Concentrations were elevated in 84% (MPO), 56% (AAT), and 100% (NEO) of children  Systemic inflammation - Concentrations were elevated in 56% (AGP) and 20% (CRP) of children - CRP was correlated with AGP (r=0.55, p<0.01)  Microbial translocation - EndoCAb values positively correlated with LAZ values - EndoCAb and Igs were consistently intercorrelated - EndoCAb inversely associated with GLP-2  2017  Kosek M et al  Causal Pathways from Enteropathogens to Environmental Enteropathy: Findings from the MAL-ED Birth Cohort Study   To assess the role of enteropathogens and other factors in growth Eight sites: Bangladesh (BGD) India (INV) Nepal (NEB) Pakistan (PKN) Brazil (BRF) Peru (PEL) South Africa (SAV) Tanzania (TZH)  Children 0-2 yo, enrolled within 17 days of birth Longitudinal cohort  n = 1253  (complete samples = 744)  Anthropometric LAZ, WAZ  Permeability L:M ratio  LMZ score AAT  Intestinal Inflammation NEO, MPO   Systemic Anthropometric - No statistical support for LMZ relating to changes in either LAZ or WAZ  - At age 1, negative association between MPO and ∆LAZ and ∆WAZ at age 1 - At both ages, negative association between AAT and ∆WAZ - Giardia was associated directly with reduced growth, but not with the fecal biomarkers.  - Higher AGP concentrations were associated with decreased ∆LAZ at both ages and ∆WAZ at age 1. Enteropathogens changed the concentration of NEO, MPO, AAT, and LMZ  Enteropathogens and gut inflammation both relate to systemic inflammation  Enteropathogens present even in the absence of diarrhoea  According to these biomarkers, EED Definition: Long-term effects on child health, stemming from host response to frequent enteric infections that alter the gut, even in the absence of diarrhoea or acute gastrointestinal illness  	 61	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments faltering from birth to two years    Exclusion:  - birth weight < 1500 g - were very ill - non-singleton - mother was < 16 yo Inflammation AGP  Permeability  - LMZ tended to be higher in children with pathogens detected, especially Cryptosporidium and Giardia - At age 1, AAT was positively associated with LMZ; MPO was negatively correlated with LMZ  Intestinal Inflammation - In a model containing all pathogen groups, all biomarkers, and LAZ and WAZ, Group II bacteria showed positive association with MPO at both ages - Group III bacteria were associated with lower AAT at age 1 and higher MPO at age 2  Systemic Inflammation - At age 2, MPO and AGP were positively correlated; NEO was negatively correlated with AGP   contributes to stunting but is not the biggest contributor   No association between intestinal permeability (as measured by L:M) and future growth -- AAT may be a more valid measure of EED   Reductions in exposure to pathogens could reduce systemic inflammation (as measured by AGP) and subsequent stunting  2017  Lee G et al  Environmental enteropathy is associated with cardiometabolic risk factors in Peruvian children  Peru site within the MAL-ED study  Children 3-5 yo  Eligible: children living a minimum of 18 mo and were still living in the study area Case-cohort  n = 303 enrolled n = 164 eligible n = 156 final  n = 154 EndoCAb Permeability 2-h sugar absorption test  20 ml dose of L:M solution, food provided after 30 minutes %L, %M, L:M ratio  Anthropometric Permeability %L and %M strongly positively correlated %M positively associated with HDL-c, Apo-AI and Apo-B %M negatively associated with VLDL-c, the TC/aHDL-c ratio, the Apo-B/Apo-AI ratio, HOMA-IR, high TG and high blood pressure No consistent associations between L:M ratio measured at any time and the Study supports the potential for a relationship between EED and the cardiometabolic profile through both inflammatory and non-inflammatory mechanisms  Limitations include relatively small sample size and that metabolic syndrome cannot be Definition: syndrome of altered small intestine structure and function characterized by partial villus atrophy and crypt hyperplasia  Cutoffs: L:M >	0.07 	 62	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments To assess the association of EED with the cardiometabolic profile among young children living in an environment of intense enteropathogen exposure  n = 155 apolipoprotein profile HAZ, WAZ, WHZ  Cardiometabolic factors Glucose TG TC HDL-c LDL-c VLDL-c HOMA-IR  Intestinal Inflammation MPO NEO AAT  Microbial translocation EndoCAb  Systemic Inflammation AGP CRP  cardiometabolic profile By 15 months of age: - 46% had L:M ratios above the 90th percentile for a healthy tropical reference population - 70% had subclinical inflammation (defined by AGP)  Anthropometric No associations between biomarkers and stunting were reported  Microbial translocation No association between EndoCAb and cardiometabolic profile No correlation reported between EndoCAb and L:M  Systemic Inflammation At 7 months, associations between: High blood pressure—CRP (OR=1.58)  High blood pressure—AGP (OR=1.57)   assessed at such an early age %L > 0.08% %M > 0.86% EndoCAb < 75GMu/ml  AGP>1 g/l    	 63	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2017   Ordiz et al  Environmental enteric dysfunction and the fecal microbiota in Malawian children  Use 16S rRNA gene sequencing to identify phylogenetic groupings of bacteria to detect dysbiosis associated with EED Rural Malawi  12-23 mo healthy children  Exclusion: diarrhoea in previous 7 days Cross-sectional nested in RCT  n = 81 Anthropometric HAZ  Permeability L:M  Bacterial genera  Children with EED at baseline: n (%) • No EED: 8 (10) • Moderate: 49 (60) • Severe: 24 (30)  Anthropometric Baseline categorization of EED (i.e. baseline L:M ratio) was associated with ∆HAZ in the subsequent 3 months (P = 0.01, Tukey-Kramer multiple comparison test)  Bacterial genera presence • 2 genera present only in no EED individuals • 51 in moderate EED • 28 in severe EED;  • 138 in all individuals regardless of EED status • 6 genera were differentially abundant in EED vs. no EED: • Megasphaera, Mitsuokella, and Sutterella were more prevalent in EED  • Succinivibrio, Klebsiella, and Clostridium_XI were less prevalent in EED  EED is not profoundly associated with fecal dysbiosis, but six genera were identified as having significantly different abundances in EED.  Study is limited by the use of fecal (colonic) bacterial colonies opposed to small bowel colonies and therefore cannot determine a causal relationship between EED and bacterial genera present Definition: characterized by small bowel villous atrophy and inflammation  EED Cutoff  No EED (< 0.15) Moderate (0.15 – 0.45) Severe (L:M > 0.45) 2017  Semba R et al  Environmental Enteric Dysfunction is Rural Malawi  Children 12-59 mo  Exclusion: presence of congenital or Cross-sectional  n = 400  n = 323 Anthropometric HAZ, WAZ  Permeability 4-h dual sugar absorption test, 1g Anthropometric HAZ, mean ± SD (p = 0.25): • EED: -2.3 ± 1.3 • no EED: -2.5 ± 1.3  WAZ, mean ± SD (p = 0.29): This study shows that EED (as measured by L:M ratio) is associated with secondary carnitine deficiency.  Gut permeability (L:M ratio) was associated with a Definition: asymptomatic condition characterized by chronic inflammation of the duodenum and jejunum and 	 64	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments Associated with Carnitine Deficiency and Altered Fatty Acid Oxidation  Use an LC-MS/MS metabolomics platform to comprehensively assess metabolites related to carnitine deficiency in individuals with EED  chronic disease or caretaker-reported diarrhoea, or were under treatment for acute malnutrition (80.7%) with EED as measured by L:M ratio  M: 5g L  L:M ratio  Serum metabolites   • EED: 0.3 ± 1.0 • no EED: 0.1 ± 1.3  Stunted (p = 0.31): • EED: n= 52 (68%) • no EED: n = 198 (61%)  Serum metabolites 77 metabolites were significantly correlated with L:M ratio to classify EED status: • 21 negatively associated • 56 positively associated   These 77 metabolites had only a modest ability to discriminate between children with and without EED, as determined by multiple machine-learning classification algorithms  Children with increased gut permeability had elevated serum levels of: • concentrations of four odd-chain fatty acids • 3-hydroxy-3-methylutarate • 3-aminoisobutyrate • trimethylamine-N-oxide • S-adenosylhomocysteine  • cystathionine • taurine • phenylacetylglutamine • 4-hydroxyphenylacetyl-glutamine serum metabolite profile corresponding to elevated serum concentrations of:  • 9 acylcarnitines • 3 intermediate metabolites associated with blocked β-oxidation of fatty acids  • 4 metabolites related to upregulation of ω-oxidation of fatty acids  Children with EED may have altered metabolism associated with an abnormal gut microbiome, as demonstrated by reduced levels of hippurate, which is normally strongly associated with diet and gut microbiome  Limitations: Cross-sectional study cannot show that the relationship between ED and carnitine deficiency is causal abnormal gut permeability  Cutoff EED: L:M ≥ 0.15  	 65	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments • phenylacetate • serotonin • homocitrulline • and more  Children with increased gut permeability had reduced serum levels of: • hippurate • 3-hydroxyhippurate • dietary polyphenolic compounds (intermediates in the pathways to hippurate) • dihydroferulic acid • catechol sulphate • citrulline • ornithine • three fat-soluble substances • and more  2017  Semba R et al  Environmental enteric dysfunction is associated with altered bile acid metabolism  To examine the potential relationship between EED and abnormalities in serum bile acid metabolism. Rural southern Malawi  Children 12-59 mo   Exclusion: presence of congenital or chronic disease or caretaker-reported diarrhoea, or were under treatment for acute malnutrition  Cross-sectional   n = 313 Permeability 4-h dual sugar absorption test, 1g M: 5g L  L:M ratio  Anthropometric HAZ, WAZ  Serum bile acids n = 244 (80%) with EED as measured by L:M ratio  Anthropometric HAZ, mean ± SD: • EED: -2.4 ± 1.3 • no EED: -2.4 ± 1.3  WAZ, mean ± SD: • EED: 0.2 ± 0.9 • no EED: 0.3 ± 0.9  The present study suggests that children with EED have alterations in bile acid metabolism. Total serum bile acids were approximately 12% lower in children with EED vs. no EED.   Malawian children had a lower proportion of TCDCA compared with studies of healthy Austrian children  Definition: clinically asymptomatic condition characterized by inflammation of the small bowel mucosa, villous atrophy, malabsorption, and increased intestinal permeability  Cutoff EED: L:M ≥ 0.15  	 66	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments Stunted (p = 0.31): • EED: n= 52 (68%) • no EED: n = 198 (61%)  Serum bile acids Total serum bile acids were significantly lower in children with EED vs. no EED (P = 0.0009)  3 most abundant serum bile acids were GCDCA, GCA, and GDCA; none of these were significantly different between EED vs. no EED groups  Serum TCDCA, TMCA, and GUDCA were less abundant but were significantly different in EED vs. no EED groups  Serum bile acid ratios, which provide the relative proportion of bile acids conjugated by taurine, were higher in children with EED vs. no EED (P < 0.0001)  Serum concentrations of unconjugated bile acids were not different between children with and without EED, which suggests that small bowel bacterial overgrowth is not a significant problem in this population   2017  Wang AZ et al  A Combined Intervention of Zinc, Multiple Micronutrients, and Albendazole Does Not Ameliorate Environmental Enteric Dysfunction or Stunting Rural Malawi  12-35 mo children who were not acutely ill, including twins  Exclusion: inability to provide a complete 4-h urine collection, recent RCT  n = 263 screened n = 254 randomly assigned to intervention groups  n = 241 Permeability 4-h L:M sugar absorption test 5g L: 1g M  Anthropometric Length, weight Intervention group Initial L:M ratio: 0.33 ∆ L:M ratio after 24 wk: 0.088 Initial length (cm): 80.9 Length 24 wk: 85.3  Placebo group Initial L:M ratio: 0.34 ∆ L:M ratio after 24 wk: 0.080 No significant differences between placebo and intervention groups with regards to L:M ratio or linear growth  Definition: subclinical condition characterized by diffuse blunting of intestinal villi and inflammation of the lamina propria  ∆ L:M ratio ≥ 0.06 is the smallest interval change that would be statistically 	 67	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments in Rural Malawian Children in a Double-Blind Randomized Controlled Trial  To assess improvements in EED (as measured by L:M and linear growth) after 12-24 weeks of combined intervention of zinc, abendazole, and multiple micronutrient powder  history of >3 loose stools/day, SAM, moderate acute malnutrition, or chronic illness  successfully completed all 3 dual-sugar absorption tests Initial length (cm): 80.4 Length 24 wk: 84.9   significant 2016  Arndt M et al  Fecal markers of environmental enteropathy and subsequent growth in Bangladeshi children  Bangladesh site of MAL-ED study  Children 0-24 mo  Exclusion:  - maternal age of < 16 years - not a singleton pregnancy  - another child already enrolled in the MAL-ED study  - severe disease requiring hospitalization before recruitment  - severe acute or chronic conditions diagnosed by a physician   Longitudinal  n = 246 (5.7% LTFU before 12 mo, 14.6% LTFU before 24 mo) Anthropometric WAZ, LAZ, WHZ  Intestinal Inflammation AAT, MPO, NEO: combined to form Kosek EE disease activity score  Anthropometric At birth, ~16% of children were stunted (LAZ < –1) • After 24 months, 78% of those stunted at birth were still stunted • After 24 months, 44% of those not stunted at birth were stunted  Intestinal Inflammation Samples considered normal in non-tropical settings: • 63% AAT • 68% MPO • 95% NEO   The Kosek EE score was negatively associated with LAZ in months 1-3 and 12–21, but not in months 3–9.   A one-unit increase in EE score in months 3-21 was associated with a High fecal MPO levels were associated with decreases in 3-month linear growth in the second year of life. Neither AAT nor NEO were associated with subsequent growth.  • The composite EE score appears to be driven by MPO levels  None of the fecal markers tested nor the EE score were significantly associated with subsequent 6-month growth Definition: subclinical intestinal disorder characterized by mucosal and systemic inflammation, reduced intestinal barrier integrity, bacterial translocation, and reduced intestinal absorptive capacity  Normal cutoff for non-tropical settings AAT < 0.27 mg/g MPO < 2,000 ng/ mL NEO < 70 nmol/L  Kosek score ranges from 0-10 	 68	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments decrease of 0.009 LAZ per 3-month period (95% CI: -0.018, 0.000)  A one-unit increase in EE score in months 12-21 was associated with a decrease of 0.013 LAZ per 3-month period (95% CI: -0.023, -0.004)  Note: When the significance level was adjusted for multiple comparisons (using either the Benjamini–Hochberg or Bonferroni method), none of the marker coefficients remained significant.  2016   Donowitz et al  Small intestine bacterial overgrowth and environmental enteropathy in Bangladeshi children  (1) To determine the prevalence of SIBO in Bangladeshi children and its association with malnutrition; (2) To determine the association of SIBO with sanitation, diarrheal disease, and EED   (Part of PROVIDE study) Urban Bangladesh  Children 2 yo  Exclusion:  WAZ < 3 SD Cross-sectional   n = 103 assessed for SIBO testing n = 90 successfully completed SIBO testing  Anthropometric LAZ  SIBO Glucose hydrogen breath test  Permeability and Absorption L:M ratio  Intestinal damage and repair REG1B  Intestinal inflammation calprotectin  Systemic SIBO - Children with SIBO had significantly worse linear growth (stunting) from enrolment to age 2 compared to those without SIBO - Diarrheal disease, WAZ, SES were not significant predictors of SIBO  Intestinal damage and repair REG1B was associated with SIBO  Intestinal inflammation Calprotectin was significantly increased in children with SIBO  Permeability and Absorption No significant different in the L:M ratio for children with or without SIBO  Systemic inflammation Findings are contradictory to other investigations which found an increase in intestinal permeability in adults with SIBO in the developed world  Lack of L:M ratio differences may indicate that SIBO and EE are separate but concomitant conditions or that EE is actually a syndrome encompassing a heterogeneous group of environmentally-derived intestinal inflammatory conditions with variable manifestations but common outcomes Definition – an inflammatory intestinal disorder of the developing world that has been implicated in growth failure and poor neurocognitive out- comes  Cutoff Normal SIBO < 12-ppm over baseline 	 69	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments  inflammation CRP, 17-plex Luminex cytokine panel, INFg, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, MCP-1, MIP-1B, TNFa  None of the markers were significantly different between the two groups 2016   Faubion WA et al  Improving the detection of EED: a lactulose, rhamnose assay of intestinal permeability in children aged under 5 years exposed to poor sanitation and hygiene   To address the limitations of the current L:M test and to assess the intestinal barrier function in infants with EED using the LR test   USA Children 2-13 mo  Zambia Children 2-36 mo  Peru Children 15-29 mo  Exclusion: concurrent illness, had diarrhea within 1 month, had been given antibiotics within 1 month Case-control  n = 131 total  USA (n=27) Zambia (n=85) Peru (n=19)  Anthropometric HAZ, WAZ  Permeability 1-h L:R test - 1000mg L: 22mg R %L, %R  Anthropometric Median (range) WAZ and HAZ: • Peru: (WAZ: −0.8 (−2.7, 0.8); HAZ: −1.8 (−3.3, −0.2)) • Zambia: (WAZ: −1.4 (−4.0, 1.1); HAZ: −2.3 (−8.5, 1.2))  • USA: (WAZ: 0.5 (−0.4, 2.1); HAZ: −0.1 (−1.8, 2.4)) HAZ and WAZ in Peruvian and Zambian cohorts were significantly different than the US cohort (p < 0.001), indicating presence of EED in the two populations  WAZ and HAZ as outcome variables: • %L significantly predicted both WAZ (p < 0.001) and HAZ (p = 0.0018) • %R did not significantly predict either WAZ or HAZ • L:R significantly predicted WAZ (p = 0.013) but failed to predict HAZ (p = 0.098)  Permeability Feasibility of LR test is indicated by completion of tests: Authors speculate that the reduced %R may be due to decreased intestinal SA, therefore recovery of lactulose alone may potentially be a more accurate marker of intestinal permeability than %R alone, LR ratio or L:M ratio  Low R2 values for both WAZ and HAZ regression models indicated that there are likely other factors that contribute to impaired growth Definition: disorder of the small bowel that is characterized by chronic intestinal inflammation, villous atrophy, malabsorption and linear growth failure 	 70	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments • Peru: 19/19 (100%) • Zambia: 73/85 (86%) • USA: 22/27 (81%)  Detectable concentrations of mannitol were present in over 90% of children in all three cohorts within the baseline urine sample, while presence of rhamnose was negligible (2.6%).   LR ratio:  • Peru: (0.75 (0.15, 5.02))  • Zambian (2.26 (0.08, 14.48))  • USA: (0.14 (0.06, 1.00))  Significant differences between Peruvian and Zambian cohorts compared to US cohort (p<0.001)  Percent (n/N) of children displaying abnormal LR ratio (compared to 95th centile from the US population as the upper limit of normal): • Peru: 53% (1/22) • Zambian 82% (10/19) • USA: 5% (60/73)  	 71	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2016  Guerrant R et al  Biomarkers of environmental enteropathy, inflammation, stunting, and impaired growth in children in northeast Brazil  1. To examine potential fecal, urinary and plasma biomarkers to determine how they associate with each other and with malnutrition  2. To determine which biomarkers best predict impaired subsequent growth Northeastern Brazil  Children 6-26 mo  Exclusion: - underlying disease - parent/guardian < 16 yo Case-cohort, matched on gender and age  n = 402 recruited  n = 375 enrolled, provided initial measurements  n = 301 final Anthropometric HAZ, WAZ   Permeability/absorption L:M, %L, %M AAT  Intestinal damage and repair REG1 i-FABP  Bacterial translocation plasma LPS IgG anti-LPS/anti-FliC IgA anti-LPS/anti-FliC  Tight junction impairment zonulin Cdn-15  Intestinal Inflammation MPO NEO  Systemic inflammation plasma SAA Anthropometric Significant associations between: • Lower WAZ with IgA anti-LPS, i-FABP at baseline • Lower HAZ with IgA anti-FLA and anti-LPS, i-FABP, citrulline, SAA at baseline • HAZ/WAZ with zonulin, children > 12 mo • growth impairment correlated with higher LPS, I-FABP and SAA • citrulline levels and weight gain primarily in girls (p < 0.001) • tryptophan and growth in boys (p = 0.010) • For urine L:M, higher values correlated (controlling for age and gender) with impaired growth (∆HAZ) (r = -0.173; p = 0.009; n = 230). • Subsequent growth was worse in those with higher AAT  Biomarkers Significant associations: • Cdn-15 with negative L:M, AAT, REG1, lipocalin-2 • KTR with MPO (p < 0.01) • KTR with CRP (p < 0.01) • KTR with citrulline, negative (p < 0.01) • MPO with AAT, LPC, REG1, kynurenine, KTR (p < 0.01) • MPO with L:M, %L, and tryptophan (negative) (p < 0.05) Many biomarkers are present in already stunted children, namely systematic IgA with anti-LPS and anti-FLA and i-FABP  Other biomarkers may help predict future stunting, including MPO, AAT, L:M, LPS, i-FABP, and SAA, as well as the interaction between MPO and NEO  Boys and girls had slightly different biomarkers, which may be due to the higher prevalence of stunting in males of the lowest SES  Biomarkers tend to cluster into 3 main groups: (1) intestinal translocation, (2) intestinal mucosal barrier disruption and inflammation, and (3) systemic inflammation  Barrier function, intestinal inflammation and systemic markers are linearly associated with each other as well as baseline stunting and ∆HAZ Cdn-15 has never been used to measure healthy gut function in individuals Definition: damage of villous architecture, caused by disruption of gut barrier function, passage of microorganisms and/or their bioproducts from the intestinal lumen to the lamina propria, and mucosal inflammation  	 72	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments sCD14 LPC citrulline tryptophan kynurenine  • REG1 correlated with AAT, MPO, and NEO (p < 0.01)  • REG1 with LPC and lactoferrin (p < 0.05) • MPO with CRP, kynurenine, and KTR (p < 0.01),  • MPO with SAA and tryptophan (negative) (p < 0.05) • interaction between MPO and NEO  Principal Component Analysis (PCA), identifying groups of biomarkers that relate to subsequent growth: • B1: LPS (translocation) • B2: L:M and i-FABP (disrupted absorptive function and epithelial cell damage) • B3: further barrier disruption with tight junction modulation (e.g. REG1 and AAT with zonulin)  Significant associations: • PCA B2 with MPO • PCA B1 with baseline HAZ  • B3 with MPO • B3 with NEO  Significant predictors of growth impairment (∆HAZ), controlling for age and gender: • HAZ at baseline (p < 0.001) • MPO (p = 0.041) • PCA B2  with EED; Cdn is inversely correlated with L:M, AAT, lipocalin-2, and REG1 and may be used as a positive biomarker of gut health  PCA analysis revealed that biomarkers of barrier disruption have significant correlation with baseline HAZ and ∆HAZ, as well as with multiple other groups of biomarkers  Conclusion: fecal MPO and AAT, and L/M, plasma LPS-neutralizing activity, I-FABP, SAA, CRP, citrulline and tryptophan provide the most promise for measuring subsequent growth failure 	 73	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2016  Kelly P et al  Endomicroscopic and Transcriptomic Analysis of Impaired Barrier Function and Malabsorption in Environmental Enteropathy  To assess the association between CLE biopsy data  Southern Zambia  Adult volunteers recruited by door-to-door invitations, focus group discussions, and individual interviews  Excluded: individuals with concurrent illness, pregnancy, use of NSAIDS or antibiotics within one month, or recent helminth infection Cross-sectional  n=49 total  n=35 HIV seronegative  n=14 HIV seropositive • n=6 on ART • n=8 not on ART  *ART = antri-retroviral therapy Anthropometric height, weight, mid upper arm circumference  Morphometry biopsy samples  Permeability: Confocal laser endomicroscopy (CLE) for biopsy  Microbial translocation: LPS LPS binding protein  Intestinal damage and repair i-FABP GLP-2  Mineral absorption: Zinc uptake  Systemic Inflammation CRP sCD14 CD163  Permeability  Watson scores: • 1: n=4 (10%)  • 2: n=6 (15%) • 3: n=31 (76%)  Plumes were positively associated with i-FABP  Morphometry All biopsies contained villous blunting, crypt lengthening, and lamina propria inflammation No significant differences between HIV and non-HIV individuals  Microbial translocation: LPS was detectable in all samples LPS was associated with: • GLP-2 (β = -0.13, n = 43; P = 0.007) • cell shedding (β = 0.83, n = 43; P = 0.035) • epithelial perimeter (β = 10136; P = 0.01)  • villus SA:volume ratio, fractional polynomial model (β = 59006; P = 0.007) • villus SA:volume ratio, linear regression model (ρ = 0.46; P = 0.003)  LPS was not associated with CRP, sCD14, CD163, or LBP  The U-shaped relationship between translocation and villus SA suggests that the reduced villus surface area in enteropathic disorders may act at least in part to mitigate the translocation burden in the presence of epithelial defects  No correlation between zinc uptake and morphometric attributes, but severe enteropathy was associated with reduced expressed of DMT-1, which regulates zinc uptake  Low circulating GLP-2 concentrations were associated with very high levels of translocation; may indicate primary endocrine failure to synthesize GLP-2 Definition: an asymptomatic disorder characterized by variance in the small intestinal mucosal architecture  Watson Score 1 = no fluorescein plumes / leakage  2 = fluorescein leakage but no microerosions 3 = fluorescein leakage with any microerosions  Zinc deficiency = plasma concentrations below 11 μmol/l 	 74	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments Intestinal Inflammation AAT   Transcriptomics Mineral absorption 21/48 (44%) participants were deficient in zinc Zinc was positively associated with GLP-2 (β = 2.70; P = 0.03)  Transcriptome analysis (n=8) n=4, very low/undetectable plumes n=4, frequent plumes 23 differentially expressed genes between the two groups  • Down-regulated genes included those involved in metal ion uptake, anti-protease activity, mucosal protection, and host defense • Up-regulated genes included those involved with inflammation  2016  Kosek M et al  Plasma tryptophan and the kynurenine-tryptophan ratio are associated with the acquisition of statural growth deficits and oral vaccine under-performance in populations with environmental enteropathy  To determine whether KTR, citrulline, and Rural Peru, rural Tanzania arms within the MAL-ED study  Children 0-2 yo, enrolled within 17 days of birth  Exclusion:  - birth weight < 1500 g - were very ill - non-singleton - mother was < 16 yo Longitudinal Cohort   n = 494 (including those LTFU over 24 mo)  Peru n = 303 enrolled n = 262 with one measurement of K, T, citrulline n = 198 retained for Anthropometric  LAZ, WAZ, WHZ  Mucosal Mass Citrulline  Permeability L:M ratio  Systemic Inflammation KTR* Plasma cytokines: • AGP • CRP (Peru only) Anthropometric  Peru:  • Mean LAZ decreased from -1.3 to    -1.9 over 24 mo • At 24 mo, ~40% of children  • 1-SD increase in tryptophan was associated with a gain in LAZ of 0.10 over the next 6 months (95% CI: 0.05–0.16, P < 0.001) • At 3 months, 1-SD increase in citrulline concentration was associated with a gain in LAZ of 0.19 over the next 6 months (95% CI: 0.07–0.31, P = 0.002);  Tanzania: • Mean LAZ decreased from -1.0 to    Tryptophan concentrations are associated with subsequent improved linear growth in children in Peru and Tanzania when measured at 3, 7, 15, and 24 mo  The relationship between tryptophan, KTR, and linear growth is stronger than that of CRP, AGP, and IL-6. This may suggest that tryptophan may be a more specific and predictive prognostic marker than these more common markers of systemic inflammation. Definition: functional disorder of the gut resulting from multiple enteric infections which result in chronic intestinal immune activation, augmented intestinal permeability, and persistent systemic immune activation  	 75	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments cytokines were associated with growth deficits in infancy and early childhood 24 mo  Tanzania n = 262 enrolled n = 219 with one measurement of K, T, citrulline n = 211 retained for 24 mo • IFN-	γ (Peru only) • IL-10 (Peru only) • IL-6 (Peru only)    -2.7 over 24 mo • At 24 mo, ~71% of children  • 1-SD increase in tryptophan was associated with a gain in LAZ of 0.13 over the next 6 months in Tanzania (95% CI = 0.03–0.22, P = 0.009) • Citrulline was not associated with growth  Neither kynurenine nor the KTR demonstrated significant relationships with LAZ   Mucosal Mass Mean citrulline level: 21.1 μM (95% CI: 12.7–32.8) Citrulline and LAZ were negatively associated at 3 mo (ρ = −0.16, P = 0.01) but not associated at any other time Citrulline was not associated with LAZ, WAZ, or WHZ at any other time.   Permeability %L was not associated with kynurenine or tryptophan, but was negatively associated with citrulline levels %M was associated with higher tryptophan and kynurenine, but not associated with citrulline    Systemic Inflammation Mean concentrations:  Tryptophan concentrations are influenced by dietary intake; given the relationship between tryptophan and growth, it may be possible to improve growth by diet diversification or by introducing enriched foods, such as protein maize, at a young age  Alternatively, intestinal microbiota—which are known to produce tryptophan and its metabolites—may play important roles in maintaining optimal tryptophan concentrations  KTR was largely driven by high tryptophan levels, but was exacerbated by low kynurenine levels   Citrulline was not a good predictor of linear growth deficits, but were inversely associated with measures of systemic inflammation (CRP, AGP, and IL-6) at all ages; this provides evidence that enterocyte function, intestinal infection, and systemic immune activation are closely linked in EED  	 76	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments • Kynurenine: 2.9 μM (95% CI: 1.8–4.5) • Tryptophan: 49.9 μM (95% CI: 22.1–74.9) • KTR: 60.6 μM (95% CI = 38.1–121.8)  • AGP: 113 (95% CI: 73-177)  In Peru, tryptophan and KTR were not associated with LAZ, WAZ, or WHZ at any time.  Kynurenine and LAZ were negatively associated at 3 mo (ρ = −0.16, P = 0.03) but not associated at any other time  In Tanzania, LAZ and WAZ were moderately inversely associated with KTR at 7, 15, and 24 mo • Spearman’s ρ for LAZ = −0.3, P < 0.0001 • Spearman’s ρ for WAZ = −0.25, P < 0.0001  Mean levels of cytokines (Peru only): • CRP: 1.05 mg/L (95% CI: 0.14–12.0) • IFN-	γ: 3.08 pg/mL (95% CI: 1.45–12.18) • IL-10: 5.53 (95% CI: 3.25–10.82) • IL-6: 1.28 (95% CI: 0.62–4.61) Correlation of CRP with AGP (95% CI = 0.54–0.68, P < 0.01) and CRP with IL-6 of similar magnitude  Strong correlation of KTR with IFN-γ 	 77	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments (0.24–0.39, P < 0.01) and KTR with IL-10 (0.27–0.38, P < 0.01)   2016  McDonald CM et al  Elevations in serum anti-flagellin and anti-LPS Igs are related to growth faltering in young Tanzanian children  To assess whether LPS- and FLA-specific immunoglobulins concentrations were associated with poor growth in children at risk of EED  Tanzania  Infants 0-18 mo  Exclusion of exposed: congenital anomalies or other conditions that would interfere with study procedures  Exclusion of unexposed:  LAZ < -2 or > 2; WAZ < -2 or > 2; no growth data were available for the review and analysis; evidence of a recent fever, (>38.58°C in the past 48 h); evidence of a systemic illness  Longitudinal cohort within RCT  n = 590 exposed, Tanzania  n = 36 unexposed, Boston Children’s Hospital (mean age: 9.5 mo, range: 5-12 mo) Anthropometric LAZ, WAZ, WHZ  Microbial translocation: LPS-specific IgA LPS-specific IgG FLA-specific IgA FLA-specific IgG Anthropometric None of the biomarkers were significantly associated with risk of stunting in either the unadjusted or adjusted models  Hazard ratios of being underweight for those with concentrations of biomarker in the highest quartile compared to those in the lowest quartile: • anti-FLA IgA: 2.02 (95% CI: 1.11, 3.67; P-trend = 0.007), • anti-LPS IgA: 1.84 (95% CI: 1.03, 3.27; P-trend = 0.02) • anti-FLA IgG: 1.94 (95% CI: 1.04, 3.62; P-trend = 0.009) • anti-LPS IgG: 2.31 (95% CI: 1.25, 4.27; P-trend = 0.01)   Microbial translocation: Mean concentrations of anti-FLA IgA, anti-FLA IgG, anti-LPS IgA, and anti-LPS IgG at 12 mo were significantly higher than those of healthy Boston children at 9.5 mo  Anti-FLA IgG, anti-LPS IgA, and anti-LPS IgG were also significantly higher There was a clear trend of increasing anti-LPS and anti-FLA IgA and IgG concentrations over the first year of life.  An increased concentration of each biomarker was associated with a significant increase in risk of underweight at 6 wk, but no association with stunting present.  Limitation: The study was ubable to quantify the amount of IgG at 6 wk of age that crossed the placenta from the mother compared with that which was produced by the infant Definition: subclinical condition of the small intestine that is characterized by villous atrophy, crypt hyperplasia, increased intestinal permeability, inflammatory cell infiltrate, and malabsorption   	 78	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments at 6 mo compared to healthy Boston babies at 9.5 mo 2016  Naylor C et al  The impact of environmental enteropathy and systemic inflammation on infant growth failure  To examine the relationship between intestinal and systemic inflammation biomarkers with poor growth in the first two years of life.   Urban Bangladesh (same population as the 2015 PROVIDE study)  Infants 0 – 18 mo  Longitudinal   n=700    Anthropometric HAZ  Intestinal inflammation: Reg1B MPO Calprotectin  Systemic inflammation: CRP Ferritin sCD14 Reg1B, MPO, and calprotectin, measured in the stool at 12 weeks of age, negatively correlated with growth  CRP, ferritin, and sCD14 strongly negatively correlated with growth  Maternal health had strong positive correlations on growth.  A model containing Reg1b, MPO, ferritin, sCD14, CRP, maternal education, and maternal height explain 46.3% of variance observed in ∆HAZ  Cluster analysis yielded three distinct clusters that lead to poor growth: • Cluster 1 = systemic inflammatory markers, including diarrhoeal burden  • Cluster 2 = enteric inflammatory markers, as well as CRP and sCD14.  • Cluster 3 = maternal health and SES status markers.   Systemic inflammation, intestinal inflammation, and maternal health may all be independent factors that lead to poor growth  Both systemic and intestinal inflammation had a negative impact on linear growth Abstract only Experimental Biology 2016 Meeting; abstract published in The FASEB Journal.   Definition: subclinical inflammation of the intestinal tract  No biomarker cutoffs provided  	 79	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2016  Ordiz M et al  Environmental Enteric Dysfunction is Associated with Poor Linear Growth and Can be Identified by Host Fecal mRNAs  To determine whether a single L:M test predicts subsequent linear growth in these rural African children and the extent to which fecal host mRNAs predict L:M Rural Malawi  Children 12-61 mo  Exclusion:  - severe malnutrition - diarrhoea within the last 3 days  - congenital abnormalities - chronic debilitating illness Longitudinal cohort  n=798    Anthropometry HAZ, WHZ  Permeability: 4-h L:M sugar absorption test -- 5g L: 1g M  Transcriptomics  Anthropometry n=140 (18%) no EED n=524 (66%) moderate EED n=134 (17%) severe EED  L:M was inversely correlated with ∆HAZ (r=0.32, P<0.001) and severe EED was associated with ∆HAZ in the subsequent 3 months (P<0.0001).  Transcriptomics 18 host mRNA transcripts were associated with L:M; 7 were identified as predictors of EED: • CDX1, HLA-DRA, MUC12, REG1A, S100A8, TNF (sensitivity = 84%, specificity = 73%) • TNF, HLA-DRA, MUC12, CD53 (sensitivity = 84%, specificity = 83%)   L:M was a significant predictor of ∆HAZ   Severe EED can be predicted by a small number of host fecal mRNAs using random forest modeling with high sensitivity and specificity   Systemic immune response: TNF, HLA-DRA  Epithelial barrier function: MUC12  Limitations: - study population age did not extend to 0-11 mo  No definition of EED provided  EE (without dysfunction part) defined as increased systemic inflammation in an asymptomatic child living in an unsanitary environment  EED Cutoff none: L:M < 0.15 moderate: 0.15< L:M< 0.45  severe: L:M > 0.45.  2016  Syed S et al  Serum anti-flagellin and anti-lipopolysaccharide immunoglobulins as predictors of growth in Pakistani infants at risk for environmental enteric dysfunction  To assess the associations between Rural Pakistan  Children 0-18 mo Prospective cohort  n = 380 Anthropometric HAZ, WAZ, WHZ  Translocation and permeability Serum anti-FLA- and anti-LPS-specific IgA  Serum anti-FLA- and anti-LPS-specific IgG At 6 months:  • higher anti-LPS IgA levels were associated with a decrease in HAZ scores over 18 months (beta = -0.29, 95% CI: -0.05 to -0.54, P = 0.009).  At 9 months:  • higher anti-FLA IgA levels were associated with a decrease in HAZ scores over 18 months (beta = -0.29, 95% CI: -0.04 to -0.55, P = 0.04) • higher anti-FLA IgG levels were EED, as measured by anti-FLA IgA and anti-LPS IgA antibodies at 6 and 9 months, is associated with declines in HAZ.   Serum levels of anti-FLA-IgA in early infancy are associated with stunting. Conference abstract only  Definition: an acquired condition of the small intestine that is linked to poor growth   	 80	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments anti-FLA and anti-LPS antibodies in children at risk of EE associated with a decrease in HAZ scores over 18 months (beta = -0.27, 95% CI: -0.01 to -0.53, P = 0.07)  anti-LPS IgG and anti-FLA IgG were not associated with ∆HAZ   Biomarkers were not associated with WAZ or WHZ scores, except for anti-LPS IgG at 6 months, with increase in WHZ over 18 months (beta = 0.26, 95% CI: -0.04 to 0.56, P = 0.01) 2016  Semba R et al  Metabolic alterations in children with environmental enteric dysfunction  Report the relationship between increased intestinal permeability and specific serum metabolites Southern Rural Malawi  12-59 mo   Excluded: children with evidence of kwashiorkor, congenital or chronic disease, caretaker-reported diarrhea, or were under treatment for malnutrition. Cross-sectional  n=315  n=68 no EED  n=247 EED (L:M ratio ≥ 0.15) Permeability: L:M  Serum metabolites: The 139 metabolites analyzed included 22 amino acids, 3 biogenic amines, 6 amino acid metabolites, 15 sphingolipids, 8 acylcarnitines, and 85 glycerophospholipids.  Systemic inflammation KTR 14 serum metabolites were significantly related with gut permeability  Negatively correlated with L:M: - tryptophan  - ornithine  - citrulline - four lysophosphatidylcholines  (lysoPC a C16:0, lysoPC a C18:0, lysoPC a C18:2, lysoPC a C20:4) - two acyl-alkyl-phosphatidylcholines (PC ae C40:1, PC ae C44:3)  -two sphingomyelins (SM C16:0, SM C16:1)   Positively correlated with L:M: - glutamate - serotonin - taurine   Adj Spearman correlations between gut permeability and the Authors suggests several serum metabolites as candidate biomarkers for EED: citrulline, ornithine,glutamate, taurine, serotonin, tryptophan, serotonin/tryptophan ratio, kynurenine/tryptophan ratio, and phos- phatidylcholines and sphingomyelins.   However, the correlations between specific serum metabolites and gut permeability were modest, which may limit their use as biomarkers. Definition: villous shortening, crypt hyperplasia, and lymphocytic infiltration  Cutoff EED: L:M ≥ 0.15  	 81	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments serotonin/tryptophan: 0.27 (P = 7.34 × 10−7) and kynurenine/tryptophan ratio: 0.25 (P = 6.44 × 10−6) 2016  Uddin M et al   Biomarkers of Environmental Enteropathy are Positively Associated with Immune Responses to an Oral Cholera Vaccine in Bangladeshi Children  To determine how host factors impact immune responses in children receiving oral cholera vaccine.  Urban Bangladesh  Children 3-14 yo  Excluded: healthy, no diarrhea in preceding 14 days; HAZ score less than -2  Case-crossover  n = 40 total  n = 20, young children (3-5 yo, mean = 4.8)  n = 20 older children (7-14 yo, mean = 10.5)  Anthropometric HAZ  Microbial translocation EndoCAb IgG LPS-specific IgA, IgG, IgM  Intestinal Inflammation MPO AAT i-FABP  Systemic Inflammation sCD14   Microbial ranslocation LPS positively associated with: • vitamin D (p = 0.02) • i-FABP (p = 0.01) • sCD14 (p = 0.07)  Intestinal Inflammation MPO positively associated with: • anti-LPS IgA (p = 0.02) • CTB IgG (p = 0.03)  Systemic Inflammation sCD14 higher in older children  Contrary to previous studies, EED, as evidenced by enteropathy biomarkers, was positively associated with increased immunogenicity to vaccine antigens   Definition: acquired syndrome, characterized by villous blunting, crypt hyperplasia, and increased intraepithelial lymphocytes and pro- inflammatory cytokine responses 	 82	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2016   Yu J et al  Environmental Enteric Dysfunction Includes a Broad Spectrum of Inflammatory Responses and Epithelial Repair Processes  To determine the association between %L and expression levels of protein coding genes (transcriptome)  Malawi  12-61 month olds residing in 1 or 6 communities in rural Malawi  Excluded: Children with chronic disability or disease, severe acute malnutrition, or receiving therapy for tuberculosis   Prospective Cohort  n=259  n=60  No EED  (%L < 0.2)  n= 157  Intermediate EED  (0.2 < %L < 0.7)  n=42 Severe EED (%L > 0.7) Anthropometric HAZ, WAZ, MUACZ  Permeability: 4-h dual sugar absorption test, 1g M: 5g L  %L  Transcriptomics 51 host RNA transcripts of the human gastrointestinal tract Permeability %L was associated with reduced linear growth (smaller ∆HAZ) in the subsequent 3-month period (P < .01)  51 transcripts were correlated significantly to %L  18 most common transcripts with EED (p-values for both Pearson and Spearman < .01)  ACSL1  AQP9*  BCL2A1  CD53  CSF3R  IFI30  IL1RN  LAPTM5  LCP1  LYN*  LYZ*  MNDA* (Pearson p = 0.012)  PIK3AP1  PLEK*  SELL*  SLC2A3  SORL1       TAGAP *also associated with ∆HAZ after normalization  Almost all of the 51 transcripts code for immunologically active proteins, such as IgG or IgE, or for cytokines that modulate the immune response; these are made in response to parasites, bacteria, and viruses  A relatively high proportion of the 18 most common transcripts showing a correlation with EED are associated with granulocyte colony-stimulating factor (G-CSF), which aids in differentiating dendritic cells and mobilizing neutrophils    Definition:  Cutoff No EED: %L < 0.2  Intermediate:   0.02 < %L < 0.7 Severe: L% > 0.7   	 83	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2015  Benzoni N et al  Plasma endotoxin core antibody concentration and linear growth are unrelated in rural Malawian children aged 2-5 years.  To examine the association of serum EndoCab versus linear growth and lactulose-mannitol assay results  Rural Malawi  Children 2-5 yo (same study population as Weisz 2012)  Excluded: children with any chronic debilitating illness, including known HIV infection or obvious congenital abnormalities, evidence of acute malnutrition, and a recent history or current case of diarrhea or hematochezia.  cross-sectional  n=388 total analyzed for L:M n=48 no EED n=340 EED Anthropometric MUACZ, WAZ, HAZ  Permeability: %L, %M, L:M  Microbial translocation: log-EndoCab Anthropometric n = 301 (78%) stunted  No significant association between log-EndoCab and HAZ (B = −0.078, P = 0.14) or change in HAZ (B = −0.018, P = 0.27)  Permeability n = 340 (88%) with EED, as determined by L:M ratio  No significant association between log-EndoCAb and log-%L (B < 0.001, P = 0.98) or log-L:M (B = 0.021, P = 0.62).  EndoCAb was not associated with linear growth nor with measurements of permeability and is therefore not a sensitive biomarker for these measurements Definition: subclinical condition with T-cell infiltration of the duodenum and jejunum, decreased villous height and increased crypt depth  Cutoff L:M > .10 is abnormal   2015  Etheredge A et al  Markers of environmental enteric dysfunction (EED) are associated with neurodevelopment in Tanzanian children  To evaluate biomarkers of gastrointestinal epithelial and barrier function associated with EED as predictors Tanzania   Children 6wk Longitudinal cohort  n = 107 Enterocyte mass citrulline  Microbial translocation and systemic immune response LPS-specific IgA LPS-specific IgG FLA-specific IgA FLA-specific IgG  Neurodevelop-mental testing Bayley Scales of At 6wk, negative association between LPS-IgA and expressive language score (p = 0.03)   At 6 months, positive association between expressive language association score and FLA-IgG (p = 0.02) and citrulline (p = 0.02)  At 12 months, negative association between citrulline and expressive language score (p = 0.04)  Higher levels of LPS-IgA at 6 weeks and citrulline at 12 months, and lower levels of FLA-IgG and citrulline at 6 months were associated with below-median language scores No definition of EED provided 	 84	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments of neurodevelopment  Infant Development-III  2015  George CM et al  Geophagy is associated with environmental enteropathy and impaired growth in children in rural Bangladesh  To determine the relationship between geophagy, EED, and stunting  Rural Bangladesh  Children 6-30 mo (median age: 17mo)  Exclusion: no chickens present in compound Longitudinal cohort  n = 324 eligible (37% LTFU)  n = 205 included in 9-month follow-up Anthropometric HAZ, WAZ, WHZ  Intestinal Inflammation AAT, MPO, NEO: combined to form EE disease activity score Calprotectin  Observed and caregiver-reported geophagy Anthropometric At 9 months, 34% had low HAZ, 27% had low WAZ, and 7% had low WHZ   Odds of stunting for those with observed geophagy was 1.81 (95% CI: 0.85, 3.89) times that of those without observed geophagy  Odds of stunting for those with caregiver-reported geophagy was 2.27 (95% CI: 1.14, 4.51) times that of those without caregiver-reported geophagy  No significant association between observed or caregiver-observed geophagy and WAZ or WHZ   Intestinal Inflammation Significant association (p < 0.05) between calprotectin and AAT; calprotectin and MPO; AAT and MPO  No significant association between baseline fecal markers of EED (calprotectin and EED activity score) in the highest versus lowest quartile and stunting or wasting  Significant association between being underweight and: • EED scores in the highest versus lowest quartile (OR: 3.73, 95% CI: Significant association between caregiver-reported geophagy in the past week and elevated EE disease activity scores and calprotectin  No significant associations found between geophagy observed during the 5-hour structured observation period and EE markers and stunting - This may be explained by short observation periods  Definition: disorder defined by abnormal intestinal morphology, reduced intestinal barrier function, and increased inflammation 	 85	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 1.38, 10.12) • AAT concentrations in the highest versus lowest quartile (OR: 3.17, 95%CI: 1.19, 8.43) 2015  Gosselin K et al  Serum citrulline does not predict stunting or environmental enteric dysfunction in Tanzanian and Malawian infants  To examine the relationship between serum citrulline and L:M, and citrulline and stunting at 18 mo   Tanzania and Malawi  Tanzania Children 6wk – 18 mo  Malawi Children 1-3 yo cohort  Tanzania n = 413  Malawi n = 102 Permeability: L:M  Mucosal mass: Citrulline  Tanzania Mean (SD) citrulline level at 6 wk = 18.0 (5.8) μmol/L.   No significant difference in hazard for children in lowest quartile of citrulline compared to other quartiles (HR = 1.07, 95%CI: 0.64, 1.78)  Malawi No significant difference in mean citrulline levels between stunted and non-stunted children (p = 0.60)  No association between L:M and citrulline (p = 0.65) or HAZ and citrulline (p = 0.31)  Citrulline did not predict subsequent stunting in Tanzania or correlate with markers of EED in Malawi. Abstract only  No definition of EED provided   2015   Lima AA et al  Risk factors, gut function biomarkers and growth deficit associated with environmental enteropathy and malnutrition: The case-control MAL-ED study in Fortaleza, Ceara, Brazil Brazil site within the MAL-ED study  Children 6-24 mo  Inclusion: WAZ = -1 Case-control  n = 402 enrolled  n = 244 preliminary analysis  (126 controls, 118 cases)  Anthropometric HAZ, WAZ  Intestinal inflammation MPO AAT Calprotectin Intestinal Inflammation Calprotectin was significantly higher in malnourished versus nourished children  Higher MPO and AAT were associated with impaired "catch-up" growth    Calprotectin, MPO, and AAT were all associated with gut inflammation and impaired growth Abstract only  No definition of EED provided 	 86	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments   To evaluate the variables related to the child and the mother, as well as environmental and socio-economic factors associated with environmental enteropathy and malnutrition 2015  Naylor C et al  Environmental Enteropathy, Oral Vaccine Failure and Growth Faltering in Infants in Bangladesh (PROVIDE study)  To examine the association between EED and impaired performance of the oral polio vaccine (OPV) and rotavirus vaccine  Urban Bangladesh  Children 0-2 yo (recruited at birth and followed for 2 years) Longitudinal cohort  n = 700 Anthropometric HAZ, WAZ, WHZ  Intestinal inflammation MPO NEO Calprotectin AAT  REG1B  Systemic inflammation cytokines  CRP ferritin sCD14   Cluster analysis yielded three distinct clusters that lead to poor growth: • Cluster 1 = systemic inflammatory markers, including diarrhoeal burden  • Cluster 2 = enteric inflammatory markers, as well as CRP and sCD14 and two of the sanitation markers (absence of an open sewer and access to a toilet/septic tank) • Cluster 3 = maternal health, HAZ at 18 weeks, and SES status markers, including income, access to treated water, and no shared toilet.   Based on cluster analysis,	REG1B, calprotectin, NEO, MPO, and AAT were considered to be biomarkers of EED   Anthropometric REG1B, MPO both negatively associated with ∆HAZ and ∆WAZ from enrolment to age 1 yo  ∆HAZ and ∆WAZ were associated with EE was associated with failure of Rotarix® and underperformance of OPV and predicted the development of malnutrition  EE and systemic inflammation biomarkers had negative associations with growth, indicating that localized enteric inflammatory damage and broad systemic inflammation both are likely to have an important effect on nutritional status   Definition: subclinical illness characterized by small intestine inflammation with shortened villi, intestinal barrier dysfunction, and reduced nutrient absorption 	 87	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments retinol binding protein, vitamin D, zinc, and ferritin  Intestinal inflammation Significant associations: • OPV2—REG1B • OPV3—REG1B, calprotectin • Rotarix IgA—AAT • Rotarix success—REG1B, NEO  Systemic inflammation • ferritin—OPV2, tetanus, pertussis, diphtheria, ∆WAZ, ∆HAZ • IL-10—Rotarix IgA • activin—diphtheria • IL-1b—∆WAZ • CRP—pertussis, ∆WAZ • sCD14—OPV1, measles, tetanus, Rotarix® success, ΔHAZ • CRP—∆HAZ   	 88	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2014  Jones KD et al   Mesalazine in the initial management of severely acutely malnourished children with environmental enteric dysfunction: A pilot randomized controlled trial  To investigate the safety and acceptability of mesalazine as a treatment for children with severe acute malnutrition (SAM)  Urban Kenya  Children 12-60 mo with SAM and stunting   Exclusion: HIV, TB, bloody diarrhea, evidence of renal or hepatic impairment, thrombocytopenia or severe anemia, if they were receiving treatment from another facility, if they had medical difficulties in normal feeding, pre-existing renal disease, asthma, hypersensitivity or salicylates, or if they were on medication known to interfere with the action of the study drug  Double-blind RCT  n = 44 (22 mesalazine, 22 placebo)  Mesalazine arm: 20/22 completed follow-up (all 22 included in final analysis)  Placebo arm: 19/22 completed follow-up (all 22 included in final analysis)  Intestinal inflammation Calprotectin  Microbial translocation  Anti-EndoCAb IgG  Systemic inflammation IFNα IFN-γ sCD14 TNFβ IL-1α, IL-1RA, IL-7, IL-8, IL-10, IL-15, IL-17a, IL-22, IL-31   Growth hormone axis IGF-1  Intestinal inflammation Baseline levels of fecal calprotectin were higher than has been reported from healthy control populations in high-income countries and sub-Saharan Africa  Anthropometric Significantly greater MUAC growth in the placebo arm to day 56   No differences in HAZ at any time (baseline, day 28, day 56) between mesalazine and placebo groups  Microbial translocation IGF-1 and EndoCAb were positively correlated with each other and negatively correlated with linear growth throughout the study (p = 0.01 and p = 0.04, Figure 3c)   Pharmacologically-mediated reduction in EED is well tolerated, providing the first clear evidence that EED is likely to be at least partly maladaptive  Because edema can alter MUAC and most of the children in this study were edematous at enrolment, the clinical significance is unclear  Mesalazine was safe to use and did not have any adverse effects, but also does not appear to significantly improve the outcomes of SAM or EED Definition: acquired syndrome of reduced intestinal barrier and absorptive function, characterised by histopathological changes of small intestinal villous atrophy and crypt hyperplasia, accompanied by lymphocytic infiltration of the lamina propria  SAM defined as MUAC <11.5 cm or bilateral pedal edema  EED was inferred on the basis of stunting and chronic inflammation (erythrocyte sedimentation rate (ESR) >20 mm/hour)   2014  Prendergast A et al  Stunting Is Characterized by Chronic Inflammation in Zimbabwe  HIV-negative mother-infant pairs (Infants 0 mo followed to 24 mo)  Case-control  n = 202 total (101 cases, 101 controls) Anthropometric HAZ, WAZ, WHZ, MUAC  Microbial translocation sCD14, IgG Anthropometric Cases had significantly lower HAZ and WAZ than controls at each time-point between birth and 18 mo  In adjusted models,  • higher levels of IGF-1 between 6 wk Stunting is influenced by both maternal and infant factors  Low-grade inflammation and the IGF-1 axis were associated in the first year of life (but not to 18 mo) Definition: villous blunting, inflammatory infiltrate and increased intestinal permeability  Cases: HAZ < -2 	 89	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments Zimbabwean Infants  To examine the relationship between the growth hormone-IGF axis and stunting as well as numerous biomarkers of EED   Exclusion: infant weight < 1500 g, acutely life-threatening conditions, mother planned to leave city after delivery EndoCAb  Intestinal damage i-FABP  Systemic Inflammation CRP, AGP, IL-6  Growth hormone axis IGF-1, IGFBP3 – 12 mo were associated with reduced odds of stunting at 18 mo (p < 0.001) • higher log10 levels of CRP and AGP between 6 wk – 12 mo were associated with increased odds of stunting (p = 0.008, p = 0.087)  There were no associations between levels of I-FABP, IL-6, sCD14 or EndoCAb and stunting  Growth hormone-IGF axis IGF-1 and IGFBP3 levels were similar for cases and controls at birth, but after 6 wk both protein levels were significantly lower in cases than controls  Inflammation versus IGF-1 • At birth, IGF-1 was strongly associated with inflammation (AGP: p < 0.001, CRP: p < 0.001, and sCD14: p = 0.031) • Negative associations were found at every subsequent time-point to 12 mo of age, but by 18 mo associations were no longer present  Microbial translocation sCD14 was higher in controls at birth and again at 18 mo, but levels in the time in between were similar between cases and controls  IgG EndoCAb levels were similar between groups throughout follow-up  Lower levels of IGF-1 are likely to mediate stunting in early life  By 18 mo, levels of IGF-1 were similar between groups — this may suggest a window of opportunity in which interventions to reduce inflammation and increase IGF-1 may improve linear growth   Infant inflammation markers were closely related to maternal inflammation at birth — this may imply that inflammation during pregnancy may “set” the infant inflammatory axis, which influences the level of IGF-1 in early life  I-FABP was not significantly associated with stunting, but levels were exceptionally high in both groups compared to healthy individuals from wealthier populations  Reduction in I-FABP in both groups between 12-18 mo may suggest that the mucosa repair beyond infancy @18mo Controls: HAZ > -0.5 @18mo  	 90	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments  Inflammation IL-6 highly elevated in cases at birth, declined over first 6 wk, then rose again after 6 wk  CRP and AGP increased from 6 wk and were higher in cases after 12 mo; however, by 18 mo they were not significantly higher (and in some instances, control levels were higher than case levels)  Analysis of mother versus infant levels • Mothers of cases had lower levels of IGF-1 (221.4, 95% CI: 239.8, 23.1)  • WAZ at birth was associated with maternal IGF-1 (R = 0.21, p = 0.003) and infant IGF-1 (R = 0.51, p < 0.0001) • HAZ at birth was not associated with maternal IGF-1 and was weakly associated with infant IGF-1 (R = 0.23, p = 0.046) • Maternal IGF-1 associated with maternal weight (p = 0.029) and height (p=0.013) • No significant association in inflammatory markers between mothers of cases and controls • Strong association between maternal and infant AGP (R = 0.52, p = 0.001), CRP (R = 0.46, P = 0.010) and sCD14 (R = 0.39, P < 0.001) • Weak association between maternal and infant IL-6 (R = 0.31, P = 0.087) 	 91	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2014  Ryan KN et al  Zinc or albendazole attenuates the progression of environmental enteropathy: A randomized controlled trial  To determine if zinc and/or albendazole improve EED, as measured by the L:M test  Rural Malawi  Asymptomatic children 1-3 yo  Exclusion: 3 or more loose stools per day, chronic debilitating illnesses such as HIV, cerebral palsy or congenital abnormalities; severe malnutrition, or children needing acute medical treatment Double-blind 3-arm RCT - zinc supplement - Albendazole supplement - Placebo  n = 234 enrolled  n = 222 analyzed (72 zinc, 73 albendazole, 77 placebo) Permeability 4-h dual sugar absorption test, 1g M: 5g L  L:M ratio, %L  Anthropometric HAZ, WAZ Permeability 95% of children in study had evidence of EE, as measured by L:M ratio  Mean baseline L:M was 0.32 – 0.18 among all children and did not differ significantly between the 3 groups   L:M ratio increased for all groups, despite interventions   ∆L:M significantly different in each intervention group compared to placebo: • Zinc: 0.03 ± 0.20 (p < 0.03) • Albendazole: 0.04 ± 0.22 (p < .04)  Anthropometric No significant differences in weight gain or linear growth among the 3 groups   No results reflecting ∆HAZ and L:M Both zinc and albendazole interventions stopped the progression of EE as measured by L:M ratio  Neither intervention had an effect on anthropometric measures; however, no long-term growth was measured Definition: subclinical condition manifested by T-cell infiltration of the small-bowel mucosa and diffuse villous atrophy  Cutoff Abnormal L:M > 0.30  Albendazole = anti-helminthic therapy   2014  Smith HE et al   Multiple micronutrient supplementation transiently ameliorates environmental enteropathy in Malawian children aged 12-35 months in a randomized controlled Rural Malawi   Children 12-35 mo  Exclusion: bloody diarrhea, inability to provide 4-h urine collection, chronic or debilitating illness, SAM, the first twin of a pair (second received Double-blind 3-arm RCT A. MN/FO B. MN/placebo C. Plcbo/plcbo  A. n = 80 enrolled, 1 LTFU, 78 L:M after 12 Permeability 4-h dual sugar absorption test, 1g M: 5g L  L:M ratio, %L  Anthropometric linear growth, weight gain Permeability No results about L:M vs HAZ  All children had abnormal L:M values at baseline • Group A: 0.44 (0.39, 0.50) • Group B: 0.49 (0.41, 0.56) • Group C: 0.43 (0.38, 0.49)  The two intervention groups had significant improvements L:M after 12 No significant difference in L:M between the intervention group and the placebo group at 12 wk (P = 0.06)  All groups had similar L:M ratios after 24 wk, suggesting the interventions were transient and limited  Definition: characterized by diffuse small bowel villous atrophy with T cell infiltration  Cutoff Clinically significant = ∆L:M > .06 units 	 92	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments clinical trial.  To test whether micronutrient and fish oil supplementation would ameliorate EED, as measured by the L:M test   same therapy but was not recorded) wk, 79 L:M after 24 wk  B. n = 80 enrolled, 2 LTFU, 78 L:M after 12 wk, 76 L:M after 24 wk  C. n = 75, enrolled, 2 LTFU, 72 L:M after 12 wk, 73 L:M after 24 wk  wk compared to baseline: • Group A: 0.35 (0.30, 0.40) • Group B: 0.37 (0.32, 0.43) • Group C: 0.38 (0.33, 0.44)  All groups had significant improvement in L:M after 24 wk compared to the baseline: • Group A: 0.35 (0.30, 0.40) • Group B: 0.38 (0.33, 0.43) • Group C: 0.37 (0.30, 0.44)  L:M ratio from 12-24 wk • Group A did not change • Group B increased by 0.01 unit  • Group C decreased by 0.01 unit  Anthropometric Significant weight gain in Group A (MN/FO) versus Group C (placebo) (p = 0.01)  No significant change in linear growth between intervention and placebo groups   The improvement in L:M in the C group after 24 wk suggests that factors other than nutrient intake may play an important role in EE.   2013  Agapova et al  Detection of low-concentration host mRNA transcripts in Malawian children at Rural Malawi  Children ages 2-5 yo  Exclusion: no diarrhea in the last 7 days, and none of Case control n = 70 34 cases, with EED  36 controls, without EED  Permeability and absorption dual-sugar absorption 1 g M: 5 g L L:M ratio  Transcriptomics  Top six potential markers of EED: - REG4 best differentiated children with increased L:M from children with normal L:M (p = 0.01) - TJP1, LAP3, REG1B, IL-22, and BDEF1 were not significantly different between children with and without EED  Human mRNA that is present in extremely low copy numbers can consistently and reproducibly be isolated and detected in a set of stool samples. Definition - diffuse villous atrophy of the small bowel associated with inflammatory T-cell infiltration, of unknown aetiology  	 93	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments risk for environmental enteropathy  To develop a method for detecting human mRNA suggestive of EE in feces  the children was known to be infected with HIV, nor had a chronic congenital condition Transcriptomics   Genes for MPO, calprotectin, TNF, IFNg, and other markers of systemic inflammation (interleukins) were not significantly different between children with and without EED     Cutoff no EED: L:M < 0.10 (Note: comparisons were made between children with L:M > 0.24 and L:M < 0.13)  2013  Kosek M et al  Fecal markers of intestinal inflammation and permeability associated with the subsequent acquisition of linear growth deficits in infants.  To clarify the ability of established markers of intestinal inflammation and permeability to predict linear growth Pilot study for MAL-ED (8 sites: BGD, INV, NEB, PKN, BRF, PEL, SAV, TZH)  *MAL-ED = Malnutrition and Enteric Diseases  Children at birth, followed to 9 mo  Exclusion: Stool samples from children with diarrhea in last 7 days or L:M testing on the day of/before stool collection were omitted  Prospective cohort  n = 661 children  (744 stool samples for which all three biomarkers were available)  Anthropometric LAZ  Intestinal Inflammation AAT, MPO, NEO: combined to form EE disease activity score Anthropometric MPO, NEO, and AAT all predicted declines in LAZ in the 6 mo following the test  A child with a max EED score (10) would be expected to lose 0.47 LAZ scores more than a child with the lowest score (0) in the six months following the test  • p < 0.01 in Brazil and Nepal • p < 0.10 in South Africa  Intestinal Inflammation All three tests predicted declines in LAZ in the 6 months following the test.  Correlations between MPO, NEO, and AAT were low (all below 0.22)   Levels of intestinal inflammation are associated with subsequent acquired deficits in linear growth in infants over 6 months   Composite score of three biomarkers was made possible by the low correlation between the three markers -- This allowed accounting for a greater degree in growth deficits than can be explained by any marker by itself  Using stool markers is markedly cheaper/more convenient than L:M urine testing   Definition: increased crypt depth, decrease in villus height, and lymphocytic infiltration 	 94	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2013  Lin A et al  Household Environmental Conditions Are Associated with Enteropathy and Impaired Growth in Rural Bangladesh  To assess the relationship between fecal environment contamination and EED, parasite burden, and growth Rural Bangladesh  0-48 mo (mean age at collection time: 35 mo)  Excluded: children with diarrhea, vomiting, or a perigenital skin infection on the day of the L:M test (n=3) Cohort  n = 136 in sample (17 LTFU)  n=119 total (66 clean environment, 53 contaminated environments)  Anthropometric: HAZ, WAZ, WHZ  Permeability: 5-h urine collection 250mg/mL L, 50mg/mL M L:M ratio  Systemic Inflammation: Total IgG   Microbial translocation EndoCab IgG  Parasitic infection Ascaris Trichuris Giardia Cryptosporidium Entamoeba hookworm Mean L:M ratio: • clean = 0.21 • contaminated = 0.31  Children from clean households (compared to contaminated households): Anthropometric • L:M strongly associated with HAZ (beta = –0.33, 95% CI: −0.62, −0.05) and WAZ scores (−0.24, 95% CI: −0.47, −0.01) • None of the anthropometric measurements were strongly associated with IgG EndoCAb or total IgG titers • 0.54 SDs (95% CI: 0.06, 1.01) higher HAZ • 22% lower number of stunted individuals (95% CI: -2%, -42%) • no other outcomes (WAZ, WHZ, HCZ, proportion wasted/underweight) were significantly associated after adjustment  Permeability • 0.32 SDs (95%CI: −0.72, 0.08) lower L:M ratio in urine Microbial translocation • 0.24 SDs (95% CI: −0.63, 0.16) lower IgG EndoCAb titers  • 0.54 SDs (95% CI: −0.95, 0.13) lower IgG EndoCAb titers, after adjusting for potential confounders  Parasitic infection Children in clean environments had higher HAZs and lower IgG EndoCAb titers than children living in contaminated households (after adjustment)  HAZ and WAZ strongly correlated with L:M ratio  Both clean and contaminated households had higher mean L:M ratios (0.21, 0.31) than healthy infants in UK   Even when children from clean households were surrounded by other households that did not meet our definition of a clean environment, these children still had dramatically lower stunting prevalence, lower levels of parasitic infection, and better gut function.  Limitations: Tests and growth measured at mean 35 months; does not take into account growth from birth Definition: disorder featuring a small bowel with abnormal morphology and physiology, including crypt hyperplasia, villous atrophy, lymphocyte infiltration into the lamina propria and epithelium, reduced mucosal surface area, and increased intestinal permeability.   *“Contaminated environment” = a household with poor water quality (median E. coli > 10CFU/ 100mL), inadequate sanitation (open defecation, open pit latrines, slabs with broken water seals, toilets that flush to somewhere else, or hanging toilets), and unhygienic handwashing conditions (a dedicated location that lacked either water or soap or the absence of a dedicated location to wash hands) 	 95	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments Children living in clean household environments had lower prevalence of all detectable parasites measured compared with children in contaminated household environments, but none of them were significant after adjustment    2013  Peterson KM et al  REG1B as a predictor of childhood stunting in Bangladesh and Peru.  To determine whether there is an association between stool REG1B concentrations with subsequent childhood growth deficit  Bangladesh and Peru  Children 0-24 mo (followed from birth) Prospective cohort  n = 222 Bangl. n = 97 Peru  Anthropometric LAZ, WAZ, WHZ  Intestinal tissue repair REG1B Anthropometric LAZ at 3 mo was significantly associated with LAZ at each subsequent month  Intestinal repair Inflated REG1B concentrations at 3 mo were significantly associated with lower LAZ at 9+, 12, 15++, 18, 21, and 24 mo +Bengali cohort only ++Peru cohort only  Higher REG1B concentrations at 3 mo were associated with future LAZ measures through 24 mo in Bengali (p = 0.006) and Peruvian (p = 0.059) cohorts  Inflated REG1B concentrations at 3 mo Higher REG1B concentrations at 3 mo were significantly associated with future LAZs in two birth cohorts  No definition of EED provided; used stunting as primary indicator of EED  	 96	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments were not associated with lower WHZ at any month  2013  van der Merwe L et al  Long-chain PUFA supplementation in rural African infants: a randomized controlled trial of effects on gut integrity, growth, and cognitive development.  To assess whether early n–3 LC-PUFA supplementation improves infant intestinal integrity, growth, and cognitive function  The Gambia, rural  Children 3 mo, followed until 9 mo  Exclusion:  Severe congenital abnormalities that could affect growth/development, infants from multiple births, HIV infection Double-blind RCT  n = 183 (92 receive treatment, 91 control)  8 (4%) LTFU (3 treatment, 5 control)  n = 172 analyzed at 9 mo (87 treatment, 85 control)  n = 155 attended follow-up at 12 mo  (79 treatment, 76 control) Anthropometric Left-side triceps, biceps, and sub-scapular skinfold thickness; MUAC; head circumference  Permeability 5-h dual-sugar absorption test  4g L : 1g M L:M ratio  Intestinal inflammation Calprotectin  Systemic inflammation CRP AGP albumin  Plasma fatty acid status plasma total lipids  Cognitive development 2-step means-end problem-solving Plasma fatty acid status FO supplementation resulted in significant increases in DHA and EPA  (This was just to measure that the individuals were actually absorbing the PUFAs)  Anthropometric (ITT analysis) • FO supplementation significantly associated with increase in MUAC (p = 0.017), tricep skinfold-thickness-for-age (p = 0.048) • No significant association between FO supplementation and linear growth (95% CI: 20.27, 0.90; P = 0.084)  Permeability • Only 5% of infants had normal L:M ratio • No significant difference in L:M ratio between groups   Systemic inflammation No significant association between FO supplementation and systemic inflammation markers   Intestinal inflammation • More than 85% of infants had calprotectin concentrations higher than healthy European references The PUFA intervention successfully increased infant n–3 plasma FA status after 5 mo of intervention  PO supplementation may cause increase in MUAC and 3 skinfold-thickness measurements   PO supplementation not associated with any other anthropometric measurements   FO supplement did not lead to reduced degrees of intestinal and systemic inflammation or reduced rates of morbidity  Definition: characterized by intestinal villous atrophy, crypt hyperplasia, and inflammatory cell invasion of the lamina propria  EPA and DHA are types of omega-3 fatty acids  Cutoff Normal L:M < 0.07 	 97	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments test single-object task attention assessment  • No significant association between FO supplementation and intestinal inflammation markers  Cognitive development No significant difference in performance between treatment groups   2013  Wessells KR et al  Associations between intestinal mucosal function and changes in plasma zinc concentration following zinc supplementation  To examine the relationship between intestinal mucosal function (as measured by L:M RATIO and plasma citrulline concentrations) and changes in zinc concentration following zinc supplementation Rural Burkina Faso  Children age 6–23 mo, currently breast-feeding  Exclusion: Hemoglobin < 60 g/L, fever, diarrhea (>3 liquid or semi-liquid stools in a 24 h period) reported in the past week, currently consuming vitamin or mineral supplements or zinc- fortified infant formulas, or demonstrate bipedal or other serious med conditions 3-armed partially masked RCT  n = 451 in original study 3 arms: 1. 5mg Zn tablet 2. 5mg liquid Zn 3. Liquid placebo  n=282 successful completion of L:M test  n=80 subgroup analysis of children with CRP concentrations < 10mg/L  Anthropometry LAZ, WAZ, WLZ  Permeability 2-h dual-sugar absorption test 4g L : 1g M L:M recovery ratio (not concentration ratio) - Note: children were allowed to eat after 1h of consuming L:M Also looked at L:C and M:C ratios  Mucosal mass Citrulline  Systemic inflammation AGP CRP HRP*  Mineral uptake Mucosal mass Citrulline positively associated with age (p = 0.02) Baseline citrulline not associated with L:M recovery ratio ∆citrulline was positively associated with WAZ over the three weeks  Permeability No consistent associations between urinary L:M recovery ratios at baseline and age or anthropometric status (HAZ)  Systemic inflammation AGP and CRP were not associated with L:M  Mineral uptake ∆PZC** was significantly greater among children who received Zn supplementation (p < 0.0001)  Negatively related to ∆PZC: • Initial plasma Zn concentration • Child age • Child HAZ • ∆WAZ Short-term Zn supplementation significantly increased PZC after 3 wk  Children with higher L:M recovery ratio at baseline had significantly smaller ∆PZC - this trend was consistent between placebo and treatment groups (i.e. zinc supplementation will increase PZC in children with or without altered permeability)  No association between L:M and citrulline, suggesting that mucosal mass may not necessarily correlate with permeability  No association between ∆PZC and baseline Citrulline/∆Citrulline Definition: chronic condition of unknown etiology, characterized by changes in small bowel morphology, including villous atrophy, hyperplasia of crypt cells and infiltration of the lamina propria by inflammatory cells  Cutoff No EED:  • L:M recovery ratio < 0.03 • L:M concentration ratio < 0.12 Alternate cutoff: • L:M recovery ratio > 0.07 • L:M concentration ratio > 0.28 Abnormal citrulline concentration < 14 µmol/L   	 98	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments zinc  • Final AGP > 1 g/L • HRP2 > 0.75 ng/mL • Time of day  Positively related to ∆PZC: • elapsed time since last breast-feed • baseline AGP > 1  Controlling for above factors, baseline L:M recovery ratio was negatively associated with ∆PZC, regardless of liquid or solid supplementation (p = 0.014)  No association between ∆PZC and L:M concentration ratio   2012  Weisz AJ et al   Abnormal gut integrity is associated with reduced linear growth in rural Malawian children  To determine the relationship between EED (as measured by the L:M test) to linear growth faltering  Rural Malawi  Children 2-5, only twins  Exclusion: Chronic debilitating illness, (e.g. HIV, congenital abnormalities), evidence of acute malnutrition, recent history of diarrhea or hematochezia  Case-cohort  n = 418 Anthropometric HAZ, WAZ, MUACZ  Permeability 4-h dual-sugar absorption test 5g L : 1g M L:M ratio, %L Anthropometric ∆HAZ associated with two %L (p < 0.05): • %L < 0.13 • %L > 0.37  ∆HAZ not associated with L:M ratio  MLR found that initial %L, WAZ, initial HAZ, number of bicycles in home, presence of animals sleeping in home, time per day clean water was brought to home, and previous treatment for malnutrition were all significant factors in predicting linear growth  Permeability This study has differing results from previous studies, which show that L:M RATIO is associated and %L is not associated - Authors mention that “differences in the pattern of the perturbation of the mannitol and lactulose excretion and L:M between different populations may represent different insults to the children’s gastrointestinal mucosa mediated by nutritional status, age, or environment” Definition: asymptomatic, diffuse villous atrophy of the small bowel associated with T-cell inflammatory infiltration and reduced intestinal integrity, leading to reduced intestinal nutrient absorption  Cutoff Abnormal: L:M > .10 	 99	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 364/418 (87%) of children had abnormal L:M ratio  2010  Kelly P et al   Gastric and intestinal barrier impairment in tropical enteropathy and HIV: Limited impact of micronutrient supplementation during a randomised controlled trial  To examine the effect of micronutrient supplementation on gut pH (study 1) and barrier function (study 2)  Urban Zambia  Adults with or without HIV  Case-cohort (within clustered RCT)  Study 1: n = 203, participants who had been taking MM or placebo for 4-9 mo  Study 2: n = 86, participants taken over the course of the 4-9 mo, some before and some after crossover (for logistic reasons)  Permeability 5-h four-sugar absorption test (0.5g xylose, 1g rhamnose, 5g lactulose, 0.2g 3-O-methyl D-glucose) Xylose recovery, RG ratio, LR ratio  Microbial translocation LPS  EndoCAb IgG/IgM  Systemic inflammation TNFR (receptor p55)  Gastric pH  Gastric pH No difference in median pH or number of individuals with hypochlorhydria between MM and placebo groups  Permeability No difference in any of the measures of permeability between MM and placebo, nor between HIV groups  Log-transformed xylose recovery negatively correlated with log-transformed anti-LPS IgG (p = 0.006)  Microbial Translocation (n = 87) No difference in LPS concentrations nor anti-LPS IgG between MM and placebo  Significant reduction of IgM in MM group compared to placebo  Systemic inflammation Correlation between log- transformed TNFRp55 and anti-LPS IgM was significant (r = 0.30, P = 0.006) – more strongly correlated than IgG  Correlation between TNFRp55 and anti-LPS IgG (r = 0.27, P = 0.01)  Little evidence to support the use of MM supplementation to reduce markers of intestinal permeability or bacterial translocation, except possibly anti-LPS IgM   Anti-LPS IgM correlated with TNF pathway activation, implying that translocation is a determinant of systemic inflammation -- Anti-LPS IgM was reduced in MM group but TNFR was not. This may imply some sort of threshold reduction in IgM before TNF pathway is affected   No definition of EED provided   Cutoff Hypochlorhydria: gastric pH > 4.0 	100	Reference and Study Outcomes of Interest Location and Target Population Design and Sample Size Measurements Results Discussion/Conclusions Comments 2010  Papadia C et al  Plasma citrulline as a quantitative biomarker of HIV-associated villous atrophy in a tropical enteropathy population  To determine if serum citrulline concentration is a reliable marker of early morphological and functional changes in HIV enteropathy Urban Zambia  Adults with tropical enteropathy (included both HIV-seronegative and seropositive, not taking ART)  Exclusion: pregnant or lactating women, individuals who had experienced diarrhea and had taken antibiotics or NSAIDS in the previous month Case-Cohort  n = 145 (44 HIV seropositive, 101 HIV seronegative)  Anthropometric MUACZ  Permeability 5-h four-sugar absorption test (0.5g xylose, 1g rhamnose, 5g lactulose, 0.2g 3-O-methyl D-glucose) Xylose recovery, RG ratio, LR ratio  Enterocyte mass Citrulline  Morphometric Villous compartment volume (VCV) Epithelial surface area (ESA) Villous height (VH) Crypt depth (CD) Villous width (VW)  Anthropometric MUACZ correlated with citrulline (p = 0.04) and were significantly reduced in all those with diarrhea (p = 0.003)  Permeability Associated with citrulline levels in both HIV positive and HIV negative patients *Note: not significant after BH correction for multiple correlations, except for xylose absorption  Morphometric VH-CD ratio significantly lower in HIV positive individuals   Enterocyte mass Citrulline concentrations significantly lower in HIV positive patients Citrulline appears to be a reliable marker of small bowel structural and functional integrity in HIV-related enteropathy  Citrulline concentration does not seem to be a sufficiently sensitive marker for rejection or viral enteritis as its values decline only in more severe intestinal mucosal damage Definition—Tropical enteropathy: asymptomatic villous atrophy of the small bowel; characterised by subclinical malabsorption and increased permeability; mild reduction in villous height, broadening of the villi and increased crypt depth Cutoff • healthy:  citrulline > 30 μmol/L • partial villous atrophy: 10< citrulline < 30 μmol/L • total villous atrophy: citrulline < 10 μmol/L 	Definitions Stunted: HAZ < –2 Underweight: WAZ < –2  Wasted: WHZ < –2 	101	 Abbreviations:  AGP, α-1-acid glycoprotein; AAT, α-1-antitrypsin; BAZ, BMI-for-age Z-score; Cdn-15, claudin-15; CRP, C-reactive protein; DHA, docosahexaenoic acid; EED, environmental enteric dysfunction; EFZ, endogenous fecal zinc; EndoCab, endotoxin core antibody; EPA, eicosapentaenoic acid; FAZ, fractional absorbed zinc; FLA, flagellin; GLP-2, glucagon-like peptide-2; GCA, glycocholic acid;  GCDCA, glycochenodexocy-cholic acid; GDCA, glycodeoxycholic acid;  GUDCA, glycoursodeoxycholic acid; HAZ, height-for-age Z-score; HEV, Hepatitis E viral infection; HIV, Human Immunodeficiency Virus; HPLC-PAD, high-performance liquid chromatography with pulsed amperometric detection; HRP, histidine-rich protein; IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; IGF-1, insulin-like growth factor-1; IGFBP3, insulin-like growth factor binding protein 3; 	102	IFN- α, interferon alpha; IFN-γ, interferon gamma; IL, interleukin;  KTR, kynurenine-tryptophan ratio; LC-MSMS, liquid chromatography-tandem mass spectrometry; L:C, lactulose-creatinine; L:M, lactulose-mannitol; L:R, lactulose-rhamnose; LPS, lipopolysaccharide; MAL-ED, Malnutrition and Enteric Disease (study) MPO, myeloperoxidase; MUACZ, mid-upper arm circumference Z-score; NEO, neopterin; NRZ, net retained zinc; PZC, plasma zinc concentration; PROVIDE, Performance of Rotavirus and Oral Polio Vaccines in Developing Countries (study); sCD14, Soluble Cluster of Differentiation 14; TAZ, total absorbed zinc TDCA, taurodeoxycholic acid;  TNFβ, tumor necrosis factor-β; TMCA, tauromurocholic acid; WAZ, weight-for-age Z-score; WHZ, weight-for-height Z-score; %L, percent lactulose permeability; %M, percent mannitol absorption; %R, percent rhamnose absorption; Appendix B - PRISMA Checklist 103 Section/topic  # Checklist item  Reported on page #  TITLE   Title  1 Identify the report as a systematic review, meta-analysis, or both.  i ABSTRACT   Structured summary  2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number.  ii-iv INTRODUCTION   Rationale  3 Describe the rationale for the review in the context of what is already known.  1-8 Objectives  4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS).  8 METHODS   Protocol and registration  5 Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number.  - Eligibility criteria  6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale.  9 Information sources  7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched.  9 Search  8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated.  9 Study selection  9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis).  10 Data collection process  10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators.  10 Data items  11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made.  10-14 Risk of bias in individual studies  12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis.  -    104 Section/topic  # Checklist item  Reported on page #  Summary measures  13 State the principal summary measures (e.g., risk ratio, difference in means).  - Synthesis of results  14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I2) for each meta-analysis.  - Risk of bias across studies  15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies).  - Additional analyses  16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified.  - RESULTS   Study selection  17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram.  19 Study characteristics  18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations.  19-20, S1 Table Risk of bias within studies  19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12).  - Results of individual studies  20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot.  Tables 1-2 Synthesis of results  21 Present results of each meta-analysis done, including confidence intervals and measures of consistency.  - Risk of bias across studies  22 Present results of any assessment of risk of bias across studies (see Item 15).  - Additional analysis  23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression [see Item 16]).  16-21 DISCUSSION   Summary of evidence  24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers).  20-33 Limitations  25 Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias).  38 Conclusions  26 Provide a general interpretation of the results in the context of other evidence, and implications for future research.  33, 39  105 FUNDING   Funding  27 Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review.  -  From:  Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 6(6): e1000097. doi:10.1371/journal.pmed1000097  For more information, visit: www.prisma-statement.org.  

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