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Environmental enteric dysfunction (EED) is a subclinical condition of intestinal inflammation, barrier dysfunction and malabsorption associated with growth faltering in children living in poverty. This study explores association of altered duodenal permeability (lactulose, rhamnose and their ratio) with higher burden of enteropathogen in the duodenal aspirate, altered histopathological findings and higher morbidity (diarrhea) that is collectively associated with linear growth faltering in children living in EED endemic setting. In a longitudinal birth cohort, 51 controls (WHZ > 0, HAZ > −1.0) and 63 cases (WHZ< -2.0, refractory to nutritional intervention) were recruited. Anthropometry and morbidity were recorded on monthly bases up to 24 months of age. Dual sugar assay of urine collected after oral administration of lactulose and rhamnose was assessed in 96 children from both the groups. Duodenal histopathology (n = 63) and enteropathogen analysis of aspirate via Taqman array card (n = 60) was assessed in only cases. Giardia was the most frequent pathogen and was associated with raised L:R ratio (p = 0.068). Gastric microscopy was more sensitive than duodenal aspirate in H. pylori detection. Microscopically confirmed H. pylori negatively correlated with HAZ at 24 months (r = −0.313, p = 0.013). Regarding histopathological parameters, goblet cell reduction significantly correlated with decline in dual sugar excretion (p< 0.05). Between cases and controls, there were no significant differences in the median (25th, 75th percentile) of urinary concentrations (μg/ml) of lactulose [27.0 (11.50, 59.50) for cases vs. 38.0 (12.0, 61.0) for controls], rhamnose [66.0 (28.0, 178.0) vs. 86.5 (29.5, 190.5)] and L:R ratio [0.47 (0.24, 0.90) vs. 0.51 (0.31, 0.71)] respectively. In multivariable regression model, 31% of variability in HAZ at 24 months of age among cases and controls was explained by final model including dual sugars. In conclusion, enteropathogen burden is associated with altered histopathological features and intestinal permeability. In cases and controls living in settings of endemic enteropathy, intestinal permeability test may predict linear growth. However, for adoption as a screening tool for EED, further validation is required due to its complex intestinal pathophysiology. Author summary: EED is a subclinical condition of compromised gut integrity secondary to frequent and repeated exposure to enteropathogens in global settings with a high prevalence of undernutrition. In this study, we reported association of gut mucosal architecture with a dual sugar intestinal permeability assay (lactulose-rhamnose) in Pakistani children. In the presence of duodenal enteropathogens, features such as chronic inflammation, intra-epithelial lymphocytosis, enterocyte injury and Paneth cell reduction were consistently observed. When comparing undernourished cases and controls living in the same setting, we found urinary excretion of the sugars was similar among groups; however, variability in HAZ among children at 24 months was partially explained by a model that includes excretion values. [ABSTRACT FROM AUTHOR]

Background: A major limitation to understanding the etiopathogenesis of environmental enteric dysfunction (EED) is the lack of a comprehensive, reproducible histologic framework for characterizing the small bowel lesions. We hypothesized that the development of such a system will identify unique histology features for EED, and that some features might correlate with clinical severity. Methods: Duodenal endoscopic biopsies from two cohorts where EED is prevalent (Pakistan, Zambia) and North American children with and without gluten sensitive enteropathy (GSE) were processed for routine hematoxylin & eosin (H&E) staining, and scanned to produce whole slide images (WSIs) which we shared among study pathologists via a secure web browser-based platform. A semi-quantitative scoring index composed of 11 parameters encompassing tissue injury and response patterns commonly observed in routine clinical practice was constructed by three gastrointestinal pathologists, with input from EED experts. The pathologists then read the WSIs using the EED histology index, and inter-observer reliability was assessed. The histology index was further used to identify within- and between-child variations as well as features common across and unique to each cohort, and those that correlated with host phenotype. Results: Eight of the 11 histologic scoring parameters showed useful degrees of variation. The overall concordance across all parameters was 96% weighted agreement, kappa 0.70, and Gwet's AC 0.93. Zambian and Pakistani tissues shared some histologic features with GSE, but most features were distinct, particularly abundance of intraepithelial lymphocytes in the Pakistani cohort, and marked villous destruction and loss of secretory cell lineages in the Zambian cohort. Conclusions: We propose the first EED histology index for interpreting duodenal biopsies. This index should be useful in future clinical and translational studies of this widespread, poorly understood, and highly consequential disorder, which might be caused by multiple contributing processes, in different regions of the world. Author summary: The study of EED has been limited by the lack of a rigorously tested, reproducible histology index that can provide insight to the pathogenesis of this entity. In this study we report the first duodenal histology index that was developed using an unbiased approach, with excellent inter-observer reproducibility, for the study of EED. The EED histology index readily identified histologic features that are common or unique to cohorts of distinct geographic locations. Incorporating the histology index into future clinical studies will provide useful insight into the pathogenesis and for intervention strategy development. [ABSTRACT FROM AUTHOR]

Environmental Enteric Dysfunction (EED) is an acquired small intestinal inflammatory condition underlying high rates of stunting in children <5 years of age in low- and middle-income countries. Children with EED are known to have repeated exposures to enteropathogens and environmental toxins that leads to malabsorptive syndrome. We aimed to characterize association of linear growth faltering with enteropathogen burden and subsequent changes in EED biomarkers. In a longitudinal birth cohort (n = 272), monthly anthropometric measurements (Length for Age Z score- LAZ) of asymptomatic children were obtained up to 18 months. Biological samples were collected at 6 and 9 months for the assessment of biomarkers. A customized TaqMan array card was used to target 40 enteropathogens in fecal samples. Linear regression was applied to study the effect of specific enteropathogen infection on change in linear growth (ΔLAZ). Presence of any pathogen in fecal sample correlated with serum flagellin IgA (6 mo, r = 0.19, p = 0.002), fecal Reg 1b (6 mo, r = 0.16, p = 0.01; 9mo, r = 0.16, p = 0.008) and serum Reg 1b (6 mo, r = 0.26, p<0.0001; 9 mo, r = 0.15, p = 0.008). At 6 months, presence of Campylobacter [β (SE) 7751.2 (2608.5), p = 0.003] and ETEC LT [β (SE) 7089.2 (3015.04), p = 0.019] was associated with increase in MPO. Giardia was associated with increase in Reg1b [β (SE) 72.189 (26.394), p = 0.006] and anti-flic IgA[β (SE) 0.054 (0.021), p = 0.0091]. Multiple enteropathogen infections in early life negatively correlated with ΔLAZ, and simultaneous changes in gut inflammatory and permeability markers. A combination vaccine targeting enteropathogens in early life could help in the prevention of future stunting. [ABSTRACT FROM AUTHOR]

Background: Environmental Enteric Dysfunction (EED) in children from low-income countries has been linked to linear growth declines. There is a critical need to identify sensitive and early EED biomarkers. Objective: Determine whether levels of antibodies against bacterial components flagellin (flic) and lipopolysaccharide (LPS) predict poor growth. Design/Methods: In a prospective birth cohort of 380 children in rural Pakistan blood and stool samples were obtained at ages 6 and 9 months. Linear mixed effects models were used to examine longitudinal associations between quartiles of anti-flic and anti-LPS antibodies and changes in LAZ, WAZ and WLZ scores. Spearman’s correlations were measured between anti-flic and anti-LPS immunoglobulins with measures of systemic/enteric inflammation and intestinal regeneration. Results: Anti-LPS IgA correlated significantly with CRP, AGP and Reg1 serum at 6mo and with MPO at 9mo. In multivariate analysis at 6mo of age, higher anti-LPS IgA levels predicted greater declines in LAZ scores over subsequent 18mo (comparing highest to lowest quartile, β (SE) change in LAZ score/year = -0.313 (0.125), p-value = 0.013). Anti-flic Ig A in the two highest quartiles measured at 9mo of age had declines in LAZ of -0.269 (0.126), p = 0.033; and -0.306 (0.129), p = 0.018 respectively, during the subsequent 18mo of life, compared to those in the lowest quartile of anti-flic IgA. Conclusions and relevance: Elevated anti-flic IgA and anti-LPS IgA antibodies at 6 and 9mo, predict declines in linear growth. Systemic and enteric inflammation correlated with anti-LPS IgA provides mechanistic considerations for potential future interventions. [ABSTRACT FROM AUTHOR]

Enteropathies such as Crohn’s disease are associated with enteric inflammation characterized by impaired TGF-β signaling, decreased expression of phosphorylated (p)-SMAD2,3 and increased expression of SMAD7 (an inhibitor of SMAD3 phosphorylation). Environmental enteropathy (EE) is an acquired inflammatory disease of the small intestine (SI), which is associated with linear growth disruption, cognitive deficits, and reduced oral vaccine responsiveness in children <5 y in resource-poor countries. We aimed to characterize EE inflammatory pathways by determining SMAD7 and p-SMAD2,3 levels (using Western blotting) in EE duodenal biopsies (N = 19 children, 7 from Pakistan, 12 from Zambia) and comparing these with healthy controls (Ctl) and celiac disease (CD) patients from Italy. Densitometric analysis of immunoblots showed that EE SI biopsies expressed higher levels of both SMAD7 (mean±SD in arbitrary units [a.u.], Ctl = 0.47±0.20 a.u., EE = 1.13±0.25 a.u., p-value = 0.03) and p-SMAD2,3 (mean±SD, Ctl = 0.38±0.14 a.u., EE = 0.60±0.10 a.u., p-value = 0.03). immunohistochemistry showed that, in EE, SMAD7 is expressed in both the epithelium and in mononuclear cells of the lamina propria (LP). In contrast, p-SMAD3 in EE is expressed much more prominently in epithelial cells than in the LP. The high SMAD7 immunoreactivity and lack of p-SMAD2,3 expression in the LP suggests defective TGF-β signaling in the LP in EE similar to a previously reported SMAD7-mediated inflammatory pathway in refractory CD and Crohn’s disease. However, Western blot densitometry showed elevated p-SMAD2,3 levels in EE, possibly suggesting a different inflammatory pathway than Crohn’s disease but more likely reflecting cumulative protein expression from across all compartments of the mucosa as opposed to the LP alone. Further studies are needed to substantiate these preliminary results and to illustrate the relationship between SMAD proteins, TGF-β signaling, and inflammatory cytokine production, all of which may be potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Objective:To assess performance of the WHO revised verbal autopsy tool for ascertaining the causes of still birth in comparison with reference standard cause of death ascertained by standardized clinical and supportive data. Methods:All stillbirths at a tertiary hospital in Karachi, Pakistan were prospectively recruited into study from August 2006- February 2008. The reference standard cause of death was established by two senior obstetricians within 48 hours using the ICD coding system. Verbal autopsy interviews using modified WHO tool were conducted by trained health workers within 2- 6 weeks of still birth and the cause of death was assigned by second panel of obstetricians. The performance was assessed in terms of sensitivity, specificity and Kappa. Results:There were 204 still births. Of these, 80.8% of antepartum and 50.5% of intrapartum deaths were correctly diagnosed by verbal autopsy. Sensitivity of verbal autopsy was highest 68.4%, (95%CI: 46-84.6) for congenital malformation followed by obstetric complication 57.6%, (95%CI: 25-84.2). The specificity for all major causes was greater than 90%. The level of agreement was high (kappa=0.72) for anomalies and moderate (k=0.4) for all major causes of still birth, except asphyxia. Conclusion:Our results suggest that verbal autopsy has reasonable validity in identifying and discriminating between causes of stillbirth in Pakistan. On the basis of these findings, we feel it has a place in resource constrained areas to inform strategic planning and mobilization of resources to attain Millennium Development Goals. [ABSTRACT FROM AUTHOR]