Mucosal genomics implicate lymphocyte activation and lipid metabolism in refractory environmental enteric dysfunction

Background & aims: Environmental enteric dysfunction (EED) limits the Sustainable Development Goals of improved childhood growth and survival. We applied mucosal genomics to advance our understanding of EED.Methods: The Study of Environmental Enteropathy and Malnutrition (SEEM) followed 416 children from birth to 24 months in a rural district in Pakistan. Biomarkers were measured at 9 months and tested for association with growth at 24 months. The duodenal methylome and transcriptome was determined in 52 undernourished SEEM participants and 42 North American controls and celiac disease patients.Results: After accounting for growth at study entry, circulating IGF-1 and ferritin predicted linear growth, whereas leptin correlated with future weight gain. The EED transcriptome exhibited suppression of antioxidant, detoxification, and lipid metabolism genes, and induction of anti-microbial response, interferon, and lymphocyte activation genes. Relative to celiac disease, suppression of antioxidant and detoxification genes and induction of anti-microbial response genes were EED-specific. At the epigenetic level, EED showed hyper-methylation of epithelial metabolism and barrier function genes, and hypo-methylation of immune response and cell proliferation genes. Duodenal co-expression modules showed association between lymphocyte proliferation and epithelial metabolic genes and histologic severity, fecal energy loss, and wasting (weight-for-length/height Z\u3c-2.0). Leptin was associated with expression of epithelial carbohydrate metabolism and stem cell renewal genes. Immune response genes were attenuated by giardia colonization.Conclusions: Children with reduced circulating IGF-1 are more likely to experience stunting. Leptin and a gene signature for lymphocyte activation and dysregulated lipid metabolism are implicated in wasting, suggesting new approaches for EED refractory to nutritional intervention

Machine learning model demonstrates stunting at birth and systemic inflammatory biomarkers as predictors of subsequent infant growth - A four-year prospective study

Background: Stunting affects up to one-third of the children in low-to-middle income countries (LMICs) and has been correlated with decline in cognitive capacity and vaccine immunogenicity. Early identification of infants at risk is critical for early intervention and prevention of morbidity. The aim of this study was to investigate patterns of growth in infants up through 48 months of age to assess whether the growth of infants with stunting eventually improved as well as the potential predictors of growth.Methods: Height-for-age z-scores (HAZ) of children from Matiari (rural site, Pakistan) at birth, 18 months, and 48 months were obtained. Results of serum-based biomarkers collected at 6 and 9 months were recorded. A descriptive analysis of the population was followed by assessment of growth predictors via traditional machine learning random forest models.Results: Of the 107 children who were followed up till 48 months of age, 51% were stunted (HAZ \u3c - 2) at birth which increased to 54% by 48 months of age. Stunting status for the majority of children at 48 months was found to be the same as at 18 months. Most children with large gains started off stunted or severely stunted, while all of those with notably large losses were not stunted at birth. Random forest models identified HAZ at birth as the most important feature in predicting HAZ at 18 months. Of the biomarkers, AGP (Alpha- 1-acid Glycoprotein), CRP (C-Reactive Protein), and IL1 (interleukin-1) were identified as strong subsequent growth predictors across both the classification and regressor models.Conclusion: We demonstrated that children most children with stunting at birth remained stunted at 48 months of age. Value was added for predicting growth outcomes with the use of traditional machine learning random forest models. HAZ at birth was found to be a strong predictor of subsequent growth in infants up through 48 months of age. Biomarkers of systemic inflammation, AGP, CRP, IL1, were also strong predictors of growth outcomes. These findings provide support for continued focus on interventions prenatally, at birth, and early infancy in children at risk for stunting who live in resource-constrained regions of the world

Promising biomarkers of environmental enteric dysfunction: a prospective cohort study in Pakistani children.

Environmental Enteric Dysfunction (EED), a syndrome characterized by chronic gut inflammation, contributes towards stunting and poor response to enteric vaccines in children in developing countries. In this study, we evaluated major putative biomarkers of EED using growth faltering as its clinical proxy. Newborns (n = 380) were enrolled and followed till 18 months with monthly anthropometry. Biomarkersassociated with gut and systemic inflammation were assessed at 6 and 9 months. Linear mixed effects model was used to determine the associations of these biomarkers with growth faltering between birth and 18 months. Fecal myeloperoxidase (neutrophil activation marker) at 6 months [β = -0.207, p = 0.005], and serum GLP 2 (enterocyte proliferation marker) at 6 and 9 months [6M: β = -0.271, p = 0.035; 9M: β = -0.267, p = 0.045] were associated with decreasing LAZ score. Ferritin at 6 and 9 months was associated with decreasing LAZ score [6M: β = -0.882, p \u3c 0.0001; 9M: β = -0.714, p \u3c 0.0001] and so was CRP [β = -0.451, p = 0.039] and AGP [β = -0.443, p = 0.012] at 9 months. Both gut specific and systemic biomarkers correlated negatively with IGF-1, but only weakly correlated, if at all with each other. We therefore conclude that EED may be contributing directly towards growth faltering, and this pathway is not entirely through the pathway of systemic inflammation

Barriers and facilitators to exclusive breastfeeding in rural Pakistan: A qualitative exploratory study

Background: Exclusive breastfeeding (EBF) of children until six months of age is considered one of the most critical interventions in tackling childhood undernutrition. EBF rates are suboptimal in Pakistan, particularly in rural areas where child undernutrition is most prevalent. This study aimed to explore barriers to EBF in a rural context of Pakistan.Methods: The study was conducted in the rural district Matiari of Sindh, Pakistan, during Jan-March 2020. We used a qualitative exploratory study design and conducted 36 focus group discussions (FGDs). Participants were purposively selected mothers who had not practiced EBF during their previous childbirth, their spouses and mothers-in-law, and lady health workers (LHWs) serving in the study catchment. FGDs were audio-recorded, transcribed, and translated into English from the local language and analysed using thematic content analysis.Results: Barriers to EBF included low awareness and cultural practices of prelacteal feeds, insufficient breast milk production, undernutrition of mothers, mothers\u27 occupation as fieldworkers, less birth spacing, low awareness about the correct technique of breastfeeding, maternal and child ailments, abnormal breasts, and influence of in-laws to start top-up feeds. Several facilitators were identified: family support, appropriate maternal diet, maternal awareness, and support in the neighborhood.Conclusion: Barriers to EBF are multifaceted in rural areas, and interventions aiming to improve adherence to EBFshould be multipronged. Awareness-raising alone might not be sufficient, and other interventions should be designed to address the barriers of maternal malnutrition, insufficient milk production, and socio-cultural practices. In addition, safe alternatives to breast milk may be necessary if breastfeeding is truly not feasible. Lack of breast milk substitutes is particularly challenging for poor rural women who cannot afford infant formula milk

Association of anti-rotavirus IgA seroconversion with growth, environmental enteric dysfunction and enteropathogens in rural Pakistani infants

Background: The underperformance of oral vaccines in children of low- and middle-income countries is partly attributable to underlying environmental enteric dysfunction (EED).Methodology: We conducted a longitudinal, community-based study to evaluate the association of oral rotavirus vaccine (Rotarix®) seroconversion with growth anthropometrics, EED biomarkers and intestinal enteropathogens in Pakistani infants. Children were enrolled between three to six months of their age based on their nutritional status. We measured serum anti-rotavirus immunoglobulin A (IgA) at enrollment and nine months of age with EED biomarkers and intestinal enteropathogens.Results: A total of 391 infants received two doses of rotavirus (RV) vaccine. 331/391 provided paired blood samples. Of these 331 children, 45% seroconverted at 9 months of age, 35% did not seroconvert and 20% were seropositive at baseline. Non-seroconverted children were more likely to be stunted, wasted and underweight at enrollment. In univariate analysis, insulin-like growth factor (IGF) concentration at 6 months were higher in seroconverters, median (25th, 75th percentile): 26.3 (16.5, 43.5) ng/ml vs. 22.5 (13.6, 36.3) ng/ml for non-seroconverters, p-value = 0.024. At nine months, fecal myeloperoxidase (MPO) concentrations were significantly lower in seroconverters, 3050(1250, 7587) ng/ml vs. 4623.3 (2189, 11650) ng/ml in non-seroconverted children, p-value = 0.017. In multivariable logistic regression analysis, alpha-1 acid glycoprotein (AGP) and IGF-1 concentrations were positively associated with seroconversion at six months. The presence of sapovirus and rotavirus in fecal samples at the time of rotavirus administration, was associated with non-seroconversion and seroconversion, respectively.Conclusion: We detected high baseline RV seropositivity and impaired RV vaccine immunogenicity in this high-risk group of children. Healthy growth, serum IGF-1 and AGP, and fecal shedding of rotavirus were positively associated with RV IgA seroconversion following immunization, whereas the presence of sapovirus was more common in non-seroconverters.Trial registration: Clinical Trials ID: NCT03588013

Standard RUTF vs. locally-made RUSF for acutely malnourished children: A quasi-experimental comparison of the impact on growth and compliance in a rural community of Pakistan

Background: The reduction in severe and moderate acute malnutrition (SAM and MAM) rates in Pakistan has been sub-optimal compared to other low-and middle-income countries (LMICs). Specially-formulated products have been designed globally to manage SAM and MAM, such as ready-to-use therapeutic food (RUTF) and ready-to-use supplementary food (RUSF), with variable efficacies. RUTF is primarily produced and patented in industrialized countries, raising supply challenges in resource-constrained regions with a high burden of acute malnutrition. RUSF minimizes costs by using locally-available ingredients while providing similar nutritional value. In this study, we compared the efficacy, side effects, and compliance of two months of supplementation with either RUTF or RUSF.Methods: Children aged nine months in the rural district of Matiari, Pakistan, with a weight-for-height z-score (WHZ) \u3c-2 received either RUTF (500 kcal sachet) for two months in 2015 or RUSF (520 kcal sachet) for two months in 2018.Results: The RUSF group had a higher height gain and mid-upper arm circumferences (MUAC) score. Higher compliance was noted with lower side effects in the RUSF group. A higher compliance rate did correlate with the growth parameters in respective groups.Conclusion: Our study found that both RUTF and RUSF partially improve the anthropometric status of acutely malnourished children, with neither being superior to the other

Distance from healthcare facilities is associated with increased morbidity of acute infection in pediatric patients in Matiari, Pakistan

The relationship between environmental factors and child health is not well understood in rural Pakistan. This study characterized the environmental factors related to the morbidity of acute respiratory infections (ARIs), diarrhea, and growth using geographical information systems (GIS) technology. Anthropometric, address and disease prevalence data were collected through the SEEM (Study of Environmental Enteropathy and Malnutrition) study in Matiari, Pakistan. Publicly available map data were used to compile coordinates of healthcare facilities. A Pearson correlation coefficient (r) was used to calculate the correlation between distance from healthcare facilities and participant growth and morbidity. Other continuous variables influencing these outcomes were analyzed using a random forest regression model. In this study of 416 children, we found that participants living closer to secondary hospitals had a lower prevalence of ARI (r = 0.154, p \u3c 0.010) and diarrhea (r = 0.228, p \u3c 0.001) as well as participants living closer to Maternal Health Centers (MHCs): ARI (r = 0.185, p \u3c 0.002) and diarrhea (r = 0.223, p \u3c 0.001) compared to those living near primary facilities. Our random forest model showed that distance has high variable importance in the context of disease prevalence. Our results indicated that participants closer to more basic healthcare facilities reported a higher prevalence of both diarrhea and ARI than those near more urban facilities, highlighting potential public policy gaps in ameliorating rural health

Quantitative morphometry and machine learning model to explore duodenal and rectal mucosal tissue of children with environmental enteric dysfunction

Environmental enteric dysfunction (EED) is a subclinical enteropathy prevalent in resource-limited settings, hypothesized to be a consequence of chronic exposure to environmental enteropathogens, resulting in malnutrition, growth failure, neurocognitive delays, and oral vaccine failure. This study explored the duodenal and colonic tissues of children with EED, celiac disease, and other enteropathies using quantitative mucosal morphometry, histopathologic scoring indices, and machine learning-based image analysis from archival and prospective cohorts of children from Pakistan and the United States. We observed villus blunting as being more prominent in celiac disease than in EED, as shorter lengths of villi were observed in patients with celiac disease from Pakistan than in those from the United States, with median (interquartile range) lengths of 81 (73, 127) µm and 209 (188, 266) µm, respectively. Additionally, per the Marsh scoring method, celiac disease histologic severity was increased in the cohorts from Pakistan. Goblet cell depletion and increased intraepithelial lymphocytes were features of EED and celiac disease. Interestingly, the rectal tissue from cases with EED showed increased mononuclear inflammatory cells and intraepithelial lymphocytes in the crypts compared with controls. Increased neutrophils in the rectal crypt epithelium were also significantly associated with increased EED histologic severity scores in duodenal tissue. We observed an overlap between diseased and healthy duodenal tissue upon leveraging machine learning image analysis. We conclude that EED comprises a spectrum of inflammation in the duodenum, as previously described, and the rectal mucosa, warranting the examination of both anatomic regions in our efforts to understand and manage EED

Nutrient dataset development via FAO/INFOODS approach for infant nutritional survey in rural Matiari, Pakistan

To accurately evaluate dietary intake, multiple resources are necessary, including serving-size modules, pictures, and questionnaires that are used to gather information during surveys. One critical component is the accessibility of food composition data at the national or regional level, which is required to determine dietary intake. Food Agriculture Organization/International Network of Food Data Systems (FAO/INFOODs) tools are useful for developing high-quality food composition data. We used these tools to create a nutrient dataset for a nutritional survey in Matiari, Sindh, and to collect dietary information through a 24-hour food recall questionnaire. The survey results indicated 540 distinct types of foods, including 291 ready-to-eat items, 84 foods used as ingredients in recipes, and 164 various composite and mixed recipes. Most food items corresponded to the national and regional Food Composition Tables (FCTs) and the Food and Nutrient Database for Dietary Studies (FNDDS) of the USDA, with the exception of recipe food data. We utilized Eurofir-recipe calculation methods to compute the recipe data. The data were homogenized and standardized utilizing EFSA and Langual™. Because of the obsolescence and inadequacy of Pakistan\u27s food composition table in assessing essential nutrients, we had to source data from various other sources. Consequently, to establish the nutrient dataset, we incorporated approximately 25 % of user data from national sources, with recipe data comprising 46 % and less than 20 % extracted from regional, U.S database, and diverse online sources. This study is the first effort in which we gathered data from reliable sources representing local eating patterns, with some exceptions. Future studies will hugely benefit from this database, especially as we face a high prevalence of undernutrition in our part of the world