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ABSTRACT Environmental enteric dysfunction (EED) is a subclinical syndrome of altered small intestinal function postulated to be an important contributor to childhood undernutrition. The role of small intestinal bacterial communities in the pathophysiology of EED is poorly defined due to a paucity of studies where there has been a direct collection of small intestinal samples from undernourished children. Sixty-three members of a Pakistani cohort identified as being acutely malnourished between 3 and 6 months of age and whose wasting (weight-for-length Z-score [WLZ]) failed to improve after a 2-month nutritional intervention underwent esophagogastroduodenoscopy (EGD). Paired duodenal luminal aspirates and duodenal mucosal biopsies were obtained from 43 children. Duodenal microbiota composition was characterized by sequencing bacterial 16S rRNA gene amplicons. Levels of bacterial taxa (amplicon sequence variants [ASVs]) were referenced to anthropometric indices, histopathologic severity in biopsies, expression of selected genes in the duodenal mucosa, and fecal levels of an immunoinflammatory biomarker (lipocalin-2). A “core” group of eight bacterial ASVs was present in the duodenal samples of 69% of participants. Streptococcus anginosus was the most prevalent, followed by Streptococcus sp., Gemella haemolysans, Streptococcus australis, Granulicatella elegans, Granulicatella adiacens, and Abiotrophia defectiva. At the time of EGD, none of the core taxa were significantly correlated with WLZ. Statistically significant correlations were documented between the abundances of Granulicatella elegans and Granulicatella adiacens and the expression of duodenal mucosal genes involved in immune responses (dual oxidase maturation factor 2, serum amyloid A, and granzyme H). These results suggest that a potential role for members of the oral microbiota in pathogenesis, notably Streptococcus, Gemella, and Granulicatella species, warrants further investigation.IMPORTANCEUndernutrition among women and children is a pressing global health problem. Environmental enteric dysfunction (EED) is a disease of the small intestine (SI) associated with impaired gut mucosal barrier function and reduced capacity for nutrient absorption. The cause of EED is ill-defined. One emerging hypothesis is that alterations in the SI microbiota contribute to EED. We performed a culture-independent analysis of the SI microbiota of a cohort of Pakistani children with undernutrition who had failed a standard nutritional intervention, underwent upper gastrointestinal tract endoscopy, and had histologic evidence of EED in their duodenal mucosal biopsies. The results revealed a shared group of bacterial taxa in their duodenums whose absolute abundances were correlated with levels of the expression of genes in the duodenal mucosa that are involved in inflammatory responses. A number of these bacterial taxa are more typically found in the oral microbiota, a finding that has potential physiologic and therapeutic implications.

Abstract The acquisition of antimicrobial resistance (AR) genes has rendered important pathogens nearly or fully unresponsive to antibiotics. It has been suggested that pathogens acquire AR traits from the gut microbiota, which collectively serve as a global reservoir for AR genes conferring resistance to all classes of antibiotics. However, only a subset of AR genes confers resistance to clinically relevant antibiotics, and, although these AR gene profiles are well-characterized for common pathogens, less is known about their taxonomic associations and transfer potential within diverse members of the gut microbiota. We examined a collection of 14,850 human metagenomes and 1666 environmental metagenomes from 33 countries, in addition to nearly 600,000 isolate genomes, to gain insight into the global prevalence and taxonomic range of clinically relevant AR genes. We find that several of the most concerning AR genes, such as those encoding the cephalosporinase CTX-M and carbapenemases KPC, IMP, NDM, and VIM, remain taxonomically restricted to Proteobacteria. Even cfiA, the most common carbapenemase gene within the human gut microbiome, remains tightly restricted to Bacteroides, despite being found on a mobilizable plasmid. We confirmed these findings in gut microbiome samples from India, Honduras, Pakistan, and Vietnam, using a high-sensitivity single-cell fusion PCR approach. Focusing on a set of genes encoding carbapenemases and cephalosporinases, thus far restricted to Bacteroides species, we find that few mutations are required for efficacy in a different phylum, raising the question of why these genes have not spread more widely. Overall, these data suggest that globally prevalent, clinically relevant AR genes have not yet established themselves across diverse commensal gut microbiota.

Abstract 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’ 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.

BackgroundThe 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.MethodsChildren aged nine months in the rural district of Matiari, Pakistan, with a weight-for-height z-score (WHZ) ResultsThe 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.ConclusionOur study found that both RUTF and RUSF partially improve the anthropometric status of acutely malnourished children, with neither being superior to the other.

IntroductionEnvironmental enteropathy is an important contributor to childhood malnutrition in the developing world. Chronic exposure to fecal pathogens leads to alteration in intestinal structure and function, resulting in impaired gut immune function, malabsorption, and growth faltering leading to environmental enteropathy.MethodsA community-based intervention study was carried out on children till 24 months of age in Matiari district, Pakistan. Blood and fecal specimens were collected from the enrolled children aged 3–6 and 9 months. A real-time PCR-based TaqMan array card (TAC) was used to detect enteropathogens.ResultsGiardia, Campylobacter spp., enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), and Cryptosporidium spp. were the most prevailing enteropathogens in terms of overall positivity at both time points. Detection of protozoa at enrollment and 9 months was negatively correlated with rate of change in height-for-age Z (ΔHAZ) scores during the first and second years of life. A positive association was found between Giardia, fecal lipocalin (LCN), and alpha 1-Acid Glycoprotein (AGP), while Campylobacter spp. showed positive associations with neopterin (NEO) and myeloperoxidase (MPO).ConclusionProtozoal colonization is associated with a decline in linear growth velocity during the first 2 years of life in children living in Environmental enteric dysfunction (EED) endemic settings. Mechanistic studies exploring the role of cumulative microbial colonization, their adaptations to undernutrition, and their influence on gut homeostasis are required to understand symptomatic enteropathogen-induced growth faltering.

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.

Abstract Background Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn’s disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community. Discussion Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals. Trial registration Retrospectively registered; clinicaltrials.gov ID NCT03588013.

The Study of Environmental Enteropathy (EE) and Malnutrition in Pakistan (SEEM) is a community intervention trial designed to understand the pathophysiology of EE and its associated clinical phenotypes in children less than two years of age. Undernutrition is the underlying cause of three million deaths worldwide annually, accounting for 45% of all deaths among children under five years of age. Linear growth stunting affects a further 165 million, especially in low- and middle-income countries. In Pakistan, an estimated one in three children are underweight and nearly half are stunted. This manuscript summarizes the challenges faced by the field team and the solutions employed for achieving our research objectives at the rural Matiari field site. Our study design involved a longitudinal collection of field data and biological samples which required constant engagement of field staff with the participating families. Challenges faced in SEEM included: 1) quality assurance parameters of anthropometry data; 2) 24-hour food recall data collection; 3) clinical management of sick enrolled children; 4) obtaining informed consent for upper gastrointestinal (UGI)endoscopy (an invasive procedure); and 5) biological sample collection (blood, urine, stool) from enrolled children. We enrolled 350 malnourished children(Weight for Height Z (WHZ) score Solutions to these challenges included good project governance, the establishment of credibility, and the development of a longitudinal bidirectional relationship embedded in trust and effective communication with the caretakers of the enrolled children. Continuous monitoring, regular training of staff, and effective teamwork were key factors that led to the successful execution of our study.

The Study of Environmental Enteropathy (EE) and Malnutrition in Pakistan (SEEM) is a community intervention trial designed to understand the pathophysiology of EE and its associated clinical phenotypes in children less than two years of age. Undernutrition is the underlying cause of three million deaths annually, accounting for 45% of all deaths among children under five years of age. Linear growth stunting affects a further 165 million. In Pakistan, where our study is focused, an estimated one in three children are underweight and nearly half are stunted. This manuscript summarizes the challenges faced by the field team and the solutions employed for achieving our research objectives at the rural field site, Matiari. Our study design involved a longitudinal collection of field data and biological samples which required constant engagement of field staff with the participating families. Challenges faced in SEEM included: 1) quality assurance parameters of anthropometry data; 2) 24-hour food recall data collection; 3) clinical management of acutely sick children enrolled in our study; 4) obtaining informed consent for upper gastrointestinal endoscopy (an invasive procedure); and 5) biological sample collection (blood, urine, stool) from malnourished children at the community level. We accomplished our goal of enrolling 350 malnourished children and 50 well-nourished children with a more than 95% success rate of collecting blood, urine and stool specimens. We collected 72% microbiome stool samples within 30 minutes of the child passing stool. We conducted an upper gastrointestinal biopsy in 63 children. Broadly, solutions to these challenges included good governance, the establishment of credibility, and the development of a longitudinal bidirectional relationship embedded in trust and effective communication with the caretakers of the children enrolled in our study. Continuous monitoring, regular training of staff, and effective teamwork were key factors that led to the successful execution of our study.