Implementation challenges from a prospective, interventional biopsy-based study of Environmental Enteropathy in rural Pakistan

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 <-2) and 50 well-nourished children(WHZ score ≥ 0 and height-for-age Z (HAZ) score ≥ -1) with 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 a UGI biopsy in 63 children. 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.

Implementation challenges from a prospective, interventional biopsy-based study of Environmental Enteropathy in rural Pakistan

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.

Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation in severe disease

Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in low- and middle income countries. It is thought to be a primary cause of most global growth-stunting cases and a key contributing factor to childhood malnutrition and diminished oral vaccine responses. While EE has been shown to be the byproduct of recurrent enteric infection, to date, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE severity by performing high-throughput single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE from Lusaka, Zambia (8 HIV negative, 3 HIV positive) and 6 adults without EE in Boston, USA. Using the resulting cellular atlas, we scored existing bulk-transcriptomic signatures of reduced villus height and decreased plasma LPS levels in EE, finding that these signatures may be driven by an increased abundance of surface mucosal cells (a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract). In addition, we identified several cell subsets whose fractional abundances associated with histological determined EE severity, small intestinal region, and HIV infection. Furthermore, by comparing distal duodenal EE samples with those from two U.S. control cohorts, we identified broadly decreased epithelial proliferative signaling, lower fractional abundances of goblet cells, and a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells but with increased pro-inflammatory cytokine expression in EE. Altogether, our work illuminates the epithelial and immune correlates of EE severity and provides new molecular targets for intervention.

The contribution of environmental enteropathy to the global problem of micronutrient deficiency

Sometimes referred to as hidden hunger, micronutrient deficiencies persist on a global scale. For some micronutrients this appears to be due to inadequate intake, for others intake may not match increased requirements. However, for most micronutrient deficiencies there is uncertainty as to the dominant driver, and the question about the contribution of malabsorption is open. Environmental enteropathy (EE), formerly referred to as tropical enteropathy and also referred to as environmental enteric dysfunction, is an asymptomatic disorder of small intestinal structure and function which is very highly prevalent in many disadvantaged populations. Recent studies of the pathology and microbiology of this disorder suggest that it is driven by very high pathogen burdens in children and adults living in insanitary environments and is characterised by major derangements of the epithelial cells of the intestinal mucosa. Transcriptomic data suggest that it may lead to impaired digestion and absorption of macronutrients. Given the very high prevalence of EE, marginal malabsorption could have large impacts at population scales. However, the relative contributions of inadequate soil and crop micronutrient contents, inadequate intake, malabsorption and increased requirements are unknown. Malabsorption may compromise attempts to improve micronutrient status, but with the exception of zinc there is currently little evidence to confirm that malabsorption contributes to micronutrient deficiency. Much further research is required to understand the role of malabsorption in hidden hunger, especially in very disadvantaged populations where these deficiencies are most prevalent.

Adaptation of the small intestine to microbial enteropathogens in Zambian children with stunting

Environmental enteropathy is a major contributor to growth faltering in millions of children in Africa and South Asia. We carried out a longitudinal, observational and interventional study in Lusaka, Zambia, of 297 children with stunting (aged 2–17 months at recruitment) and 46 control children who had good growth (aged 1–5 months at recruitment). Control children contributed data only at baseline. Children were provided with nutritional supplementation of daily cornmeal-soy blend, an egg and a micronutrient sprinkle, and were followed up to 24 months of age. Children whose growth did not improve over 4–6 months of nutritional supplementation were classified as having non-responsive stunting. We monitored microbial translocation from the gut lumen to the bloodstream in the cohort with non-responsive stunting (n = 108) by measuring circulating lipopolysaccharide (LPS), LPS-binding protein and soluble CD14 at baseline and when non-response was declared. We found that microbial translocation decreased with increasing age, such that LPS declined in 81 (75%) of 108 children with non-responsive stunting, despite sustained pathogen pressure and ongoing intestinal epithelial damage. We used confocal laser endomicroscopy and found that mucosal leakiness also declined with age. However, expression of brush border enzyme, nutrient transporter and mucosal barrier genes in intestinal biopsies did not change with age or correlate with biomarkers of microbial translocation. We propose that environmental enteropathy arises through adaptation to pathogen-mediated epithelial damage. Although environmental enteropathy reduces microbial translocation, it does so at the cost of impaired growth. The reduced epithelial surface area imposed by villus blunting may explain these findings.

Safety and Ethics in Endoscopic Studies in Children: Evidence From the BEECH Study in Zambia

Background Environmental enteropathy is an example of a poorly-understood intestinal disorder affecting millions of children worldwide, characterized by malabsorption and stunting. Although there is increasing interest in non-invasive means of assessing intestinal structure and function, the potential value of intestinal biopsy for histology, immunostaining, RNA sequencing and epigenetic work means that endoscopic biopsy remains extremely valuable. We here report our experience in the BEECH (Biomarkers of Environmental Enteropathy in CHildren) study of stunting in Zambia, in the belief that it may help address the knowledge gap regarding the safety of endoscopic biopsy in vulnerable young children. Methods We report our experience of safety in 119 children undergoing endoscopic biopsy in the BEECH study in Lusaka Children’s Hospital, Lusaka, and discuss ethical considerations in this light. Results Upper gastrointestinal endoscopy was performed on children with stunting (length-for-age z score -2 or less) not responsive to nutritional interventions. Conscious sedation was provided by anaesthetists. Of 119 children, 5 (4%) developed transient desaturation, but no serious adverse events were experienced; no clinical, demographic or anaesthetic characteristics were identified as predictive of desaturation. Two children derived clinically useful information from the endoscopy, one life-saving. Of 105 lactase tests, 59 (54%) showed hypolactasia. Discussion Children with stunting underwent endoscopy safely, and some derived clinical benefit. Safety and the possibility of clinical benefit are usually felt to be preconditions for the ethical justification for endoscopy for research in children, and we believe that these conditions were met in this study.

Formula prebiotik berbasis pangan lokal dapat meningkatkan z-skor PB/U pada anak stunting

Stunting occurs in a chronic small intestinal inflammation syndrome, called pediatric environmental enteropathy, resulting in increased intestinal permeability and the entry of immune cells into the intestinal epithelium. The purpose of this study was to study the types of prebiotic food formulas and their effects on LAZ in stunting children. This type of research is a quasi-experimental design with a pretest-posttest control group design. Held in Sei Ambawang District, Kubu Raya Regency 2019. Samples of stunting children aged 12-24 months were 40 children. The sampling technique was used for purposive sampling. The intervention given is prebiotic formula food given for 30 days. Data processing for LAZ, nutrient intake, and t-independent test statistical analysis each using WHO Anthro 2005, Nutri survey, and SPSS software. The results showed there were differences in LAZ before and after in the group given MF-2 food formula in stunting children (p= 0,034), whereas in the group who received MF-1 showed no difference (p= 0,114). Based on LAZ differences between the two groups after the intervention with independent t-test did not show significant differences (p > 0,05). In conclusion, MF-1 and MF-2 did not differ in increasing LAZ in stunting children, but each group showed MF-2 had an effect on increasing LAZ.