Study of Environmental Enteropathy and Malnutrition (SEEM) in Pakistan: protocols for biopsy based biomarker discovery and validation.

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 < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority. METHODS: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 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 .

A shared group of bacterial taxa in the duodenal microbiota of undernourished Pakistani children with environmental enteric dysfunction.

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.

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) <-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.

Impact of diarrhoea and acute respiratory infection on environmental enteric dysfunction and growth of malnourished children in Pakistan: a longitudinal cohort study.

Background: Diarrhoea and acute respiratory infections (ARI) are assumed to be major drivers of growth and likely contribute to environmental enteric dysfunction (EED), which is a precursor to childhood malnutrition. In the present study, we checked the correlation between diarrhoeal/ARI burden and EED using a novel duodenal histological index. Methods: Between November 2017 and July 2019, a total of 365 infants with weight-for-height Z scores (WHZ score) of <-2 were enrolled, and 51 infants with WHZ scores of >0 and height-for-age Z scores (HAZ scores) of >-1 were selected as age-matched healthy controls. Morbidity was assessed weekly and categorised as the total number of days with diarrhoea and acute respiratory infection (ARI) from enrolment until two years of age and was further divided into four quartiles in ascending order. Findings: The HAZ declined until two years of age regardless of morbidity burden, and WHZ and weight-for-age Z scores (WAZ scores) were at their lowest at six months. Sixty-three subjects who had a WHZ score <-2 and failed to respond to nutritional and educational interventions were further selected at 15 months to investigate their EED histological scores with endoscopy further. EED histological scores of the subjects were higher with increasing diarrhoeal frequency yet remained statistically insignificant (p = 0.810). Interpretation: There was not a clear correlation between diarrhoea and ARI frequency with growth faltering, however, children with the highest frequency of diarrhoea had the highest EED histological scores and growth faltering. Funding: Bill and Melinda Gates Foundation and The National Institutes of 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.

Clinically relevant antibiotic resistance genes are linked to a limited set of taxa within gut microbiome worldwide.

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.

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' 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.

Impact of enteropathogens on faltering growth in a resource-limited setting.

Introduction: Environmental 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. Methods: A 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. Results: Giardia, 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). Conclusion: Protozoal 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.

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.

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 < 0.010) and diarrhea (r = 0.228, p < 0.001) as well as participants living closer to Maternal Health Centers (MHCs): ARI (r = 0.185, p < 0.002) and diarrhea (r = 0.223, p < 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.

Gut integrity and duodenal enteropathogen burden in undernourished children with environmental enteric dysfunction.

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.