Potential determinants of low circulating glucagon-like peptide 2 concentrations in Zambian children with non-responsive stunting.

NEW FINDINGS: What is the central question of this study? Non-responsive stunting is characterised by a progressive decline of circulating glucagon-like peptide 2: what are the possible causes of this decline? What is the main finding and its importance? In contrast with the established loss of Paneth and goblet cells in environmental enteropathy, there was no evidence of a parallel loss of enteroendocrine cells as seen by positive tissue staining for chromogranin A. Transcriptomic and genomic analyses showed evidence of genetic transcripts that could account for some of the variability seen in circulating glucagon-like peptide 2 values. ABSTRACT: Nutrient sensing determines digestive and hormonal responses following nutrient ingestion. We have previously reported decreased levels of glucagon-like peptide 2 (GLP-2) in children with stunting. Here we demonstrate the presence of enteroendocrine cells in stunted children and explore potential pathways that may be involved in reduced circulating levels of GLP-2. At the time of performing diagnostic endoscopies for non-responsive stunted children, intestinal biopsies were collected for immunofluorescence staining of enteroendocrine cells and transcriptomic analysis. Circulating levels of GLP-2 were also measured and correlated with transcriptomic data. An exploratory genome-wide association study (GWAS) was conducted on DNA samples (n = 158) to assess genetic contribution to GLP-2 variability. Intestinal tissue sections collected from non-responsive stunted children stained positive for chromogranin A (88/89), alongside G-protein-coupled receptors G-protein receptor 119 (75/87), free fatty acid receptor 3 (76/89) and taste 1 receptor 1 (39/45). Transcriptomic analysis found three pathways correlated with circulating GLP-2: sugar metabolism, epithelial transport, and barrier function, which likely reflect downstream events following receptor-ligand interaction. GWAS analysis revealed potential genetic contributions to GLP-2 half-life and receptor binding. Enteroendocrine cell loss was not identified in stunted Zambian children as has been observed for goblet and Paneth cells. Transcriptomic analysis suggests that GLP-2 has pleiotrophic actions on the intestinal mucosa in malnutrition, but further work is needed to dissect pathways leading to perturbations in nutrient sensing.

Reduced Fractional Absorption of Zinc in Children With Environmental Enteropathy in Zambia.

OBJECTIVES: We measured fractional absorption of zinc (FAZ) in children with environmental enteropathy (EE) and carried out transcriptomic analysis of biopsies from these children in order to compare FAZ to histology of intestinal biopsies, expression of zinc transporter genes, and biomarkers of enteropathy. METHODS: Fractional absorption of a standardized aqueous dose of zinc was measured by a dual isotope ratio technique in a cohort of children ages between 9 and 24 months in Lusaka, Zambia, who all had non-responsive stunting. Gene expression analysis was carried out on biopsies through RNA sequencing using an Illumina HiSeq2000 platform. RESULTS: All 33 children had histological features of environmental enteropathy and plasma zinc concentrations below the lower limit of normal. Measured FAZ ranged from 0.18 to 0.93; all values >0.55 were observed in girls. FAZ was negatively correlated with faecal myeloperoxidase (MPO) (ρ = -0.51, n = 17; P = 0.04) and faecal calprotectin (ρ = -0.50, n = 16; P = 0.05), but not blood biomarkers. Of 41 genes with known roles in zinc metabolism, only three metallothionein genes were significantly correlated with FAZ. CONCLUSIONS: Zinc homeostasis is impaired in children with environmental enteropathy, and was inversely correlated with mucosal inflammation. Reduced FAZ without specific changes in expression of most zinc transporter genes could be explained by reduced absorptive surface area due to villus/microvillus atrophy.

Histo-Blood Group Antigens, Enteropathogen Carriage and Environmental Enteropathy in Stunted Zambian Children.

OBJECTIVES: Stunting, the most common form of childhood undernutrition, is associated with environmental enteropathy (EE). Enteric infections are believed to play a role in the pathophysiology of EE and stunting though the exact mechanism remains undetermined. The FUT2 (secretor) and FUT3 (Lewis) genes have been shown to be associated with some symptomatic enteric infections in both children and adults. These genes are responsible for the presence of histo-blood group antigens (HBGAs) in various secretions and epithelial surfaces.We evaluated whether the secretor and Lewis status influences asymptomatic enteric infections and thus EE severity on duodenal biopsies of stunted children. METHODS: In this case-control study, we used saliva samples to determine the secretor and Lewis status of stunted children (cases, n = 113) enrolled in a nutritional rehabilitation program and from their well-nourished counterparts (controls, n = 42). Where available, saliva was also collected from the mothers. Baseline stool samples were used to detect asymptomatic enteropathogen carriage. Duodenal biopsies were collected from a subgroup of stunted children (n = 77) who had an upper gastrointestinal endoscopy done as part of the evaluation process for their non-response to nutritional therapy. RESULTS: The proportion of secretors was similar between the cases and the controls (82% vs 81%, P = 0.81). The stunted children had significantly higher rates of carrying multiple enteropathogens, but this was not associated with their secretor status nor that of their mothers. The secretor status was also not associated with mucosal morphometry of duodenal biopsies. CONCLUSION: This case-control analysis in Zambian children does not support the hypothesis that fucosylation status determines asymptomatic enteropathogen carriage in stunting.

Recovery of children following hospitalisation for complicated severe acute malnutrition.

Nutritional recovery and hospital readmission following inpatient management of complicated severe acute malnutrition (SAM) are poorly characterised. We aimed to ascertain patterns and factors associated with hospital readmission, nutritional recovery and morbidity, in children discharged from hospital following management of complicated SAM in Zambia and Zimbabwe over 52-weeks posthospitalization. Multivariable Fine-Gray subdistribution hazard models, with death and loss to follow-up as competing risks, were used to identify factors associated with hospital readmission; negative binomial regression to assess time to hospitalisation and ordinal logistic regression to model factors associated with nutritional recovery. A total of 649 children (53% male, median age 18.2 months) were discharged to continue community nutritional rehabilitation. All-cause hospital readmission was 15.4% (95% CI 12.7, 18.6) over 52 weeks. Independent risk factors for time to readmission were cerebral palsy (adjusted subhazard ratio (aSHR): 2.96, 95% CI 1.56, 5.61) and nonoedematous SAM (aSHR: 1.64, 95%CI 1.03, 2.64). Unit increases in height-for-age Z-score (HAZ) (aSHR: 0.82, 95% CI 0.71, 0.95) and enrolment in Zambia (aSHR: 0.52, 95% CI 0.28, 0.97) were associated with reduced subhazard of time to readmission. Young age, SAM at discharge, nonoedematous SAM and cerebral palsy were associated with poor nutritional recovery throughout follow-up. Collectively, nonoedematous SAM, ongoing SAM at discharge, cerebral palsy and low HAZ are independent risk factors for readmission and poor nutritional recovery following complicated SAM. Children with these high-risk features should be prioritised for additional convalescent care to improve long-term outcomes.

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.

Glucagon-like Peptide 2 Concentrations Vary in Zambian Children During Diarrhoea, in Malnutrition and Seasonally.

OBJECTIVES: Glucagon-like peptide 2 (GLP-2) is a 33 amino acid peptide hormone released from enteroendocrine L-cells following nutrient ingestion. It has been shown to exert trophic effects on the gut. We set out to measure GLP-2 concentrations in blood in children with diarrhoea and malnutrition. METHODS: GLP-2 levels were measured in blood samples collected from 5 different groups of children (n = 324) at different time points: those with acute diarrhoea, during illness and 3 weeks after recovery; persistent diarrhoea and severe acute malnutrition; controls contemporaneous for diarrhoea; stunted children from the community; and controls contemporaneous for the stunted children. Stool biomarkers and pathogen analysis were carried out on the children with stunting. RESULTS: GLP-2 concentrations were higher during acute diarrhoea (median 3.1 ng/mL, interquartile range 2.1, 4.4) than on recovery (median 1.8, interquartile range 1.4, 3.1; P = 0.001), but were not elevated in children with persistent diarrhoea and severe acute malnutrition. In stunted children, there was a progressive decline in GLP-2 levels from 3.2 ng/mL (1.9, 4.9) to 1.0 (0.0, 2.0; P < 0.001) as the children became more stunted. Measures of seasonality (rainfall, temperature,Food Price Index, and Shiga toxin-producing Escherichia coli) were found to be significantly associated with GLP-2 concentrations in multivariable analysis. We also found a correlation between stool inflammatory biomarkers and GLP-2. CONCLUSIONS: In diarrhoea, GLP-2 levels increased in acute but not persistent diarrhoea. Malnutrition was associated with reduced concentrations. GLP-2 displayed seasonal variation consistent with variations in nutrient availability.

Children infected by human herpesvirus 6B with febrile seizures are more likely to develop febrile status epilepticus: A case-control study in a referral hospital in Zambia.

BACKGROUND: Human herpesvirus 6B (HHV-6B) is the causative agent of Roseola infantum, and has also been suggested to play a role in the pathogenesis of febrile seizures in young children, a percentage of whom go on to develop febrile status epilepticus (FSE), but the existing data is conflicting and inconclusive. HHV-6A is a distinct species, rarely detected in most parts of the world, but prior studies suggest a higher prevalence in febrile African children. We describe a case-control study comparing the frequency of HHV-6A and/or HHV-6B infections in children with febrile seizures (including FSE) and a control group of febrile children without seizures. METHODS: We recruited children aged 6 to 60 months admitted with a febrile illness with (cases) or without (controls) seizures presenting within 48 hours of commencement of fever. Three milliliters of whole blood was centrifuged and plasma stored at -80°C for pooled screening for HHV-6B and HHV-6A by Taqman real-time polymerase chain reaction. RESULTS: 102 cases and 95 controls were recruited. The prevalence of HHV-6B DNA detection did not differ significantly between cases (5.8% (6/102)) and controls (10.5% (10/95)) but HHV-6B infection was associated with FSE (OR, 15; 95% CI, [1.99-120]; P= 0.009). HHV-6A was not detected. CONCLUSION: Prevalence of HHV-6B was similar among cases and controls. Within the FS group, HHV-6B infection was associated with FSE, suggesting HHV-6B infections could play a role in the pathogenesis of FSE.

Anthropometry relationship with duodenal histologic features of children with environmental enteric dysfunction: a multicenter cross-sectional study.

BACKGROUND: Environmental enteric dysfunction (EED) is a precursor of growth faltering in children living in impoverished conditions who are frequently exposed to environmental toxins and enteropathogens, leading to small bowel inflammatory, malabsorptive, and permeability derangements and low-grade chronic systemic inflammation. OBJECTIVES: We explored the association between anthropometrics and duodenal histologic features of EED among children from 3 lower middle-income country centers. METHODS: In this cross-sectional study, Pakistani children (n = 63) with wasting, Bangladesh children (n = 116) with stunting or at risk for stunting (height-for-age Z score [HAZ] <-1 but ≥-2), and Zambian children (n = 108) with wasting or stunting received nutritional intervention. Children with anthropometric status refractory to intervention underwent endoscopy. Linear regression models included anthropometric around endoscopy, scores of histology parameters, and a global index score of EED-the total score percent-5 (TSP-5). Multivariable models were adjusted for center, age, sex, and histology slide quality. RESULTS: Intersite variation was observed while exploring the association between anthropometrics and the TSP-5; for example, Pakistani children had the worst HAZ, yet their median TSP-5 score was lower than that of the other 2 centers. Even within each site, no overall pattern of higher TSP-5 score was observed with worsening HAZ. During univariate analysis, TSP-5 (coefficient: 0.01; 95% confidence interval [CI]: 0, 0.02), goblet cell depletion (coefficient: 0.22; 95% CI: 0.06, 0.37), and Paneth cell depletion (coefficient: 0.14; 95% CI: 0.01, 0.27) were associated with HAZ scores; however, they lost statistical significance in the multivariable models, with study center most strongly confounding the relationships seen in univariate models between anthropometry and histology. CONCLUSIONS: This study contributes a crucial negative finding that duodenal morphological features did not associate with anthropometric phenotypes; hence, anthropometric measurements may not be a suitable outcome measure for use in EED trials. Trial outcomes may need to be defined by combining the functional and structural elements of the gut to monitor EED.

Biomarker relationships with small bowel histopathology among malnourished children with environmental enteric dysfunction in a multicountry cohort study.

BACKGROUND: Validated biomarkers could catalyze environmental enteric dysfunction (EED) research. OBJECTIVES: Leveraging an EED histology scoring system, this multicountry analysis examined biomarker associations with duodenal histology features among children with EED. We also examined differences in 2-h compared with 1-h urine collections in the lactulose rhamnose (LR) dual sugar test. METHODS: Three cohorts of undernourished children unresponsive to nutrition intervention underwent esophagogastroduodenoscopy and duodenal biopsies. Histopathology scores were compared to fecal calprotectin (CAL), myeloperoxidase (MPO), neopterin (NEO), and urinary LR ratio and lactulose percentage recovery. Log-transformed biomarkers were used in linear regressions adjusted for age, center, and sample collection-biopsy time interval in multivariable models. RESULTS: Data on >1 biomarker were available for 120 Bangladeshi (CAL, MPO, NEO, and LR), 63 Pakistani (MPO, NEO, and LR), and 63 Zambian children (CAL). Median age at endoscopy was similar (19 mo) across centers. Median sample collection prior to endoscopy was consistent with each center's study design: 2 wk in Bangladesh (urine and stool) and Zambia (stool), and 6 (urine) and 11 (stool) mo in Pakistan. In multivariable models, intraepithelial lymphocytes were associated with CAL (exponentiated [exp.] coefficient: 1.19; 95% confidence interval [CI]: 1, 1.41), intramucosal Brunner's glands with MPO (exp. coefficient: 1.33; 95% CI: 1.05, 1.69) and NEO (exp. coefficient: 1.37; 95% CI: 1.1, 1.7), and chronic inflammation with NEO (exp. coefficient: 1.61; 95% CI: 1.17, 2.17). Intraepithelial lymphocytes were associated with lactulose % recovery (exp. coefficient: 1.22; 95% CI: 1.05, 1.41). LR recovery was substantially lower in 1-h collections than in 2-h collections. CONCLUSIONS: Four commonly used markers of enteric dysfunction were associated with specific histologic features. One-hour urine collection may be insufficient to reflect small bowel permeability in LR testing. While acknowledging the challenges with obtaining relevant tissue, these findings form the basis for further EED biomarker validation research.

Enteric pathogens relationship with small bowel histologic features of environmental enteric dysfunction in a multicountry cohort study.

BACKGROUND: Environmental Enteric Dysfunction (EED) is an acquired disorder of asymptomatic altered gut function, the etiology of which is unknown. EED is postulated to be a major contributor to growth faltering in early childhood in regions where early-life enteropathogenic carriage is prevalent. Few studies have examined the critical organ (the upper small bowel) with enteropathogens in the evolution of small bowel disease. OBJECTIVES: The objective of this study was to determine if fecal enteropathogenic detection predicts subsequent EED histology. METHODS: Fecal samples were obtained from undernourished children aged <2 y without diarrhea enrolled in 3 cohort studies, who failed nutritional intervention and subsequently underwent endoscopy. Duodenal biopsies from 245 (Bangladesh n = 120, Pakistan n = 57, and Zambia n = 68) children were scored using a semiquantitative histologic grading protocol. Thirteen enteropathogens were sought in common across the 3 centers using TaqMan array cards (TAC) (Bangladesh and Pakistan) and the Luminex platform (Zambia). An additional 18 pathogens and 32 virulence loci were sought by TAC and included in sensitivity analyses restricted to TAC data. RESULTS: Multivariable linear regressions adjusting for study center, age at stool collection, and stool-to-biopsy interval demonstrated the following: 1) an association of norovirus and Shigella detection with subsequent enterocyte injury [ß 0.2 (95% CI: 0.1, 0.3); P = 0.002 and ß 0.2 (95% CI: 0.0, 0.3); P = 0.008, respectively], 2) association of Campylobacter with intraepithelial lymphocytes [ß 0.2 (95% CI: 0.0, 0.4); P = 0.046], and 3) association of Campylobacter and enterotoxigenic Escherichia coli with a summative EED histopathology index score [ß 4.2 (95% CI: 0.8, 7.7); P = 0.017 and ß 3.9 (95% CI: 0.5, 7.3); P = 0.027, respectively]. All but 2 of these associations (Shigella-enterocyte injury and Campylobacter-index score) were also demonstrated in TAC-only sensitivity analyses, which identified additional associations between other pathogens, pathogen burden, or virulence loci primarily with the same histologic parameters. CONCLUSIONS: The detection of some enteropathogens in asymptomatic infections is associated with subsequent EED histopathology. These novel findings offer a basis for future EED etiology and pathogenesis studies.

NAD+ precursors and bile acid sequestration treat preclinical refractory environmental enteric dysfunction.

Environmental enteric dysfunction (EED) is a diffuse small bowel disorder associated with poor growth, inadequate responses to oral vaccines, and nutrient malabsorption in millions of children worldwide. We identify loss of the small intestinal Paneth and goblet cells that are critical for innate immunity, reduced villous height, increased bile acids, and dysregulated nicotinamide adenine dinucleotide (NAD+) synthesis signaling as potential mechanisms underlying EED and which also correlated with diminished length-for-age z score. Isocaloric low-protein diet (LPD) consumption in mice recapitulated EED histopathology and transcriptomic changes in a microbiota-independent manner, as well as increases in serum and fecal bile acids. Children with refractory EED harbor single-nucleotide polymorphisms in key enzymes involved in NAD+ synthesis. In mice, deletion of Nampt, the gene encoding the rate-limiting enzyme in the NAD+ salvage pathway, from intestinal epithelium also reduced Paneth cell function, a deficiency that was further aggravated by LPD. Separate supplementation with NAD+ precursors or bile acid sequestrant partially restored LPD-associated Paneth cell defects and, when combined, fully restored all histopathology defects in LPD-fed mice. Therapeutic regimens that increase protein and NAD+ contents while reducing excessive bile acids may benefit children with refractory EED.

Inflammation and epithelial repair predict mortality, hospital readmission, and growth recovery in complicated severe acute malnutrition.

Severe acute malnutrition (SAM) is the most high-risk form of undernutrition, particularly when children require hospitalization for complications. Complicated SAM is a multisystem disease with high inpatient and postdischarge mortality, especially in children with comorbidities such as HIV; however, the underlying pathogenesis of complicated SAM is poorly understood. Targeted multiplex biomarker analysis in children hospitalized with SAM (n = 264) was conducted on plasma samples, and inflammatory markers were assessed on stool samples taken at recruitment, discharge, and 12 to 24 and 48 weeks after discharge from three hospitals in Zimbabwe and Zambia. Compared with adequately nourished controls (n = 173), we found that at baseline, complicated SAM was characterized by systemic, endothelial, and intestinal inflammation, which was exacerbated by HIV infection. This persisted over 48 weeks despite nutritional recovery and was associated with children's outcomes. Baseline plasma concentrations of vascular endothelial growth factor, glucagon-like peptide-2, and intestinal fatty acid-binding protein were independently associated with lower mortality or hospital readmission over the following 48 weeks. Following principal components analysis of baseline biomarkers, higher scores of a component representing growth factors was associated with greater weight-for-height z score recovery and lower mortality or hospital readmission over the 48 weeks. Conversely, components representing higher gut and systemic inflammation were associated with higher mortality or hospital readmission. These findings highlight the interplay between inflammation, which damages tissues, and growth factors, which mediate endothelial and epithelial regeneration, and support further studies investigating interventions to reduce inflammation and promote epithelial repair as an approach to reducing mortality and improving nutritional recovery.

Malnutrition enteropathy in Zambian and Zimbabwean children with severe acute malnutrition: A multi-arm randomized phase II trial.

Malnutrition underlies almost half of all child deaths globally. Severe Acute Malnutrition (SAM) carries unacceptable mortality, particularly if accompanied by infection or medical complications, including enteropathy. We evaluated four interventions for malnutrition enteropathy in a multi-centre phase II multi-arm trial in Zambia and Zimbabwe and completed in 2021. The purpose of this trial was to identify therapies which could be taken forward into phase III trials. Children of either sex were eligible for inclusion if aged 6-59 months and hospitalised with SAM (using WHO definitions: WLZ <-3, and/or MUAC <11.5 cm, and/or bilateral pedal oedema), with written, informed consent from the primary caregiver. We randomised 125 children hospitalised with complicated SAM to 14 days treatment with (i) bovine colostrum (n = 25), (ii) N-acetyl glucosamine (n = 24), (iii) subcutaneous teduglutide (n = 26), (iv) budesonide (n = 25) or (v) standard care only (n = 25). The primary endpoint was a composite of faecal biomarkers (myeloperoxidase, neopterin, α1-antitrypsin). Laboratory assessments, but not treatments, were blinded. Per-protocol analysis used ANCOVA, adjusted for baseline biomarker value, sex, oedema, HIV status, diarrhoea, weight-for-length Z-score, and study site, with pre-specified significance of P < 0.10. Of 143 children screened, 125 were randomised. Teduglutide reduced the primary endpoint of biomarkers of mucosal damage (effect size -0.89 (90% CI: -1.69,-0.10) P = 0.07), while colostrum (-0.58 (-1.4, 0.23) P = 0.24), N-acetyl glucosamine (-0.20 (-1.01, 0.60) P = 0.67), and budesonide (-0.50 (-1.33, 0.33) P = 0.32) had no significant effect. All interventions proved safe. This work suggests that treatment of enteropathy may be beneficial in children with complicated malnutrition. The trial was registered at ClinicalTrials.gov with the identifier NCT03716115.

Genetic variation in environmental enteropathy and stunting in Zambian children: A pilot genome wide association study using the H3Africa chip.

PURPOSE: Stunting is known to be heavily influenced by environmental factors, so the genetic contribution has received little attention. Here we report an exploration of genetic influences in stunted Zambian children with environmental enteropathy. METHOD: Children with stunting (LAZ < -2) were enrolled and given nutritional therapy. Those that were non-responsive to therapy were designated as cases, and children with good growth (LAZ > -1) from the same community as controls. Blood and stool samples were taken to measure biomarkers of intestinal inflammation, epithelial damage, and microbial translocation. Single nucleotide polymorphism array genotyping was carried out on saliva samples using the H3Africa consortium array. RESULTS: Genome wide associations were analysed in 117 cases and 41 controls. While no significant associations with stunting were observed at P<5x10-8, likely due to the small sample size, interesting associations were observed at lower thresholds. SNPs associated with stunting were in genomic regions known to modulate neuronal differentiation and fatty acid biosynthesis. SNPs associated with increased microbial translocation were associated with non-integrin membrane ECM interactions, tight junctions, hemostasis, and G-alpha signalling events. SNPs associated with increased inflammation were associated with, ECM interactions, purine metabolism, axon guidance, and cell motility. SNPs negatively associated with inflammation overlapped genes involved in semaphoring interactions. We explored the existing coeliac disease risk HLA genotypes and found present: DQ2.5 (7.5%), DQ8 (3.5%) and DQ2.2 (3.8%); however, no children were positive for coeliac antibodies. We detected HLA-DRB:1301 and HLA-C:1802 with high odds ratios and P<0.05 in stunted children compared to controls. CONCLUSION: Genetic variations associated with stunting and the enteropathy underlying it, include variants associated with multiple pathways relating to gene expression, glycosylation, nerve signalling, and sensing of the nutritional and microbiological milieu.

Risk factors for inpatient mortality among children with severe acute malnutrition in Zimbabwe and Zambia.

BACKGROUND/OBJECTIVES: Malnutrition underlies 45% of deaths in children under-5 years annually. Children hospitalised with complicated severe acute malnutrition (SAM) have unacceptably high mortality. We aimed to identify variables from early hospital admission (baseline factors) independently associated with inpatient mortality in this cohort to identify those most at risk. SUBJECTS/METHODS: Observational study of 745 children aged 0-59 months admitted with complicated SAM at three hospitals in Zimbabwe/Zambia. Children underwent anthropometry and clinical assessment by a study physician within 72 h of enrolment, and caregivers provided sociodemographic data. Children were followed-up daily until discharge/death. A multivariable survival analysis identified the baseline factors independently associated with mortality. RESULTS: 70/745 (9.4%) children died in hospital. Age between 6-23 months [aHR 6.53, 95%CI 2.24-19.02], higher mid-upper arm circumference [aHR 0.73, 95%CI 0.59-0.89], presence of oedema [aHR 2.22, 95%CI 1.23-4.05], shock [aHR 8.18, 95%CI 3.79-17.65], sepsis [aHR 3.13, 95%CI 1.44-6.80], persistent diarrhoea [aHR 2.27, 95%CI 1.18-4.37], lack of a toilet at home [aHR 4.35, 95%CI 1.65-11.47], and recruitment at one Harare site [aHR 0.38, 95%CI 0.18-0.83] were all independently associated with inpatient mortality. Oedematous children had a significantly higher birthweight [2987 g vs 2757 g, p < 0.001] than those without oedema; higher birthweight was weakly associated with mortality [aHR 1.50 95%CI 0.97-2.31]. CONCLUSIONS: Children with oedema, low MUAC, baseline infections, shock and lack of home sanitation had a significantly increased risk of inpatient mortality following hospitalisation for complicated SAM. Children with high-risk features may require additional care. A better understanding of the pathophysiology of SAM is needed to identify adjunctive interventions.

Severe acute malnutrition promotes bacterial binding over proinflammatory cytokine secretion by circulating innate immune cells.

Children with severe acute malnutrition (SAM) have high infectious mortality and morbidity, implicating defects in their immune defenses. We hypothesized that circulating innate immune cells from children (0 to 59 months) hospitalized with SAM in Zambia and Zimbabwe (n = 141) have distinct capacity to respond to bacteria relative to adequately nourished healthy controls (n = 92). SAM inpatients had higher neutrophil and monocyte Escherichia coli binding capacity but lower monocyte activation and proinflammatory mediator secretion in response to lipopolysaccharide or heat-killed Salmonella typhimurium than controls. Among SAM cases, wasting severity was negatively associated with cytokine secretion, children with HIV had lower monocyte activation, and the youngest children released the least myeloperoxidase upon stimulation. Inpatient bacterial binding capacity and monocyte activation were associated with higher odds of persistent SAM at discharge, a risk factor for subsequent mortality. Thus, SAM shifts innate immune cell function, favoring bacterial containment over proinflammatory activation, which may contribute to health deficits after discharge.

Epithelial Abnormalities in the Small Intestine of Zambian Children With Stunting.

Background: Environmental enteropathy (EE) contributes to impaired linear growth (stunting), in millions of children worldwide. We have previously reported that confocal laser endomicroscopy (CLE) shows fluorescein leaking from blood to gut lumen in vivo in adults and children with EE. We set out to identify epithelial lesions which might explain this phenomenon in Zambian children with stunting non-responsive to nutritional support. Methods: We performed confocal laser endomicroscopy (CLE) in 75 children and collected intestinal biopsies for histology in 91 children. CLE videos were evaluated, employing the Watson score to determine severity of leakiness. Morphometry was carried out on well-orientated mucosa and 3 biopsies were examined by electron microscopy. Results: Confocal laser endomicroscopy demonstrated substantial leakage from circulation to gut lumen in 73 (97%) children. Histology consistently showed characteristic changes of EE: villus blunting, lamina propria and epithelial inflammation, and depletion of secretory cells (Paneth cells and goblet cells). Epithelial abnormalities included marked variability in epithelial height, disorganised and shortened microvilli, dilated intercellular spaces, pseudostratification, formation of synechiae between epithelium on adjacent villi, crypt destruction, and abundant destructive lesions which may correspond to the microerosions identified on CLE. Conclusion: Epithelial abnormalities were almost universal in Zambian children with non-responsive stunting, including epithelial microerosions, cell-cell adhesion anomalies, and defects in secretory cells which may all contribute to impairment of mucosal barrier function and microbial translocation.

Selenium status in adults and children in Lusaka, Zambia.

Background: Selenium (Se) is a trace element found in many foodstuffs and critical for antioxidant and immune functions. Widespread Se deficiency has been noted in populations of some sub-Saharan African countries including Ethiopia and Malawi. As a first step towards developing a fuller understanding of problems with the availability of Se in the diet in Lusaka province, Zambia, we measured plasma Se in adults and children in this geographic area. Methods: Total plasma Se was measured using inductively coupled plasma optical emission spectrometry (ICP-OES) in several groups of adults recruited to various pre-existing studies, including those of high and low socioeconomic status (SES) and pregnant women, and children with a range of nutritional states (healthy, stunted or wasted). Results: A total of 660 plasma samples from 391 adults and 269 children were included. Adults had a median plasma Se concentration of 0.27 µmol/l (IQR 0.14-0.43). Concentrations consistent with deficiency (<0.63 µmol/l) were found in 83% of adults. Low SES was associated with low plasma Se among adults, [OR 0.1; 95% CI 0.1-0.3, p < 0.0001]. Among the children, 24% had plasma Se less than 0.41 µmol/l. There was a statistically significant positive correlation between plasma Se and age among children, Spearman's rho 0.47, p < 0.0001. Conclusions: These data suggest that Se deficiency is widespread in Lusaka province and could in part be related to socio-economic status. Supplementation or agronomic biofortification may therefore be needed.

13C-sucrose breath test for the non-invasive assessment of environmental enteropathy in Zambian adults.

Objectives: Environmental enteropathy (EE) is a subclinical disorder highly prevalent in tropical and disadvantaged populations and is thought to play a role in growth faltering in children, poor responses to oral vaccines, and micronutrient deficiencies. This study aims to evaluate the potential of a non-invasive breath test based on stable isotopes for evaluation of impaired digestion and absorption of sucrose in EE. Methods: We optimized a 13C-sucrose breath test (13C-SBT) in 19 young adults in Glasgow, United Kingdom. In a further experiment (in 18 adults) we validated the 13C-SBT using Reducose, an intestinal glucosidase inhibitor. We then compared the 13C-SBT to intestinal mucosal morphometry, immunostaining for sucrose-isomaltase (SI) expression, and SI activity in 24 Zambian adults with EE. Results: Fully labeled sucrose (0.3 mg/kg) provided clear breath enrichment signals over 2-3 h in both British and Zambian adults, more than fivefold higher than naturally enriched sucrose. Reducose dramatically impaired 13C-sucrose digestion, reducing 4 h 13CO2 breath recovery by > 50%. Duodenal biopsies in Zambian adults confirmed the presence of EE, and SI immunostaining was present in 16/24 adults. The kinetics of 13CO2 evolution were consistently faster in participants with detectable SI immunostaining. Although sucrase activity was strongly correlated with villus height (r = 0.72; P < 0.05) after adjustment for age, sex and body mass index, there were no correlations between 13C-SBT and villus height or measured sucrase activity in pinch biopsies. Conclusion: A 13C-SBT was developed which was easy to perform, generated clear enrichment of 13CO2 in breath samples, and clearly reports sucrase activity. Further work is needed to validate it and understand its applications in evaluating EE.

Colostrum Therapy for Human Gastrointestinal Health and Disease.

There is increasing awareness that a broad range of gastrointestinal diseases, and some systemic diseases, are characterized by failure of the mucosal barrier. Bovine colostrum is a complex biological fluid replete with growth factors, nutrients, hormones, and paracrine factors which have a range of properties likely to contribute to mucosal healing in a wide range of infective, inflammatory, and injury conditions. In this review, we describe the anatomy and physiology of the intestinal barrier and how it may fail. We survey selected diseases in which disordered barrier function contributes to disease pathogenesis or progression, and review the evidence for or against efficacy of bovine colostrum in management. These disorders include enteropathy due to non-steroidal anti-inflammatory drugs (NSAIDs), inflammatory bowel disease (IBD), necrotizing enterocolitis, infectious diarrhea, intestinal failure, and damage due to cancer therapy. In animal models, bovine colostrum benefits NSAID enteropathy, IBD, and intestinal failure. In human trials, there is substantial evidence of efficacy of bovine colostrum in inflammatory bowel disease and in infectious diarrhea. Given the robust scientific rationale for using bovine colostrum as a promoter of mucosal healing, further work is needed to define its role in therapy.