Effects of Zinc Supplementation on Metabolomic Profiles in Tanzanian Infants: A Randomized Trial.

BACKGROUND: Provision of zinc supplementation to young children has been associated with reduced infectious morbidity and better growth outcomes. However, the metabolic pathways underlying these outcomes are unclear, and metabolomic data from humans undergoing zinc supplementation, particularly infants, are generally lacking. OBJECTIVES: This study aimed to examine the effect of zinc supplementation on metabolic profiles in Tanzanian infants aged 6 wk and 6 mo. METHODS: Blood samples were collected at age 6 wk and 6 mo from 50 Tanzanian infants who were enrolled in a randomized placebo-controlled trial of zinc supplementation (5 mg oral daily). Metabolomic analysis using an ultrahigh-performance liquid chromatography/tandem mass spectroscopy platform was performed to identify potential metabolomic profiles and biomarkers associated with zinc supplementation. Principal component analysis (PCA) was used to summarize metabolomic data from all samples. Two-way repeated measures analysis of variance with compound symmetry covariance structures were used to compare metabolome levels over time between infants in the 2 treatment arms. RESULTS: In PCA, the samples tended to be more separated by child age (6 wk compared with 6 mo) than by zinc supplementation status. We found that zinc supplementation affected a variety of metabolites associated with amino acid, lipid, nucleotide, and xenobiotic metabolism, including indoleacetate in the tryptophan metabolism pathway; 3-methoxytrosine and 4-hydrxoyphenylphruvate in the tyrosine pathway; eicosanedioate, 2-aminooctanoate, and N-acetyl-2-aminooctanoate in the fatty acid pathway; and N6-succinyladenosine in the purine metabolism pathway. Compared to the relatively small number of metabolites associated with zinc supplements, many infant metabolites changed significantly from age 6 wk to 6 mo. CONCLUSIONS: Zinc supplementation, despite having overall clinical benefits, appears to induce limited metabolomic changes in blood metabolites in young infants. Future larger studies may be warranted to further examine metabolic pathways associated with zinc supplementation. The parent trial was registered at clinicaltrials.gov as NCT00421668.

Assessing environmental enteric dysfunction via multiplex assay and its relation to growth and development among HIV-exposed uninfected Tanzanian infants.

BACKGROUND: Environmental enteric dysfunction (EED) may contribute to poor growth and development in young children. While validated EED biomarkers are currently lacking, multiplex assays are able to capture multiple domains of the condition. The purpose of this exploratory study was to examine the relationship between biomarkers of EED and subsequent growth and development among Tanzanian HIV-exposed uninfected (HEU) infants. METHODOLOGY: We enrolled 467 infants of mothers living with HIV who had participated in a trial of vitamin D3 supplementation during pregnancy. Infant serum samples collected at 6 weeks (n = 365) and 6 months (n = 266) were analyzed for anti-flagellin and anti-lipopolysaccharide (LPS) IgA and IgG via ELISA as well as the 11-plex Micronutrient and EED Assessment Tool (MEEDAT), which incorporates two biomarkers of EED [intestinal fatty acid-binding protein (I-FABP) and soluble CD14 (sCD14)]. Outcomes were 12-month growth [length-for-age z-score (LAZ), weight-for-length z-score (WLZ), and weight-for-age z-score (WAZ)] and development [Caregiver Reported Early Development Instruments (CREDI) z-scores] and were assessed using linear regression. FINDINGS: In primary analyses, higher quartiles of 6-month anti-LPS IgG concentrations were significantly associated with lower LAZ at 12 months (ptrend = 0.040). In secondary analyses, higher log2-transformed 6-week anti-flagellin IgA and 6-month anti-LPS IgA concentrations were significantly associated with lower LAZ at 12 months. No associations were observed between I-FABP or sCD14 and infant growth. However, higher log2-transformed 6-week sCD14 concentrations were significantly associated with lower overall CREDI z-scores, while higher log2-transformed 6-month I-FABP concentrations were significantly associated with higher overall CREDI z-scores. CONCLUSIONS: Unlike anti-flagellin and anti-LPS Igs, MEEDAT's biomarkers of EED (I-FABP and sCD14) were not associated with subsequent linear growth among HEU infants in Tanzania. The relationship between EED and infant development warrants further study.

Biomarkers of environmental enteric dysfunction and adverse birth outcomes: An observational study among pregnant women living with HIV in Tanzania.

BACKGROUND: Environmental enteric dysfunction (EED) may contribute to adverse birth outcomes in low-resource settings. We examined the associations of EED biomarkers with birth outcomes in pregnant women living with human immunodeficiency virus in Dar es Salaam, Tanzania. METHODS: We performed a cohort study of 706 HIV-infected pregnant women. Maternal serum samples collected at 32 weeks gestation were analyzed for markers of EED (anti-flagellin and anti-LPS immunoglobulins, intestinal fatty acid-binding protein [I-FABP] and soluble CD14), systemic inflammation (C-reactive protein and α1-acid glycoprotein [AGP]), and growth hormone resistance (insulin-like growth factor 1 [IGF-1] and fibroblast growth factor 21 [FGF21]. Associations of biomarkers categorized into quartiles with birth outcomes (birthweight, gestational duration, birthweight-for-gestational age, and stillbirth) were assessed using linear and log-binomial regression models adjusted for multiple sociodemographic and clinical variables. FINDINGS: Maternal EED biomarkers were not significantly associated with birthweight, gestation duration, or birthweight-for-gestational age. However, higher quintiles of I-FABP concentrations were associated with greater risk of stillbirth (ptrend=0·02). Higher AGP was associated with lower birthweight and was associated with increased risk of small-for-gestational age births. Higher IGF-1 was associated with higher birthweight and birthweight-for-gestational age while higher FGF21 was associated with shorter gestation and higher risk of preterm birth. INTERPRETATION: Maternal biomarkers of EED, systemic inflammation, and growth hormones were differentially associated with birth outcomes. Biomarkers of EED may be useful to identify pregnant women at risk of adverse birth outcomes, but further research is needed to confirm these findings and elucidate biological mechanisms. FUNDING: National Institutes of Health.

Aflatoxin exposure in pregnant women of mixed status of human immunodeficiency virus infection and rate of gestational weight gain: a Ugandan cohort study.

OBJECTIVES: To examine the association between aflatoxin (AF) exposure during pregnancy and rate of gestational weight gain (GWG) in a sample of pregnant women of mixed HIV status in Gulu, northern Uganda. METHODS: 403 pregnant women were included (133 HIV-infected on antiretroviral therapy (ART), 270 HIV-uninfected). Women's weight, height and socio-demographic characteristics were collected at baseline (~19 weeks' gestation); weight was assessed at each follow-up visit. Serum was collected at baseline and tested for aflatoxin B1 -lysine adduct (AFB-lys) levels using high-performance liquid chromatography (HPLC). Linear mixed-effects models were used to examine the association between AFB-lys levels and rate of GWG. RESULTS: AFB-lys levels (detected in 98.3% of samples) were higher among HIV-infected pregnant women than HIV-uninfected pregnant women [median (interquartile range): 4.8 (2.0, 15.0) vs. 3.5 (1.6, 6.1) pg/mg of albumin, P < 0.0001]. Adjusting for HIV status, a one-log increase in aflatoxin levels was associated with a 16.2 g per week lower rate of GWG (P = 0.028). The association between AFB-lys and the rate of GWG was stronger and significant only among HIV-infected women on ART [-25.7 g per week per log (AFB-lys), P = 0.009 for HIV-infected women vs. -7.5 g per week per log (AFB-lys), P = 0.422 for HIV-uninfected women]. CONCLUSIONS: Pregnant women with higher levels of AF exposure had lower rates of GWG. The association was stronger for HIV-infected women on ART, suggesting increased risk.

OBJECTIFS: Examiner l'association entre l'exposition à l'aflatoxine (AF) pendant la grossesse et le taux de gain de poids pendant la grossesse (GWG) dans un échantillon de femmes enceintes de statut mixte VIH à Gulu, dans le nord de l' Ouganda. MÉTHODES: 403 femmes enceintes ont été incluses (133 infectées par le VIH sous traitement antirétroviral (ART), 270 non infectées par le VIH). Le poids, la taille et les caractéristiques sociodémographiques des femmes ont été collectés au départ (~19 semaines de gestation); le poids a été évalué à chaque visite de suivi. Le sérum a été recueilli au départ et testé pour les niveaux d'adduit d'aflatoxine B1 -lysine (AFB-lys) en utilisant la chromatographie liquide à haute performance (HPLC). Des modèles linéaires d'effets mixtes ont été utilisés pour examiner l'association entre les niveaux de AFB-lys et le taux de GWG. RÉSULTATS: Les niveaux de AFB-lys (détectés dans 98,3% des échantillons) étaient plus élevés chez les femmes enceintes infectées par le VIH que chez celles enceintes non infectées par le VIH [médiane (intervalle interquartile): 4,8 (2,0, 15,0) vs 3,5 (1,6, 6,1) pg/mg d'albumine, P <0,0001]. En ajustant pour le statut VIH, une augmentation d'un log des niveaux d'aflatoxine était associée à un taux de GWG inférieur de 16,2 g par semaine (P = 0,028). L'association entre AFB-lys et le taux de GWG était plus forte et significative seulement chez les femmes infectées par le VIH sous ART [-25,7 g par semaine et par log (AFB-lys), P = 0,009 pour les femmes infectées par le VIH contre -7,5 g par semaine et par log (AFB-lys), P = 0,422 pour les femmes non infectées par le VIH]. CONCLUSIONS: Les femmes enceintes présentant des niveaux plus élevés d'exposition à l'AF avaient des taux de GWG plus faibles. L'association était plus forte pour les femmes infectées par le VIH sous ART, ce qui suggère un risque accru.

Predictors of low birth weight and preterm birth in rural Uganda: Findings from a birth cohort study.

BACKGROUND: Approximately 20.5 million infants were born weighing <2500 g (defined as low birthweight or LBW) in 2015, primarily in low- and middle-income countries (LMICs). Infants born LBW, including those born preterm (<37 weeks gestation), are at increased risk for numerous consequences, including neonatal mortality and morbidity as well as suboptimal health and nutritional status later in life. The objective of this study was to identify predictors of LBW and preterm birth among infants in rural Uganda. METHODS: Data were derived from a prospective birth cohort study conducted from 2014-2016 in 12 districts across northern and southwestern Uganda. Birth weights were measured in triplicate to the nearest 0.1 kg by trained enumerators within 72 hours of delivery. Gestational age was calculated from the first day of last menstrual period (LMP). Associations between household, maternal, and infant characteristics and birth outcomes (LBW and preterm birth) were assessed using bivariate and multivariable logistic regression with stepwise, backward selection analyses. RESULTS: Among infants in the study, 4.3% were born LBW (143/3,337), and 19.4% were born preterm (744/3,841). In multivariable analysis, mothers who were taller (>150 cm) (adjusted Odds Ratio (aOR) = 0.42 (95% CI = 0.24, 0.72)), multigravida (aOR = 0.62 (95% CI = 0.39, 0.97)), or with adequate birth spacing (>24 months) (aOR = 0.60 (95% CI = 0.39, 0.92)) had lower odds of delivering a LBW infant Mothers with severe household food insecurity (aOR = 1.84 (95% CI = 1.22, 2.79)) or who tested positive for malaria during pregnancy (aOR = 2.06 (95% CI = 1.10, 3.85)) had higher odds of delivering a LBW infant. In addition, in multivariable analysis, mothers who resided in the Southwest (aOR = 0.64 (95% CI = 0.54, 0.76)), were ≥20 years old (aOR = 0.76 (95% CI = 0.61, 0.94)), with adequate birth spacing (aOR = 0.76 (95% CI = 0.63, 0.93)), or attended ≥4 antenatal care (ANC) visits (aOR = 0.56 (95% CI = 0.47, 0.67)) had lower odds of delivering a preterm infant; mothers who were neither married nor cohabitating (aOR = 1.42 (95% CI = 1.00, 2.00)) or delivered at home (aOR = 1.25 (95% CI = 1.04, 1.51)) had higher odds. CONCLUSIONS: In rural Uganda, severe household food insecurity, adolescent pregnancy, inadequate birth spacing, malaria infection, suboptimal ANC attendance, and home delivery represent modifiable risk factors associated with higher rates of LBW and/or preterm birth. Future studies on interventions to address these risk factors may be warranted.

Risk factors for mortality among Tanzanian infants and children.

BACKGROUND: During the era of the Millennium Development Goals, under 5 mortality rates decreased significantly worldwide; however, reductions were not equally distributed. Children in sub-Saharan Africa still account for more than 50% of the world's annual childhood deaths among children under 5 years of age. Understanding upstream risk factors for mortality among children may reduce the large burden of childhood mortality in sub-Saharan Africa. Our objective was to identify risk factors for mortality among infants and children in Tanzania. METHODS: We conducted a secondary analysis of data pooled from two randomized-controlled micronutrient supplementation trials. A total of 4787 infants were enrolled in the two trials (n = 2387 HIV-exposed and n = 2400 HIV-unexposed). Predictors of mortality were assessed using unadjusted and adjusted hazard ratios (aHRs). RESULTS: There were 307 total deaths, 262 (11%) among children who were HIV-exposed and 45 (2%) among children who were HIV-unexposed (P < 0.001). The most common cause of death was respiratory diseases (n = 109, 35.5%). Causes of death did not significantly differ between HIV-exposed and HIV-unexposed children. In adjusted regression analyses, children with birth weight <2500 g (aHR 1.75, 95% CI 1.21-2.54), Apgar score of ≤7 at 5 min (aHR 2.16, 95% CI 1.29-3.62), or who were HIV-exposed but not infected (aHR 3.35, 95% CI 2.12-5.28) or HIV-infected (aHR 27.56, 95% CI 17.43-43.58) had greater risk of mortality. CONCLUSIONS: Infection with HIV, low birthweight, or low Apgar scores were associated with higher mortality risk. Early identification and modification of determinants of mortality among infants and children may be the first step to reducing such deaths.

Markers of Environmental Enteric Dysfunction Are Associated with Poor Growth and Iron Status in Rural Ugandan Infants.

BACKGROUND: Environmental enteric dysfunction (EED), characterized by altered intestinal permeability/inflammation, microbial translocation, and systemic inflammation (SI), may be a significant contributor to micronutrient deficiencies and poor growth in infants from low-resource settings. OBJECTIVE: We examined associations among EED, SI, growth, and iron status at 6 mo of age. METHODS: We performed a cross-sectional analysis of 6-mo-old infants (n = 548) enrolled in a Ugandan birth-cohort study (NCT04233944). EED was assessed via serum concentrations of anti-flagellin and anti- LPS immunoglobulins (Igs); SI was assessed via serum concentrations of ɑ1-acid glycoprotein (AGP) and C-reactive protein (CRP); iron status was assessed via serum concentrations of hemoglobin (Hb), soluble transferrin receptor (sTfR), and ferritin. Associations were assessed using adjusted linear regression analysis. RESULTS: At 6 mo, ∼35% of infants were stunted [length-for-age z score (LAZ) < -2] and ∼53% were anemic [hemoglobin (Hb) <11.0 g/dL]. Nearly half (∼46%) had elevated AGP (>1 g/L) and ∼30% had elevated CRP (>5 mg/L). EED and SI biomarkers were significantly correlated (r = 0.142-0.193, P < 0.001 for all). In adjusted linear regression models, which included adjustments for SI, higher anti-flagellin IgA, anti-LPS IgA, and anti-LPS IgG concentrations were each significantly associated with lower LAZ [ß (95% CI): -0.21 (-0.41, 0.00), -0.23 (-0.44, -0.03), and -0.33 (-0.58, -0.09)]. Furthermore, higher anti-flagellin IgA, anti-flagellin IgG, and anti-LPS IgA concentrations were significantly associated with lower Hb [ß (95% CI): -0.24 (-0.45, -0.02), -0.58 (-1.13, 0.00), and -0.26 (-0.51, 0.00)] and higher anti-flagellin IgG and anti-LPS IgG concentrations were significantly associated with higher sTfR [ß (95% CI): 2.31 (0.34, 4.28) and 3.13 (0.75, 5.51)]. CONCLUSIONS: EED is associated with both low LAZ and iron status in 6-mo-old infants. Further research on the mechanisms by which EED affects growth and micronutrient status is warranted.

Markers of Systemic Inflammation and Environmental Enteric Dysfunction Are Not Reduced by Zinc or Multivitamins in Tanzanian Infants: A Randomized, Placebo-Controlled Trial.

OBJECTIVE: To examine whether daily zinc and/or multivitamin supplementation reduce biomarkers of environmental enteric dysfunction (EED), systemic inflammation, or markers of growth in a sample of infants from Dar es Salaam, Tanzania. STUDY DESIGN: Subgroup analysis of infants participating in a randomized, double-blind, placebo-controlled trial received daily oral supplementation of zinc, multivitamins, zinc + multivitamins, or placebo for 18 months starting at 6 weeks of age. EED (anti-flagellin and anti-lipopolysaccharide immunoglobulins), systemic inflammation (C-reactive protein and alpha-1-acid glycoprotein), and growth biomarkers (insulin-like growth factor-1 and insulin-like growth factor binding protein-3) were measured via enzyme-linked immunosorbent assay in a subsample of 590 infants at 6 weeks and 6 months of age. EED biomarkers also were measured in 162 infants at 12 months of age. RESULTS: With the exception of anti-lipopolysaccharide IgG concentrations, which were significantly greater in infants who received multivitamins compared with those who did not (1.41 ± 0.61 vs 1.26 ± 0.65, P = .006), and insulin-like growth factor binding protein-3 concentrations, which were significantly lower in children who received zinc compared with those who did not (981.13 ± 297.59 vs 1019.10 ± 333.01, P = .03), at 6 months of age, we did not observe any significant treatment effects of zinc or multivitamins on EED, systemic inflammation, or growth biomarkers. CONCLUSIONS: Neither zinc nor multivitamin supplementation ameliorated markers of EED or systemic inflammation during infancy. Other interventions should be prioritized for future trials. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00421668.

Maternal aflatoxin exposure during pregnancy and adverse birth outcomes in Uganda.

Aflatoxins are toxic metabolites of Aspergillus moulds and are widespread in the food supply, particularly in low- and middle-income countries. Both in utero and infant exposure to aflatoxin B1 (AFB1 ) have been linked to poor child growth and development. The objective of this prospective cohort study was to investigate the association between maternal aflatoxin exposure during pregnancy and adverse birth outcomes, primarily lower birth weight, in a sample of 220 mother-infant pairs in Mukono district, Uganda. Maternal aflatoxin exposure was assessed by measuring the serum concentration of AFB1 -lysine (AFB-Lys) adduct at 17.8 ± 3.5 (mean ± SD)-week gestation using high-performance liquid chromatography. Anthropometry and birth outcome characteristics were obtained within 48 hr of delivery. Associations between maternal aflatoxin exposure and birth outcomes were assessed using multivariable linear regression models adjusted for confounding factors. Median maternal AFB-Lys level was 5.83 pg/mg albumin (range: 0.71-95.60 pg/mg albumin, interquartile range: 3.53-9.62 pg/mg albumin). In adjusted linear regression models, elevations in maternal AFB-Lys levels were significantly associated with lower weight (adj-ß: 0.07; 95% CI: -0.13, -0.003; p = 0.040), lower weight-for-age z-score (adj-ß: -0.16; 95% CI: -0.30, -0.01; p = 0.037), smaller head circumference (adj-ß: -0.26; 95% CI: -0.49, -0.02; p = 0.035), and lower head circumference-for-age z-score (adj-ß: -0.23; 95% CI: -0.43, -0.03; p = 0.023) in infants at birth. Overall, our data suggest an association between maternal aflatoxin exposure during pregnancy and adverse birth outcomes, particularly lower birth weight and smaller head circumference, but further research is warranted.

Unsafe Drinking Water Is Associated with Environmental Enteric Dysfunction and Poor Growth Outcomes in Young Children in Rural Southwestern Uganda.

Environmental enteric dysfunction (EED), a subclinical disorder of the small intestine, and poor growth are associated with living in poor water, sanitation, and hygiene (WASH) conditions, but specific risk factors remain unclear. Nested within a birth cohort study, this study investigates relationships among water quality, EED, and growth in 385 children living in southwestern Uganda. Water quality was assessed using a portable water quality test when children were 6 months, and safe water was defined as lacking Escherichia coli contamination. Environmental enteric dysfunction was assessed using the lactulose:mannitol (L:M) test at 12-16 months. Anthropometry and covariate data were extracted from the cohort study, and associations were assessed using linear and logistic regression models. Less than half of the households (43.8%) had safe water, and safe versus unsafe water did not correlate with improved versus unimproved water source. In adjusted linear regression models, children from households with safe water had significantly lower log-transformed (ln) L:M ratios (ß: -0.22, 95% confidence interval (CI): -0.44, -0.00) and significantly higher length-for-age (ß: 0.29, 95% CI: 0.00, 0.58) and weight-for-age (ß: 0.20, 95% CI: 0.05, 0.34) Z-scores at 12-16 months. Furthermore, in adjusted linear regression models, ln L:M ratios at 12-16 months significantly decreased with increasing length-for-age Z-scores at birth, 6 months, and 9 months (ß: -0.05, 95% CI: -0.10, -0.004; ß: -0.06, 95% CI: -0.11, -0.006; and ß: -0.05, 95% CI: -0.09, -0.005, respectively). Overall, our data suggest that programs seeking to improve nutrition should address poor WASH conditions simultaneously, particularly related to household drinking water quality.

Biomarkers of maternal environmental enteric dysfunction are associated with shorter gestation and reduced length in newborn infants in Uganda.

Background: Adverse birth outcomes, including preterm birth and stunting at birth, have long-term health implications. The relation between adverse birth outcomes and chronic, asymptomatic gastrointestinal inflammation (environmental enteric dysfunction-EED) is poorly understood. Objective: We aimed to examine the relation between maternal EED and adverse birth outcomes in a sample of pregnant Ugandan women and their newborn infants. Design: We conducted a prospective cohort study in Mukono, Uganda. A total of 258 pregnant women were enrolled at their first prenatal visit (∼18 weeks of gestation). EED was measured by urinary lactulose:mannitol (L:M) ratio and serum concentrations of antibodies to the bacterial components flagellin and LPS. Covariates were obtained from survey data collected at 2 time points. Associations were assessed through the use of unadjusted and adjusted simple linear regression models. Results: Complete birth outcome data were recorded for 220 infants within 48 h of delivery. Mean ± SD gestational age was 39.7 ± 2.1 wk, and 7% were born preterm. Mean ± SD length and length-for-age z score (LAZ) at birth were 48.1 ± 3.2 cm and -0.44 ± 1.07, respectively. L:M ratio was not associated with any birth outcome. In adjusted models, higher concentrations of natural log-transformed anti-flagellin immunoglobin G (IgG) and anti-LPS IgG were significantly associated with shorter length of gestation (ß: -0.89 wk; 95% CI: -1.77, -0.01 wk, and ß: -1.01 wk; 95% CI: -1.87, -0.17 wk, respectively) and with reduced length (ß: -0.80 cm; 95% CI: -1.55, -0.05 cm, and ß: -0.79 cm; 95% CI: -1.54, -0.04 cm, respectively) and LAZ at birth (ß -0.44 z score; 95% CI: -0.83, -0.05, and ß: -0.40 z score; 95% CI: -0.79, -0.01, respectively). Conclusion: Maternal anti-flagellin and anti-LPS IgG concentrations in pregnancy, but not L:M ratio, were associated with shorter gestation and reduced infant length at birth. Further research on the relation between maternal EED and birth outcomes is warranted.