Pathobiome driven gut inflammation in Pakistani children with environmental enteric dysfunction

Environmental Enteric Dysfunction (EED) is an acquired small intestinal inflammatory condition underlying high rates of stunting in children \u3c5 years of age in low- and middle-income countries. Children with EED are known to have repeated exposures to enteropathogens and environmental toxins that leads to malabsorptive syndrome. We aimed to characterize association of linear growth faltering with enteropathogen burden and subsequent changes in EED biomarkers. In a longitudinal birth cohort (n = 272), monthly anthropometric measurements (Length for Age Z score- LAZ) of asymptomatic children were obtained up to 18 months. Biological samples were collected at 6 and 9 months for the assessment of biomarkers. A customized TaqMan array card was used to target 40 enteropathogens in fecal samples. Linear regression was applied to study the effect of specific enteropathogen infection on change in linear growth (ΔLAZ). Presence of any pathogen in fecal sample correlated with serum flagellin IgA (6 mo, r = 0.19, p = 0.002), fecal Reg 1b (6 mo, r = 0.16, p = 0.01; 9mo, r = 0.16, p = 0.008) and serum Reg 1b (6 mo, r = 0.26, p\u3c0.0001; 9 mo, r = 0.15, p = 0.008). At 6 months, presence of Campylobacter [β (SE) 7751.2 (2608.5), p = 0.003] and ETEC LT [β (SE) 7089.2 (3015.04), p = 0.019] was associated with increase in MPO. Giardia was associated with increase in Reg1b [β (SE) 72.189 (26.394), p = 0.006] and antiflic IgA[β (SE) 0.054 (0.021), p = 0.0091]. Multiple enteropathogen infections in early life negatively correlated with ΔLAZ, and simultaneous changes in gut inflammatory and permeability markers. A combination vaccine targeting enteropathogens in early life could help in the prevention of future stuntin

Nasopharyngeal carriage of Streptococcus pneumoniae in children under 5 years of age before introduction of pneumococcal vaccine (PCV10) in urban and rural districts in Pakistan

Background: Benefits of pneumococcal conjugate vaccine programs have been linked to the vaccine’s ability to disrupt nasopharyngeal carriage and transmission. The 10-valent pneumococcal vaccine (PCV10) was included in the Expanded Program on Immunization (EPI) in Sindh, Pakistan in February 2013. This study was carried out immediately before PCV10 introduction to establish baseline pneumococcal carriage and prevalent serotypes in young children and to determine if carriage differed in urban and rural communities.Methods: Nasopharyngeal specimens were collected from a random sample of children 3-11 and 12-59 months of age in an urban community (Karachi) and children 3-11 months of age in a rural community (Matiari). Samples were processed in a research laboratory in Karachi. Samples were transported in STGG media, enriched in Todd Hewitt broth, rabbit serum and yeast extract, cultured on 5% sheep blood agar, and serotyped using the CDC standardized sequential multiplex PCR assay. Serotypes were categorized into PCV10-type and non-vaccine types.Results: We enrolled 670 children. Pneumococci were detected in 73.6% and 79.5 % of children in the infant group in Karachi and Matiari, respectively, and 78.2% of children 12 to 59 months of age in Karachi. In infants, 38. 9% and 33.5% of those carrying pneumococci in Karachi and Matiari, respectively, had PCV10 types. In the older age group in Karachi, the proportion was 30.7%, not significantly different from infants. The most common serotypes were 6A, 23F, 19A, 6B and 19F.Conclusion: We found that about 3 of 4 children carried pneumococci, and this figure did not vary with age group or urban or rural residence. Planned annual surveys in the same communities will inform change in carriage of PCV10 serotype pneumococci after the introduction and uptake of PCV10 in these communitie

Serum anti-flagellin and anti-lipopolysaccharide immunoglobulins as predictors of linear growth faltering in Pakistani infants at risk for environmental enteric dysfunction

Background: Environmental Enteric Dysfunction (EED) in children from low-income countries has been linked to linear growth declines. There is a critical need to identify sensitive and early EED biomarkers.Objective: Determine whether levels of antibodies against bacterial components flagellin (flic) and lipopolysaccharide (LPS) predict poor growth.Design/Methods: In a prospective birth cohort of 380 children in rural Pakistan blood and stool samples were obtained at ages 6 and 9 months. Linear mixed effects models were used to examine longitudinal associations between quartiles of anti-flic and anti-LPS antibodies and changes in LAZ, WAZ and WLZ scores. Spearman\u27s correlations were measured between anti-flic and anti-LPS immunoglobulins with measures of systemic/enteric inflammation and intestinal regeneration.Results: Anti-LPS IgA correlated significantly with CRP, AGP and Reg1 serum at 6mo and with MPO at 9mo. In multivariate analysis at 6mo of age, higher anti-LPS IgA levels predicted greater declines in LAZ scores over subsequent 18mo (comparing highest to lowest quartile, β (SE) change in LAZ score/year = -0.313 (0.125), p-value = 0.013). Anti-flic Ig A in the two highest quartiles measured at 9mo of age had declines in LAZ of -0.269 (0.126), p = 0.033; and -0.306 (0.129), p = 0.018 respectively, during the subsequent 18mo of life, compared to those in the lowest quartile of anti-flic IgA.Conclusions and Relevance: Elevated anti-flic IgA and anti-LPS IgA antibodies at 6 and 9mo, predict declines in linear growth. Systemic and enteric inflammation correlated with anti-LPS IgA provides mechanistic considerations for potential future interventions

Effect of provision of daily zinc and iron with several micronutrients on growth and morbidity among young children in Pakistan: a cluster-randomised trial.

BACKGROUND: Powders containing iron and other micronutrients are recommended as a strategy to prevent nutritional anaemia and other micronutrient deficiencies in children. We assessed the effects of provision of two micronutrient powder formulations, with or without zinc, to children in Pakistan. METHODS: We did a cluster randomised trial in urban and rural sites in Sindh, Pakistan. A baseline survey identified 256 clusters, which were randomly assigned (within urban and rural strata, by computer-generated random numbers) to one of three groups: non-supplemented control (group A), micronutrient powder without zinc (group B), or micronutrient powder with 10 mg zinc (group C). Children in the clusters aged 6 months were eligible for inclusion in the study. Powders were to be given daily between 6 and 18 months of age; follow-up was to age 2 years. Micronutrient powder sachets for groups B and C were identical except for colour; investigators and field and supervisory staff were masked to composition of the micronutrient powders until trial completion. Parents knew whether their child was receiving supplementation, but did not know whether the powder contained zinc. Primary outcomes were growth, episodes of diarrhoea, acute lower respiratory tract infection, fever, and incidence of admission to hospital. This trial is registered with ClinicalTrials.gov, number NCT00705445. RESULTS: The trial was done between Nov 1, 2008, and Dec 31, 2011. 947 children were enrolled in group A clusters, 910 in group B clusters, and 889 in group C clusters. Micronutrient powder administration was associated with lower risk of iron-deficiency anaemia at 18 months compared with the control group (odds ratio [OR] for micronutrient powder without zinc=0·20, 95% CI 0·11-0·36; OR for micronutrient powder with zinc=0·25, 95% CI 0·14-0·44). Compared with the control group, children in the group receiving micronutrient powder without zinc gained an extra 0·31 cm (95% CI 0·03-0·59) between 6 and 18 months of age and children receiving micronutrient powder with zinc an extra 0·56 cm (0·29-0·84). We recorded strong evidence of an increased proportion of days with diarrhoea (p=0·001) and increased incidence of bloody diarrhoea (p=0·003) between 6 and 18 months in the two micronutrient powder groups, and reported chest indrawing (p=0·03). Incidence of febrile episodes or admission to hospital for diarrhoea, respiratory problems, or febrile episodes did not differ between the three groups. INTERPRETATION: Use of micronutrient powders reduces iron-deficiency anaemia in young children. However, the excess burden of diarrhoea and respiratory morbidities associated with micronutrient powder use and the very small effect on growth recorded suggest that a careful assessment of risks and benefits must be done in populations with malnourished children and high diarrhoea burdens. FUNDING: Bill & Melinda Gates Foundation

Infant and Maternal Characteristics at six and nine months^*.

<p>Infant and Maternal Characteristics at six and nine months^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193768#t001fn002" target="_blank">*</a>}.</p

Median LAZ, WLZ and WAZ scores in Pakistani infants over 18 months of follow-up.

<p>Dashed lines show q1 and q3 quartiles. Abbreviations: LAZ = Length-for-Age Z score, WLZ = Weight-for-Length Z score and WAZ = Weight-for-Age Z score.</p

Anti-Flagellin and anti-LPS immunoglobulin concentrations in Pakistani infants.

<p>Closed shapes indicate means±SDs in Pakistani infants over the follow-up period. Open shapes indicate means±SDs values in 36 healthy Boston infants with a mean age of 9.5 months. n = 380 for all immunoglobulins in Pakistani infants at 6 and 9 months of age; n = 36 for all immunoglobulins in Boston infants at 9.5 months of age. **** p< 0.0001, ** p< 0.01 and * p< 0.05 for comparisons of mean biomarker concentrations in Pakistani infants with those of healthy Boston infants (unpaired t tests).</p

Correlation coefficient matrix of immunoglobulins to flagellin and lipolysaccharide with measures of systemic inflammation and enteric inflammation & regeneration.

<p>Correlation coefficient matrix of immunoglobulins to flagellin and lipolysaccharide with measures of systemic inflammation and enteric inflammation & regeneration.</p

The association of anti-flagellin and anti-lipopolysaccharide immunoglobulin concentrations at 6 and 9 months with annual Z score changes for length using linear mixed effects models.

<p>The association of anti-flagellin and anti-lipopolysaccharide immunoglobulin concentrations at 6 and 9 months with annual Z score changes for length using linear mixed effects models.</p