Momodou K Darboe

Isolation of Helicobacter pylori from human faeces

Helicobacter pylori is arguably the commonest chronic infection in man. However, its route of transmission is unknown. We have isolated viable H pylori from the faeces of an infected individual from The Gambia. The organism was cultured on selective media after concentration of faecal bacteria by centrifugation in a buffer equilibrated with a microaerophilic gas mixture. Growth characteristics, microscopic appearances, and enzyme activities were the same as those of a typical gastric isolate of H pylori. Protein preparations derived from the new isolate and the typical strain were antigenically similar, and had very similar electrophoretic profiles (including two major protein bands of 62 and 26 kDa, corresponding to the urease enzyme subunits). With the same technique, organisms with the colony morphology, growth requirements, enzyme activities, and microscopic appearances of H pylori were isolated from the faeces of 9 of 23 randomly selected children aged 3-27 months from a Gambian village with a high prevalence of H pylori infection in early life. Faecal-oral transmission is probably important in the spread of infection in such communities.

Effectiveness of an early supplementation scheme of high-dose vitamin A versus standard WHO protocol in Gambian mothers and infants: a randomised controlled trial

BACKGROUND Most developing countries have adopted a standard WHO dosing schedule for vitamin A supplementation. However, in 2002 the International Vitamin A Consultative Group (IVACG) Annecy Accord recommended a new high-dose regimen for mothers and infants. Our aim was to test whether the new high-dose regimen of vitamin A supplementation would increase maternal and infant plasma vitamin A, reduce infant Helicobacter pylori infection and nasopharyngeal pneumococcal carriage, and improve infant gut epithelial integrity. METHODS In an area of moderate vitamin A deficiency in rural Gambia, 220 mother-infant pairs were enrolled in a randomised double-blind trial between September, 2001, and October, 2004, that compared the IVACG high dose with the WHO dose. The primary endpoints were levels of maternal and infant plasma vitamin A, H pylori infection, pneumococcal carriage, and gut epithelial integrity. The trial is registered as ISRCTN 98554309. FINDINGS 197 infants completed follow-up to 12 months (99 high dose and 98 WHO dose). There were no adverse events at dosing. No differences were found in the primary outcomes for high-dose versus WHO schedule: maternal vitamin A concentration at 2 months +0.02 micromol/L (95% CI -0.10 to 0.15); infant vitamin A at 5 months +0.01 micromol/L (-0.06 to 0.08); H pylori infection at 12 months -0.3% (-14.7 to 14.2); maternal pneumococcal carriage at 12 months -2.0% (-13.7 to 9.7); infant pneumococcal carriage at 12 months -4.1% (-15.8 to 7.6); infant gut mucosal damage at 12 months 5.2% (-8.7 to 19.2). There were more clinic attendances by the high-dose group in the first 6 months of life (p=0.018). INTERPRETATION Our results do not lend support to the proposal to increase the existing WHO standard dosing schedule for vitamin A in areas of moderate vitamin A deficiency. Caution is urged for future studies because trials have shown possible adverse effects of higher doses of vitamin A, and potential negative interactions with the expanded programme on immunisation (EPI) vaccines.

A randomized trial to investigate the effects of pre-natal and infant nutritional supplementation on infant immune development in rural Gambia: the ENID trial: Early Nutrition and Immune Development

BackgroundRecent observational research indicates that immune development may be programmed by nutritional exposures early in life. Such findings require replication from trials specifically designed to assess the impact of nutritional intervention during pregnancy on infant immune development. The current trial seeks to establish: (a) which combination of protein-energy (PE) and multiple-micronutrient (MMN) supplements would be most effective; and (b) the most critical periods for intervention in pregnancy and infancy, for optimal immune development in infancy.Methods/DesignThe ENID Trial is a 2 x 2 x 2 factorial randomized, partially blind trial to assess whether nutritional supplementation to pregnant women (from < 20 weeks gestation to term) and their infants (from 6 to 12 months of age) can enhance infant immune development. Eligible pregnant women from the West Kiang region of The Gambia (pregnancy dated by ultrasound examination) are randomized on entry to 4 intervention groups (Iron-folate (FeFol = standard care), multiple micronutrients (MMN), protein-energy (PE), PE + MMN). Women are visited at home weekly for supplement administration and morbidity assessment and seen at MRC Keneba at 20 and 30 weeks gestation for a detailed antenatal examination, including ultrasound. At delivery, cord blood and placental samples are collected, with detailed infant anthropometry collected within 72 hours. Infants are visited weekly thereafter for a morbidity questionnaire. From 6 to 12 months of age, infants are further randomized to a lipid-based nutritional supplement, with or without additional MMN. The primary outcome measures of this study are thymic development during infancy, and antibody response to vaccination. Measures of cellular markers of immunity will be made in a selected sub-cohort. Subsidiary studies to the main trial will additionally assess the impact of supplementation on infant growth and development to 24 months of age.DiscussionThe proposed trial is designed to test whether nutritional repletion can enhance early immune development and, if so, to help determine the most efficacious form of nutritional support. Where there is evidence of benefit from a specific intervention/combination of interventions, future research should focus on refining the supplements to achieve the optimal, most cost-effective balance of interventions for improved health outcomes.Trial registrationISRCTN49285450

The dynamics of nasopharyngeal streptococcus pneumoniae carriage among rural Gambian mother-infant pairs

BackgroundStreptococcus pneumoniae is an important cause of community acquired pneumonia, sepsis, meningitis and otitis media globally and has been incriminated as a major cause of serious childhood bacterial infections in The Gambia. Better understanding of the dynamics of transmission and carriage will inform control strategies.MethodsThis study was conducted among 196 mother-infant pairs recruited at birth from six villages in the West Kiang region of The Gambia. Nasopharyngeal swabs were collected from mother-infant pairs at birth (within 12 hours of delivery), 2, 5 and 12 months. Standard techniques of culture were used to identify carriage and serotype S. pneumoniae.ResultsOf 46 serotypes identified, the 6 most common, 6A, 6B, 14, 15, 19F and 23F, accounted for 67.3% of the isolates from infants. Carriage of any serotype among infants rose from 1.5% at birth to plateau at approximately 80% by 2 m (prevalence at 2 m = 77%; 5 m = 86%; 12 m = 78%). Likewise, maternal carriage almost doubled in the first 2 months post-partum and remained elevated for the next 10 m (prevalence at birth = 13%; 2 m = 24%; 5 m = 22%; 12 m = 21%). Carriage was significantly seasonal in both infants and mothers with a peak in December and lowest transmission in August. The total number of different serotypes we isolated from each infant varied and less than would be expected had the serotypes assorted independently. In contrast, this variability was much as expected among mothers. The half-life of a serotype colony was estimated to be 1.90 m (CI95%: 1.66-2.21) in infants and 0.75 m (CI95%: 0.55-1.19) in mothers. While the odds for a serotype to be isolated from an infant increased by 9-fold if it had also been isolated from the mother, the population attributable fraction (PAF) of pneumococcal carriage in infants due to maternal carriage was only 9.5%. Some marked differences in dynamics were observed between vaccine and non-vaccine serotypes.ConclusionsColonisation of the nasopharynx in Gambian infants by S. pneumoniae is rapid and highly dynamic. Immunity or inter-serotype competition may play a role in the dynamics. Reducing mother-infant transmission would have a minimal effect on infant carriage.

Commentary: Challenging public health orthodoxies—prophesy or heresy?

In 1633, after many years of skirmishing with the Catholic Church over his support for Copernicus’ heliocentric theory of the universe, Galileo was finally sentenced by the inquisition to prison and religious penances. In a formal ceremony at the church of Santa Maria Sofia Minerva, he was forced to abjure his errors, and spent the rest of his life under house arrest in Sienna. The prophet had been convicted as a heretic. Without, yet, wishing to confer the status of prophet on Peter Aaby and his disciples based in Guinea Bissau, there are significant parallels in their persistent challenges to some of the deepest rooted public health orthodoxies of the present day. Aaby has a long history of interrogating datasets in a way that others have failed to do and coming up with some uncomfortable findings. For many years he has been in conflict with WHO and many vaccinologists concerning his beliefs that childhood vaccines can have non-specific downstream effects on mortality (sometimes positive, but often negative) that have been ignored by mainstream research.1–4 More recently his team have even challenged, on the basis of a randomized trial, the value of WHOs recommendation that exclusive breastfeeding should continue to 6 months.5 Some may consider that house arrest is too lenient, even in Guinea Bissau instead of Sienna. Christine Stabell Benn, working within Aabys group, has for several years been pursuing another line of heresy, namely that it may be beneficial to withhold vitamin A supplementation (VAS) from infant girls in developing countries, at least until they have had their first measles vaccination.6–8 In this issue of IJE,9 the Bissau team summarize the evidence in support of their belief that co-administration of vitamin A with diphtheria–tetanus–pertussis vaccine (DTP) results in negative interactions that lead to higher subsequent mortality; an effect confined to girls. They then take this a step further by drawing on data from two very large trials of zinc and iron-plus-folic-acid supplementation from Nepal10,11 and Zanzibar (Pemba).12,13 Using these data they argue that these trials also reveal important sex and age-differential effects of preventive treatment with micronutrients with a ‘tendency’ for detrimental effects in infants especially among girls.9 They conclude that micronutrient supplementation policies might have to differ for boys and girls, and that in their view ‘all evidence suggests that currently infant girls have little to gain from micronutrients’.9 Could it really be true that in populations such as rural West Africa (where anaemia rates can exceed 90% and where >70% of young children can have plasma retinol levels <0.7 μmol/l14), we can advocate that micronutrients should be withheld from girls? Is it really possible that marginal nutrient deficiencies might be protective where infections are highly prevalent, as recently postulated for iron?15 Only a few years ago such a view, which is in direct conflict with policy recommendations from WHO,16,17 the International Vitamin A Consultative Group (IVACG)18 and the International Nutritional Anemias Consultative Group (INACG),19 would have been condemned as heresy. However, the recent findings from the Pemba trial12 that supplementation with iron-plus-folic-acid caused a significant increase in serious adverse events and mortality leading to the trials termination by its Data and Safety Monitoring Board have brought a renewed humility, and a recognition that there can be complex interactions between micronutrients, pathogens and immunity.20 The state of our collective ignorance has been made painfully apparent.21 The evidence garnered by Benn and colleagues in support of their central contention that there are negative interactions between VAS and DTP vaccination in girls is somewhat fragmentary and often based on non-significant trends. Some of it is vulnerable to possible ascertainment biases related both to records of deaths and of vaccination status; criticisms of which the Guinea Bissau group are well aware,22,23 and which they endeavour to incorporate into their analyses. The evidence is complex and has been recently considered by a group of experts (including Benn and Aaby) convened at the London School of Hygiene & Tropical Medicine (LSHTM). It would be presumptive of us to offer a definite adjudication on the Benn thesis, or to pre-empt any report that may emerge from the LSHTM group. However, we offer the following recommendations: There is sufficient circumstantial evidence for the Benn/Aaby thesis to be taken seriously; as has recently occurred at the LSHTM meeting. Those public health nutritionists or vaccinologists who tend towards the evangelical would be wise to leave room for scepticism and open debate. One of the major aims of the London meeting was to gather together additional existing datasets that may be able to contribute further evidence for or against the Benn/Aaby hypothesis. Such datasets need to be very large given the welcome fact that infant and child mortality rates are declining in most countries and we may be searching for relatively rare endpoints and modest effect sizes. Impartial third-party re-analysis of such datasets should be expedited and, in view of the latest suggestion that the negative interactions may extend to supplementation with zinc and iron-plus-folic-acid, it is hoped that the Johns’ Hopkins group have already been asked to contribute the data from their Nepal and Zanzibar trials 10–13 so that the appropriate re-analysis can be done. It would be unethical to conduct a prospective 4-arm trial in which infants were randomized with and without vitamin A and with and without DTP, but it might still be ethically acceptable to conduct a trial in which the vitamin A treatment was randomized (depending on the ethics committees level of conviction concerning the Benn/Aaby hypothesis). However, much of the evidence accrued so far has come from areas with very high mortality and any research team initiating a new trial would have a moral responsibility to put in place better health surveillance and care, which, by reducing the number of serious adverse events, would probably require any such trial to be so large as to be prohibitively costly irrespective of any ethical arguments. Therefore the prospects of obtaining RCT standard evidence are remote. Aabys contention that vaccines can have potent and long-lasting unintended effects has important implications for the design and analysis of vaccine trials. Since post-marketing (Phase 4) data surveillance is not currently practicable in most developing country settings, there is a responsibility to try and assess such outcomes at the Phase 3 stage with long-term follow-up and assessment of multiple outcomes, not just those related to the target disease. There is an urgent need to introduce a greater sophistication and more modern immunological techniques in order to understand the complex interactions between micronutrients and vaccines. Although community-wide VAS has been repeatedly proven to reduce all cause mortality in children, we know virtually nothing about the mechanism by which it saves lives.21,24 It is striking that Benn and colleagues are able to muster very little in the way of supportive mechanistic theories as to why there may be negative interactions and why these might be sex-specific. Even if large trials with mortality outcomes are not possible, it should be feasible to learn more about the putative interactions by means of very detailed investigations into how DTP and vitamin A status interact to modulate the downstream effects of the vaccine (for instance in relation to suggestions that it may exaggerate sex-specific T-helper cell biases). Contrary to the frequently cited statement that ‘it would be unethical’ to perform trials in which vitamin A (or iron) were withheld in a control group, we believe that recent findings have re-established an ethical equipoise and that it is now essential to conduct such trials. In the light of the concerns about vitamin A and DTP, and in view of the fact that at least two studies have shown that high doses of vitamin A can be associated with poorer clinical outcomes,8,25 and that measles vaccination appears to cancel out any adverse consequences of VAS + DTP,9,26 it may be wise to modify the current WHO advice concerning the timing of first VAS in infants. Currently the recommendation is ‘after 6 months’.17 A prudent approach might to be recommend ‘with, or soon after, measles vaccination’. Public health nutritionists have generally moved on from single micronutrient interventions (vitamin A, iron, zinc and iodine) and are busily exploring the efficacy of multiple micronutrients in the form of Sprinkles® or lipid-based nutrient supplements (LNS). The inclusion of long-chain polyunsaturated fatty acids, which are already known to have potentially significant immunomodulatory properties,27 is being considered in LNS formulation. What effects might these other micronutrients have on vaccine responses? These should also be studied using, in the first instance, careful assays of their proximal effects on immune function. Conflict of interest: None declared.

Breast milk sodium content in rural Gambian women: between- and within-women variation in the first 6 months after delivery

It has been suggested that infancy is a particularly sensitive period with respect to the effect of dietary sodium on future risk of hypertension. One difficulty of researching the effects of early sodium intake on later health is accurately measuring sodium intake from breast milk. In observational studies, sodium content has been calculated by estimating breast milk volume consumed and assuming a fixed sodium concentration for all women at all times (a standardised measure). The objectives of this study were to investigate the variation in breast milk sodium concentration in the first 6 months postpartum within women and test whether the pattern of change in sodium concentration differs between women. The study population was 197 rural Gambian women. Multilevel models were used to investigate whether the sodium content of breast milk changed over time within and between women. Fractional polynomials were used to identify the best-fitting functions of age to be included in the within and between variance functions. Sodium levels decreased with time; the reduction was initially rapid (levels decreasing by 17.7% between 30 and 60 days after delivery). Immediately after birth, there was substantial variation in breast milk sodium content between women but this reduced with time. Our results suggest that it is not appropriate to use a standardised measure of breast milk sodium content when direct measurement is possible - particularly when there is a research interest in measuring sodium intake in very early infancy.

Following the World Health Organization's Recommendation of Exclusive Breastfeeding to 6 Months of Age Does Not Impact the Growth of Rural Gambian Infants.

Background: The WHO recommends exclusive breastfeeding (EBF) for the first 6 mo of life. Objective: The objective of this study was to assess the benefit of EBF to age 6 mo on growth in a large sample of rural Gambian infants at high risk of undernutrition. Methods: Infants with growth monitoring from birth to 2 y of age (n = 756) from the ENID (Early Nutrition and Immune Development) trial were categorized as exclusively breastfed if only breast milk and no other liquids or foods were given. EBF status was entered into confounder-adjusted multilevel models to test associations with growth trajectories by using >11,000 weight-for-age (WAZ), length-for-age (LAZ), and weight-for-length (WLZ) z score observations. Results: Thirty-two percent of infants were exclusively breastfed to age 6 mo. The mean age of discontinuation of EBF was 5.2 mo, and growth faltering started at ∼3.5 mo of age. Some evidence for a difference in WAZ and WHZ was found between infants who were exclusively breastfed to age 6 mo (EBF-6) and those who were not (nEBF-6), at 6 and 12 mo of age, with EBF-6 children having a higher mean z score. The differences in z scores between the 2 groups were small in magnitude (at 6 mo of age: 0.147 WAZ; 95% CI: −0.001, 0.293 WAZ; 0.189 WHZ; 95% CI: 0.038, 0.341 WHZ). No evidence for a difference between EBF-6 and nEBF-6 infants was observed for LAZ at any time point (6, 12, and 24 mo of age). Furthermore, a higher mean WLZ at 3 mo of age was associated with a subsequent higher mean age at discontinuation of EBF, which implied reverse causality in this setting (coefficient: 0.060; 95% CI: 0.008, 0.120). Conclusion: This study suggests that EBF to age 6 mo has limited benefit to the growth of rural Gambian infants. This trial was registered at http://www.isrctn.com as ISRCTN49285450.

Association of prenatal lipid‐based nutritional supplementation with fetal growth in rural Gambia

Abstract Prenatal supplementation with protein‐energy (PE) and/or multiple‐micronutrients (MMNs) may improve fetal growth, but trials of lipid‐based nutritional supplements (LNSs) have reported inconsistent results. We conducted a post‐hoc analysis of non‐primary outcomes in a trial in Gambia, with the aim to test the associations of LNS with fetal growth and explore how efficacy varies depending on nutritional status. The sample comprised 620 pregnant women in an individually randomized, partially blinded trial with four arms: (a) iron and folic acid (FeFol) tablet (usual care, referent group), (b) MMN tablet, (c) PE LNS, and (d) PE + MMN LNS. Analysis of variance examined unadjusted differences in fetal biometry z‐scores at 20 and 30 weeks and neonatal anthropometry z‐scores, while regression tested for modification of intervention‐outcome associations by season and maternal height, body mass index, and weight gain. Despite evidence of between‐arm differences in some fetal biometry, z‐scores at birth were not greater in the intervention arms than the FeFol arm (e.g., birth weight z‐scores: FeFol −0.71, MMN −0.63, PE −0.64, PE + MMN −0.62; group‐wise p = .796). In regression analyses, intervention associations with birth weight and head circumference were modified by maternal weight gain between booking and 30 weeks gestation (e.g., PE + MMN associations with birth weight were +0.462 z‐scores (95% CI [0.097, 0.826]) in the highest quartile of weight gain but –0.099 z‐scores (−0.459, 0.260) in the lowest). In conclusion, we found no strong evidence that a prenatal LNS intervention was associated with better fetal growth in the whole sample.

The effects of maternal iron deficiency on infant fibroblast growth factor-23 and mineral metabolism.

Fibroblast growth factor-23 (FGF23), a phosphate(Phos)-regulating hormone, is abnormally elevated in hypophosphataemic syndromes and an elevated FGF23 is a predictor of mortality in kidney disease. Recent findings suggest iron deficiency as a potential mediator of FGF23 expression and murine studies have shown in utero effects of maternal iron deficiency on offspring FGF23 and phosphate metabolism. Our aim was to investigate the impact of maternal iron status on infant FGF23 and mineral metabolites over the first 2 years of life. Infants born to mothers with normal (NIn = 25,) and low (LIn = 25) iron status during pregnancy, from a mother-infant trial (ISRCTN49285450) in rural Gambia, West Africa, had blood and plasma samples analysed at 12, 24, 52, 78 and 104 weeks (wk) of age. Circulating intact-FGF23 (I-FGF23), Phos, total alkaline phosphatase (TALP) and haemoglobin (Hb) decreased and estimated glomerular filtration rate increased over time [all P ≤ 0.0001)]. C-terminal-FGF23 (C-FGF23) and TALP were significantly higher in LI compared with NI, from 52 wk for C-FGF23 [Beta coefficient (SE) 18.1 (0.04) %, P = 0.04] and from 24 wk for TALP [44.7 (29.6) U/L, P = 0.04]. Infant Hb was the strongest negative predictor of C-FGF23 concentration [− 21% (4%) RU/mL, P ≤ 0.0001], Phos was the strongest positive predictor of I-FGF23 [32.0(3.9) pg/mL, P ≤ 0.0001] and I-FGF23 did not predict C-FGF23 over time [− 0.5% (0.5%), P = 0.3]. In conclusion, this study suggests that poor maternal iron status is associated with a higher infant C-FGF23 and TALP but similar I-FGF23 concentrations in infants and young children. These findings further highlight the likely public health importance of preventing iron deficiency during pregnancy. Whether or not children who are born to iron deficient mothers have persistently high concentrations of these metabolites and are more likely to be at risk of impaired bone development and pre-disposed to rickets requires further research.

Hormonal Correlates and Predictors of Nutritional Recovery in Malnourished African Children.

ABSTRACT Background Malnourished children show variable growth responses to nutritional rehabilitation. We aimed to investigate whether these differences could be explained by variations in growth and energy-regulating hormones. Methods Quasi-experimental study: Children aged 6–24 months in rural Gambia were recruited to controls if weight-for-height z-score (WHZ) > −2 (n = 22), moderate acute malnutrition if WHZ < −2 and > −3 (n = 18) or severe acute malnutrition if WHZ < −3 (n = 20). Plasma hormone and salivary CRP levels were determined by ELISA. Results In univariable analyses, increases in weight-for-age z-score (WAZ) in malnourished children were positively correlated with insulin (F-ratio 7.8, p = 0.006), C-peptide (F-ratio 12.2, p < 0.001) and cortisol (F-ratio 5.0, p = 0.03). In multivariable analysis, only baseline C-peptide (F-ratio 7.6, p = 0.009) predicted the changes in WAZ over 28 days of interventions. Conclusion In rural Gambian, malnourished children, although it cannot be used in isolation, baseline C-peptide was a predictor of future response to rehabilitation.