Conor P. Doherty

Hepcidin is the major predictor of erythrocyte iron incorporation in anemic African children

Iron supplementation strategies in the developing world remain controversial because of fears of exacerbating prevalent infectious diseases. Understanding the conditions in which iron will be absorbed and incorporated into erythrocytes is therefore important. We studied Gambian children with either postmalarial or nonmalarial anemia, who were given oral iron supplements daily for 30 days. Supplements administered on days 1 and 15 contained the stable iron isotopes 57Fe and 58Fe, respectively, and erythrocyte incorporation was measured in blood samples drawn 14 days later. We investigated how the iron-regulatory hormone hepcidin and other inflammatory/iron-related indices, all measured on the day of isotope administration, correlated with erythrocyte iron incorporation. In univariate analyses, hepcidin, ferritin, C-reactive protein, and soluble transferrin receptor (sTfR) strongly predicted incorporation of 57Fe given on day 1, while hepcidin, ferritin, and sTfR/log ferritin correlated with 58Fe incorporation. In a final multivariate model, the most consistent predictor of erythrocyte isotope incorporation was hepcidin. We conclude that under conditions of competing signals (anemia, iron deficiency, and infection), hepcidin powerfully controls use of dietary iron. We suggest that low-cost point-of-care hepcidin assays would aid iron supplementation programs in the developing world.

Expression of the Iron Hormone Hepcidin Distinguishes Different Types of Anemia in African Children

The iron hormone hepcidin correctly identifies African children in whom iron supplementation is most likely to be beneficial. Hepcidin Guides Iron Supplementation in African Children Anemia affects 300 million preschool children worldwide and has multiple causes including iron deficiency or infection. Dietary iron supplementation is used to combat anemia, but substantial concerns exist that iron can be harmful, in part by promoting infection. Iron is likely to preferentially benefit children with iron deficiency anemia, but identification of such children is challenging. Hepcidin is the hormone that regulates body iron levels and inhibits absorption of iron from the diet. Hepcidin concentrations are generally low in iron deficiency but are raised in iron-replete individuals and are also increased by infection. In a new study, Pasricha et al. set out to investigate whether hepcidin measurements would enable targeting of interventions to children who need iron but who are also able to absorb it. They tested this by measuring hepcidin in three cohorts of preschool African children from The Gambia and Tanzania. Single cutoffs of hepcidin concentrations efficiently identified children with iron deficiency, distinguished between iron deficiency anemia and anemia due to infection and inflammation, and predicted which children would incorporate >20% of an oral iron dose into their red blood cells. Thus, hepcidin is a critical determinant of iron homeostasis and may be a useful marker to guide diagnosis of anemia and enable screen-and-treat iron supplementation programs. Childhood anemia is a major global health problem resulting from multiple causes. Iron supplementation addresses iron deficiency anemia but is undesirable for other types of anemia and may exacerbate infections. The peptide hormone hepcidin governs iron absorption; hepcidin transcription is mediated by iron, inflammation, and erythropoietic signals. However, the behavior of hepcidin in populations where anemia is prevalent is not well established. We show that hepcidin measurements in 1313 African children from The Gambia and Tanzania (samples taken in 2001 and 2008, respectively) could be used to identify iron deficiency anemia. A retrospective secondary analysis of published data from 25 Gambian children with either postmalarial or nonmalarial anemia demonstrated that hepcidin measurements identified individuals who incorporated >20% oral iron into their erythrocytes. Modeling showed that this sensitivity of hepcidin expression at the population level could potentially enable simple groupings of individuals with anemia into iron-responsive and non–iron-responsive subtypes and hence could guide iron supplementation for those who would most benefit.

Iron metabolism and malaria.

Recent evidence from a large, randomized, controlled trial has suggested that the universal administration of iron to children in malaria-endemic areas is associated with an increase in adverse health outcomes. The purpose of this paper is to summarize the available ecologic and intervention trials related to iron and malaria in children, and to set these against current knowledge of the biology of host–pathogen interactions involving iron metabolism. We conclude that, although not fully consistent, the balance of evidence confirms that administration of iron (usually in combination with folic acid) increases the incidence of malaria when given without prophylaxis and in the absence of universal access to treatment. The mechanisms by which additional iron can benefit the parasite are far from clear. There is evidence to suggest that the apparent detrimental effect of iron supplementation may vary according to levels of antecedent iron status, the presence of hemoglobinopathies and glucose-6-phosphate dehydrogenase (G6PD) deficiency, and other host genetic variants, such as variants in haptoglobin. The effects of malaria on host iron metabolism are also reviewed and reveal that the key cause of malaria-induced anemia is a maldistribution of iron and suppression of erythropoiesis rather than an exacerbation of gross iron deficiency. We tentatively conclude that, if it is to be recommended, universal iron supplementation in malarious areas should only be considered in conjunction with some form of prophylaxis (e.g., intermittent preventive therapy [IPT]) or in the context of good health services with ready access to facilities for malaria diagnosis and treatment. An alternative approach would be to screen for anemia and target supplementation only to anemic children. With regard to treatment, there is good evidence that iron supplementation should be withheld until the treatment schedule is complete, both because iron may inhibit treatment and because the absorption of oral iron is blocked by the inflammatory response.

Iron incorporation and post-malaria anaemia

Background Iron supplementation is employed to treat post-malarial anaemia in environments where iron deficiency is common. Malaria induces an intense inflammatory reaction that stalls reticulo-endothelial macrophagal iron recycling from haemolysed red blood cells and inhibits oral iron absorption, but the magnitude and duration of these effects are unclear. Methodology/Principal Findings We examined the red blood cell incorporation of oral administered stable isotopes of iron and compared incorporation between age matched 18 to 36 months old children with either anaemia post-malaria (n = 37) or presumed iron deficiency anaemia alone (n = 36). All children were supplemented for 30 days with 2 mg/kg elemental iron as liquid iron sulphate and administered 57Fe and 58Fe on days 1 and 15 of supplementation respectively. 57Fe and58Fe incorporation were significantly reduced (8% vs. 28%: p<0.001 and 14% vs. 26%: p = 0.045) in the malaria vs. non-malaria groups. There was a significantly greater haemoglobin response in the malaria group at both day 15 (p = 0.001) and 30 (p<0.000) with a regression analysis estimated greater change in haemoglobin of 7.2 g/l (s.e. 2.0) and 10.1 g/l (s.e. 2.5) respectively. Conclusion/Significance Post-malaria anaemia is associated with a better haemoglobin recovery despite a significant depressant effect on oral iron incorporation which may indicate that early erythropoetic iron need is met by iron recycling rather than oral iron. Supplemental iron administration is of questionable utility within 2 weeks of clinical malaria in children with mild or moderate anaemia.

Haplotype Association between Haptoglobin (Hp2) and Hp Promoter SNP (A-61C) May Explain Previous Controversy of Haptoglobin and Malaria Protection

Background Malaria is one of the strongest recent selective pressures on the human genome, as evidenced by the high levels of varying haemoglobinopathies in human populations–despite the increased risk of mortality in the homozygous states. Previously, functional polymorphisms of Hp, coded by the co-dominant alleles Hp1 and Hp2, have been variously associated with several infectious diseases, including malaria susceptibility. Methodology/Principal Findings Risk of a clinical malarial episode over the course of a malarial transmission season was assessed using active surveillance in a cohort of Gambian children aged 10–72 months. We report for the first time that the major haplotype for the A-61C mutant allele in the promoter of haptoglobin (Hp)–an acute phase protein that clears haemoglobin released from haemolysis of red cells–is associated with protection from malarial infection in older children, (children aged ≥36 months, >500 parasites/ul and temperature >37.5°C; OR = 0.42; [95% CI 0.24–0.73] p = 0.002) (lr test for interaction, <36 vs ≥36 months, p = 0.014). Protection was also observed using two other definitions, including temperature >37.5°C, dipstick positive, plus clinical judgement of malaria blinded to dipstick result (all ages, OR = 0.48, [95% CI 0.30–0.78] p = 0.003; ≥36 months, OR = 0.31, [95% CI 0.15–0.62] p = 0.001). A similar level of protection was observed for the known protective genetic variant, sickle cell trait (HbAS). Conclusions/Significance We propose that previous conflicting results between Hp phenotypes/genotypes and malaria susceptibility may be explained by differing prevalence of the A-61C SNP in the populations studied, which we found to be highly associated with the Hp2 allele. We report the -61C allele to be associated with decreased Hp protein levels (independent of Hp phenotype), confirming in vitro studies. Decreased Hp expression may lead to increased oxidant stress and increased red cell turnover, and facilitate the development of acquired immunity, similar to a mechanism suggested for sickle cell trait.

Comparison of an enzyme immunoassay for the detection of Helicobacter pylori antigens in the faeces with the urea breath test.

BACKGROUND Current diagnostic tests for Helicobacter pylori are invasive (endoscopy) or indirect (urea breath test, serology). AIMS To evaluate a new enzyme immunoassay (EIA) which detects H pyloriantigens in faeces, by comparing its sensitivity and specificity in children with the 13C urea breath test (UBT). METHODS A total of 119 children underwent a UBT and provided a faecal sample for antigen testing within seven days. After an overnight fast each child provided a pretest breath sample, and samples at 30 and 40 minutes after ingestion of 100 mg 13C labelled urea. 13C enrichment of breath was measured by isotope ratio mass spectrometry. Faeces were stored at −70°C until antigen testing, using the EIA. Samples were read spectrophotometrically at 450 nm and results were interpreted using recommended cut offs of optical density <0.14 as negative, ⩾0.16 as positive, with ⩾0.14 and <0.16 representing equivocal results. Sensitivity and specificity were calculated using the manufacturers cut off compared with UBT. RESULTS Sensitivity and specificity were 88% and 82%, respectively. Negative and positive predictive values were 97% and 58%. CONCLUSIONS The EIA is an alternative, non-invasive, and easy to use method for the detection ofH pylori in children. Its high negative predictive value suggests a role in screening out uninfected children.

Micronutrient supplementation and infection: a double-edged sword?

Single-nutrient supplementation targeted at specific population groups has become an increasingly popular strategy to combat micronutrient malnutrition. This population-based approach is a pragmatic response to limited resources and assumes that, within a targeted group, the diagnosis of the nutrient deficiency is secure, its prevalence is clinically significant, and the benefits of supplementation outweigh the risks. The introduction of routine vitamin A supplementation to children in developing countries has encouraged this approach and the development of further micronutrient supplementation programs. The population-based approach, by definition, ignores individual variation in response caused by either genetic differences or clinical conditions, such as the presence of ongoing infection. The acute inflammatory response is a systemic adaptation that optimizes the immune response to an infection. Cytokines, such as interleukins (IL) 1 and 2 and tumour necrosis factor are released by leukocytes, macrophages, and mast cells in response to tissue injury and act on an array of cells to increase vascular permeability, to activate complement, and to promote chemotaxis. Plasma protein concentrations may increase, as with the positive acute-phase proteins C-reactive protein, serum amyloid A, -1-antitrypsin, fibrinogen, and ferritin, or they may decrease, as wth the negative acutephase proteins albumin, prealbumin, and retinol-binding protein. Inflammation also is associated with dramatic changes in plasma micronutrient concentrations. Changes in the micronutrient milieu may be the host’s attempt to optimize immune function and to deprive invading organisms of essential micronutrients for replication. Iron and zinc are important in the immune response, and different plasma concentrations are associated with either immunostimulatory or suppressant effects. Intracellular pathogens compete with host cells for available micronutrients. Either micronutrient deficiency or supplementation can impair metabolic adaptation to maintain an optimal microenvironment during inflammation. Immune dysfunction may result from this or from genetic variations in cellular mechanisms that control micronutrient flux and competition. In this review, we first describe the key roles of iron and zinc in immune function and the effect of inflammation on their plasma concentrations. We next discuss the insights gained from clinical studies of children in developing countries in whom the metabolic response of plasma iron and zinc to inflammation has been disturbed by either deficiency or supplementation. Finally, we outline some newly characterized cellular mechanisms involved in transporting and in competing for these ions and demonstrate their clinical relevance. In reviewing the literature on iron or zinc supplementation and infection, we emphasize the variability of the immune response to supplementation and attempt to identify groups in which this variability is high.

Iron delocalisation in the pathogenesis of malarial anaemia.

There is consensus that the pathophysiology of malaria-associated anaemia is multifactorial, but the precise mechanisms behind many of the haematological changes during malaria remain unclear. In this review, we attempt to build a composite picture of the pathophysiology of malarial anaemia using evidence from experimental, human and animal studies. We propose that cytokine- and hepcidin-mediated iron delocalisation, a principal mechanism in the anaemia of inflammation, plays an important role in the aetiology of malarial anaemia, and can explain some of the clinical and laboratory findings. These mechanisms interact with other aetiological determinants, such as dietary iron and micronutrient supply, helminth load, other infections and genetic variation, in determining the severity and associated features of anaemia. We suggest that iron delocalisation as a mechanism for malarial anaemia could be exploited for the development of alternative therapeutic strategies for post-malaria anaemia.

Haptoglobin genotype, anaemia and malaria in Gambian children

Objective  To retest our previous finding that the haptoglobin (Hp) 22 genotype is associated with seasonal anaemia, and to investigate the role of malaria in this effect.

Haptoglobin and Sickle Cell Polymorphisms and Risk of Active Trachoma in Gambian Children

Background Susceptibility and resistance to trachoma, the leading infectious cause of blindness, have been associated with a range of host genetic factors. In vitro studies of the causative organism, Chlamydia trachomatis, demonstrate that iron availability regulates its growth, suggesting that host genes involved in regulating iron status and/or availability may modulate the risk of trachoma. The objective was to investigate whether haptoglobin (Hp) haplotypes constructed from the functional polymorphism (Hp1/Hp2) plus the functional promoter SNPs -61A-C (rs5471) and -101C-G (rs5470), or sickle cell trait (HbAS, rs334) were associated with risk of active trachoma when stratified by age and sex, in rural Gambian children. Methodology and Principal Findings In two cross sectional surveys of children aged 6–78 months (n = 836), the prevalence of the clinical signs of active trachoma was 21.4%. Within boys, haplotype E (-101G, -61A, Hp1), containing the variant allele of the -101C-G promoter SNP, was associated with a two-fold increased risk of active trachoma (OR = 2.0 [1.17–3.44]). Within girls, an opposite association was non-significant (OR = 0.58 [0.32–1.04]; P = 0.07) and the interaction by sex was statistically significant (P = 0.001). There was no association between trachoma and HbAS. Conclusions These data indicate that genetic variation in Hp may affect susceptibility to active trachoma differentially by sex in The Gambia.