br Materials and Methods br Discussion Both malaria

Materials and Methods
Discussion Both malaria and ID are important public health problems in African children. In the current study 12% of children were parasitaemic and 46% had ID. Hepcidin concentrations fell rapidly in infancy and then more slowly with increasing age, while female infants had higher concentrations than males. We found that asymptomatic malaria was associated with significantly elevated hepcidin concentrations, which were proportional to parasite density and modified by age, inflammation, and the presence of ID. Furthermore, hepcidin was positively associated with antibody titres to P. falciparum antigens. Concentrations fell rapidly and then slowly after treatment of clinical malaria, but were not altered by non-malarial febrile illnesses. Nevertheless, hepcidin concentrations did not predict the subsequent risk of malaria or other febrile illnesses. We found a non-linear association between age and hepcidin. In agreement with a study in 3–12-month old Zimbabwean infants (Mupfudze et al., 2014), hepcidin concentrations were the highest in the youngest children and decreased dramatically in infancy (Fig. 2A). We add to this that hepcidin concentrations are the highest in the first 3months of life. However, in diindolylmethane to European studies, which showed either no change or an increase in hepcidin with age (Sdogou et al., 2015; Cangemi et al., 2013), we found a slow decline in hepcidin with increasing age. These findings might be explained by age-specific differences in the strength of hepcidin stimuli (such as iron stores, erythropoietic drive, and inflammation) (Fig. 3), and we hypothesize that different environmental conditions may influence hepcidin signalling at varying ages in European children. In agreement with a study in Kenyan infants (Jaeggi et al., 2013), we found that hepcidin concentrations were about twice as high in female infants compared to males. Ferritin, sTfR and CRP explained 37·5%, 37·3%, and 10·2% of the variance in hepcidin respectively (p<0·0005 for each). In agreement with a study in Indonesian school children (de Mast et al., 2010), asymptomatic malaria was also associated with an increase in plasma hepcidin (explaining 7% of variance; p<0·0005), both in the presence and absence of inflammation, suggesting that malaria regulates hepcidin via non-inflammatory, as well as inflammatory pathways (Armitage et al., 2009). We add that hepcidin levels are proportional to parasite density in asymptomatic infection, as observed in febrile malaria (Howard et al., 2007; Casals-Pascual et al., 2012), but not in severe infection (Burte et al., 2013). Hepcidin concentrations were markedly higher in younger compared to older parasitized children, suggesting that iron absorption is more likely to be impaired in younger children. Finally, among children with parasitaemia, hepcidin levels were markedly lower in those with ID than those without ID, suggesting that malaria-mediated up-regulating stimuli may be overruled by iron demand and erythropoietic stimuli down-regulating hepcidin synthesis. Thus, children with ID and parasitaemia might be in danger of receiving iron if hepcidin-guided supplementation was implemented without malaria screening. Antibodies to malaria antigens are sensitive biomarkers of population-level malaria exposure and may be useful surveillance tools for malaria control and elimination (Elliott et al., 2014). We investigated whether malaria antibodies might reflect malaria-induced hepcidin levels. We found that plasma hepcidin concentrations were strongly positively associated with antibodies to P. falciparum antigens in age-adjusted models. For AMA1 and MSP2 we found interactions between age and antibody titres so that in younger children a small increase in antibody titres was associated with a large increase in hepcidin concentrations, while in older children increased antibody titres had little effect on hepcidin. This agrees with our study showing that these antibodies act as measures of exposure in younger children with lower immunity and as measures of protective immunity in older children with higher antibody titres (Stanisic et al., 2015). By contrast antibodies to schizont extract were strongly positively associated with hepcidin concentrations regardless of age, which might be explained by their shorter half-life and high rates of sero-reversion at all ages (Ondigo et al., 2014). Taken together, these data suggest that sero-surveillance tools may be of use to identify population-level malaria-induced hepcidin, particularly in younger children, with relevance to programmes to restore healthy iron status.