Effect of preventive supplementation with zinc and other micronutrients on malaria and diarrhoeal morbidity in African children

Background: Zinc is important for innate and adaptive immune responses to infection. Preventive zinc supplementation has been shown to reduce the incidence of acute diarrhoea by 20%. Few trials have evaluated its effect against malaria. Because trial results for both outcomes are inconsistent, research priorities must shift from studies to measure efficacy to identifying factors that determine the magnitude of the effect of zinc supplementation. We hypothesized that protection by zinc supplementation depends on concomitant supplementation with other nutrients. Objectives: Specific objectives were: a) to assess the effect of supplementation with zinc, alone or in combination with other nutrients, on the rates of malaria (primary objective); b) to assess intervention effects on rates of diarrhoea and other common diseases; c) to identify factors that determine the magnitude of the effect of the interventions. Our studies also provided an opportunity to assess effects of α+-thalassaemia on malaria and malaria-associated anaemia. This haemoglobin disorder is highly prevalent in eastern Africa and that has recently been reported to protect against severe malaria. Methods: In a highly malaria-endemic area in rural Tanzania, we randomised children (n=612) aged 6-60 months with height-for-age z-score ≤ –1.5 SD to daily supplementation with: a) zinc, vitamins and other mineral elements (‘multi-nutrients’); b) zinc; c) multi-nutrients without zinc; or d) placebo. Those with Plasmodium infection at baseline were treated. Field staff and participants were blinded to treatment. Sick children were detected and evaluated in a research clinic. The primary outcome, an episode of malaria, was pre-defined as current Plasmodium antigenaemia in children with guardian-reported fever and any of the following: a) confirmed fever (axillary temperature ≥ 37.5 °C), or b) unconfirmed fever with inflammation (whole blood C-reactive protein concentrations ≥ 8 mg/L), separated by at least 14 days from a previous malaria episode. Results: The primary analysis included 1,572 episodes of malaria and 526 child-years of observation. The prevalence of zinc deficiency (plasma zinc concentration inflammation (plasma C-reactive protein concentration prevalence was dramatically reduced by zinc supplementation. We found no evidence that concurrent supplementation with multi-nutrients influenced the magnitude of the effect of zinc on rates of malaria or diarrhoea, so that marginal effects will be presented in the remainder of this summary. Although we found no evidence that zinc alone protected against malaria, it reduced rates of diarrhoea by 24% (95% CI: 4%–40%) and of episodes of fever without localising signs by 25% (4%–43%), two disorders with mutually exclusive case definitions. We found no effect of multi-nutrients on the overall rate of malaria episodes, regardless the case definition used, but the effect estimate was likely underestimated by children becoming asymptomatically infected in the course of the intervention period. In the first 100 days of intervention, and in the analysis of first events, supplementation with multi-nutrients, with or without zinc, increased the hazard of malaria by one-third. In addition, subgroup analysis indicated that this effect depended strongly on age and iron status at baseline, with rates of episodes with parasite densities > 10,000 parasites/ μL increasing by 27 % (1%-61%) and 53% (11%–111%) in the youngest children (6-17 months) and in children with iron deficiency, whilst there was no evident effect in older children or those without iron deficiency (p-values for interaction: 0.02 and 0.007). Despite the increase in malaria rates, the children who had the lowest haemoglobin concentrations during malaria (those aged 6-17 months) were better able to maintain their haemoglobin concentrations when having received multi-nutrients. Direct epidemiological evidence is lacking, however, if and under what conditions the higher haemoglobin concentrations during malaria (and expected reduced risk of death due to severe malarial anemia) outweigh the possible increase in other potentially lethal disease manifestations. Multi-nutrient supplementation seemed to increase the rate of diarrhoea by 19% (–6% to 50%). Subgroup analysis indicated that this effect depended on Giardia intestinalis infection at baseline (p-values for interaction: 0.03): in those without multi-nutrients, infection was associated with a reduction in rates of diarrhoea by 68% (34%-85%), whilst there was no evidence for such protection in those receiving multi-nutrients. Similar effect modification was found for fever without localizing signs. Of 612 children in the trial, 50% had normal genotype, whilst 41% and 9% were heterozygote and homozygous, respectively, for α+-thalassaemia. We found no evidence of group differences in malaria rates between genotypes. Subgroup analysis suggested, however, that the effect of α+-thalassemia depended on age. Thus in children below 18 months, malaria rates were increased by 30% (2%–65%) in heterozygotes, whereas they were decreased by 20% (5%–32%) in older children (p-value for interaction: 0.001). Similar patterns were found for homozygotes, even though estimates were less precise due the smaller numbers of children in this age class. Based on data from a pilot survey and a study in Kenya, we found that children with α+- thalassaemia (particularly homozygotes) were protected against the decline in haemoglobin concentration associated with mild to asymptomatic infections, particularly when these infections were accompanied by inflammation. Interpretation and conclusions for policies: We found no evidence that addition of vitamins and other mineral elements increased the health benefits of zinc supplements. The beneficial effects of zinc described in this thesis strengthen the case for scaling up zinc interventions in deficient populations of African children, without concerns that it will cause adverse effects due to malaria. Multi-nutrient supplementation may be unsafe in malaria-endemic areas, particularly in young children with iron deficiency. Thus the recommendation by the World Health Organization that iron supplements should be administered routinely to iron-deficient infants in settings with adequate access to anti-malarial treatment is insufficiently supported by evidence and should be reconsidered. Our results underscore that supplementation or home fortification, even when targeting deficient subgroups in settings with access to adequate primary care, should not be recommended in malariaendemic areas until their safety has been demonstrated. <br/

An Unattended Mask makes an Attended Target Disappear

In pattern masking, the target and mask are presented at the same location and follow one another very closely in time. When the observer attends to the target, he or she must also attend to the mask, as the switching time for attention is quite slow. In a series of experiments, we present mask–target–mask sequences staggered in time and location (Cavanagh, Holcombe, & Chou, 2008) that allow participants to attentively track the target location without attending to the masks. The results show that the strength of masking is on average unaffected by the removal of attention from the masks. Moreover, after isolating the target location perceptually with moving attention, it is clear that the target, when at threshold, has not been degraded or integrated with a persisting mask but it has vanished. We also show that the strength of masking is unaffected by the lateral spacing between adjacent target and mask sequences until the spacing is so large that the apparent motion driving the attentive tracking breaks down. Finally, we compare the effect of the pre- and postmask and find that the premask is responsible for the larger part of the masking

An unattended mask makes an attended target disappear

In pattern masking, the target and mask are presented at the same location and follow one another very closely in time. When the observer attends to the target, he or she must also attend to the mask, as the switching time for attention is quite slow. In a series of experiments, we present mask-target-mask sequences staggered in time and locatio

Tumour necrosis factor allele variants and their association with the occurrence and severity of malaria in African children: a longitudinal study.

BACKGROUND: Tumour necrosis factor (TNF) is central to the immune response to Plasmodium infection. Its plasma concentration is influenced by allele variants in the promoter region of TNF. The study's objectives were to assess TNF allele variants (TNF(-1031), TNF(-308)): (1) modulation of malaria rates in young Tanzanian children; (2) modulation of the severity of malaria as indicated by haemoglobin concentrations at the time of presentation with febrile episodes; and (3) the association between Plasmodium infection and haemoglobin concentration in symptomless parasite carriers. METHODS: Data from a placebo-controlled trial in which 612 Tanzanian children aged 6-60 months with height-for-age z-score in the range -3 SD to 1.5 SD was utilised. Those with Plasmodium infection at baseline were treated with artemether-lumefantrine. An episode of malaria was predefined as current Plasmodium infection with an inflammatory response (axillary temperature ≥37.5°C or whole blood C-reactive protein concentration ≥8 mg/L) in children reported sick. Linkage disequilibrium (LD) pattern assessment as well as haplotype analysis was conducted using HAPLOVIEW. Cox regression models used in the primary analysis accounted for multiple episodes per child. RESULTS: Genotyping of 94.9% (581/612) children for TNF(-1031) (TNF(-1031)T>C); allele frequency was 0.39. Corresponding values for rs1800629 (TNF(-308)G>A) were 95.4% (584/612) and 0.17. Compared to the wild type genotype (TT), malaria rates were increased in the TNF -1031CC genotype (hazard ratio, HR [95% CI]: 1.41 [1.01‒1.97] and 1.31 [0.97‒1.76] for crude analysis and adjusting for pre-specified baseline factors, respectively) but decreased in those with the TNF(-308)AA genotype (corresponding HR: 0.13 [0.02‒0.63] and 0.16 [0.04‒0.67]). These associations were weaker when analysing first episodes of malaria (P value -0.59 and 0.38, respectively). No evidence that allele variants of TNF(-1031) and TNF(-308) affected haemoglobin concentration at first episode of malaria, or that they modified the association between Plasmodium infection and haemoglobin concentrations at baseline was observed. CONCLUSION: In this cohort of Tanzanian children, the TNF (-1031)CC genotype was associated with increased rates of malarial episodes, whereas the TNF(-308)AA genotype was associated with decreased rates

Effect of Preventive Supplementation with Zinc and other Micronutrients on Non-Malarial Morbidity in Tanzanian Pre-School Children: A Randomized Trial.

The efficacy of preventive zinc supplementation against diarrhea and respiratory illness may depend on simultaneous supplementation with other micronutrients. We aimed to assess the effect of supplementation with zinc and multiple micronutrients on diarrhea and other causes of non-malarial morbidity. Rural Tanzanian children (n = 612) aged 6-60 months and with height-for-age z-score < -1.5 SD were randomized to daily supplementation with zinc (10 mg) alone, multi-nutrients without zinc, multi-nutrients with zinc, or placebo. Children were followed for an average of 45 weeks. During follow-up, we recorded morbidity episodes. We found no evidence that concurrent supplementation with multi-nutrients influenced the magnitude of the effect of zinc on rates of diarrhea, respiratory illness, fever without localizing signs, or other illness (guardian-reported illness with symptoms involving skin, ears, eyes and abscesses, but excluding trauma or burns). Zinc supplementation reduced the hazard rate of diarrhea by 24% (4%-40%). By contrast, multi-nutrients seemed to increase this rate (HR; 95% CI: 1.19; 0.94-1.50), particularly in children with asymptomatic Giardia infection at baseline (2.03; 1.24-3.32). Zinc also protected against episodes of fever without localizing signs (0.75; 0.57-0.96), but we found no evidence that it reduced the overall number of clinic visits. We found no evidence that the efficacy of zinc supplements in reducing diarrhea rates is enhanced by concurrent supplementation with other micronutrients. By reducing rates of fever without localizing signs, supplementation with zinc may reduce inappropriate drug use with anti-malarial medications and antibiotics. ClinicalTrials.gov NCT00623857

Effect of α+-thalassaemia on episodes of fever due to malaria and other causes: a community-based cohort study in Tanzania

It is controversial to what degree α(+)-thalassaemia protects against episodes of uncomplicated malaria and febrile disease due to infections other than Plasmodium. In Tanzania, in children aged 6-60 months and height-for-age z-score < -1.5 SD (n = 612), rates of fevers due to malaria and other causes were compared between those with heterozygous or homozygotes α(+)-thalassaemia and those with a normal genotype, using Cox regression models that accounted for multiple events per child. The overall incidence of malaria was 3.0/child-year (1, 572/526 child-years); no differences were found in malaria rates between genotypes (hazard ratios, 95% CI: 0.93, 0.82-1.06 and 0.91, 0.73-1.14 for heterozygotes and homozygotes respectively, adjusted for baseline factors that were predictive for outcome). However, this association strongly depended on age: among children aged 6-17 months, those with α(+)-thalassaemia experienced episodes more frequently than those with a normal genotype (1.30, 1.02-1.65 and 1.15, 0.80-1.65 for heterozygotes and homozygotes respectively), whereas among their peers aged 18-60 months, α(+)-thalassaemia protected against malaria (0.80, 0.68-0.95 and 0.78, 0.60-1.03; p-value for interaction 0.001 and 0.10 for hetero- and homozygotes respectively). No effect was observed on non-malarial febrile episodes. In this population, the association between α(+)-thalassaemia and malaria depends on age. Our data suggest that protection by α(+)-thalassaemia is conferred by more efficient acquisition of malaria-specific immunity

Alterations in early cytokine-mediated immune responses to Plasmodium falciparum infection in Tanzanian children with mineral element deficiencies: a cross-sectional survey

BACKGROUND: Deficiencies in vitamins and mineral elements are important causes of morbidity in developing countries, possibly because they lead to defective immune responses to infection. The aim of the study was to assess the effects of mineral element deficiencies on early innate cytokine responses to Plasmodium falciparum malaria. METHODS: Peripheral blood mononuclear cells from 304 Tanzanian children aged 6-72 months were stimulated with P. falciparum-parasitized erythrocytes obtained from in vitro cultures. RESULTS: The results showed a significant increase by 74% in geometric mean of TNF production in malaria-infected individuals with zinc deficiency (11% to 240%; 95% CI). Iron deficiency anaemia was associated with increased TNF production in infected individuals and overall with increased IL-10 production, while magnesium deficiency induced increased production of IL-10 by 46% (13% to 144%) in uninfected donors. All donors showed a response towards IL-1beta production, drawing special attention for its possible protective role in early innate immune responses to malaria. CONCLUSIONS: In view of these results, the findings show plasticity in cytokine profiles of mononuclear cells reacting to malaria infection under conditions of different micronutrient deficiencies. These findings lay the foundations for future inclusion of a combination of precisely selected set of micronutrients rather than single nutrients as part of malaria vaccine intervention programmes in endemic countries

Associations between Common Variants in Iron-Related Genes with Haematological Traits in Populations of African Ancestry.

BACKGROUND: Large genome-wide association (GWA) studies of European ancestry individuals have identified multiple genetic variants influencing iron status. Studies on the generalizability of these associations to African ancestry populations have been limited. These studies are important given interethnic differences in iron status and the disproportionate burden of iron deficiency among African ancestry populations. METHODS: We tested the associations of 20 previously identified iron status-associated single nucleotide polymorphisms (SNPs) in 628 Kenyans, 609 Tanzanians, 608 South Africans and 228 African Americans. In each study, we examined the associations present between 20 SNPs with ferritin and haemoglobin, adjusting for age, sex and CRP levels. RESULTS: In the meta analysis including all 4 African ancestry cohorts, we replicated previously reported associations with lowered haemoglobin concentrations for rs2413450 (β = -0.19, P = 0.02) and rs4820268 (β = -0.16, P = 0.04) in TMPRSS6. An association with increased ferritin concentrations was also confirmed for rs1867504 in TF (β = 1.04, P = <0.0001) in the meta analysis including the African cohorts only. CONCLUSIONS: In all meta analyses, we only replicated 4 of the 20 single nucleotide polymorphisms reported to be associated with iron status in large GWA studies of European ancestry individuals. While there is now evidence for the associations of a number of genetic variants with iron status in both European and African ancestry populations, the considerable lack of concordance highlights the importance of continued ancestry-specific studies to elucidate the genetic underpinnings of iron status in ethnically diverse populations

Effect of nutrient deficiencies on in vitro Th1 and Th2 cytokine response of peripheral blood mononuclear cells to Plasmodium falciparum infection

BACKGROUND: An appropriate balance between pro-inflammatory and anti-inflammatory cytokines that mediate innate and adaptive immune responses is required for effective protection against human malaria and to avoid immunopathology. In malaria endemic countries, this immunological balance may be influenced by micronutrient deficiencies. METHODS: Peripheral blood mononuclear cells from Tanzanian preschool children were stimulated in vitro with Plasmodium falciparum-parasitized red blood cells to determine T-cell responses to malaria under different conditions of nutrient deficiencies and malaria status. RESULTS: The data obtained indicate that zinc deficiency is associated with an increase in TNF response by 37%; 95% CI: 14% to 118% and IFN-gamma response by 74%; 95% CI: 24% to 297%. Magnesium deficiency, on the other hand, was associated with an increase in production of IL-13 by 80%; 95% CI: 31% to 371% and a reduction in IFN-gamma production. These results reflect a shift in cytokine profile to a more type I cytokine profile and cell-cell mediated responses in zinc deficiency and a type II response in magnesium deficiency. The data also reveal a non-specific decrease in cytokine production in children due to iron deficiency anaemia that is largely associated with malaria infection status. CONCLUSIONS: The pathological sequels of malaria potentially depend more on the balance between type I and type II cytokine responses than on absolute suppression of these cytokines and this balance may be influenced by a combination of micronutrient deficiencies and malaria status

Plasma concentration of parasite DNA as a measure of disease severity in falciparum malaria.

In malaria-endemic areas, Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children (111 with uncomplicated malaria and 113 with severe malaria) and 211 Asian adults (100 with uncomplicated malaria and 111 with severe malaria) presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples