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/

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

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 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

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

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

Effect of Supplementation with Zinc and Other Micronutrients on Malaria in Tanzanian Children: A Randomised Trial

Hans Verhoef and colleagues report findings from a randomized trial conducted among Tanzanian children at high risk for malaria. Children in the trial received either daily oral supplementation with either zinc alone, multi-nutrients without zinc, multi-nutrients with zinc, or placebo. The investigators did not find evidence from this study that zinc or multi-nutrients protected against malaria episodes

Effect of Manganese in Test Media on In Vitro Susceptibility of Enterobacteriaceae and Acinetobacter baumannii to Tigecycline

Item does not contain fulltextWe assessed the effect of increasing manganese concentrations in test media (0.001 to 1,024 mg/liter) on MICs of tigecycline. For both broth microdilution (BMD) and Etests, this effect was negligible for physiological concentrations, but MICs increased when concentrations exceeded 8 mg/liter. Susceptibility testing should be performed on media with standardized low manganese content.1 september 201

Protection against Diarrhea Associated with Giardia intestinalis Is Lost with Multi-Nutrient Supplementation: A Study in Tanzanian Children

Giardia intestinalis is a well-known cause of diarrhea in industrialized countries. In children in developing countries, asymptomatic infections are common and their role as cause of diarrhea has been questioned. In a cohort of rural Tanzanian pre-school children, we assessed the association between the presence of Giardia at baseline and subsequent diarrhea risk. The study was conducted in the context of a randomised trial assessing the effect of supplementation with zinc and other micro-nutrients on malaria, and half of the children daily received a multi-nutrient supplement. Surprisingly, we found that the presence of Giardia at baseline was associated with a substantial reduction in diarrhea risk. Multivariate statistical analysis showed that this protection could not be explained by differences in age or walking distance to the dispensary between children with and without Giardia. Because we cannot exclude that children differed in other (unmeasured) characteristics, we cannot draw firm conclusions about the causality of the observed association, but our findings support the view that the parasite is not an important cause of diarrhea in highly endemic settings. Striking was that the Giardia-associated protection was lost when children received multi-nutrients. Our data do not provide information about the mechanisms involved, but suggest that multi-nutrients may influence the compositionor pathogenicity of intestinal biota

α-Thalassemia Impairs the Cytoadherence of Plasmodium falciparum-Infected Erythrocytes

α-Thalassemia results from decreased production of α-globin chains that make up part of hemoglobin tetramers (Hb; α(2)β(2)) and affects up to 50% of individuals in some regions of sub-Saharan Africa. Heterozygous (-α/αα) and homozygous (-α/-α) genotypes are associated with reduced risk of severe Plasmodium falciparum malaria, but the mechanism of this protection remains obscure. We hypothesized that α-thalassemia impairs the adherence of parasitized red blood cells (RBCs) to microvascular endothelial cells (MVECs) and monocytes--two interactions that are centrally involved in the pathogenesis of severe disease.We obtained P. falciparum isolates directly from Malian children with malaria and used them to infect αα/αα (normal), -α/αα and -α/-α RBCs. We also used laboratory-adapted P. falciparum clones to infect -/-α RBCs obtained from patients with HbH disease. Following a single cycle of parasite invasion and maturation to the trophozoite stage, we tested the ability of parasitized RBCs to bind MVECs and monocytes. Compared to parasitized αα/αα RBCs, we found that parasitized -α/αα, -α/-α and -/-α RBCs showed, respectively, 22%, 43% and 63% reductions in binding to MVECs and 13%, 33% and 63% reductions in binding to monocytes. α-Thalassemia was associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite's main cytoadherence ligand and virulence factor, on the surface of parasitized RBCs.Parasitized α-thalassemic RBCs show PfEMP1 display abnormalities that are reminiscent of those on the surface of parasitized sickle HbS and HbC RBCs. Our data suggest a model of malaria protection in which α-thalassemia ameliorates the pro-inflammatory effects of cytoadherence. Our findings also raise the possibility that other unstable hemoglobins such as HbE and unpaired α-globin chains (in the case of β-thalassemia) protect against life-threatening malaria by a similar mechanism