Linking human Dead end 1 (DND1) variants to male infertility employing zebrafish embryos.

STUDY QUESTION: Is the vertebrate protein Dead end (DND1) a causative factor for human infertility and can novel in vivo assays in zebrafish help in evaluating this? SUMMARY ANSWER: Combining patient genetic data with functional in vivo assays in zebrafish reveals a possible role for DND1 in human male fertility. WHAT IS KNOWN ALREADY: About 7% of the male population is affected by infertility but linking specific gene variants to the disease is challenging. The function of the DND1 protein was shown to be critical for germ cell development in several model organisms but a reliable and cost-effective method for evaluating the activity of the protein in the context of human male infertility is still missing. STUDY DESIGN, SIZE, DURATION: Exome data from 1305 men included in the Male Reproductive Genomics cohort were examined in this study. A total of 1114 of the patients showed severely impaired spermatogenesis but were otherwise healthy. Eighty-five men with intact spermatogenesis were included in the study as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: We screened the human exome data for rare, stop-gain, frameshift, splice site, as well as missense variants in DND1. The results were validated by Sanger sequencing. Immunohistochemical techniques and, when possible, segregation analyses were performed for patients with identified DND1 variants. The amino acid exchange in the human variant was mimicked at the corresponding site of the zebrafish protein. Using different aspects of germline development in live zebrafish embryos as biological assays, we examined the activity level of these DND1 protein variants. MAIN RESULTS AND THE ROLE OF CHANCE: In human exome sequencing data, we identified four heterozygous variants in DND1 (three missense and one frameshift variant) in five unrelated patients. The function of all of the variants was examined in the zebrafish and one of those was studied in more depth in this model. We demonstrate the use of zebrafish assays as a rapid and effective biological readout for evaluating the possible impact of multiple gene variants on male fertility. This in vivo approach allowed us to assess the direct impact of the variants on germ cell function in the context of the native germline. Focusing on the DND1 gene, we find that zebrafish germ cells, expressing orthologs of DND1 variants identified in infertile men, failed to arrive correctly at the position where the gonad develops and exhibited defects in cell fate maintenance. Importantly, our analysis facilitated the evaluation of single nucleotide variants, whose impact on protein function is difficult to predict, and allowed us to distinguish variants that do not affect the protein's activity from those that strongly reduce it and could thus potentially be the primary cause for the pathological condition. These aberrations in germline development resemble the testicular phenotype of azoospermic patients. LIMITATIONS, REASONS FOR CAUTION: The pipeline we present requires access to zebrafish embryos and to basic imaging equipment. The notion that the activity of the protein in the zebrafish-based assays is relevant for the human homolog is well supported by previous knowledge. Nevertheless, the human protein may differ in some respects from its homologue in zebrafish. Thus, the assay should be considered only one of the parameters used in defining DND1 variants as causative or non-causative for infertility. WIDER IMPLICATIONS OF THE FINDINGS: Using DND1 as an example, we have shown that the approach described in this study, relying on bridging between clinical findings and fundamental cell biology, can help to establish links between novel human disease candidate genes and fertility. In particular, the power of the approach we developed is manifested by the fact that it allows the identification of DND1 variants that arose de novo. The strategy presented here can be applied to different genes in other disease contexts. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the German Research Foundation, Clinical Research Unit, CRU326 'Male Germ Cells'. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Effect of three years' seasonal malaria chemoprevention on molecular markers of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine and amodiaquine in Ouelessebougou, Mali.

BACKGROUND: In 2012, seasonal malaria chemoprevention (SMC) was recommended as policy for malaria control by the World Health Organization (WHO) in areas of highly seasonal malaria transmission across the Sahel sub-region in Africa along with monitoring of drug resistance. We assessed the long-term impact of SMC on Plasmodium falciparum resistance to sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) over a 3-year period of SMC implementation in the health district of Ouelessebougou, Mali. METHODS: In 8 randomly selected sub-districts of Ouelessebougou, Mali, children aged 0-5 years were randomly selected during cross-sectional surveys at baseline (August 2014) and 1, 2 and 3 years post-SMC, at the beginning and end of the malaria transmission season. Blood smears and blood spots on filter paper were obtained and frequencies of mutation in P. falciparum genes related to resistance to SP and AQ (Pfdhfr, Pfdhps, Pfmdr1, and Pfcrt) were assessed by PCR amplification on individual samples and PCR amplification followed by deep sequencing on pooled (by site and year) samples. RESULTS: At each survey, approximately 50-100 individual samples were analysed by PCR amplification and a total of 1,164 samples were analysed by deep sequencing with an average read depth of 18,018-36,918 after pooling by site and year. Most molecular markers of resistance did not increase in frequency over the period of study (2014-2016). After 3 years of SMC, the frequencies of Pfdhps 540E, Pfdhps 437G and Pfcrt K76T remained similar compared to baseline (4.0 vs 1.4%, p = 0.41; 74.5 vs 64.6%, p = 0.22; 71.3 vs 67.4%, p = 0.69). Nearly all samples tested carried Pfdhfr 59R, and this proportion remained similar 3 years after SMC implementation (98.8 vs 100%, p = 1). The frequency of Pfmdr1 N86Y increased significantly over time from 5.6% at baseline to 18.6% after 3 years of SMC (p = 0.016). Results of pooled analysis using deep sequencing were consistent with those by individual analysis with standard PCR, but also indicated for the first time the presence of mutations at the Pfdhps A581G allele at a frequency of 11.7% after 2 years of SMC, as well as the Pfdhps I431V allele at frequencies of 1.6-9.3% following 1 and 2 years of SMC, respectively. CONCLUSION: Two and 3 years of SMC implementation were associated with increased frequency of the Pfmdr1 N86Y mutation but not Pfdhps 540E, Pfdhps 437G and Pfcrt K76T. The first-time detection of the Pfdhps haplotype bearing the I431V and A581G mutations in Mali, even at low frequency, warrants further long-term surveillance.

Malaria Infection Is Common and Associated With Perinatal Mortality and Preterm Delivery Despite Widespread Use of Chemoprevention in Mali: An Observational Study 2010 to 2014.

BACKGROUND: In malaria-endemic areas, pregnant women and especially first-time mothers are more susceptible to Plasmodium falciparum. Malaria diagnosis is often missed during pregnancy, because many women with placental malaria remain asymptomatic or have submicroscopic parasitemia, masking the association between malaria and pregnancy outcomes. Severe maternal anemia and low birthweight deliveries are well-established sequelae, but few studies have confirmed the relationship between malaria infection and severe outcomes like perinatal mortality in high transmission zones. METHODS: Pregnant women of any gestational age enrolled at antenatal clinic into a longitudinal cohort study in Ouelessebougou, Mali, an area of high seasonal malaria transmission. Follow-up visits included scheduled and unscheduled visits throughout pregnancy. Blood smear microscopy and polymerase chain reaction (PCR) analysis were employed to detect both microscopic and submicroscopic infections, respectively. Intermittent preventative treatment in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) was documented and prompt treatment regardless of symptoms given upon malaria diagnosis. RESULTS: Of the 1850 women followed through delivery, 72.6% of women received 2 or more IPTp-SP doses, 67.2% of women experienced at least 1 infection between enrollment up to and including delivery. Malaria infection increased the risks of stillbirth (adjusted hazard ratio [aHR] 3.87, 95% confidence interval [CI]: 1.18-12.71) and preterm delivery (aHR 2.41, 95% CI: 1.35-4.29) in primigravidae, and early neonatal death (death within 7 days) in secundigravidae and multigravidae (aHR 6.30, 95% CI: 1.41-28.15). CONCLUSIONS: Malaria treatment after diagnosis, alongside IPTp-SP, is insufficient to prevent malaria-related stillbirth, early neonatal death and preterm delivery (PTD). Although IPTp-SP was beneficial in Mali during the study period, new tools are needed to improve pregnancy outcomes. CLINICAL TRIALS REGISTRATION: NCT01168271.

Malaria is a cause of iron deficiency in African children.

Malaria and iron deficiency (ID) are common and interrelated public health problems in African children. Observational data suggest that interrupting malaria transmission reduces the prevalence of ID1. To test the hypothesis that malaria might cause ID, we used sickle cell trait (HbAS, rs334 ), a genetic variant that confers specific protection against malaria2, as an instrumental variable in Mendelian randomization analyses. HbAS was associated with a 30% reduction in ID among children living in malaria-endemic countries in Africa (n = 7,453), but not among individuals living in malaria-free areas (n = 3,818). Genetically predicted malaria risk was associated with an odds ratio of 2.65 for ID per unit increase in the log incidence rate of malaria. This suggests that an intervention that halves the risk of malaria episodes would reduce the prevalence of ID in African children by 49%.

Effect of 4 years of seasonal malaria chemoprevention on the acquisition of antibodies to Plasmodium falciparum antigens in Ouelessebougou, Mali.

BACKGROUND: More than 200 million people live in areas of highly seasonal malaria transmission where Seasonal Malaria Chemoprevention (SMC) with sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) was recommended in 2012 by WHO. This strategy is now implemented widely and protected more than 19 million children in 2018. It was previously reported that exposure to SMC reduced antibody levels to AMA1, MSP-142 and CSP, but the duration of exposure to SMC up to three 3 years, had no effect on antibody levels to MSP-142 and CSP. METHODS: In 2017, a cross-sectional survey was carried out 1 month after the last dose of SMC had been given to children aged 4-5 years randomly selected from areas where SMC had been given for 2 or 4 years during the malaria transmission season. A total of 461 children were enrolled, 242 children in areas where SMC had been implemented for 4 years and 219 children in areas where SMC had been implemented for 2 years. Antibody extracted from dry blood spots was used to measure IgG levels to the malaria antigens CSP, MSP-142 and AMA1 by ELISA. RESULTS: The prevalence of antibodies to MSP-142 was similar in children who had received SMC for 4 years compared to those who had received SMC for only 2 years (85.1 vs 86.0%, ajusted odd ratio (aOR) = 1.06, 95% confidence intervals (CI 0.62-1.80), p = 0.80). The prevalence of antibodies to AMA-1 and to CSP was not lower in children who received SMC for 4 years compared to those who had received SMC for only 2 years (95.3 vs 88.8%, aOR = 3.16, 95% CI 1.44-6.95, p = 0.004 for AMA-1; and 91.2 vs 81.9%, aOR = 3.14, 95% CI 1.70-5.76, p < 0.001 for CSP). Median antibody levels for anti-MSP-142 IgG were not significatively inferior in children who had received SMC for four rather than 2 years (0.88 (IQR: 0.64-1.15) and 0.95 ((0.68-1.15), respectively), anti-CSP (1.30 (1.00-1.56) and 1.17 (0.87-1.47)), and anti-AMA-1 (1.45 (1.24-1.68) and 1.41 (1.17-1.64)). CONCLUSION: In an area of high seasonal malaria transmission, children who had received SMC for 4 years did not had lower seropositivity or antibody levels to AMA1, MSP-142 and CSP compared to children who had received SMC for only 2 years suggesting that children who have received SMC for 4 years may not be more at risk of malaria after the cessation of SMC than children who have received SMC for a shorter period.

Impact of seasonal malaria chemoprevention on hospital admissions and mortality in children under 5 years of age in Ouelessebougou, Mali.

BACKGROUND: Seasonal malaria chemoprevention is widely implemented in Sahel and sub-Sahel countries in Africa. Few studies have assessed the impact of the SMC on hospital admission and death when it is implemented in the health system. This retrospective study assessed the impact of seasonal malaria chemoprevention (SMC) on hospitalizations and deaths of children under 5 years of age during the second year of implementation of SMC in the health district of Ouelessebougou in Mali. METHODS: In February 2017, a survey was conducted to assess hospital admissions and deaths in children under 5 years of age in two health sub-districts where SMC was implemented in 2015 and two health sub-districts where SMC was not implemented. The survey reviewed deaths and hospitalizations of children under 5, in the four health sub-districts. The crude and specific incidence rates of hospitalizations and deaths were determined in both groups and expressed per 1000 children per year. A negative binomial regression model and a Cox model were used to estimate the relative risks of hospitalization and death after adjusting for confounders. The R software was used for data analysis. RESULTS: A total of 6638 children under 5 years of age were surveyed, 2759 children in the SMC intervention areas and 3879 children in the control areas. All causes mortality rate per 1000 person-years was 8.29 in the control areas compared to 3.63 in the intervention areas; age and gender adjusted mortality rate ratio 0.44 (95% CI 0.22-0.91), p = 0.027. The incidence rate of all causes hospital admissions was 19.60 per 1000 person-years in the intervention group compared to 33.45 per 1000 person-years in the control group, giving an incidence rate ratio (IRR) adjusted for age and gender of 0.61 (95% CI 0.44-0.84), p = 0.003. CONCLUSION: The implementation of SMC was associated with a substantial reduction in hospital admissions and all-cause mortality. Trial registration ClinicalTrials.gov NCT02646410.

Effect of Seasonal Malaria Chemoprevention on Immune Markers of Exhaustion and Regulation.

BACKGROUND: Seasonal malaria chemoprevention (SMC) is a novel strategy to reduce malaria infections in children. Infection with Plasmodium falciparum results in immune dysfunction characterized by elevated expression of markers associated with exhaustion, such as PD1 and LAG3, and regulatory CD4+FOXP3+ T cells. METHODS: In the current study, the impact of seasonal malaria chemoprevention on malaria-induced immune dysfunction, as measured by markers associated with exhaustion and regulatory T cells, was explored by flow cytometry. RESULTS: Children that received seasonal malaria chemoprevention had fewer malaria episodes and showed significantly lower fold changes in CD4+PD1+ and CD4+PD1+LAG3+ compared to those that did not receive SMC. Seasonal malaria chemoprevention had no observable effect on fold changes in CD8 T cells expressing PD1 or CD160. However, children receiving SMC showed greater increases in CD4+FOXP3+ T regulatory cells compared to children not receiving SMC. CONCLUSIONS: These results provide important insights into the dynamics of malaria-induced changes in the CD4 T-cell compartment of the immune system and suggest that the reduction of infections due to seasonal malaria chemoprevention may also prevent immune dysfunction. CLINICAL TRIALS REGISTRATION: NCT02504918.

Optimal mode for delivery of seasonal malaria chemoprevention in Ouelessebougou, Mali: A cluster randomized trial.

BACKGROUND: Seasonal malaria chemoprevention (SMC), the administration of complete therapeutic courses of antimalarials to children aged 3-59 months during the malaria transmission season, is a new strategy recommended by the World Health Organization (WHO) for malaria control in Sahelian countries such as Mali with seasonal transmission. The strategy is a highly cost-effective approach to reduce malaria burden in these areas. Despite the substantial benefits of SMC on malaria infection and disease, the optimal approach to deliver SMC remains to be determined. While fixed-point delivery (FPD) and non-directly observed treatment (NDOT) by community health workers are logistically attractive, these need to be evaluated and compared to other modes of delivery for maximal coverage. METHODS: To determine the optimal mode fixed-point (FPD) vs door-to-door delivery (DDD); directly observed treatment (DOT) vs. non- directly observed treatment (NDOT)), 31 villages in four health sub-districts were randomized to receive three rounds of SMC with Sulfadoxine-pyrimethamine plus Amodiaquine (SP+AQ) at monthly intervals using one of the following methods: FPD+DOT; FPD+NDOT; DDD+DOT; DDD+NDOT. The primary endpoint was SMC coverage assessed by cross-sectional survey of 2,035 children at the end of intervention period. RESULTS: Coverage defined as the proportion of children who received all three days of SMC treatment during the three monthly rounds based information collected by interview (primary endpoint) was significantly higher in children who received SMC using DDD 74% (95% CI 69% - 80%) compared to FPD 60% (95% CI 50% - 70%); p = 0.009. It was similar in children who received SMC using DOT or NDOT 65%, (95% CI 55% - 76%) versus 68% (95% CI 57% - 79%); p = 0.72. CONCLUSIONS: In summary, door-to-door delivery of SMC provides better coverage than FPD. Directly observed therapy, which requires more time and resources, did not improve coverage with SMC. TRIAL REGISTRATION: ClinicalTrials.gov NCT02646410.

The Vertebrate Protein Dead End Maintains Primordial Germ Cell Fate by Inhibiting Somatic Differentiation.

Maintaining cell fate relies on robust mechanisms that prevent the differentiation of specified cells into other cell types. This is especially critical during embryogenesis, when extensive cell proliferation, patterning, and migration events take place. Here we show that vertebrate primordial germ cells (PGCs) are protected from reprogramming into other cell types by the RNA-binding protein Dead end (Dnd). PGCs knocked down for Dnd lose their characteristic morphology and adopt various somatic cell fates. Concomitantly, they gain a gene expression profile reflecting differentiation into cells of different germ layers, in a process that we could direct by expression of specific cell-fate determinants. Importantly, we visualized these events within live zebrafish embryos, which provide temporal information regarding cell reprogramming. Our results shed light on the mechanisms controlling germ cell fate maintenance and are relevant for the formation of teratoma, a tumor class composed of cells from more than one germ layer.

Systemic Inflammatory Response to Malaria During Pregnancy Is Associated With Pregnancy Loss and Preterm Delivery.

BACKGROUND: Pregnancy malaria (PM) is associated with a proinflammatory immune response characterized by increased levels of cytokines and chemokines such as tumor necrosis factor-α, interferon-γ, interleukin 10 (IL-10), and CXCL9. These changes are associated with poor outcomes including low birthweight delivery and maternal anemia. However, it is unknown if inflammatory pathways during malaria are related to pregnancy loss and preterm delivery (PTD). METHODS: Cytokine and chemokine levels were measured in maternal peripheral blood at enrollment, gestational week 30-32, and delivery, and in placental blood, of 638 women during a longitudinal cohort study in Ouelessebougou, Mali. Plasmodium falciparum infection was assessed by blood smear microscopy at all visits. RESULTS: PM was associated with increased levels of cytokines and chemokines including IL-10 and CXCL9. In a competing risks model adjusted for known covariates, high CXCL9 levels measured in the peripheral blood during pregnancy were associated with increased risk of pregnancy loss and PTD. At delivery, high IL-10 levels in maternal blood were associated with an increase in pregnancy loss, and increased IL-1ß levels in placental blood were associated with pregnancy loss and PTD. CONCLUSIONS: PM is associated with increased proinflammatory cytokine and chemokine levels in placental and maternal peripheral blood. Systemic inflammatory responses to malaria during pregnancy predict increased risk of pregnancy loss and PTD. CLINICAL TRIALS REGISTRATION: NCT01168271.

Effect of seasonal malaria chemoprevention on the acquisition of antibodies to Plasmodium falciparum antigens in Ouelessebougou, Mali.

BACKGROUND: Seasonal malaria chemoprevention (SMC) is a new strategy to reduce malaria burden in young children in Sahelian countries. It consists of the administration of full treatment courses of sulfadoxine-pyrimethamine plus amodiaquine to children at monthly intervals during the malaria season. However, it is not clear if there is a cumulative effect of SMC over time on acquisition of antibodies to malaria antigens. METHODS: A cross-sectional serosurvey was carried out 1 month after the last dose of SMC in 2016. Children aged 3-4 years were randomly selected from areas where SMC was given for 1, 2 or 3 years during the malaria season. Children in the areas where SMC had been implemented for 1 year but who failed to receive SMC were used as comparison group. Antibody extracted from dry blood spots was used to measure IgG levels to CSP, MSP-142 and AMA1. RESULTS: The prevalence of antibodies to AMA-1 were high and similar in children who received SMC for 1, 2 or 3 years and also when compared to those who never received SMC (96.3 vs 97.5%, adjusted OR = 0.99, 95% CI 0.33-2.97, p = 0.99). The prevalence of antibodies to MSP-142 and to CSP were similar in children that received SMC for 1, 2 or 3 years, but were lower in these children compared to those who did not receive SMC (87.1 vs 91.2%, adjusted OR = 0.55, 95% CI 0.29-1.01, p = 0.05 for MSP-142; 79.8 vs 89.2%, adjusted OR = 0.52, 95% CI 0.30-0.90, p = 0.019 for CSP). CONCLUSIONS: SMC reduced seropositivity to MSP-142 and CSP, but the duration of SMC did not further reduce seropositivity. Exposure to SMC did not reduce the seropositivity to AMA1.

Designing a VAR2CSA-based vaccine to prevent placental malaria.

Placental malaria (PM) due to Plasmodium falciparum is a major cause of maternal, fetal and infant mortality, but the mechanisms of pathogenesis and protective immunity are relatively well-understood for this condition, providing a path for vaccine development. P. falciparum parasites bind to chondroitin sulfate A (CSA) to sequester in the placenta, and women become resistant over 1-2 pregnancies as they acquire antibodies that block adhesion to CSA. The protein VAR2CSA, a member of the PfEMP1 variant surface antigen family, mediates parasite adhesion to CSA, and is the leading target for a vaccine to prevent PM. Obstacles to PM vaccine development include the large size (∼ 350 kD), high cysteine content, and sequence variation of VAR2CSA. A number of approaches have been taken to identify the combination of VAR2CSA domains and alleles that can induce broadly active antibodies that block adhesion of heterologous parasite isolates to CSA. This review summarizes these approaches, which have examined VAR2CSA fragments for binding activity, antigenicity with naturally acquired antibodies, and immunogenicity in animals for inducing anti-adhesion or surface-reactive antibodies. Two products are expected to enter human clinical studies in the near future based on N-terminal VAR2CSA fragments that have high binding affinity for CSA, and additional proteins preferentially expressed by placental parasites are also being examined for their potential contribution to a PM vaccine.

Chemokine-Dependent pH Elevation at the Cell Front Sustains Polarity in Directionally Migrating Zebrafish Germ Cells.

Directional cell migration requires cell polarization with respect to the distribution of the guidance cue. Cell polarization often includes asymmetric distribution of response components as well as elements of the motility machinery. Importantly, the function and regulation of most of these molecules are known to be pH dependent. Intracellular pH gradients were shown to occur in certain cells migrating in vitro, but the functional relevance of such gradients for cell migration and for the response to directional cues, particularly in the intact organism, is currently unknown. In this study, we find that primordial germ cells migrating in the context of the developing embryo respond to the graded distribution of the chemokine Cxcl12 by establishing elevated intracellular pH at the cell front. We provide insight into the mechanisms by which a polar pH distribution contributes to efficient cell migration. Specifically, we show that Carbonic Anhydrase 15b, an enzyme controlling the pH in many cell types, including metastatic cancer cells, is expressed in migrating germ cells and is crucial for establishing and maintaining an asymmetric pH distribution within them. Reducing the level of the protein and thereby erasing the pH elevation at the cell front resulted in abnormal cell migration and impaired arrival at the target. The basis for the disrupted migration is found in the stringent requirement for pH conditions in the cell for regulating contractility, for the polarization of Rac1 activity, and hence for the formation of actin-rich structures at the leading edge of the migrating cells.

Fetal origins of malarial disease: cord blood cytokines as risk markers for pediatric severe malarial anemia.

BACKGROUND: Severe malarial anemia (SMA) remains a major cause of pediatric illness and mortality in Sub-Saharan Africa. Here we test the hypothesis that prenatal exposures, reflected by soluble inflammatory mediators in cord blood, can condition an individual's susceptibility to SMA. METHODS: In a Tanzanian birth cohort (n = 743), we measured cord blood concentrations of tumor necrosis factor (TNF), TNF receptors I and II (TNF-RI and TNF-RII), interleukin (IL)-1ß, IL-4, IL-5, IL-6, IL-10, and interferon gamma (IFN-γ). After adjusting for conventional covariates, we calculated the hazard ratios (HR) for time to first SMA event with log(e) cytokine concentrations dichotomized at the median, by quartile, and per standard deviation (SD) increase. RESULTS: Low levels of TNF, TNF-RI, IL-1ß, and IL-5 and high levels of TNF-RII were associated statistically significantly and respectively with approximately 3-fold, 2-fold, 8-fold, 4-fold, and 3-fold increased risks of SMA (Hb < 50 g/L). TNF, TNF-RI, and IL-1ß concentrations were inversely and log-linearly associated with SMA risk; the HR (95% confidence interval [CI]) per 1-SD increase were respectively 0.81 (.65, 1.02), 0.76 (.62, .92), and 0.50 (.40, .62). CONCLUSIONS: These data suggest that proinflammatory cytokine levels at birth are inversely associated with SMA risk and support the hypothesis that pediatric malarial disease has fetal origins.

Implications of malaria on iron deficiency control strategies.

The populations in greatest need of iron supplementation are also those at greatest risk of malaria: pregnant women and young children. Iron supplementation has been shown to increase malaria risk in these groups in numerous studies, although this effect is likely diminished by factors such as host immunity, host iron status, and effective malaria surveillance and control. Conversely, the risk of anemia is increased by malaria infections and preventive measures against malaria decrease anemia prevalence in susceptible populations without iron supplementation. Studies have shown that subjects with malaria experience diminished absorption of orally administered iron, so that as a consequence, iron supplementation may have generally reduced efficacy in malarious populations. A possible mechanistic link between malaria, poor absorption of iron, and anemia is provided by recent research on hepcidin, the human iron control hormone. Our improved understanding of iron metabolism may contribute to the control of malaria and the treatment of anemia. Malaria surveillance and control are necessary components of programs to control iron deficiency and may enhance the efficacy of iron supplementation.

CXC ligand 9 response to malaria during pregnancy is associated with low-birth-weight deliveries.

Placental infection with Plasmodium falciparum is associated with increased levels of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), and previous studies have associated increased levels of these cytokines with low birth weight (LBW), especially for malaria-infected primigravidae. To define the contribution of TNF-α and IFN-γ networks to placental-malaria-associated LBW, we measured chemokines induced by TNF-α and IFN-γ and related them to birth weight in a birth cohort of 782 mother-infant pairs residing in an area of P. falciparum holoendemicity in Tanzania. Among primigravidae, levels of CCL2, CXC ligand 9 (CXCL9), and CXCL13 were significantly higher during malaria infection in both the placenta and peripheral blood. Placental CXCL9 and CXCL13 levels were also higher in placental blood from secundigravidae and multigravidae. In multivariate analyses adjusted for known predictors of birth weight, malaria-infected primigravidae with placental CXCL9 levels in the lowest tertile gave birth to babies who weighed 610 g more than babies born to mothers with high CXCL9 levels. CXCL9 expression is induced by IFN-γ, and the strong association between birth weight and placental CXCL9 is consistent with previous observations relating IFN-γ to poor pregnancy outcomes.

Iron deficiency protects against severe Plasmodium falciparum malaria and death in young children.

BACKGROUND: Iron supplementation may increase malaria morbidity and mortality, but the effect of naturally occurring variation in iron status on malaria risk is not well studied. METHODS: A total of 785 Tanzanian children living in an area of intense malaria transmission were enrolled at birth, and intensively monitored for parasitemia and illness including malaria for up to 3 years, with an average of 47 blood smears. We assayed plasma samples collected at routine healthy-child visits, and evaluated the impact of iron deficiency (ID) on future malaria outcomes and mortality. RESULTS: ID at routine, well-child visits significantly decreased the odds of subsequent parasitemia (23% decrease, P < .001) and subsequent severe malaria (38% decrease, P = .04). ID was also associated with 60% lower all-cause mortality (P = .04) and 66% lower malaria-associated mortality (P = .11). When sick visits as well as routine healthy-child visits are included in analyses (average of 3 iron status assays/child), ID reduced the prevalence of parasitemia (6.6-fold), hyperparasitemia (24.0-fold), and severe malaria (4.0-fold) at the time of sample collection (all P < .001). CONCLUSIONS: Malaria risk is influenced by physiologic iron status, and therefore iron supplementation may have adverse effects even among children with ID. Future interventional studies should assess whether treatment for ID coupled with effective malaria control can mitigate the risks of iron supplementation for children in areas of malaria transmission.

Pregnancy malaria: cryptic disease, apparent solution

Malaria during pregnancy can be severe in non-immune women, but in areas of stable transmission, where women are semi-immune and often asymptomatic during infection, malaria is an insidious cause of disease and death for mothers and their offspring. Sequelae, such as severe anaemia and hypertension in the mother and low birth weight and infant mortality in the offspring, are often not recognised as consequences of infection. Pregnancy malaria, caused by Plasmodium falciparum, is mediated by infected erythrocytes (IEs) that bind to chondroitin sulphate A and are sequestered in the placenta. These parasites have a unique adhesion phenotype and distinct antigenicity, which indicates that novel targets may be required for development of an effective vaccine. Women become resistant to malaria as they acquire antibodies against placental IE, which leads to higher haemoglobin levels and heavier babies. Proteins exported from the placental parasites have been identified, including both variant and conserved antigens, and some of these are in preclinical development for vaccines. A vaccine that prevents P. falciparum malaria in pregnant mothers is feasible and would potentially save hundreds of thousands of lives each year.

Intermittent treatment to prevent pregnancy malaria does not confer benefit in an area of widespread drug resistance.

BACKGROUND: Millions of African women receive sulfadoxine-pyrimethamine (SP) as intermittent preventive treatment during pregnancy (IPTp) to avoid poor outcomes that result from malaria. However, parasites resistant to SP are widespread in parts of Africa, and IPTp may perversely exacerbate placental infections that contain SP-resistant parasites. METHODS: The study used a cross-sectional design. We determined IPTp use in a delivery cohort of 880 pregnant women in Muheza, Tanzania, by report and by plasma sulfa measurements, and we examined its effects on maternal and fetal delivery outcomes. RESULTS: In the overall cohort, IPTp was not associated with decreased odds of placental malaria or with increased mean maternal hemoglobin or mean birth weight. Unexpectedly, IPTp was associated with decreased cord hemoglobin level and increased risk of fetal anemia, which may be related to in utero SP exposure. CONCLUSIONS: IPTp does not improve overall pregnancy outcomes in Muheza, Tanzania, where SP-resistant parasites predominate and may increase the odds of fetal anemia. As parasite resistance increases in a community, the overall effect of IPTp may transition from net benefit to neutral or net harm.