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A blood test that captures cumulative exposure over time and assesses levels of naturally acquired immunity (NAI) would provide a critical tool to monitor the impact of interventions to reduce malaria transmission and broaden our understanding of how NAI develops around the world as a function of age and exposure. This article describes a collaborative effort in multiple International Centers of Excellence in Malaria Research (ICEMRs) to develop such tests using malaria-specific antibody responses as biosignatures of transmission and immunity. The focus is on the use of Plasmodium falciparum and Plasmodium vivax protein microarrays to identify a panel of the most informative antibody responses in diverse malaria-endemic settings representing an unparalleled spectrum of malaria transmission and malaria species mixes before and after interventions to reduce malaria transmission.

Tools to reliably measure Plasmodium falciparum (Pf) exposure in individuals and communities are needed to guide and evaluate malaria control interventions. Serologic assays can potentially produce precise exposure estimates at low cost; however, current approaches based on responses to a few characterized antigens are not designed to estimate exposure in individuals. Pf-specific antibody responses differ by antigen, suggesting that selection of antigens with defined kinetic profiles will improve estimates of Pf exposure. To identify novel serologic biomarkers of malaria exposure, we evaluated responses to 856 Pf antigens by protein microarray in 186 Ugandan children, for whom detailed Pf exposure data were available. Using data-adaptive statistical methods, we identified combinations of antibody responses that maximized information on an individual's recent exposure. Responses to three novel Pf antigens accurately classified whether an individual had been infected within the last 30, 90, or 365 d (cross-validated area under the curve = 0.86-0.93), whereas responses to six antigens accurately estimated an individual's malaria incidence in the prior year. Cross-validated incidence predictions for individuals in different communities provided accurate stratification of exposure between populations and suggest that precise estimates of community exposure can be obtained from sampling a small subset of that community. In addition, serologic incidence predictions from cross-sectional samples characterized heterogeneity within a community similarly to 1 y of continuous passive surveillance. Development of simple ELISA-based assays derived from the successful selection strategy outlined here offers the potential to generate rich epidemiologic surveillance data that will be widely accessible to malaria control programs.

Background Resistance of malaria parasites to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) is increasing in prevalence in Africa. Combination therapy can both improve treatment and provide important public health benefits if it curbs the spread of parasites harbouring resistance genes. Thus, drug combinations must be identified which minimise gametocyte emergence in treated cases, and so prevent selective transmission of parasites resistant to any of the partner drugs. Methods and Findings In a randomised controlled trial, 497 children with uncomplicated falciparum malaria were treated with CQ and SP (three doses and one dose respectively; n = 91), or six doses of artemether in fixed combination with lumefantrine (co-artemether [Coartem, Riamet]) (n = 406). Carriage rates of Plasmodium falciparum gametocytes and trophozoites were measured 7, 14, and 28 d after treatment. The infectiousness of venous blood from 29 children carrying P. falciparum gametocytes 7 d after treatment was tested by membrane-feeding of Anopheles mosquitoes. Children treated with co-artemether were significantly less likely to carry gametocytes within the 4 weeks following treatment than those receiving CQ/SP (30 of 378 [7.94%] versus 42 of 86 [48.8%]; p < 0.0001). Carriers in the co-artemether group harboured gametocytes at significantly lower densities, for shorter periods (0.3 d versus 4.2 d; p < 0.0001) and were less infectious to mosquitoes at day 7 (p < 0.001) than carriers who had received CQ/SP. Conclusions Co-artemether is highly effective at preventing post-treatment transmission of P. falciparum. Our results suggest that co-artemether has specific activity against immature sequestered gametocytes, and has the capacity to minimise transmission of drug-resistant parasites.

Abstract Background Countries aiming for malaria elimination require a detailed understanding of the current intensity of malaria transmission within their national borders. National household sample surveys are now being used to define infection prevalence but these are less efficient in areas of exceptionally low endemicity. Here we present the results of a national malaria indicator survey in the Republic of Djibouti, the first in sub-Saharan Africa to combine parasitological and serological markers of malaria, to evaluate the extent of transmission in the country and explore the potential for elimination. Methods A national cross-sectional household survey was undertaken from December 2008 to January 2009. A finger prick blood sample was taken from randomly selected participants of all ages to examine for parasitaemia using rapid diagnostic tests (RDTs) and confirmed using Polymerase Chain Reaction (PCR). Blood spots were also collected on filter paper and subsequently used to evaluate the presence of serological markers (combined AMA-1 and MSP-119) of Plasmodium falciparum exposure. Multivariate regression analysis was used to determine the risk factors for P. falciparum infection and/or exposure. The Getis-Ord G-statistic was used to assess spatial heterogeneity of combined infections and serological markers. Results A total of 7151 individuals were tested using RDTs of which only 42 (0.5%) were positive for P. falciparum infections and confirmed by PCR. Filter paper blood spots were collected for 5605 individuals. Of these 4769 showed concordant optical density results and were retained in subsequent analysis. Overall P. falciparum sero-prevalence was 9.9% (517/4769) for all ages; 6.9% (46/649) in children under the age of five years; and 14.2% (76/510) in the oldest age group (≥ 50 years). The combined infection and/or antibody prevalence was 10.5% (550/4769) and varied from 8.1% to 14.1% but overall regional differences were not statistically significant (χ2 = 33.98, p = 0.3144). Increasing age (p < 0.001) and decreasing household wealth status (p < 0.001) were significantly associated with increasing combined P. falciparum infection and/or antibody prevalence. Significant P. falciparum hot spots were observed in Dikhil region. Conclusion Malaria transmission in the Republic of Djibouti is very low across all regions with evidence of micro-epidemiological heterogeneity and limited recent transmission. It would seem that the Republic of Djibouti has a biologically feasible set of pre-conditions for elimination, however, the operational feasibility and the potential risks to elimination posed by P. vivax and human population movement across the sub-region remain to be properly established.

Abstract Background Insecticide-treated nets (ITNs) and indoor residual spraying (IRS) represent the front-line tools for malaria vector control globally, but are optimally effective where the majority of baseline transmission occurs indoors. In the surveyed area of rural southern Tanzania, bed net use steadily increased over the last decade, reducing malaria transmission intensity by 94%. Methods Starting before bed nets were introduced (1997), and then after two milestones of net use had been reached-75% community-wide use of untreated nets (2004) and then 47% use of ITNs (2009)-hourly biting rates of malaria vectors from the Anopheles gambiae complex and Anopheles funestus group were surveyed. Results In 1997, An. gambiae s.l. and An. funestus mosquitoes exhibited a tendency to bite humans inside houses late at night. For An. gambiae s.l., by 2009, nocturnal activity was less (p = 0.0018). At this time, the sibling species composition of the complex had shifted from predominantly An. gambiae s.s. to predominantly An. arabiensis. For An. funestus, by 2009, nocturnal activity was less (p = 0.0054) as well as the proportion biting indoors (p < 0.0001). At this time, An. funestus s.s. remained the predominant species within this group. As a consequence of these altered feeding patterns, the proportion (mean ± standard error) of human contact with mosquitoes (bites per person per night) occurring indoors dropped from 0.99 ± 0.002 in 1997 to 0.82 ± 0.008 in 2009 for the An. gambiae complex (p = 0.0143) and from 1.00 ± An. funestus complex (p = 0.0004) over the same time period. Conclusions High usage of ITNs can dramatically alter African vector populations so that intense, predominantly indoor transmission is replaced by greatly lowered residual transmission, a greater proportion of which occurs outdoors. Regardless of the underlying mechanism, the residual, self-sustaining transmission will respond poorly to further insecticidal measures within houses. Additional vector control tools which target outdoor biting mosquitoes at the adult or immature stages are required to complement ITNs and IRS.

Abstract Background Malaria incidence has been reported to be falling in several countries in sub-Saharan Africa in recent years. This fall appears to have started before the widespread introduction of insecticide-treated nets. In the new era of calls to eliminate and eradicate malaria in sub-Saharan Africa, exploring possible causes for this fall seem pertinent. Presentation of the hypothesis The authors explore an argument that presumptive treatment of fever cases as malaria may have played a role in reducing transmission of malaria by the prophylactic effect of antimalarials and their widespread use. This strategy, which is already in practise is termed Opportunistic Presumptive Treatment (OPT). Testing the hypothesis Further comparison of epidemiological indicators between areas with OPT and more targeted treatment is required. If data suggest a benefit of OPT, combining long acting antimalarials that have an anti-gametocyticidal activity component plus using high levels of vector control measures may reduce transmission, prevent resistant strains spreading and be easily implemented. Implications of the hypothesis OPT is practised widely by presumptive treatment of fever in health facilities and home management of fever. Improving diagnosis using rapid diagnostic tests and thus reducing the number of doses of antimalarials given may have counter intuitive effects on transmission in the context of elimination of malaria in high to moderate transmission settings.

Abstract The development of acquired protective immunity to Plasmodium falciparum infection in young African children is considered in the context of three current strategies for malaria prevention: insecticide-impregnated bed nets or curtains, anti-sporozoite vaccines and intermittent preventive therapy. Evidence is presented that each of these measures may permit attenuated P. falciparum blood-stage infections, which do not cause clinical malaria but can act as an effective blood-stage "vaccine". It is proposed that the extended serum half-life, and rarely considered liver-stage prophylaxis provided by the anti-folate combination sulphadoxine-pyrimethamine frequently lead to such attenuated infections in high transmission areas, and thus contribute to the sustained protection from malaria observed among children receiving the combination as intermittent preventative therapy or for parasite clearance in vaccine trials.

Significant selection pressure has been exerted on the genomes of human populations exposed to Plasmodium falciparum infection, resulting in the acquisition of mechanisms of resistance against severe malarial disease. Many host genetic factors, including sickle cell trait, have been associated with reduced risk of developing severe malaria, but do not account for all of the observed phenotypic variation. Identification of novel inherited risk factors relies upon high-resolution genome-wide association studies (GWAS). We present findings of a GWAS of severe malaria performed in a Tanzanian population (n = 914, 15.2 million SNPs). Beyond the expected association with the sickle cell HbS variant, we identify protective associations within two interleukin receptors (IL-23R and IL-12RBR2) and the kelch-like protein KLHL3 (all P<10−6), as well as near significant effects for Major Histocompatibility Complex (MHC) haplotypes. Complementary analyses, based on detecting extended haplotype homozygosity, identified SYNJ2BP, GCLC and MHC as potential loci under recent positive selection. Through whole genome sequencing of an independent Tanzanian cohort (parent-child trios n = 247), we confirm the allele frequencies of common polymorphisms underlying associations and selection, as well as the presence of multiple structural variants that could be in linkage with these SNPs. Imputation of structural variants in a region encompassing the glycophorin genes on chromosome 4, led to the characterisation of more than 50 rare variants, and individually no strong evidence of associations with severe malaria in our primary dataset (P>0.3). Our approach demonstrates the potential of a joint genotyping-sequencing strategy to identify as-yet unknown susceptibility loci in an African population with well-characterised malaria phenotypes. The regions encompassing these loci are potential targets for the design of much needed interventions for preventing or treating malarial disease. [ABSTRACT FROM AUTHOR]

X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers. [ABSTRACT FROM AUTHOR]

Malaria is a global public health challenge, with drug resistance a major barrier to disease control and elimination. To meet the urgent need for better treatments and vaccines, a deeper knowledge of Plasmodium biology and malaria epidemiology is required. An improved understanding of the genomic variation of malaria parasites, especially the most virulent Plasmodium falciparum (Pf) species, has the potential to yield new insights in these areas. High-throughput sequencing and genotyping is generating large amounts of genomic data across multiple parasite populations. The resulting ability to identify informative variants, particularly single-nucleotide polymorphisms (SNPs), will lead to the discovery of intra- and inter-population differences and thus enable the development of genetic barcodes for diagnostic assays and clinical studies. Knowledge of genetic variability underlying drug resistance and other differential phenotypes will also facilitate the identification of novel mutations and contribute to surveillance and stratified medicine applications. The PlasmoView interactive web-browsing tool enables the research community to visualise genomic variation and annotation (eg, biological function) in a geographic setting. The first release contains over 600 000 high-quality SNPs in 631 Pf isolates from laboratory strains and four malaria-endemic regions (West Africa, East Africa, Southeast Asia and Oceania). [ABSTRACT FROM AUTHOR]

Background: Many studies report associations between human genetic factors and immunity to malaria but few have been reliably replicated. These studies are usually country-specific, use small sample sizes and are not directly comparable due to differences in methodologies. This study brings together samples and data collected from multiple sites across Africa and Asia to use standardized methods to look for consistent genetic effects on anti-malarial antibody levels., Methods: Sera, DNA samples and clinical data were collected from 13,299 individuals from ten sites in Senegal, Mali, Burkina Faso, Sudan, Kenya, Tanzania, and Sri Lanka using standardized methods. DNA was extracted and typed for 202 Single Nucleotide Polymorphisms with known associations to malaria or antibody production, and antibody levels to four clinical grade malarial antigens [AMA1, MSP1, MSP2, and (NANP)4] plus total IgE were measured by ELISA techniques. Regression models were used to investigate the associations of clinical and genetic factors with antibody levels., Results: Malaria infection increased levels of antibodies to malaria antigens and, as expected, stable predictors of anti-malarial antibody levels included age, seasonality, location, and ethnicity. Correlations between antibodies to blood-stage antigens AMA1, MSP1 and MSP2 were higher between themselves than with antibodies to the (NANP)4 epitope of the pre-erythrocytic circumsporozoite protein, while there was little or no correlation with total IgE levels. Individuals with sickle cell trait had significantly lower antibody levels to all blood-stage antigens, and recessive homozygotes for CD36 (rs321198) had significantly lower anti-malarial antibody levels to MSP2., Conclusion: Although the most significant finding with a consistent effect across sites was for sickle cell trait, its effect is likely to be via reducing a microscopically positive parasitaemia rather than directly on antibody levels. However, this study does demonstrate a framework for the feasibility of combining data from sites with heterogeneous malaria transmission levels across Africa and Asia with which to explore genetic effects on anti-malarial immunity.