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Host genetic factors can confer resistance against malaria1, raising the question of whether this has led to evolutionary adaptation of parasite populations. Here we searched for association between candidate host and parasite genetic variants in 3,346 Gambian and Kenyan children with severe malaria caused by Plasmodium falciparum. We identified a strong association between sickle haemoglobin (HbS) in the host and three regions of the parasite genome, which is not explained by population structure or other covariates, and which is replicated in additional samples. The HbS-associated alleles include nonsynonymous variants in the gene for the acyl-CoA synthetase family member2–4PfACS8 on chromosome 2, in a second region of chromosome 2, and in a region containing structural variation on chromosome 11. The alleles are in strong linkage disequilibrium and have frequencies that covary with the frequency of HbS across populations, in particular being much more common in Africa than other parts of the world. The estimated protective effect of HbS against severe malaria, as determined by comparison of cases with population controls, varies greatly according to the parasite genotype at these three loci. These findings open up a new avenue of enquiry into the biological and epidemiological significance of the HbS-associated polymorphisms in the parasite genome and the evolutionary forces that have led to their high frequency and strong linkage disequilibrium in African P. falciparum populations.

Performance of the malaria Rapid Diagnostic Test (RDT) OptiMal-IT® was evaluated in Mauritania where malaria is low and dependent on a short transmission season. Slide microscopy was considered as the reference method of diagnosis. Febrile patients with suspected malaria were recruited from six health facilities, 3 urban and 3 rural, during two periods (December 2011 to February 2012, and August 2012 to March 2013). Overall, 780 patients were sampled, with RDT and thick blood film microscopy results being obtained for 759 of them. Out of 774 slides examined, of which 200 were positive, P. falciparum and P. vivax mono-infections were detected in 63.5% (127) and 29.5% (59), while P. falciparum/P. vivax coinfections were detected in 7% (14). Both species were observed in all study sites, although in significantly different proportions. The proportions of thick blood film and OptiMal-IT® RDT positive individuals was 26.3% and 30.3% respectively. Sensitivity and specificity of OptiMal-IT® RDT were 89% [95% CI, 84.7-93.3] and 91.1% [88.6-93.4]. Positives and negative predictive values were 78.1% [72.2-83.7] and 95.9% [94.1-97.5]. These diagnostic values are similar to those generally reported elsewhere, and support the use of RDTs as the main diagnostic tool for malaria in Mauritanian health facilities. In the future, choice of RDTs to be used must take account of thermostability in a hot, dry environment and their ability to detect P. falciparum and P. vivax.

methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genomewide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome. The genetic diversity and evolutionary plasticity of P. falciparum are major obstacles for malaria elimination. New forms of resistance against antimalarial drugs are continually emerging 1,2 , and new forms of antigenic variation are a critical point of vulnerability for future malaria vaccines. Effective tools are needed to detect evolutionary changes in the parasite population and to monitor the spread of genetic variants that affect malaria control. Here we describe the use of deep sequencing to analyse P. falciparum diversity, using blood samples from patients with malaria. The P. falciparum genome has several unusual features that greatly complicate sequence analysis, such as extreme AT bias, large tracts of nonunique sequence and several large families of intensely polymorphic genes 3 . Our aim was therefore not to determine the entire genome sequence of individual field samples—which would be prohibitively expensive with current technologies—but to define an initial set of single nucleotide polymorphisms (SNPs) distributed across the P. falciparum genome, whose genotype can be ascertained with confidence in parasitized blood samples by deep sequencing. An additional complication in the analysis of P. falciparum genome variation is that the billions of haploid parasites that infect a single individual can be a complex mixture of genetic types. Previous studies 4–8 have largely focused on laboratory-adapted parasite clones, but the within-host diversity of natural infections is of fundamental biological interest. Parasites in the blood replicate asexually, but when they are taken up in the blood meal of an Anopheles mosquito they undergo sexual mating. If the parasites in the blood are of diverse genetic types, this process of sexual mating can generate novel recombinant forms. Deep sequencing provides new ways of investigating within-host diversity and the role of sexual recombination in parasite evolution.

Information on the period during which infants lose their maternally derived antibodies to malaria and begin to acquire naturally their own immune responses against parasite antigens is crucial for understanding when malaria vaccines may be best administered. This study investigated the rates of decline and acquisition of serum antibody isotypes IgG1, IgG2, IgG3, IgG4, IgM and IgA to Plasmodium falciparum antigens apical membrane antigen (AMA1), merozoite surface proteins (MSP1-19, MSP2 and MSP3) in a birth cohort of 53 children living in an urban area in the Gambia, followed over the first 3 years of life (sampled at birth, 4, 9, 18 and 36 months). Antigen-specific maternally transferred antibody isotypes of all IgG subclasses were detected at birth and were almost totally depleted by 4 months of age. Acquisition of specific antibody isotypes to the antigens began with IgM, followed by IgG1 and IgA. Against the MSP2 antigen, IgG1 but not IgG3 responses were observed in the children, in contrast with the maternally derived antibodies to this antigen that were mostly IgG3. This confirms that IgG subclass responses to MSP2 are strongly dependent on age or previous malaria experience, polarized towards IgG1 early in life and to IgG3 in older exposed individuals.

Longitudinal cohort studies are important to describe the dynamics of naturally acquired antibody response profiles to defined Plasmodium falciparum malaria antigens relative to clinical malaria episodes. In children under 7 years of age in The Gambia, serum IgG responses were measured to P. falciparum merozoite antigens AMA1, EBA175, MSP1(19), MSP2 and crude schizont extract, over a 10-month period. Persistence of antibody responses was measured in 152 children during the dry season when there was virtually no malaria transmission, and 103 children were monitored for new episodes of clinical malaria during the subsequent wet season when transmission occurred. Children who experienced clinical malaria had lower antibody levels at the start of the study than those who remained free from malaria. Associations between dry season antibody persistence and subsequent wet season antibody levels suggested robust immunological memory responses. Mean antibody levels to all antigens were elevated by the end of the wet season in children who experienced clinical malaria; each of these children had a boosted antibody response to at least one antigen. In all children, antibody avidities were lower against MSP2 than other antigens, a difference that did not change throughout the study period or in relation to clinical malaria episodes.

Similarity between two individuals in the combination of genetic markers along their chromosomes indicates shared ancestry and can be used to identify historical connections between different population groups due to admixture. We use a genome-wide, haplotype-based, analysis to characterise the structure of genetic diversity and gene-flow in a collection of 48 sub-Saharan African groups. We show that coastal populations experienced an influx of Eurasian haplotypes over the last 7000 years, and that Eastern and Southern Niger-Congo speaking groups share ancestry with Central West Africans as a result of recent population expansions. In fact, most sub-Saharan populations share ancestry with groups from outside of their current geographic region as a result of gene-flow within the last 4000 years. Our in-depth analysis provides insight into haplotype sharing across different ethno-linguistic groups and the recent movement of alleles into new environments, both of which are relevant to studies of genetic epidemiology. DOI: http://dx.doi.org/10.7554/eLife.15266.001, eLife digest Our genomes contain a record of historical events. This is because when groups of people are separated for generations, the DNA sequence in the two groups’ genomes will change in different ways. Looking at the differences in the genomes of people from the same population can help researchers to understand and reconstruct the historical interactions that brought their ancestors together. The mixing of two populations that were previously separate is known as admixture. Africa as a continent has few written records of its history. This means that it is somewhat unknown which important movements of people in the past generated the populations found in modern-day Africa. Busby et al. have now attempted to use DNA to look into this and reconstruct the last 4000 years of genetic history in African populations. As has been shown in other regions of the world, the new analysis showed that all African populations are the result of historical admixture events. However, Busby et al. could characterize these events to unprecedented level of detail. For example, multiple ethnic groups from The Gambia and Mali all show signs of sharing the same set of ancestors from West Africa, Europe and Asia who mixed around 2000 years ago. Evidence of a migration of people from Central West Africa, known as the Bantu expansion, could also be detected, and was shown to carry genes to the south and east. An important next step will be to now look at the consequences of the observed gene-flow, and ask if it has contributed to spreading beneficial, or detrimental, mutations around Africa. DOI: http://dx.doi.org/10.7554/eLife.15266.002

Background Genomic differentiation between Anopheles gambiae and Anopheles coluzzii - the major malaria vectors in sub-Saharan Africa - is localized into large “islands” toward the centromeres of chromosome-X and the two autosomes. Linkage disequilibrium between these genomic islands was first detected between species-specific polymorphisms within ribosomal DNA genes (IGS-rDNA) on the X-chromosome and a single variant at position 702 of intron 1 (Int-1702) of the para Voltage-Gated Sodium Channel (VGSC) gene on chromosome arm 2 L. Intron-1 sequence data from West and Central Africa revealed two clearly distinct and species-specific haplogroups, each characterized by very low polymorphism, which has been attributed to a selective sweep. The aim of this study was to analyse Int-1 sequence diversity in A. gambiae and A. coluzzii populations from the Far-West of their range, in order to assess whether this selective-sweep signature could persist in a zone of high interspecific hybridization. Methods A 531 bp region of VGSC Int-1 was sequenced in 21 A. coluzzii, 31 A. gambiae, and 12 hybrids from The Gambia and Guinea Bissau, located within the Far-West geographical region, and in 53 A. gambiae s.l. samples from the rest of the range. Results Far-West samples exhibit dramatic Int-1 polymorphism, far higher within each country than observed throughout the rest of the species range. Moreover, patterning of haplotypes within A. coluzzii confirms previous evidence of a macro-geographic subdivision into a West and a Central African genetic cluster, and reveals a possible genetic distinction of A. coluzzii populations from the Far-West. Conclusions The results suggest a relaxation of selective pressures acting across the VGSC gene region in the hybrid zone. Genetic differentiation in the Far-West could be attributable to a founder effect within A. coluzzii, with subsequent extensive gene flow with secondarily-colonizing A. gambiae, potentially yielding a novel insight on the dynamic processes impacting genetic divergence of these key malaria vectors. Electronic supplementary material The online version of this article (doi:10.1186/s12936-014-0522-1) contains supplementary material, which is available to authorized users.

Plasmodium parasites, the causative agents of malaria, are responsible for a significant burden of disease worldwide. Understanding populations and parasite biology is critical to developing effective control mechanisms and questions relating to these have been addressed in this thesis using whole genome and transcriptome sequencing. In Southeast Asia, the zoonotic species Plasmodium knowlesi now causes a significant amount of malarial disease, particularly in Malaysia. Clinical isolates of P. knowlesi from peninsular Malaysia were whole genome sequenced and single nucleotide polymorphisms were used to inform population genomic analyses. This work confirmed the existence of a third, divergent population of P. knowlesi in peninsular Malaysia and uncovered evidence of selection acting on these parasites. In sub-Saharan Africa, P. falciparum is responsible for virtually all malarial disease. Clinical isolates sampled from West Africa were investigated using whole transcriptome sequencing of ex vivo parasites, focusing on a gene known as mspdbl2, which is a possible marker of gametocytogenesis. Schizonts from clinical isolates were assessed for MSPDBL2 expression by immunofluorescence assays and were whole transcriptome sequenced. Analysis of gene expression was carried out correlating to expression of MSPDBL2, revealing enrichment of genes with known or suspected involvement in gametocytogenesis. A limiting factor of this investigation was the very low amount of material available from ex vivo culture. An alternative avenue for investigating limiting material is through single-cell sequencing. One method for single-cell transcriptomics was trialled in this thesis and extensive optimisation resulted in the sequencing of transcriptomes from a small number of P. falciparum schizonts, offering a future methodology for investigating gene expression using very few parasites.

A highly efficacious vaccine against the malaria parasite Plasmodium falciparum is needed. Repeat sequences are common in P. falciparum proteins and some are known immune targets. Short read sequence data are available for thousands of parasite isolates, but aligning and assembling repetitive sequences remains a challenge. A combined mapping and de novo assembly approach was developed to resolve highly complex and polymorphic allelic repeat sequences in the merozoite protein MSP-1. This approach gave an unbiased call of allele frequencies and full repeat sequence for a majority of clinical isolates tested. These data were used to design polyvalent hybrid sequences that would containing motifs from multiple alleles. Potential construct designs representing a greater spectrum of sequence diversity than that of previously designed polyvalent hybrid antigens were generated. Assays of mechanisms of antibody mediated inhibition of parasite growth are needed to identify which antigen sequences are functional targets of immunity. Such assays are hard to standardise and would be benefitted by availability of human monoclonal antibody reagents. As an approach towards obtaining these, a technique was developed using a tetramerised P. falciparum MSP-1 recombinant antigen to isolate cognate B-cells from the blood of exposed Ghanaian adult donors. Despite lower than expected viability of cryopreserved samples, 82 memory antigen specific B-cells were successfully isolated by single cell flow sorting of lymphocytes from 16 donors. Complimentary DNA encoding both the heavy and light chain immunoglobulin variable regions was sequenced and analysed for two of these cells, revealing some distinct features. This is the first time a tetrameric antigen has been used to isolate human B-cells recognising a P. falciparum antigen, demonstrating their potential for use in the study of malarial immunity. The modest numbers of specific B-cells sorted from cryopreserved samples encourage the application of this approach to freshly obtained samples.