Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe 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 genome-wide 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.

PlasmoView: a web-based resource to visualise global Plasmodium falciparum genomic variation.

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).

Reduced risk of Plasmodium vivax malaria in Papua New Guinean children with Southeast Asian ovalocytosis in two cohorts and a case-control study.

BACKGROUND: The erythrocyte polymorphism, Southeast Asian ovalocytosis (SAO) (which results from a 27-base pair deletion in the erythrocyte band 3 gene, SLC4A1Δ27) protects against cerebral malaria caused by Plasmodium falciparum; however, it is unknown whether this polymorphism also protects against P. vivax infection and disease. METHODS AND FINDINGS: The association between SAO and P. vivax infection was examined through genotyping of 1,975 children enrolled in three independent epidemiological studies conducted in the Madang area of Papua New Guinea. SAO was associated with a statistically significant 46% reduction in the incidence of clinical P. vivax episodes (adjusted incidence rate ratio [IRR] = 0.54, 95% CI 0.40-0.72, p<0.0001) in a cohort of infants aged 3-21 months and a significant 52% reduction in P. vivax (blood-stage) reinfection diagnosed by PCR (95% CI 22-71, p = 0.003) and 55% by light microscopy (95% CI 13-77, p = 0.014), respectively, in a cohort of children aged 5-14 years. SAO was also associated with a reduction in risk of P. vivax parasitaemia in children 3-21 months (1,111/µl versus 636/µl, p = 0.011) and prevalence of P. vivax infections in children 15-21 months (odds ratio [OR] = 0.39, 95% CI 0.23-0.67, p = 0.001). In a case-control study of children aged 0.5-10 years, no child with SAO was found among 27 cases with severe P. vivax or mixed P. falciparum/P. vivax malaria (OR = 0, 95% CI 0-1.56, p = 0.11). SAO was associated with protection against severe P. falciparum malaria (OR = 0.38, 95% CI 0.15-0.87, p = 0.014) but no effect was seen on either the risk of acquiring blood-stage infections or uncomplicated episodes with P. falciparum. Although Duffy antigen receptor expression and function were not affected on SAO erythrocytes compared to non-SAO children, high level (>90% binding inhibition) P. vivax Duffy binding protein-specific binding inhibitory antibodies were observed significantly more often in sera from SAO than non-SAO children (SAO, 22.2%; non-SAO, 6.7%; p = 0.008). CONCLUSIONS: In three independent studies, we observed strong associations between SAO and protection against P. vivax malaria by a mechanism that is independent of the Duffy antigen. P. vivax malaria may have contributed to shaping the unique host genetic adaptations to malaria in Asian and Oceanic populations. Please see later in the article for the Editors' Summary.

Evidence that the erythrocyte invasion ligand PfRh2 is a target of protective immunity against Plasmodium falciparum malaria.

Abs targeting blood-stage Ags of Plasmodium falciparum are important in acquired immunity to malaria, but major targets remain unclear. The P. falciparum reticulocyte-binding homologs (PfRh) are key ligands used by merozoites during invasion of erythrocytes. PfRh2a and PfRh2b are functionally important members of this family and may be targets of protective immunity, but their potential role in human immunity has not been examined. We expressed eight recombinant proteins covering the entire PfRh2 common region, as well as PfRh2a- and PfRh2b-specific regions. Abs were measured among a cohort of 206 Papua New Guinean children who were followed prospectively for 6 mo for reinfection and malaria. At baseline, Abs were associated with increasing age and active infection. High levels of IgG to all PfRh2 protein constructs were strongly associated with protection from symptomatic malaria and high-density parasitemia. The predominant IgG subclasses were IgG1 and IgG3, with little IgG2 and IgG4 detected. To further understand the significance of PfRh2 as an immune target, we analyzed PfRh2 sequences and found that polymorphisms are concentrated in an N-terminal region of the protein and seem to be under diversifying selection, suggesting immune pressure. Cluster analysis arranged the sequences into two main groups, suggesting that many of the haplotypes identified may be antigenically similar. These findings provide evidence suggesting that PfRh2 is an important target of protective immunity in humans and that Abs act by controlling blood-stage parasitemia and support its potential for vaccine development.

An open dataset of Plasmodium vivax genome variation in 1,895 worldwide samples.

This report describes the MalariaGEN Pv4 dataset, a new release of curated genome variation data on 1,895 samples of Plasmodium vivax collected at 88 worldwide locations between 2001 and 2017. It includes 1,370 new samples contributed by MalariaGEN and VivaxGEN partner studies in addition to previously published samples from these and other sources. We provide genotype calls at over 4.5 million variable positions including over 3 million single nucleotide polymorphisms (SNPs), as well as short indels and tandem duplications. This enlarged dataset highlights major compartments of parasite population structure, with clear differentiation between Africa, Latin America, Oceania, Western Asia and different parts of Southeast Asia. Each sample has been classified for drug resistance to sulfadoxine, pyrimethamine and mefloquine based on known markers at the dhfr, dhps and mdr1 loci. The prevalence of all of these resistance markers was much higher in Southeast Asia and Oceania than elsewhere. This open resource of analysis-ready genome variation data from the MalariaGEN and VivaxGEN networks is driven by our collective goal to advance research into the complex biology of P. vivax and to accelerate genomic surveillance for malaria control and elimination.

Naturally acquired Duffy-binding protein-specific binding inhibitory antibodies confer protection from blood-stage Plasmodium vivax infection.

Individuals residing in malaria-endemic regions acquire protective immunity after repeated infection with malaria parasites; however, mechanisms of protective immunity and their immune correlates are poorly understood. Blood-stage infection with Plasmodium vivax depends completely on interaction of P. vivax Duffy-binding protein (PvDBP) with the Duffy antigen on host erythrocytes. Here, we performed a prospective cohort treatment/reinfection study of children (5-14 years) residing in a P. vivax-endemic region of Papua New Guinea (PNG) in which children were cleared of blood-stage infection and then examined biweekly for reinfection for 25 weeks. To test the hypothesis that naturally acquired binding inhibitory antibodies (BIAbs) targeting PvDBP region II (PvDBPII) provide protection against P. vivax infection, we used a quantitative receptor-binding assay to distinguish between antibodies that merely recognize PvDBP and those that inhibit binding to Duffy. The presence of high-level BIAbs (>90% inhibition of PvDBPII-Duffy binding, n = 18) before treatment was associated with delayed time to P. vivax reinfection diagnosed by light microscopy (P = 0.02), 55% reduced risk of P. vivax reinfection (Hazard's ratio = 0.45, P = 0.04), and 48% reduction in geometric mean P. vivax parasitemia (P < 0.001) when compared with children with low-level BIAbs (n = 148). Further, we found that stable, high-level BIAbs displayed strain-transcending inhibition by reducing reinfection with similar efficiency of PNG P. vivax strains characterized by six diverse PvDBPII haplotypes. These observations demonstrate a functional correlate of protective immunity in vivo and provide support for developing a vaccine against P. vivax malaria based on PvDBPII.

Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally.

BACKGROUND: Malaria in humans is caused by apicomplexan parasites belonging to 5 species of the genus Plasmodium. Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease is not known. Dimorphism in defined genes has led to P. ovale parasites being divided into classic and variant types. We hypothesized that these dimorphs represent distinct parasite species. METHODS: Multilocus sequence analysis of 6 genetic characters was carried out among 55 isolates from 12 African and 3 Asia-Pacific countries. RESULTS: Each genetic character displayed complete dimorphism and segregated perfectly between the 2 types. Both types were identified in samples from Ghana, Nigeria, São Tomé, Sierra Leone, and Uganda and have been described previously in Myanmar. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts. CONCLUSIONS: We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.

Association between naturally acquired antibodies to erythrocyte-binding antigens of Plasmodium falciparum and protection from malaria and high-density parasitemia.

BACKGROUND: Antibodies targeting blood stage antigens are important in protection against malaria, but the principle targets remain unclear. Erythrocyte-binding antigens (EBAs) are important erythrocyte invasion ligands used by merozoites and may be targets of protective immunity, but there are limited data examining their potential importance. METHODS: We examined antibodies among 206 Papua New Guinean children who were treated with antimalarials at enrollment and observed prospectively for 6 months for reinfection and malaria. Immunoglobulin (Ig) G, IgG subclasses, and IgM to different regions of EBA175, EBA140, and EBA181 expressed as recombinant proteins were assessed in comparison with several other merozoite antigens. RESULTS: High levels of IgG to each of the EBAs were strongly associated with protection from symptomatic malaria and high density parasitemia, but not with risk of reinfection per se. The predominant IgG subclasses were either IgG1 or IgG3, depending on the antigen. The predominance of IgG1 versus IgG3 reflected structural features of specific regions of the proteins. IgG3 was most strongly associated with protection, even for those antigens that had an IgG1 predominant response. CONCLUSIONS: The EBAs appear important targets of acquired protective immunity. These findings support their further development as vaccine candidates.

Strain-specific duffy binding protein antibodies correlate with protection against infection with homologous compared to heterologous plasmodium vivax strains in Papua New Guinean children.

Individuals repeatedly infected with malaria acquire protection from infection and disease; immunity is thought to be primarily antibody-mediated and directed to blood-stage infection. Merozoite surface proteins involved in the invasion of host erythrocytes are likely targets of protective antibodies. We hypothesized that Papua New Guinean children (n = 206) who acquire high antibody levels to two Plasmodium vivax merozoite proteins, Duffy binding protein region II (PvDBPII) and the 19-kDa C-terminal region of P. vivax merozoite surface protein 1 (PvMSP1(19)), would have a delay in the time to reinfection following treatment to clear all blood-stage malaria infections. Ninety-four percent of the children were reinfected with P. vivax during biweekly follow-ups for 6 months. Since PvDBPII is polymorphic, we examined whether individuals acquired strain-specific immunity to PvDBPII. Children with high antibody levels to a prevalent PvDBPII allele (O) were associated with a delay in the time to reinfection with the same variant of P. vivax by 25% compared to parasites expressing other PvDBPII alleles (age-adjusted hazard ratio, 0.75 [95% confidence interval, 0.56 to 1.00 by Cox regression]) and 39% lower incidence density parasitemia (P = 0.01). Two other prevalent alleles (AH and P) showed a similar trend of 16% and 18% protection, respectively, against parasites with the same PvDBPII allele and reduced incidence density parasitemia. Antibodies directed to PvDBPII PNG-P and -O were both associated with a 21 to 26% reduction in the risk of P. vivax infections with higher levels of parasitemia (>150 parasites/mul), respectively. There was no association with high antibody levels to PvMSP1(19) and a delay in the time to P. vivax reinfection. Thus, anti-PvDBPII antibodies are associated with strain-specific immunity to P. vivax and support the use of PvDBPII for a vaccine against P. vivax.

Immunoglobulin G subclass-specific responses against Plasmodium falciparum merozoite antigens are associated with control of parasitemia and protection from symptomatic illness.

Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear. Different malaria antigens induce distinct immunoglobulin G (IgG) subclass responses, but the importance of different responses in protective immunity from malaria is not known and the factors determining subclass responses in vivo are poorly understood. We examined IgG and IgG subclass responses to the merozoite antigens MSP1-19 (the 19-kDa C-terminal region of merozoite surface protein 1), MSP2 (merozoite surface protein 2), and AMA-1 (apical membrane antigen 1), including different polymorphic variants of these antigens, in a longitudinal cohort of children in Papua New Guinea. IgG1 and IgG3 were the predominant subclasses of antibodies to each antigen, and all antibody responses increased in association with age and exposure without evidence of increasing polarization toward one subclass. The profiles of IgG subclasses differed somewhat for different alleles of MSP2 but not for different variants of AMA-1. Individuals did not appear to have a propensity to make a specific subclass response irrespective of the antigen. Instead, data suggest that subclass responses to each antigen are generated independently among individuals and that antigen properties, rather than host factors, are the major determinants of IgG subclass responses. High levels of AMA-1-specific IgG3 and MSP1-19-specific IgG1 were strongly predictive of a reduced risk of symptomatic malaria and high-density P. falciparum infections. However, no antibody response was significantly associated with protection from parasitization per se. Our findings have major implications for understanding human immunity and for malaria vaccine development and evaluation.

Cellular tumor necrosis factor, gamma interferon, and interleukin-6 responses as correlates of immunity and risk of clinical Plasmodium falciparum malaria in children from Papua New Guinea.

The role of early to intermediate Plasmodium falciparum-induced cellular responses in the development of clinical immunity to malaria is not well understood, and such responses have been proposed to contribute to both immunity and risk of clinical malaria episodes. To investigate whether P. falciparum-induced cellular responses are able to function as predictive correlates of parasitological and clinical outcomes, we conducted a prospective cohort study of children (5 to 14 years of age) residing in a region of Papua New Guinea where malaria is endemic Live, intact P. falciparum-infected red blood cells were applied to isolated peripheral blood mononuclear cells obtained at baseline. Cellular cytokine production, including production of interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor (TNF) (formerly tumor necrosis factor alpha), and gamma interferon (IFN-gamma), was measured, and the cellular source of key cytokines was investigated. Multicytokine models revealed that increasing P. falciparum-induced IL-6 production was associated with an increased incidence of P. falciparum clinical episodes (incidence rate ratio [IRR], 1.75; 95% confidence interval [CI], 1.20 to 2.53), while increasing P. falciparum-induced TNF and IFN-gamma production was associated with a reduced incidence of clinical episodes (IRR for TNF, 0.55 [95% CI, 0.38 to 0.80]; IRR for IFN-gamma, 0.71 [95% CI, 0.55 to 0.90]). Furthermore, we found that monocytes/macrophages and gammadelta-T cells are important for the P. falciparum-induced production of IL-6 and TNF. Early to intermediate cellular cytokine responses to P. falciparum may therefore be important correlates of immunity and risk of symptomatic malaria episodes and thus warrant detailed investigation in relation to the development and implementation of effective vaccines.

Association of early interferon-gamma production with immunity to clinical malaria: a longitudinal study among Papua New Guinean children.

BACKGROUND: Elucidating the cellular and molecular basis of naturally acquired immunity to Plasmodium falciparum infection would assist in developing a rationally based malaria vaccine. Innate, intermediate, and adaptive immune mechanisms are all likely to contribute to immunity. Interferon-gamma (IFN-gamma) has been implicated in both protection against and the pathogenesis of malaria in humans. In addition, considerable heterogeneity exists among rapid IFN-gamma responses to P. falciparum in malaria-naive donors. The question remains whether similar heterogeneity is observed in malaria-exposed individuals and whether high, medium, or low IFN-gamma responsiveness is differentially associated with protective immunity or morbidity. METHODS: A 6-month longitudinal cohort study involving 206 school-aged Papua New Guinean children was performed. Peripheral blood mononuclear cells collected at baseline were exposed to live P. falciparum-infected erythrocytes. Early IFN-gamma responses were measured, and IFN-gamma-expressing cells were characterized by flow cytometry. IFN-gamma responsiveness was then tested for associations with parasitological and clinical outcome variables. RESULTS: Malaria-specific heterogeneity in early IFN-gamma responsiveness was observed among children. High-level early IFN-gamma responses were associated with protection from high-density and clinical P. falciparum infections. Parasite-induced early IFN-gamma was predominantly derived from gammadelta T cells (68% of which expressed the natural killer marker CD56) and alphabeta T cells, whereas natural killer cells and other cells made only minor contributions. The expression of CD56 in malaria-responsive, IFN-gamma-expressing gammadelta T cells correlated with IFN-gamma responsiveness. CONCLUSIONS: High, early IFN-gamma production by live parasite-stimulated peripheral blood mononuclear cells is a correlate of immunity to symptomatic malaria in Papua New Guinean children, and natural killer-like gammadelta T cells may contribute to protection.

Enhanced detection of gametocytes by magnetic deposition microscopy predicts higher potential for Plasmodium falciparum transmission.

BACKGROUND: Aggregated haemozoin crystals within malaria-infected erythrocytes confer susceptibility of parasitized cells to a magnetic field. Here the utility of this method for diagnosis of human malaria is evaluated in a malaria-endemic region of Papua New Guinea (PNG). METHODS AND FINDINGS: Individuals with Plasmodium falciparum malaria symptoms (n = 55) provided samples for conventional blood smear (CBS) and magnetic deposition microscopy (MDM) diagnosis. Standard Giemsa staining and light microscopy was performed to evaluate all preparations. Plasmodium falciparum parasitaemia observed on MDM slides was consistently higher than parasitaemia observed by (CBS) for ring (CBS = 2.6 vs. MDM = 3.4%; t-test P-value = 0.13), trophozoite (CBS = 0.5 vs. MDM = 1.6%; t-test P-value = 0.01), schizont (CBS = 0.003 vs. MDM = 0.1%; t-test P-value = 0.08) and gametocyte (CBS = 0.001 vs. MDM = 0.4%; t-test P-value = 0.0002) parasitaemias. Gametocyte prevalence determined by CBS compared to MDM increased from 7.3% to 45%, respectively. CONCLUSION: MDM increased detection sensitivity of P. falciparum-infected, haemozoin-containing erythrocytes from infected humans while maintaining detection of ring-stage parasites. Gametocyte prevalence five-fold higher than observed by CBS suggests higher malaria transmission potential in PNG endemic sites compared to previous estimates.

Host erythrocyte polymorphisms and exposure to Plasmodium falciparum in Papua New Guinea.

BACKGROUND: The protection afforded by human erythrocyte polymorphisms against the malaria parasite, Plasmodium falciparum, has been proposed to be due to reduced ability of the parasite to invade or develop in erythrocytes. If this were the case, variable levels of parasitaemia and rates of seroconversion to infected-erythrocyte variant surface antigens (VSA) should be seen in different host genotypes. METHODS: To test this hypothesis, P. falciparum parasitaemia and anti-VSA antibody levels were measured in a cohort of 555 asymptomatic children from an area of intense malaria transmission in Papua New Guinea. Linear mixed models were used to investigate the effect of alpha+-thalassaemia, complement receptor-1 and south-east Asian ovalocytosis, as well as glucose-6-phosphate dehydrogenase deficiency and ABO blood group on parasitaemia and age-specific seroconversion to VSA. RESULTS: No host polymorphism showed a significant association with both parasite prevalence/density and age-specific seroconversion to VSA. CONCLUSION: Host erythrocyte polymorphisms commonly found in Papua New Guinea do not effect exposure to blood stage P. falciparum infection. This contrasts with data for sickle cell trait and highlights that the above-mentioned polymorphisms may confer protection against malaria via distinct mechanisms.

Low prevalence of an acute phase response in asymptomatic children from a malaria-endemic area of Papua New Guinea.

Levels of C-reactive protein (CRP), a classic marker for the acute phase response (APR), were measured in children with asymptomatic malaria infection in the Amele region of Papua New Guinea (PNG). Despite the presence of parasitemia, the prevalence of CRP levels consistent with an APR (CRP > 10 microg/mL) was very low (< 10%). Splenomegaly was significantly associated with increased parasitemia (P < 0.001) and CRP levels (P < 0.001), highlighting the importance of splenomegaly as an indicator of recent high density infection in this population. Multivariate analysis showed that CRP levels were significantly associated with splenomegaly, fever, hemoglobin, and age (P < or = 0.002). CRP levels also increased with increasing parasitemia (P < 0.001) but remained < 3.5 microg/mL. The low levels of CRP indicate that children in the Amele modulate inflammation associated with malaria.

The risk of malarial infections and disease in Papua New Guinean children.

In a treatment re-infection study of 206 Papua New Guinean school children, we examined risk of reinfection and symptomatic malaria caused by different Plasmodium species. Although children acquired a similar number of polymerase chain reaction-detectable Plasmodium falciparum and P. vivax infections in six months of active follow-up (P. falciparum = 5.00, P. vivax = 5.28), they were 21 times more likely to develop symptomatic P. falciparum malaria (1.17/year) than P. vivax malaria (0.06/year). Children greater than nine years of age had a reduced risk of acquiring P. vivax infections of low-to-moderate (>150/microL) density (adjusted hazard rate [AHR] = 0.65 and 0.42), whereas similar reductions in risk with age of P. falciparum infection was only seen for parasitemias > 5,000/microL (AHR = 0.49) and symptomatic episodes (AHR = 0.51). Infection and symptomatic episodes with P. malariae and P. ovale were rare. By nine years of age, children have thus acquired almost complete clinical immunity to P. vivax characterized by a very tight control of parasite density, whereas the acquisition of immunity to symptomatic P. falciparum malaria remained incomplete. These observations suggest that different mechanisms of immunity may be important for protection from these malaria species.

Microsatellite polymorphism within pfcrt provides evidence of continuing evolution of chloroquine-resistant alleles in Papua New Guinea.

BACKGROUND: Polymorphism in the pfcrt gene underlies Plasmodium falciparum chloroquine resistance (CQR), as sensitive strains consistently carry lysine (K), while CQR strains carry threonine (T) at the codon 76. Previous studies have shown that microsatellite (MS) haplotype variation can be used to study the evolution of CQR polymorphism and to characterize intra- and inter-population dispersal of CQR in Papua New Guinea (PNG). METHODS: Here, following identification of new polymorphic MS in introns 2 and 3 within the pfcrt gene (msint2 and msint3, respectively), locus-by-locus and haplotype heterozygosity (H) analyses were performed to determine the distribution of this intronic polymorphism among pfcrt chloroquine-sensitive and CQR alleles. RESULTS: For MS flanking the pfcrt CQR allele, H ranged from 0.07 (B5M77, -18 kb) to 0.094 (9B12, +2 kb) suggesting that CQ selection pressure was responsible for strong homogenisation of this gene locus. In a survey of 206 pfcrt-SVMNT allele-containing field samples from malaria-endemic regions of PNG, H for msint2 was 0.201. This observation suggests that pfcrt msint2 exhibits a higher level of diversity than what is expected from the analyses of pfcrt flanking MS. Further analyses showed that one of the three haplotypes present in the early 1980's samples has become the predominant haplotype (frequency = 0.901) in CQR parasite populations collected after 1995 from three PNG sites, when CQR had spread throughout malaria-endemic regions of PNG. Apparent localized diversification of pfcrt haplotypes at each site was also observed among samples collected after 1995, where minor CQR-associated haplotypes were found to be unique to each site. CONCLUSION: In this study, a higher level of diversity at MS loci within the pfcrt gene was observed when compared with the level of diversity at pfcrt flanking MS. While pfcrt (K76T) and its immediate flanking region indicate homogenisation in PNG CQR parasite populations, pfcrt intronic MS variation provides evidence that the locus is still evolving. Further studies are needed to determine whether these intronic MS introduce the underlying genetic mechanisms that may generate pfcrt allelic diversity.

Association of haptoglobin levels with age, parasite density, and haptoglobin genotype in a malaria-endemic area of Gabon.

Haptoglobin (Hp) levels were investigated in relation to host genotype in a malaria-endemic area in Gabon. A cross-sectional study of 1-12-year-old children was conducted in the rainy season, a period of high malaria transmission, to examine this relationship. Variables that influenced Hp levels were Hp genotype, location, and age interacting with parasite density. At low parasite densities, there was a negative correlation between Hp levels and age. At higher densities, there was a positive correlation with age. This suggests that in the presence of greater parasite-induced hemolysis, older children are capable of increased production of Hp. Sickle cell trait and ABO blood group was not associated with Hp levels in this population.

Haptoglobin levels are associated with haptoglobin genotype and alpha+ -Thalassemia in a malaria-endemic area.

Haptoglobin (Hp) is an acute phase protein that removes free hemoglobin (Hb) released during hemolysis. Hp has also been shown to be toxic for malaria parasites. alpha(+)-Thalassemia is a hemoglobinopathy that results in subclinical hemolytic anemia. alpha(+)-Thassemia homozygosity confers protection against severe malarial disease by an as yet unidentified mechanism. Hp levels were measured in a serial cross-sectional survey of children in Madang Province, Papua New Guinea (PNG). Hp levels were related to age, Hp genotype, Hb levels, parasitemia, splenomegaly, and alpha(+)-thalassemia genotype. Surprisingly, children who were homozygous for alpha(+) -thalassemia had significantly higher levels of Hp than did heterozygotes, after controlling for relevant confounders. We suggest that this is the result of either reduced mean cell Hb associated with alpha(+) -thalassemia homozygosity or an elevated IL-6-dependent acute phase response.

Genomic analysis of local variation and recent evolution in Plasmodium vivax.

The widespread distribution and relapsing nature of Plasmodium vivax infection present major challenges for the elimination of malaria. To characterize the genetic diversity of this parasite in individual infections and across the population, we performed deep genome sequencing of >200 clinical samples collected across the Asia-Pacific region and analyzed data on >300,000 SNPs and nine regions of the genome with large copy number variations. Individual infections showed complex patterns of genetic structure, with variation not only in the number of dominant clones but also in their level of relatedness and inbreeding. At the population level, we observed strong signals of recent evolutionary selection both in known drug resistance genes and at new loci, and these varied markedly between geographical locations. These findings demonstrate a dynamic landscape of local evolutionary adaptation in the parasite population and provide a foundation for genomic surveillance to guide effective strategies for control and elimination of P. vivax.