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A research priority for Plasmodium vivax malaria is to improve our understanding of the spatial distribution of risk and its relationship with the burden of P. vivax disease in human populations. The aim of the research outlined in this article is to provide a contemporary evidence-based map of the global spatial extent of P. vivax malaria, together with estimates of the human population at risk (PAR) of any level of transmission in 2009. The most recent P. vivax case-reporting data that could be obtained for all malaria endemic countries were used to classify risk into three classes: malaria free, unstable (<0.1 case per 1,000 people per annum (p.a.)) and stable (> or =0.1 case per 1,000 p.a.) P. vivax malaria transmission. Risk areas were further constrained using temperature and aridity data based upon their relationship with parasite and vector bionomics. Medical intelligence was used to refine the spatial extent of risk in specific areas where transmission was reported to be absent (e.g., large urban areas and malaria-free islands). The PAR under each level of transmission was then derived by combining the categorical risk map with a high resolution population surface adjusted to 2009. The exclusion of large Duffy negative populations in Africa from the PAR totals was achieved using independent modelling of the gene frequency of this genetic trait. It was estimated that 2.85 billion people were exposed to some risk of P. vivax transmission in 2009, with 57.1% of them living in areas of unstable transmission. The vast majority (2.59 billion, 91.0%) were located in Central and South East (CSE) Asia, whilst the remainder were located in America (0.16 billion, 5.5%) and in the Africa+ region (0.10 billion, 3.5%). Despite evidence of ubiquitous risk of P. vivax infection in Africa, the very high prevalence of Duffy negativity throughout Central and West Africa reduced the PAR estimates substantially. After more than a century of development and control, P. vivax remains more widely distributed than P. falciparum and is a potential cause of morbidity and mortality amongst the 2.85 billion people living at risk of infection, the majority of whom are in the tropical belt of CSE Asia. The probability of infection is reduced massively across Africa by the frequency of the Duffy negative trait, but transmission does occur on the continent and is a concern for Duffy positive locals and travellers. The final map provides the spatial limits on which the endemicity of P. vivax transmission can be mapped to support future cartographic-based burden estimations.

Radiation-attenuated Plasmodium sporozoites (RAS) are the only vaccine shown to induce sterilizing protection against malaria in both humans and rodents. Importantly, these \"whole-parasite\" vaccines are currently under evaluation in human clinical trials. Studies with inbred mice reveal that RAS-induced CD8 T cells targeting liver-stage parasites are critical for protection. However, the paucity of defined T cell epitopes for these parasites has precluded precise understanding of the specific characteristics of RAS-induced protective CD8 T cell responses. Thus, it is not known whether quantitative or qualitative differences in RAS-induced CD8 T cell responses underlie the relative resistance or susceptibility of immune inbred mice to sporozoite challenge. Moreover, whether extraordinarily large CD8 T cell responses are generated and required for protection following RAS immunization, as has been described for CD8 T cell responses following single-antigen subunit vaccination, remains unknown. Here, we used surrogate T cell activation markers to identify and track whole-parasite, RAS-vaccine-induced effector and memory CD8 T cell responses. Our data show that the differential susceptibility of RAS-immune inbred mouse strains to Plasmodium berghei or P. yoelii sporozoite challenge does not result from host- or parasite-specific decreases in the CD8 T cell response. Moreover, the surrogate activation marker approach allowed us for the first time to evaluate CD8 T cell responses and protective immunity following RAS-immunization in outbred hosts. Importantly, we show that compared to a protective subunit vaccine that elicits a CD8 T cell response to a single epitope, diversifying the targeted antigens through whole-parasite RAS immunization only minimally, if at all, reduced the numerical requirements for memory CD8 T cell-mediated protection. Thus, our studies reveal that extremely high frequencies of RAS-induced memory CD8 T cells are required, but may not suffice, for sterilizing anti-Plasmodial immunity. These data provide new insights into protective CD8 T cell responses elicited by RAS-immunization in genetically diverse hosts, information with relevance to developing attenuated whole-parasite vaccines.

Malaria rapid diagnostic tests (RDTs) offer significant potential to improve the diagnosis of malaria, and are playing an increasing role in malaria case management, control and elimination. Peru, along with other South American countries, is moving to introduce malaria RDTs as components of malaria control programmes supported by the Global Fund for AIDS, TB and malaria. The selection of the most suitable malaria RDTs is critical to the success of the programmes. Eight of nine microscopy positive P. falciparum samples collected in Iquitos, Peru tested negative or weak positive using HRP2-detecting RDTs. These samples were tested for the presence of pfhrp2 and pfhrp3 and their flanking genes by PCR, as well as the presence of HRP proteins by ELISA. To investigate for geographic extent of HRP-deleted parasites and their temporal occurrence a retrospective study was undertaken on 148 microscopy positive P. falciparum samples collected in different areas of the Amazon region of Peru. Eight of the nine isolates lacked the pfhrp2 and/or pfhrp3 genes and one or both flanking genes, and the absence of HRP was confirmed by ELISA. The retrospective study showed that 61 (41%) and 103 (70%) of the 148 samples lacked the pfhrp2 or pfhrp3 genes respectively, with 32 (21.6%) samples lacking both hrp genes. This is the first documentation of P. falciparum field isolates lacking pfhrp2 and/or pfhrp3. The high frequency and wide distribution of different parasites lacking pfhrp2 and/or pfhrp3 in widely dispersed areas in the Peruvian Amazon implies that malaria RDTs targeting HRP2 will fail to detect a high proportion of P. falciparum in malaria-endemic areas of Peru and should not be used. RDTs detecting parasite LDH or aldolase and quality microscopy should be use for malaria diagnosis in this region. There is an urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighbouring countries.

A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05-0.5 parasites/mL whereas specific kDNA tests detected 5.10(-3) par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3-94.4%, specificity of 85-95%, accuracy of 86.8-89.5% and kappa index of 0.7-0.8 compared to consensus PCR reports of the 16 good performing tests and 63-69%, 100%, 71.4-76.2% and 0.4-0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

Human movement is a key behavioral factor in many vector-borne disease systems because it influences exposure to vectors and thus the transmission of pathogens. Human movement transcends spatial and temporal scales with different influences on disease dynamics. Here we develop a conceptual model to evaluate the importance of variation in exposure due to individual human movements for pathogen transmission, focusing on mosquito-borne dengue virus. We develop a model showing that the relevance of human movement at a particular scale depends on vector behavior. Focusing on the day-biting Aedes aegypti, we illustrate how vector biting behavior combined with fine-scale movements of individual humans engaged in their regular daily routine can influence transmission. Using a simple example, we estimate a transmission rate (R(0)) of 1.3 when exposure is assumed to occur only in the home versus 3.75 when exposure at multiple locations--e.g., market, friend's--due to movement is considered. Movement also influences for which sites and individuals risk is greatest. For the example considered, intriguingly, our model predicts little correspondence between vector abundance in a site and estimated R(0) for that site when movement is considered. This illustrates the importance of human movement for understanding and predicting the dynamics of a disease like dengue. To encourage investigation of human movement and disease, we review methods currently available to study human movement and, based on our experience studying dengue in Peru, discuss several important questions to address when designing a study. Human movement is a critical, understudied behavioral component underlying the transmission dynamics of many vector-borne pathogens. Understanding movement will facilitate identification of key individuals and sites in the transmission of pathogens such as dengue, which then may provide targets for surveillance, intervention, and improved disease prevention.

CD4+CD25+Foxp3+ regulatory T cells (Tregs) regulate disease-associated immunity and excessive inflammatory responses, and numbers of CD4+CD25+Foxp3+ Tregs are increased during malaria infection. The mechanisms governing their generation, however, remain to be elucidated. In this study we investigated the role of commonly accepted factors for Foxp3 induction, TCR stimulation and cytokines such as IL-2, TGFβ and IL-10, in the generation of human CD4+CD25+Foxp3+ T cells by the malaria parasite Plasmodium falciparum. Using a co-culture system of malaria-infected red blood cells (iRBCs) and peripheral blood mononuclear cells from healthy individuals, we found that two populations of Foxp3hi and Foxp3int CD4+CD25hi T cells with a typical Treg phenotype (CTLA-4+, CD127low, CD39+, ICOS+, TNFRII+) were induced. Pro-inflammatory cytokine production was confined to the Foxp3int subset (IFNγ, IL-4 and IL-17) and inversely correlated with high relative levels of Foxp3hi cells, consistent with Foxp3hi CD4 T cell–mediated inhibition of parasite-induced effector cytokine T cell responses. Both Foxp3hi and Foxp3int cells were derived primarily from proliferating CD4+CD25− T cells with a further significant contribution from CD25+Foxp3+ natural Treg cells to the generation of the Foxp3hi subset. Generation of Foxp3hi, but not Foxp3int, cells specifically required TGFβ1 and IL-10. Add-back experiments showed that monocytes expressing increased levels of co-stimulatory molecules were sufficient for iRBC-mediated induction of Foxp3 in CD4 T cells. Foxp3 induction was driven by IL-2 from CD4 T cells stimulated in an MHC class II–dependent manner. However, transwell separation experiments showed that direct contact of monocytes with the cells that acquire Foxp3 expression was not required. This novel TCR-independent and therefore antigen-non specific mechanism for by-stander CD4+CD25hiFoxp3+ cell induction is likely to reflect a process also occurring in vivo as a consequence of immune activation during malaria infection, and potentially a range of other infectious diseases. Infection with the malaria parasite Plasmodium falciparum affects 300–600 million people each year. Regulatory T cells (Tregs) expressing the transcription factor Foxp3, which drives genes involved in immunosuppression, are specialized immune cells that can inhibit both protective and harmful inflammatory responses during malaria. While Treg numbers are increased during malaria infection, little is known about how they are induced by the parasite. We addressed this question using an in vitro culture system to model the interaction of the malaria parasite with human immune cells. We found that the parasite induced soluble immune mediators, including the T cell growth-factor IL-2 and the regulatory proteins IL-10 and TGFβ, which drive the induction and expansion of Tregs. These Tregs expressed high levels of Foxp3 and suppressed the production of inflammation and protective immunity-driving mediators by concurrently induced effector T cells. Importantly, we demonstrate that induction of Tregs by the malaria parasite did not necessarily require direct contact with antigen-presenting cells. Our findings suggest that the parasite induces Tregs in an antigen non-specific manner, which may explain why malarial immunosuppression is not confined to malaria-specific immune responses, and provide new insights into the mechanisms governing Treg induction during malaria infection, and potentially other infectious diseases.

Toxoplasma gondii is a natural intracellular protozoal pathogen of mice and other small mammals. After infection, the parasite replicates freely in many cell types (tachyzoite stage) before undergoing a phase transition and encysting in brain and muscle (bradyzoite stage). In the mouse, early immune resistance to the tachyzoite stage is mediated by the family of interferon-inducible immunity-related GTPases (IRG proteins), but little is known of the nature of this resistance. We reported earlier that IRG proteins accumulate on intracellular vacuoles containing the pathogen, and that the vacuolar membrane subsequently ruptures. In this report, live-cell imaging microscopy has been used to follow this process and its consequences in real time. We show that the rupture of the vacuole is inevitably followed by death of the intracellular parasite, shown by its permeability to cytosolic protein markers. Death of the parasite is followed by the death of the infected cell. The death of the cell has features of pyronecrosis, including membrane permeabilisation and release of the inflammatory protein, HMGB1, but caspase-1 cleavage is not detected. This sequence of events occurs on a large scale only following infection of IFNγ-induced cells with an avirulent strain of T. gondii, and is reduced by expression of a dominant negative mutant IRG protein. Cells infected by virulent strains rarely undergo necrosis. We did not find autophagy to play any role in the key steps leading to the death of the parasite. We conclude that IRG proteins resist infection by avirulent T. gondii by a novel mechanism involving disruption of the vacuolar membrane, which in turn ultimately leads to the necrotic death of the infected cell.

Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species with excellent analytical tools available.

The var gene encoded hyper-variable Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates cytoadhesion of infected erythrocytes to human endothelium. Antibodies blocking cytoadhesion are important mediators of malaria immunity acquired by endemic populations. The development of a PfEMP1 based vaccine mimicking natural acquired immunity depends on a thorough understanding of the evolved PfEMP1 diversity, balancing antigenic variation against conserved receptor binding affinities. This study redefines and reclassifies the domains of PfEMP1 from seven genomes. Analysis of domains in 399 different PfEMP1 sequences allowed identification of several novel domain classes, and a high degree of PfEMP1 domain compositional order, including conserved domain cassettes not always associated with the established group A-E division of PfEMP1. A novel iterative homology block (HB) detection method was applied, allowing identification of 628 conserved minimal PfEMP1 building blocks, describing on average 83% of a PfEMP1 sequence. Using the HBs, similarities between domain classes were determined, and Duffy binding-like (DBL) domain subclasses were found in many cases to be hybrids of major domain classes. Related to this, a recombination hotspot was uncovered between DBL subdomains S2 and S3. The VarDom server is introduced, from which information on domain classes and homology blocks can be retrieved, and new sequences can be classified. Several conserved sequence elements were found, including: (1) residues conserved in all DBL domains predicted to interact and hold together the three DBL subdomains, (2) potential integrin binding sites in DBLα domains, (3) an acylation motif conserved in group A var genes suggesting N-terminal N-myristoylation, (4) PfEMP1 inter-domain regions proposed to be elastic disordered structures, and (5) several conserved predicted phosphorylation sites. Ideally, this comprehensive categorization of PfEMP1 will provide a platform for future studies on var/PfEMP1 expression and function.

One of the criteria to objectively prioritize merozoite antigens for malaria vaccine development is the demonstration that naturally acquired antibodies are associated with protection from malaria. However, published evidence of the protective effect of these antibodies is conflicting. We performed a systematic review with meta-analysis of prospective cohort studies examining the association between anti-merozoite immunoglobin (Ig) G responses and incidence of Plasmodium falciparum malaria. Two independent researchers searched six databases and identified 33 studies that met predefined inclusion and quality criteria, including a rigorous definition of symptomatic malaria. We found that only five studies were performed outside sub-Saharan Africa and that there was a deficiency in studies investigating antibodies to leading vaccine candidates merozoite surface protein (MSP)-1(42) and erythrocyte binding antigen (EBA)-175. Meta-analyses of most-studied antigens were conducted to obtain summary estimates of the association between antibodies and incidence of P. falciparum malaria. The largest effect was observed with IgG to MSP-3 C terminus and MSP-1(19) (responders versus nonresponders, 54%, 95% confidence interval [CI] [33%-68%] and 18% [4%-30%] relative reduction in risk, respectively) and there was evidence of a dose-response relationship. A tendency towards protective risk ratios (RR<1) was also observed for individual study estimates for apical membrane antigen (AMA)-1 and glutamate-rich protein (GLURP)-R0. Pooled estimates showed limited evidence of a protective effect for antibodies to MSP-1 N-terminal regions or MSP-1-EGF (epidermal growth factor-like modules). There was no significant evidence for the protective effect for MSP-2 (responders versus nonresponders pooled RR, MSP-2(FC27) 0.82, 95% CI 0.62-1.08, p = 0.16 and MSP-2(3D7) 0.92, 95% CI 0.75-1.13, p = 0.43). Heterogeneity, in terms of clinical and methodological diversity between studies, was an important issue in the meta-analysis of IgG responses to merozoite antigens. These findings are valuable for advancing vaccine development by providing evidence supporting merozoite antigens as targets of protective immunity in humans, and to help identify antigens that confer protection from malaria. Further prospective cohort studies that include a larger number of lead antigens and populations outside Africa are greatly needed to ensure generalizability of results. The reporting of results needs to be standardized to maximize comparability of studies. We therefore propose a set of guidelines to facilitate the uniform reporting of malaria immuno-epidemiology observational studies. Please see later in the article for the Editors' Summary.