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Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance., It is unclear whether new antimalarial compounds may rapidly lose effectiveness in the field because of parasite resistance. Here, Corey et al. investigate the acquisition of drug resistance and the extent to which common resistance mechanisms decrease susceptibility to a diverse set of 50 antimalarial compounds.

MMV007564 is a novel antimalarial benzimidazolyl piperidine chemotype identified in cellular screens. To identify the genetic determinant of MMV007564 resistance, parasites were cultured in the presence of the compound to generate resistant lines. Whole genome sequencing revealed distinct mutations in the gene named Plasmodium falciparum cyclic amine resistance locus (pfcarl), encoding a conserved protein of unknown function. Mutations in pfcarl are strongly associated with resistance to a structurally unrelated class of compounds, the imidazolopiperazines, including KAF156, currently in clinical trials. Our data demonstrate that pfcarl mutations confer resistance to two distinct compound classes, benzimidazolyl piperidines and imidazolopiperazines. However, MMV007564 and the imidazolopiperazines, KAF156 and GNF179, have different timings of action in the asexual blood stage and different potencies against the liver and sexual blood stages. These data suggest that pfcarl is a multidrug-resistance gene rather than a common target for benzimidazolyl piperidines and imidazolopiperazines.

When deprived of nutrients, trophozoites of the eye pathogen Acanthamoeba castellanii make a cyst wall, which contains cellulose and has two layers connected by cone-shaped ostioles. We recently showed chitin is also present and identified three sets of lectins, which localize to the ectocyst layer (Jonah lectin) or the endocyst layer and ostioles (Luke and Leo lectins). To determine how the cyst wall is made, we examined encysting protists using structured illumination microscopy, probes for glycopolymers, and tags for lectins. In the first stage (3 to 9 hr), cellulose, chitin, and a Jonah lectin were each made in dozens of encystation-specific vesicles. In the second stage (12 to 18 hr), a primordial wall contained both glycopolymers and Jonah lectin, while small, flat ostioles were outlined by a Luke lectin. In the third stage (24 to 36 hr), an ectocyst layer enriched in Jonah lectin was connected to an endocyst layer enriched in Luke and Leo lectins by large, conical ostioles. Jonah and Luke lectins localized to the same places in mature cyst walls (72 hr) independent of the timing of expression. The Jonah lectin and the glycopolymer bound by the lectin were accessible in the ectocyst layer of mature walls. In contrast, Luke and Leo lectins and glycopolymers bound by the lectins were mostly inaccessible in the endocyst layer and ostioles. These results show that cyst wall formation is a tightly choreographed event, in which glycopolymers and lectins combine to form a mature wall with a protected endocyst layer.ImportanceWhile the cyst wall of Acanthamoeba castellanii, cause of eye infections, contains cellulose like plants and chitin like fungi, it is a temporary, protective structure, analogous to spore coats of bacteria. We showed here that, unlike plants and fungi, A. castellanii makes cellulose and chitin in encystation-specific vesicles. The outer and inner layers of cyst walls, which resemble the primary and secondary walls of plant cells, respectively, are connected by unique structures (ostioles) that synchronously develop from small, flat circles to large, conical structures. Cyst wall proteins, which are lectins that bind cellulose and chitin, localize to inner or outer layers independent of the timing of expression. Because of its abundance and accessibility in the outer layer, the Jonah lectin is an excellent target for diagnostic antibodies. A description of the sequence of events during cyst wall development is a starting point for mechanistic studies of its assembly.

Background Acanthamoeba castellanii, which causes keratitis and blindness in under-resourced countries, is an emerging pathogen worldwide, because of its association with contact lens use. The wall makes cysts resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Methodology/Principal findings Transmission electron microscopy and structured illumination microscopy (SIM) showed purified cyst walls of A. castellanii retained an outer ectocyst layer, an inner endocyst layer, and conical ostioles that connect them. Mass spectrometry showed candidate cyst wall proteins were dominated by three families of lectins (named here Jonah, Luke, and Leo), which bound well to cellulose and less well to chitin. An abundant Jonah lectin, which has one choice-of-anchor A (CAA) domain, was made early during encystation and localized to the ectocyst layer of cyst walls. An abundant Luke lectin, which has two carbohydrate-binding modules (CBM49), outlined small, flat ostioles in a single-layered primordial wall and localized to the endocyst layer and ostioles of mature walls. An abundant Leo lectin, which has two unique domains with eight Cys residues each (8-Cys), localized to the endocyst layer and ostioles. The Jonah lectin and glycopolymers, to which it binds, were accessible in the ectocyst layer. In contrast, Luke and Leo lectins and the glycopolymers, to which they bind, were mostly inaccessible in the endocyst layer and ostioles. Conclusions/Significance The most abundant A. castellanii cyst wall proteins are three sets of lectins, which have carbohydrate-binding modules that are conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique to Acanthamoebae (8-Cys of Leo). Cyst wall formation is a tightly choreographed event, in which lectins and glycopolymers combine to form a mature wall with a protected endocyst layer. Because of its accessibility in the ectocyst layer, an abundant Jonah lectin is an excellent diagnostic target., Author summary A half century ago, investigators identified cellulose in the Acanthamoeba cyst wall, which has two layers and conical ostioles that connect them. Here we showed cyst walls contain three large sets of cellulose-binding lectins, which localize to the ectocyst layer (a Jonah lectin) or to the endocyst layer and ostioles (Luke and Leo lectins). We used the lectins to establish a sequence for cyst wall assembly when trophozoites are starved and encyst. In the first stage, a Jonah lectin and glycopolymers were present in dozens of distinct vesicles. In the second stage, a primordial wall contained small, flat ostioles outlined by a Luke lectin. In the third stage, a Jonah lectin remained in the ectocyst layer, while Luke and Leo lectins moved to the endocyst layer and ostioles. A description of the major events during cyst wall development is a starting point for mechanistic studies of its assembly.

Acanthamoeba castellanii, cause of keratitis and blindness, is an emerging pathogen because of its association with contact lens use. The cyst wall contributes to pathogenesis as cysts are resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Here we used structured illumination microscopy (SIM) and probes for glycopolymers to show that purified cyst walls ofA. castellaniiretain endocyst and ectocyst layers and conical structures (ostioles) that connect them. Mass spectrometry showed candidate cyst wall proteins (CWPs) are dominated by three families of lectins (named here Luke, Leo, and Jonah), because each binds to microcrystalline cellulose +/- chitin. Luke lectins contain two or three carbohydrate-binding modules (CBM49), which were first identified in a tomato cellulase. Leo lectins have two unique domains with eight Cys residues each (8-Cys) +/- a Thr-, Lys-, and His-rich spacer. Jonah lectins contain one or three choice-of-anchor A (CAA) domains previously of unknown function. Representative members of each family were tagged with green fluorescent protein (GFP) and expressed under their own promoters in transfected parasites. A representative Jonah lectin with one CAA domain is made early during encystation and localizes to the ectocyst layer. In contrast, Leo and Luke lectins are made later and localize to the endocyst layer and ostioles. Probes for CWPs (anti-GFP antibodies) and for glycopolymers (maltose-binding protein-fusions with CWPs) suggest Jonah lectin and the glycopolymers to which it binds are accessible in the ectocyst layer, while Luke and Leo lectins and their epitopes are mostly inaccessible in the ectocyst layer and ostioles. In summary, the most abundantA. castellaniiCWPs are three sets of lectins, which have conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique carbohydrate-binding modules (8-Cys of Jonah).Author summaryFifty years ago, the cyst wall ofAcanthamoeba castellaniiwas shown to contain cellulose and have an ectocyst layer, an endocyst layer, and conical ostioles that attach them. The goals here were to identify abundant cyst wall proteins (CWPs) and begin to determine how the wall is assembled. We used wheat germ agglutinin to show cyst walls also contain chitin fibrils. When trophozoites are starved of nutrients, they become immotile and make CWPs and glycopolymers in dozens of small vesicles. The primordial cyst wall is composed of a single, thin layer containing cellulose, chitin, and an abundant CWP we called Jonah. The primordial wall also has small, flat ostioles that contain another abundant CWP we called Luke. Jonah (the best candidate for diagnostic antibodies) is accessible in the ectocyst layer of mature cyst walls, while Luke and a third abundant CWP we termed Leo are present but mostly inaccessible in the endocyst layer and ostioles. WhileA. castellaniicyst walls contain cellulose (like plants) and chitin (like fungi), the glycopolymers are made in vesicles rather than at the plasma membrane, and the CWPs (Luke, Leo, and Jonah lectins) are unique to the protist.

Severe malaria syndromes are precipitated by Plasmodium falciparum parasites binding to endothelial receptors on the vascular lining. This binding is mediated by members of the highly variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. We have previously identified a subset of PfEMP1 proteins associated with severe malaria and found that the receptor for these PfEMP1 variants is endothelial protein C receptor (EPCR). The binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDR) of the subtypes α1.1 and α1.4 to α1.8. In this study, we investigated the acquisition of anti-CIDR antibodies using plasma samples collected in four study villages with different malaria transmission intensities in northeastern Tanzania during a period with a decline in malaria transmission. We show that individuals exposed to high levels of malaria transmission acquire antibodies to EPCR-binding CIDR domains early in life and that these antibodies are acquired more rapidly than antibodies to other CIDR domains. The rate by which antibodies to EPCR-binding CIDR domains are acquired in populations in areas where malaria is endemic is determined by the malaria transmission intensity, and on a population level, the antibodies are rapidly lost if transmission is interrupted. This indicates that sustained exposure is required to maintain the production of the antibodies.

Dissecting Plasmodium drug resistance Malaria is a deadly disease with no effective vaccine. Physicians thus depend on antimalarial drugs to save lives, but such compounds are often rendered ineffective when parasites evolve resistance. Cowell et al. systematically studied patterns of Plasmodium falciparum genome evolution by analyzing the sequences of clones that were resistant to diverse antimalarial compounds across the P. falciparum life cycle (see the Perspective by Carlton). The findings identify hitherto unrecognized drug targets and drug-resistance genes, as well as additional alleles in known drug-resistance genes. Science , this issue p. 191 ; see also p. 159

Chemogenetic characterization throughin vitroevolution combined with whole genome analysis is a powerful tool to discover novel antimalarial drug targets and identify drug resistance genes. Our comprehensive genome analysis of 262Plasmodium falciparumparasites treated with 37 diverse compounds reveals how the parasite evolves to evade the action of small molecule growth inhibitors. This detailed data set revealed 159 gene amplifications and 148 nonsynonymous changes in 83 genes which developed during resistance acquisition. Using a new algorithm, we show that gene amplifications contribute to 1/3 of drug resistance acquisition events. In addition to confirming known multidrug resistance mechanisms, we discovered novel multidrug resistance genes. Furthermore, we identified promising new drug target-inhibitor pairs to advance the malaria elimination campaign, including: thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This deep exploration of theP. falciparumresistome and drug-able genome will guide future drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms of the deadliest malaria parasite.One Sentence SummaryWhole genome sequencing reveals howPlasmodium falciparumevolves resistance to diverse compounds and identifies new antimalarial drug targets.

Objective. To identify factors associated with perinatal mortality in northeastern Tanzania. Design. Prospective cohort study. Setting. Northeastern Tanzania. Population. 872 mothers and their newb ...

Malaria remains a major threat, in sub-Saharan Africa primarily, and the most deadly infections are those with Plasmodium falciparum. Pregnancy-associated malaria is a clinically important complication of infection; it results from a unique interaction between proteoglycans in the placental intervillous space and parasite antigens. Both placental and chondroitin sulphate A-selected parasites have high-level transcripts of a unique var gene named var2csa. However, VAR2CSA has not been consistently found by proteomic analysis of placental parasites. Contrary to this, we found VAR2CSA expressed on the surface of infected erythrocytes from placenta. Importantly, this was achieved with cross-reactive antibodies against VAR2CSA.

The var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family is responsible for antigenic variation and sequestration of infected erythrocytes during malaria. We have previously grouped the 60 PfEMP1 variants of P. falciparum clone 3D7 into groups A and B/A (category A) and groups B, B/C, and C (category non-A). Expression of category A molecules is associated with severe malaria, and that of category non-A molecules is associated with uncomplicated malaria and asymptomatic infection. Here we assessed cross-reactivity among 60 different recombinant PfEMP1 domains derived from clone 3D7 by using a competition enzyme-linked immunosorbent assay and a pool of plasma from 63 malaria-exposed Tanzanian individuals. We conclude that naturally acquired antibodies are largely directed toward epitopes varying between different domains with a few, mainly category A, domains sharing cross-reactive antibody epitopes. Identification of groups of serological cross-reacting molecules is pivotal for the development of vaccines based on PfEMP1.

The widespread emergence of Plasmodium falciparum (Pf) strains resistant to frontline agents has fuelled the search for fast-acting agents with novel mechanism of action. Here, we report the discovery and optimization of novel antimalarial compounds, the triaminopyrimidines (TAPs), which emerged from a phenotypic screen against the blood stages of Pf. The clinical candidate (compound 12) is efficacious in a mouse model of Pf malaria with an ED99, The emergence of resistant Plasmodium strains fuels the search for new antimalarials. Here, the authors present a new class of potent antimalarial compounds, the triaminopyrimidines, that display low toxicity and long half-life in animal models.

Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure-activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED90 of 28.6 mg·kg(-1)) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.

Sequestration of Plasmodium falciparum-infected erythrocytes in host blood vessels is a key triggering event in the pathogenesis of severe childhood malaria, which is responsible for about one million deaths every year. Sequestration is mediated by specific interactions between members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins was unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology and the development of new malaria interventions. © 2013 Macmillan Publishers Limited. All rights reserved.

To identify factors associated with perinatal mortality in northeastern Tanzania.Prospective cohort study.Northeastern Tanzania. Population. 872 mothers and their newborns.Pregnant women were screened for factors possibly associated with perinatal mortality, including preeclampsia, small-for-gestational age, preterm delivery, anemia, and health-seeking behavior. Fetal growth was monitored using ultrasound. Finally, the specific causes of the perinatal deaths were evaluated.Perinatal mortality.Forty-six deaths occurred. Key factors associated with perinatal mortality were preterm delivery (adjusted odds ratio (OR) 14.47, 95% confidence interval (CI) 3.23-64.86, p0.001), small-for-gestational age (adjusted OR 3.54, 95%CI 1.18-10.61, p = 0.02), and maternal anemia (adjusted OR 10.34, 95%CI 1.89-56.52, p = 0.007). Adherence to the antenatal care program (adjusted OR 0.027, 95%CI 0.003-0.26, p = 0.002) protected against perinatal mortality. The cause of death in 43% of cases was attributed to complications related to labor and specifically to intrapartum asphyxia (30%) and neonatal infection (13%). Among the remaining deaths, 27% (7/26) were attributed to preeclampsia and 23% (6/26) to small-for-gestational age. Of these, 54% (14/26) were preterm.Preeclampsia, small-for-gestational age and preterm delivery were key risk factors and causes of perinatal mortality in this area of Tanzania. Maternal anemia was also strongly associated with perinatal mortality. Furthermore, asphyxia accounted for a large proportion of the perinatal deaths. Interventions should target the prevention and handling of these conditions in order to reduce perinatal mortality.

The clinical outcome of Plasmodium falciparum infections ranges from asymptomatic parasitemia to severe malaria syndromes associated with high mortality. The virulence of P. falciparum infections is associated with the type of P. falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the surface of infected erythrocytes to anchor these to the vascular lining. Although var2csa , the var gene encoding the PfEMP1 associated with placental malaria, was discovered in 2003, the identification of the var /PfEMP1 variants associated with severe malaria in children has remained elusive. To identify var /PfEMP1 variants associated with severe disease outcome, we compared var transcript levels in parasites from 88 children with severe malaria and 40 children admitted to the hospital with uncomplicated malaria. Transcript analysis was performed by RT-quantitative PCR using a set of 42 primer pairs amplifying var subtype-specific loci covering most var /PfEMP1 subtypes. In addition, we characterized the near-full-length sequence of the most prominently expressed var genes in three patients diagnosed with severe anemia and/or cerebral malaria. The combined analysis showed that severe malaria syndromes, including severe anemia and cerebral malaria, are associated with high transcript levels of PfEMP1 domain cassette 8-encoding var genes. Transcript levels of group A var genes, including genes encoding domain cassette 13, were also significantly higher in patients with severe syndromes compared with those with uncomplicated malaria. This study specifies the var /PfEMP1 types expressed in severe malaria in children, and thereby provides unique targets for future efforts to prevent and treat severe malaria infections.

Background Members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion antigen family are major contributors to the pathogenesis of P. falciparum malaria infections. The PfEMP1-encoding var genes are among the most diverse sequences in nature, but three genes, var1, var2csa and var3 are found conserved in most parasite genomes. The most severe forms of malaria disease are caused by parasites expressing a subset of antigenically conserved PfEMP1 variants. Thus the ubiquitous and conserved VAR3 PfEMP1 is of particular interest to the research field. Evidence of VAR3 expression on the infected erythrocyte surface has never been presented, and var3 genes have been proposed to be transcribed and expressed differently from the rest of the var gene family members. Methods In this study, parasites expressing VAR3 PfEMP1 were generated using anti-VAR3 antibodies and the var transcript and PfEMP1 expression profiles of the generated parasites were investigated. The IgG reactivity by plasma from children living in malaria-endemic Tanzania was tested to parasites and recombinant VAR3 protein. Parasites from hospitalized children were isolated and the transcript level of var3 was investigated. Results Var3 is transcribed and its protein product expressed on the surface of infected erythrocytes. The VAR3-expressing parasites were better recognized by children´s IgG than a parasite line expressing a Group B var gene. Two in 130 children showed increased recognition of parasites expressing VAR3 and to the recombinant VAR3 protein after a malaria episode and the isolated parasites showed high levels of var3 transcripts. Conclusions Collectively, the presented data suggest that var3 is transcribed and its protein product expressed on the surface of infected erythrocytes in the same manner as seen for other var genes both in vitro and in vivo. Only very few children exhibit seroconversion to VAR3 following a malaria episode requiring hospitalization, supporting the previous conclusion drawn from var3 transcript analysis of parasites collected from children hospitalized with malaria, that VAR3 is not associated with severe anaemia or cerebral malaria syndromes in children.

Malaria during pregnancy in Plasmodium falciparum endemic regions is a major cause of mortality and severe morbidity. VAR2CSA is the parasite ligand responsible for sequestration of Plasmodium falciparum infected erythrocytes to the receptor chondroitin sulfate A (CSA) in the placenta and is the leading candidate for a placental malaria vaccine. Antibodies induced in rats against the recombinant DBL4e domain of VAR2CSA inhibit the binding of a number of laboratory and field parasite isolates to CSA. In this study, we used a DBL4e peptide-array to identify epitopes targeted by DBL4e-specific antibodies that inhibit CSA-binding of infected erythrocytes. We identified three regions of overlapping peptides which were highly antigenic. One peptide region distinguished itself particularly by showing a clear difference in the binding profile of highly parasite blocking IgG compared to the IgG with low capacity to inhibit parasite adhesion to CSA. This region was further characterized and together these results suggest that even though antibodies against the synthetic peptides which cover this region did not recognize native protein, the results using the mutant domain suggest that this linear epitope might be involved in the induction of inhibitory antibodies induced by the recombinant DBL4e domain.

Background Accurate diagnosis and prompt treatment of pregnancy-associated malaria (PAM) are key aspects in averting adverse pregnancy outcomes. Microscopy is the gold standard in malaria diagnosis, but it has limited detection and availability. When used appropriately, rapid diagnostic tests (RDTs) could be an ideal diagnostic complement to microscopy, due to their ease of use and adequate sensitivity in detecting even sub-microscopic infections. Polymerase chain reaction (PCR) is even more sensitive, but it is mainly used for research purposes. The accuracy and reliability of RDTs in diagnosing PAM was evaluated using microscopy and PCR. Methods A cohort of pregnant women in north-eastern Tanzania was followed throughout pregnancy for detection of plasmodial infection using venous and placental blood samples evaluated by histidine rich protein 2 (HRP-2) and parasite lactate dehydrogenase (pLDH) based RDTs (Parascreen™) or HRP-2 only (Paracheck Pf® and ParaHIT®f), microscopy and nested Plasmodium species diagnostic PCR. Results From a cohort of 924 pregnant women who completed the follow up, complete RDT and microscopy data was available for 5,555 blood samples and of these 442 samples were analysed by PCR. Of the 5,555 blood samples, 49 ((proportion and 95% confidence interval) 0.9% [0.7 -1.1]) samples were positive by microscopy and 91 (1.6% [1.3-2.0]) by RDT. Forty-six (50.5% [40.5 - 60.6]) and 45 (49.5% [39.4 – 59.5]) of the RDT positive samples were positive and negative by microscopy, respectively, whereas nineteen (42.2% [29.0 - 56.7]) of the microscopy negative, but RDT positive, samples were positive by PCR. Three (0.05% [0.02 - 0.2]) samples were positive by microscopy but negative by RDT. 351 of the 5,461 samples negative by both RDT and microscopy were tested by PCR and found negative. There was no statistically significant difference between the performances of the different RDTs. Conclusions Microscopy underestimated the real burden of malaria during pregnancy and RDTs performed better than microscopy in diagnosing PAM. In areas where intermittent preventive treatment during pregnancy may be abandoned due to low and decreasing malaria risk and instead replaced with active case management, screening with RDT is likely to identify most infections in pregnant women and out-performs microscopy as a diagnostic tool.

Acquired protection from Plasmodium falciparum placental malaria, a major cause of maternal, fetal, and infant morbidity, is mediated by IgG specific for the P. falciparum erythrocyte membrane protein 1 variant VAR2CSA. This protein enables adhesion of P. falciparum-infected erythrocytes to chondroitin sulfate A in the intervillous space. Although interclonal variation of the var2csa gene is lower than that among var genes in general, VAR2CSA-specific Abs appear to target mainly polymorphic epitopes. This has raised doubts about the feasibility of VAR2CSA-based vaccines. We used eight human monoclonal IgG Abs from affinity-matured memory B cells of P. falciparum-exposed women to study interclonal variation and functional importance of Ab epitopes among placental and peripheral parasites from East and West Africa. Most placental P. falciparum isolates were labeled by several mAbs, whereas peripheral isolates from children were essentially nonreactive. The mAb reactivity of peripheral isolates from pregnant women indicated that some were placental, whereas others had alternative sequestration foci. Most of the mAbs were comparable in their reactivity with bound infected erythrocytes (IEs) and recombinant VAR2CSA and interfered with IE and/or VAR2CSA binding to chondroitin sulfate A. Pair-wise mAb combinations were more inhibitory than single mAbs, and all of the mAbs together was the most efficient combination. Each mAb could opsonize IEs for phagocytosis, and a combination of the eight mAbs caused phagocytosis similar to that of plasma IgG-opsonized IEs. We conclude that functionally important Ab epitopes are shared by the majority of polymorphic VAR2CSA variants, which supports the feasibility of VAR2CSA-based vaccines against placental malaria.

Background A subset of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1SM) is involved in the cytoadherence of P. falciparum-infected red blood cells (iRBC) contributing to the pathogenesis of severe disease among young children in malaria endemic areas. The PfEMP1SM are encoded by group A var genes that are composed of a more constrained range of amino acid sequences than groups B and C var genes encoding PfEMP1UM associated with uncomplicated malaria. Also, unlike var genes from groups B and C, those from group A do not have sequences consistent with CD36 binding – a major cytoadhesion phenotype of P. falciparum isolates. Methods A 3D7 PfEMP1SM sub-line (3D7SM) expressing VAR4 (PFD1235w/MAL8P1.207) was selected for binding to CD36. The protein expression of this parasite line was monitored by surface staining of iRBC using VAR4-specific antibodies. The serological phenotype of the 3D7SM parasites was determined by flow cytometry using malaria semi-immune and immune plasma and transcription of the 59 var genes in 3D7 were analysed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using var-specific primers. Results A selection-induced increased adhesion of 3D7SM iRBC to CD36 resulted in a reduced var4 transcription and VAR4 surface expression. Conclusion VAR4 is not involved in CD36 adhesion. The current findings are consistent with the notion that CD36 adhesion is not associated with particular virulent parasite phenotypes, such as those believed to be exhibited by VAR4 expressing parasites.

Plasmodium falciparum variant surface antigens (VSA) are targets of protective immunity to malaria. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and repetitive interspersed family (RIFIN) proteins are encoded by the two variable multigene families, var and rif genes, respectively. Whereas PfEMP1s are known to mediate cytoadhesion, the function of RIFINs is unknown. The sequence diversity and organisation of rif genes of the P. falciparum clones 3D7, HB3, DD2, and IT/FCR3 were investigated using a tree-building method which allowed sub-grouping of RIFINs into distinct groups. Two novel rif gene groups, rifA1 and rifA2, containing inter-genomic conserved rif genes, were identified. All rifA1 genes were orientated head-to-head with a neighbouring Group A var gene whereas rifA2 was present in all parasite genomes as a single copy gene with a unique 5' untranslated region. Rif transcript levels were determined in two different parasite lines, 3D7-Lib and NF54-VAR2CSA, expressing VSA associated with severe malaria in children and pregnant women, respectively. The 3D7-Lib showed high transcript levels of Group A var and neighbouring rif genes, whereas rifA2 was found highly transcribed in the VAR2CSA-expressing parasite line. In addition, two rif genes were found transcribed at early and late intra-erythrocyte stages independently of var gene transcription. Rif genes are organised in groups and inter-genomic conserved gene families, suggesting that RIFIN sub-groups may have different functional capacities. This conclusion is experimentally supported by group-specific rif transcription in parasites with different VSA and PfEMP1 expression phenotypes.

Variant surface antigens (VSA) on the surface of Plasmodium falciparum -infected red blood cells play a major role in the pathogenesis of malaria and are key targets for acquired immunity. The best-characterized VSA belong to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. In areas where P. falciparum is endemic, parasites causing severe malaria and malaria in young children with limited immunity tend to express semiconserved PfEMP1 molecules encoded by group A var genes. Here we investigated antibody responses of Tanzanians who were 0 to 19 years old to PF11_0008, a group A PfEMP1. PF11_0008 has previously been found to be highly transcribed in a nonimmune Dutch volunteer experimentally infected with NF54 parasites. A high proportion of the Tanzanian donors had antibodies against recombinant PF11_0008 domains, and in children who were 4 to 9 years old the presence of antibodies to the PF11_0008 CIDR2β domain was associated with reduced numbers of malaria episodes. These results indicate that homologues of PF11_0008 are present in P. falciparum field isolates and suggest that PF11_0008 CIDR2β-reactive antibodies might be involved in protection against malaria episodes.

In Plasmodium falciparum–endemic areas, pregnancy-associated malaria (PAM) is an important health problem. The condition is precipitated by accumulation of parasite-infected erythrocytes (IEs) in the placenta, and this process is mediated by parasite-encoded variant surface antigens (VSA) binding to chondroitin sulfate A (CSA). Parasites causing PAM express unique VSA types, VSAPAM, which can be serologically classified as sex specific and parity dependent. It is sex specific because men from malaria-endemic areas do not develop VSAPAM antibodies; it is parity dependent because women acquire anti-VSAPAM immunoglobulin (Ig) G as a function of parity. Previously, it was shown that transcription of var2csa is up-regulated in placental parasites and parasites selected for CSA binding. Here, we show the following: (a) that VAR2CSA is expressed on the surface of CSA-selected IEs; (b) that VAR2CSA is recognized by endemic plasma in a sex-specific and parity-dependent manner; (c) that high anti-VAR2CSA IgG levels can be found in pregnant women from both West and East Africa; and (d) that women with high plasma levels of anti-VAR2CSA IgG give birth to markedly heavier babies and have a much lower risk of delivering low birth weight children than women with low levels.

Item does not contain fulltext Parasite-encoded variant surface antigens (VSAs) like the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in nonimmune patients tend to express a restricted subset of VSA (VSA(SM)) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSA(UM)). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSA(UM) to in vitro-selected sublines expressing VSA(SM) to identify PfEMP1 responsible for the VSA(SM) phenotype. Expression of VSA(SM) was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var genes, such as PFD1235w/MAL7P1.1, appear to be involved in the pathogenesis of severe disease and are thus attractive candidates for a vaccine against life-threatening P. falciparum malaria.

Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is approximately 25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum-infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID(50) values for SM/LCPL-PLC activities and the parasite growth at 3-5 microM and approximately 7 microM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.

Background Despite some problems related to accuracy and applicability of malaria rapid diagnostic tests (RDTs), they are currently the best option in areas with limited laboratory services for improving case management through parasitological diagnosis and reducing over-treatment. This study was conducted in areas with declining malaria burden to assess; 1) the accuracy of RDTs when used at different community settings, 2) the impact of using RDTs on anti-malarial dispensing by community-owned resource persons (CORPs) and 3) adherence of CORPs to treatment guidelines by providing treatment based on RDT results. Methods Data were obtained from: 1) a longitudinal study of passive case detection of fevers using CORPs in six villages in Korogwe; and 2) cross-sectional surveys (CSS) in six villages of Korogwe and Muheza districts, north-eastern, Tanzania. Performance of RDTs was compared with microscopy as a gold standard, and factors affecting their accuracy were explored using a multivariate logistic regression model. Results Overall sensitivity and specificity of RDTs in the longitudinal study (of 23,793 febrile cases; 18,154 with microscopy and RDTs results) were 88.6% and 88.2%, respectively. In the CSS, the sensitivity was significantly lower (63.4%; χ2 = 367.7, p < 0.001), while the specificity was significantly higher (94.3%; χ2 = 143.1, p < 0.001) when compared to the longitudinal study. As determinants of sensitivity of RDTs in both studies, parasite density of < 200 asexual parasites/μl was significantly associated with high risk of false negative RDTs (OR≥16.60, p < 0.001), while the risk of false negative test was significantly lower among cases with fever (axillary temperature ≥37.5°C) (OR ≤ 0.63, p ≤ 0.027). The risk of false positive RDT (as a determinant of specificity) was significantly higher in cases with fever compared to afebrile cases (OR≥2.40, p < 0.001). Using RDTs reduced anti-malarials dispensing from 98.9% to 32.1% in cases aged ≥5 years. Conclusion Although RDTs had low sensitivity and specificity, which varied widely depending on fever and parasite density, using RDTs reduced over-treatment with anti-malarials significantly. Thus, with declining malaria prevalence, RDTs will potentially identify majority of febrile cases with parasites and lead to improved management of malaria and non-malaria fevers.

Background Parasites causing severe malaria in non-immune patients express a restricted subset of variant surface antigens (VSA), which are better recognized by immune sera than VSA expressed during non-severe disease in semi-immune individuals. The most prominent VSA are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which is expressed on the surface of infected erythrocytes where it mediates binding to endothelial receptors. Thus, severe malaria may be caused by parasites expressing PfEMP1 variants that afford parasites optimal sequestration in immunologically naïve individuals and high effective multiplication rates. Methods var gene transcription was analysed using real time PCR and PfEMP1 expression by western blots as well as immune plasma recognition of parasite cultures established from non-immune volunteers shortly after infection with NF54 sporozoites. Results In cultures representing the first generation of parasites after hepatic release, all var genes were transcribed, but GroupA var genes were transcribed at the lowest levels. In cultures established from second or third generation blood stage parasites of volunteers with high in vivo parasite multiplication rates, the var gene transcription pattern differed markedly from the transcription pattern of the cultures representing first generation parasites. This indicated that parasites expressing specific var genes, mainly belonging to group A and B, had expanded more effectively in vivo compared to parasites expressing other var genes. The differential expression of PfEMP1 was confirmed at the protein level by immunoblot analysis. In addition, serological typing showed that immune sera more often recognized second and third generation parasites than first generation parasites. Conclusion In conclusion, the results presented here support the hypothesis that parasites causing severe malaria express a subset of PfEMP1, which bestows high parasite growth rates in individuals with limited pre-existing immunity.

Protection against Plasmodium falciparum malaria is largely mediated by IgG against surface Ags such as the erythrocyte membrane protein 1 family (PfEMP1) responsible for antigenic variation and sequestration of infected erythrocytes. PfEMP1 molecules can be divided into groups A, B/A, B, C, and B/C. We have previously suggested that expression of groups A and B/A PfEMP1 is associated with severe disease and that Abs to these molecules are acquired earlier in life than Abs to PfEMP1 belonging to groups B, B/C, and C PfEMP1. In this study, we compared the acquisition of IgG to 20 rPfEMP1 domains derived from 3D7 in individuals living under markedly different malaria transmission intensity and were unable to find differences in the Ab acquisition rate to PfEMP1 of different groupings (A, B, or C) or domain type (α, β, γ, δ, ε, or x). Abs were acquired early in life in individuals living in the high transmission village and by the age of 2–4 years most individuals had Abs against most constructs. This level of reactivity was found at the age of 10–20 years in the medium transmission village and was never reached by individuals living under low transmission. Nevertheless, the sequence by which individuals acquired Abs to particular constructs was largely the same in the three villages. This indicates that the pattern of PfEMP1 expression by parasites transmitted at the different sites was similar, suggesting that PfEMP1 expression is nonrandom and shaped by host-parasite relationship factors operating at all transmission intensities.