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Find video protocols related to scientific articles indexed in Pubmed.
Effects of mefloquine use on Plasmodium vivax multidrug resistance.
Emerging Infect. Dis.
PUBLISHED: 10-02-2014
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Numerous studies have indicated a strong association between amplification of the multidrug resistance-1 gene and in vivo and in vitro mefloquine resistance of Plasmodium falciparum. Although falciparum infection usually is not treated with mefloquine, incorrect diagnosis, high frequency of undetected mixed infections, or relapses of P. vivax infection triggered by P. falciparum infections expose non-P. falciparum parasites to mefloquine. To assess the consequences of such unintentional treatments on P. vivax, we studied variations in number of Pvmdr-1 (PlasmoDB accession no. PVX_080100, NCBI reference sequence NC_009915.1) copies worldwide in 607 samples collected in areas with different histories of mefloquine use from residents and from travelers returning to France. Number of Pvmdr-1 copies correlated with drug use history. Treatment against P. falciparum exerts substantial collateral pressure against sympatric P. vivax, jeopardizing future use of mefloquine against P. vivax. A drug policy is needed that takes into consideration all co-endemic species of malaria parasites.
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Comparative analysis of IgG responses to Plasmodium falciparum MSP1p19 and PF13-DBL1?1 using ELISA and a magnetic bead-based duplex assay (MAGPIX®-Luminex) in a Senegalese meso-endemic community.
Malar. J.
PUBLISHED: 08-13-2014
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Numerous Plasmodium falciparum antigens elicit humoral responses in humans living in endemic areas. Use of multiplex assays is a convenient approach to monitor the antibody response against multiple antigens, but to integrate multiplex assay-derived data with datasets, generated previously using ELISA, comparative studies are needed. This work compares antibody responses to two P. falciparum antigens monitored using both technologies.
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The rise and fall of malaria in a West African rural community, Dielmo, Senegal, from 1990 to 2012: a 22 year longitudinal study.
Lancet Infect Dis
PUBLISHED: 05-06-2014
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A better understanding of the effect of malaria control interventions on vector and parasite populations, acquired immunity, and burden of the disease is needed to guide strategies to eliminate malaria from highly endemic areas. We monitored and analysed the changes in malaria epidemiology in a village community in Senegal, west Africa, over 22 years.
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A novel Plasmodium-specific prodomain fold regulates the malaria drug target SUB1 subtilase.
Nat Commun
PUBLISHED: 03-06-2014
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The Plasmodium subtilase SUB1 plays a pivotal role during the egress of malaria parasites from host hepatocytes and erythrocytes. Here we report the crystal structure of full-length SUB1 from the human-infecting parasite Plasmodium vivax, revealing a bacterial-like catalytic domain in complex with a Plasmodium-specific prodomain. The latter displays a novel architecture with an amino-terminal insertion that functions as a 'belt', embracing the catalytic domain to further stabilize the quaternary structure of the pre-protease, and undergoes calcium-dependent autoprocessing during subsequent activation. Although dispensable for recombinant enzymatic activity, the SUB1 'belt' could not be deleted in Plasmodium berghei, suggesting an essential role of this domain for parasite development in vivo. The SUB1 structure not only provides a valuable platform to develop new anti-malarial candidates against this promising drug target, but also defines the Plasmodium-specific 'belt' domain as a key calcium-dependent regulator of SUB1 during parasite egress from host cells.
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Reduced erythrocyte deformability associated with hypoargininemia during Plasmodium falciparum malaria.
Sci Rep
PUBLISHED: 01-21-2014
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The mechanisms underlying reduced red blood cell (RBC) deformability during Plasmodium falciparum (Pf) malaria remain poorly understood. Here, we explore the possible involvement of the L-arginine and nitric oxide (NO) pathway on RBC deformability in Pf-infected patients and parasite cultures. RBC deformability was reduced during the acute attack (day0) and returned to normal values upon convalescence (day28). Day0 values correlated with plasma L-arginine levels (r = 0.69; p = 0.01) and weakly with parasitemia (r = -0.38; p = 0.006). In vitro, day0 patient's plasma incubated with ring-stage cultures at 41°C reduced RBC deformability, and this effect correlated strongly with plasma L-arginine levels (r = 0.89; p < 0.0001). Moreover, addition of exogenous L-arginine to the cultures increased deformability of both Pf-free and trophozoite-harboring RBCs. NO synthase activity, evidenced in Pf-infected RBCs, induced L-arginine-dependent NO production. These data show that hypoargininemia during P. falciparum malaria may altogether impair NO production and reduce RBC deformability, particularly at febrile temperature.
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An exported kinase (FIKK4.2) that mediates virulence-associated changes in Plasmodium falciparum-infected red blood cells.
Int. J. Parasitol.
PUBLISHED: 01-16-2014
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Alteration of the adhesive and mechanical properties of red blood cells caused by infection with the malaria parasite Plasmodium falciparum underpin both its survival and extreme pathogenicity. A unique family of parasite putative exported kinases, collectively called FIKK (Phenylalanine (F) - Isoleucine (I) - Lysine (K) - Lysine (K)), has recently been implicated in these pathophysiological processes, however, their precise function in P. falciparum-infected red blood cells or their likely role in malaria pathogenesis remain unknown. Here, for the first time, we demonstrate that one member of the FIKK family, FIKK4.2, can function as an active kinase and is localised in a novel and distinct compartment of the parasite-infected red blood cell which we have called K-dots. Notably, targeted disruption of the gene encoding FIKK4.2 (fikk4.2) dramatically alters the parasite's ability to modify and remodel the red blood cells in which it multiplies. Specifically, red blood cells infected with fikk4.2 knockout parasites were significantly less rigid and less adhesive when compared with red blood cells infected with normal parasites from which the transgenic clones had been derived, despite expressing similar levels of the major cytoadhesion ligand, PfEMP1, on the red blood cell surface. Notably, these changes were accompanied by dramatically altered knob-structures on infected red blood cells that play a key role in cytoadhesion which is responsible for much of the pathogenesis associated with falciparum malaria. Taken together, our data identifies FIKK4.2 as an important kinase in the pathogenesis of P. falciparum malaria and strengthens the attractiveness of FIKK kinases as targets for the development of novel next-generation anti-malaria drugs.
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Association of antibody responses to the conserved Plasmodium falciparum merozoite surface protein 5 with protection against clinical malaria.
PLoS ONE
PUBLISHED: 01-01-2014
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Plasmodium falciparum merozoite surface protein 5 (PfMSP5) is an attractive blood stage vaccine candidate because it is both exposed to the immune system and well conserved. To evaluate its interest, we investigated the association of anti-PfMSP5 IgG levels, in the context of responses to two other conserved Ags PfMSP1p19 and R23, with protection from clinical episodes of malaria in cross-sectional prospective studies in two different transmission settings.
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De Novo Assembly of a Field Isolate Genome Reveals Novel Plasmodium vivax Erythrocyte Invasion Genes.
PLoS Negl Trop Dis
PUBLISHED: 12-01-2013
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Recent sequencing of Plasmodium vivax field isolates and monkey-adapted strains enabled characterization of SNPs throughout the genome. These analyses relied on mapping short reads onto the P. vivax reference genome that was generated using DNA from the monkey-adapted strain Salvador I. Any genomic locus deleted in this strain would be lacking in the reference genome sequence and missed in previous analyses. Here, we report de novo assembly of a P. vivax field isolate genome. Out of 2,857 assembled contigs, we identify 362 contigs, each containing more than 5 kb of contiguous DNA sequences absent from the reference genome sequence. These novel P. vivax DNA sequences account for 3.8 million nucleotides and contain 792 predicted genes. Most of these contigs contain members of multigene families and likely originate from telomeric regions. Interestingly, we identify two contigs containing predicted protein coding genes similar to known Plasmodium red blood cell invasion proteins. One gene encodes the reticulocyte-binding protein gene orthologous to P. cynomolgi RBP2e and P. knowlesi NBPXb. The second gene harbors all the hallmarks of a Plasmodium erythrocyte-binding protein, including conserved Duffy-binding like and C-terminus cysteine-rich domains. Phylogenetic analysis shows that this novel gene clusters separately from all known Plasmodium Duffy-binding protein genes. Additional analyses showing that this gene is present in most P. vivax genomes and transcribed in blood-stage parasites suggest that P. vivax red blood cell invasion mechanisms may be more complex than currently understood. The strategy employed here complements previous genomic analyses and takes full advantage of next-generation sequencing data to provide a comprehensive characterization of genetic variations in this important malaria parasite. Further analyses of the novel protein coding genes discovered through de novo assembly have the potential to identify genes that influence key aspects of P. vivax biology, including alternative mechanisms of human erythrocyte invasion.
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Whole Genome Sequencing of Field Isolates Reveals a Common Duplication of the Duffy Binding Protein Gene in Malagasy Plasmodium vivax Strains.
PLoS Negl Trop Dis
PUBLISHED: 11-01-2013
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Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes.
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A key role for Plasmodium subtilisin-like SUB1 protease in egress of malaria parasites from host hepatocytes.
J. Biol. Chem.
PUBLISHED: 10-02-2013
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In their mammalian host, Plasmodium parasites have two obligatory intracellular development phases, first in hepatocytes and subsequently in erythrocytes. Both involve an orchestrated process of invasion into and egress from host cells. The Plasmodium SUB1 protease plays a dual role at the blood stage by enabling egress of the progeny merozoites from the infected erythrocyte and priming merozoites for subsequent erythrocyte invasion. Here, using conditional mutagenesis in P. berghei, we show that SUB1 plays an essential role at the hepatic stage. Stage-specific sub1 invalidation during prehepatocytic development showed that SUB1-deficient parasites failed to rupture the parasitophorous vacuole membrane and to egress from hepatocytes. Furthermore, mechanically released parasites were not adequately primed and failed to establish a blood stage infection in vivo. The critical involvement of SUB1 in both pre-erythrocytic and erythrocytic developmental phases qualifies SUB1 as an attractive multistage target for prophylactic and therapeutic anti-Plasmodium intervention strategies.
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Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies.
Lancet Infect Dis
PUBLISHED: 09-11-2013
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Artemisinin resistance in Plasmodium falciparum lengthens parasite clearance half-life during artemisinin monotherapy or artemisinin-based combination therapy. Absence of in-vitro and ex-vivo correlates of artemisinin resistance hinders study of this phenotype. We aimed to assess whether an in-vitro ring-stage survival assay (RSA) can identify culture-adapted P falciparum isolates from patients with slow-clearing or fast-clearing infections, to investigate the stage-dependent susceptibility of parasites to dihydroartemisinin in the in-vitro RSA, and to assess whether an ex-vivo RSA can identify artemisinin-resistant P falciparum infections.
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A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.
Nature
PUBLISHED: 07-15-2013
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Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (K13-propeller) with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.
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[Plasmodium falciparum susceptibility to antimalarial drugs: global data issued from the Pasteur Institutes international network].
Med Sci (Paris)
PUBLISHED: 07-12-2013
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Malaria research units within the Institut Pasteur international network (RIIP-Palu) located in Africa, in South-East Asia and in South America, work for many years in close collaboration with the National malaria control programmes. Relying on technical platforms with well-equipped laboratories and scientific expertise, they are at the forefront of research on the antimalarial drug resistance by working together for training young scientists and developping similar protocols allowing comprehensive comparisons. Including fundamental and operational researches, they conduct regional and international projects which aim (1) to detect the emergence of antimalarial drugs resistant parasites and to evaluate their spatio-temporal distribution, (2) to develop in vitro and molecular tools, (3) to identify epidemiological factors involved in the emergence and the spread of antimalarial drugs resistant parasites and (4) to understand the molecular and cellular mechanisms implicated in resistance. In this review, will be presented methodological approaches and data obtained since 2000.
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In Silico screening on the three-dimensional model of the Plasmodium vivax SUB1 protease leads to the validation of a novel anti-parasite compound.
J. Biol. Chem.
PUBLISHED: 05-07-2013
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Widespread drug resistance calls for the urgent development of new antimalarials that target novel steps in the life cycle of Plasmodium falciparum and Plasmodium vivax. The essential subtilisin-like serine protease SUB1 of Plasmodium merozoites plays a dual role in egress from and invasion into host erythrocytes. It belongs to a new generation of attractive drug targets against which specific potent inhibitors are actively searched. We characterize here the P. vivax SUB1 enzyme and show that it displays a typical auto-processing pattern and apical localization in P. vivax merozoites. To search for small PvSUB1 inhibitors, we took advantage of the similarity of SUB1 with bacterial subtilisins and generated P. vivax SUB1 three-dimensional models. The structure-based virtual screening of a large commercial chemical compounds library identified 306 virtual best hits, of which 37 were experimentally confirmed inhibitors and 5 had Ki values of <50 ?M for PvSUB1. Interestingly, they belong to different chemical families. The most promising competitive inhibitor of PvSUB1 (compound 2) was equally active on PfSUB1 and displayed anti-P. falciparum and Plasmodium berghei activity in vitro and in vivo, respectively. Compound 2 inhibited the endogenous PfSUB1 as illustrated by the inhibited maturation of its natural substrate PfSERA5 and inhibited parasite egress and subsequent erythrocyte invasion. These data indicate that the strategy of in silico screening of three-dimensional models to select for virtual inhibitors combined with stringent biological validation successfully identified several inhibitors of the PvSUB1 enzyme. The most promising hit proved to be a potent cross-inhibitor of PlasmodiumSUB1, laying the groundwork for the development of a globally active small compound antimalarial.
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Surface area loss and increased sphericity account for the splenic entrapment of subpopulations of Plasmodium falciparum ring-infected erythrocytes.
PLoS ONE
PUBLISHED: 02-21-2013
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Ex vivo perfusion of human spleens revealed innate retention of numerous cultured Plasmodium falciparum ring-infected red blood cells (ring-iRBCs). Ring-iRBC retention was confirmed by a microsphiltration device, a microbead-based technology that mimics the mechanical filtering function of the human spleen. However, the cellular alterations underpinning this retention remain unclear. Here, we use ImageStream technology to analyze infected RBCs morphology and cell dimensions before and after fractionation with microsphiltration. Compared to fresh normal RBCs, the mean cell membrane surface area loss of trophozoite-iRBCs, ring-iRBCs and uninfected co-cultured RBCs (uRBCs) was 14.2% (range: 8.3-21.9%), 9.6% (7.3-12.2%) and 3.7% (0-8.4), respectively. Microsphilters retained 100%, ?50% and 4% of trophozoite-iRBCs, ring-iRBCs and uRBCs, respectively. Retained ring-iRBCs display reduced surface area values (estimated mean, range: 17%, 15-18%), similar to the previously shown threshold of surface-deficient RBCs retention in the human spleen (surface area loss: >18%). By contrast, ring-iRBCs that successfully traversed microsphilters had minimal surface area loss and normal sphericity, suggesting that these parameters are determinants of their retention. To confirm this hypothesis, fresh normal RBCs were exposed to lysophosphatidylcholine to induce a controlled loss of surface area. This resulted in a dose-dependent retention in microsphilters, with complete retention occurring for RBCs displaying >14% surface area loss. Taken together, these data demonstrate that surface area loss and resultant increased sphericity drive ring-iRBC retention in microsphilters, and contribute to splenic entrapment of a subpopulation of ring-iRBCs. These findings trigger more interest in malaria research fields, including modeling of infection kinetics, estimation of parasite load, and analysis of risk factors for severe clinical forms. The determination of the threshold of splenic retention of ring-iRBCs has significant implications for diagnosis (spleen functionality) and drug treatment (screening of adjuvant therapy targeting ring-iRBCs).
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Red blood cell polymorphism and susceptibility to Plasmodium vivax.
Adv. Parasitol.
PUBLISHED: 02-07-2013
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Resistance to Plasmodium vivax blood-stage infection has been widely recognised to result from absence of the Duffy (Fy) blood group from the surface of red blood cells (RBCs) in individuals of African descent. Interestingly, recent studies from different malaria-endemic regions have begun to reveal new perspectives on the association between Duffy gene polymorphism and P. vivax malaria. In Papua New Guinea and the Americas, heterozygous carriers of a Duffy-negative allele are less susceptible to P. vivax infection than Duffy-positive homozygotes. In Brazil, studies show that the Fy(a) antigen, compared to Fy(b), is associated with lower binding to the P. vivax Duffy-binding protein and reduced susceptibility to vivax malaria. Additionally, it is interesting that numerous studies have now shown that P. vivax can infect RBCs and cause clinical disease in Duffy-negative people. This suggests that the relationship between P. vivax and the Duffy antigen is more complex than customarily described. Evidence of P. vivax Duffy-independent red cell invasion indicates that the parasite must be evolving alternative red cell invasion pathways. In this chapter, we review the evidence for P. vivax Duffy-dependent and Duffy-independent red cell invasion. We also consider the influence of further host gene polymorphism associated with malaria endemicity on susceptibility to vivax malaria. The interaction between the parasite and the RBC has significant potential to influence the effectiveness of P. vivax-specific vaccines and drug treatments. Ultimately, the relationships between red cell polymorphisms and P. vivax blood-stage infection will influence our estimates on the population at risk and efforts to eliminate vivax malaria.
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High number of previous Plasmodium falciparum clinical episodes increases risk of future episodes in a sub-group of individuals.
PLoS ONE
PUBLISHED: 02-06-2013
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There exists great disparity in the number of clinical P. falciparum episodes among children of the same age and living in similar conditions. The epidemiological determinants of such disparity are unclear. We used a data-mining approach to explore a nineteen-year longitudinal malaria cohort study dataset from Senegal and identify variables associated with increased risk of malaria episodes. These were then verified using classical statistics and replicated in a second cohort. In addition to age, we identified a novel high-risk group of children in whom the history of P. falciparum clinical episodes greatly increased risk of further episodes. Age and a high number of previous falciparum clinical episodes not only play major roles in explaining the risk of P. falciparum episodes but also are risk factors for different groups of people. Combined, they explain the majority of falciparum clinical attacks. Contrary to what is widely believed, clinical immunity to P. falciparum does not de facto occur following many P. falciparum clinical episodes. There exist a sub-group of children who suffer repeated clinical episodes. In addition to posing an important challenge for population stratification during clinical trials, this sub-group disproportionally contributes to the disease burden and may necessitate specific prevention and control measures.
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Screening and evaluation of inhibitors of Plasmodium falciparum merozoite egress and invasion using cytometry.
Methods Mol. Biol.
PUBLISHED: 01-31-2013
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Drug discovery programs heavily rely on assays adequately monitoring the activity of the drug on the -parasite stage targeted. So far, assays used to screen molecules active against Plasmodium falciparum parasites have mostly been based on measuring growth inhibition of asexual blood stages. We have developed a robust protocol allowing for monitoring parasite egress at the late schizont stage and subsequent erythrocyte invasion. This cytometry-based methodology uses nucleic acid labelling by the dye YOYO-1 and synchronized in vitro culture of P.falciparum exposed to inhibitors during the late phase of the intraerythrocytic cycle and the reinvasion process. This cytometry-based method is quick, accurate and allows for distinguishing egress from reinvasion on thousands of events. The throughput is also increased, as the assay can be scaled up for medium throughput screening for compounds that inhibit either the egress of merozoites or their entry into host erythrocytes.
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Microsphiltration: a microsphere matrix to explore erythrocyte deformability.
Methods Mol. Biol.
PUBLISHED: 01-31-2013
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The altered deformability of erythrocytes infected with Plasmodium falciparum is central in malaria -pathogenesis, as it influences the hemodynamic properties of the infected cell and its retention in the spleen. Exported parasite proteins, as well as the shape and volume of the parasite itself, influence the deformability of the infected erythrocyte. To explore changes in erythrocyte deformability, we have developed a new method, called microsphiltration, based on filtration of erythrocytes through a mixture of metal microspheres that mimic the geometry of inter-endothelial splenic slits. As P. falciparum develops in its host cell, the retention rates observed in microspheres correlate with the progressive decrease of erythrocyte deformability and with the retention rates in the spleen. The yields of microsphiltration separation allow for molecular analyses of subpopulations with distinct mechanical phenotypes.
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Plasmodium falciparum STEVOR proteins impact erythrocyte mechanical properties.
Blood
PUBLISHED: 11-21-2011
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Infection of erythrocytes with the human malaria parasite, Plasmodium falciparum, results in dramatic changes to the host cell structure and morphology. The predicted functional localization of the STEVOR proteins at the erythrocyte surface suggests that they may be involved in parasite-induced modifications of the erythrocyte membrane during parasite development. To address the biologic function of STEVOR proteins, we subjected a panel of stevor transgenic parasites and wild-type clonal lines exhibiting different expression levels for stevor genes to functional assays exploring parasite-induced modifications of the erythrocyte membrane. Using this approach, we show that stevor expression impacts deformability of the erythrocyte membrane. This process may facilitate parasite sequestration in deep tissue vasculature.
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Control of Plasmodium falciparum erythrocytic cycle: ?? T cells target the red blood cell-invasive merozoites.
Blood
PUBLISHED: 11-01-2011
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The control of Plasmodium falciparum erythrocytic parasite density is essential for protection against malaria, because it prevents pathogenesis and progression toward severe disease. P falciparum blood-stage parasite cultures are inhibited by human V?9V?2 ?? T cells, but the underlying mechanism remains poorly understood. Here, we show that both intraerythrocytic parasites and the extracellular red blood cell-invasive merozoites specifically activate V?9V?2 T cells in a ?? T cell receptor-dependent manner and trigger their degranulation. In contrast, the ?? T cell-mediated antiparasitic activity only targets the extracellular merozoites. Using perforin-deficient and granulysin-silenced T-cell lines, we demonstrate that granulysin is essential for the in vitro antiplasmodial process, whereas perforin is dispensable. Patients infected with P falciparum exhibited elevated granulysin plasma levels associated with high levels of granulysin-expressing V?2(+) T cells endowed with parasite-specific degranulation capacity. This indicates in vivo activation of V?9V?2 T cells along with granulysin triggering and discharge during primary acute falciparum malaria. Altogether, this work identifies V?9V?2 T cells as unconventional immune effectors targeting the red blood cell-invasive extracellular P falciparum merozoites and opens novel perspectives for immune interventions harnessing the antiparasitic activity of V?9V?2 T cells to control parasite density in malaria patients.
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Malaria morbidity and pyrethroid resistance after the introduction of insecticide-treated bednets and artemisinin-based combination therapies: a longitudinal study.
Lancet Infect Dis
PUBLISHED: 08-17-2011
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Substantial reductions in malaria have been reported in several African countries after distribution of insecticide-treated bednets and the use of artemisinin-based combination therapies (ACTs). Our aim was to assess the effect of these policies on malaria morbidity, mosquito populations, and asymptomatic infections in a west African rural population.
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Artesunate tolerance in transgenic Plasmodium falciparum parasites overexpressing a tryptophan-rich protein.
Antimicrob. Agents Chemother.
PUBLISHED: 04-04-2011
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Due to their rapid, potent action on young and mature intraerythrocytic stages, artemisinin derivatives are central to drug combination therapies for Plasmodium falciparum malaria. However, the evidence for emerging parasite resistance/tolerance to artemisinins in southeast Asia is of great concern. A better understanding of artemisinin-related drug activity and resistance mechanisms is urgently needed. A recent transcriptome study of parasites exposed to artesunate led us to identify a series of genes with modified levels of expression in the presence of the drug. The gene presenting the largest mRNA level increase, Pf10_0026 (PArt), encoding a hypothetical protein of unknown function, was chosen for further study. Immunodetection with PArt-specific sera showed that artesunate induced a dose-dependent increase of the protein level. Bioinformatic analysis showed that PArt belongs to a Plasmodium-specific gene family characterized by the presence of a tryptophan-rich domain with a novel hidden Markov model (HMM) profile. Gene disruption could not be achieved, suggesting an essential function. Transgenic parasites overexpressing PArt protein were generated and exhibited tolerance to a spike exposure to high doses of artesunate, with increased survival and reduced growth retardation compared to that of wild-type-treated controls. These data indicate the involvement of PArt in parasite defense mechanisms against artesunate. This is the first report of genetically manipulated parasites displaying a stable and reproducible decreased susceptibility to artesunate, providing new possibilities to investigate the parasite response to artemisinins.
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Ethical issues in human genomics research in developing countries.
BMC Med Ethics
PUBLISHED: 03-18-2011
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Genome-wide association studies (GWAS) provide a powerful means of identifying genetic variants that play a role in common diseases. Such studies present important ethical challenges. An increasing number of GWAS is taking place in lower income countries and there is a pressing need to identify the particular ethical challenges arising in such contexts. In this paper, we draw upon the experiences of the MalariaGEN Consortium to identify specific ethical issues raised by such research in Africa, Asia and Oceania.
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Structure of a Plasmodium falciparum PfEMP1 rosetting domain reveals a role for the N-terminal segment in heparin-mediated rosette inhibition.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-14-2011
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The human malaria parasite Plasmodium falciparum can cause infected red blood cells (iRBC) to form rosettes with uninfected RBC, a phenotype associated with severe malaria. Rosetting is mediated by a subset of the Plasmodium falciparum membrane protein 1 (PfEMP1) variant adhesins expressed on the infected host-cell surface. Heparin and other sulfated oligosaccharides, however, can disrupt rosettes, suggesting that therapeutic approaches to this form of severe malaria are feasible. We present a structural and functional study of the N-terminal domain of PfEMP1 from the VarO variant comprising the N-terminal segment (NTS) and the first DBL domain (DBL1?(1)), which is directly implicated in rosetting. We demonstrate that NTS-DBL1?(1)-VarO binds to RBC and that heparin inhibits this interaction in a dose-dependent manner, thus mimicking heparin-mediated rosette disruption. We have determined the crystal structure of NTS-DBL1?(1), showing that NTS, previously thought to be a structurally independent component of PfEMP1, forms an integral part of the DBL1? domain. Using mutagenesis and docking studies, we have located the heparin-binding site, which includes NTS. NTS, unique to the DBL ?-class domain, is thus an intrinsic structural and functional component of the N-terminal VarO domain. The specific interaction observed with heparin opens the way for developing antirosetting therapeutic strategies.
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Investigating the host binding signature on the Plasmodium falciparum PfEMP1 protein family.
PLoS Pathog.
PUBLISHED: 03-01-2011
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The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family plays a central role in antigenic variation and cytoadhesion of P. falciparum infected erythrocytes. PfEMP1 proteins/var genes are classified into three main subfamilies (UpsA, UpsB, and UpsC) that are hypothesized to have different roles in binding and disease. To investigate whether these subfamilies have diverged in binding specificity and test if binding could be predicted by adhesion domain classification, we generated a panel of 19 parasite lines that primarily expressed a single dominant var transcript and assayed binding against 12 known host receptors. By limited dilution cloning, only UpsB and UpsC var genes were isolated, indicating that UpsA var gene expression is rare under in vitro culture conditions. Consequently, three UpsA variants were obtained by rosette purification and selection with specific monoclonal antibodies to create a more representative panel. Binding assays showed that CD36 was the most common adhesion partner of the parasite panel, followed by ICAM-1 and TSP-1, and that CD36 and ICAM-1 binding variants were highly predicted by adhesion domain sequence classification. Binding to other host receptors, including CSA, VCAM-1, HABP1, CD31/PECAM, E-selectin, Endoglin, CHO receptor "X", and Fractalkine, was rare or absent. Our findings identify a category of larger PfEMP1 proteins that are under dual selection for ICAM-1 and CD36 binding. They also support that the UpsA group, in contrast to UpsB and UpsC var genes, has diverged from binding to the major microvasculature receptor CD36 and likely uses other mechanisms to sequester in the microvasculature. These results demonstrate that CD36 and ICAM-1 have left strong signatures of selection on the PfEMP1 family that can be detected by adhesion domain sequence classification and have implications for how this family of proteins is specializing to exploit hosts with varying levels of anti-malaria immunity.
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Allelic diversity of the Plasmodium falciparum erythrocyte membrane protein 1 entails variant-specific red cell surface epitopes.
PLoS ONE
PUBLISHED: 01-27-2011
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The clonally variant Plasmodium falciparum PfEMP1 adhesin is a virulence factor and a prime target of humoral immunity. It is encoded by a repertoire of functionally differentiated var genes, which display architectural diversity and allelic polymorphism. Their serological relationship is key to understanding the evolutionary constraints on this gene family and rational vaccine design. Here, we investigated the Palo Alto/VarO and IT4/R29 and 3D7/PF13_003 parasites lines. VarO and R29 form rosettes with uninfected erythrocytes, a phenotype associated with severe malaria. They express an allelic Cys2/group A NTS-DBL1?(1) PfEMP1 domain implicated in rosetting, whose 3D7 ortholog is encoded by PF13_0003. Using these three recombinant NTS-DBL1?(1) domains, we elicited antibodies in mice that were used to develop monovariant cultures by panning selection. The 3D7/PF13_0003 parasites formed rosettes, revealing a correlation between sequence identity and virulence phenotype. The antibodies cross-reacted with the allelic domains in ELISA but only minimally with the Cys4/group B/C PFL1955w NTS-DBL1?. By contrast, they were variant-specific in surface seroreactivity of the monovariant-infected red cells by FACS analysis and in rosette-disruption assays. Thus, while ELISA can differentiate serogroups, surface reactivity assays define the more restrictive serotypes. Irrespective of cumulated exposure to infection, antibodies acquired by humans living in a malaria-endemic area also displayed a variant-specific surface reactivity. Although seroprevalence exceeded 90% for each rosetting line, the kinetics of acquisition of surface-reactive antibodies differed in the younger age groups. These data indicate that humans acquire an antibody repertoire to non-overlapping serotypes within a serogroup, consistent with an antibody-driven diversification pressure at the population level. In addition, the data provide important information for vaccine design, as production of a vaccine targeting rosetting PfEMP1 adhesins will require engineering to induce variant-transcending responses or combining multiple serotypes to elicit a broad spectrum of immunity.
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Combined effects of Gm or Km immunoglobulin allotypes and age on antibody responses to Plasmodium falciparum VarO rosetting variant in Benin.
Microbes Infect.
PUBLISHED: 01-21-2011
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Clinical protection of Beninese children against Plasmodium falciparum malaria was shown to be influenced by immunoglobulin (IG) Gm and Km allotypes, and related to seroreactivity with the rosette-forming VarO-antigenic variant. IgG to the VarO-infected erythrocyte surface, IgG1 and IgG3 to PfEMP1-NTS-DBL1?(1)-VarO were higher in the under 4-year-old children carrying the Gm 5,6,13,14;1,17 phenotype. In contrast, surface-reactive IgG, total IgG, IgG1 and IgG3 to NTS-DBL1?(1)- and DBL2?C2-VarO domains were lower in the above 4-year-old children harbouring the Km1 allotype. These data outline an age-related association of antibodies against malaria antigens and IG allotype distribution.
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The sensing of poorly deformable red blood cells by the human spleen can be mimicked in vitro.
Blood
PUBLISHED: 12-16-2010
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Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as a critical determinant of pathogenesis in hereditary spherocytosis, malaria, and other RBC disorders. Using an ex vivo perfusion system, we had previously shown that retention of Plasmodium falciparum-infected RBCs (Pf-RBCs) occur in the splenic red pulp, upstream from the sinus wall. To experimentally replicate the mechanical sensing of RBCs by the splenic microcirculation, we designed a sorting device where a mixture of 5- to 25-?m-diameter microbeads mimics the geometry of narrow and short interendothelial splenic slits. Heated RBCs, Pf-RBCs, and RBCs from patients with hereditary spherocytosis were retained in the microbead layer, without hemolysis. The retention rates of Pf-RBCs were similar in microbeads and in isolated perfused human spleens. These in vitro results directly confirm the importance of the mechanical sensing of RBCs by the human spleen. In addition, rigid and deformable RBC subpopulations could be separated and characterized at the molecular level, and the device was used to deplete a stored RBC population from its subpopulation of rigid RBCs. This experimental approach may contribute to a better understanding of the role of the spleen in the pathogenesis of inherited and acquired RBC disorders.
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The pathogenesis of Plasmodium falciparum malaria in humans: insights from splenic physiology.
Blood
PUBLISHED: 09-17-2010
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Clinical manifestations of Plasmodium falciparum infection are induced by the asexual stages of the parasite that develop inside red blood cells (RBCs). Because splenic microcirculatory beds filter out altered RBCs, the spleen can innately clear subpopulations of infected or uninfected RBC modified during falciparum malaria. The spleen appears more protective against severe manifestations of malaria in naïve than in immune subjects. The spleen-specific pitting function accounts for a large fraction of parasite clearance in artemisinin-treated patients. RBC loss contributes to malarial anemia, a clinical form associated with subacute progression, frequent splenomegaly, and relatively low parasitemia. Stringent splenic clearance of ring-infected RBCs and uninfected, but parasite-altered, RBCs, may altogether exacerbate anemia and reduce the risks of severe complications associated with high parasite loads, such as cerebral malaria. The age of the patient directly influences the risk of severe manifestations. We hypothesize that coevolution resulting in increased splenic clearance of P. falciparum-altered RBCs in children favors the survival of the host and, ultimately, sustained parasite transmission. This analysis of the RBC-spleen dynamic interactions during P falciparum infection reflects both data and hypotheses, and provides a framework on which a more complete immunologic understanding of malaria pathogenesis may be elaborated.
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The humoral response to Plasmodium falciparum VarO rosetting variant and its association with protection against malaria in Beninese children.
Malar. J.
PUBLISHED: 08-06-2010
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The capacity of Plasmodium falciparum-infected erythrocytes to bind uninfected erythrocytes (rosetting) is associated with severe malaria in African children. Rosetting is mediated by a subset of the variant surface antigens PfEMP1 targeted by protective antibody responses. Analysis of the response to rosette-forming parasites and their PfEMP1 adhesive domains is essential for understanding the acquisition of protection against severe malaria. To this end, the antibody response to a rosetting variant was analysed in children recruited with severe or uncomplicated malaria or asymptomatic P. falciparum infection.
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Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2.
MAbs
PUBLISHED: 06-29-2010
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Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity, and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novels anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis.
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Chloroquine clinical failures in P. falciparum malaria are associated with mutant Pfmdr-1, not Pfcrt in Madagascar.
PLoS ONE
PUBLISHED: 06-22-2010
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Molecular studies have demonstrated that mutations in the Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) play a major role in chloroquine resistance, while mutations in P. falciparum multidrug resistance gene (Pfmdr-1) act as modulator. In Madagascar, the high rate of chloroquine treatment failure (44%) appears disconnected from the overall level of in vitro CQ susceptibility (prevalence of CQ-resistant parasites <5%) or Pfcrt mutant isolates (<1%), strongly contrasting with sub-Saharan African countries. Previous studies showed a high frequency of Pfmdr-1 mutant parasites (>60% of isolates), but did not explore their association with P. falciparum chloroquine resistance. To document the association of Pfmdr-1 alleles with chloroquine resistance in Madagascar, 249 P. falciparum samples collected from patients enrolled in a chloroquine in vivo efficacy study were genotyped in Pfcrt/Pfmdr-1 genes as well as the estimation of the Pfmdr-1 copy number. Except 2 isolates, all samples displayed a wild-type Pfcrt allele without Pfmdr-1 amplification. Chloroquine treatment failures were significantly associated with Pfmdr-1 86Y mutant codon (OR?=?4.6). The cumulative incidence of recurrence of patients carrying the Pfmdr-1 86Y mutation at day 0 (21 days) was shorter than patients carrying Pfmdr-1 86N wild type codon (28 days). In an independent set of 90 selected isolates, in vitro susceptibility to chloroquine was not associated with Pfmdr-1 polymorphisms. Analysis of two microsatellites flanking Pfmdr-1 allele showed that mutations occurred on multiple genetic backgrounds. In Madagascar, Pfmdr-1 polymorphism is associated with late chloroquine clinical failures and unrelated with in vitro susceptibility or Pfcrt genotype. These results highlight the limits of the current in vitro tests routinely used to monitor CQ drug resistance in this unique context. Gaining insight about the mechanisms that regulate polymorphism in Pfmdr1 remains important, particularly regarding the evolution and spread of Pfmdr-1 alleles in P. falciparum populations under changing drug pressure which may have important consequences in terms of antimalarial use management.
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Heritability of the human infectious reservoir of malaria parasites.
PLoS ONE
PUBLISHED: 05-28-2010
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Studies on human genetic factors associated with malaria have hitherto concentrated on their role in susceptibility to and protection from disease. In contrast, virtually no attention has been paid to the role of human genetics in eliciting the production of parasite transmission stages, the gametocytes, and thus enhancing the spread of disease.
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Identification of a role for the PfEMP1 semi-conserved head structure in protein trafficking to the surface of Plasmodium falciparum infected red blood cells.
Cell. Microbiol.
PUBLISHED: 05-05-2010
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Transport of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) variants to the red blood cell (RBC) surface enables malarial parasite evasion of host immunity by modifying the antigenic and adhesive properties of infected RBCs. In this study, we applied the Bxb1 integrase system to integrate transgenes encoding truncated PfEMP1-GFP fusions into cytoadherent A4 parasites and characterize their surface transport requirements. Our studies revealed that the semi-conserved head structure of PfEMP1 proteins, in combination with the predicted transmembrane region and cytoplasmic tail, encodes sufficient information for RBC surface display. In contrast, miniPfEMP1 proteins with truncated head structures were exported to the RBC cytoplasm but were not detected at the RBC surface by flow cytometry or immuno-electron microscopy. We demonstrated the absence of a mechanistic barrier to having native and miniPfEMP1 proteins displayed simultaneously at the RBC surface. However, surface-exposed miniPfEMP1 proteins did not convey cytoadherence properties to their host cells, implicating potential steric considerations in host-receptor interactions or the need for multiple domains to mediate cell binding. This study establishes a new system to investigate PfEMP1 transport and demonstrates that the PfEMP1 semi-conserved head structure is under selection for protein transport, in addition to its known roles in adhesion.
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Identification of a mutant PfCRT-mediated chloroquine tolerance phenotype in Plasmodium falciparum.
PLoS Pathog.
PUBLISHED: 04-01-2010
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Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ), the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1) suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2) implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3) uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt.
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Origins of the recent emergence of Plasmodium falciparum pyrimethamine resistance alleles in Madagascar.
Antimicrob. Agents Chemother.
PUBLISHED: 03-22-2010
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The combination of sulfadoxine-pyrimethamine is recommended for use as intermittent preventive treatment of malaria during pregnancy and is deployed in Africa. The emergence and the spread of resistant parasites are major threats to such an intervention. We have characterized the Plasmodium falciparum dhfr (pfdhfr) haplotypes and flanking microsatellites in 322 P. falciparum isolates collected from the Comoros Islands and Madagascar. One hundred fifty-six (48.4%) carried the wild-type pfdhfr allele, 19 (5.9%) carried the S108N single-mutation allele, 30 (9.3%) carried the I164L single-mutation allele, 114 (35.4%) carried the N51I/C59R/S108N triple-mutation allele, and 3 (1.0%) carried the N51I/C59R/S108N/I164L quadruple-mutation allele. Microsatellite analysis showed the introduction from the Comoros Islands of the ancestral pfdhfr triple mutant allele of Asian origin and its spread in Madagascar. Evidence for the emergence on multiple occasions of the I164L single-mutation pfdhfr allele in Madagascar was also obtained. Thus, the conditions required to generate mutants with quadruple mutations are met in Madagascar, representing a serious threat to current drug policy.
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Plasmodium vivax clinical malaria is commonly observed in Duffy-negative Malagasy people.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-15-2010
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Malaria therapy, experimental, and epidemiological studies have shown that erythrocyte Duffy blood group-negative people, largely of African ancestry, are resistant to erythrocyte Plasmodium vivax infection. These findings established a paradigm that the Duffy antigen is required for P. vivax erythrocyte invasion. P. vivax is endemic in Madagascar, where admixture of Duffy-negative and Duffy-positive populations of diverse ethnic backgrounds has occurred over 2 millennia. There, we investigated susceptibility to P. vivax blood-stage infection and disease in association with Duffy blood group polymorphism. Duffy blood group genotyping identified 72% Duffy-negative individuals (FY*B(ES)/*B(ES)) in community surveys conducted at eight sentinel sites. Flow cytometry and adsorption-elution results confirmed the absence of Duffy antigen expression on Duffy-negative erythrocytes. P. vivax PCR positivity was observed in 8.8% (42/476) of asymptomatic Duffy-negative people. Clinical vivax malaria was identified in Duffy-negative subjects with nine P. vivax monoinfections and eight mixed Plasmodium species infections that included P. vivax (4.9 and 4.4% of 183 participants, respectively). Microscopy examination of blood smears confirmed blood-stage development of P. vivax, including gametocytes. Genotyping of polymorphic surface and microsatellite markers suggested that multiple P. vivax strains were infecting Duffy-negative people. In Madagascar, P. vivax has broken through its dependence on the Duffy antigen for establishing human blood-stage infection and disease. Further studies are necessary to identify the parasite and host molecules that enable this Duffy-independent P. vivax invasion of human erythrocytes.
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Decreased in vitro susceptibility of Plasmodium falciparum isolates to artesunate, mefloquine, chloroquine, and quinine in Cambodia from 2001 to 2007.
Antimicrob. Agents Chemother.
PUBLISHED: 03-01-2010
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This study describes the results of in vitro antimalarial susceptibility assays and molecular polymorphisms of Plasmodium falciparum isolates from Cambodia. The samples were collected from patients enrolled in therapeutic efficacy studies (TES) conducted by the Cambodian National Malaria Control Program for the routine efficacy monitoring of artemisinin-based combination therapy (ACT) (artesunate-mefloquine and artemether-lumefantrine combinations). The isolates (n = 2,041) were obtained from nine sentinel sites during the years 2001 to 2007. Among these, 1,588 were examined for their in vitro susceptibilities to four antimalarials (artesunate, mefloquine, chloroquine, and quinine), and 851 isolates were genotyped for single nucleotide polymorphisms (SNPs). The geometric means of the 50% inhibitory concentrations (GMIC(50)s) of the four drugs tested were significantly higher for isolates from western Cambodia than for those from eastern Cambodia. GMIC(50)s for isolates from participants who failed artesunate-mefloquine therapy were significantly higher than those for patients who were cured (P, <0.001). In vitro correlation of artesunate with the other drugs was observed. The distributions of the SNPs differed between eastern and western Cambodia, suggesting different genetic backgrounds of the parasite populations in these two parts of the country. The GMIC(50)s of the four drugs tested increased significantly in eastern Cambodia during 2006 to 2007. These results are worrisome, because they may signal deterioration of the efficacy of artesunate-mefloquine beyond the Cambodian-Thai border.
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Chronic infection during placental malaria is associated with up-regulation of cycloxygenase-2.
Malar. J.
PUBLISHED: 02-09-2010
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Placental malaria (PM) is associated with poor foetal development, but the pathophysiological processes involved are poorly understood. Cyclooxygenase (COX) and lipoxygenase (LOX) which convert fatty acids to prostaglandins and leukotrienes, play important roles in pregnancy and foetal development. COX-2, currently targeted by specific drugs, plays a dual role as it associates with both pre-eclampsia pathology and recovery during infection. The role of COX during PM was questioned by quantifying at delivery COX-1, COX-2, 15-LOX, and IL-10 expression in two groups of malaria infected and uninfected placenta.
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Parasite polymorphism and severe malaria in Dakar (Senegal): a West African urban area.
PLoS ONE
PUBLISHED: 02-01-2010
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Transmission of malaria in West African urban areas is low and healthcare facilities are well organized. However, malaria mortality remains high. We conducted a survey in Dakar with the general objective to establish who died from severe malaria (SM) in urban areas (particularly looking at the age-groups) and to compare parasite isolates associated with mild or severe malaria.
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Geographic structuring of the Plasmodium falciparum sarco(endo)plasmic reticulum Ca2+ ATPase (PfSERCA) gene diversity.
PLoS ONE
PUBLISHED: 01-22-2010
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Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp), of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC(50) for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates). This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte.
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Biochemical and biophysical characterisation of DBL1alpha1-varO, the rosetting domain of PfEMP1 from the VarO line of Plasmodium falciparum.
Mol. Biochem. Parasitol.
PUBLISHED: 01-04-2010
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Rosetting of erythrocytes infected with Plasmodium falciparum is frequently observed in children with severe malaria. This adhesion phenomenon has been linked to the DBL1alpha domain of P. falciparum erythrocyte membrane protein 1 (PfEMP1) in three laboratory clones: FCR3S1.2, IT4R29 and Palo Alto varO. Here, we compare the soluble recombinant NTS-DBL1alpha(1)-varO domain (NTS: N-terminal segment) obtained from E. coli, Pichia pastoris and baculovirus/insect cell expression systems. In each case, the presence of NTS was necessary for obtaining a soluble product. Successful expression in E. coli required maltose-binding protein as an N-terminal fusion partner. Each expression system produced an identical, correctly folded protein, as judged by biochemical and biophysical characterisations, and by the capacity to elicit antibodies that react with the surface of VarO-infected erythrocytes and disrupt VarO rosettes. Binding studies using surface plasmon resonance (SPR) techniques showed that NTS-DBL1alpha(1) produced in E. coli binds to heparin with micromolar affinity. IC(50) constants for other sulphated oligosaccharides were determined using SPR by measuring their competitive binding to the soluble protein in the presence of immobilized heparin. The affinity to NTS-DBL1alpha(1) was related to the degree of sulphation of the oligosaccharide, although the position of the sulphate groups on the sugar rings was also important. VarO rosettes could be disrupted by sulphated oligosaccharides with an efficacy that correlated with their binding affinity to recombinant NTS-DBL1alpha(1). Thus high yields of soluble NTS-DBL1alpha(1) with native conformation have been produced, opening novel perspectives for both structure-function studies and vaccine development.
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Plasmodium falciparum drug resistance in Madagascar: facing the spread of unusual pfdhfr and pfmdr-1 haplotypes and the decrease of dihydroartemisinin susceptibility.
Antimicrob. Agents Chemother.
PUBLISHED: 08-24-2009
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The aim of this study was to provide the first comprehensive spatiotemporal picture of Plasmodium falciparum resistance in various geographic areas in Madagascar. Additional data about the antimalarial resistance in the neighboring islands of the Comoros archipelago were also collected. We assessed the prevalence of pfcrt, pfmdr-1, pfdhfr, and pfdhps mutations and the pfmdr-1 gene copy number in 1,596 P. falciparum isolates collected in 26 health centers (20 in Madagascar and 6 in the Comoros Islands) from 2006 to 2008. The in vitro responses to a panel of drugs by 373 of the parasite isolates were determined. The results showed (i) unusual profiles of chloroquine susceptibility in Madagascar, (ii) a rapid rise in the frequency of parasites with both the pfdhfr and the pfdhps mutations, (iii) the alarming emergence of the single pfdhfr 164L genotype, and (iv) the progressive loss of the most susceptible isolates to artemisinin derivatives. In the context of the implementation of the new national policy for the fight against malaria, continued surveillance for the detection of P. falciparum resistance in the future is required.
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Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers.
Malar. J.
PUBLISHED: 04-29-2009
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Several strategies are currently deployed in many countries in the tropics to strengthen malaria control toward malaria elimination. To measure the impact of any intervention, there is a need to detect malaria properly. Mostly, decisions still rely on microscopy diagnosis. But sensitive diagnosis tools enabling to deal with a large number of samples are needed. The molecular detection approach offers a much higher sensitivity, and the flexibility to be automated and upgraded.
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Retention of erythrocytes in the spleen: a double-edged process in human malaria.
Curr. Opin. Hematol.
PUBLISHED: 04-23-2009
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Splenomegaly is frequent in acute or chronic forms of Plasmodium falciparum malaria, and splenectomy is associated with more frequent fever and parasitaemia. A novel role for the spleen in malaria is indicated by recent epidemiological and experimental data, bringing about a novel paradigm on severe malaria pathogenesis.
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Population diversity and antibody selective pressure to Plasmodium falciparum MSP1 block2 locus in an African malaria-endemic setting.
BMC Microbiol.
PUBLISHED: 03-10-2009
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Genetic evidence for diversifying selection identified the Merozoite Surface Protein1 block2 (PfMSP1 block2) as a putative target of protective immunity against Plasmodium falciparum. The locus displays three family types and one recombinant type, each with multiple allelic forms differing by single nucleotide polymorphism as well as sequence, copy number and arrangement variation of three amino acid repeats. The family-specific antibody responses observed in endemic settings support immune selection operating at the family level. However, the factors contributing to the large intra-family allelic diversity remain unclear. To address this question, population allelic polymorphism and sequence variant-specific antibody responses were studied in a single Senegalese rural community where malaria transmission is intense and perennial.
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FlexiChip package: an universal microarray with a dedicated analysis software for high-thoughput SNPs detection linked to anti-malarial drug resistance.
Malar. J.
PUBLISHED: 03-02-2009
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A number of molecular tools have been developed to monitor the emergence and spread of anti-malarial drug resistance to Plasmodium falciparum. One of the major obstacles to the wider implementation of these tools is the absence of practical methods enabling high throughput analysis. Here a new Zip-code array is described, called FlexiChip, linked to a dedicated software program, which largely overcomes this problem.
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Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia.
Malar. J.
PUBLISHED: 01-12-2009
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The combination of artesunate and mefloquine was introduced as the national first-line treatment for Plasmodium falciparum malaria in Cambodia in 2000. However, recent clinical trials performed at the Thai-Cambodian border have pointed to the declining efficacy of both artesunate-mefloquine and artemether-lumefantrine. Since pfmdr1 modulates susceptibility to mefloquine and artemisinin derivatives, the aim of this study was to assess the link between pfmdr1 copy number, in vitro susceptibility to individual drugs and treatment failure to combination therapy.
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Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia.
Antimicrob. Agents Chemother.
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The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.
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Whole genome sequencing of field isolates provides robust characterization of genetic diversity in Plasmodium vivax.
PLoS Negl Trop Dis
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An estimated 2.85 billion people live at risk of Plasmodium vivax transmission. In endemic countries vivax malaria causes significant morbidity and its mortality is becoming more widely appreciated, drug-resistant strains are increasing in prevalence, and an increasing number of reports indicate that P. vivax is capable of breaking through the Duffy-negative barrier long considered to confer resistance to blood stage infection. Absence of robust in vitro propagation limits our understanding of fundamental aspects of the parasites biology, including the determinants of its dormant hypnozoite phase, its virulence and drug susceptibility, and the molecular mechanisms underlying red blood cell invasion.
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Pf155/RESA protein influences the dynamic microcirculatory behavior of ring-stage Plasmodium falciparum infected red blood cells.
Sci Rep
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Proteins exported by Plasmodium falciparum to the red blood cell (RBC) membrane modify the structural properties of the parasitized RBC (Pf-RBC). Although quasi-static single cell assays show reduced ring-stage Pf-RBCs deformability, the parameters influencing their microcirculatory behavior remain unexplored. Here, we study the dynamic properties of ring-stage Pf-RBCs and the role of the parasite protein Pf155/Ring-Infected Erythrocyte Surface Antigen (RESA). Diffraction phase microscopy revealed RESA-driven decreased Pf-RBCs membrane fluctuations. Microfluidic experiments showed a RESA-dependent reduction in the Pf-RBCs transit velocity, which was potentiated at febrile temperature. In a microspheres filtration system, incubation at febrile temperature impaired traversal of RESA-expressing Pf-RBCs. These results show that RESA influences ring-stage Pf-RBCs microcirculation, an effect that is fever-enhanced. This is the first identification of a parasite factor influencing the dynamic circulation of young asexual Pf-RBCs in physiologically relevant conditions, offering novel possibilities for interventions to reduce parasite survival and pathogenesis in its human host.
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Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting.
PLoS Pathog.
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The ABO blood group influences susceptibility to severe Plasmodium falciparum malaria. Recent evidence indicates that the protective effect of group O operates by virtue of reduced rosetting of infected red blood cells (iRBCs) with uninfected RBCs. Rosetting is mediated by a subgroup of PfEMP1 adhesins, with RBC binding being assigned to the N-terminal DBL1?? domain. Here, we identify the ABO blood group as the main receptor for VarO rosetting, with a marked preference for group A over group B, which in turn is preferred to group O RBCs. We show that recombinant NTS-DBL1?? and NTS-DBL1??-CIDR1? reproduce the VarO-iRBC blood group preference and document direct binding to blood group trisaccharides by surface plasmon resonance. More detailed RBC subgroup analysis showed preferred binding to group A?, weaker binding to groups A? and B, and least binding to groups A(x) and O. The 2.8 Å resolution crystal structure of the PfEMP1-VarO Head region, NTS-DBL1??-CIDR1?, reveals extensive contacts between the DBL1?? and CIDR1? and shows that the NTS-DBL1?? hinge region is essential for RBC binding. Computer docking of the blood group trisaccharides and subsequent site-directed mutagenesis localized the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBL??. RBC binding involves residues that are conserved between rosette-forming PfEMP1 adhesins, opening novel opportunities for intervention against severe malaria. By deciphering the structural basis of blood group preferences in rosetting, we provide a link between ABO blood grouppolymorphisms and rosette-forming adhesins, consistent with the selective role of falciparum malaria on human genetic makeup.
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Possible association of the Plasmodium falciparum T1526C resa2 gene mutation with severe malaria.
Malar. J.
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Plasmodium falciparum exports proteins that remodel the erythrocyte membrane. One such protein, called Pf155/RESA (RESA1) contributes to parasite fitness, optimizing parasite survival during febrile episodes. Resa1 gene is a member of a small family comprising three highly related genes. Preliminary evidence led to a search for clues indicating the involvement of RESA2 protein in the pathophysiology of malaria. In the present study, cDNA sequence of resa2 gene was obtained from two different strains. The proportion of P. falciparum isolates having a non-stop T1526C mutation in resa2 gene was evaluated and the association of this genotype with severity of malaria was investigated.
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A switch in infected erythrocyte deformability at the maturation and blood circulation of Plasmodium falciparum transmission stages.
Blood
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Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission.
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Quantitative assessment of sensing and sequestration of spherocytic erythrocytes by the human spleen.
Blood
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Splenic sequestration of RBCs with reduced surface area and cellular deformability has long been recognized as contributing to pathogenesis of several RBC disorders, including hereditary spherocytosis. However, the quantitative relationship between the extent of surface area loss and splenic entrapment remains to be defined. To address this issue, in the present study, we perfused ex vivo normal human spleens with RBCs displaying various degrees of surface area loss and monitored the kinetics of their splenic retention. Treatment with increasing concentrations of lysophosphatidylcholine resulted in a dose-dependent reduction of RBC surface area at constant volume, increased osmotic fragility, and decreased deformability. The degree of splenic retention of treated RBCs increased with increasing surface area loss. RBCs with a > 18% average surface area loss (> 27% reduced surface area-to-volume ratio) were rapidly and completely entrapped in the spleen. Surface-deficient RBCs appeared to undergo volume loss after repeated passages through the spleen and escape from splenic retention. The results of the present study for the first time define the critical extent of surface area loss leading to splenic entrapment and identify an adaptive volume regulation mechanism that allows spherocytic RBCs to prolong their life span in circulation. These results have significant implications for understanding the clinical heterogeneity of RBC membrane disorders.
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A new and frequent human T-cell leukemia virus indeterminate Western blot pattern: epidemiological determinants and PCR results in central African inhabitants.
J. Clin. Microbiol.
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Human T-cell leukemia virus (HTLV) indeterminate Western blot (WB) serological patterns are frequently observed in plasma/serum from persons living in intertropical areas. In the framework of ongoing projects on HTLV-1/2 and related viruses in Central Africa, we systematically analyzed plasma from villagers living in South Cameroon by WB. The group included 1,968 individuals (mean age, 44 years; age range, 5 to 90 years; 978 women/990 men), both Bantus (1,165) and Pygmies (803). Plasma samples were tested by WB analysis (MPD HTLV Blot 2.4) and interpreted according to the manufacturers instructions. Only clear bands were considered in the analysis. Among the 1,968 plasma samples, 38 (1.93%) were HTLV-1, 13 (0.66%) were HTLV-2, and 6 (0.3%) were HTLV WB seropositive. Furthermore, 1,292 (65.65%) samples were WB sero-indeterminate, including 104 (5.28%) with an HTLV-1 Gag-indeterminate pattern (HGIP) and 68 (3.45%) with a peculiar yet unreported pattern exhibiting mostly a strong shifted GD21 and a p28. The other 619 (31.45%) samples were either WB negative or exhibited other patterns, mostly with unique p19 or p24 bands. DNA, extracted from peripheral blood buffy coat, was subjected to PCR using several primer pairs known to detect HTLV-1/2/3/4. Most DNAs from HTLV-1- and HTLV-seropositive individuals were PCR positive. In contrast, all the others, from persons with HTLV-2, HGIP, new WB, and other indeterminate patterns, were PCR negative. Epidemiological determinant analysis of the persons with this new peculiar WB pattern revealed that seroprevalence was independent from age, sex, or ethnicity, thus resembling the indeterminate profile HGIP rather than HTLV-1. Moreover, this new pattern persists over time.
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Discordant temporal evolution of Pfcrt and Pfmdr1 genotypes and Plasmodium falciparum in vitro drug susceptibility to 4-aminoquinolines after drug policy change in French Guiana.
Antimicrob. Agents Chemother.
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Analysis of the evolution of drug target genes under changing drug policy is needed to assist monitoring of Plasmodium falciparum drug resistance in the field. Here we genotype Pfcrt and Pfdmr1 of 700 isolates collected in French Guiana from 2000 (5 years after withdrawal of chloroquine) to 2008, i.e., the period when the artemether-lumefantrine combination was progressively introduced and mefloquine was abandoned. Gene sequencing showed fixation of the 7G8-type Pfcrt SMVNT resistance haplotype and near fixation of the NYCDY Pfdmr1 haplotype. Pfdmr1 gene copy number correlated with 50% inhibitory concentrations of mefloquine and halofantrine (r = 0.64 and 0.47, respectively, n = 547); its temporal changes paralleled changes in in vitro mefloquine susceptibility. However, the molecular parameters studied did not account for the regained in vitro susceptibility to chloroquine and showed a poor correlation with susceptibility to artemether, lumefantrine, or quinine. Identification of novel markers of resistance to these antimalarials is needed in this South American area.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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