Academic literature on the topic 'Malaria, cerebral. Genetic markers. Plasmodium falciparum'

The vast majority of malaria patients worldwide are currently treated with combination therapy comprising one of the artemisinin family of drugs, characterised by rapid action and short plasma half-life, co-formulated with a longer-lasting drug from the amino aryl-alcohol or quinoline families. There is now a widely perceived threat to treatment efficacy, as reduced susceptibility to rapid artemisinin clearance in vivo has become prevalent among populations of Plasmodium falciparum in the Greater Mekong subregion since 2008. In vitro and in vivo drug selection studies, heterologous cell expression experiments and genetic epidemiology have identified many candidate markers of reduced ring-stage susceptibility to artemisinin. Certain variants of the P. falciparum pfk13 gene, which encodes a kelch domain protein implicated in the unfolded protein response, are strongly associated with slow parasite clearance by artemisinin in the Mekong subregion. However, anomalies in the epidemiological association of pfk13 variants with true treatment failure in vivo and the curious cell-cycle stage specificity of this phenotype in vitro warrant exploration in some depth. Taken together, available data suggest that the emergence of P. falciparum expressing K13 variants has not yet precipitated a public health emergency. Alternative candidate markers of artemisinin susceptibility are also described, as K13-independent treatment failure has been observed in African P. falciparum and in the rodent malaria parasite Plasmodium chabaudi.

Plasmodium falciparum, the most malignant human malaria, is responsible for 2-3 million deaths annually. These infections often involve blockage of the cerebral microvasculature by P. falciparum-infected erythrocytes (Fig. 1). This aspect is considered the major factor in the pathogenesis of cerebral malaria.Upon invasion of the erythrocyte, P. falciparum immediately begins to remodel the infected erythrocyte. The adherence points of infected erythrocytes, termed knobs (Fig. 2 and 3), contain antigenically diverse 200-350kDa surface proteins (PfEMPl; Fig. 4). The PfEMPl variant surface proteins are encoded by a large and extremely diverse family of genes (var), and switches in the expression of var genes account for rapid changes in the antigenic and adhesive properties of P. falciparum-inkcted erythrocytes (2.4% per generation). Switches in the PfEMPl expression may not only affect the phenotype of the parasite strain but may also change its sequestration to endothelial cells. Genetic reorganization in this protein can lead to binding any of the following endothelial cell receptors; ICAM-1, CD36, thrombospondin, chondroitin sulfate (Fig. 5),2 ELAM-1, or VCAM-1.

There is introduced the analysis of molecular-genetic mechanisms of tropical malaria pathogenesis and P. falciparum virulence. It is shown, that pathogenesis of tropical malaria is associated with the properties of red blood cells membrane surface (RBCs or erythrocytes) that are infected by P. falciparum. There are «knobs structures» on membrane surface infected RBCs. Knobs structures contains a complex of P. falciparum proteins – PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1). PfEMP1 is associated with virulence of P. falciparum. Complex PfEMP1 has difficult polymorphous structure. Domains of PfEMP1 are able to associate with different cell receptors. Virulence`s individual components of the main factor are selectively sensitive to different tissues and organs. The severity of the clinical malaria infection course depends on the complex structure PfEMP1 of malaria parasites. Composition of polypeptide PfEMP1 is determined by var-complex. Nowadays there are 60 variants of var-complex. Regulation of gene expression, forming part of the var-complex, is carried out on a molecular-genetic level, cellular level, tissue level. Modern research in this area are aimed to explore genes polymorphism of the virulence`s main factor, to identify mechanism of its differential expression. Search of molecular – genetic markers is relevant to develop methods of gene diagnostic and malaria vaccine.

Background Host factors, including host genetic variation, have been shown to influence the outcome of Plasmodium falciparum infection. Genome-wide linkage studies have mapped mild malaria resistance genes on chromosome 6p21, whereas NCR3-412 polymorphism (rs2736191) lying within this region was found to be associated with mild malaria. Methods Blood samples were taken from 188 Plasmodium falciparum malaria patients (76 mild malaria patients, 85 cerebral malaria patients, and 27 severe non-cerebral malaria patients). NCR3-412 (rs2736191) was analysed by sequencing, and haematological parameters were measured. Finally, their association with clinical phenotypes was assessed. Results We evidenced an association of thrombocytopenia with both cerebral malaria and severe non-cerebral malaria, and of an association of high leukocyte count with cerebral malaria. Additionally, we found no association of NCR3-412 with either cerebral malaria, severe non-cerebral malaria, or severe malaria after grouping cerebral malaria and severe non-cerebral malaria patients. Conclusions Our results suggest that NCR3 genetic variation has no effect, or only a small effect on the occurrence of severe malaria, although it has been strongly associated with mild malaria. We discuss the biological meaning of these results. Besides, we confirmed the association of thrombocytopenia and high leukocyte count with severe malaria phenotypes.

ABSTRACT Sequestration of Plasmodium falciparum-infected erythrocytes is a pathological feature of fatal cerebral malaria. P. falciparum is genetically diverse among, and often within, patients. Preferential sequestration of certain genotypes might be important in pathogenesis. We compared circulating parasites with parasites sequestered in the brain, spleen, liver, and lung in the same Malawian children with fatal malaria, classifying serotypes using antibodies to merozoite surface proteins 1 and 2 and immunofluorescence in order to differentiate parasites and to quantify the proportions of each serotype. We found (i) similar distributions of various serotypes in different tissues and (ii) concordance between parasite serotypes in peripheral blood and parasite serotypes in tissues. No serotypes predominated in the brain in cerebral malaria, and parasites belonging to a single serotype did not cluster within individual vessels or within single tissues. These findings do not support the hypothesis that cerebral malaria is caused by cerebral sequestration of certain virulent types.

ABSTRACT To explore the hypothesis that susceptibility to cerebral malaria is influenced by genetic variation in endothelial nitric oxide synthase (eNOS), we genotyped three commonly defined polymorphic loci of eNOS, Glu298→Asp, intron 4 variable number of tandem repeat region, and T-786→C, in 244 patients (mean age, 36.2 years) with mild malaria and 194 patients (mean age, 35.6 years) with severe malaria belonging to same ethnic group in Orissa, an eastern Indian state. We found that there was an association of the Glu298→Asp substitution (P = 0.0037; odds ratio, 1.95; 95% confidence interval, 1.2 to 3.0) and a single unique haplotype defined by “C-b-Asp” (P corrected = 0.0024) for protection against cerebral malaria. Further, the median plasma level of nitrite-nitrate was found to be increased in individuals with the Glu298→Asp substitution and was significantly higher in the mild malaria group (P ≤ 0.0001), but the increase was not significant in the severe malaria group (P = 0.0528). These findings suggest that the Glu298→Asp substitution and the “C-b-Asp” haplotype may enhance eNOS expression and NO production, which leads to protection against cerebral malaria. These findings may increase our understanding of the pathogenesis of malaria.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Multiple, selectively neutral genetic markers are the most appropriate tools for analysis of parasite population structure and epidemiology, but yet existing methods for characterization of malaria field samples utilize a limited number of antigen encoding genes, which appear to be under strong selection. We describe protocols for characterization of 12 microsatellite markers from finger-prick blood samples infected with Plasmodium falciparum. A two-step, heminested strategy was used to amplify all loci, and products were visualized by fluorescent end-labelling of internal primers. This procedure allows amplification from low levels of template, while eliminating the problem of spurious products due to primer carry over from the primary round of PCR. The loci can be conveniently multiplexed, while accurate sizing and quantification of PCR products can be automated using the GENOTYPER software. The primers do not amplify co-infecting malaria species such as P. vivax and P. malariae. To demonstrate the utility of these markers, we characterized 57 infected finger-prick blood samples from the village of Mebat in Papua New Guinea for all 12 loci, and all samples were genotyped a second time to measure reproducibility. Numbers of alleles per locus range from 4 to 10 in this population, while heterozygosities range from 0·21 to 0·87. Reproducibility (measured as concordance between predominant alleles detected in replicate samples) ranged from 92 to 98% for the 12 loci. The composition of PCR products from infections containing multiple malaria clones could also be defined using strict criteria and scored in a highly repeatable manner.

The emergence and spread of drug resistance is a problem hindering malaria elimination in Southeast Asia. In this study, genetic variations in drug resistance markers of Plasmodium falciparum were determined in parasites from asymptomatic populations located in three geographically dispersed townships of Myanmar by PCR and sequencing. Mutations in dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps), chloroquine resistance transporter (pfcrt), multidrug resistance protein 1 (pfmdr1), multidrug resistance-associated protein 1 (pfmrp1), and Kelch protein 13 (k13) were present in 92.3%, 97.6%, 84.0%, 98.8%, and 68.3% of the parasites, respectively. The pfcrt K76T, pfmdr1 N86Y, pfmdr1 I185K, and pfmrp1 I876V mutations were present in 82.7%, 2.5%, 87.5%, and 59.8% isolates, respectively. The most prevalent haplotypes for pfdhfr, pfdhps, pfcrt and pfmdr1 were 51I/59R/108N/164L, 436A/437G/540E/581A, 74I/75E/76T/220S/271E/326N/356T/371I, and 86N/130E/184Y/185K/1225V, respectively. In addition, 57 isolates had three different point mutations (K191T, F446I, and P574L) and three types of N-terminal insertions (N, NN, NNN) in the k13 gene. In total, 43 distinct haplotypes potentially associated with multidrug resistance were identified. These findings demonstrate a high prevalence of multidrug-resistant P. falciparum in asymptomatic infections from diverse townships in Myanmar, emphasizing the importance of targeting asymptomatic infections to prevent the spread of drug-resistant P. falciparum.

Le paludisme à P. falciparum est l’une des causes majeures de mortalité et de morbidité dans le monde. Ce parasite est responsable de plusieurs manifestations cliniques allant du portage asymptomatique et infections non compliquées aigüe au paludisme grave et compliqué, tel que le neuropaludisme. Nous avons émis l’hypothèse que l’expression différentielle des gènes contribue à la variation phénotypique de parasites, entraînant des interactions spécifiques avec l’hôte, qui à son tour déterminent le type de manifestations cliniques du paludisme. L’objectif principal de cette étude était d’identifier les facteurs génétiques de P. falciparum impliqués dans la pathogenèse du neuropaludisme. Ceci a été réalisé par l’analyse complète du transcriptome d’isolats provenant d’enfants camerounais porteurs asymptomatiques (PA) ou atteints d’accès simple (AS) ou de neuropaludisme (NP). Le transcriptome du clone non sélectionnée (3D7) et la lignée sélectionnée (3D7-Lib) a été également analysé. Les résultats ont montré la surexpression de plusieurs gènes chez des isolats provenant d’enfants atteints de neuropaludisme et chez la lignée 3D7-Lib, par rapport à ceux provenant d’enfants asymptomatiques et 3D7, respectivement. L’analyse de l’ontologie de gène indique que les gènes potentiellement impliqués dans la pathogenèse, la cytoadhérence et l’agrégation des érythrocytes sont surreprésentés parmi les gènes surexprimés chez les isolats de CM et 3D7-Lib. Les résultats les plus marquants étaient la surexpression des gènes var (groups A et B) portant les domaines cassettes DC4, DC5, DC8 et DC13 et les gènes avoisinants rif chez les isolats de NP et la lignée 3D7-Lib, par rapport aux isolats de PA et au clone non sélectionné 3D7, respectivement. Le rôle joué par ces gènes dans la virulence parasitaire est lié à la cytoadhérence, c’est-à-dire la capacité de leurs protéines exprimées à interagir entre les érythrocytes parasités et les récepteurs endothéliaux post capillaires. Parmi ces récepteurs, le CD36 et inter cellular adhesion molecule 1 (ICAM-1) ont été les plus couramment utilisés par les isolats. L’étude sur l’implication de ces deux récepteurs, ainsi que celle des ligands PfEMP-1, dans la pathogenèse du neuropaludisme devrait être approfondie poursuivie. Nous avons analysé le phénotype de cytoadhérence et les profils de transcription des variantes de Pfemp-1 des isolats frais provenant des enfants béninois atteints de NP ou AS à l’aide du test d’adhérence statique aux récepteurs CD36, ICAM-1 et CSPG et au moyen de RT-PCR quantitative pour les groupes A, B, var2, var3, DC8 et DC13. Nos résultats montrent que le niveau de cytoadhérence des parasites associés au neuropaludisme au CD36 est significativement plus important que celui des parasites associés à l’accès simple. En outre, nous n’avons pas trouvé de différence significative entre la cytoadhérence des isolats de deux groupes cliniques à ICAM-1 et au CSPG. En outre, les niveaux d’expression des groupes var A, B, var2, var3 et du DC8 et DC13 sont plus élevés chez les isolats associés au neuropaludisme que chez les isolats associés à l’accès simple. Nos résultats montrent également que, chez les parasites provenant de NP le haut niveau de cytoadhérence des parasites au CD36 est corrélé au niveau de l’expression de groupe B de gènes var. En revanche, les profils d’expression des groupes spécifiques du gène var et le phénotype de cytoadhérence aux récepteurs ICAM-1 et CSPG n’étaient pas corrélés. Nos résultats suggèrent un rôle important du récepteur CD36 et des protéines codées par les variantes de PfEMP-1 codées par le groupe B dans la pathogenèse du neuropaludisme<br>Plasmodium falciparum infection is a major cause of mortality and morbidity worldwide. This parasite is involved in several clinical manifestations, ranging from asymptomatic carriage and acute uncomplicated to severe and complicated malaria, including cerebral malaria. We hypothesized that differential gene expression contributes to phenotypic variation of parasites leading to specific interaction with the host which induces several clinical categories of malaria. The principal aim of this study was to identify parasite genetic factors implicated in the pathogenesis of cerebral malaria. We investigated the whole transcriptome of parasites isolated from Cameroonian children with asymptomatic (AM), uncomplicated (UM) and cerebral malaria (CM). We also investigated the transcriptome of 3D7 clone and the selected 3D7-Lib line. Our results revealed the up-regulation of several genes in CM isolates and 3D7-Lib line compared to AM isolates and 3D7 clone respectively. Gene ontology analysis indicates an over-representation of genes implicated in pathogenesis, cytoadherence, and erythrocyte aggregation among up-regulated genes in CM and 3D7-Lib. The most remarkable outcomes were the up-regulation of UPS A and B var genes containing architectural Domains Cassettes DC4, DC5, DC8, and DC13 and their neighboring rif genes in isolates from CM and 3D7-Lib line, compared with isolates from AM and the unselected 3D7 line, respectively. The involvement of these genes in parasite virulence rises from the ability of their encoded proteins to mediate cytoadherence of infected erythrocytes to post-capillary endothelial receptors. Of these receptors, CD36 and Inter Cellular Adhesion Molecule-1 (ICAM-1) were found as the most commonly used by the isolates. The implication of these two receptors, as well as that of PfEMP-1 ligands in the pathogenesis of CM needs to be more elucidated. We examined the adhesive phenotype and the transcription patterns of Pfemp-1 variants of fresh isolates from Beninese children with CM or UM malaria by static binding assay to CD36, ICAM-1 and CSPG and RT-qPCR for groups A, B, var2, var3, DC8, and DC13. Our findings showed that isolates from CM patients bind more to CD36 than those from UM cases. No differences were observed in binding levels to ICAM-1 or CSPG between these two groups. Furthermore, CM isolates transcribed groups A, B, var2, var3, DC8 and DC13 of var genes at higher levels than UM isolates. Interestingly, the high transcription levels of group B in CM parasites correlated with their higher level of binding to CD36. In contrary, the expression profiles of a specific var group and the binding phenotype of isolates to ICAM-1 and to CSPG were not correlated. Our findings support the implication of CD36 along with PfEMP-1 variants encoded by group B in cerebral malaria pathogenesis

A malária é responsável por cerca de 207 milhões de casos e 627 mil óbitos em todo o mundo. No Brasil, em 2013, foram registrados mais de 167 mil casos. Um dos grandes desafios para o controle da doença é o desenvolvimento de resistência aos antimaláricos. Dentre as cinco espécies que podem causar malária em seres humanos, Plasmodium falciparum é a que apresenta maior habilidade de desenvolver resistência a quase todas as classes de medicamentos utilizados no tratamento. Estudos com marcadores moleculares têm associado mutações em diversos genes à resistência aos antimaláricos. A mutação K76T no gene pfcrt é relacionada à resistência à cloroquina. Mutações em pfmdr1 e aumento de seu número de cópias foram associados à resistência a diversos antimaláricos, como quinino, mefloquina e derivados de artemisinina. A resistência aos antifolatos é associada a mutações nos genes pfdhfr e pfdhps. Mutações nos genes pfATPase6 e pfAP2-u têm sido relacionadas à diminuição de sensibilidade à artemisinina. O monitoramento dessas mutações e suas associações às respostas in vivo e in vitro aos antimaláricos pode contribuir para a escolha de terapêutica para o controle da doença. O objetivo deste estudo foi analisar o perfil genético relacionado à resistência em P. falciparum ao longo de 27 anos. Foram analisadas amostras de P. falciparum coletadas no período entre 1984 e 2011, de pacientes atendidos em serviços de saúde nos estados do Pará e São Paulo, com infecções provenientes principalmente de países da América do Sul e África. A análise do gene pfcrt mostrou frequência de 100% da mutação K76T nas amostras de países da América do Sul ao longo das décadas analisadas. Este resultado é concordante com o fenótipo de resistência à cloroquina observado em 100% das amostras testadas in vitro para sensibilidade a este antimalárico. Amostras de países africanos apresentaram o genótipo selvagem em infecções únicas ou mistas. Com relação ao gene pfmdr1, a análise do códon 86 mostrou surgimento do mutante 86Y na última década em amostras da América do Sul e ocorrência de alelos selvagens e mutantes em amostras da África. Na análise do códon 1246 foi observada predominância do mutante 1246Y nas amostras sul-americanas e do selvagem D1246 nas africanas. Com relação ao gene pfdhfr, as amostras brasileiras apresentaram frequência de 100% dos mutantes 51I e 108N. O mutante 59R não foi observado no período 1980-1990, mas estava presente em 22,6% das amostras de 2000-2010. A análise do gene pfdhps das amostras brasileiras mostrou frequência de 100% do mutante 437G em todas as décadas e diminuição da frequência de 540E no período 2000-2010 em relação aos anteriores. O sequenciamento do gene pfATPase6 revelou a ocorrência de mutações que já haviam sido relatadas anteriormente, porém seu papel na resistência aos derivados de artemisinina ainda não foi elucidado. Na análise do gene pfAP2-? foram observadas duas novas mutações. Não foram observadas associações entre as mutações estudadas e a resposta in vivo e in vitro à mefloquina, quinino e derivados de artemisinina. Este estudo permitiu a avaliação do perfil genético de P. falciparum, com análise de marcadores moleculares relacionados à resistência a diversos antimaláricos, em amostras coletadas ao longo de 27 anos em que o parasito foi submetido à pressão de diferentes esquemas terapêuticos adotados no Brasil<br>Malaria is responsible for 207 million cases and 627 thousand deaths worldwide. In Brazil, in 2013, more than 167 thousand cases were reported. The major challenge for malaria control is the emergence and spread of resistance to antimalarials. Among the five species able to cause malaria in humans, Plasmodium falciparum presents ability to develop resistance to almost all classes of drugs for malaria treatment. Studies with molecular markers have associated mutations in several genes to antimalarial resistance. The mutation K76T in pfcrt is related to chloroquine resistance. Polymorphisms in pfmdr1 and increased copy number were associated to several antimalarials resistance, such as quinine, mefloquine and artemisinin derivatives. Resistance to antifolates is associated to mutations in pfdhfr and pfdhps genes. Mutations in pfATPase6 and pfAP2-u were linked to decreased sensibility to artemisinins. The monitoring of these mutations and their association with in vivo and in vitro responses may contribute to the choice of therapeutics for malaria control. The aim of the present study was to analyze the genetic profile related to resistance in P. falciparum over twenty-seven years. Samples collected from 1984 to 2011 from patients enrolled at health facilities in Para and Sao Paulo States were assessed. The origin of infections was mainly from South America and Africa countries. Pfcrt analysis showed that all samples from South America countries harbored the K76T mutation over the four decades, in agreement with the resistant phenotypic response of samples tested in vitro for chloroquine. African samples presented the wild type in mixed or single infections. Regarding to the pfmdr1 gene, the emergence of the mutant 86Y was observed in the last decade in South American samples and occurrence of both, mutant and wild type, in African ones. The analysis of 1246 showed only the mutant 1246Y in South American samples and the wild type D1246 in samples from Africa. Regarding to pfdhfr gene, the frequency of mutants 51I and 108N was 100% in Brazilian samples. The mutant 59R was not observed in the period 1980-1990 and it was present in 22.6% in samples from 2000-2010. The mutant 437G of pfdhps gene was observed in 100% of Brazilian samples in all decades and the frequency of mutant 540E decreased in the period 2000-2010 when compared to 1980-1990. The sequence analysis of pfATPase6 gene showed mutations previous described, but their role in artemisinin resistance is not well defined. Two novel mutations were observed in pfAP2-? gene. No association between the polymorphisms studied and in vivo or in vitro responses to mefloquine, quinine and artemisinin derivatives was observed. This study enabled the knowledge of P. falciparum genetic profile regarding to mutations related to resistance in samples collected over twenty-seven years, when the parasite was exposed to selective pressure of several therapeutic schemes adopted in Brazil

Malaria, the severe vector-borne disease has embedded serious consequences on mankind since ages, causing deterioration of health, leading to deaths. The causative parasite has a wide distribution aligned from tropical to subtropical regions. Out of all the five species Plasmodium vivax and Plasmodium falciparum have registered about more than 600 million cases worldwide. Throughout the decades, identification of various antimalarial drugs, targets, preventive measures and advancement of vaccines were achieved. The key to executing malaria elimination is the appropriate laboratory diagnosis. Development includes positive scientific judgments for a vaccine, advanced progress of 3 non-pyrethroid insecticides, novel genetic technologies, possibilities to alter malaria parasite mediation by the mosquito, identification of drug resistance markers, initiation of Plasmodium vivax liver stage assessment, perspective to mathematical modeling and screening for active ingredients for drugs and insecticides. Although the last century witnessed many successful programs with scientific progress, however, this was matched with notable obstacles. The mutation in the genes has changed the overall gameplay of eradication. This chapter aims to examine the numerous experimental and theoretical works that have been established in the last two decades along with the ongoing methodologies consisting of detailed explanations necessary for the establishment of new targets and drugs.