Full Text Available We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.
Tamborrini, Marco; Stoffel, Sabine A; Westerfeld, Nicole
In clinical trials, immunopotentiating reconstituted influenza virosomes (IRIVs) have shown great potential as a versatile antigen delivery platform for synthetic peptides derived from Plasmodium falciparum antigens. This study describes the immunogenicity of a virosomally-formulated recombinant...... fusion protein comprising domains of the two malaria vaccine candidate antigens MSP3 and GLURP....
Full Text Available The association between P. falciparum eba-175, ama-1, and msp-3 polymorphism in the pathogenicity of malaria disease was investigated. We therefore compared the prevalence of different alleles between symptomatic and asymptomatic malarial children under five years of age living in Burkina Faso. Blood filter papers were collected during the 2008 malaria transmission season from 228 symptomatic and 199 asymptomatic children under five years of age. All patients were living in the rural area of Saponé at about 50 km from Ouagadougou, the capital city of Burkina Faso. P. falciparum parasite DNA was extracted using QIAGEN kits and the alleles diversity was assessed by a nested PCR. PCR products were then digested by restriction enzymes based on already described polymorphic regions of the eba-175, ama-1, and msp-3 genes. The individual alleles eba-175_FCR3 and msp-3_K1 frequencies were statistically higher (p0.05. The comparative analysis of P. falciparum genotypes indicated that the polymorphism in eba-175 and msp-3 genotypes varied between asymptomatic and symptomatic clinical groups and may contribute to the pathogenesis of malaria.
David Tiga Kangoye
Full Text Available Children below six months are reported to be less susceptible to clinical malaria. Maternally derived antibodies and foetal haemoglobin are important putative protective factors. We examined antibodies to Plasmodium falciparum merozoite surface protein 3 (MSP3 and glutamate-rich protein (GLURP, in children in their first two years of life in Burkina Faso and their risk of malaria.A cohort of 140 infants aged between four and six weeks was recruited in a stable transmission area of south-western Burkina Faso and monitored for 24 months by active and passive surveillance. Malaria infections were detected by examining blood smears using light microscopy. Enzyme-linked immunosorbent assay was used to quantify total Immunoglobulin G to Plasmodium falciparum antigens MSP3 and two regions of GLURP (R0 and R2 on blood samples collected at baseline, three, six, nine, 12, 18 and 24 months. Foetal haemoglobin and variant haemoglobin fractions were measured at the baseline visit using high pressure liquid chromatography.A total of 79.6% of children experienced one or more episodes of febrile malaria during monitoring. Antibody titres to MSP3 were prospectively associated with an increased risk of malaria while antibody responses to GLURP (R0 and R2 did not alter the risk. Antibody titres to MSP3 were higher among children in areas of high malaria risk. Foetal haemoglobin was associated with delayed first episode of febrile malaria and haemoglobin CC type was associated with reduced incidence of febrile malaria.We did not find any evidence of association between titres of antibodies to MSP3, GLURP-R0 or GLURP-R2 as measured by enzyme-linked immunosorbent assay and early protection against malaria, although anti-MSP3 antibody titres may reflect increased exposure to malaria and therefore greater risk. Foetal haemoglobin was associated with protection against febrile malaria despite the study limitations and its role is therefore worthy further investigation.
Full Text Available Abstract Background In clinical trials, immunopotentiating reconstituted influenza virosomes (IRIVs have shown great potential as a versatile antigen delivery platform for synthetic peptides derived from Plasmodium falciparum antigens. This study describes the immunogenicity of a virosomally-formulated recombinant fusion protein comprising domains of the two malaria vaccine candidate antigens MSP3 and GLURP. Methods The highly purified recombinant protein GMZ2 was coupled to phosphatidylethanolamine and the conjugates incorporated into the membrane of IRIVs. The immunogenicity of this adjuvant-free virosomal formulation was compared to GMZ2 formulated with the adjuvants Montanide ISA 720 and Alum in three mouse strains with different genetic backgrounds. Results Intramuscular injections of all three candidate vaccine formulations induced GMZ2-specific antibody responses in all mice tested. In general, the humoral immune response in outbred NMRI mice was stronger than that in inbred BALB/c and C57BL/6 mice. ELISA with the recombinant antigens demonstrated immunodominance of the GLURP component over the MSP3 component. However, compared to the Al(OH3-adjuvanted formulation the two other formulations elicited in NMRI mice a larger proportion of anti-MSP3 antibodies. Analyses of the induced GMZ2-specific IgG subclass profiles showed for all three formulations a predominance of the IgG1 isotype. Immune sera against all three formulations exhibited cross-reactivity with in vitro cultivated blood-stage parasites. Immunofluorescence and immunoblot competition experiments showed that both components of the hybrid protein induced IgG cross-reactive with the corresponding native proteins. Conclusion A virosomal formulation of the chimeric protein GMZ2 induced P. falciparum blood stage parasite cross-reactive IgG responses specific for both MSP3 and GLURP. GMZ2 thus represents a candidate component suitable for inclusion into a multi-valent virosomal
Full Text Available Abstract Background Clinical profiling of two components for a synthetic peptide-based virosomal malaria vaccine has yielded promising results, encouraging the search for additional components for inclusion in a final multi-valent vaccine formulation. This report describes the immunological characterization of linear and cyclized synthetic peptides comprising amino acids 211-237 of Plasmodium falciparum merozoite surface protein (MSP-3. Methods These peptides were coupled to phosphatidylethanolamine (PE; the conjugates were intercalated into immunopotentiating reconstituted influenza virosomes (IRIVs and then used for immunizations in mice to evaluate their capacity to elicit P. falciparum cross-reactive antibodies. Results While all MSP-3-derived peptides were able to elicit parasite-binding antibodies, stabilization of turn structures by cyclization had no immune-enhancing effect. Therefore, further pre-clinical profiling was focused on FB-12, a PE conjugate of the linear peptide. Consistent with the immunological results obtained in mice, all FB-12 immunized rabbits tested seroconverted and consistently elicited antibodies that interacted with blood stage parasites. It was observed that a dose of 50 μg was superior to a dose of 10 μg and that influenza pre-existing immunity improved the immunogenicity of FB-12 in rabbits. FB-12 production was successfully up-scaled and the immunogenicity of a vaccine formulation, produced according to the rules of Good Manufacturing Practice (GMP, was tested in mice and rabbits. All animals tested developed parasite-binding antibodies. Comparison of ELISA and IFA titers as well as the characterization of a panel of anti-FB-12 monoclonal antibodies indicated that at least the majority of antibodies specific for the virosomally formulated synthetic peptide were parasite cross-reactive. Conclusion These results reconfirm the suitability of IRIVs as a carrier/adjuvant system for the induction of strong humoral
Amoah, L. E.; Nuvor, S. V.; Obboh, E. K.
patients living in the Central Region of Ghana and to determine whether naturally occurring antibody levels against P. falciparum GLURP (PF3D7-1035300) and MSP3 (PF3D7-1035400) antigens are associated with decreased parasite load. Methods: Dried filter paper blood blots were obtained from children......Background: Plasmodium falciparum genetic diversity and multiplicity of infection (MOI) are parasite features that have been suggested to influence the acquisition of protective immunity against malaria. This study sought to assess the relationship between MOI and parasite density (PD) in malaria...... and adults diagnosed with uncomplicated P. falciparum malaria. Microscopy was used to estimate P. falciparum parasite density and polymerase chain reaction (PCR) amplification of the polymorphic regions of msp1 (PF3D7-0930300) and msp2 (PF3D7-0206800) was used for parasite genotyping and MOI determination...
Segeja Method D
Full Text Available Abstract Background Development and deployment of an effective malaria vaccine would complement existing malaria control measures. A blood stage malaria vaccine candidate, Merozoite Surface Protein-3 (MSP3, produced as a long synthetic peptide, has been shown to be safe in non-immune and semi-immune adults. A phase Ib dose-escalating study was conducted to assess the vaccine's safety and immunogenicity in children aged 12 to 24 months in Korogwe, Tanzania (ClinicalTrials.gov number: NCT00469651. Methods This was a double-blind, randomized, controlled, dose escalation phase Ib trial, in which children were given one of two different doses of the MSP3 antigen (15 μg or 30 μg or a control vaccine (Engerix B. Children were randomly allocated either to the MSP3 candidate malaria vaccine or the control vaccine administered at a schedule of 0, 1, and 2 months. Immunization with lower and higher doses was staggered for safety reasons starting with the lower dose. The primary endpoint was safety and reactogenicity within 28 days post-vaccination. Blood samples were obtained at different time points to measure immunological responses. Results are presented up to 84 days post-vaccination. Results A total of 45 children were enrolled, 15 in each of the two MSP3 dose groups and 15 in the Engerix B group. There were no important differences in reactogenicity between the two MSP3 groups and Engerix B. Grade 3 adverse events were infrequent; only five were detected throughout the study, all of which were transient and resolved without sequelae. No serious adverse event reported was considered to be related to MSP3 vaccine. Both MSP3 dose regimens elicited strong cytophilic IgG responses (subclasses IgG1 and IgG3, the isotypes involved in the monocyte-dependant mechanism of Plasmodium falciparum parasite-killing. The titers reached are similar to those from African adults having reached a state of premunition. Furthermore, vaccination induced seroconversion in
Full Text Available BACKGROUND: Two related merozoite surface proteins, MSP3 and MSP6, have previously been identified as targets of antibody-dependent cellular inhibition (ADCI, a protective mechanism against Plasmodium falciparum malaria. Both MSP3 and MSP6 share a common characteristic small N-terminal signature amino-acid stretch (NLRNA/G, a feature similar to MSP3-like orthologs identified in other human and primate malaria parasites. METHODS/RESULTS: This signature amino-acid sequence led to the identification of eight ORFs contiguously located on P. falciparum chromosome 10. Our subsequent investigations on their expression, localization, sequence conservation, epitope sharing, immunogenicity and the functional role of antibodies in defense are reported here. Six members of P. falciparum MSP3-multigene family share similar sequence organization within their C-terminal regions, are simultaneously expressed as merozoite surface proteins and are highly conserved among parasite isolates. Each of these proteins is a target of naturally occurring antibodies effective at parasite killing in ADCI assays. Moreover, both naturally occurring antibodies and those generated by immunization display cross-reactivity with other members of the family and exhibit varied binding avidities. CONCLUSIONS/SIGNIFICANCE: The unusual characteristics of the MSP3 multi-gene family lead us to hypothesize that the simultaneous expression of targets eliciting cross-reactive antibody responses capable of controlling parasite densities could represent an immune process selected through evolution to maintain homeostasis between P. falciparum and human hosts; a process that allows the continuous transmission of the parasite without killing the host. Our observations also have practical consequences for vaccine development by suggesting MSP3 vaccine efficacy might be improved when combined with the various C-terminus regions of the MSP3 family members to generate a wider range of antibodies
Amoah, Linda E; Acquah, Festus K; Ayanful-Torgby, Ruth
the transmission of malaria to an uninfected person. This study aimed at examining the dynamics of naturally acquired immune responses against the asexual and sexual forms of Plasmodium falciparum as well as assessing differences in the multiplicity of infection (MOI) in asymptomatic Ghanaian children living...... in two communities with varying malaria transmission intensities. METHODS: School children aged between 6 and 12 years were recruited from Obom, a high malaria prevalence setting and Abura, a low malaria prevalence setting and enrolled in monthly multiple cross sectional surveys between February and May...... 2015. Filter paper blood blots (DBS) as well as thick and thin blood smears were made from finger-pricked blood at each visit. Plasmodium falciparum parasite prevalence was determined by microscopy and PCR. Serum eluted from the DBS were used to assess anti-Pfs230 (sexual stage) and anti-MSP3 (asexual...
Coetzer Theresa L
Full Text Available Abstract Background The development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised. Methods P. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database. Results Following four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library. Conclusion The construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.
Sawaswong, Vorthon; Simpalipan, Phumin; Siripoon, Napaporn; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn
Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.
Nourein, Amal B.; Abass, Mohammed A.; Nugud, Abdel Hameed D.; El Hassan, Ibrahim; Snow, Robert W.; Noor, Abdisalan M.
Background: Identifying the location and size of residual foci of infections is critical where malaria elimination is the primary goal. Here the spatial heterogeneity of Plasmodium falciparum infections within the urban extent of Khartoum state in Sudan is investigated using data from cross-sectional surveys undertaken from 1999 to 2008 to inform the Khartoum Malaria Free Initiative (KMFI).Methods: From 1999-2008 the KMFI undertook cross-sectional surveys of 256 clusters across 203 random sam...
Zhang, Jin; Bowling, John J; Smithson, David; Clark, Julie; Jacob, Melissa R; Khan, Shabana I; Tekwani, Babu L; Connelly, Michele; Samoylenko, Vladimir; Ibrahim, Mohamed A; Zaki, Mohamed A; Wang, Mei; Hester, John P; Tu, Ying; Jeffries, Cynthia; Twarog, Nathaniel; Shelat, Anang A; Walker, Larry A; Muhammad, Ilias; Guy, R Kiplin
A diverse library of pre-fractionated plant extracts, generated by an automated high-throughput system, was tested using an in vitro anti-malarial screening platform to identify known or new natural products for lead development. The platform identifies hits on the basis of in vitro growth inhibition of Plasmodium falciparum and counter-screens for cytotoxicity to human foreskin fibroblast or embryonic kidney cell lines. The physical library was supplemented by early-stage collection of analytical data for each fraction to aid rapid identification of the active components within each screening hit. A total of 16,177 fractions from 1300 plants were screened, identifying several P. falciparum inhibitory fractions from 35 plants. Although individual fractions were screened for bioactivity to ensure adequate signal in the analytical characterizations, fractions containing less than 2.0 mg of dry weight were combined to produce combined fractions (COMBIs). Fractions of active COMBIs had EC50 values of 0.21-50.28 and 0.08-20.04 µg/mL against chloroquine-sensitive and -resistant strains, respectively. In Berberis thunbergii, eight known alkaloids were dereplicated quickly from its COMBIs, but berberine was the most-active constituent against P. falciparum. The triterpenoids α-betulinic acid and β-betulinic acid of Eugenia rigida were also isolated as hits. Validation of the anti-malarial discovery platform was confirmed by these scaled isolations from B. thunbergii and E. rigida. These results demonstrate the value of curating and exploring a library of natural products for small molecule drug discovery. Attention given to the diversity of plant species represented in the library, focus on practical analytical data collection, and the use of counter-screens all facilitate the identification of anti-malarial compounds for lead development or new tools for chemical biology.
Full Text Available BACKGROUND: Studies to identify phenotypically-associated polymorphisms in the Plasmodium falciparum 23 Mb genome will require a dense array of marker loci. It was considered promising to undertake initial allelic association studies to prospect for virulence polymorphisms in Thailand, as the low endemicity would allow higher levels of linkage disequilibrium (LD than would exist in more highly endemic areas. METHODOLOGY/PRINCIPAL FINDINGS: Assessment of LD was first made with 11 microsatellite loci widely dispersed in the parasite genome, and 16 microsatellite loci covering a approximately 140 kb region of chromosome 2 (an arbitrarily representative non-telomeric part of the genome, in a sample of 100 P. falciparum isolates. The dispersed loci showed minimal LD (Index of Association, I(S (A = 0.013, P = 0.10, while those on chromosome 2 showed significant LD values mostly between loci <5 kb apart. A disease association study was then performed comparing parasites in 113 severe malaria cases and 245 mild malaria controls. Genotyping was performed on almost all polymorphisms in the binding domains of three erythrocyte binding antigens (eba175, eba140 and eba181, and repeat sequence polymorphisms approximately 2 kb apart in each of three reticulocyte binding homologues (Rh1, Rh2a/b, and Rh4. Differences between cases and controls were seen for (i codons 388-90 in eba175, and (ii a repeat sequence centred on Rh1 codon 667. CONCLUSIONS/SIGNIFICANCE: Allelic association studies on P. falciparum require dense genotypic markers, even in a population of only moderate endemicity that has more extensive LD than highly endemic populations. Disease-associated polymorphisms in the eba175 and Rh1 genes encode differences in the middle of previously characterised erythrocyte binding domains, marking these for further investigation.
Jeremy Ryan De Silva
Full Text Available Malaria remains a major health threat in many parts of the globe and causes high mortality and morbidity with 214 million cases of malaria occurring globally in 2015. Recent studies have outlined potential diagnostic markers and vaccine candidates one of which is the merozoite surface protein (MSP-3. In this study, novel recombinant Plasmodium knowlesi MSP-3 was cloned, expressed and purified in an Escherichia coli system. Subsequently, the recombinant protein was evaluated for its sensitivity and specificity. The recombinant pkMSP-3 protein reacted with sera from patients with P. knowlesi infection in both Western blot (61% and ELISA (100%. Specificity-wise, pkMSP-3 did not react with healthy donor sera in either assay and only reacted with a few non-malarial parasitic patient sera in the ELISA assay (3 of 49. In conclusion, sensitivity and specificity of pkMSP-3 was found to be high in the ELISA and Western Blot assay and thus utilising both assays in tandem would provide the best sero-diagnostic result for P. knowlesi infection.
Full Text Available Block II of Plasmodium vivax merozoite surface protein 3α (PvMSP3α is conserved and has been proposed as a potential candidate for a malaria vaccine. The present study aimed to compare sequence diversity in PvMSP3a block II at a local microgeographic scale in a village as well as from larger geographic regions (countries and worldwide. Blood samples were collected from asymptomatic carriers of P. vivax in a village at the western border of Thailand and PvMSP3α was amplified and sequenced. For population genetic analysis, 237 PvMSP3α block II sequences from eleven P. vivax endemic countries were analyzed. PvMSP3α sequences from 20 village-level samples revealed two length variant types with one type containing a large deletion in block I. In contrast, block II was relatively conserved; especially, some non-synonymous mutations were extensively shared among 11 parasite populations. However, the majority of the low-frequency synonymous variations were population specific. The conserved pattern of nucleotide diversity in block II sequences was probably due to functional/structural constraints, which were further supported by the tests of neutrality. Notably, a small region in block II that encodes a predicted B cell epitope was highly polymorphic and showed signs of balancing selection, signifying that this region might be influenced by the immune selection and may serve as a starting point for designing multi-antigen/stage epitope based vaccines against this parasite.
Preston, Mark D.
Malaria is a major public health problem that is actively being addressed in a global eradication campaign. Increased population mobility through international air travel has elevated the risk of re-introducing parasites to elimination areas and dispersing drug-resistant parasites to new regions. A simple genetic marker that quickly and accurately identifies the geographic origin of infections would be a valuable public health tool for locating the source of imported outbreaks. Here we analyse the mitochondrion and apicoplast genomes of 711 Plasmodium falciparum isolates from 14 countries, and find evidence that they are non-recombining and co-inherited. The high degree of linkage produces a panel of relatively few single-nucleotide polymorphisms (SNPs) that is geographically informative. We design a 23-SNP barcode that is highly predictive (?92%) and easily adapted to aid case management in the field and survey parasite migration worldwide. 2014 Macmillan Publishers Limited. All rights reserved.
Reagan M Mogire
Full Text Available Malaria causes about half a million deaths annually, with Plasmodium falciparum being responsible for 90% of all the cases. Recent reports on artemisinin resistance in Southeast Asia warrant urgent discovery of novel drugs for the treatment of malaria. However, most bioactive compounds fail to progress to treatments due to safety concerns. Drug repositioning offers an alternative strategy where drugs that have already been approved as safe for other diseases could be used to treat malaria. This study screened approved drugs for antimalarial activity using an in silico chemogenomics approach prior to in vitro verification. All the P. falciparum proteins sequences available in NCBI RefSeq were mined and used to perform a similarity search against DrugBank, TTD and STITCH databases to identify similar putative drug targets. Druggability indices of the potential P. falciparum drug targets were obtained from TDR targets database. Functional amino acid residues of the drug targets were determined using ConSurf server which was used to fine tune the similarity search. This study predicted 133 approved drugs that could target 34 P. falciparum proteins. A literature search done at PubMed and Google Scholar showed 105 out of the 133 drugs to have been previously tested against malaria, with most showing activity. For further validation, drug susceptibility assays using SYBR Green I method were done on a representative group of 10 predicted drugs, eight of which did show activity against P. falciparum 3D7 clone. Seven had IC50 values ranging from 1 μM to 50 μM. This study also suggests drug-target association and hence possible mechanisms of action of drugs that did show antiplasmodial activity. The study results validate the use of proteome-wide target similarity approach in identifying approved drugs with activity against P. falciparum and could be adapted for other pathogens.
Nourein, Amal B; Abass, Mohammed A; Nugud, Abdel Hameed D; El Hassan, Ibrahim; Snow, Robert W; Noor, Abdisalan M
Identifying the location and size of residual foci of infections is critical where malaria elimination is the primary goal. Here the spatial heterogeneity of Plasmodium falciparum infections within the urban extent of Khartoum state in Sudan is investigated using data from cross-sectional surveys undertaken from 1999 to 2008 to inform the Khartoum Malaria Free Initiative (KMFI). From 1999-2008 the KMFI undertook cross-sectional surveys of 256 clusters across 203 random samples of residential blocks in the urban Khartoum state in September of each year. Within sampled blocks, at least five persons, including at least one child under the age of five years, were selected from each household. Blood smears were collected from the sampled individuals to examine the presence of P. falciparum parasites. Residential blocks were mapped. Data were analysed for spatial clustering using the Bernoulli model and the significance of clusters were tested using the Kulldorff scan statistic. A total of 128,510 malaria slide examinations were undertaken during the study period. In 1999, overall prevalence was 2.5%, rising to 3.2% in 2000 and consistently staying below 1% in subsequent years. From 2006, over 90% of all surveyed clusters reported no infections. Spatial clustering of infections was present in each year but not statistically significant in the years 2001, 2002, 2004 and 2008. Spatial clusters of high infection were often located at the junction of the Blue and White Niles. Persisting foci of malaria infection in Khartoum are likely to distort wide area assessments and disproportionately affect future transmission within the city limits. Improved investments in surveillance that combines both passive and active case detection linked to a geographic information system and a more detailed analysis of the location and stability of foci should be undertaken to facilitate and track malaria elimination in the state of Khartoum.
Amal B Nourein
Full Text Available Identifying the location and size of residual foci of infections is critical where malaria elimination is the primary goal. Here the spatial heterogeneity of Plasmodium falciparum infections within the urban extent of Khartoum state in Sudan is investigated using data from cross-sectional surveys undertaken from 1999 to 2008 to inform the Khartoum Malaria Free Initiative (KMFI.From 1999-2008 the KMFI undertook cross-sectional surveys of 256 clusters across 203 random samples of residential blocks in the urban Khartoum state in September of each year. Within sampled blocks, at least five persons, including at least one child under the age of five years, were selected from each household. Blood smears were collected from the sampled individuals to examine the presence of P. falciparum parasites. Residential blocks were mapped. Data were analysed for spatial clustering using the Bernoulli model and the significance of clusters were tested using the Kulldorff scan statistic.A total of 128,510 malaria slide examinations were undertaken during the study period. In 1999, overall prevalence was 2.5%, rising to 3.2% in 2000 and consistently staying below 1% in subsequent years. From 2006, over 90% of all surveyed clusters reported no infections. Spatial clustering of infections was present in each year but not statistically significant in the years 2001, 2002, 2004 and 2008. Spatial clusters of high infection were often located at the junction of the Blue and White Niles.Persisting foci of malaria infection in Khartoum are likely to distort wide area assessments and disproportionately affect future transmission within the city limits. Improved investments in surveillance that combines both passive and active case detection linked to a geographic information system and a more detailed analysis of the location and stability of foci should be undertaken to facilitate and track malaria elimination in the state of Khartoum.
Ponder, Elizabeth L; Albrow, Victoria E; Leader, Brittany A; Békés, Miklós; Mikolajczyk, Jowita; Fonović, Urša Pečar; Shen, Aimee; Drag, Marcin; Xiao, Junpeng; Deu, Edgar; Campbell, Amy J; Powers, James C; Salvesen, Guy S; Bogyo, Matthew
Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum. Copyright © 2011 Elsevier Ltd. All rights reserved.
Carey, Maureen A; Papin, Jason A; Guler, Jennifer L
Malaria remains a major public health burden and resistance has emerged to every antimalarial on the market, including the frontline drug, artemisinin. Our limited understanding of Plasmodium biology hinders the elucidation of resistance mechanisms. In this regard, systems biology approaches can facilitate the integration of existing experimental knowledge and further understanding of these mechanisms. Here, we developed a novel genome-scale metabolic network reconstruction, iPfal17, of the asexual blood-stage P. falciparum parasite to expand our understanding of metabolic changes that support resistance. We identified 11 metabolic tasks to evaluate iPfal17 performance. Flux balance analysis and simulation of gene knockouts and enzyme inhibition predict candidate drug targets unique to resistant parasites. Moreover, integration of clinical parasite transcriptomes into the iPfal17 reconstruction reveals patterns associated with antimalarial resistance. These results predict that artemisinin sensitive and resistant parasites differentially utilize scavenging and biosynthetic pathways for multiple essential metabolites, including folate and polyamines. Our findings are consistent with experimental literature, while generating novel hypotheses about artemisinin resistance and parasite biology. We detect evidence that resistant parasites maintain greater metabolic flexibility, perhaps representing an incomplete transition to the metabolic state most appropriate for nutrient-rich blood. Using this systems biology approach, we identify metabolic shifts that arise with or in support of the resistant phenotype. This perspective allows us to more productively analyze and interpret clinical expression data for the identification of candidate drug targets for the treatment of resistant parasites.
Healer, Julie; Chiu, Chris Y; Hansen, Diana S
Malaria is one the most serious infectious diseases with over 200 million clinical cases annually. Most cases of the severe disease are caused by Plasmodium falciparum. The blood stage of Plasmodium parasite is entirely responsible for malaria-associated pathology. The population most susceptible to severe malaria are children under the age of 5, with low levels of immunity. It is only after many years of repeated exposure that individuals living in endemic areas develop clinical immunity. This form of protection prevents clinical episodes by substantially reducing parasite burden. Naturally acquired immunity predominantly targets blood-stage parasites with antibody responses being the main mediators of protection. The targets of clinical immunity are the extracellular merozoite and the infected erythrocyte surface, with the extremely diverse PfEMP1 proteins the main target here. This observation provides a strong rationale that an effective anti-malaria vaccine targeting blood-stage parasites is achievable. Thus the identification of antigenic targets of naturally acquired immunity remains an important step towards the formulation of novel vaccine combinations before testing their efficacy in clinical trials. This review summarizes the main findings to date defining antigenic targets present on the extracellular merozoite associated with naturally acquired immunity to P. falciparum malaria.
Escalante, A A; Lal, A A; Ayala, F J
We have studied the genetic polymorphism at 10 Plasmodium falciparum loci that are considered potential targets for specific antimalarial vaccines. The polymorphism is unevenly distributed among the loci; loci encoding proteins expressed on the surface of the sporozoite or the merozoite (AMA-1, CSP, LSA-1, MSP-1, MSP-2, and MSP-3) are more polymorphic than those expressed during the sexual stages or inside the parasite (EBA-175, Pfs25, PF48/45, and RAP-1). Comparison of synonymous and nonsyno...
Turner, Louise; Wang, Christian W; Lavstsen, Thomas
diverse merozoite antigens. The acquisition of a broad and strain transcendent repertoire of PfEMP1 antibodies may reflect a parasite strategy of expressing most or all PfEMP1 variants at liver release optimizing the likelihood of survival and establishment of chronic infections in the new host....
Full Text Available BACKGROUND: Surrogate markers of protective immunity to malaria in humans are needed to rationalize malaria vaccine discovery and development. In an effort to identify such markers, and thereby provide a clue to the complex equation malaria vaccine development is facing, we investigated the relationship between protection acquired through exposure in the field with naturally occurring immune responses (i.e., induced by the parasite to molecules that are considered as valuable vaccine candidates. METHODS AND FINDINGS: We analyzed, under comparative conditions, the antibody responses of each of six isotypes to five leading malaria vaccine candidates in relation to protection acquired by exposure to natural challenges in 217 of the 247 inhabitants of the African village of Dielmo, Senegal (96 children and 121 older adolescents and adults. The status of susceptibility or resistance to malaria was determined by active case detection performed daily by medical doctors over 6 y from a unique follow-up study of this village. Of the 30 immune responses measured, only one, antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3, was strongly associated with clinical protection against malaria in all age groups, i.e., independently of age. This immunological parameter had a higher statistical significance than the sickle cell trait, the strongest factor of protection known against Plasmodium falciparum. A single determination of antibody was significantly associated with the clinical outcome over six consecutive years in children submitted to massive natural parasite challenges by mosquitoes (over three parasite inoculations per week. Finally, the target epitopes of these antibodies were found to be fully conserved. CONCLUSIONS: Since anti-MSP3 IgG3 antibodies can naturally develop along with protection against P. falciparum infection in young children, our results provide the encouraging indication that these antibodies should be
Development of nuclear DNA markers for evolutionary studies in. Plasmodium falciparum. CELIA THOMAS, SNEH SHALINI, N. RAGHAVENDRA, MEENAKSHI CHOUDHARY, ANJU VERMA,. HEMA JOSHI, A. P. DASH and APARUP DAS*. National Institute of Malaria Research (ICMR), 22 Sham Nath Marg, Delhi 110 054, ...
Full Text Available There is no approved vaccine for malaria, and precisely how human antibody responses to malaria parasite components and potential vaccine molecules are developed and maintained remains poorly defined. In this study, antibody anamnestic or memory response elicited by a single episode of P. falciparum infection was investigated.This study involved 362 malaria patients aged between 6 months to 60 years, of whom 19% were early-diagnosed people living with HIV/AIDS (PLWHA. On the day malaria was diagnosed and 42 days later, blood specimens were collected. Parasite density, CD4+ cells, and antibodies specific to synthetic peptides representing antigenic regions of the P. falciparum proteins GLURP, MSP3 and HRPII were measured.On the day of malaria diagnosis, Immunoglobulin (IgG antibodies against GLURP, MSP3 and HRP II peptides were present in the blood of 75%, 41% and 60% of patients, respectively. 42 days later, the majority of patients had boosted their serum IgG antibody more than 1.2 fold. The increase in level of IgG antibody against the peptides was not affected by parasite density at diagnosis. The median CD4+ cell counts of PLWHAs and HIV negative individuals were not statistically different, and median post-infection increases in anti-peptide IgG were similar in both groups of patients.In the majority (70% of individuals, an infection of P. falciparum elicits at least 20% increase in level of anti-parasite IgG. This boost in anti-P. falciparum IgG is not affected by parasite density on the day of malaria diagnosis, or by HIV status.
green fluorescent protein (GFP). GFP signals were examined at 44 hr post- inoculation, corresponding to mature liver stage parasites. From our first...falciparum, P. cynomolgi, asexual blood stages, liver stages, high-throughput screen, drug assays, cell culture, transfection, green fluorescent ...Impact 7 5. Changes /Problems 8 6. Products 8 7. Participants & Other Collaborating Organizations 8 8. Special Reporting Requirements 12, 13 9
Mamo, Hassen; Esen, Meral; Ajua, Anthony; Theisen, Michael; Mordmüller, Benjamin; Petros, Beyene
In Ethiopia, the general population is vulnerable to unpredictable epidemics of Plasmodium falciparum malaria. However, there is little information on the anti-malaria immune profile of the population in the endemic regions of the country. The study was designed to investigate the nature of humoral immune response to malaria in two ethnic groups in two endemic localities: Shewa Robit in north, and Boditi in south Ethiopia which are characterized by varying levels of malaria transmission and altitude. In a cross-sectional study, the study participants were diagnosed for malaria infection microscopically and by the rapid diagnostic test (RDT). Sera were tested by using enzyme-linked immunosorbent assay (ELISA) for total immunoglobulin (Ig) G against P. falciparum blood-stage vaccine candidate GMZ2 and its subunits (Glutamate-rich protein (GLURP-R0), merozoite surface protein 3 (MSP3); as well as IgG subclasses against GLURP-R0 and MSP3. Whereas 23(8.6%) blood smear-positive cases for P. falciparum were detected in Boditi, all Shewa Robit study participants had no detectable P. falciparum infection. In both localities, total IgG prevalence and levels to GMZ2 were significantly higher than the response to the component domains indicating the strong recognition of GMZ2 by antibodies acquired through natural exposure. Total IgG and subclass prevalence and levels were higher in Shewa Robit than Boditi, suggesting difference in the intensity of malaria transmission in the two localities and/or genetic differences between the two populations in their response to the antigens. In both study sites, IgG subclass levels to GLURP-R0 were significantly higher than that to MSP3 for all corresponding subclasses in most individuals, indicating the higher relative antigenicity and probably protective potential of GLURP-R0 compared to MSP3. Against both GLURP-R0 and MSP3, the ratio of cytophilic to noncytophilic antibodies was >1 in the majority of the study participants, in both
Mamo, Hassen; Esen, Meral; Ajua, Anthony
. The significantly higher antibody prevalence and level detected against GMZ2 compared to either of its subunits separately, in naturally exposed populations, suggests the synergistic effect of GLURP-R0 and MSP3 and that GMZ2 could be a more relevant blood-stage malaria vaccine candidate than the individual......ABSTRACT: BACKGROUND: In Ethiopia, the general population is vulnerable to unpredictable epidemics of Plasmodium falciparum malaria. However, there is little information on the anti-malaria immune profile of the population in the endemic regions of the country. METHODS: The study was designed...... for malaria infection microscopically and by the rapid diagnostic test (RDT). Sera were tested by using enzyme-linked immunosorbent assay (ELISA) for total immunoglobulin (Ig) G against P. falciparum blood-stage vaccine candidate GMZ2 and its subunits (Glutamate-rich protein (GLURP-R0), merozoite surface...
Larissa Rodrigues Gomes
Full Text Available Anti-glycosylphosphatidylinositol (GPI antibodies (Abs may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-19 in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown in the Angolan patients. In addition, our data show that PvMSP1-19may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.
Paloque, Lucie; Ramadani, Arba P; Mercereau-Puijalon, Odile; Augereau, Jean-Michel; Benoit-Vical, Françoise
Plasmodium falciparum resistance to artemisinins, the most potent and fastest acting anti-malarials, threatens malaria elimination strategies. Artemisinin resistance is due to mutation of the PfK13 propeller domain and involves an unconventional mechanism based on a quiescence state leading to parasite recrudescence as soon as drug pressure is removed. The enhanced P. falciparum quiescence capacity of artemisinin-resistant parasites results from an increased ability to manage oxidative damage and an altered cell cycle gene regulation within a complex network involving the unfolded protein response, the PI3K/PI3P/AKT pathway, the PfPK4/eIF2α cascade and yet unidentified transcription factor(s), with minimal energetic requirements and fatty acid metabolism maintained in the mitochondrion and apicoplast. The detailed study of these mechanisms offers a way forward for identifying future intervention targets to fend off established artemisinin resistance.
Ellekvist, Peter; Colding, Hanne
to identify, clone and characterise a number of these transport proteins from the parasite. Since the P. falciparum transport proteins differ from their human homologues, they may provide potential drug targets in the treatment of malaria. An example of a P. falciparum transport protein which seems promising...
Yooseph, Shibu; Kirkness, Ewen F; Tran, Tuan M; Harkins, Derek M; Jones, Marcus B; Torralba, Manolito G; O'Connell, Elise; Nutman, Thomas B; Doumbo, Safiatou; Doumbo, Ogobara K; Traore, Boubacar; Crompton, Peter D; Nelson, Karen E
In humans it is unknown if the composition of the gut microbiota alters the risk of Plasmodium falciparum infection or the risk of developing febrile malaria once P. falciparum infection is established. Here we collected stool samples from a cohort composed of 195 Malian children and adults just prior to an intense P. falciparum transmission season. We assayed these samples using massively parallel sequencing of the 16S ribosomal RNA gene to identify the composition of the gut bacterial communities in these individuals. During the ensuing 6-month P. falciparum transmission season we examined the relationship between the stool microbiota composition of individuals in this cohort and their prospective risk of both P. falciparum infection and febrile malaria. Consistent with prior studies, stool microbial diversity in the present cohort increased with age, although the overall microbiota profile was distinct from cohorts in other regions of Africa, Asia and North America. Age-adjusted Cox regression analysis revealed a significant association between microbiota composition and the prospective risk of P. falciparum infection; however, no relationship was observed between microbiota composition and the risk of developing febrile malaria once P. falciparum infection was established. These findings underscore the diversity of gut microbiota across geographic regions, and suggest that strategic modulation of gut microbiota composition could decrease the risk of P. falciparum infection in malaria-endemic areas, potentially as an adjunct to partially effective malaria vaccines.
Background: The ability of the host immune system to efficiently clear Plasmodium falciparum parasites during a malaria infection ... infection, in an attempt to identify immunological signs indicative of the development of natural immunity against malaria in Ibadan, ..... Stephens R, Langhorne J. Priming of CD4+ T cells and.
Full Text Available The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF, red blood cells infected with Plasmodium falciparum (EPfs, and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA, and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.Epítopes de antígenos compartidos entre Plasmodium falciparum y Anopheles albimanus fueron identificados. Diferentes grupos de conejos fueron inmunizados con: extracto crudo de mosquito hembra de An. albimanus (EAaH, glóbulos rojos infectados con P. falciparum (EPfs y la vacuna antimalárica sintética SPf66. Los anticuerpos policlonales producidos en conejos fueron evaluados por ELISA, inmunoensayo simultáneo de múltiples antígenos (MABA e Immunoblotting. Todos los extractos resultaron inmunogénicos cuando se evaluaron por ELISA, MABA e Immunoblotting. Diez moléculas fueron identificadas en los mosquitos hembras y diez en los antígenos de
Bryan, Donna; Silva, Nilupa; Rigsby, Peter; Dougall, Thomas; Corran, Patrick; Bowyer, Paul W; Ho, Mei Mei
At a World Health Organization (WHO) sponsored meeting it was concluded that there is an urgent need for a reference preparation that contains antibodies against malaria antigens in order to support serology studies and vaccine development. It was proposed that this reference would take the form of a lyophilized serum or plasma pool from a malaria-endemic area. In response, an immunoassay standard, comprising defibrinated human plasma has been prepared and evaluated in a collaborative study. A pool of human plasma from a malaria endemic region was collected from 140 single plasma donations selected for reactivity to Plasmodium falciparum apical membrane antigen-1 (AMA-1) and merozoite surface proteins (MSP-1 19 , MSP-1 42 , MSP-2 and MSP-3). This pool was defibrinated, filled and freeze dried into a single batch of ampoules to yield a stable source of naturally occurring antibodies to P. falciparum. The preparation was evaluated by an enzyme-linked immunosorbent assay (ELISA) in a collaborative study with sixteen participants from twelve different countries. This anti-malaria human serum preparation (NIBSC Code: 10/198) was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in October 2014, as the first WHO reference reagent for anti-malaria (Plasmodium falciparum) human serum with an assigned arbitrary unitage of 100 units (U) per ampoule. Analysis of the reference reagent in a collaborative study has demonstrated the benefit of this preparation for the reduction in inter- and intra-laboratory variability in ELISA. Whilst locally sourced pools are regularly use for harmonization both within and between a few laboratories, the presence of a WHO-endorsed reference reagent should enable optimal harmonization of malaria serological assays either by direct use of the reference reagent or calibration of local standards against this WHO reference. The intended uses of this reference reagent, a multivalent preparation, are (1) to allow cross
Ramasamy, R; Ramasamy, M; Yasawardena, S
There is considerable interest in using merozoite proteins in a vaccine against falciparum malaria. Observations that antibodies to merozoite surface proteins block invasion are a basis for optimism. This article draws attention to important and varied aspects of how antibodies to Plasmodium
Liu, Weimin; Li, Yingying; Learn, Gerald H; Rudicell, Rebecca S; Robertson, Joel D; Keele, Brandon F; Ndjango, Jean-Bosco N; Sanz, Crickette M; Morgan, David B; Locatelli, Sabrina; Gonder, Mary K; Kranzusch, Philip J; Walsh, Peter D; Delaporte, Eric; Mpoudi-Ngole, Eitel; Georgiev, Alexander V; Muller, Martin N; Shaw, George M; Peeters, Martine; Sharp, Paul M; Rayner, Julian C; Hahn, Beatrice H
Plasmodium falciparum is the most prevalent and lethal of the malaria parasites infecting humans, yet the origin and evolutionary history of this important pathogen remain controversial. Here we develop a single-genome amplification strategy to identify and characterize Plasmodium spp. DNA sequences in faecal samples from wild-living apes. Among nearly 3,000 specimens collected from field sites throughout central Africa, we found Plasmodium infection in chimpanzees (Pan troglodytes) and western gorillas (Gorilla gorilla), but not in eastern gorillas (Gorilla beringei) or bonobos (Pan paniscus). Ape plasmodial infections were highly prevalent, widely distributed and almost always made up of mixed parasite species. Analysis of more than 1,100 mitochondrial, apicoplast and nuclear gene sequences from chimpanzees and gorillas revealed that 99% grouped within one of six host-specific lineages representing distinct Plasmodium species within the subgenus Laverania. One of these from western gorillas comprised parasites that were nearly identical to P. falciparum. In phylogenetic analyses of full-length mitochondrial sequences, human P. falciparum formed a monophyletic lineage within the gorilla parasite radiation. These findings indicate that P. falciparum is of gorilla origin and not of chimpanzee, bonobo or ancient human origin.
Full Text Available BACKGROUND: Areas endemic for Plasmodium falciparum, hepatitis B virus (HBV and hepatitis C virus (HCV overlap in many parts of sub-Saharan Africa. HBV and HCV infections develop in the liver, where takes place the first development stage of P. falciparum before its further spread in blood. The complex mechanisms involved in the development of hepatitis may potentially influence the development of the liver stage of malaria parasites. Understanding the molecular mechanisms of these interactions could provide new pathophysiological insights for treatment strategies in Malaria. METHODOLOGY: We studied a cohort of 319 individuals living in a village where the three infections are prevalent. The patients were initially given a curative antimalarial treatment and were then monitored for the emergence of asexual P. falciparum forms in blood, fortnightly for one year, by microscopy and polymerase chain reaction. PRINCIPAL FINDINGS: At inclusion, 65 (20.4% subjects had detectable malaria parasites in blood, 36 (11.3% were HBV chronic carriers, and 61 (18.9% were HCV chronic carriers. During follow-up, asexual P. falciparum forms were detected in the blood of 203 patients. The median time to P. falciparum emergence in blood was respectively 140 and 120 days in HBV- and HBV+ individuals, and 135 and 224 days in HCV- and HCV+ individuals. HCV carriage was associated with delayed emergence of asexual P. falciparum forms in blood relative to patients without HCV infection. CONCLUSIONS: This pilot study represents first tentative evidence of a potential epidemiological interaction between HBV, HCV and P. falciparum infections. Age is an important confounding factor in this setting however multivariate analysis points to an interaction between P. falciparum and HCV at the hepatic level with a slower emergence of P. falciparum in HCV chronic carriers. More in depth analysis are necessary to unravel the basis of hepatic interactions between these two pathogens
Feagin, Jean E; Harrell, Maria Isabel; Lee, Jung C; Coe, Kevin J; Sands, Bryan H; Cannone, Jamie J; Tami, Germaine; Schnare, Murray N; Gutell, Robin R
The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. The identification of 14 additional small mitochondrial transcripts from P. falciparum and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered.
Le Roch Karine G
Full Text Available Abstract Background With the sequence of the Plasmodium falciparum genome and several global mRNA and protein life cycle expression profiling projects now completed, elucidating the underlying networks of transcriptional control important for the progression of the parasite life cycle is highly pertinent to the development of new anti-malarials. To date, relatively little is known regarding the specific mechanisms the parasite employs to regulate gene expression at the mRNA level, with studies of the P. falciparum genome sequence having revealed few cis-regulatory elements and associated transcription factors. Although it is possible the parasite may evoke mechanisms of transcriptional control drastically different from those used by other eukaryotic organisms, the extreme AT-rich nature of P. falciparum intergenic regions (~90% AT presents significant challenges to in silico cis-regulatory element discovery. Results We have developed an algorithm called Gene Enrichment Motif Searching (GEMS that uses a hypergeometric-based scoring function and a position-weight matrix optimization routine to identify with high-confidence regulatory elements in the nucleotide-biased and repeat sequence-rich P. falciparum genome. When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from P. falciparum life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative cis-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis. Among these candidates were novel motifs, as well as many of the elements for which biological experimental evidence already exists in the Plasmodium literature. To provide evidence for the biological relevance of a cell invasion-related element predicted by GEMS, reporter gene and electrophoretic mobility shift assays
Full Text Available Malaria is caused by infection with protozoan parasites belonging to the genus Plasmodium, which have arguably exerted the greatest selection pressure on humans in the history of our species. Besides humans, different Plasmodium parasites infect a wide range of animal hosts, from marine invertebrates to primates. On the other hand, individual Plasmodium species show high host specificity. The extraordinary evolution of Plasmodium probably began when a free-living red algae turned parasitic, and culminated with its ability to thrive inside a human red blood cell. Studies on the African apes generated new data on the evolution of malaria parasites in general and the deadliest human-specific species, Plasmodium falciparum, in particular. Initially, it was hypothesized that P. falciparum descended from the chimpanzee malaria parasite P. reichenowi, after the human and the chimp lineage diverged about 6 million years ago. However, a recently identified new species infecting gorillas, unexpectedly showed similarity to P. falciparum and was therefore named P. praefalciparum. That finding spurred an alternative hypothesis, which proposes that P. falciparum descended from its gorilla rather than chimp counterpart. In addition, the gorilla-to-human host shift may have occurred more recently (about 10 thousand years ago than the theoretical P. falciparum-P. reichenowi split. One of the key aims of the studies on Plasmodium evolution is to elucidate the mechanisms that allow the incessant host shifting and retaining the host specificity, especially in the case of human-specific species. Thorough understanding of these phenomena will be necessary to design effective malaria treatment and prevention strategies.
Nancy O Duah
Full Text Available It is important to understand the extent to which genetic factors regulate acquired immunity to common infections. A classical twin study design is useful to estimate the heritable component of variation in measurable immune parameters.This study assessed the relative heritability of different plasma antibody isotypes and subclasses (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE naturally acquired to P. falciparum blood stage antigens AMA1, MSP1-19, MSP2 (two allelic types and MSP3 (two allelic types. Separate analyses were performed on plasma from 213 pairs of Gambian adult twins, 199 child twin pairs sampled in a dry season when there was little malaria transmission, and another set of 107 child twin pairs sampled at the end of the annual wet season when malaria was common. There were significantly positive heritability (h(2 estimates for 48% (20/42 of the specific antibody assays (for the seven isotypes and subclasses to the six antigens tested among the adults, 48% (20/42 among the children in the dry season and 31% (13/42 among the children in the wet season. In children, there were significant heritability estimates for IgG4 reactivity against each of the antigens, and this subclass had higher heritability than the other subclasses and isotypes. In adults, 75% (15/20 of the significantly heritable antigen-specific isotype responses were attributable to non-HLA class II genetic variation, whereas none showed a significant HLA contribution.Genome-wide approaches are now warranted to map the major genetic determinants of variable antibody isotype and subclass responses to malaria, alongside evaluation of their impact on infection and disease. Although plasma levels of IgG4 to malaria antigens are generally low, the exceptionally high heritability of levels of this subclass in children deserves particular investigation.
Simon I Hay
Full Text Available Efficient allocation of resources to intervene against malaria requires a detailed understanding of the contemporary spatial distribution of malaria risk. It is exactly 40 y since the last global map of malaria endemicity was published. This paper describes the generation of a new world map of Plasmodium falciparum malaria endemicity for the year 2007.A total of 8,938 P. falciparum parasite rate (PfPR surveys were identified using a variety of exhaustive search strategies. Of these, 7,953 passed strict data fidelity tests for inclusion into a global database of PfPR data, age-standardized to 2-10 y for endemicity mapping. A model-based geostatistical procedure was used to create a continuous surface of malaria endemicity within previously defined stable spatial limits of P. falciparum transmission. These procedures were implemented within a Bayesian statistical framework so that the uncertainty of these predictions could be evaluated robustly. The uncertainty was expressed as the probability of predicting correctly one of three endemicity classes; previously stratified to be an informative guide for malaria control. Population at risk estimates, adjusted for the transmission modifying effects of urbanization in Africa, were then derived with reference to human population surfaces in 2007. Of the 1.38 billion people at risk of stable P. falciparum malaria, 0.69 billion were found in Central and South East Asia (CSE Asia, 0.66 billion in Africa, Yemen, and Saudi Arabia (Africa+, and 0.04 billion in the Americas. All those exposed to stable risk in the Americas were in the lowest endemicity class (PfPR2-10 5 to or = 40% areas. High endemicity was widespread in the Africa+ region, where 0.35 billion people are at this level of risk. Most of the rest live at intermediate risk (0.20 billion, with a smaller number (0.11 billion at low stable risk.High levels of P. falciparum malaria endemicity are common in Africa. Uniformly low endemic levels are
Hay, Simon I; Guerra, Carlos A; Gething, Peter W; Patil, Anand P; Tatem, Andrew J; Noor, Abdisalan M; Kabaria, Caroline W; Manh, Bui H; Elyazar, Iqbal R F; Brooker, Simon; Smith, David L; Moyeed, Rana A; Snow, Robert W
Efficient allocation of resources to intervene against malaria requires a detailed understanding of the contemporary spatial distribution of malaria risk. It is exactly 40 y since the last global map of malaria endemicity was published. This paper describes the generation of a new world map of Plasmodium falciparum malaria endemicity for the year 2007. A total of 8,938 P. falciparum parasite rate (PfPR) surveys were identified using a variety of exhaustive search strategies. Of these, 7,953 passed strict data fidelity tests for inclusion into a global database of PfPR data, age-standardized to 2-10 y for endemicity mapping. A model-based geostatistical procedure was used to create a continuous surface of malaria endemicity within previously defined stable spatial limits of P. falciparum transmission. These procedures were implemented within a Bayesian statistical framework so that the uncertainty of these predictions could be evaluated robustly. The uncertainty was expressed as the probability of predicting correctly one of three endemicity classes; previously stratified to be an informative guide for malaria control. Population at risk estimates, adjusted for the transmission modifying effects of urbanization in Africa, were then derived with reference to human population surfaces in 2007. Of the 1.38 billion people at risk of stable P. falciparum malaria, 0.69 billion were found in Central and South East Asia (CSE Asia), 0.66 billion in Africa, Yemen, and Saudi Arabia (Africa+), and 0.04 billion in the Americas. All those exposed to stable risk in the Americas were in the lowest endemicity class (PfPR2-10 5 to or = 40%) areas. High endemicity was widespread in the Africa+ region, where 0.35 billion people are at this level of risk. Most of the rest live at intermediate risk (0.20 billion), with a smaller number (0.11 billion) at low stable risk. High levels of P. falciparum malaria endemicity are common in Africa. Uniformly low endemic levels are found in the
Pratt-Riccio, Lilian Rose; Perce-da-Silva, Daiana de Souza; Lima-Junior, Josué da Costa
The genetic diversity displayed by Plasmodium falciparum, the most deadly Plasmodium species, is a significant obstacle for effective malaria vaccine development. In this study, we identified genetic polymorphisms in P. falciparum glutamate-rich protein (GLURP), which is currently being tested in...
Jessica K O'Hara
Full Text Available Nicotinamide adenine dinucleotide (NAD+ is an essential metabolite utilized as a redox cofactor and enzyme substrate in numerous cellular processes. Elevated NAD+ levels have been observed in red blood cells infected with the malaria parasite Plasmodium falciparum, but little is known regarding how the parasite generates NAD+. Here, we employed a mass spectrometry-based metabolomic approach to confirm that P. falciparum lacks the ability to synthesize NAD+ de novo and is reliant on the uptake of exogenous niacin. We characterized several enzymes in the NAD+ pathway and demonstrate cytoplasmic localization for all except the parasite nicotinamidase, which concentrates in the nucleus. One of these enzymes, the P. falciparum nicotinate mononucleotide adenylyltransferase (PfNMNAT, is essential for NAD+ metabolism and is highly diverged from the human homolog, but genetically similar to bacterial NMNATs. Our results demonstrate the enzymatic activity of PfNMNAT in vitro and demonstrate its ability to genetically complement the closely related Escherichia coli NMNAT. Due to the similarity of PfNMNAT to the bacterial enzyme, we tested a panel of previously identified bacterial NMNAT inhibitors and synthesized and screened twenty new derivatives, which demonstrate a range of potency against live parasite culture. These results highlight the importance of the parasite NAD+ metabolic pathway and provide both novel therapeutic targets and promising lead antimalarial compounds.
Bracchi-Ricard, V; Nguyen, K T; Zhou, Y; Rajagopalan, P T; Chakrabarti, D; Pei, D
Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Until recently, PDF has been thought as an enzyme unique to the bacterial kingdom. Searches of the genomic DNA databases identified several genes that encode proteins of high sequence homology to bacterial PDF from eukaryotic organisms. The cDNA encoding Plasmodium falciparum PDF (PfPDF) has been cloned and overexpressed in Escherichia coli. The recombinant protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is inhibited by specific PDF inhibitors. Western blot analysis indicated expression of mature PfPDF in trophozoite, schizont, and segmenter stages of intraerythrocytic development. These results provide strong evidence that a functional PDF is present in P. falciparum. In addition, PDF inhibitors inhibited the growth of P. falciparum in the intraerythrocytic culture. (c)2001 Elsevier Science.
Mok, Sachel; Liong, Kek-Yee; Lim, Eng-How; Huang, Ximei; Zhu, Lei; Preiser, Peter Rainer; Bozdech, Zbynek
Drug resistance in Plasmodium falciparum remains a challenge for the malaria eradication programmes around the world. With the emergence of artemisinin resistance, the efficacy of the partner drugs in the artemisinin combination therapies (ACT) that include quinoline-based drugs is becoming critical. So far only few resistance markers have been identified from which only two transmembrane transporters namely PfMDR1 (an ATP-binding cassette transporter) and PfCRT (a drug-metabolite transporter) have been experimentally verified. Another P. falciparum transporter, the ATP-binding cassette containing multidrug resistance-associated protein (PfMRP2) represents an additional possible factor of drug resistance in P. falciparum. In this study, we identified a parasite clone that is derived from the 3D7 P. falciparum strain and shows increased resistance to chloroquine, mefloquine and quinine through the trophozoite and schizont stages. We demonstrate that the resistance phenotype is caused by a 4.1 kb deletion in the 5′ upstream region of the pfmrp2 gene that leads to an alteration in the pfmrp2 transcription and thus increased level of PfMRP2 protein. These results also suggest the importance of putative promoter elements in regulation of gene expression during the P. falciparum intra-erythrocytic developmental cycle and the potential of genetic polymorphisms within these regions to underlie drug resistance. PMID:24372851
Choveaux David L
Full Text Available Abstract Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369, containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds.
Tan, Lii Lian; Lau, Tiek Ying; Timothy, William; Prabakaran, Dhanaraj
Chloroquine resistance (CQR) in falciparum malaria was identified to be associated with several mutations in the chloroquine resistance transporter gene (pfcrt) that encodes the transmembrane transporter in digestive vacuole membrane of the parasite. This study aimed to investigate the point mutations across the full-length pfcrt in Plasmodium falciparum isolates in Sabah, Malaysia. A total of 31 P. falciparum positive samples collected from Keningau, Kota Kinabalu, and Kudat, Sabah, were analyzed. pfcrt was PCR amplified and cloned prior to sequence analysis. This study showed that all the previously described 10 point mutations associated with CQR at codons 72, 74, 75, 76, 97, 220, 271, 326, 356, and 371 were found with different prevalence. Besides, two novel point mutations, I166V and H273N, were identified with 22.5% and 19.3%, respectively. Three haplotypes, namely, CVMNK (29%), CVIET (3.2%), and SVMNT (67.7%), were identified. High prevalence of SVMNT among P. falciparum isolates from Sabah showed that these isolates are closer to the P. falciparum isolates from Papua New Guinea rather than to the more proximal Southeast Asian CVIET haplotype. Full-length analysis of pfcrt showed that chloroquine resistant P. falciparum in Sabah is still prevalent despite the withdrawal of chloroquine usage since 1979.
Gething Peter W
Full Text Available Abstract Background Transmission intensity affects almost all aspects of malaria epidemiology and the impact of malaria on human populations. Maps of transmission intensity are necessary to identify populations at different levels of risk and to evaluate objectively options for disease control. To remain relevant operationally, such maps must be updated frequently. Following the first global effort to map Plasmodium falciparum malaria endemicity in 2007, this paper describes the generation of a new world map for the year 2010. This analysis is extended to provide the first global estimates of two other metrics of transmission intensity for P. falciparum that underpin contemporary questions in malaria control: the entomological inoculation rate (PfEIR and the basic reproductive number (PfR. Methods Annual parasite incidence data for 13,449 administrative units in 43 endemic countries were sourced to define the spatial limits of P. falciparum transmission in 2010 and 22,212 P. falciparum parasite rate (PfPR surveys were used in a model-based geostatistical (MBG prediction to create a continuous contemporary surface of malaria endemicity within these limits. A suite of transmission models were developed that link PfPR to PfEIR and PfR and these were fitted to field data. These models were combined with the PfPR map to create new global predictions of PfEIR and PfR. All output maps included measured uncertainty. Results An estimated 1.13 and 1.44 billion people worldwide were at risk of unstable and stable P. falciparum malaria, respectively. The majority of the endemic world was predicted with a median PfEIR of less than one and a median PfRc of less than two. Values of either metric exceeding 10 were almost exclusive to Africa. The uncertainty described in both PfEIR and PfR was substantial in regions of intense transmission. Conclusions The year 2010 has a particular significance as an evaluation milestone for malaria global health policy. The
Lindsey S Garver
Full Text Available Immune responses mounted by the malaria vector Anopheles gambiae are largely regulated by the Toll and Imd (immune deficiency pathways via the NF-kappaB transcription factors Rel1 and Rel2, which are controlled by the negative regulators Cactus and Caspar, respectively. Rel1- and Rel2-dependent transcription in A. gambiae has been shown to be particularly critical to the mosquito's ability to manage infection with the rodent malaria parasite Plasmodium berghei. Using RNA interference to deplete the negative regulators of these pathways, we found that Rel2 controls resistance of A. gambiae to the human malaria parasite Plasmodium falciparum, whereas Rel 1 activation reduced infection levels. The universal relevance of this defense system across Anopheles species was established by showing that caspar silencing also prevents the development of P. falciparum in the major malaria vectors of Asia and South America, A. stephensi and A. albimanus, respectively. Parallel studies suggest that while Imd pathway activation is most effective against P. falciparum, the Toll pathway is most efficient against P. berghei, highlighting a significant discrepancy between the human pathogen and its rodent model. High throughput gene expression analyses identified a plethora of genes regulated by the activation of the two Rel factors and revealed that the Toll pathway played a more diverse role in mosquito biology than the Imd pathway, which was more immunity-specific. Further analyses of key anti-Plasmodium factors suggest they may be responsible for the Imd pathway-mediated resistance phenotype. Additionally, we found that the fitness cost caused by Rel2 activation through caspar gene silencing was undetectable in sugar-fed, blood-fed, and P. falciparum-infected female A. gambiae, while activation of the Toll pathway's Rel1 had a major impact. This study describes for the first time a single gene that influences an immune mechanism that is able to abort
Ellekvist, Peter; Ricke, Christina Høier; Litman, Thomas
concentrations of K(+) when inside the erythrocyte and low concentrations when in plasma. In the recently published genome of P. falciparum, we have identified a gene, pfkch1, encoding a potential K(+) channel, which to some extent resembles the big-conductance (BK) K(+) channel. We have cloned the approximately......In most living cells, K(+) channels are important for the generation of the membrane potential and for volume regulation. The parasite Plasmodium falciparum, which causes malignant malaria, must be able to deal with large variations in the ambient K(+) concentration: it is exposed to high...... could be a potential drug target....
Full Text Available Abstract The protozoan Plasmodium falciparum has a complex life cycle in which asexual multiplication in the vertebrate host alternates with an obligate sexual reproduction in the anopheline mosquito. Apart from the apparent recombination advantages conferred by sex, P. falciparum has evolved a remarkable biology and adaptive phenotypes to insure its transmission despite the dangers of sex. This review mainly focuses on the current knowledge on commitment to sexual development, gametocytogenesis and the evolutionary significance of various aspects of gametocyte biology. It goes further than pure biology to look at the strategies used to improve successful transmission. Although gametocytes are inevitable stages for transmission and provide a potential target to fight malaria, they have received less attention than the pathogenic asexual stages. There is a need for research on gametocytes, which are a fascinating stage, responsible to a large extent for the success of P. falciparum.
Wanaguru, Madushi; Liu, Weimin; Hahn, Beatrice H; Rayner, Julian C; Wright, Gavin J
Plasmodium falciparum, the cause of almost all human malaria mortality, is a member of the Laverania subgenus which infects African great apes. Interestingly, Laverania parasites exhibit strict host specificity in their natural environment: P. reichenowi, P. billcollinsi, and P. gaboni infect only chimpanzees; P. praefalciparum, P. blacklocki, and P. adleri are restricted to gorillas, and P. falciparum is pandemic in humans. The molecular mechanism(s) responsible for these host restrictions are not understood, although the interaction between the parasite blood-stage invasion ligand EBA175 and the host erythrocyte receptor Glycophorin-A (GYPA) has been implicated previously. We reexamined the role of the EBA175-GYPA interaction in host tropism using recombinant proteins and biophysical assays and found that EBA175 orthologs from the chimpanzee-restricted parasites P. reichenowi and P. billcollinsi both bound to human GYPA with affinities similar to that of P. falciparum, suggesting that the EBA175-GYPA interaction is unlikely to be the sole determinant of Laverania host specificity. We next investigated the contribution of the recently discovered Reticulocyte-binding protein Homolog 5 (RH5)-Basigin (BSG) interaction in host-species selectivity and found that P. falciparum RH5 bound chimpanzee BSG with a significantly lower affinity than human BSG and did not bind gorilla BSG, mirroring the known host tropism of P. falciparum. Using site-directed mutagenesis, we identified residues in BSG that are responsible for the species specificity of PfRH5 binding. Consistent with the essential role of the PfRH5-BSG interaction in erythrocyte invasion, we conclude that species-specific differences in the BSG receptor provide a molecular explanation for the restriction of P. falciparum to its human host.
Abba, Katharine; Kirkham, Amanda J; Olliaro, Piero L; Deeks, Jonathan J; Donegan, Sarah; Garner, Paul; Takwoingi, Yemisi
In settings where both Plasmodium vivax and Plasmodium falciparum infection cause malaria, rapid diagnostic tests (RDTs) need to distinguish which species is causing the patients' symptoms, as different treatments are required. Older RDTs incorporated two test lines to distinguish malaria due to P. falciparum, from malaria due to any other Plasmodium species (non-falciparum). These RDTs can be classified according to which antibodies they use: Type 2 RDTs use HRP-2 (for P. falciparum) and aldolase (all species); Type 3 RDTs use HRP-2 (for P. falciparum) and pLDH (all species); Type 4 use pLDH (fromP. falciparum) and pLDH (all species).More recently, RDTs have been developed to distinguish P. vivax parasitaemia by utilizing a pLDH antibody specific to P. vivax. To assess the diagnostic accuracy of RDTs for detecting non-falciparum or P. vivax parasitaemia in people living in malaria-endemic areas who present to ambulatory healthcare facilities with symptoms suggestive of malaria, and to identify which types and brands of commercial test best detect non-falciparum and P. vivax malaria. We undertook a comprehensive search of the following databases up to 31 December 2013: Cochrane Infectious Diseases Group Specialized Register; MEDLINE; EMBASE; MEDION; Science Citation Index; Web of Knowledge; African Index Medicus; LILACS; and IndMED. Studies comparing RDTs with a reference standard (microscopy or polymerase chain reaction) in blood samples from a random or consecutive series of patients attending ambulatory health facilities with symptoms suggestive of malaria in non-falciparum endemic areas. For each study, two review authors independently extracted a standard set of data using a tailored data extraction form. We grouped comparisons by type of RDT (defined by the combinations of antibodies used), and combined in meta-analysis where appropriate. Average sensitivities and specificities are presented alongside 95% confidence intervals (95% CI). We included 47 studies
Abba, Katharine; Kirkham, Amanda J; Olliaro, Piero L; Deeks, Jonathan J; Donegan, Sarah; Garner, Paul; Takwoingi, Yemisi
Background In settings where both Plasmodium vivax and Plasmodium falciparum infection cause malaria, rapid diagnostic tests (RDTs) need to distinguish which species is causing the patients' symptoms, as different treatments are required. Older RDTs incorporated two test lines to distinguish malaria due to P. falciparum, from malaria due to any other Plasmodium species (non-falciparum). These RDTs can be classified according to which antibodies they use: Type 2 RDTs use HRP-2 (for P. falciparum) and aldolase (all species); Type 3 RDTs use HRP-2 (for P. falciparum) and pLDH (all species); Type 4 use pLDH (fromP. falciparum) and pLDH (all species). More recently, RDTs have been developed to distinguish P. vivax parasitaemia by utilizing a pLDH antibody specific to P. vivax. Objectives To assess the diagnostic accuracy of RDTs for detecting non-falciparum or P. vivax parasitaemia in people living in malaria-endemic areas who present to ambulatory healthcare facilities with symptoms suggestive of malaria, and to identify which types and brands of commercial test best detect non-falciparum and P. vivax malaria. Search methods We undertook a comprehensive search of the following databases up to 31 December 2013: Cochrane Infectious Diseases Group Specialized Register; MEDLINE; EMBASE; MEDION; Science Citation Index; Web of Knowledge; African Index Medicus; LILACS; and IndMED. Selection criteria Studies comparing RDTs with a reference standard (microscopy or polymerase chain reaction) in blood samples from a random or consecutive series of patients attending ambulatory health facilities with symptoms suggestive of malaria in non-falciparum endemic areas. Data collection and analysis For each study, two review authors independently extracted a standard set of data using a tailored data extraction form. We grouped comparisons by type of RDT (defined by the combinations of antibodies used), and combined in meta-analysis where appropriate. Average sensitivities and
IgG antibodies to endothelial protein C receptor-binding Cysteine-rich interdomain region domains of Plasmodium falciparum erythrocyte membrane protein 1 are acquired early in life in individuals exposed to malaria
Turner, Louise; Lavstsen, Thomas; Mmbando, Bruno P
Severe malaria syndromes are precipitated by Plasmodium falciparum parasites binding to endothelial receptors on the vascular lining. This binding is mediated by members of the highly variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. We have previously identified a subset of Pf...
Background Plasmodium falciparum malaria remains a leading health problem in Africa and its control is seriously challenged by drug resistance. Although resistance to the sulphadoxine-pyrimethamine (SP) is widespread, this combination remains an important component of malaria control programmes as intermittent preventive therapy (IPT) for pregnant women and children. In Angola, resistance patterns have been poorly characterized, and IPT has been employed for pregnant women since 2006. The aim of this study was to assess the prevalence of key antifolate resistance mediating polymorphisms in the pfdhfr and pfdhps genes in P. falciparum samples from Angola. Methods Plasmodium falciparum samples collected in Luanda, in 2007, were genotyped by amplification and DNA forward and reverse sequencing of the pfdhfr and pfdhps genes. Results The most prevalent polymorphisms identified were pfdhfr 108N (100%), 51I (93%), 59R (57%) and pfdhps 437G (93%). Resistance-mediating polymorphisms in pfdhps less commonly observed in West Africa were also identified (540E in 10%, 581G in 7% of samples). Conclusion This study documents an important prevalence of 4 P. falciparum polymorphisms that predicts an antifolate resistance in Luanda. Further, some samples presented additional mutations associated to high-level resistance. These results suggest that the use of SP for IPT may no longer be warranted in Angola. PMID:21864379
The present study was carried out to investigate the relationship between blood group types and P. falciparum malaria, as well as malaria preventive measures. The venous blood specimens were collected, processed, Giemsa-stained and examined microscopically. ABO groups were determined by agglutination test using ...
Their platelet counts were evaluated using the auto-analyser Sysmex KX-21N. Results: The overall prevalence of thrombocytopenia was 5.0%, but it was higher in children with severe malaria. None of the children in the control group had thrombocytopenia. Conclusion: The prevalence of thrombocytopenia in falciparum ...
Pukrittayakamee, S.; Krishna, S.; ter Kuile, F.; Wilaiwan, O.; Williamson, D. H.; White, N. J.
We investigated the integrity of the gluconeogenic pathway in severe malaria using alanine metabolism as a measure. Alanine disposition and liver blood flow, assessed by indocyanine green (ICG) clearance, were measured simultaneously in 10 patients with falciparum malaria (six severe and four
Plasmodium falciparum malaria is one of the most important parasitic diseases affecting sub-Saharan Africa, despite the availability of interventions. It exerts tremendous socio-economic and medical burden on the continent, particularly in under five children and pregnant women. In this review, we have attempted to ...
Resistance patterns of plasmodium falciparum malaria to chloroquine in Kampala, Uganda. ... Sixty three (65.6%) patients showed clinical improvement, 29 (30.2%) deteriorated and four (4.2%) had no change. Adequate parasitogical response was seen in 71 (74 %), moderate in four (4.2%) and poor in 21 (21.8%) patients.
Microthrombotic complications are the best described; however, a number of cases of thrombosis involving larger vessels have been published in the literature. Herein, we describe the case of a woman with malaria associated with massive pulmonary embolism. Keywords: Falciparum, malaria, pulmonary embolism ...
Agrawal, Sonia; Moser, Kara A; Morton, Lindsay; Cummings, Michael P; Parihar, Ankita; Dwivedi, Ankit; Shetty, Amol C; Drabek, Elliott F; Jacob, Christopher G; Henrich, Philipp P; Parobek, Christian M; Jongsakul, Krisada; Huy, Rekol; Spring, Michele D; Lanteri, Charlotte A; Chaorattanakawee, Suwanna; Lon, Chanthap; Fukuda, Mark M; Saunders, David L; Fidock, David A; Lin, Jessica T; Juliano, Jonathan J; Plowe, Christopher V; Silva, Joana C; Takala-Harrison, Shannon
Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine. Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset. Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity. Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: email@example.com.
Full Text Available BACKGROUND: Mortality from severe pediatric falciparum malaria appears low in Oceania but Plasmodium vivax is increasingly recognized as a cause of complications and death. The features and prognosis of mixed Plasmodium species infections are poorly characterized. Detailed prospective studies that include accurate malaria diagnosis and detection of co-morbidities are lacking. METHODS AND FINDINGS: We followed 340 Papua New Guinean (PNG children with PCR-confirmed severe malaria (77.1% P. falciparum, 7.9% P. vivax, 14.7% P. falciparum/vivax hospitalized over a 3-year period. Bacterial cultures were performed to identify co-incident sepsis. Clinical management was under national guidelines. Of 262 children with severe falciparum malaria, 30.9%, 24.8% and 23.2% had impaired consciousness, severe anemia, and metabolic acidosis/hyperlactatemia, respectively. Two (0.8% presented with hypoglycemia, seven (2.7% were discharged with neurologic impairment, and one child died (0.4%. The 27 severe vivax malaria cases presented with similar phenotypic features to the falciparum malaria cases but respiratory distress was five times more common (P=0.001; one child died (3.7%. The 50 children with P. falciparum/vivax infections shared phenotypic features of mono-species infections, but were more likely to present in deep coma and had the highest mortality (8.0%; P=0.003 vs falciparum malaria. Overall, bacterial cultures were positive in only two non-fatal cases. 83.6% of the children had alpha-thalassemia trait and seven with coma/impaired consciousness had South Asian ovalocytosis (SAO. CONCLUSIONS: The low mortality from severe falciparum malaria in PNG children may reflect protective genetic factors other than alpha-thalassemia trait/SAO, good nutrition, and/or infrequent co-incident sepsis. Severe vivax malaria had similar features but severe P. falciparum/vivax infections were associated with the most severe phenotype and worst prognosis.
Associations between an IgG3 polymorphism in the binding domain for FcRn, transplacental transfer of malaria-specific IgG3, and protection against Plasmodium falciparum malaria during infancy: A birth cohort study in Benin.
Full Text Available Transplacental transfer of maternal immunoglobulin G (IgG to the fetus helps to protect against malaria and other infections in infancy. Recent studies have emphasized the important role of malaria-specific IgG3 in malaria immunity, and its transfer may reduce the risk of malaria in infancy. Human IgGs are actively transferred across the placenta by binding the neonatal Fc receptor (FcRn expressed within the endosomes of the syncytiotrophoblastic membrane. Histidine at position 435 (H435 provides for optimal Fc-IgG binding. In contrast to other IgG subclasses, IgG3 is highly polymorphic and usually contains an arginine at position 435, which reduces its binding affinity to FcRn in vitro. The reduced binding to FcRn is associated with reduced transplacental transfer and reduced half-life of IgG3 in vivo. Some haplotypes of IgG3 have histidine at position 435. This study examines the hypotheses that the IgG3-H435 variant promotes increased transplacental transfer of malaria-specific antibodies and a prolonged IgG3 half-life in infants and that its presence correlates with protection against clinical malaria during infancy.In Benin, 497 mother-infant pairs were included in a longitudinal birth cohort. Both maternal and cord serum samples were assayed for levels of IgG1 and IgG3 specific for MSP119, MSP2 (both allelic families, 3D7 and FC27, MSP3, GLURP (both regions, R0 and R2, and AMA1 antigens of Plasmodium falciparum. Cord:maternal ratios were calculated. The maternal IgG3 gene was sequenced to identify the IgG3-H435 polymorphism. A multivariate logistic regression was used to examine the association between maternal IgG3-H435 polymorphism and transplacental transfer of IgG3, adjusting for hypergammaglobulinemia, maternal malaria, and infant malaria exposure. Twenty-four percent of Beninese women living in an area highly endemic for malaria had the IgG3-H435 allele (377 women homozygous for the IgG3-R435 allele, 117 women heterozygous for the Ig
Full Text Available Abstract Background Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes. The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusion This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.
Shivendra G. Tewari
Full Text Available Chloroquine, long the default first-line treatment against malaria, is now abandoned in large parts of the world because of widespread drug-resistance in Plasmodium falciparum. In spite of its importance as a cost-effective and efficient drug, a coherent understanding of the cellular mechanisms affected by chloroquine and how they influence the fitness and survival of the parasite remains elusive. Here, we used a systems biology approach to integrate genome-scale transcriptomics to map out the effects of chloroquine, identify targeted metabolic pathways, and translate these findings into mechanistic insights. Specifically, we first developed a method that integrates transcriptomic and metabolomic data, which we independently validated against a recently published set of such data for Krebs-cycle mutants of P. falciparum. We then used the method to calculate the effect of chloroquine treatment on the metabolic flux profiles of P. falciparum during the intraerythrocytic developmental cycle. The model predicted dose-dependent inhibition of DNA replication, in agreement with earlier experimental results for both drug-sensitive and drug-resistant P. falciparum strains. Our simulations also corroborated experimental findings that suggest differences in chloroquine sensitivity between ring- and schizont-stage P. falciparum. Our analysis also suggests that metabolic fluxes that govern reduced thioredoxin and phosphoenolpyruvate synthesis are significantly decreased and are pivotal to chloroquine-based inhibition of P. falciparum DNA replication. The consequences of impaired phosphoenolpyruvate synthesis and redox metabolism are reduced carbon fixation and increased oxidative stress, respectively, both of which eventually facilitate killing of the parasite. Our analysis suggests that a combination of chloroquine (or an analogue and another drug, which inhibits carbon fixation and/or increases oxidative stress, should increase the clearance of P. falciparum
Molina-Cruz, Alvaro; Zilversmit, Martine M; Neafsey, Daniel E; Hartl, Daniel L; Barillas-Mury, Carolina
Plasmodium falciparum malaria remains a devastating public health problem. Recent discoveries have shed light on the origin and evolution of Plasmodium parasites and their interactions with their vertebrate and mosquito hosts. P. falciparum malaria originated in Africa from a single horizontal transfer between an infected gorilla and a human, and became global as the result of human migration. Today, P. falciparum malaria is transmitted worldwide by more than 70 different anopheline mosquito species. Recent studies indicate that the mosquito immune system can be a barrier to malaria transmission and that the P. falciparum Pfs47 gene allows the parasite to evade mosquito immune detection. Here, we review the origin and globalization of P. falciparum and integrate this history with analysis of the biology, evolution, and dispersal of the main mosquito vectors. This new perspective broadens our understanding of P. falciparum population structure and the dispersal of important parasite genetic traits.
Tamar E Carter
Full Text Available Hispaniola, comprising Haiti and the Dominican Republic, has been identified as a candidate for malaria elimination. However, incomplete surveillance data in Haiti hamper efforts to assess the impact of ongoing malaria control interventions. Characteristics of the genetic diversity of Plasmodium falciparum populations can be used to assess parasite transmission, which is information vital to evaluating malaria elimination efforts. Here we characterize the genetic diversity of P. falciparum samples collected from patients at seven sites in Haiti using 12 microsatellite markers previously employed in population genetic analyses of global P. falciparum populations. We measured multiplicity of infections, level of genetic diversity, degree of population geographic substructure, and linkage disequilibrium (defined as non-random association of alleles from different loci. For low transmission populations like Haiti, we expect to see few multiple infections, low levels of genetic diversity, high degree of population structure, and high linkage disequilibrium. In Haiti, we found low levels of multiple infections (12.9%, moderate to high levels of genetic diversity (mean number of alleles per locus = 4.9, heterozygosity = 0.61, low levels of population structure (highest pairwise Fst = 0.09 and no clustering in principal components analysis, and moderate linkage disequilibrium (ISA = 0.05, P<0.0001. In addition, population bottleneck analysis revealed no evidence for a reduction in the P. falciparum population size in Haiti. We conclude that the high level of genetic diversity and lack of evidence for a population bottleneck may suggest that Haiti's P. falciparum population has been stable and discuss the implications of our results for understanding the impact of malaria control interventions. We also discuss the relevance of parasite population history and other host and vector factors when assessing transmission intensity from genetic diversity data.
Rougeron, Virginie; Tiedje, Kathryn E; Chen, Donald S; Rask, Thomas S; Gamboa, Dionicia; Maestre, Amanda; Musset, Lise; Legrand, Eric; Noya, Oscar; Yalcindag, Erhan; Renaud, François; Prugnolle, Franck; Day, Karen P
Strong founder effects resulting from human migration out of Africa have led to geographic variation in single nucleotide polymorphisms (SNPs) and microsatellites (MS) of the malaria parasite, Plasmodium falciparum . This is particularly striking in South America where two major founder populations of P. falciparum have been identified that are presumed to have arisen from the transatlantic slave trade. Given the importance of the major variant surface antigen of the blood stages of P. falciparum as both a virulence factor and target of immunity, we decided to investigate the population genetics of the genes encoding " Plasmodium falciparum Erythrocyte Membrane Protein 1" ( Pf EMP1) among several countries in South America, in order to evaluate the transmission patterns of malaria in this continent. Deep sequencing of the DBLα domain of var genes from 128 P. falciparum isolates from five locations in South America was completed using a 454 high throughput sequencing protocol. Striking geographic variation in var DBLα sequences, similar to that seen for SNPs and MS markers, was observed. Colombia and French Guiana had distinct var DBLα sequences, whereas Peru and Venezuela showed an admixture. The importance of such geographic variation to herd immunity and malaria vaccination is discussed.
Carter, Tamar E.; Boulter, Alexis; Existe, Alexandre; Romain, Jean R.; St. Victor, Jean Yves; Mulligan, Connie J.; Okech, Bernard A.
Antimalarial drugs are a key tool in malaria elimination programs. With the emergence of artemisinin resistance in southeast Asia, an effort to identify molecular markers for surveillance of resistant malaria parasites is underway. Non-synonymous mutations in the kelch propeller domain (K13-propeller) in Plasmodium falciparum have been associated with artemisinin resistance in samples from southeast Asia, but additional studies are needed to characterize this locus in other P. falciparum populations with different levels of artemisinin use. Here, we sequenced the K13-propeller locus in 82 samples from Haiti, where limited government oversight of non-governmental organizations may have resulted in low-level use of artemisinin-based combination therapies. We detected a single-nucleotide polymorphism (SNP) at nucleotide 1,359 in a single isolate. Our results contribute to our understanding of the global genomic diversity of the K13-propeller locus in P. falciparum populations. PMID:25646258
Full Text Available Abstract: Malaria remains a major cause of morbidity and mortality in India. Plasmodium falciparum remains the main culprit although cases with vivax malaria are on the rise. Severe malaria as defined by the WHO criteria has high rate of complications and mortality. In our study we recruited microscopy positive falciparum and vivax malaria patients. Haematological and biochemical laboratory investigations were carried out in recruited patients. Both parameters were found to be significantly derailed in falciparum cases as compared to vivax. A direct correlation has been observed between kidney function tests (serum creatinine,serum urea and direct bilirubin levels across all cases of malaria. Hence these parameters can be used to identify and monitor the progress of cases of severe malaria as significant proportion of patients fulfilled the criteria of severe malaria in the cohort.
Abdi, Abdirahman; Yu, Lu; Goulding, David; Rono, Martin K.; Bejon, Philip; Choudhary, Jyoti; Rayner, Julian
Background: Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. Methods: Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate that had been adapted to in vitro culture for a relatively shorter period, and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). Results: We show that P. falciparum extracellular vesicles ( PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer’s clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that PfEVs may play a role in parasite-host interactions. Comparison of this dataset with previously published datasets helps to define a core secretome present in PfEVs. Conclusions: P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify new targets for interventions or diagnostics. PMID:28944300
Lilburn Timothy G
Full Text Available Abstract Background Malaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome in the malaria parasite Plasmodium falciparum and its sibling species 123, providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum. Results We investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database 4, and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H system 5, blood stage microarray experiments 678, proteomics 9101112, literature text mining, and sequence homology analysis. Seventy-seven (77 out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs. These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins, range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide
Artemisinin derivatives constitute a key component of the present-day treatment for Plasmodium falciparum malaria. Resistance with artemisinins is generally associated with S769N point mutation in the sarco-endoplasmic reticulumdependant ATPase6 (SERCA ATPase6) gene of Plasmodium falciparum, few studies have ...
Chloroquine is still a first-line antimalarial drug in uncomplicated falciparum malaria. Increasing resistance to chloroquine has been reported in many parts of Nigeria. Clinical and parasitological responses and classes of resistance to chloroquine in falciparum malaria in five communities in Delta region, southern Nigeria ...
Saito, Fumiji; Hirayasu, Kouyuki; Satoh, Takeshi
, but the immune regulatory mechanisms used by P. falciparum remain largely unknown. Here we show that P. falciparum uses immune inhibitory receptors to achieve immune evasion. RIFIN proteins are products of a polymorphic multigene family comprising approximately 150-200 genes per parasite genome...
Infectious Diseases and Environmental Health Research Group, Department of ... Conclusion: There is high genetic diversity in MSP – 2 and GLURP allelic families of Plasmodium falciparum isolates from Okelele. Health Centre, Ilorin, Nigeria. Keywords: Plasmodium falciparum, Merozoite Surface Protein, genetic diversity.
Background: Plasmodium falciparum, the most dangerous malaria parasite species to humans remains an important public health concern in Okelele, a rural community in Ilorin, Kwara State, Nigeria. There is however little information about the genetic diversity of Plasmodium falciparum in Nigeria. Objective: To determine ...
Malaria during pregnancy poses a substantial risk to mother and foetus especially an infection with Plasmodium falciparum. This study was undertaken to assess the prevalence of falciparum malaria among pregnant women in Aba South Local Government Area, Abia State, south-east Nigeria. Blood samples from 432 ...
McGready, R.; Brockman, A.; Cho, T.; Cho, D.; van Vugt, M.; Luxemburger, C.; Chongsuphajaisiddhi, T.; White, N. J.; Nosten, F.
Since no effective malaria prevention measures have been identified for pregnant women living on the western border of Thailand, prompt diagnosis and efficient treatment are paramount, although drug resistance in Plasmodium falciparum has narrowed the treatment options. An open randomized comparison
Taylor, B.J.; Lanke, K.; Banman, S.L.; Morlais, I.; Morin, M.J.; Bousema, T.; Rijpma, S.R.; Yanow, S.K.
We describe a novel one-step reverse transcriptase real-time PCR (direct RT-PCR) for Plasmodium falciparum malaria parasites that amplifies RNA targets directly from blood. We developed the assay to identify gametocyte-specific transcripts in parasites from patient blood samples, as a means of
Wijayalath, Wathsala; Majji, Sai; Villasante, Eileen F; Brumeanu, Teodor D; Richie, Thomas L; Casares, Sofia
Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored. Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes. Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3-5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge. DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates
Oppenheim, Sara J; Rosenfeld, Jeffrey A; DeSalle, Rob
The persistent and growing gap between the availability of sequenced genomes and the ability to assign functions to sequenced genes led us to explore ways to maximize the information content of automated annotation for studies of anopheline mosquitos. Specifically, we use genome content analysis of a large number of previously sequenced anopheline mosquitos to follow the loss and gain of protein families over the evolutionary history of this group. The importance of this endeavor lies in the potential for comparative genomic studies between Anopheles and closely related non-vector species to reveal ancestral genome content dynamics involved in vector competence. In addition, comparisons within Anopheles could identify genome content changes responsible for variation in the vectorial capacity of this family of important parasite vectors. The competence and capacity of P. falciparum vectors do not appear to be phylogenetically constrained within the Anophelinae. Instead, using ancestral reconstruction methods, we suggest that a previously unexamined component of vector biology, anopheline nucleotide metabolism, may contribute to the unique status of anophelines as P. falciparum vectors. While the fitness effects of nucleotide co-option by P. falciparum parasites on their anopheline hosts are not yet known, our results suggest that anopheline genome content may be responding to selection pressure from P. falciparum. Whether this response is defensive, in an attempt to redress improper nucleotide balance resulting from P. falciparum infection, or perhaps symbiotic, resulting from an as-yet-unknown mutualism between anophelines and P. falciparum, is an open question that deserves further study. Clearly, there is a wealth of functional information to be gained from detailed manual genome annotation, yet the rapid increase in the number of available sequences means that most researchers will not have the time or resources to manually annotate all the sequence data they
Wanaguru, Madushi; Liu, Weimin; Hahn, Beatrice H.; Rayner, Julian C.; Wright, Gavin J.
Plasmodium falciparum, the cause of almost all human malaria mortality, is a member of the Laverania subgenus which infects African great apes. Interestingly, Laverania parasites exhibit strict host specificity in their natural environment: P. reichenowi, P. billcollinsi, and P. gaboni infect only chimpanzees; P. praefalciparum, P. blacklocki, and P. adleri are restricted to gorillas, and P. falciparum is pandemic in humans. The molecular mechanism(s) responsible for these host restrictions are not understood, although the interaction between the parasite blood-stage invasion ligand EBA175 and the host erythrocyte receptor Glycophorin-A (GYPA) has been implicated previously. We reexamined the role of the EBA175–GYPA interaction in host tropism using recombinant proteins and biophysical assays and found that EBA175 orthologs from the chimpanzee-restricted parasites P. reichenowi and P. billcollinsi both bound to human GYPA with affinities similar to that of P. falciparum, suggesting that the EBA175–GYPA interaction is unlikely to be the sole determinant of Laverania host specificity. We next investigated the contribution of the recently discovered Reticulocyte-binding protein Homolog 5 (RH5)–Basigin (BSG) interaction in host-species selectivity and found that P. falciparum RH5 bound chimpanzee BSG with a significantly lower affinity than human BSG and did not bind gorilla BSG, mirroring the known host tropism of P. falciparum. Using site-directed mutagenesis, we identified residues in BSG that are responsible for the species specificity of PfRH5 binding. Consistent with the essential role of the PfRH5–BSG interaction in erythrocyte invasion, we conclude that species-specific differences in the BSG receptor provide a molecular explanation for the restriction of P. falciparum to its human host. PMID:24297912
Whitman, Timothy J; Coyne, Philip E; Magill, Alan J; Blazes, David L; Green, Michael D; Milhous, Wilbur K; Burgess, Timothy H; Freilich, Daniel; Tasker, Sybil A; Azar, Ramzy G; Endy, Timothy P; Clagett, Christopher D; Deye, Gregory A; Shanks, G Dennis; Martin, Gregory J
In 2003, 44 U.S. Marines were evacuated from Liberia with either confirmed or presumed Plasmodium falciparum malaria. An outbreak investigation showed that only 19 (45%) used insect repellent, 5 (12%) used permethrin-treated clothing, and none used bed netting. Adherence with weekly mefloquine (MQ) was reported by 23 (55%). However, only 4 (10%) had serum MQ levels high enough to correlate with protection (> 794 ng/mL), and 9 (22%) had evidence of steady-state kinetics (MQ carboxy metabolite/MQ > 3.79). Tablets collected from Marines met USP identity and dissolution specifications for MQ. Testing failed to identify P. falciparum isolates with MQ resistance. This outbreak resulted from under use of personal protective measures and inadequate adherence with chemophrophylaxis. It is essential that all international travelers make malaria prevention measures a priority, especially when embarking to regions of the world with high transmission intensity such as west Africa..
Lai, Shengjie; Wardrop, Nicola A.; Huang, Zhuojie; Bosco, Claudio; Sun, Junling; Bird, Tomas; Wesolowski, Amy; Zhou, Sheng; Zhang, Qian; Zheng, Canjun; Li, Zhongjie; Tatem, Andrew J.; Yu, Hongjie
Plasmodium falciparum malaria importation from Africa to China is rising with increasing Chinese overseas investment and international travel. Identifying networks and drivers of this phenomenon as well as the contributors to high case-fatality rate is a growing public health concern to enable efficient response. From 2011-2015, 8653 P. falciparum cases leading to 98 deaths (11.3 per 1000 cases) were imported from 41 sub-Saharan countries into China, with most cases (91.3%) occurring in labour-related Chinese travellers. Four strongly connected groupings of origin African countries with destination Chinese provinces were identified, and the number of imported cases was significantly associated with the volume of air passengers to China (P = 0.006), parasite prevalence in Africa (P relationship with parasite importation. Risk factors for deaths from imported cases were related to the capacity of malaria diagnosis and diverse socioeconomic factors. The spatial heterogeneity uncovered, principal drivers explored, and risk factors for mortality found in the rising rates of P. falciparum malaria importation to China can serve to refine malaria elimination strategies and the management of cases, and high risk groups and regions should be targeted.
Full Text Available The two organelles, apicoplast and mitochondrion, of the malaria parasite Plasmodium falciparum have unique morphology in liver and blood stages; they undergo complex branching and looping prior to division and segregation into daughter merozoites. Little is known about the molecular processes and proteins involved in organelle biogenesis in the parasite. We report the identification of an AAA+/FtsH protease homolog (PfFtsH1 that exhibits ATP- and Zn(2+-dependent protease activity. PfFtsH1 undergoes processing, forms oligomeric assemblies, and is associated with the membrane fraction of the parasite cell. Generation of a transfectant parasite line with hemagglutinin-tagged PfFtsH1, and immunofluorescence assay with anti-PfFtsH1 Ab demonstrated that the protein localises to P. falciparum mitochondria. Phylogenetic analysis and the single transmembrane region identifiable in PfFtsH1 suggest that it is an i-AAA like inner mitochondrial membrane protein. Expression of PfFtsH1 in Escherichia coli converted a fraction of bacterial cells into division-defective filamentous forms implying a sequestering effect of the Plasmodium factor on the bacterial homolog, indicative of functional conservation with EcFtsH. These results identify a membrane-associated mitochondrial AAA+/FtsH protease as a candidate regulatory protein for organelle biogenesis in P. falciparum.
Lucas Carmen M
Full Text Available Abstract Background Several of the intended Plasmodium falciparum vaccine candidate antigens are highly polymorphic and could render a vaccine ineffective if their antigenic sites were not represented in the vaccine. In this study, characterization of genetic variability was performed in major B and T-cell epitopes within vaccine candidate antigens in isolates of P. falciparum from Peru. Methods DNA sequencing analysis was completed on 139 isolates of P. falciparum collected from endemic areas of the Amazon basin in Loreto, Peru from years 1998 to 2006. Genetic diversity was determined in immunological important regions in circumsporozoite protein (CSP, merozoite surface protein-1 (MSP-1, apical membrane antigen-1 (AMA-1, liver stage antigen-1 (LSA-1 and thrombospondin-related anonymous protein (TRAP. Alleles identified by DNA sequencing were aligned with the vaccine strain 3D7 and DNA polymorphism analysis and FST study-year pairwise comparisons were done using the DnaSP software. Multilocus analysis (MLA was performed and average of expected heterozygosity was calculated for each loci and haplotype over time. Results Three different alleles for CSP, seven for MSP-1 Block 2, one for MSP-1 Block 17, three for AMA-1 and for LSA-1 each and one for TRAP were identified. There were 24 different haplotypes in 125 infections with complete locus typing for each gene. Conclusion Characterization of the genetic diversity in Plasmodium isolates from the Amazon Region of Peru showed that P. falciparum T and B cell epitopes in these antigens have polymorphisms more similar to India than to Africa. These findings are helpful in the formulation of a vaccine considering restricted repertoire populations.
Cuesta Astroz, Yesid; Segura Latorre, Cesar
Malaria is a parasitic disease that has a high impact on public health in developing countries. The sequencing of the plasmodium falciparum genome and the development of proteomics have enabled a breakthrough in understanding the biology of the parasite. Proteomics have allowed to characterize qualitatively and quantitatively the parasite s expression of proteins and has provided information on protein expression under conditions of stress induced by antimalarial. Given the complexity of their life cycle, this takes place in the vertebrate host and mosquito vector. It has proven difficult to characterize the protein expression during each stage throughout the infection process in order to determine the proteome that mediates several metabolic, physiological and energetic processes. Two dimensional electrophoresis, liquid chromatography and mass spectrometry have been useful to assess the effects of antimalarial on parasite protein expression and to characterize the proteomic profile of different p. falciparum stages and organelles. The purpose of this review is to present state of the art tools and advances in proteomics applied to the study of malaria, and to present different experimental strategies used to study the parasite's proteome in order to show the advantages and disadvantages of each one.
Barman, Bhupen; Bhattacharya, Prasanta Kumar; Lynrah, Kryshan G; Ete, Tony; Issar, Neel Kanth
Malaria is one of the most common protozoan diseases, especially in tropical countries. The clinical manifestation of malaria, especially falciparum malaria varies from mild acute febrile illness to life threatening severe systemic complications involving one or more organ systems. We would like to report a case of complicated falciparum malaria involving cerebral, renal, hepatic system along with acute pancreatitis. The patient was successfully treated with anti malarial and other supportive treatment. To the best of our knowledge there are very few reports of acute pancreatitis due to malaria. Falciparum malaria therefore should be added to the list of infectious agents causing acute pancreatitis especially in areas where malaria is endemic.
Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A. (McMaster U.); (Melbourne); (Toronto); (Deakin); (HWMRI)
The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.
Full Text Available The most severe form of malaria in humans is caused by the protozoan parasite Plasmodium falciparum. The invasive form of malaria parasites is termed a merozoite and it employs an array of parasite proteins that bind to the host cell to mediate invasion. In Plasmodium falciparum, the erythrocyte binding-like (EBL and reticulocyte binding-like (Rh protein families are responsible for binding to specific erythrocyte receptors for invasion and mediating signalling events that initiate active entry of the malaria parasite. Here we have addressed the role of the cytoplasmic tails of these proteins in activating merozoite invasion after receptor engagement. We show that the cytoplasmic domains of these type 1 membrane proteins are phosphorylated in vitro. Depletion of PfCK2, a kinase implicated to phosphorylate these cytoplasmic tails, blocks P. falciparum invasion of red blood cells. We identify the crucial residues within the PfRh4 cytoplasmic domain that are required for successful parasite invasion. Live cell imaging of merozoites from these transgenic mutants show they attach but do not penetrate erythrocytes implying the PfRh4 cytoplasmic tail conveys signals important for the successful completion of the invasion process.
Wadi, Ishan; Pillai, C Radhakrishna; Anvikar, Anupkumar R; Sinha, Abhinav; Nath, Mahendra; Valecha, Neena
Malaria remains a global health problem despite availability of effective tools. For malaria elimination, drugs targeting sexual stages of Plasmodium falciparum need to be incorporated in treatment regimen along with schizonticidal drugs to interrupt transmission. Primaquine is recommended as a transmission blocking drug for its effect on mature gametocytes but is not extensively utilized because of associated safety concerns among glucose-6-phosphate dehydrogenase (G6PD) deficient patients. In present work, methylene blue, which is proposed as an alternative to primaquine is investigated for its gametocytocidal activity amongst Indian field isolates. An effort has been made to establish Indian field isolates of P. falciparum as in vitro model for gametocytocidal drugs screening. Plasmodium falciparum isolates were adapted to in vitro culture and induced to gametocyte production by hypoxanthine and culture was enriched for gametocyte stages using N-acetyl-glucosamine. Gametocytes were incubated with methylene blue for 48 h and stage specific gametocytocidal activity was evaluated by microscopic examination. Plasmodium falciparum field isolates RKL-9 and JDP-8 were able to reproducibly produce gametocytes in high yield and were used to screen gametocytocidal drugs. Methylene blue was found to target gametocytes in a concentration dependent manner by either completely eliminating gametocytes or rendering them morphologically deformed with mean IC 50 (early stages) as 424.1 nM and mean IC 50 (late stages) as 106.4 nM. These morphologically altered gametocytes appeared highly degenerated having shrinkage, distortions and membrane deformations. Field isolates that produce gametocytes in high yield in vitro can be identified and used to screen gametocytocidal drugs. These isolates should be used for validation of gametocytocidal hits obtained previously by using lab adapted reference strains. Methylene blue was found to target gametocytes produced from Indian field
Carter, Tamar E; Malloy, Halley; Existe, Alexandre; Memnon, Gladys; St Victor, Yves; Okech, Bernard A; Mulligan, Connie J
Hispaniola, comprising Haiti and the Dominican Republic, has been identified as a candidate for malaria elimination. However, incomplete surveillance data in Haiti hamper efforts to assess the impact of ongoing malaria control interventions. Characteristics of the genetic diversity of Plasmodium falciparum populations can be used to assess parasite transmission, which is information vital to evaluating malaria elimination efforts. Here we characterize the genetic diversity of P. falciparum samples collected from patients at seven sites in Haiti using 12 microsatellite markers previously employed in population genetic analyses of global P. falciparum populations. We measured multiplicity of infections, level of genetic diversity, degree of population geographic substructure, and linkage disequilibrium (defined as non-random association of alleles from different loci). For low transmission populations like Haiti, we expect to see few multiple infections, low levels of genetic diversity, high degree of population structure, and high linkage disequilibrium. In Haiti, we found low levels of multiple infections (12.9%), moderate to high levels of genetic diversity (mean number of alleles per locus = 4.9, heterozygosity = 0.61), low levels of population structure (highest pairwise Fst = 0.09 and no clustering in principal components analysis), and moderate linkage disequilibrium (ISA = 0.05, PHaiti. We conclude that the high level of genetic diversity and lack of evidence for a population bottleneck may suggest that Haiti's P. falciparum population has been stable and discuss the implications of our results for understanding the impact of malaria control interventions. We also discuss the relevance of parasite population history and other host and vector factors when assessing transmission intensity from genetic diversity data.
Epidemiologia de la malaria falciparum complicada: estudio de casos y controles en Tumaco y Turbo, Colombia, 2003 The epidemiology of complicated falciparum malaria: case and controls study in Tumaco and Turbo, Colombia, 2003
Alberto Tobón C.
Full Text Available OBJETIVOS: Identificar aspectos del hospedero, del parásito y del ambiente asociados con ocurrencia de malaria por Plasmodium falciparum complicada. MÉTODOS: Estudio de casos y controles en pacientes de Tumaco y Turbo (Colombia aplicando los criterios de complicación de la Organización Mundial de la Salud. RESULTADOS: Entre noviembre 2002 y julio 2003 se captaron 64 casos (malaria complicada y 135 controles (malaria no complicada. Las complicaciones fueron: hiperparasitemia (40%, falla hepática (36%, síndrome dificultad respiratoria aguda (7%, falla renal (4%, trombocitopenia grave (3%, anemia grave (2%, malaria cerebral (2% e hipoglicemia grave (1%. Se encontraron como factores de riesgo para malaria falciparum complicada: a Los antecedentes de malaria falciparum durante el último año fueron menores en los casos (OR= 7.0 (1.2-43.6 P=0.019; b Mayor uso previo de antimaláricos en los casos (OR=2.2 (1.1-4.4 P=0.031 y c mayor uso de cloroquina en los casos (OR=7.4 (1.1-7.8 P=0.017. Se hallaron los alelos MAD-20 y K1 del gen msp1 y FC-27 e IC-1 del gen msp2, cuya distribución de frecuencias fue similar entre casos y controles, aunque el alelo K1 mostró una variación importante entre grupos (casos: 9.4%, controles: 3.5%. La frecuencia de "signos de peligro" fue significativamente mayor en los casos (OR= 3.3, (1.5-7.4 P=0.001. Los criterios de complicación malárica de la Organización Mundial de la Salud se comparan con otros y se discuten algunas implicaciones. CONCLUSIÓN: Se identificaron como factores de riesgo para malaria falciparum complicada, la ausencia de antecedentes de malaria falciparum en el último año y el uso de antimaláricos antes de llegar al hospital.OBJECTIVES: Aimed at identifying host and parasite aspects associated to the presence of Plasmodium falciparum complicated malaria. METHODS: Case and controls study in patients from Tumaco and Turbo (Colombia. We used the World Health Organization criteria to assess the
Full Text Available Understanding naturally acquired immune responses to Plasmodium in India is key to improving malaria surveillance and diagnostic tools. Here we describe serological profiling of immune responses at three sites in India by probing protein microarrays consisting of 515 Plasmodium vivax and 500 Plasmodium falciparum proteins with 353 plasma samples. A total of 236 malaria-positive (symptomatic and asymptomatic plasma samples and 117 malaria-negative samples were collected at three field sites in Raurkela, Nadiad, and Chennai. Indian samples showed significant seroreactivity to 265 P. vivax and 373 P. falciparum antigens, but overall seroreactivity to P. vivax antigens was lower compared to P. falciparum antigens. We identified the most immunogenic antigens of both Plasmodium species that were recognized at all three sites in India, as well as P. falciparum antigens that were associated with asymptomatic malaria. This is the first genome-scale analysis of serological responses to the two major species of malaria parasite in India. The range of immune responses characterized in different endemic settings argues for targeted surveillance approaches tailored to the diverse epidemiology of malaria across the world.
Saharan Africa and they increase the prevalence of anaemia in pregnancy with resultant poor pregnancy outcomes. This study was carried out to assess the impact of Plasmodium falciparum and hookworm infections on.
Dekker, E.; Romijn, J. A.; Ekberg, K.; Wahren, J.; van Thien, H.; Ackermans, M. T.; Thuy, L. T.; Chandramouli, V.; Kager, P. A.; Landau, B. R.; Sauerwein, H. P.
Although glucose production is increased in severe malaria, the influence of uncomplicated malaria on glucose production is unknown. Therefore, we measured in eight adult Vietnamese patients with uncomplicated falciparum malaria and eight healthy Vietnamese controls glucose production (by infusion
Hijar, Gisely; Padilla, Carlos; Marquiño, Wilmer; Falconi, Eduardo; Montoya, Ysabel
Eight genotypes of Plasmodium falciparum were detected after analysing blood samples obtained from 30 Peruvian jungle-dwelling patients in Loreto, a high transmission area for P. falciparum, using amplification of the polymorphic marker gene GLURP (glutamate-rich protein). Genotypes I (GLURP450) and VIII (GLURP800) were the most common (15/30 and 13/30, respectively). This single copy gene showed 15 patients to be infected with a single genotype of P. falciparum; the other 15 were infected with mixed genotypes, one of them with 4 genotypes. These findings are compatible with a high genetic complexity of P. falciparum. Further investigations are needed, using this and other markers, in order to design malaria control measures in Peru.
Barber Bridget E
Full Text Available Abstract Background In areas co-endemic for multiple Plasmodium species, correct diagnosis is crucial for appropriate treatment and surveillance. Species misidentification by microscopy has been reported in areas co-endemic for vivax and falciparum malaria, and may be more frequent in regions where Plasmodium knowlesi also commonly occurs. Methods This prospective study in Sabah, Malaysia, evaluated the accuracy of routine district and referral hospital-based microscopy, and microscopy performed by an experienced research microscopist, for the diagnosis of PCR-confirmed Plasmodium falciparum, P. knowlesi, and Plasmodium vivax malaria. Results A total of 304 patients with PCR-confirmed Plasmodium infection were enrolled, including 130 with P. knowlesi, 122 with P. falciparum, 43 with P. vivax, one with Plasmodium malariae and eight with mixed species infections. Among patients with P. knowlesi mono-infection, routine and cross-check microscopy both identified 94 (72% patients as “P. malariae/P. knowlesi”; 17 (13% and 28 (22% respectively were identified as P. falciparum, and 13 (10% and two (1.5% as P. vivax. Among patients with PCR-confirmed P. falciparum, routine and cross-check microscopy identified 110/122 (90% and 112/118 (95% patients respectively as P. falciparum, and 8/122 (6.6% and 5/118 (4.2% as “P. malariae/P. knowlesi”. Among those with P. vivax, 23/43 (53% and 34/40 (85% were correctly diagnosed by routine and cross-check microscopy respectively, while 13/43 (30% and 3/40 (7.5% patients were diagnosed as “P. malariae/P. knowlesi”. Four of 13 patients with PCR-confirmed P. vivax and misdiagnosed by routine microscopy as “P. malariae/P. knowlesi” were subsequently re-admitted with P. vivax malaria. Conclusions Microscopy does not reliably distinguish between P. falciparum, P. vivax and P. knowlesi in a region where all three species frequently occur. Misdiagnosis of P. knowlesi as both P. vivax and P. falciparum, and
Full Text Available Abstract Background Falciparum malaria is the most deadly among the four main types of human malaria. Although great success has been achieved since the launch of the National Malaria Control Programme in 1955, malaria remains a serious public health problem in China. This paper aimed to analyse the geographic distribution, demographic patterns and time trends of falciparum malaria in China. Methods The annual numbers of falciparum malaria cases during 1992–2003 and the individual case reports of each clinical falciparum malaria during 2004–2005 were extracted from communicable disease information systems in China Center for Diseases Control and Prevention. The annual number of cases and the annual incidence were mapped by matching them to corresponding province- and county-level administrative units in a geographic information system. The distribution of falciparum malaria by age, gender and origin of infection was analysed. Time-series analysis was conducted to investigate the relationship between the falciparum malaria in the endemic provinces and the imported falciparum malaria in non-endemic provinces. Results Falciparum malaria was endemic in two provinces of China during 2004–05. Imported malaria was reported in 26 non-endemic provinces. Annual incidence of falciparum malaria was mapped at county level in the two endemic provinces of China: Yunnan and Hainan. The sex ratio (male vs. female for the number of cases in Yunnan was 1.6 in the children of 0–15 years and it reached 5.7 in the adults over 15 years of age. The number of malaria cases in Yunnan was positively correlated with the imported malaria of concurrent months in the non-endemic provinces. Conclusion The endemic area of falciparum malaria in China has remained restricted to two provinces, Yunnan and Hainan. Stable transmission occurs in the bordering region of Yunnan and the hilly-forested south of Hainan. The age and gender distribution in the endemic area is
Barman, Bhupen; Bhattacharya, Prasanta Kumar; Lynrah, Kryshan G; Ete, Tony; Issar, Neel Kanth
Malaria is one of the most common protozoan diseases, especially in tropical countries. The clinical manifestation of malaria, especially falciparum malaria varies from mild acute febrile illness to life threatening severe systemic complications involving one or more organ systems. We would like to report a case of complicated falciparum malaria involving cerebral, renal, hepatic system along with acute pancreatitis. The patient was successfully treated with anti malarial and other supportive...
Grillet, María-Eugenia; El Souki, Mayida; Laguna, Francisco; León, José Rafael
We investigated the periodicity of Plasmodium vivax and P. falciparum incidence in time-series of malaria data (1990-2010) from three endemic regions in Venezuela. In particular, we determined whether disease epidemics were related to local climate variability and regional climate anomalies such as the El Niño Southern Oscillation (ENSO). Malaria periodicity was found to exhibit unique features in each studied region. Significant multi-annual cycles of 2- to about 6-year periods were identified. The inter-annual variability of malaria cases was coherent with that of SSTs (ENSO), mainly at temporal scales within the 3-6 year periods. Additionally, malaria cases were intensified approximately 1 year after an El Niño event, a pattern that highlights the role of climate inter-annual variability in the epidemic patterns. Rainfall mediated the effect of ENSO on malaria locally. Particularly, rains from the last phase of the season had a critical role in the temporal dynamics of Plasmodium. The malaria-climate relationship was complex and transient, varying in strength with the region and species. By identifying temporal cycles of malaria we have made a first step in predicting high-risk years in Venezuela. Our findings emphasize the importance of analyzing high-resolution spatial-temporal data to better understand malaria transmission dynamics. Copyright © 2013 Elsevier B.V. All rights reserved.
BACKGROUND: Achieving adequate antimalarial drug exposure is essential for curing malaria. Day 7 blood or plasma lumefantrine concentrations provide a simple measure of drug exposure that correlates well with artemether-lumefantrine efficacy. However, the 'therapeutic' day 7 lumefantrine concentr......BACKGROUND: Achieving adequate antimalarial drug exposure is essential for curing malaria. Day 7 blood or plasma lumefantrine concentrations provide a simple measure of drug exposure that correlates well with artemether-lumefantrine efficacy. However, the 'therapeutic' day 7 lumefantrine......-lumefantrine for uncomplicated Plasmodium falciparum malaria, to define therapeutic day 7 lumefantrine concentrations and identify patient factors that substantially alter these concentrations. A systematic review of PubMed, Embase, Google Scholar, ClinicalTrials.gov and conference proceedings identified all relevant studies...... lumefantrine concentrations ≥200 ng/ml and high cure rates in most uncomplicated malaria patients. Three groups are at increased risk of treatment failure: very young children (particularly those underweight-for-age); patients with high parasitemias; and patients in very low transmission intensity areas...
Full Text Available BACKGROUND: Natural immunity to Plasmodium falciparum has been widely studied, but its effects on parasite dynamics are poorly understood. Acquisition and clearance rates of untreated infections are key elements of the dynamics of malaria, but estimating these parameters is challenging because of frequent super-infection and imperfect detectability of parasites. Consequently, information on effects of host immune status or age on infection dynamics is fragmentary. METHODS: An age-stratified cohort of 347 individuals from Northern Ghana was sampled six times at 2 month intervals. High-throughput capillary electrophoresis was used to genotype the msp-2 locus of all P. falciparum infections detected by PCR. Force of infection (FOI and duration were estimated for each age group using an immigration-death model that allows for imperfect detection of circulating parasites. RESULTS: Allowing for imperfect detection substantially increased estimates of FOI and duration. Effects of naturally acquired immunity on the FOI and duration would be reflected in age dependence in these indices, but in our cohort data FOI tended to increase with age in children. Persistence of individual parasite clones was characteristic of all age-groups. Duration peaked in 5-9 year old children (average duration 319 days, 95% confidence interval 318;320. CONCLUSIONS: The main age-dependence is on parasite densities, with only small age-variations in the FOI and persistence of infections. This supports the hypothesis that acquired immunity controls transmission mainly by limiting blood-stage parasite densities rather than changing rates of acquisition or clearance of infections.
Full Text Available Abstract Background The Plasmodium falciparum genome (3D7 strain published in 2002, revealed ~5,400 genes, mostly based on in silico predictions. Experimental data is therefore required for structural and functional assessments of P. falciparum genes and expression, and polymorphic data are further necessary to exploit genomic information to further qualify therapeutic target candidates. Here, we undertook a large scale analysis of a P. falciparum FcB1-schizont-EST library previously constructed by suppression subtractive hybridization (SSH to study genes expressed during merozoite morphogenesis, with the aim of: 1 obtaining an exhaustive collection of schizont specific ESTs, 2 experimentally validating or correcting P. falciparum gene models and 3 pinpointing genes displaying protein polymorphism between the FcB1 and 3D7 strains. Results A total of 22,125 clones randomly picked from the SSH library were sequenced, yielding 21,805 usable ESTs that were then clustered on the P. falciparum genome. This allowed identification of 243 protein coding genes, including 121 previously annotated as hypothetical. Statistical analysis of GO terms, when available, indicated significant enrichment in genes involved in "entry into host-cells" and "actin cytoskeleton". Although most ESTs do not span full-length gene reading frames, detailed sequence comparison of FcB1-ESTs versus 3D7 genomic sequences allowed the confirmation of exon/intron boundaries in 29 genes, the detection of new boundaries in 14 genes and identification of protein polymorphism for 21 genes. In addition, a large number of non-protein coding ESTs were identified, mainly matching with the two A-type rRNA units (on chromosomes 5 and 7 and to a lower extent, two atypical rRNA loci (on chromosomes 1 and 8, TARE subtelomeric regions (several chromosomes and the recently described telomerase RNA gene (chromosome 9. Conclusion This FcB1-schizont-EST analysis confirmed the actual expression of 243
Holder Anthony A
Full Text Available Abstract Background The evasion of host immune response by the human malaria parasite Plasmodium falciparum has been linked to expression of a range of variable antigens on the infected erythrocyte surface. Several genes are potentially involved in this process with the var, rif and stevor multigene families being the most likely candidates and coding for rapidly evolving proteins. The high sequence diversity of proteins encoded by these gene families may have evolved as an immune evasion strategy that enables the parasite to establish long lasting chronic infections. Previous findings have shown that the hypervariable region (HVR of STEVOR has significant sequence diversity both within as well as across different P. falciparum lines. However, these studies did not address whether or not there are ancestral stevor that can be found in different parasites. Methods DNA and RNA sequences analysis as well as phylogenetic approaches were used to analyse the stevor sequence repertoire and diversity in laboratory lines and Kilifi (Kenya fresh isolates. Results Conserved stevor genes were identified in different P. falciparum isolates from different global locations. Consistent with previous studies, the HVR of the stevor gene family was found to be highly divergent both within and between isolates. Importantly phylogenetic analysis shows some clustering of stevor sequences both within a single parasite clone as well as across different parasite isolates. Conclusion This indicates that the ancestral P. falciparum parasite genome already contained multiple stevor genes that have subsequently diversified further within the different P. falciparum populations. It also confirms that STEVOR is under strong selection pressure.
Barrera, Sandra Milena; Pérez, Manuel Alberto; Knudson, Angélica; Nicholls, Rubén Santiago; Guerra, Angela Patricia
The genetic diversity of Plasmodium falciparum has been one of the major obstacles for the success of anti-malaria drug therapy. It provides the parasite an ability to evade the host's immune response by generating changes in its antigenic composition and resistance to antimalarial drugs. The genetic diversity of P.falciparum was characterized in 4 Colombian localities through the analysis of polymorphic genes. Eighty-one samples were obtained from patients with uncomplicated P. falciparum malaria and screened for polymorphic variants of msp1, msp2 (merozoite surface proteins) and glurp (glutamate-rich protein) with a multiplex PCR assay. The geographic regions sampled were Tierralta (Córdoba), in northwestern Colombia and in the Orinoco river watershed of eastern Colombia--Inírida (Guainía), La Carpa (Guaviare), and Casuarito (Vichada). The MAD20 variant was detected in all samples analyzed for the msp1 gene. For the msp2 gene, the IC allelic family was found in 96.3% of the samples as compared to 4.9% of the samples with the FC family. Both families showed size polymorphism with bands between 467 and 513 basepairs (bp) for IC and 286 and 300 bp for FC. PCR products of differing sizes were detected for the glurp gene and grouped into 5 size classes: I (600-699 bp) 2.5%, II (700-799 bp) 19.8%, III (800-899 bp) 72.8%, IV (900-999 bp) 1.2% and V (1000-1099 bp) 3.7%. The msp1 molecular marker did not provide information for differentiating P. falciparum parasite populations. The msp2 gene was more suitable for studying the genetic diversity, however, further studies are required to identify polymorphisms within the two allelic families. The glurp gene showed a great genetic diversity of circulating P. falciparum populations, and suggested that this gene may be useful for distinguishing between recrudescence and reinfection.
Full Text Available Abstract Background The objective of this study was to identify demographic, environmental and socioeconomic risk factors and spatial patterns of Plasmodium falciparum parasitaemia in a high endemicity area of Africa, and to specify how this information can facilitate improved malaria control at the district level. Methods A questionnaire was administered to about 4,000 schoolchildren in 55 schools in western Côte d'Ivoire to determine children's socioeconomic status and their habit of sleeping under bed nets. Environmental data were obtained from satellite images, digitized ground maps and a second questionnaire addressed to school directors. Finger prick blood samples were collected and P. falciparum parasitaemia determined under a microscope using standardized, quality-controlled methods. Bayesian variogram models were utilized for spatial risk modelling and mapping of P. falciparum parasitaemia at non-sampled locations, assuming stationary and non-stationary underlying spatial dependence. Results Two-thirds of the schoolchildren were infected with P. falciparum and the mean parasitaemia among infected children was 959 parasites/μl of blood. Age, socioeconomic status, not sleeping under a bed net, coverage rate with bed nets and environmental factors (e.g., normalized difference vegetation index, rainfall, land surface temperature and living in close proximity to standing water were significantly associated with the risk of P. falciparum parasitaemia. After accounting for spatial correlation, age, bed net coverage, rainfall during the main malaria transmission season and distance to rivers remained significant covariates. Conclusion It is argued that a massive increase in bed net coverage, particularly in villages in close proximity to rivers, in concert with other control measures, is necessary to bring malaria endemicity down to intermediate or low levels.
Pava, Zuleima; Noviyanti, Rintis; Handayuni, Irene; Trimarsanto, Hidayat; Trianty, Leily; Burdam, Faustina H; Kenangalem, Enny; Utami, Retno A S; Tirta, Yusrifar K; Coutrier, Farah; Poespoprodjo, Jeanne R; Price, Ric N; Marfurt, Jutta; Auburn, Sarah
Genetic analyses of Plasmodium have potential to inform on transmission dynamics, but few studies have evaluated this on a local spatial scale. We used microsatellite genotyping to characterise the micro-epidemiology of P. vivax and P. falciparum diversity to inform malaria control strategies in Timika, Papua Indonesia. Genotyping was undertaken on 713 sympatric P. falciparum and P. vivax isolates from a cross-sectional household survey and clinical studies conducted in Timika. Standard population genetic measures were applied, and the data was compared to published data from Kalimantan, Bangka, Sumba and West Timor. Higher diversity (HE = 0.847 vs 0.625; p = 0.017) and polyclonality (46.2% vs 16.5%, p<0.001) were observed in P. vivax versus P. falciparum. Distinct P. falciparum substructure was observed, with two subpopulations, K1 and K2. K1 was comprised solely of asymptomatic infections and displayed greater relatedness to isolates from Sumba than to K2, possibly reflecting imported infections. The results demonstrate the greater refractoriness of P. vivax versus P. falciparum to control measures, and risk of distinct parasite subpopulations persisting in the community undetected by passive surveillance. These findings highlight the need for complimentary new surveillance strategies to identify transmission patterns that cannot be detected with traditional malariometric methods.
Full Text Available Abstract Background Hidden Markov Models (HMMs are a powerful tool for protein domain identification. The Pfam database notably provides a large collection of HMMs which are widely used for the annotation of proteins in new sequenced organisms. In Pfam, each domain family is represented by a curated multiple sequence alignment from which a profile HMM is built. In spite of their high specificity, HMMs may lack sensitivity when searching for domains in divergent organisms. This is particularly the case for species with a biased amino-acid composition, such as P. falciparum, the main causal agent of human malaria. In this context, fitting HMMs to the specificities of the target proteome can help identify additional domains. Results Using P. falciparum as an example, we compare approaches that have been proposed for this problem, and present two alternative methods. Because previous attempts strongly rely on known domain occurrences in the target species or its close relatives, they mainly improve the detection of domains which belong to already identified families. Our methods learn global correction rules that adjust amino-acid distributions associated with the match states of HMMs. These rules are applied to all match states of the whole HMM library, thus enabling the detection of domains from previously absent families. Additionally, we propose a procedure to estimate the proportion of false positives among the newly discovered domains. Starting with the Pfam standard library, we build several new libraries with the different HMM-fitting approaches. These libraries are first used to detect new domain occurrences with low E-values. Second, by applying the Co-Occurrence Domain Discovery (CODD procedure we have recently proposed, the libraries are further used to identify likely occurrences among potential domains with higher E-values. Conclusion We show that the new approaches allow identification of several domain families previously absent in
Kang, Jung-Mi; Lee, Jinyoung; Moe, Mya; Jun, Hojong; Lê, Hương Giang; Kim, Tae Im; Thái, Thị Lam; Sohn, Woon-Mok; Myint, Moe Kyaw; Lin, Khin; Shin, Ho-Joon; Kim, Tong-Soo; Na, Byoung-Kuk
Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is one of leading blood stage malaria vaccine candidates. However, genetic variation and antigenic diversity identified in global PfAMA-1 are major hurdles in the development of an effective vaccine based on this antigen. In this study, genetic structure and the effect of natural selection of PfAMA-1 among Myanmar P. falciparum isolates were analysed. Blood samples were collected from 58 Myanmar patients with falciparum malaria. Full-length PfAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. PfAMA-1 sequence of each isolate was sequenced. Polymorphic characteristics and effect of natural selection were analysed with using DNASTAR, MEGA4, and DnaSP programs. Polymorphic nature and natural selection in 459 global PfAMA-1 were also analysed. Thirty-seven different haplotypes of PfAMA-1 were identified in 58 Myanmar P. falciparum isolates. Most amino acid changes identified in Myanmar PfAMA-1 were found in domains I and III. Overall patterns of amino acid changes in Myanmar PfAMA-1 were similar to those in global PfAMA-1. However, frequencies of amino acid changes differed by country. Novel amino acid changes in Myanmar PfAMA-1 were also identified. Evidences for natural selection and recombination event were observed in global PfAMA-1. Among 51 commonly identified amino acid changes in global PfAMA-1 sequences, 43 were found in predicted RBC-binding sites, B-cell epitopes, or IUR regions. Myanmar PfAMA-1 showed similar patterns of nucleotide diversity and amino acid polymorphisms compared to those of global PfAMA-1. Balancing natural selection and intragenic recombination across PfAMA-1 are likely to play major roles in generating genetic diversity in global PfAMA-1. Most common amino acid changes in global PfAMA-1 were located in predicted B-cell epitopes where high levels of nucleotide diversity and balancing natural selection were found. These results highlight the
Miotto, Olivo; Amato, Roberto; Ashley, Elizabeth A; MacInnis, Bronwyn; Almagro-Garcia, Jacob; Amaratunga, Chanaki; Lim, Pharath; Mead, Daniel; Oyola, Samuel O; Dhorda, Mehul; Imwong, Mallika; Woodrow, Charles; Manske, Magnus; Stalker, Jim; Drury, Eleanor; Campino, Susana; Amenga-Etego, Lucas; Thanh, Thuy-Nhien Nguyen; Tran, Hien Tinh; Ringwald, Pascal; Bethell, Delia; Nosten, Francois; Phyo, Aung Pyae; Pukrittayakamee, Sasithon; Chotivanich, Kesinee; Chuor, Char Meng; Nguon, Chea; Suon, Seila; Sreng, Sokunthea; Newton, Paul N; Mayxay, Mayfong; Khanthavong, Maniphone; Hongvanthong, Bouasy; Htut, Ye; Han, Kay Thwe; Kyaw, Myat Phone; Faiz, Md Abul; Fanello, Caterina I; Onyamboko, Marie; Mokuolu, Olugbenga A; Jacob, Christopher G; Takala-Harrison, Shannon; Plowe, Christopher V; Day, Nicholas P; Dondorp, Arjen M; Spencer, Chris C A; McVean, Gilean; Fairhurst, Rick M; White, Nicholas J; Kwiatkowski, Dominic P
We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population. PMID:25599401
Rono, Martin K; Nyonda, Mary A; Simam, Joan J; Ngoi, Joyce M; Mok, Sachel; Kortok, Moses M; Abdullah, Abdullah S; Elfaki, Mohammed M; Waitumbi, John N; El-Hassan, Ibrahim M; Marsh, Kevin; Bozdech, Zbynek; Mackinnon, Margaret J
Success in eliminating malaria will depend on whether parasite evolution outpaces control efforts. Here, we show that Plasmodium falciparum parasites (the deadliest of the species causing human malaria) found in low-transmission-intensity areas have evolved to invest more in transmission to new hosts (reproduction) and less in within-host replication (growth) than parasites found in high-transmission areas. At the cellular level, this adaptation manifests as increased production of reproductive forms (gametocytes) early in the infection at the expense of processes associated with multiplication inside red blood cells, especially membrane transport and protein trafficking. At the molecular level, this manifests as changes in the expression levels of genes encoding epigenetic and translational machinery. Specifically, expression levels of the gene encoding AP2-G-the transcription factor that initiates reproduction-increase as transmission intensity decreases. This is accompanied by downregulation and upregulation of genes encoding HDAC1 and HDA1-two histone deacetylases that epigenetically regulate the parasite's replicative and reproductive life-stage programmes, respectively. Parasites in reproductive mode show increased reliance on the prokaryotic translation machinery found inside the plastid-derived organelles. Thus, our dissection of the parasite's adaptive regulatory architecture has identified new potential molecular targets for malaria control.
Miotto, Olivo; Amato, Roberto; Ashley, Elizabeth A; MacInnis, Bronwyn; Almagro-Garcia, Jacob; Amaratunga, Chanaki; Lim, Pharath; Mead, Daniel; Oyola, Samuel O; Dhorda, Mehul; Imwong, Mallika; Woodrow, Charles; Manske, Magnus; Stalker, Jim; Drury, Eleanor; Campino, Susana; Amenga-Etego, Lucas; Thanh, Thuy-Nhien Nguyen; Tran, Hien Tinh; Ringwald, Pascal; Bethell, Delia; Nosten, Francois; Phyo, Aung Pyae; Pukrittayakamee, Sasithon; Chotivanich, Kesinee; Chuor, Char Meng; Nguon, Chea; Suon, Seila; Sreng, Sokunthea; Newton, Paul N; Mayxay, Mayfong; Khanthavong, Maniphone; Hongvanthong, Bouasy; Htut, Ye; Han, Kay Thwe; Kyaw, Myat Phone; Faiz, Md Abul; Fanello, Caterina I; Onyamboko, Marie; Mokuolu, Olugbenga A; Jacob, Christopher G; Takala-Harrison, Shannon; Plowe, Christopher V; Day, Nicholas P; Dondorp, Arjen M; Spencer, Chris C A; McVean, Gilean; Fairhurst, Rick M; White, Nicholas J; Kwiatkowski, Dominic P
We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.
Mahamar, Almahamoudou; Attaher, Oumar; Swihart, Bruce; Barry, Amadou; Diarra, Bacary S; Kanoute, Moussa B; Cisse, Kadidia B; Dembele, Adama B; Keita, Sekouba; Gamain, Benoît; Gaoussou, Santara; Issiaka, Djibrilla; Dicko, Alassane; Duffy, Patrick E; Fried, Michal
P. falciparum virulence is related to adhesion and sequestration of infected erythrocytes (IE) in deep vascular beds, but the endothelial receptors involved in severe malaria remain unclear. In the largest ever study of clinical isolates, we surveyed adhesion of freshly collected IE from children under 5 years of age in Mali to identify novel vascular receptors, and examined the effects of host age, hemoglobin type, blood group and severe malaria on levels of IE adhesion to a panel of endothelial receptors. Several novel molecules, including integrin α3β1, VE-cadherin, ICAM-2, junctional adhesion molecule-B (JAM-B), laminin, and cellular fibronectin, supported binding of IE from children. Severe malaria was not significantly associated with levels of IE adhesion to any of the 19 receptors. Hemoglobin AC, which reduces severe malaria risk, reduced IE binding to the receptors CD36 and integrin α5β1, while hemoglobin AS did not modify IE adhesion to any receptors. Blood groups A, AB and B significantly reduced IE binding to ICAM-1. Severe malaria risk varies with age, but age significantly impacted the level of IE binding to only a few receptors: IE binding to JAM-B decreased with age, while binding to CD36 and integrin α5β1 significantly increased with age.
Full Text Available Malaria remains a major health problem worldwide. All clinical symptoms of malaria are attributed to the asexual blood stages of the parasite life cycle. Proteins resident in apical organelles and present on the surface of P. falciparum merozoites are considered promising candidates for the development of blood stage malaria vaccines. In the present study, we have identified and characterized a microneme associated antigen, PfMA [PlasmoDB Gene ID: PF3D7_0316000, PFC0700c]. The gene was selected by applying a set of screening criteria such as transcriptional upregulation at late schizogony, inter-species conservation and the presence of signal sequence or transmembrane domains. The gene sequence of PfMA was found to be conserved amongst various Plasmodium species. We experimentally demonstrated that the transcript for PfMA was expressed only in the late blood stages of parasite consistent with a putative role in erythrocyte invasion. PfMA was localized by immunofluorescence and immuno-electron microscopy to be in the micronemes, an apical organelle of merozoites. The functional role of the PfMA protein in erythrocyte invasion was identified as a parasite adhesin involved in direct attachment with the target erythrocyte. PfMA was demonstrated to bind erythrocytes in a sialic acid independent, chymotrypsin and trypsin resistant manner and its antibodies inhibited P. falciparum erythrocyte invasion. Invasion of erythrocytes is a complex multistep process that involves a number of redundant ligand-receptor interactions many of which still remain unknown and even uncharacterized. Our work has identified and characterized a novel P. falciparum adhesin involved in erythrocyte invasion.
Sugith Babu Badugu
Full Text Available The eukaryotic Meiotic Recombination protein 11 (Mre11 plays pivotal roles in the DNA damage response (DDR. Specifically, Mre11 senses and signals DNA double strand breaks (DSB and facilitates their repair through effector proteins belonging to either homologous recombination (HR or non-homologous end joining (NHEJ repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11 that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11. Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N. PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarily conserved component of the DDR in Plasmodium.
Reuter, Stephanie E; Upton, Richard N; Evans, Allan M; Navaratnam, Visweswaran; Olliaro, Piero L
The determination of dosing regimens for the treatment of malaria is largely empirical and thus a better understanding of the pharmacokinetic/pharmacodynamic properties of antimalarial agents is required to assess the adequacy of current treatment regimens and identify sources of suboptimal dosing that could select for drug-resistant parasites. Mefloquine is a widely used antimalarial, commonly given in combination with artesunate. Mefloquine pharmacokinetics was assessed in 24 healthy adults and 43 patients with Plasmodium falciparum malaria administered mefloquine in combination with artesunate. Population pharmacokinetic modelling was conducted using NONMEM. A two-compartment model with a single transit compartment and first-order elimination from the central compartment most adequately described mefloquine concentration-time data. The model incorporated population parameter variability for clearance (CL/F), central volume of distribution (VC/F) and absorption rate constant (KA) and identified, in addition to body weight, malaria infection as a covariate for VC/F (but not CL/F). Monte Carlo simulations predict that falciparum malaria infection is associated with a shorter elimination half-life (407 versus 566 h) and T>MIC (766 versus 893 h). This is the first known population pharmacokinetic study to show falciparum malaria to influence mefloquine disposition. Protein binding, anaemia and other factors may contribute to differences between healthy individuals and patients. As VC/F is related to the earlier portion of the concentration-time profiles, which occurs during acute malaria, and CL/F is more related to the terminal phase during convalescence after treatment, this may explain why malaria was found to be a covariate for VC/F but not CL/F. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: firstname.lastname@example.org.
Reteng, Patrick; Vrisca, Visia; Sukarno, Inka; Djarkoni, Ilham Habib; Kalangi, Jane Angela; Jacobs, George Eduardo; Runtuwene, Lucky Ronald; Eshita, Yuki; Maeda, Ryuichiro; Suzuki, Yutaka; Mongan, Arthur Elia; Warouw, Sarah Maria; Yamagishi, Junya; Tuda, Josef
Malaria still poses one of the major threats to human health. Development of effective antimalarial drugs has decreased this threat; however, the emergence of drug-resistant Plasmodium falciparum, a cause of Malaria, is disconcerting. The antimalarial drug chloroquine has been effectively used, but resistant parasites have spread worldwide. Interestingly, the withdrawal of the drug reportedly leads to an increased population of susceptible parasites in some cases. We examined the prevalence of genomic polymorphisms in a malaria parasite P. falciparum, associated with resistance to an antimalarial drug chloroquine, after the withdrawal of the drug from Indonesia. Blood samples were collected from 95 malaria patients in North Sulawesi, Indonesia, in 2010. Parasite DNA was extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for pfcrt and pfmdr1. In parallel, multiplex amplicon sequencing for the same genes was carried out with Illumina MiSeq. Of the 59 cases diagnosed as P. falciparum infection by microscopy, PCR-RFLP analysis clearly identified the genotype 76T in pfcrt in 44 cases. Sequencing analysis validated the identified genotypes in the 44 cases and demonstrated that the haplotype in the surrounding genomic region was exclusively SVMNT. Results of pfmdr1 were successfully obtained for 51 samples, where the genotyping results obtained by the two methods were completely consistent. In pfmdr1, the 86Y mutant genotype was observed in 45 cases (88.2%). Our results suggest that the prevalence of the mutated genotypes remained dominant even 6 years after the withdrawal of chloroquine from this region. Diversified haplotype of the resistance-related locus, potentially involved in fitness costs, unauthorized usage of chloroquine, and/or a short post-withdrawal period may account for the observed high persistence of prevalence.
Full Text Available Plasmodium falciparum is the causative agent of deadly malaria disease. It is an intracellular eukaryote and completes its multi-stage life cycle spanning the two hosts viz, mosquito and human. In order to habituate within host environment, parasite conform several strategies to evade host immune responses such as surface antigen polymorphism or modulation of host immune system and it is mediated by secretion of proteins from parasite to the host erythrocyte and beyond, collectively known as, malaria secretome. In this review, we will discuss about the deployment of parasitic secretory protein in mechanism implicated for immune evasion, protein trafficking, providing virulence, changing permeability and cyto-adherence of infected erythrocyte. We will be covering the possibilities of developing malaria secretome as a drug/vaccine target. This gathered information will be worthwhile in depicting a well-organized picture for host-pathogen interplay during the malaria infection and may also provide some clues for development of novel anti-malarial therapies.
Mwangi Jonathan M
Full Text Available Abstract Background Plasmodium falciparum is usually asynchronous during in vitro culture. Although various synchronization methods are available, they are not able to narrow the range of ages of parasites. A newly developed method is described that allows synchronization of parasites to produce cultures with an age range as low as 30 minutes. Methods Trophozoites and schizonts are enriched using Plasmion. The enriched late stage parasites are immobilized as a monolayer onto plastic Petri dishes using concanavalin A. Uninfected erythrocytes are placed onto the monolayer for a limited time period, during which time schizonts on the monolayer rupture and the released merozoites invade the fresh erythrocytes. The overlay is then taken off into a culture flask, resulting in a highly synchronized population of parasites. Results Plasmion treatment results in a 10- to 13-fold enrichment of late stage parasites. The monolayer method results in highly synchronized cultures of parasites where invasion has occurred within a very limited time window, which can be as low as 30 minutes. The method is simple, requiring no specialized equipment and relatively cheap reagents. Conclusions The new method for parasite synchronization results in highly synchronized populations of parasites, which will be useful for studies of the parasite asexual cell cycle.
Full Text Available Malaria is responsible for estimated 584,000 deaths in 2013. Researchers are working on new drugs and medicinal herbs due to drug resistance that is a major problem facing them; the search is on for new medicinal herbs. Cinnamon is the bark of a tree with reported antiparasitic effects. Metabonomics is the simultaneous study of all the metabolites in biological fluids, cells, and tissues detected by high throughput technology. It was decided to determine the mechanism of the effect of aqueous extract of cinnamon on the metabolome of Plasmodium falciparum in vitro using 1HNMR spectroscopy. Prepared aqueous extract of cinnamon was added to a culture of Plasmodium falciparum 3D7 and its 50% inhibitory concentration determined, and, after collection, their metabolites were extracted and 1HNMR spectroscopy by NOESY method was done. The spectra were analyzed by chemometric methods. The differentiating metabolites were identified using Human Metabolome Database and the metabolic cycles identified by Metaboanalyst. 50% inhibitory concentration of cinnamon on Plasmodium falciparum was 1.25 mg/mL with p<0.001. The metabolites were identified as succinic acid, glutathione, L-aspartic acid, beta-alanine, and 2-methylbutyryl glycine. The main metabolic cycles detected were alanine and aspartame and glutamate pathway and pantothenate and coenzyme A biosynthesis and lysine biosynthesis and glutathione metabolism, which are all important as drug targets.
Full Text Available Abstract Background Malaria caused by protozoan parasites of the genus Plasmodium spp. is a major health burden in tropical countries. The development of new control tools, including vaccines and drugs, is urgently needed. The availability of genome sequences from several malaria parasite species provides a basis on which to identify new potential intervention targets. Database mining for orthologs to the Plasmodium falciparum trophozoite protein R45, a vaccine candidate, led us identify a new gene family. Results Orthologs to the P. falciparum trophozoite protein R45 were detected exclusively in protozoan parasites of the phylum Apicomplexa, including several Plasmodium spp., Toxoplasma gondii and Cryptosporidium parvum. All family members are hybrid genes with a conserved C-terminal protein kinase domain of a novel type, recently called FIKK kinase, associated with a non conserved N-terminal region without any known functional signature. While a single copy gene was detected in most species, considerable gene expansion was observed in P. falciparum and its closest phylogenic relative P. reichenowi, with 20 and six copies, respectively, each with a distinct N-terminal domain. Based on full length protein sequence, pairs of orthologs were observed in closely related species, such as P. berghei and P.y. yoelii, P. vivax and P. knowlesi, or P. reichenowi and P. falciparum. All 20 P. falciparum paralogs possess a canonical Plasmodium export element downstream of a signal / anchor sequence required for exportation outside the parasitophorous vacuole. This is consistent with the reported association of the trophozoite protein R45, the only paralog characterised to date, with the infected red blood cell membrane. Interestingly, most genes are located in the subtelomeric region of chromosomes, in association with other multigene families contributing to the remodelling of the infected red blood cell membrane, in particular the ring erythrocyte surface
Williams, John; Njie, Fanta; Cairns, Matthew
BACKGROUND: Non-Plasmodium falciparum malaria infections are found in many parts of sub-Saharan Africa but little is known about their importance in pregnancy. METHODS: Blood samples were collected at first antenatal clinic attendance from 2526 women enrolled in a trial of intermittent screening...... of non-falciparum malaria as well as falciparum malaria. The outcome of pregnancy did not differ between women with a non-falciparum malaria infection and those who were not infected with malaria at first ANC attendance. CONCLUSIONS: Non-falciparum infections were infrequent in the populations studied...
Full Text Available Abstract Background In the fight against malaria, the discovery of chemical compounds with a novel mode of action and/or chemistry distinct from currently used drugs is vital to counteract the parasite’s known ability to develop drug resistance. Another desirable aspect is efficacy against gametocytes, the sexual developmental stage of the parasite which enables the transmission through Anopheles vectors. Using a chemical rescue approach, we previously identified compounds targeting Plasmodium falciparum coenzyme A (CoA synthesis or utilization, a promising target that has not yet been exploited in anti-malarial drug development. Results We report on the outcomes of a series of biological tests that help to define the species- and stage-specificity, as well as the potential targets of these chemically diverse compounds. Compound activity against P. falciparum gametocytes was determined to assess stage-specificity and transmission-reducing potential. Against early stage gametocytes IC50 values ranging between 60 nM and 7.5 μM were obtained. With the exception of two compounds with sub-micromolar potencies across all intra-erythrocytic stages, activity against late stage gametocytes was lower. None of the compounds were specific pantothenate kinase inhibitors. Chemical rescue profiling with CoA pathway intermediates demonstrated that most compounds acted on either of the two final P. falciparum CoA synthesis enzymes, phosphopantetheine adenylyltransferase (PPAT or dephospho CoA kinase (DPCK. The most active compound targeted either phosphopantothenoylcysteine synthetase (PPCS or phosphopantothenoylcysteine decarboxylase (PPCDC. Species-specificity was evaluated against Trypanosoma cruzi and Trypanosoma brucei brucei. No specific activity against T. cruzi amastigotes was observed; however three compounds inhibited the viability of trypomastigotes with sub-micromolar potencies and were confirmed to act on T. b. brucei CoA synthesis. Conclusions
Hviid, L; Theander, T G; Abu-Zeid, Y A
Sixteen patients suffering from acute Plasmodium falciparum malaria were studied. All were residents of an area of unstable malaria-transmission in Eastern Sudan. Blood-samples were drawn at diagnosis, and 7 and 30 days later. Blood-samples from thirteen donors, drawn outside the malaria...... convalescence. Five donors examined by fluorescence-activated cell sorting (FACS) showed no increase in surface expression of IL-2 receptor on peripheral lymphocytes. The data indicate that acute P. falciparum malaria causes a depletion of antigen-reactive T-cells from the peripheral circulation, probably due...
Jakobsen, P H; Bygbjerg, I C; Theander, T G
. falciparum malaria compared to the levels during acute disease. Thus, both soluble Hb and haptoglobin appear to be markers of recent P. falciparum infections. Very high levels of CRP protein were measured in some of the malaria patients at the day of treatment while lower levels were recorded 7 and 30 days...... after treatment. Soluble Hb levels were associated with malariometric parameters in a similar fashion to haptoglobin. The new Mab-based assay for measuring soluble Hb in the peripheral blood of malaria patients may be useful for future epidemiological studies of malaria....
Hogh, B; Petersen, E; Dziegiel, Morten Hanefeld
A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain two...... areas of repeated amino acid sequences. Antibodies against recombinant GLURP489-1271, as well as against a synthetic peptide corresponding to GLURP899-916, and against a synthetic peptide representing the major glutamate rich repeat sequence from the P. falciparum ring erythrocyte surface antigen (Pf155...
Lau, Tiek Ying; Sylvi, Mersumpin; William, Timothy
The sulphadoxine/pyrimethamine (SDX/PYR) combination had been chosen to treat uncomplicated falciparum malaria in Malaysia for more than 30 years. Non-silent mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are responsible for the resistance to pyrimethamine and sulphadoxine, respectively. This study reports the mutational analysis of pfdhfr and pfdhps in single Plasmodium falciparum infection isolates from the interior division of Sabah, Malaysian Borneo. A total of 22 P. falciparum single infection isolates collected from two districts of the interior division of Sabah from February to November 2010 were recruited for the mutational study of pfdhfr and pfdhps. Both genes were amplified by nested PCR prior to DNA sequencing and mutational analysis. A total of three pfdhfr and four pfdhps alleles were identified. The most prevalent pfdhfr allele is ANRNL (86%) involving triple mutation at position 108(S to N), 59(C to R) and 164(I to L). In pfdhps, two novel alleles, SGTGA (73%) and AAKAA (5%) were identified. Alleles involving triple mutation in both pfdhfr (ANRNL) and pfdhps (SGTGA), which were absent in Sabah in a study conducted about 15 years ago, are now prevalent. High prevalence of mutations in SDX/PYR associated drug resistance genes are reported in this study. This mutational study of pfdhps and pfdhfr indicating that SDX/PYR should be discontinued in this region.
Parobek, Christian M; Parr, Jonathan B; Brazeau, Nicholas F; Lon, Chanthap; Chaorattanakawee, Suwanna; Gosi, Panita; Barnett, Eric J; Norris, Lauren D; Meshnick, Steven R; Spring, Michele D; Lanteri, Charlotte A; Bailey, Jeffrey A; Saunders, David L; Lin, Jessica T; Juliano, Jonathan J
Plasmodium falciparum in western Cambodia has developed resistance to artemisinin and its partner drugs, causing frequent treatment failure. Understanding this evolution can inform the deployment of new therapies. We investigated the genetic architecture of 78 falciparum isolates using whole-genome sequencing, correlating results to in vivo and ex vivo drug resistance and exploring the relationship between population structure, demographic history, and partner drug resistance. Principle component analysis, network analysis and demographic inference identified a diverse central population with three clusters of clonally expanding parasite populations, each associated with specific K13 artemisinin resistance alleles and partner drug resistance profiles which were consistent with the sequential deployment of artemisinin combination therapies in the region. One cluster displayed ex vivo piperaquine resistance and mefloquine sensitivity with a high rate of in vivo failure of dihydroartemisinin-piperaquine. Another cluster displayed ex vivo mefloquine resistance and piperaquine sensitivity with high in vivo efficacy of dihydroartemisinin-piperaquine. The final cluster was clonal and displayed intermediate sensitivity to both drugs. Variations in recently described piperaquine resistance markers did not explain the difference in mean IC90 or clinical failures between the high and intermediate piperaquine resistance groups, suggesting additional loci may be involved in resistance. The results highlight an important role for partner drug resistance in shaping the P. falciparum genetic landscape in Southeast Asia and suggest that further work is needed to evaluate for other mutations that drive piperaquine resistance. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Nunes Miriam CS
Full Text Available Abstract Background Fourier transforms and their associated power spectra are used for detecting periodicities and protein-coding genes and is generally regarded as a well established technique. Many of the periodicities which have been found with this method are quite well understood such as the periodicity of 3 nt which is associated to codon usage. But what is the origin of the peculiar frequency multiples k/21 which were reported for a tiny section of chromosome 2 in P. falciparum? Are these present in other chromosomes and perhaps in related organisms? And how should we interpret fractional periodicities in genomes? Results We applied the binary indicator power spectrum to all chromosomes of P. falciparum, and found that the frequency overtones k/21 are present only in non-coding sections. We did not find such frequency overtones in any other related genomes. Furthermore, the frequency overtones were identified as artifacts of the way the genome is encoded into a numerical sequence, that is, they are frequency aliases. By choosing a different way to encode the sequence the overtones do not appear. In view of these results, we revisited early applications of this technique to proteins where frequency overtones were reported. Conclusions Some authors hinted recently at the possibility of mapping artifacts and frequency aliases in power spectra. However, in the case of P. falciparum the frequency aliases are particularly strong and can mask the 1/3 frequency which is used for gene detecting. This shows that albeit being a well known technique, with a long history of application in proteins, few researchers seem to be aware of the problems represented by frequency aliases.
El Bakkouri, Majida; Pow, Andre; Mulichak, Anne; Cheung, Kevin L Y; Artz, Jennifer D; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F; Goodman, C Dean; McFadden, Geoffrey I; Ortega, Joaquin; Hui, Raymond; Houry, Walid A
The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum. Copyright © 2010 Elsevier Ltd. All rights reserved.
Full Text Available BACKGROUND: Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant. METHODS AND FINDINGS: Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum-infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation. CONCLUSION: This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory.
Full Text Available Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant.Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum-infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation.This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory.
Douglas, Nicholas M.; Lampah, Daniel A.; Kenangalem, Enny; Simpson, Julie A.; Poespoprodjo, Jeanne R.; Sugiarto, Paulus; Anstey, Nicholas M.; Price, Ric N.
Background The burden of anemia attributable to non-falciparum malarias in regions with Plasmodium co-endemicity is poorly documented. We compared the hematological profile of patients with and without malaria in southern Papua, Indonesia. Methods and Findings Clinical and laboratory data were linked for all patients presenting to a referral hospital between April 2004 and December 2012. Data were available on patient demographics, malaria diagnosis, hemoglobin concentration, and clinical outcome, but other potential causes of anemia could not be identified reliably. Of 922,120 patient episodes (837,989 as outpatients and 84,131 as inpatients), a total of 219,845 (23.8%) were associated with a hemoglobin measurement, of whom 67,696 (30.8%) had malaria. Patients with P. malariae infection had the lowest hemoglobin concentration (n = 1,608, mean = 8.93 [95% CI 8.81–9.06]), followed by those with mixed species infections (n = 8,645, mean = 9.22 [95% CI 9.16–9.28]), P. falciparum (n = 37,554, mean = 9.47 [95% CI 9.44–9.50]), and P. vivax (n = 19,858, mean = 9.53 [95% CI 9.49–9.57]); p-value for all comparisons anemia (hemoglobin anemia (adjusted odds ratio [AOR] 3.25 [95% CI 2.99–3.54]); AORs for severe anaemia associated with P. falciparum, P. vivax, and P. malariae were 2.11 (95% CI 2.00–2.23), 1.87 (95% CI 1.74–2.01), and 2.18 (95% CI 1.76–2.67), respectively, panemia was attributable to non-falciparum infections compared with 15.1% (95% CI 13.9%–16.3%) for P. falciparum monoinfections. Patients with severe anemia had an increased risk of death (AOR = 5.80 [95% CI 5.17–6.50]; panemia in early infancy, mixed P. vivax/P. falciparum infections are associated with a greater hematological impairment than either species alone, and in adulthood P. malariae, although rare, is associated with the lowest hemoglobin concentration. These findings highlight the public health importance of integrated genus-wide malaria
Malarial parasite P. falciparum, an apicomplexan protozoan has a 23.3 MB nuclear genome and encodes ~ 5600 transcripts. The genetic diversity of the parasite within and across geographical zones is a challenge to gene expression studies which are essential for understanding of disease process, outcome and developing markers for diagnostics and prognostics. Here, we describe the strategy involved in designing a custom P. falciparum 15K array using the Agilent platform and Genotypic\\'s Right Design methodology to study the transcriptome of Indian field isolates for which genome sequence information is limited. The array contains probes representing genome sequences of two distinct geographical isolates (i.e. 3D7 and HB3) and sub-telomeric var gene sequences of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts through a 244K array experimentation. Array performance for the 15K array, was evaluated and validated using RNA materials from P. falciparum clinical isolates. A large percentage (91%) of the represented transcripts was detected from Indian P. falciparum patient isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains. The 15K cross strain P. falciparum array has shown good efficiency in detecting transcripts from P. falciparum parasite samples isolated from patients. The low parasite loads and presence of host RNA makes arrays a preferred platform for gene expression studies over RNA-Seq.
Alan Kang-Wai Mu
Full Text Available Malaria is caused by parasitic protozoans of the genus Plasmodium and is one of the most prevalent infectious diseases in tropical and subtropical regions. For this reason, effective and practical diagnostic methods are urgently needed to control the spread of malaria. The aim of the current study was to identify a panel of new malarial markers, which could be used to diagnose patients infected with various Plasmodium species, including P. knowlesi, P. vivax and P. falciparum. Sera from malaria-infected patients were pooled and compared to control sera obtained from healthy individuals using the isobaric tags for relative and absolute quantitation (iTRAQ technique. Mass spectrometry was used to identify serum proteins and quantify their relative abundance. We found that the levels of several proteins were increased in pooled serum from infected patients, including cell adhesion molecule-4 and C-reactive protein. In contrast, the serum concentration of haptoglobin was reduced in malaria-infected individuals, which we verified by western blot assay. Therefore, these proteins might represent infectious markers of malaria, which could be used to develop novel diagnostic tools for detecting P. knowlesi, P. vivax and P. falciparum. However, these potential malarial markers will need to be validated in a larger population of infected individuals.
Diallo, Mamadou A; Badiane, Aida S; Diongue, Khadim; Deme, Awa; Lucchi, Naomi W; Gaye, Marie; Ndiaye, Tolla; Ndiaye, Mouhamadou; Sene, Louise K; Diop, Abdoulaye; Gaye, Amy; Ndiaye, Yaye D; Samb, Diama; Yade, Mamadou S; Ndir, Omar; Udhayakumar, Venkatachalam; Ndiaye, Daouda
Plasmodium ovale is rarely described in Senegal. A case of clinical malaria due to P. ovale wallikeri in West Central of Senegal is reported. A 34-year-old male baker in Dakar, with no significant previous medical history, was admitted to a health clinic with fever and vomiting. Fever had been lasting for 4 days with peaks every 48 h. As monospecific Plasmodium falciparum HRP-2 RDT was negative, he was treated with antibiotics. However, owing to persisting symptoms, he was referred to the emergency unit of the Youssou Mbargane Diop Hospital, Dakar, Senegal. Clinical examination found impaired general condition. All other physical examinations were normal. Laboratory tests showed anaemia (haemoglobin 11.4 g/dl), severe thrombocytopaenia (platelets 30 × 10(9)/mm(3)), leukopenia (3650/mm(3)), lymphocytopenia (650/mm(3)). Renal function was normal as indicated by creatininaemia and uraemia (11 mg/l and 0.25 g/l, respectively) and liver enzymes were slightly elevated (aspartate aminotransferase 77 UI/l and alanine aminotransferase 82 UI/l). Blood smear evaluations in Parasitology Laboratory of Aristide Le Dantec Hospital showed malaria parasites of the species P. ovale with a 0.08 % parasitaemia. Molecular confirmation was done by real time PCR targeting the 18S rRNA gene. The P. ovale infection was further analysed to species level targeting the potra gene and was identified as P. ovale wallikeri. According to the hospital's malaria treatment guidelines for severe malaria, treatment consisted of intravenous quinine at hour 0 (start of treatment) and 24 h after initial treatment, followed by artemether-lumefantrine 24 h later. A negative microscopy was noted on day 3 post-treatment and the patient reported no further symptoms. Malaria due to non-falciparum species is probably underestimated in Senegal. RDTs specific to non-falciparum species and/or pan specific RDTs should be included as tools of diagnosis to fight against malaria in Senegal. In addition
Wang, Bo; Pakpour, Nazzy; Napoli, Eleonora; Drexler, Anna; Glennon, Elizabeth K K; Surachetpong, Win; Cheung, Kong; Aguirre, Alejandro; Klyver, John M; Lewis, Edwin E; Eigenheer, Richard; Phinney, Brett S; Giulivi, Cecilia; Luckhart, Shirley
Fruit flies and mammals protect themselves against infection by mounting immune and metabolic responses that must be balanced against the metabolic needs of the pathogens. In this context, p38 mitogen-activated protein kinase (MAPK)-dependent signaling is critical to regulating both innate immunity and metabolism during infection. Accordingly, we asked to what extent the Asian malaria mosquito Anopheles stephensi utilizes p38 MAPK signaling during infection with the human malaria parasite Plasmodium falciparum. A. stephensi p38 MAPK (AsP38 MAPK) was identified and patterns of signaling in vitro and in vivo (midgut) were analyzed using phospho-specific antibodies and small molecule inhibitors. Functional effects of AsP38 MAPK inhibition were assessed using P. falciparum infection, quantitative real-time PCR, assays for reactive oxygen species and survivorship under oxidative stress, proteomics, and biochemical analyses. The genome of A. stephensi encodes a single p38 MAPK that is activated in the midgut in response to parasite infection. Inhibition of AsP38 MAPK signaling significantly reduced P. falciparum sporogonic development. This phenotype was associated with AsP38 MAPK regulation of mitochondrial physiology and stress responses in the midgut epithelium, a tissue critical for parasite development. Specifically, inhibition of AsP38 MAPK resulted in reduction in mosquito protein synthesis machinery, a shift in glucose metabolism, reduced mitochondrial metabolism, enhanced production of mitochondrial reactive oxygen species, induction of an array of anti-parasite effector genes, and decreased resistance to oxidative stress-mediated damage. Hence, P. falciparum-induced activation of AsP38 MAPK in the midgut facilitates parasite infection through a combination of reduced anti-parasite immune defenses and enhanced host protein synthesis and bioenergetics to minimize the impact of infection on the host and to maximize parasite survival, and ultimately, transmission
The haemoglobin measurement was done with the haematology analyzer, Sysmex KX-21N. Malaria parasites were enumerated and the presence of malaria pigment noted. Identification of P. falciparum was done. Statistical tests used were odds ratio and chi square at a significance level of p < 0.05. Results: 24.3% of the ...
Sulfadoxine-pyrimethamine (SP), the current first line antimalarial drug in Tanzania, is compromised by evolution and spread of mutations in the parasite's dhfr and dhps genes. In the present study we established the baseline frequencies of Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate ...
India being a tropical country, parasitic infections especially with Plasmodium species are very common in this region. The present case report is that of Plasmodium vivax, Plasmodium falciparum and dengue co‑infection in a 6 months pregnant lady who was timely diagnosed and appropriately treated followed by a ...
De Vries, Jutte J. C.; Van Assen, Sander; Mulder, André B.; Kampinga, Greetje A.
An adult traveler presented with fever and malaise after returning from Sierra Leone. Young trophozoites of Plasmodium falciparum were seen in a blood smear, with parasitemia being 10%. Moreover, blood cultures drawn on admission signaled as "positive" after 1 day of incubation, but no bacteria were
In addition, surgical gloves.were worn during the entire procedure to. protect both the operator and the isolates. Thick and thin blood smears were prepared from each blood sample, stained with Giemsa's solution and examined micro- scopically for the presence of P. falciparum.. Isolates were considered to be unsuitable for ...
Malaria and Human Immunodeficiency Virus (HIV) infections are major public health problems in Sub-Saharan Africa. Their overlapping geographical distribution and co-existence often result into high morbidity and mortality. This study was designed to establish the prevalence of Plasmodium falciparum malaria among HIV ...
Sep 11, 2009 ... B (1996) Plasma alpha-tocopherol retinol and carotenoids in children with falciparum malaria. Am.J.Clin.Nutr. 64: 94-100. 13. Kremsner, P. G., Greve, B., Lell, B.,. Luckner, D and Schmid, D. (2000). Malarial anaemia in African children associated with high oxygen radial production. Lancet 355: 40-41. 14.
Background Malaria parasites are frequently trans- mitted by unscreened blood transfusions in Africa. Pathogen reduction methods in whole blood would thus greatly improve blood safety. We aimed to determine the efficacy of riboflavin plus irradiation for treatment of whole blood infected with Plasmodium falciparum.
The in-vitro sensitivity of P. falciparum to quinine, mefloquine and halofantrine encourages the use of these drugs as alternative in case of chloroquine treatment failure. Nevertheless, it is important to maintain and to extend malaria and drug sensitivity surveillance in Madagascar. (East African Medical Journal: 2002 79(5): ...
Briolant, Sébastien; Almeras, Lionel; Belghazi, Maya; Boucomont-Chapeaublanc, Elodie; Wurtz, Nathalie; Fontaine, Albin; Granjeaud, Samuel; Fusaï, Thierry; Rogier, Christophe; Pradines, Bruno
The emergence of Plasmodium falciparum resistance to most anti-malarial compounds has highlighted the urgency to develop new drugs and to clarify the mechanisms of anti-malarial drugs currently used. Among them, doxycycline is used alone for malaria chemoprophylaxis or in combination with quinine or artemisinin derivatives for malaria treatment. The molecular mechanisms of doxycycline action in P. falciparum have not yet been clearly defined, particularly at the protein level. A proteomic approach was used to analyse protein expression changes in the schizont stage of the malarial parasite P. falciparum following doxycycline treatment. A comparison of protein expression between treated and untreated protein samples was performed using two complementary proteomic approaches: two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and isobaric tagging reagents for relative and absolute quantification (iTRAQ). After doxycycline treatment, 32 and 40 P. falciparum proteins were found to have significantly deregulated expression levels by 2D-DIGE and iTRAQ methods, respectively. Although some of these proteins have been already described as being deregulated by other drug treatments, numerous changes in protein levels seem to be specific to doxycycline treatment, which could perturb apicoplast metabolism. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm this hypothesis. In this study, a specific response to doxycycline treatment was distinguished and seems to involve mitochondrion and apicoplast organelles. These data provide a starting point for the elucidation of drug targets and the discovery of mechanisms of resistance to anti-malarial compounds.
Resistance of P. falciparum to the common and cheap antimalarial drugs chloroquine and sulpadoxyne-pyrimethamine has a profound public health impact in malaria endemic areas like Nigeria. This increasing drug resistance has necessitated change in antimalarial therapy in. Africa. In view of this, the World Health.
Wrenger, Carsten; Mueller, Ingrid B.; Schifferdecker, Anna J.; Jain, Rishabh; Jordanova, Rositsa; Groves, Matthew R.
Aspartate aminotransferases (AspATs; EC 18.104.22.168) catalyze the conversion of aspartate and α-ketoglutarate into oxaloacetate and glutamate and are key enzymes in the nitrogen metabolism of all organisms. Recent findings suggest that the plasmodial enzyme [Plasmodium falciparum aspartate
Hogh, B; Petersen, E; Dziegiel, Morten Hanefeld
A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain two...
Schmidt, Christoph Q; Kennedy, Alexander T; Tham, Wai-Hong
Malaria remains one of the world's deadliest diseases. Plasmodium falciparum is responsible for the most severe and lethal form of human malaria. P. falciparum's life cycle involves two obligate hosts: human and mosquito. From initial entry into these hosts, malaria parasites face the onslaught of the first line of host defence, the complement system. In this review, we discuss the complex interaction between complement and malaria infection in terms of hosts immune responses, parasite survival and pathogenesis of severe forms of malaria. We will focus on the role of complement receptor 1 and its associated polymorphisms in malaria immune complex clearance, as a mediator of parasite rosetting and as an entry receptor for P. falciparum invasion. Complement evasion strategies of P. falciparum parasites will also be highlighted. The sexual forms of the malaria parasites recruit the soluble human complement regulator Factor H to evade complement-mediated killing within the mosquito host. A novel evasion strategy is the deployment of parasite organelles to divert complement attack from infective blood stage parasites. Finally we outline the future challenge to understand the implications of these exploitation mechanisms in the interplay between successful infection of the host and pathogenesis observed in severe malaria. Copyright © 2015 Elsevier Ltd. All rights reserved.
Lunev, Sergey; Bosch, Soraya S.; Batista, Fernando de Assis; Wrenger, Carsten; Groves, Matthew R.
The de novo pyrimidine-biosynthesis pathway of Plasmodium falciparum is a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is unable to take up pyrimidines from the host. Direct (or indirect) inhibition of de
Egwang, T G; Apio, B; Riley, E; Okello, D
To establish Plasmodium falciparum malariometric indices in a field study site in Apac district, northern Uganda. A community-based cross sectional survey. Atopi Parish, Apac district, Uganda, 1995. One thousand two hundred and thirty four volunteers aged below one and ninety years. P. falciparum parasitaemia rates and parasite density, splenomegaly, bednet use and chloroquine consumption. All subjects with P. falciparum positive smears were treated with chloroquine. The population prevalence of parasitaemia was 62.1% with the predominant species being P. falciparum (100%) and P. malariae in the minority (3.5%); P. ovale was not seen. The prevalence of parasitaemia in subjects older than 20 years and in those under ten years was 36% and 85%, respectively. The geometric mean parasite density started to decline by the age of six years. The splenomegaly rate in subjects over the age of 12 years and in those under nine years was 19.8% and 63.1%, respectively. Bednet use and chloroquine consumption was low. Interestingly, the reported use of chloroquine in the week immediately preceding the study was more frequent in children under two years old than in the rest of the population. Malaria transmission in Atopi Parish in northern Uganda is hyperendemic and age-related acquired anti-parasite immunity seems to appear by seven years of age.
Price, R. N.; Simpson, J. A.; Nosten, F.; Luxemburger, C.; Hkirjaroen, L.; ter Kuile, F.; Chongsuphajaisiddhi, T.; White, N. J.
The factors contributing to anemia in falciparum malaria were characterized in 4,007 prospectively studied patients on the western border of Thailand. Of these, 727 patients (18%) presented with anemia (haematocrit < 30%), and 1% (55 of 5,253) required blood transfusion. The following were found to
Mar 18, 2008 ... ISSN 1684–5315 © 2008 Academic Journals. Full Length Research Paper. Variation of nitric oxide levels in imported Plasmodium falciparum malaria episodes. De Sousa, Karina*, Silva, Marcelo S. and Tavira, Luís T. Instituto de Higiene e Medicina Tropical, Centro de Malária e outras Doenças Tropicais, ...
We have initiated such a study and presented herewith the results from the in silico understanding of a seventh chromosomal region of the malarial parasite Plasmodium falciparum encompassing the antigenic var genes (coding pfemp1) and the drug-resistant gene pfcrt located at a specified region of the chromosome 7.
This study was undertaken to establish data on the effect of acute falciparum malaria on plasma levels some biochemical parameters in the pathology of malaria in Nigeria children. We estimated the levels of. Na+, K+, HCO3, Ca++ , inorganic PO4 =, bilirubin, total protein, albumin, urea, creatinine and glucose in the plasma ...
Mockenhaupt, Frank P.; Ehrhardt, Stephan; Eggelte, Teunis A.; Markert, Miriam; Anemana, Sylvester; Otchwemah, Rowland; Bienzle, Ulrich
In 366 Ghanaian children with symptomatic Plasmodium falciparum malaria, low haemoglobin levels and severe anaemia were associated with a high multiplicity of infection (MOI) and with distinct merozoite surface protein alleles. High MOI not only reflects premunition but may also contribute to
Background: Acute malarial anemia remains a major public health problem. Hepcidin, the major hormone controlling the availability of iron, is raised during acute and asymptomatic parasitemia. Understanding the role and mechanism of raised hepcidin and so reduced iron availability during infection is critical to establish evidence-based guidelines for management of malaria anemia. Our recent clinical evidence suggests a potential role of IL-10 in the regulation of hepcidin in patients with acute P. falciparum malaria. Methods: We have measured secretion of hepcidin by primary macrophages and the hepatoma cell line HepG2 stimulated with IL-10, IL-6 and Plasmodium falciparum-infected erythrocytes. Findings: We have observed that IL-10 and IL-6 production increased in primary macrophages when these cells were co-cultured with Plasmodium falciparum-infected erythrocytes. We found that IL-10 induced hepcidin secretion in primary macrophages in a dose-dependent manner but not in HepG2 cells. These effects were mediated through signal transducer and activator of transcription (STAT) 3-phosphorylation and completely abrogated by a specific STAT3 inhibitor. Conclusion: IL-10 can directly regulate hepcidin in primary macrophages but not in HepG2 cells. This effect can be modulated by Plasmodium falciparum. The results are consistent with a role for IL-10 in modulating iron metabolism during acute phase of infection. 2014 Huang et al.
Nielsen, H; Theander, T G
The release of superoxide anion from blood monocytes was studied in eight patients with acute primary attack P. falciparum malaria. Before treatment a significant enhancement of the oxidative burst prevailed, which contrasts with previous findings of a depressed monocyte chemotactic responsiveness...
Tuikue Ndam, Nicaise; Bischoff, Emmanuel; Proux, Caroline
BACKGROUND: Pregnancy-associated malaria (PAM) causing maternal anemia and low birth weight is among the multiple manifestations of Plasmodium falciparum malaria. Infected erythrocytes (iEs) can acquire various adhesive properties that mediate the clinical severity of malaria. Recent advances...
Fine-scale genetic characterization of Plasmodium falciparum chromosome 7 encompassing the antigenic var and the drug-resistant pfcrt genes. RUCHI BAJAJ1, SUJATA MOHANTY2, A. P. DASH1 and APARUP DAS1, ∗. 1Evolutionary Genomics and Bioinformatics Laboratory, National Institute of Malaria Research,.
Dr Oboro VO
ABSTRACT. Background: drug resistant malaria is spreading inexorably to areas with drug sensitive malaria parasites. This study compared the in vitro sensitivities of Plasmodium falciparum fresh parasite isolates, to some standard antimalarial drugs, in Makurdi and Masaka located over 300 km apart, in north central.
This study was carried out to determine the efficiency of two reagents, RNAlater and RNAwiz, for their ability to stabilize Plasmodium falciparum RNA in infected whole blood and saponin lysed parasite pellets for use in DNA microarrays. Eight infected blood samples were stored in each of the reagents, and RNA extracted at ...
Hogh, B; Petersen, E; Dziegiel, Morten Hanefeld
A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain t...
Changing the malaria epidemiology will affect the genetic diversity of Plasmodium falciparum. We studied the association between diversity at the merozoite surface protein 2 loci and the severity of disease in childhood malaria in two populations and at different time periods in Ibadan, southwest Nigeria. Population A ...
Jensen, Anja T R; Magistrado, Pamela; Sharp, Sarah
Parasite-encoded variant surface antigens (VSAs) like the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria...... in nonimmune patients tend to express a restricted subset of VSA (VSA(SM)) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSA(UM)). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSA(UM) to in vitro-selected sublines expressing VSA......(SM) to identify PfEMP1 responsible for the VSA(SM) phenotype. Expression of VSA(SM) was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var...
Tessema, Sofonias K; Monk, Stephanie L; Schultz, Mark B; Tavul, Livingstone; Reeder, John C; Siba, Peter M; Mueller, Ivo; Barry, Alyssa E
Plasmodium falciparum malaria is a major global health problem that is being targeted for progressive elimination. Knowledge of local disease transmission patterns in endemic countries is critical to these elimination efforts. To investigate fine-scale patterns of malaria transmission, we have compared repertoires of rapidly evolving var genes in a highly endemic area. A total of 3680 high-quality DBLα-sequences were obtained from 68 P. falciparum isolates from ten villages spread over two distinct catchment areas on the north coast of Papua New Guinea (PNG). Modelling of the extent of var gene diversity in the two parasite populations predicts more than twice as many var gene alleles circulating within each catchment (Mugil = 906; Wosera = 1094) than previously recognized in PNG (Amele = 369). In addition, there were limited levels of var gene sharing between populations, consistent with local parasite population structure. Phylogeographic analyses demonstrate that while neutrally evolving microsatellite markers identified population structure only at the catchment level, var gene repertoires reveal further fine-scale geospatial clustering of parasite isolates. The clustering of parasite isolates by village in Mugil, but not in Wosera was consistent with the physical and cultural isolation of the human populations in the two catchments. The study highlights the microheterogeneity of P. falciparum transmission in highly endemic areas and demonstrates the potential of var genes as markers of local patterns of parasite population structure. © 2014 John Wiley & Sons Ltd.
Rosalynn L Ord
Full Text Available BACKGROUND: We present the first population genetic analysis of homologous loci from two sympatric human malaria parasite populations sharing the same human hosts, using full-length sequences of ama1 genes from Plasmodium vivax and P. falciparum collected in the Venezuelan Amazon. METHODOLOGY/PRINCIPAL FINDINGS: Significant differences between the two species were found in genetic diversity at the ama1 locus, with 18 distinct haplotypes identified among the 73 Pvama1 sequences obtained, compared to 6 unique haplotypes from 30 Pfama1 sequences, giving overall diversity estimates of h = 0.9091, and h = 0.538 respectively. Levels of recombination were also found to differ between the species, with P. falciparum exhibiting very little recombination across the 1.77 kb sequence. In contrast, analysis of patterns of nucleotide substitutions provided evidence that polymorphisms in the ama1 gene of both species are maintained by balancing selection, particularly in domain I. The two distinct population structures observed are unlikely to result from different selective forces acting upon the two species, which share both human and mosquito hosts in this setting. Rather, the highly structured P. falciparum population appears to be the result of a population bottleneck, while the much less structured P. vivax population is likely to be derived from an ancient pool of diversity, as reflected in a larger estimate of effective population size for this species. Greatly reduced mosquito transmission in 1997, due to low rainfall prior to the second survey, was associated with far fewer P. falciparum infections, but an increase in P. vivax infections, probably due to hypnozoite activation. CONCLUSIONS/SIGNIFICANCE: The relevance of these findings to putative competitive interactions between these two important human pathogen species is discussed. These results highlight the need for future control interventions to employ strategies targeting each of the parasite
Clinical presentation of severe malaria due plasmodiun falciparum. casecontrol study in Tumaco and Turbo (Colombia. Clínica de la malaria complicada debida a P. falciparum Estudio de casos y controles en Tumaco y Turbo (Colombia
Jaime Carmona Fonseca
Full Text Available Background: Latin American studies on severe falciparum malaria are scarce, therefore, the pattern of complications of the region is uknown. Objectives. To identify characterize severe malaria in patients from Tumaco (Nariño and Turbo (Antioquia in Colombia. Methods. The 2000 World Health Organization criteria for complicated malaria were applied in a cases and controls study. Results. 64 cases (P falciparum complicated malaria and 135 controls (P falciparum uncomplicated malaria were included. The time of evolution of the disease (mean 5.6 days in cases and 5.9 in the controls and the frequency of most symptoms were similar in both groups (p>0.05. However, respiratory distress and jaundice was more frequent in the cases (p<0.05. The mean glycemia and creatinina values were similar in both groups; hemoglobin and platelet count were lower in the cases (p<0.05 when compared to controls. On the other hand, blood ureic nitrogen, aspartatoaminotransferase, and total and direct bilirrubin were lower in controls (p<0.05. The frequency of complications in the cases was as follows: hyperparasitaemia 48%, liver dysfunction 44%, acute respiratory distress syndrome 9%, kidney failure 6%, severe thrombocytopenia 5%, severe anemia 3%, cerebral malaria 3% and severe hipoglycemia 2%. The WHO criteria for severe malaria were compared with others and the implications are discussed. Antecedentes y problema: son muy pocos los estudios latinoamericanos sobre malaria por Plasmodium falciparum (P falciparum complicada y se requiere estudiarla para identificar un patrón propio. OBJETIVOS. Identificar las complicaciones presentes en pacientes de Tumaco (Nariño y Turbo (Antioquia en Colombia, con malaria por P falciparum. MÉTODOS. Diseño de casos y controles. Se aplicaron los criterios diagnósticos de complicación OMS-2000 (Organización Mundial de la Salud. RESULTADOS. Se captaron 64 casos (con malaria por P. falciparum complicada y 135 controles (con malaria por
Guerra, Ángela Patricia; Calvo, Eliana Patricia; Wasserman, Moisés; Chaparro-Olaya, Jacqueline
The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies. To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8). The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice. The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies. The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.
Van Rooijen Nico
Full Text Available Abstract Background Mice with genetic deficiencies in adaptive immunity are used for the grafting of human cells or pathogens, to study human diseases, however, the innate immune responses to xenografts in these mice has received little attention. Using the NOD/SCID Plasmodium falciparum mouse model an analysis of innate defences responsible for the substantial control of P. falciparum which remains in such mice, was performed. Methods NOD/SCID mice undergoing an immunomodulatory protocol that includes, clodronate-loaded liposomes to deplete macrophages and an anti-polymorphonuclear leukocytes antibody, were grafted with human red blood cells and P. falciparum. The systematic and kinetic analysis of the remaining innate immune responses included the number and phenotype of peripheral blood leukocytes as well as inflammatory cytokines/chemokines released in periphery. The innate responses towards the murine parasite Plasmodium yoelii were used as a control. Results Results show that 1 P. falciparum induces a strong inflammation characterized by an increase in circulating leukocytes and the release of inflammatory cytokines; 2 in contrast, the rodent parasite P. yoelii, induces a far more moderate inflammation; 3 human red blood cells and the anti-inflammatory agents employed induce low-grade inflammation; and 4 macrophages seem to bear the most critical function in controlling P. falciparum survival in those mice, whereas polymorphonuclear and NK cells have only a minor role. Conclusions Despite the use of an immunomodulatory treatment, immunodeficient NOD/SCID mice are still able to mount substantial innate responses that seem to be correlated with parasite clearance. Those results bring new insights on the ability of innate immunity from immunodeficient mice to control xenografts of cells of human origin and human pathogens.
Sylla, Khadime; Tine, Roger Clément Kouly; Ndiaye, Magatte; Sow, Doudou; Sarr, Aïssatou; Mbuyi, Marie Louise Tshibola; Diouf, Ibrahima; Lô, Amy Colé; Abiola, Annie; Seck, Mame Cheikh; Ndiaye, Mouhamadou; Badiane, Aïda Sadikh; N'Diaye, Jean Louis A; Ndiaye, Daouda; Faye, Oumar; Dieng, Thérèse; Dieng, Yémou; Ndir, Oumar; Gaye, Oumar; Faye, Babacar
In Senegal, a significant decrease of malaria transmission intensity has been noted the last years. Parasitaemia has become lower and, therefore, more difficult to detect by microscopy. In the context of submicroscopic parasitaemia, it has become relevant to rely on relevant malaria surveillance tools to better document malaria epidemiology in such settings. Serological markers have been proposed as an essential tool for malaria surveillance. This study aimed to evaluate the sero-epidemiological situation of Plasmodium falciparum malaria in two sentinel sites in Senegal. Cross-sectional surveys were carried out in Velingara (south Senegal) and Keur Soce (central Senegal) between September and October 2010. Children under 10 years old, living in these areas, were enrolled using two-level, random sampling methods. P. falciparum infection was diagnosed using microscopy. P. falciparum antibodies against circumsporozoite protein (CSP), apical membrane protein (AMA1) and merozoite surface protein 1_42 (MSP1_42) were measured by ELISA method. A stepwise logistic regression analysis was done to assess factors associated with P. falciparum antibodies carriage. A total of 1,865 children under 10 years old were enrolled. The overall falciparum malaria prevalence was 4.99% with high prevalence in Velingara of 10.03% compared to Keur Soce of 0.3%. Symptomatic malaria cases (fever associated with parasitaemia) represented 17.37%. Seroprevalence of anti-AMA1, anti-MSP1_42 and anti-CSP antibody was 38.12, 41.55 and 40.38%, respectively. The seroprevalence was more important in Velingara and increased with age, active malaria infection and area of residence. The use of serological markers can contribute to improved malaria surveillance in areas with declining malaria transmission. This study provided useful baseline information about the sero-epidemiological situation of malaria in Senegal and can contribute to the identification of malaria hot spots in order to concentrate
Magistrado, Pamela A; Lusingu, John; Vestergaard, Lasse S
where P. falciparum is endemic, parasites causing severe malaria and malaria in young children with limited immunity tend to express semiconserved PfEMP1 molecules encoded by group A var genes. Here we investigated antibody responses of Tanzanians who were 0 to 19 years old to PF11_0008, a group A Pf......Variant surface antigens (VSA) on the surface of Plasmodium falciparum-infected red blood cells play a major role in the pathogenesis of malaria and are key targets for acquired immunity. The best-characterized VSA belong to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. In areas...... of antibodies to the PF11_0008 CIDR2beta domain was associated with reduced numbers of malaria episodes. These results indicate that homologues of PF11_0008 are present in P. falciparum field isolates and suggest that PF11_0008 CIDR2beta-reactive antibodies might be involved in protection against malaria...
Full Text Available Imported malaria in France is characterized by various clinical manifestations observed in a heterogeneous population of patients such as travelers/expatriates and African migrants. In this population, host factors and parasite biomass associated with severe imported malaria are poorly known.From data collected by the Centre National de Référence du Paludisme, we identified epidemiological, demographic and biological features including parasite biomass and anti-plasmodial antibody levels (negative, positive and strongly positive serology associated with different disease severity groups (very severe, moderately severe, and uncomplicated malaria in 3 epidemiological groups (travelers/expatriates, first- and second-generation migrants.Age, ethnicity, absence of prior infection with P. falciparum, antibody levels, plasma PfHRP2 levels, total and circulating parasite biomass were related to severe malaria onset. Sequestered parasite biomass tended to be increased in very severe malaria, and was strongly correlated to the antibody level of the host.Prior exposure to P. falciparum is associated with high anti-plasmodial antibody levels which influence clinical presentation of imported malaria and its correlated circulating and sequestered parasite burden.
Surachetpong, Win; Singh, Naresh; Cheung, Kong Wai; Luckhart, Shirley
Malaria is caused by infection with intraerythrocytic protozoa of the genus Plasmodium that are transmitted by Anopheles mosquitoes. Although a variety of anti-parasite effector genes have been identified in anopheline mosquitoes, little is known about the signaling pathways that regulate these responses during parasite development. Here we demonstrate that the MEK-ERK signaling pathway in Anopheles is controlled by ingested human TGF-beta1 and finely tunes mosquito innate immunity to parasite infection. Specifically, MEK-ERK signaling was dose-dependently induced in response to TGF-beta1 in immortalized cells in vitro and in the A. stephensi midgut epithelium in vivo. At the highest treatment dose of TGF-beta1, inhibition of ERK phosphorylation increased TGF-beta1-induced expression of the anti-parasite effector gene nitric oxide synthase (NOS), suggesting that increasing levels of ERK activation negatively feed back on induced NOS expression. At infection levels similar to those found in nature, inhibition of ERK activation reduced P. falciparum oocyst loads and infection prevalence in A. stephensi and enhanced TGF-beta1-mediated control of P. falciparum development. Taken together, our data demonstrate that malaria parasite development in the mosquito is regulated by a conserved MAPK signaling pathway that mediates the effects of an ingested cytokine.
Surachetpong, Win; Singh, Naresh; Cheung, Kong Wai; Luckhart, Shirley
Malaria is caused by infection with intraerythrocytic protozoa of the genus Plasmodium that are transmitted by Anopheles mosquitoes. Although a variety of anti-parasite effector genes have been identified in anopheline mosquitoes, little is known about the signaling pathways that regulate these responses during parasite development. Here we demonstrate that the MEK-ERK signaling pathway in Anopheles is controlled by ingested human TGF-β1 and finely tunes mosquito innate immunity to parasite infection. Specifically, MEK-ERK signaling was dose-dependently induced in response to TGF-β1 in immortalized cells in vitro and in the A. stephensi midgut epithelium in vivo. At the highest treatment dose of TGF-β1, inhibition of ERK phosphorylation increased TGF-β1-induced expression of the anti-parasite effector gene nitric oxide synthase (NOS), suggesting that increasing levels of ERK activation negatively feed back on induced NOS expression. At infection levels similar to those found in nature, inhibition of ERK activation reduced P. falciparum oocyst loads and infection prevalence in A. stephensi and enhanced TGF-β1-mediated control of P. falciparum development. Taken together, our data demonstrate that malaria parasite development in the mosquito is regulated by a conserved MAPK signaling pathway that mediates the effects of an ingested cytokine. PMID:19343212
Charles, Macarthur; Das, Sanchita; Daniels, Rachel; Kirkman, Laura; Delva, Glavdia G.; Destine, Rodney; Escalante, Ananias; Villegas, Leopoldo; Daniels, Noah M.; Shigyo, Kristi; Volkman, Sarah K.; Pape, Jean W.
Hispaniola is the only Caribbean island to which Plasmodium falciparum malaria remains endemic. Resistance to the antimalarial drug chloroquine has rarely been reported in Haiti, which is located on Hispaniola, but the K76T pfcrt (P. falciparum chloroquine resistance transporter) gene mutation that confers chloroquine resistance has been detected intermittently. We analyzed 901 patient samples collected during 2006–2009 and found 2 samples showed possible mixed parasite infections of genetically chloroquine-resistant and -sensitive parasites. Direct sequencing of the pfcrt resistance locus and single-nucleotide polymorphism barcoding did not definitively identify a resistant population, suggesting that sustained propagation of chloroquine-resistant parasites was not occurring in Haiti during the study period. Comparison of parasites from Haiti with those from Colombia, Panama, and Venezuela reveals a geographically distinct population with highly related parasites. Our findings indicate low genetic diversity in the parasite population and low levels of chloroquine resistance in Haiti, raising the possibility that reported cases may be of exogenous origin. PMID:27089479
M El Bakkouri; A Pow; A Mulichak; K Cheung; J Artz; M Amani; S Fell; T de Koning-Ward; C Goodman; et al.
The Clpchaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clpchaperones and proteases in the humanmalariaparasitePlasmodiumfalciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clpchaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.
Magistrado, Pamela A; Lusingu, John; Vestergaard, Lasse S
Variant surface antigens (VSA) on the surface of Plasmodium falciparum-infected red blood cells play a major role in the pathogenesis of malaria and are key targets for acquired immunity. The best-characterized VSA belong to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. In are...
Cavanagh, David R; Kocken, Clemens H M; White, John H; Cowan, Graeme J M; Samuel, Kay; Dubbeld, Martin A; Voorberg-van der Wel, Annemarie; Thomas, Alan W; McBride, Jana S; Arnot, David E
The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals.
Lilian Rose Pratt-Riccio
Full Text Available The genetic diversity displayed by Plasmodium falciparum, the most deadly Plasmodium species, is a significant obstacle for effective malaria vaccine development. In this study, we identified genetic polymorphisms in P. falciparum glutamate-rich protein (GLURP, which is currently being tested in clinical trials as a malaria vaccine candidate, from isolates found circulating in the Brazilian Amazon at variable transmission levels. The study was performed using samples collected in 1993 and 2008 from rural villages situated near Porto Velho, in the state of Rondônia. DNA was extracted from 126 P. falciparum-positive thick blood smears using the phenol-chloroform method and subjected to a nested polymerase chain reaction protocol with specific primers against two immunodominant regions of GLURP, R0 and R2. Only one R0 fragment and four variants of the R2 fragment were detected. No differences were observed between the two time points with regard to the frequencies of the fragment variants. Mixed infections were uncommon. Our results demonstrate conservation of GLURP-R0 and limited polymorphic variation of GLURP-R2 in P. falciparum isolates from individuals living in Porto Velho. This is an important finding, as genetic polymorphisms in B and T-cell epitopes could have implications for the immunological properties of the antigen.
Abba, Katharine; Deeks, Jonathan J; Olliaro, Piero; Naing, Cho-Min; Jackson, Sally M; Takwoingi, Yemisi; Donegan, Sarah; Garner, Paul
Rapid diagnostic tests (RDTs) for Plasmodium falciparum malaria use antibodies to detect either HRP-2 antigen or pLDH antigen, and can improve access to diagnostics in developing countries. To assess the diagnostic accuracy of RDTs for detecting P. falciparum parasitaemia in persons living in endemic areas who present to ambulatory healthcare facilities with symptoms suggestive of malaria by type and brand. We undertook a comprehensive search of the following databases: Cochrane Infectious Diseases Group Specialized Register; MEDLINE; EMBASE; MEDION; Science Citation Index; Web of Knowledge; African Index Medicus; LILACS; IndMED; to January 14, 2010. Studies comparing RDTs with a reference standard (microscopy or polymerase chain reaction) in blood samples from a random or consecutive series of patients attending ambulatory health facilities with symptoms suggestive of malaria in P. falciparum endemic areas. For each study, a standard set of data was extracted independently by two authors, using a tailored data extraction form. Comparisons were grouped hierarchically by target antigen, and type and brand of RDT, and combined in meta-analysis where appropriate. We identified 74 unique studies as eligible for this review and categorized them according to the antigens they detected. Types 1 to 3 include HRP-2 (from P. falciparum) either by itself or with other antigens. Types 4 and 5 included pLDH (from P. falciparum) either by itself or with other antigens. In comparisons with microscopy, we identified 71 evaluations of Type 1 tests, eight evaluations of Type 2 tests and five evaluations of Type 3 tests. In meta-analyses, average sensitivities and specificities (95% CI) were 94.8% (93.1% to 96.1%) and 95.2% (93.2% to 96.7%) for Type 1 tests, 96.0% (94.0% to 97.3%) and 95.3% (87.3% to 98.3%) for Type 2 tests, and 99.5% (71.0% to 100.0%) and 90.6% (80.5% to 95.7%) for Type 3 tests, respectively. Overall for HRP-2, the meta-analytical average sensitivity and
Full Text Available Abstract Background In Cambodia, malaria transmission is low and most cases occur in forested areas. Sero-epidemiological techniques can be used to identify both areas of ongoing transmission and high-risk groups to be targeted by control interventions. This study utilizes repeated cross-sectional data to assess the risk of being malaria sero-positive at two consecutive time points during the rainy season and investigates who is most likely to sero-convert over the transmission season. Methods In 2005, two cross-sectional surveys, one in the middle and the other at the end of the malaria transmission season, were carried out in two ecologically distinct regions in Cambodia. Parasitological and serological data were collected in four districts. Antibodies to Plasmodium falciparum Glutamate Rich Protein (GLURP and Plasmodium vivax Merozoite Surface Protein-119 (MSP-119 were detected using Enzyme Linked Immunosorbent Assay (ELISA. The force of infection was estimated using a simple catalytic model fitted using maximum likelihood methods. Risks for sero-converting during the rainy season were analysed using the Classification and Regression Tree (CART method. Results A total of 804 individuals participating in both surveys were analysed. The overall parasite prevalence was low (4.6% and 2.0% for P. falciparum and 7.9% and 6.0% for P. vivax in August and November respectively. P. falciparum force of infection was higher in the eastern region and increased between August and November, whilst P. vivax force of infection was higher in the western region and remained similar in both surveys. In the western region, malaria transmission changed very little across the season (for both species. CART analysis for P. falciparum in the east highlighted age, ethnicity, village of residence and forest work as important predictors for malaria exposure during the rainy season. Adults were more likely to increase their antibody responses to P. falciparum during the
Ray, Sandipan; Renu, Durairaj; Srivastava, Rajneesh; Gollapalli, Kishore; Taur, Santosh; Jhaveri, Tulip; Dhali, Snigdha; Chennareddy, Srinivasarao; Potla, Ankit; Dikshit, Jyoti Bajpai; Srikanth, Rapole; Gogtay, Nithya; Thatte, Urmila; Patankar, Swati; Srivastava, Sanjeeva
This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites Plasmodium falciparum and P. vivax to obtain mechanistic insights about disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with falciparum malaria (FM) (n = 20), vivax malaria (VM) (n = 17) and healthy controls (HC) (n = 20) were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC). Compared to HC, 30 and 31 differentially expressed and statistically significant (p<0.05) serum proteins were identified in FM and VM respectively, and almost half (46.2%) of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC) were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC) curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates
Full Text Available This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites Plasmodium falciparum and P. vivax to obtain mechanistic insights about disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with falciparum malaria (FM (n = 20, vivax malaria (VM (n = 17 and healthy controls (HC (n = 20 were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC. Compared to HC, 30 and 31 differentially expressed and statistically significant (p<0.05 serum proteins were identified in FM and VM respectively, and almost half (46.2% of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates
Dziegiel, Morten Hanefeld; Borre, M B; Petersen, E
This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactos......This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta...
Full Text Available Emergence of rapid drug resistance to existing antimalarial drugs in Plasmodium falciparum has created the need for prediction of novel targets as well as leads derived from original molecules with improved activity against a validated drug target. The malaria parasite has a plant plastid-like apicoplast. To overcome the problem of falciparum malaria, the metabolic pathways in parasite apicoplast have been used as antimalarial drug targets. Among several pathways in apicoplast, isoprenoid biosynthesis is one of the important pathways for parasite as its multiplication in human erythrocytes requires isoprenoids. Therefore targeting this pathway and exploring leads with improved activity is a highly attractive approach. This report has explored progress towards the study of proteins and inhibitors of isoprenoid biosynthesis pathway. For more comprehensive analysis, antimalarial drug-protein interaction has been covered.
Skinner-Adams, Tina S; Stack, Colin M; Trenholme, Katharine R; Brown, Chris L; Grembecka, Jolanta; Lowther, Jonathan; Mucha, Artur; Drag, Marcin; Kafarski, Pawel; McGowan, Sheena; Whisstock, James C; Gardiner, Donald L; Dalton, John P
The neutral aminopeptidases M1 alanyl aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) of the human malaria parasite Plasmodium falciparum are targets for the development of novel anti-malarial drugs. Although the functions of these enzymes remain unknown, they are believed to act in the terminal stages of haemoglobin degradation, generating amino acids essential for parasite growth and development. Inhibitors of both enzymes are lethal to P. falciparum in culture and kill the murine malaria P. chabaudi in vivo. Recent biochemical, structural and functional studies provide the substrate specificity and mechanistic binding data needed to guide the development of more potent anti-malarial drugs. Together with biological studies, these data form the rationale for choosing PfM1AAP and PfM17LAP as targets for anti-malarial development. Copyright 2009 Elsevier Ltd. All rights reserved.
Singh, Shubhra; Puri, S K; Srivastava, Kumkum
A comparative efficacy of four antibiotics, plasmocin (macrolid), Biomyc-1, -2, (tetracycline), and Biomyc-3, and Mycoplasma Removing Agent (quinolone derivatives) was determined for elimination of mycoplasma from Plasmodium falciparum culture. Presence of mycoplasma was detected using enzyme-PCR-based mycoplasma detection kit and survival of malaria parasite was determined in Giemsa's stained smear made from treated and untreated cultures. It was observed that a combination of Biomyc-1 and -2 killed malaria parasites within 24 h, whereas plasmocin and Biomyc-3 caused slow death of malaria parasite stretched over a period of 6 days. The only compound which did not kill malaria parasite and eradicated mycoplasma from P. falciparum culture was observed to be MRA.
Full Text Available Malaria in pregnancy is a serious public health problem in tropical areas. Frequently, the placenta is infected by accumulation of Plasmodium falciparum-infected erythrocytes in the intervillous space. Falciparum malaria acts during pregnancy by a range of mechanisms, and chronic or repeated infection and co-infections have insidious effects. The susceptibility of pregnant women to malaria is due to both immunological and humoral changes. Until a malaria vaccine becomes available, the deleterious effects of malaria in pregnancy can be avoided by protection against infection and prompt treatment with safe, effective antimalarial agents; however, concurrent infections such as with HIV and helminths during pregnancy are jeopardizing malaria control in sub-Saharan Africa.
Goel, Suchi; Palmkvist, Mia; Moll, Kirsten
Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum–encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs—preferentiall......Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum–encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs......—preferentially of blood group A—to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population....
Helegbe, Gideon K; Goka, Bamenla Q; Kurtzhals, Jørgen
BACKGROUND: Severe anaemia (SA), intravascular haemolysis (IVH) and respiratory distress (RD) are severe forms of Plasmodium falciparum malaria, with RD reported to be of prognostic importance in African children with malarial anaemia. Complement factors have been implicated in the mechanism......55)] in children with discrete clinical forms of P. falciparum malaria. The relationship between the findings and clinical parameters including coma, haemoglobin (Hb) levels and RD were investigated. RESULTS: Of the 484 samples tested, 131(27%) were positive in DCT, out of which 115/131 (87.8%) were...... malaria, possibly through induction of erythrophagocytosis and haemolysis. In contrast to other studies, this study did not find association between levels of the complement...
Full Text Available Abstract Artemisinin-based combination therapies (ACTs are recommended for use against uncomplicated malaria in areas of multi-drug resistant malaria, such as sub-Saharan Africa. However, their long-term usefulness in these high transmission areas remains unclear. It has been suggested that documentation of the S769N PfATPase6 mutations may indicate an emergence of artemisinin resistance of Plasmodium falciparum in the field. The present study assessed PfATPase6 mutations (S769N and A623E in 615 asymptomatic P. falciparum infections in Tanzania but no mutant genotype was detected. This observation suggests that resistance to artemisinin has not yet been selected in Tanzania, supporting the Ministry of Health's decision to adopt artemether+lumefantrine as first-line malaria treatment. The findings recommend further studies to assess PfATPase6 mutations in sentinel sites and verify their usefulness in monitoring emergency of ACT resistance.
Esen, Meral; Kremsner, Peter G; Schleucher, Regina
Malaria is a major public health problem in Sub-Saharan Africa. In highly endemic regions infants, children and pregnant women are mostly affected. An effective malaria vaccine would complement existing malaria control strategies because it can be integrated in existing immunization programs easily......-immune individuals. Ten, 30 and 100 microg of GMZ2 were well tolerated in 30 healthy malaria-naïve German volunteers when given three times in monthly intervals. Antigen-specific antibodies as well as memory B-cells were induced and detectable throughout the one year follow-up of the study. We conclude that GMZ2...... is a safe and immunogenic malaria vaccine candidate suitable for further clinical development....
Dwivedi, Ankit; Reynes, Christelle; Kuehn, Axel; Roche, Daniel B; Khim, Nimol; Hebrard, Maxim; Milanesi, Sylvain; Rivals, Eric; Frutos, Roger; Menard, Didier; Mamoun, Choukri Ben; Colinge, Jacques; Cornillot, Emmanuel
Plasmodium falciparum malaria is one of the most widespread parasitic infections in humans and remains a leading global health concern. Malaria elimination efforts are threatened by the emergence and spread of resistance to artemisinin-based combination therapy, the first-line treatment of malaria. Promising molecular markers and pathways associated with artemisinin drug resistance have been identified, but the underlying molecular mechanisms of resistance remains unknown. The genomic data from early period of emergence of artemisinin resistance (2008-2011) was evaluated, with aim to define k13 associated genetic background in Cambodia, the country identified as epicentre of anti-malarial drug resistance, through characterization of 167 parasite isolates using a panel of 21,257 SNPs. Eight subpopulations were identified suggesting a process of acquisition of artemisinin resistance consistent with an emergence-selection-diffusion model, supported by the shifting balance theory. Identification of population specific mutations facilitated the characterization of a core set of 57 background genes associated with artemisinin resistance and associated pathways. The analysis indicates that the background of artemisinin resistance was not acquired after drug pressure, rather is the result of fixation followed by selection on the daughter subpopulations derived from the ancestral population. Functional analysis of artemisinin resistance subpopulations illustrates the strong interplay between ubiquitination and cell division or differentiation in artemisinin resistant parasites. The relationship of these pathways with the P. falciparum resistant subpopulation and presence of drug resistance markers in addition to k13, highlights the major role of admixed parasite population in the diffusion of artemisinin resistant background. The diffusion of resistant genes in the Cambodian admixed population after selection resulted from mating of gametocytes of sensitive and resistant
Full Text Available Abstract Background The emergence of Plasmodium falciparum resistance to most anti-malarial compounds has highlighted the urgency to develop new drugs and to clarify the mechanisms of anti-malarial drugs currently used. Among them, doxycycline is used alone for malaria chemoprophylaxis or in combination with quinine or artemisinin derivatives for malaria treatment. The molecular mechanisms of doxycycline action in P. falciparum have not yet been clearly defined, particularly at the protein level. Methods A proteomic approach was used to analyse protein expression changes in the schizont stage of the malarial parasite P. falciparum following doxycycline treatment. A comparison of protein expression between treated and untreated protein samples was performed using two complementary proteomic approaches: two-dimensional fluorescence difference gel electrophoresis (2D-DIGE and isobaric tagging reagents for relative and absolute quantification (iTRAQ. Results After doxycycline treatment, 32 and 40 P. falciparum proteins were found to have significantly deregulated expression levels by 2D-DIGE and iTRAQ methods, respectively. Although some of these proteins have been already described as being deregulated by other drug treatments, numerous changes in protein levels seem to be specific to doxycycline treatment, which could perturb apicoplast metabolism. Quantitative reverse transcription polymerase chain reaction (RT-PCR was performed to confirm this hypothesis. Conclusions In this study, a specific response to doxycycline treatment was distinguished and seems to involve mitochondrion and apicoplast organelles. These data provide a starting point for the elucidation of drug targets and the discovery of mechanisms of resistance to anti-malarial compounds.
Estimated higher ratios of IFN-γ/IL-10 and IFN-γ/IL-12 were also observed in the symptomatic children while the asymptomatic controls had higher IL-12/IL-10 ratio. The mean concentration levels of anti-P. falciparum IgG1, IgG2, IgG3 antibodies were statistically significantly higher in the individuals >5 years of age than <5 ...
Myat P Kyaw
Full Text Available Plasmodium falciparum resistance to artemisinins, the first line treatment for malaria worldwide, has been reported in western Cambodia. Resistance is characterized by significantly delayed clearance of parasites following artemisinin treatment. Artemisinin resistance has not previously been reported in Myanmar, which has the highest falciparum malaria burden among Southeast Asian countries.A non-randomized, single-arm, open-label clinical trial of artesunate monotherapy (4 mg/kg daily for seven days was conducted in adults with acute blood-smear positive P. falciparum malaria in Kawthaung, southern Myanmar. Parasite density was measured every 12 hours until two consecutive negative smears were obtained. Participants were followed weekly at the study clinic for three additional weeks. Co-primary endpoints included parasite clearance time (the time required for complete clearance of initial parasitemia, parasite clearance half-life (the time required for parasitemia to decrease by 50% based on the linear portion of the parasite clearance slope, and detectable parasitemia 72 hours after commencement of artesunate treatment. Drug pharmacokinetics were measured to rule out delayed clearance due to suboptimal drug levels.The median (range parasite clearance half-life and time were 4.8 (2.1-9.7 and 60 (24-96 hours, respectively. The frequency distributions of parasite clearance half-life and time were bimodal, with very slow parasite clearance characteristic of the slowest-clearing Cambodian parasites (half-life longer than 6.2 hours in approximately 1/3 of infections. Fourteen of 52 participants (26.9% had a measurable parasitemia 72 hours after initiating artesunate treatment. Parasite clearance was not associated with drug pharmacokinetics.A subset of P. falciparum infections in southern Myanmar displayed markedly delayed clearance following artemisinin treatment, suggesting either emergence of artemisinin resistance in southern Myanmar or spread
Lopez, Ana Cecilia; Ortiz, Andres; Coello, Jorge; Sosa-Ochoa, Wilfredo; Torres, Rosa E Mejia; Banegas, Engels I; Jovel, Irina; Fontecha, Gustavo A
Understanding the population structure of Plasmodium species through genetic diversity studies can assist in the design of more effective malaria control strategies, particularly in vaccine development. Central America is an area where malaria is a public health problem, but little is known about the genetic diversity of the parasite's circulating species. This study aimed to investigate the allelic frequency and molecular diversity of five surface antigens in field isolates from Honduras. Five molecular markers were analysed to determine the genotypes of Plasmodium vivax and Plasmodium falciparum from endemic areas in Honduras. Genetic diversity of ama-1, msp-1 and csp was investigated for P. vivax, and msp-1 and msp-2 for P. falciparum. Allelic frequencies were calculated and sequence analysis performed. A high genetic diversity was observed within Plasmodium isolates from Honduras. A different number of genotypes were elucidated: 41 (n = 77) for pvama-1; 23 (n = 84) for pvcsp; and 23 (n = 35) for pfmsp-1. Pvcsp sequences showed VK210 as the only subtype present in Honduran isolates. Pvmsp-1 (F2) was the most polymorphic marker for P. vivax isolates while pvama-1 was least variable. All three allelic families described for pfmsp-1 (n = 30) block 2 (K1, MAD20, and RO33), and both allelic families described for the central domain of pfmsp-2 (n = 11) (3D7 and FC27) were detected. However, K1 and 3D7 allelic families were predominant. All markers were randomly distributed across the country and no geographic correlation was found. To date, this is the most complete report on molecular characterization of P. vivax and P. falciparum field isolates in Honduras with regards to genetic diversity. These results indicate that P. vivax and P. falciparum parasite populations are highly diverse in Honduras despite the low level of transmission.
Eisenhut, Michael; Omari, Aika; MacLehose, Harriet G
Background In children with malaria caused by Plasmodium falciparum, quinine administered rectally may be easier to use and less painful than intramuscular or intravenous administration. The objective of this review was to compare the effectiveness of intrarectal with intravenous or intramuscular quinine for treating falciparum malaria. Methods All randomized and quasi-randomized controlled trials comparing intrarectal with intramuscular or intravenous quinine for treating people with falciparum malaria located through the following sources were included: Cochrane Infectious Diseases Group Specialized Register, CENTRAL, MEDLINE, EMBASE, LILACS and CINAHL. Trial quality was assessed and data, including adverse event data, were extracted. Dichotomous data were analysed using odds ratios and continuous data using weighted mean difference. Results Eight randomized controlled trials (1,247 children) fulfilled the inclusion criteria. The same principal investigator led seven of the trials. Five compared intrarectal with intravenous quinine, and six compared intrarectal with intramuscular treatment. No statistically significant difference was detected for death, parasite clearance by 48 hours and seven days, parasite and fever clearance time, coma recovery time, duration of hospitalization and time before drinking began. One trial (898 children) reported that intrarectal was less painful than intramuscular administration. Conclusion No difference in the effect on parasites and clinical illness was detected for the use of intrarectal quinine compared with other routes, but most trials were small. Pain during application may be less with intrarectal quinine. Further larger trials, in patients with severe malaria and in adults, are required before the intrarectal route could be recommended. PMID:15904520
Minodier, Philippe; Noël, Guilhem; Tall, Mamadou; Retornaz, Karine; Piarroux, Renaud; Parzy, Daniel; Ranque, Stephane
Children with uncomplicated Plasmodium falciparum imported malaria are treated with various antimalarial regimens including mefloquine depending on national guidelines. Little is known regarding mefloquine treatment efficacy in this setting. In this prospective study, children 3 months to 16 years of age admitted in a tertiary hospital emergency ward in France with uncomplicated P. falciparum malaria were treated with oral mefloquine. Each dose was given with an antiemetic. Between 2004 and 2009, 95 children were evaluated. In all, 94% had traveled in the Indian Ocean region (Comoros and Madagascar); 79% used a malaria chemoprophylaxis, but none was fully compliant with World Health Organization recommended regimens. Main clinical features at admission were fever (91%), vomiting (44%), and headaches (44%). Hemoglobin mefloquine, and no relapse was noted within 45 days after admission. One Plasmodium vivax relapse occurred 6 months later. Vomiting within 1 hour after dosing occurred in 20% of children. Significant features associated with early vomiting by univariate analysis were a weight ≤ 15 kg, C-reactive protein ≥ 50 mg/L, and parasitemia ≥ 1%, but only low weight was significant by multivariate analysis. Mefloquine is an effective treatment for uncomplicated imported P. falciparum malaria in children returning from countries with low mefloquine resistance. Early vomiting after mefloquine dosing is frequent, especially in children < 15 kg of weight, but a second dose can be given successfully.
Full Text Available Abstract Background In Plasmodium falciparum, resistance to chloroquine (CQ is conferred by a K to T mutation at amino acid position 76 (K76T in the P. falciparum CQ transporter (PfCRT. To date, at least 15 pfcrt genotypes, which are represented by combinations of five amino acids at positions 72-76, have been described in field isolates from various endemic regions. To identify novel mutant pfcrt genotypes and to reveal the genetic relatedness of pfcrt genotypes, a large-scale survey over a wide geographic area was performed. Methods Sequences for exon 2 in pfcrt, including known polymorphic sites at amino acid positions 72, 74, 75 and 76, were obtained from 256 P. falciparum isolates collected from eight endemic countries in Asia (Bangladesh, Cambodia, Lao P.D.R., the Philippines and Thailand, Melanesia (Papua New Guinea and Vanuatu and Africa (Ghana. A haplotype network was constructed based on six microsatellite markers located -29 kb to 24 kb from pfcrt in order to examine the genetic relatedness among mutant pfcrt genotypes. Results In addition to wild type (CVMNK at positions 72-76, four mutant pfcrt were identified; CVIET, CVIDT, SVMNT and CVMNT (mutated amino acids underlined. Haplotype network revealed that there were only three mutant pfcrt lineages, originating in Indochina, Philippines and Melanesia. Importantly, the Indochina lineage contained two mutant pfcrt genotypes, CVIET (n = 95 and CVIDT (n = 14, indicating that CVIDT shares a common origin with CVIET. Similarly, one major haplotype in the Melanesian lineage contained two pfcrt genotypes; SVMNT (n = 71 and CVMNT (n = 3. In Africa, all mutant pfcrt genotypes were the CVIET of the Indochina lineage, probably resulting from the intercontinental migration of CQ resistance from Southeast Asia. Conclusions The number of CQ-mutant lineages observed in this study was identical to that found in previous studies. This supports the hypothesis that the emergence of novel CQ resistance
Full Text Available Abstract Background Genetic mapping is a powerful method to identify mutations that cause drug resistance and other phenotypic changes in the human malaria parasite Plasmodium falciparum. For efficient mapping of a target gene, it is often necessary to genotype a large number of polymorphic markers. Currently, a community effort is underway to collect single nucleotide polymorphisms (SNP from the parasite genome. Here we evaluate polymorphism detection accuracy of a high-density 'tiling' microarray with 2.56 million probes by comparing single feature polymorphisms (SFP calls from the microarray with known SNP among parasite isolates. Results We found that probe GC content, SNP position in a probe, probe coverage, and signal ratio cutoff values were important factors for accurate detection of SFP in the parasite genome. We established a set of SFP calling parameters that could predict mSFP (SFP called by multiple overlapping probes with high accuracy (≥ 94% and identified 121,087 mSFP genome-wide from five parasite isolates including 40,354 unique mSFP (excluding those from multi-gene families and ~18,000 new mSFP, producing a genetic map with an average of one unique mSFP per 570 bp. Genomic copy number variation (CNV among the parasites was also cataloged and compared. Conclusion A large number of mSFP were discovered from the P. falciparum genome using a high-density microarray, most of which were in clusters of highly polymorphic genes at chromosome ends. Our method for accurate mSFP detection and the mSFP identified will greatly facilitate large-scale studies of genome variation in the P. falciparum parasite and provide useful resources for mapping important parasite traits.
Frosch, Anne EP; John, Chandy C
Effective Plasmodium falciparum immunity requires a precisely timed and balanced response of inflammatory and anti-inflammatory immune regulators. These responses begin with innate immune effectors and are modulated over the course of an infection and between episodes to limit inflammation. To date, there are no effective immunomodulatory therapies for severe malaria. Some of the most potent immunomodulators are naturally occurring infections, including helminthic and chronic viral infections. This review examines malaria coinfection with these organisms, and their impact on malaria morbidity and immune responses. Overall, there is compelling evidence to suggest that chronic coinfections can modulate deleterious malaria-specific immune responses, suggesting that therapeutic agents may be effective if utilized early in infection. Examination of the mechanisms of these effects may serve as a platform to identify more targeted and effective malaria immunomodulatory therapeutics. PMID:23241191
Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro
Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.
Datta, Suvro Sankha; Mukherjee, Somnath; Bhattacharya, Prasun; Mukherjee, Krishnendu
A 58 year old lady presented with high grade fever, pallor, abdominal pain, loss of appetite and swelling of legs. She was subsequently diagnosed with SLE along with infection of Plasmodium falciparum malaria. She was clinically pale and advised for two units of packed red cell transfusion. One of the two units was incompatible, so only one unit was issued. Subsequently, DAT and auto control were positive. Later antibody specificity was identified, which came out to be anti JK-a. Because of recent transfusion 2 weeks back, her antigenic phenotype could not be elicited. Though we could not make out whether this antibody was the result of pregnancy or transfusion induced allo anti-JK-a or SLE induced auto anti JK-a, this antibody is highly clinically significant from transfusion point of view.
Tosh, K; Kilbey, B
Part of the topoisomerase I (TopoI)-encoding gene from Plasmodium falciparum (Pf) was isolated by PCR from cDNA using oligodeoxyribonucleotides modelled on the highly conserved regions of sequence from other species. The entire TopoI gene was obtained by screening a Pf K1 HindIII-EcoRI genomic library in lambda NM1149 with a random-labeled heterologous probe from the Saccharomyces cerevisiae TopoI gene. DNA sequence analysis revealed an open reading frame of 2520 nt encoding a deduced protein of 839 amino acids (aa) with no detectable introns. The Pf TopoI aa sequence has about 40% identity with most eukaryotic TopoI homologues. The gene is located as a single copy on chromosome 5 and Northern analysis identified a transcript of 3.8 kb.
Full Text Available A despeito dos avanços no tratamento e das campanhas de prevenção e de controle da malária nos distintos continentes nos quais a moléstia grassa, a entidade mórbida permanece com significativa relevância no mundo contemporâneo. O Plasmodium falciparum é o grande responsável pela malária grave, caracterizada por distúrbios em diferentes órgãos e sistemas, com possibilidade de evolução ao óbito. Embora incipientes, os estudos proteômicos na malária têm trazido boas perspectivas para melhor compreensão dos aspectos biológicos do Plasmodium, assim como dos mecanismos fisiopatológicos, diagnósticos, terapêuticos e profiláticos da enfermidade. Desse modo, o objetivo do presente artigo é apresentar uma breve revisão das aplicações da análise proteômica na malária por P. falciparum.Despite advances in treatment and campaigns for prevention and control of malaria on the various continents where it is still rampant, this disease remains significantly relevant to the contemporary world. Plasmodium falciparum is the organism that is mainly responsible for severe malaria, which is characterized by disturbances in different organs and systems, with possibly fatal outcomes. Although incipient, proteomic studies of malaria have yielded favorable prospects for elucidating the biological aspects of Plasmodium as well as the pathophysiological, diagnostic, prophylactic, and therapeutic mechanisms of the disease. Thus, the aim of the present article is to present a brief review of the applications of proteomic analysis in P. falciparum malaria.
Jorge E Suarez
Full Text Available The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720 which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD were found to be critical for peptide binding to erythrocytes.
Full Text Available The mechanisms of stage-specific gene regulation in the malaria parasite Plasmodium falciparum are largely unclear, with only a small number of specific regulatory transcription factors (AP2 family having been identified. In particular, the transcription factors that function in the intraerythrocytic stage remain to be elucidated. Previously, as a model case for stage-specific transcription in the P. falciparum intraerythrocytic stage, we analyzed the transcriptional regulation of pf1-cys-prx, a trophozoite/schizont-specific gene, and suggested that some nuclear factors bind specifically to the cis-element of pf1-cys-prx and enhance transcription. In the present study, we purified nuclear factors from parasite nuclear extract by 5 steps of chromatography, and identified a factor termed PREBP. PREBP is not included in the AP2 family, and is a novel protein with four K-homology (KH domains. The KH domain is known to be found in RNA-binding or single-stranded DNA-binding proteins. PREBP is well conserved in Plasmodium species and partially conserved in phylum Apicomplexa. To evaluate the effects of PREBP overexpression, we used a transient overexpression and luciferase assay combined approach. Overexpression of PREBP markedly enhanced luciferase expression under the control of the pf1-cys-prx cis-element. These results provide the first evidence of a novel transcription factor that activates the gene expression in the malaria parasite intraerythrocytic stage. These findings enhance our understanding of the evolution of specific transcription machinery in Plasmodium and other eukaryotes.
Baldwin, Michael; Russo, Crystal; Li, Xuerong; Chishti, Athar H.
Highlights: • PfSPP is an ER resident protease. • PfSPP is expressed both as a monomer and dimer. • The signal peptide of HSP101 is the first known substrate of PfSPP. • Reduced PfSPP activity may significantly affect ER homeostasis. - Abstract: Previously we described the identification of a Plasmodium falciparum signal peptide peptidase (PfSPP) functioning at the blood stage of malaria infection. Our studies also demonstrated that mammalian SPP inhibitors prevent malaria parasite growth at the late-ring/early trophozoite stage of intra-erythrocytic development. Consistent with its role in development, we tested the hypothesis that PfSPP functions at the endoplasmic reticulum of P.falciparum where it cleaves membrane-bound signal peptides generated following the enzyme activity of signal peptidase. The localization of PfSPP to the endoplasmic reticulum was confirmed by immunofluorescence microscopy and immunogold electron microscopy. Biochemical analysis indicated the existence of monomer and dimer forms of PfSPP in the parasite lysate. A comprehensive bioinformatics screen identified several candidate PfSPP substrates in the parasite genome. Using an established transfection based in vivo luminescence assay, malaria heat shock protein 101 (HSP101) was identified as a substrate of PfSPP, and partial inhibition of PfSPP correlated with the emergence of gametocytes. This finding unveils the first known substrate of PfSPP, and provides new perspectives for the function of intra-membrane proteolysis at the erythrocyte stage of malaria parasite life cycle
Baldwin, Michael; Russo, Crystal; Li, Xuerong [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Chishti, Athar H., E-mail: email@example.com [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Sackler School of Graduate Biomedical Sciences, Programs in Physiology, Pharmacology, and Microbiology, Tufts University School of Medicine, Boston, MA 02111 (United States)
Highlights: • PfSPP is an ER resident protease. • PfSPP is expressed both as a monomer and dimer. • The signal peptide of HSP101 is the first known substrate of PfSPP. • Reduced PfSPP activity may significantly affect ER homeostasis. - Abstract: Previously we described the identification of a Plasmodium falciparum signal peptide peptidase (PfSPP) functioning at the blood stage of malaria infection. Our studies also demonstrated that mammalian SPP inhibitors prevent malaria parasite growth at the late-ring/early trophozoite stage of intra-erythrocytic development. Consistent with its role in development, we tested the hypothesis that PfSPP functions at the endoplasmic reticulum of P.falciparum where it cleaves membrane-bound signal peptides generated following the enzyme activity of signal peptidase. The localization of PfSPP to the endoplasmic reticulum was confirmed by immunofluorescence microscopy and immunogold electron microscopy. Biochemical analysis indicated the existence of monomer and dimer forms of PfSPP in the parasite lysate. A comprehensive bioinformatics screen identified several candidate PfSPP substrates in the parasite genome. Using an established transfection based in vivo luminescence assay, malaria heat shock protein 101 (HSP101) was identified as a substrate of PfSPP, and partial inhibition of PfSPP correlated with the emergence of gametocytes. This finding unveils the first known substrate of PfSPP, and provides new perspectives for the function of intra-membrane proteolysis at the erythrocyte stage of malaria parasite life cycle.
Lavstsen, Thomas; Turner, Louise; Saguti, Fredy
The clinical outcome of Plasmodium falciparum infections ranges from asymptomatic parasitemia to severe malaria syndromes associated with high mortality. The virulence of P. falciparum infections is associated with the type of P. falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the...
Bello, Shaibu O; Chika, Aminu; AbdulGafar, Jimoh O
The purpose of this study is to summarize the available data on the efficacy of Artesunate plus Amodiaquine (AS+AQ) versus Artemether -Lumefantrine (AL) for the treatment of uncomplicated Plasmodium falciparum malaria in sub-Saharan Africa using uncorrected parasitaemia as a clinically relevant endpoint. Studies and conference abstracts identified through Pubmed, Medline, Embase, Ansinet, AJOL, Bioline, Cochrane Infectious Diseases Group trials register, The Cochrane Controlled Trials Registe...
Avery, Vicky M; Bashyam, Sridevi; Burrows, Jeremy N; Duffy, Sandra; Papadatos, George; Puthukkuti, Shyni; Sambandan, Yuvaraj; Singh, Shivendra; Spangenberg, Thomas; Waterson, David; Willis, Paul
In view of the need to continuously feed the pipeline with new anti-malarial agents adapted to differentiated and more stringent target product profiles (e.g., new modes of action, transmission-blocking activity or long-duration chemo-protection), a chemical library consisting of more than 250,000 compounds has been evaluated in a blood-stage Plasmodium falciparum growth inhibition assay and further assessed for chemical diversity and novelty. The selection cascade used for the triaging of hits from the chemical library started with a robust three-step in vitro assay followed by an in silico analysis of the resulting confirmed hits. Upon reaching the predefined requirements for selectivity and potency, the set of hits was subjected to computational analysis to assess chemical properties and diversity. Furthermore, known marketed anti-malarial drugs were co-clustered acting as 'signposts' in the chemical space defined by the hits. Then, in cerebro evaluation of the chemical structures was performed to identify scaffolds that currently are or have been the focus of anti-malarial medicinal chemistry programmes. Next, prioritization according to relaxed physicochemical parameters took place, along with the search for structural analogues. Ultimately, synthesis of novel chemotypes with desired properties was performed and the resulting compounds were subsequently retested in a P. falciparum growth inhibition assay. This screening campaign led to a 1.25% primary hit rate, which decreased to 0.77% upon confirmatory repeat screening. With the predefined potency (EC₅₀ 10) criteria, 178 compounds progressed to the next steps where chemical diversity, physicochemical properties and novelty assessment were taken into account. This resulted in the selection of 15 distinct chemical series. A selection cascade was applied to prioritize hits resulting from the screening of a medium-sized chemical library against blood-stage P. falciparum. Emphasis was placed on chemical
Kurtzhals, J A; Reimert, C M; Tette, E
To assess the eosinophil response to Plasmodium falciparum infection a cohort of initially parasite-free Ghanaian children was followed for 3 months. Seven of nine children who acquired an asymptomatic P. falciparum infection showed increase in eosinophil counts, while a decrease was found in sev...
McGready, R.; Cho, T.; Samuel, N. N.; Villegas, L.; Brockman, A.; van Vugt, M.; Looareesuwan, S.; White, N. J.; Nosten, F.
In areas where multidrug-resistant Plasmodium falciparum (MDR-Pf) is prevalent, only quinine is known to be safe and effective in pregnant women. On the western border of Thailand, 7 days of supervised quinine (30 mg/kg daily) cures two-thirds of P. falciparum-infected women in the 2nd and 3rd
Lloyd, Yukie M.; Ngati, Elise P.; Salanti, Ali
Antibody-mediated phagocytosis is an important immune effector mechanism against Plasmodium falciparum-infected erythrocytes (IE); however, current phagocytosis assays use IE collected from infected individuals or from in vitro cultures of P. falciparum, making them prone to high variation. A sim...
Ocholla, Harold; Preston, Mark D; Mipando, Mwapatsa
BACKGROUND: Selection by host immunity and antimalarial drugs has driven extensive adaptive evolution in Plasmodium falciparum and continues to produce ever-changing landscapes of genetic variation. METHODS: We performed whole-genome sequencing of 69 P. falciparum isolates from Malawi and used...
Peek, Ron; van Gool, Tom; Panchoe, Daynand; Greve, Sophie; Bus, Ellen; Resida, Lesley
Plasmodium falciparum in Suriname was studied for the presence of drug resistance and genetic variation in blood samples of 86 patients with symptomatic malaria. Drug resistance was predicted by determining point mutations in the chloroquine resistance marker of the P. falciparum chloroquine
Dekker, E.; Hellerstein, M. K.; Romijn, J. A.; Neese, R. A.; Peshu, N.; Endert, E.; Marsh, K.; Sauerwein, H. P.
To evaluate glucose kinetics in children with falciparum malaria, basal glucose production and gluconeogenesis and an estimate of the flux of the gluconeogenic precursors were measured in Kenyan children with uncomplicated falciparum malaria before (n = 11) and during infusion of alanine (1.5
Invasive pulmonary aspergillosis is an extraordinary rare in the immunocompetent host. Falciparum malaria contributes to high morbidity and mortality of malaria infection cases in the world. The impairments of both humoral and cellular immunity could be the reason of invasive pulmonary aspergillosis in falciparum malaria infection. Forty-nine years old patient came with fever, jaundice, pain in the right abdomen, after visiting a remote area in Africa about one month before admission. Blood films and rapid test were positive for Plasmodium falciparum. After malaria therapy in five days, consciousness was altered into somnolence and intubated with respiratory deterioration. Invasive pulmonary aspergillosis after falciparum malaria infection is life-threatening. There should be awareness of physicians of invasive pulmonary aspergillosis in falciparum malaria infection.
Mejia Torres, Rosa Elena; Banegas, Engels Ilich; Mendoza, Meisy; Diaz, Cesar; Bucheli, Sandra Tamara Mancero; Fontecha, Gustavo A; Alam, Md Tauqeer; Goldman, Ira; Udhayakumar, Venkatachalam; Zambrano, Jose Orlinder Nicolas
Chloroquine (CQ) is officially used for the primary treatment of Plasmodium falciparum malaria in Honduras. In this study, the therapeutic efficacy of CQ for the treatment of uncomplicated P. falciparum malaria in the municipality of Puerto Lempira, Gracias a Dios, Honduras was evaluated using the Pan American Health Organization-World Health Organization protocol with a follow-up of 28 days. Sixty-eight patients from 6 months to 60 years of age microscopically diagnosed with uncomplicated P. falciparum malaria were included in the final analysis. All patients who were treated with CQ (25 mg/kg over 3 days) cleared parasitemia by day 3 and acquired no new P. falciparum infection within 28 days of follow-up. All the parasite samples sequenced for CQ resistance mutations (pfcrt) showed only the CQ-sensitive genotype (CVMNK). This finding shows that CQ remains highly efficacious for the treatment of uncomplicated P. falciparum malaria in Gracias a Dios, Honduras.
Zhu, Xi-Guang; She, Guang-Song
To explore the feasible prevention and control measures for imported falciparum malaria. The epidemic situation of imported falciparum malaria and status of labor export were investigated by using the epidemic statistical method, epidemiological survey and follow-up survey. There were 46 direct network reported cases of imported falciparum malaria in Jiangdu District in 2011, and they were all export laborers to Africa, being aged from 30-49 years, Totally 28 patients of them had the history of disease in foreign countries, and 29 cases were in the same labor export services company. A total of 444 migrant labors were investigated and 24 patients were diagnosed falciparum malaria. In order to prevent and control imported falciparum malaria, we should strengthen the standard management of labor export services market and health education.
Melissa D Conrad
Full Text Available The emergence of resistance to artemisinin derivatives in Southeast Asia, manifested as delayed clearance of Plasmodium falciparum following treatment with artemisinins, is a major concern. Recently, the artemisinin resistance phenotype was attributed to mutations in portions of a P. falciparum gene (PF3D7_1343700 encoding kelch (K13 propeller domains, providing a molecular marker to monitor the spread of resistance. The P. falciparum cysteine protease falcipain-2 (FP2; PF3D7_1115700 has been shown to contribute to artemisinin action, as hemoglobin degradation is required for potent drug activity, and a stop mutation in the FP2 gene was identified in parasites selected for artemisinin resistance. Although delayed parasite clearance after artemisinin-based combination therapy (ACT has not yet been noted in Uganda and ACTs remain highly efficacious, characterizing the diversity of these genes is important to assess the potential for resistance selection and to provide a baseline for future surveillance. We therefore sequenced the K13-propeller domain and FP2 gene in P. falciparum isolates from children previously treated with ACT in Uganda, including samples from 2006-7 (n = 49 and from 2010-12 (n = 175. Using 3D7 as the reference genome, we identified 5 non-synonymous polymorphisms in the K13-propeller domain (133 isolates and 35 in FP2 (160 isolates; these did not include the polymorphisms recently associated with resistance after in vitro selection or identified in isolates from Asia. The prevalence of K13-propeller and FP2 polymorphisms did not increase over time, and was not associated with either time since prior receipt of an ACT or the persistence of parasites ≥2 days following treatment with an ACT. Thus, the K13-propeller and FP2 polymorphisms associated with artemisinin resistance are not prevalent in Uganda, and we did not see evidence for selection of polymorphisms in these genes.
Simpalipan, Phumin; Pattaradilokrat, Sittiporn; Harnyuttanakorn, Pongchai
Antigen-detecting rapid diagnostic tests (RDTs) have been recommended by the World Health Organization for use in remote areas to improve malaria case management. Lactate dehydrogenase (LDH) of Plasmodium falciparum is one of the main parasite antigens employed by various commercial RDTs. It has been hypothesized that the poor detection of LDH-based RDTs is attributed in part to the sequence diversity of the gene. To test this, the present study aimed to investigate the genetic diversity of the P. falciparum ldh gene in Thailand and to construct the map of LDH sequence diversity in P. falciparum populations worldwide. The ldh gene was sequenced for 50 P. falciparum isolates in Thailand and compared with hundreds of sequences from P. falciparum populations worldwide. Several indices of molecular variation were calculated, including the proportion of polymorphic sites, the average nucleotide diversity index (π), and the haplotype diversity index (H). Tests of positive selection and neutrality tests were performed to determine signatures of natural selection on the gene. Mean genetic distance within and between species of Plasmodium ldh was analysed to infer evolutionary relationships. Nucleotide sequences of P. falciparum ldh could be classified into 9 alleles, encoding 5 isoforms of LDH. L1a was the most common allelic type and was distributed in P. falciparum populations worldwide. Plasmodium falciparum ldh sequences were highly conserved, with haplotype and nucleotide diversity values of 0.203 and 0.0004, respectively. The extremely low genetic diversity was maintained by purifying selection, likely due to functional constraints. Phylogenetic analysis inferred the close genetic relationship of P. falciparum to malaria parasites of great apes, rather than to other human malaria parasites. This study revealed the global genetic variation of the ldh gene in P. falciparum, providing knowledge for improving detection of LDH-based RDTs and supporting the candidacy of
Mukherjee, Angana; Bopp, Selina; Magistrado, Pamela; Wong, Wesley; Daniels, Rachel; Demas, Allison; Schaffner, Stephen; Amaratunga, Chanaki; Lim, Pharath; Dhorda, Mehul; Miotto, Olivo; Woodrow, Charles; Ashley, Elizabeth A; Dondorp, Arjen M; White, Nicholas J; Wirth, Dyann; Fairhurst, Rick; Volkman, Sarah K
Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin-piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance. Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA 0-3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates. Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA 0-3h = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance. This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine
Echeverry Diego F
Full Text Available Abstract Background Resistance to chloroquine and antifolate drugs has evolved independently in South America, suggesting that genotype - phenotype studies aimed at understanding the genetic basis of resistance to these and other drugs should be conducted in this continent. This research was conducted to better understand the population structure of Colombian Plasmodium falciparum in preparation for such studies. Results A set of 384 SNPs were genotyped in blood spot DNA samples from 447 P. falciparum infected subjects collected over a ten year period from four provinces of the Colombian Pacific coast to evaluate clonality, population structure and linkage disequilibrium (LD. Most infections (81% contained a single predominant clone. These clustered into 136 multilocus genotypes (MLGs, with 32% of MLGs recovered from multiple (2 – 28 independent subjects. We observed extremely low genotypic richness (R = 0.42 and long persistence of MLGs through time (median = 537 days, range = 1 – 2,997 days. There was a high probability (>5% of sampling parasites from the same MLG in different subjects within 28 days, suggesting caution is needed when using genotyping methods to assess treatment success in clinical drug trials. Panmixia was rejected as four well differentiated subpopulations (FST = 0.084 - 0.279 were identified. These occurred sympatrically but varied in frequency within the four provinces. Linkage disequilibrium (LD decayed more rapidly (r2 = 0.17 for markers Conclusions We conclude that Colombian populations have several advantages for association studies, because multiple clone infections are uncommon and LD decays over the scale of one or a few genes. However, the extensive population structure and low genotype richness will need to be accounted for when designing and analyzing association studies.
Hessel, Audrey; Raynal, Bertrand; England, Patrick; Cohen, Jacques H.; Bertrand, Olivier; Peyrard, Thierry; Bentley, Graham A.; Lewit-Bentley, Anita; Mercereau-Puijalon, Odile
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 DBL1α1 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-DBL1α1 and NTS-DBL1α1-CIDR1γ 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 A1, weaker binding to groups A2 and B, and least binding to groups Ax and O. The 2.8 Å resolution crystal structure of the PfEMP1-VarO Head region, NTS-DBL1α1-CIDR1γ, reveals extensive contacts between the DBL1α1 and CIDR1γ and shows that the NTS-DBL1α1 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-DBLα1. 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. PMID:22807674
De, Sai Lata; Stanisic, Danielle I; van Breda, Karin; Bellete, Bernadette; Harris, Ivor; McCallum, Fiona; Edstein, Michael D; Good, Michael F
Malaria is a disease caused by a protozoan of the Plasmodium genus and results in 0.5-0.7million deaths per year. Increasing drug resistance of the parasite and insecticide resistance of mosquitoes necessitate alternative control measures. Numerous vaccine candidates have been identified but none have been able to induce robust, long-lived protection when evaluated in malaria endemic regions. Rodent studies have demonstrated that chemically attenuated blood stage parasites can persist at sub-patent levels and induce homologous and heterologous protection against malaria. Parasite-specific cellular responses were detected, with protection dependent on CD4+ T cells. To investigate this vaccine approach for Plasmodium falciparum, we characterised the persistence and immunogenicity of chemically attenuated P. falciparum FVO strain parasites (CAPs) in non-splenectomised Aotus nancymaae monkeys following administration of a single dose. Control monkeys received either normal red blood cells or wild-type parasites followed by drug treatment. Chemical attenuation was performed using tafuramycin A, which irreversibly binds to DNA. CAPs were detected in the peripheral blood for up to 2days following inoculation as determined by thick blood smears, and for up to 8days as determined by quantitative PCR. Parasite-specific IgG was not detected in monkeys that received CAPs; however, in vitro parasite-specific T cell proliferation was observed. Following challenge, the CAP monkeys developed an infection; however, one CAP monkey and the infection and drug-cure monkeys showed partial or complete resistance. These experiments lay the groundwork for further assessment of CAPs as a potential vaccine against malaria. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.
Newton Paul N
Full Text Available Abstract Background Artemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates. Results In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC. In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype. Conclusions The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis
Background: Experimentally studying the transmission of the malaria parasite and its regulating factors requires availability of human blood donors carrying infectious gametocytes. The difficulty of identifying gametocyte carriers from the community is often limited due to financial and human resources constraints.
Willie, Nigani; Mehlotra, Rajeev K; Howes, Rosalind E; Rakotomanga, Tovonahary A; Ramboarina, Stephanie; Ratsimbasoa, Arsène C; Zimmerman, Peter A
Plasmodium falciparum histidine-rich protein 2 (PfHRP2) forms the basis of many current malaria rapid diagnostic tests (RDTs). However, the parasites lacking part or all of the pfhrp2 gene do not express the PfHRP2 protein and are, therefore, not identifiable by PfHRP2-detecting RDTs. We evaluated the performance of the SD Bioline Malaria Ag P.f/Pan RDT together with pfhrp2 variation in Madagascar. Genomic DNA isolated from 260 patient blood samples were polymerase chain reaction (PCR)-amplified for the parasite 18S rRNA and pfhrp2 genes. Post-PCR ligation detection reaction-fluorescent microsphere assay (LDR-FMA) was performed for the identification of parasite species. Plasmodium falciparum histidine-rich protein 2 amplicons were sequenced. Polymerase chain reaction diagnosis of patient samples showed that 29% (75/260) were infected and P. falciparum was present in 95% (71/75) of these PCR-positive samples. Comparing RDT and P. falciparum detection by LDR-FMA, eight samples were RDT negative but P. falciparum positive (false negatives), all of which were pfhrp2 positive. The sensitivity and specificity of the RDT were 87% and 90%, respectively. Seventy-three samples were amplified for pfhrp2 , from which nine randomly selected amplicons were sequenced, yielding 13 sequences. Amplification of pfhrp2 , combined with RDT analysis and P. falciparum detection by LDR-FMA, showed that there was no indication of pfhrp2 deletion. Sequence analysis of pfhrp2 showed that the correlation between pfhrp2 sequence structure and RDT detection rates was unclear. Although the observed absence of pfhrp2 deletion from the samples screened here is encouraging, continued monitoring of the efficacy of the SD Bioline Malaria Ag P.f/Pan RDT for malaria diagnosis in Madagascar is warranted.
Barfod, Lea; Dobrilovic, Tina; Magistrado, Pamela
Acquired protection from Plasmodium falciparum placental malaria, a major cause of maternal, fetal, and infant morbidity, is mediated by IgG specific for the P. falciparum erythrocyte membrane protein 1 variant VAR2CSA. This protein enables adhesion of P. falciparum-infected erythrocytes...... monoclonal IgG Abs from affinity-matured memory B cells of P. falciparum-exposed women to study interclonal variation and functional importance of Ab epitopes among placental and peripheral parasites from East and West Africa. Most placental P. falciparum isolates were labeled by several mAbs, whereas...
Isba, Rachel; Zani, Babalwa; Gathu, Michael; Sinclair, David
Background The World Health Organization (WHO) recommends artemisinin-based combination therapy (ACT) for treating people with Plasmodium falciparum malaria. Five combinations are currently recommended, all administered over three days. Artemisinin-naphthoquine is a new combination developed in China, which is being marketed as a one-day treatment. Although shorter treatment courses may improve adherence, the WHO recommends at least three days of the short-acting artemisinin component to eliminate 90% P. falciparum parasites in the bloodstream, before leaving the longer-acting partner drug to clear the remaining parasites. Objectives To evaluate the efficacy and safety of the artemisinin-naphthoquine combination for treating adults and children with uncomplicated P. falciparum malaria. Search methods We searched the Cochrane Infectious Diseases Group Specialized Register; Cochrane Central Register of Controlled Trials (CENTRAL) published in The Cochrane Library; MEDLINE; EMBASE; and LILACS up to January 2015. We also searched the metaRegister of Controlled Trials (mRCT) using 'malaria' and 'arte* OR dihydroarte*' as search terms. Selection criteria Randomized controlled trials comparing artemisinin-naphthoquine combinations with established WHO-recommended ACTs for the treatment of adults and children with uncomplicated malaria due to P. falciparum. Data collection and analysis Two review authors independently assessed trials for eligibility and risk of bias, and extracted data. We analysed primary outcomes in line with the WHO 'Protocol for assessing and monitoring antimalarial drug efficacy' and compared drugs using risk ratios (RR) and 95% confidence intervals (CI). Secondary outcomes were effects on gametocytes, haemoglobin, and adverse events. We assessed the quality of evidence using the GRADE approach. Main results Four trials, enrolling 740 adults and children, met the inclusion criteria. Artemisinin-naphthoquine was administered as a single dose (two
Amoah, Linda Eva; Opong, Akua; Ayanful-Torgby, Ruth; Abankwa, Joana; Acquah, Festus K
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder that results in impaired enzyme activity. Although G6PD deficiency is globally distributed it is more prevalent in malaria-endemic countries. Several mutations have been identified in the G6PD gene, which alter enzyme activity. The G6PD genotype predominantly found in sub-Saharan Africa is the G6PDB (G6PD376A) with (G6PD376G) and G6PDA- (G6PD376G/202A, G6PD376G/542T, G6PD376G/680T and G6PD376G/968C) occurring at lower frequencies. The aim of this study was to identify the prevalence of G6PD deficiency and asymptomatic Plasmodium falciparum carriage in children living in southern Ghana and determine whether G6PD deficiency influences asymptomatic carriage of P. falciparum parasites. Blood samples were obtained once a month from 170 healthy Ghanaian school children aged between 5 and 12 years from Basic schools in two communities Obom and Abura with similar rainfall patterns and malaria peak seasons. G6PD enzyme activity was assessed using the qualitative G6PD RDT kit (AccessBIO). G6PD genotyping and asymptomatic parasite carriage was determined by PCR followed by restriction fragment length polymorphism (RFLP) of DNA extracted from dried blood spots. The only sub-Saharan G6PD A- allele detected was the A376G/G202A found in 12.4 % (21/170), of the children and distributed as 4.1 % (7/170) A-, 1.8 % (3/170) A-/A- homozygous deficient males and females and 6.5 % (11/170) A/A- and B/A- heterozygous deficient females. Phenotypically, 10.6 % (15/142) of the children were G6PD deficient. The asymptomatic carriage of P. falciparum by PCR was 50, 29.4, 38.2 and 38.8 % over the months of February through May 2015, respectively, and 28.8, 22.4, 25.9 and 5.9 % by microscopy during the same periods. G6PD deficiency was significantly associated with a lowered risk of PCR-estimated asymptomatic P. falciparum carriage in children during the off peak malaria season in Southern Ghana.
Nzila, Alexis; Mberu, Eddy; Bray, Pat; Kokwaro, Gilbert; Winstanley, Peter; Marsh, Kevin; Ward, Steve
Resistance to drugs can result from changes in drug transport, and this resistance can sometimes be overcome by a second drug that modifies the transport mechanisms of the cell. This strategy has been exploited to partly reverse resistance to chloroquine in Plasmodium falciparum. Studies with human tumor cells have shown that probenecid can reverse resistance to the antifolate methotrexate, but the potential for reversal of antifolate resistance has not been studied in P. falciparum. In the present study we tested the ability of probenecid to reverse antifolate resistance in P. falciparum in vitro. Probenecid, at concentrations that had no effect on parasite viability alone (50 μM), was shown to increase the sensitivity of a highly resistant parasite isolate to the antifolates pyrimethamine, sulfadoxine, chlorcycloguanil, and dapsone by seven-, five-, three-, and threefold, respectively. The equivalent effects against an antifolate-sensitive isolate were activity enhancements of approximately 3-, 6-, 1.2-, and 19-fold, respectively. Probenecid decreased the level of uptake of radiolabeled folic acid, suggesting a transport-based mechanism linked to folate salvage. When probenecid was tested with chloroquine, it chemosensitized the resistant isolate to chloroquine (i.e., enhanced the activity of chloroquine). This enhancement of activity was associated with increased levels of chloroquine accumulation. In conclusion, we have shown that probenecid can chemosensitize malaria parasites to antifolate compounds via a mechanism linked to reduced folate uptake. Notably, this effect is observed in both folate-sensitive and -resistant parasites. In contrast to the activities of antifolate compounds, the effect of probenecid on chloroquine sensitivity was selective for chloroquine-resistant parasites (patent P407595GB [W. P. Thompson & Co., Liverpool, United Kingdom] has been filed to protect this intellectual property). PMID:12821454
Full Text Available Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV, with a parasitophorous vacuole membrane (PVM separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2 contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1. PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process.
Chattopadhyay, Rana; Majam, Victoria F; Kumar, Sanjai
Transfusion-transmitted malaria remains a serious concern for blood safety. Viable Plasmodium parasites must be present in human blood to transmit malaria, but their survival in blood over time stored under refrigeration has never been carefully investigated. We spiked leukoreduced normal human blood with Plasmodium falciparum (3D7 strain) asexual ring-stage parasites and stored it at 4 °C for 28 days, taking samples at different days intervals. We evaluated the samples for parasitemia by blood film microscopy and by culturing red blood cells (RBCs) to allow further development of parasites. We observed a significant reduction in parasitemia (0.5% vs. 0.12%) after only 1 day in storage at 4 °C. Thereafter, reduction in parasitemia was relatively gradual. Microscopically detectable parasites were present even after 28 days of storage. However, after storing for more than 14 days at 4 °C, parasites no longer replicated when cultured in vitro. Although the storage of asexual blood-stage P. falciparum parasites at 4 °C is detrimental to their survival (a 7.1-fold reduction in parasitemia after 14 days in storage), parasites remained microscopically detectable for 28 days, the end time point of our study. Further in vitro and in vivo studies will be needed to confirm loss of viability of P. falciparum after 14 days in storage, but our initial efforts repeatedly failed to show maturation and development of the parasites in cultured RBCs after that time. © 2010 American Association of Blood Banks.
Sharling, Lisa; Enevold, Anders; Sowa, Kordai M P
erythrocyte surface. However, several studies have found that parasite adhesion to placental receptors can be markedly less sensitive to trypsin. This study investigates whether chondroitin sulphate A (CSA) binding parasites express trypsin-resistant variant surface antigens (VSA) that bind female....... falciparum infected erythrocytes selected for adhesion to CSA were found to express trypsin-resistant VSA that are the target of naturally acquired antibodies from pregnant women living in a malaria endemic region of Ghana. However in vitro adhesion to CSA and HA was relatively trypsin sensitive. An improved...... labelling technique for the detection of VSA expressed by CSA binding isolates has also been described. CONCLUSION: The VSA expressed by CSA binding P. falciparum isolates are currently considered potential targets for a vaccine against PAM. This study identifies discordance between the trypsin sensitivity...
Kumar, Sanjai; Epstein, Judith E; Richie, Thomas L; Nkrumah, Francis K; Soisson, Lorraine; Carucci, Daniel J; Hoffman, Stephen L
Scientists from several organizations worldwide are working together to develop a multistage, multigene DNA-based vaccine against Plasmodium falciparum malaria. This collaborative vaccine development effort is named Multi-Stage DNA-based Malaria Vaccine Operation. An advisory board of international experts in vaccinology, malariology and field trials provides the scientific oversight to support the operation. This article discusses the rationale for the approach, underlying concepts and the pre-clinical development process, and provides a brief outline of the plans for the clinical testing of a multistage, multiantigen malaria vaccine based on DNA plasmid immunization technology.
Full Text Available We studied all consensus sequences within the four least 'variable blocks' (VB present in the DBL6ε domain of VAR2CSA, the protein involved in the adhesion of infected red blood cells by Plasmodium falciparum that causes the Pregnancy-Associated Malaria (PAM. Characterising consensus sequences with respect to recognition of antibodies and percentage of responders among pregnant women living in areas where P. falciparum is endemic allows the identification of the most antigenic sequences within each VB. When combining these consensus sequences among four serotypes from VB1 or VB5, the most often recognized ones are expected to induce pan-reactive antibodies recognizing VAR2CSA from all plasmodial strains. These sequences are of main interest in the design of an immunogenic molecule. Using a similar approach than for DBL6ε, we studied the five other DBL and the CIDRpam from VAR2CSA, and again identified VB segments with highly conserved consensus sequences. In addition, we identified consensus sequences in other var genes expressed by non-PAM parasites. This finding paves the way for vaccine design against other pathologies caused by P. falciparum.
Fanello, C; Onyamboko, M; Lee, S J; Woodrow, C; Setaphan, S; Chotivanich, K; Buffet, P; Jauréguiberry, S; Rockett, K; Stepniewska, K; Day, N P J; White, N J; Dondorp, A M
Parenteral artesunate is the treatment of choice for severe malaria. Recently, haemolytic anaemia occurring 1 to 3 weeks after artesunate treatment of falciparum malaria has been reported in returning travellers in temperate countries. To assess these potential safety concerns in African children, in whom most deaths from malaria occur, an open-labelled, randomized controlled trial was conducted in Kinshasa, Democratic Republic of Congo. 217 children aged between 6 months and 14 years with acute uncomplicated falciparum malaria and parasite densities over 100,000/μL were randomly allocated to intravenous artesunate or quinine, hospitalized for 3 days and then followed for 42 days. The immediate reduction in haemoglobin was less with artesunate than with quinine: median (IQR) fall at 72 h 1.4 g/dL (0.90-1.95) vs. 1.7 g/dL (1.10-2.40) (p = 0.009). This was explained by greater pitting then recirculation of once infected erythrocytes. Only 5% of patients (in both groups) had a ≥ 10% reduction in haemoglobin after day 7 (p = 0.1). One artesunate treated patient with suspected concomitant sepsis had a protracted clinical course and required a blood transfusion on day 14. Clinically significant delayed haemolysis following parenteral artesunate is uncommon in African children hospitalised with acute falciparum malaria and high parasitaemias. ClinicalTrials.gov ; Identifier: NCT02092766 (18/03/2014).
Full Text Available Abstract Malaria during pregnancy, particularly Plasmodium falciparum malaria, has been linked to increased morbidity and mortality, which must be reduced by both preventive measures and effective case management. The World Health Organization (WHO recommends artemisinin-based combination therapy (ACT to treat uncomplicated falciparum malaria during the second and third trimesters of pregnancy, and quinine plus clindamycin during the first trimester. However, the national policies of many African countries currently recommend quinine throughout pregnancy. Therefore, the aim of this article is to provide a summary of the available data on the safety and efficacy of artemether-lumefantrine (AL in pregnancy. An English-language search identified 16 publications from 1989 to October 2011 with reports of artemether or AL exposure in pregnancy, including randomized clinical trials, observational studies and systematic reviews. Overall, there were 1,103 reports of AL use in pregnant women: 890 second/third trimester exposures; 212 first trimester exposures; and one case where the trimester of exposure was not reported. In the second and third trimesters, AL was not associated with increased adverse pregnancy outcomes as compared with quinine or sulphadoxine-pyrimethamine, showed improved tolerability relative to quinine, and its efficacy was non-inferior to quinine. There is evidence to suggest that the pharmacokinetics of anti-malarial drugs may change in pregnancy, although the impact on efficacy and safety needs to be studied further, especially since the majority of studies report high cure rates and adequate tolerability. As there are fewer reports of AL safety in the first trimester, additional data are required to assess the potential to use AL in the first trimester. Though the available safety and efficacy data support the use of AL in the second and third trimesters, there is still a need for further information. These findings reinforce the
Full Text Available In Africa, infant susceptibility to Plasmodium falciparum malaria increases substantially as fetal hemoglobin (HbF and maternal immune IgG disappear from circulation. During the first few months of life, however, resistance to malaria is evidenced by extremely low parasitemias, the absence of fever, and the almost complete lack of severe disease. This resistance has previously been attributed in part to poor parasite growth in HbF-containing red blood cells (RBCs. A specific role for maternal immune IgG in infant resistance to malaria has been hypothesized but not yet identified.We found that P. falciparum parasites invade and develop normally in fetal (cord blood, CB RBCs, which contain up to 95% HbF. However, these parasitized CB RBCs are impaired in their binding to human microvascular endothelial cells (MVECs, monocytes, and nonparasitized RBCs--cytoadherence interactions that have been implicated in the development of high parasite densities and the symptoms of malaria. Abnormal display of the parasite's cytoadherence antigen P. falciparum erythrocyte membrane protein-1 (PfEMP-1 on CB RBCs accounts for these findings and is reminiscent of that on HbC and HbS RBCs. IgG purified from the plasma of immune Malian adults almost completely abolishes the adherence of parasitized CB RBCs to MVECs.Our data suggest a model of malaria protection in which HbF and maternal IgG act cooperatively to impair the cytoadherence of parasitized RBCs in the first few months of life. In highly malarious areas of Africa, an infant's contemporaneous expression of HbC or HbS and development of an immune IgG repertoire may effectively reconstitute the waning protective effects of HbF and maternal immune IgG, thereby extending the malaria resistance of infancy into early childhood.
Al-Mekhlafi, Abdulsalam M; Mahdy, Mohammed A K; Al-Mekhlafi, Hesham M; Azazy, Ahmed A; Fong, Mun Yik
Malaria remains a significant health problem in Yemen with Plasmodium falciparum being the predominant species which is responsible for 90% of the malaria cases. Despite serious concerns regarding increasing drug resistance, chloroquine is still used for the prevention and treatment of malaria in Yemen. This study was carried out to determine the prevalence of choloroquine resistance (CQR) of P. falciparum isolated from Yemen based on the pfcrt T76 mutation. A cross-sectional study was carried out among 511 participants from four governorates in Yemen. Blood samples were screened using microscopic and species-specific nested PCR based on the 18S rRNA gene to detect and identify Plasmodium species. Blood samples positive for P. falciparum were used for detecting the pfcrt T76 mutation using nested-PCR. The prevalence of pfcrt T76 mutation was 81.5% (66 of 81 isolates). Coastal areas/foothills had higher prevalence of pfcrt T76 mutation compared to highland areas (90.5% vs 71.8%) (p = 0.031). The pfcrt T76 mutation had a significant association with parasitaemia (p = 0.045). Univariate analysis shows a significant association of pfcrt T76 mutation with people aged > 10 years (OR = 9, 95% CI = 2.3 - 36.2, p = 0.001), low household income (OR = 5, 95% CI = 1.3 - 19.5, p = 0.027), no insecticide spray (OR = 3.7, 95% CI = 1.16 - 11.86, p = 0.025) and not sleeping under insecticide treated nets (ITNs) (OR = 4.8, 95% CI = 1.38 - 16.78, p = 0.01). Logistic regression model confirmed age > 10 years and low household income as predictors of pfcrt T76 mutation in Yemen P. falciparum isolates. The high prevalence of pfcrt T76 mutation in Yemen could be a predictive marker for the prevalence of P. falciparum CQR. This finding shows the necessity for an in-vivo therapeutic efficacy test for CQ. P. falciparum CQR should be addressed in the national strategy to control malaria.
Preston, M.D.; Campino, S.; Assefa, S.A.; Echeverry, D.F.; Ocholla, H.; Amambua-Ngwa, A.; Stewart, L.B.; Conway, D.J.; Borrmann, S.; Michon, P.; Zongo, I.; Ouedraogo, J.B.; Djimde, A.A.; Doumbo, O.K.; Nosten, F.; Pain, A.; Bousema, T.; Drakeley, C.J.; Fairhurst, R.M.; Sutherland, C.J.; Roper, C.; Clark, T.G.
Malaria is a major public health problem that is actively being addressed in a global eradication campaign. Increased population mobility through international air travel has elevated the risk of re-introducing parasites to elimination areas and dispersing drug-resistant parasites to new regions. A
Dwivedi, Ankit; Khim, Nimol; Reynes, Christelle; Ravel, Patrice; Ma, Laurence; Tichit, Magali; Bourchier, Christiane; Kim, Saorin; Dourng, Dany; Khean, Chanra; Chim, Pheaktra; Siv, Sovannaroth; Frutos, Roger; Lek, Dysoley; Mercereau-Puijalon, Odile; Ariey, Frédéric; Menard, Didier; Cornillot, Emmanuel
Western Cambodia is recognized as the epicentre of emergence of Plasmodium falciparum multi-drug resistance. The emergence of artemisinin resistance has been observed in this area since 2008-2009 and molecular signatures associated to artemisinin resistance have been characterized in k13 gene. At present, one of the major threats faced, is the possible spread of Asian artemisinin resistant parasites over the world threatening millions of people and jeopardizing malaria elimination programme efforts. To anticipate the diffusion of artemisinin resistance, the identification of the P. falciparum population structure and the gene flow among the parasite population in Cambodia are essential. To this end, a mid-throughput PCR-LDR-FMA approach based on LUMINEX technology was developed to screen for genetic barcode in 533 blood samples collected in 2010-2011 from 16 health centres in malaria endemics areas in Cambodia. Based on successful typing of 282 samples, subpopulations were characterized along the borders of the country. Each 11-loci barcode provides evidence supporting allele distribution gradient related to subpopulations and gene flow. The 11-loci barcode successfully identifies recently emerging parasite subpopulations in western Cambodia that are associated with the C580Y dominant allele for artemisinin resistance in k13 gene. A subpopulation was identified in northern Cambodia that was associated to artemisinin (R539T resistant allele of k13 gene) and mefloquine resistance. The gene flow between these subpopulations might have driven the spread of artemisinin resistance over Cambodia.
Hiasindh Ashmi Antony
Full Text Available The emergence and distribution of drug resistance in malaria are serious public health concerns in tropical and subtropical regions of the world. However, the molecular mechanism of drug resistance remains unclear. In the present study, we performed a high-throughput RNA-Seq to identify and characterize the differentially expressed genes between the chloroquine (CQ sensitive (3D7 and resistant (Dd2 strains of Plasmodium falciparum. The parasite cells were cultured in the presence and absence of CQ by in vitro method. Total RNA was isolated from the harvested parasite cells using TRIzol, and RNA-Seq was conducted using an Illumina HiSeq 2500 sequencing platform with paired-end reads and annotated using Tophat. The transcriptome analysis of P. falciparum revealed the expression of ~5000 genes, in which ~60% of the genes have unknown function. Cuffdiff program was used to identify the differentially expressed genes between the CQ-sensitive and resistant strains. Here, we furnish a detailed description of the experimental design, procedure, and analysis of the transcriptome sequencing data, that have been deposited in the National Center for Biotechnology Information (accession nos. PRJNA308455 and GSE77499.
Denise L. Doolan
Full Text Available Studies in mice have shown that immunity to malaria sporozoites is mediated primarily by citotoxic T lymphocytes (CTL specific for epitopes within the circumsporozoite (CS protein. Humans, had never been shown to generate CTL against any malaria or other parasite protein. The design of a sub-unit vaccine for humans ralies on the epitopes recognized by CTL being identified and polymorphisms therein being defined. We have developed a novel technique using an entire series of overlapping synthetic peptides to define the epitopes of the Plasmodium falciparum CS protein recognized by human CTL and have analyzed the sequence variation of the protein with respect to the identified CTL epitopic domain. We have demonstrated that some humans can indeed generate CTL. against the P. falciparum CS protein. Furthermore, the extent of variation observed for the CTL recognition domain is finite and the combination of peptides necessary for inclusion in a polyvalent vaccine may be small. If ways can be found to increase immune responsiveness, then a vaccine designed to stimulate CS protein-specific CTL activity may prevent malaria.
Pease, Brittany N; Huttlin, Edward L; Jedrychowski, Mark P; Talevich, Eric; Harmon, John; Dillman, Timothy; Kannan, Natarajan; Doerig, Christian; Chakrabarti, Ratna; Gygi, Steven P; Chakrabarti, Debopam
During asexual intraerythrocytic development, Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the Plasmodium asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase-substrate pairs operative in various signaling networks in the parasite.
Douglas, Nicholas M; Lampah, Daniel A; Kenangalem, Enny; Simpson, Julie A; Poespoprodjo, Jeanne R; Sugiarto, Paulus; Anstey, Nicholas M; Price, Ric N
The burden of anemia attributable to non-falciparum malarias in regions with Plasmodium co-endemicity is poorly documented. We compared the hematological profile of patients with and without malaria in southern Papua, Indonesia. Clinical and laboratory data were linked for all patients presenting to a referral hospital between April 2004 and December 2012. Data were available on patient demographics, malaria diagnosis, hemoglobin concentration, and clinical outcome, but other potential causes of anemia could not be identified reliably. Of 922,120 patient episodes (837,989 as outpatients and 84,131 as inpatients), a total of 219,845 (23.8%) were associated with a hemoglobin measurement, of whom 67,696 (30.8%) had malaria. Patients with P. malariae infection had the lowest hemoglobin concentration (n = 1,608, mean = 8.93 [95% CI 8.81-9.06]), followed by those with mixed species infections (n = 8,645, mean = 9.22 [95% CI 9.16-9.28]), P. falciparum (n = 37,554, mean = 9.47 [95% CI 9.44-9.50]), and P. vivax (n = 19,858, mean = 9.53 [95% CI 9.49-9.57]); p-value for all comparisons malaria, those with mixed Plasmodium infection were at greatest risk of severe anemia (adjusted odds ratio [AOR] 3.25 [95% CI 2.99-3.54]); AORs for severe anaemia associated with P. falciparum, P. vivax, and P. malariae were 2.11 (95% CI 2.00-2.23), 1.87 (95% CI 1.74-2.01), and 2.18 (95% CI 1.76-2.67), respectively, pcause of severe anemia in early infancy, mixed P. vivax/P. falciparum infections are associated with a greater hematological impairment than either species alone, and in adulthood P. malariae, although rare, is associated with the lowest hemoglobin concentration. These findings highlight the public health importance of integrated genus-wide malaria control strategies in areas of Plasmodium co-endemicity.
Kumar, Shiva; Mudeppa, Devaraja G; Sharma, Ambika; Mascarenhas, Anjali; Dash, Rashmi; Pereira, Ligia; Shaik, Riaz Basha; Maki, Jennifer N; White, John; Zuo, Wenyun; Tuljapurkar, Shripad; Duraisingh, Manoj T; Gomes, Edwin; Chery, Laura; Rathod, Pradipsinh K
Previous whole genome comparisons of Plasmodium falciparum populations have not included collections from the Indian subcontinent, even though two million Indians contract malaria and about 50,000 die from the disease every year. Stratification of global parasites has revealed spatial relatedness of parasite genotypes on different continents. Here, genomic analysis was further improved to obtain country-level resolution by removing var genes and intergenic regions from distance calculations. P. falciparum genomes from India were found to be most closely related to each other. Their nearest neighbors were from Bangladesh and Myanmar, followed by Thailand. Samples from the rest of Southeast Asia, Africa and South America were increasingly more distant, demonstrating a high-resolution genomic-geographic continuum. Such genome stratification approaches will help monitor variations of malaria parasites within South Asia and future changes in parasite populations that may arise from in-country and cross-border migrations. Copyright Â© 2016 The Author(s). Published by Elsevier B.V. All rights reserved.
Looareesuwan, S.; Ho, M.; Wattanagoon, Y.; White, N.J.; Warrell, D.A.; Bunnag, D.; Harinasuta, T.; Wyler, D.J.
Plasmodium-infected erythrocytes lose their normal deformability and become susceptible to splenic filtration. In animal models, this is one mechanism of antimalarial defense. To assess the effect of acute falciparum malaria on splenic filtration, we measured the clearance of heated /sup 51/Cr-labeled autologous erythrocytes in 25 patients with acute falciparum malaria and in 10 uninfected controls. Two groups of patients could be distinguished. Sixteen patients had splenomegaly, markedly accelerated clearance of the labeled erythrocytes (clearance half-time, 8.4 +/- 4.4 minutes (mean +/- SD) vs. 62.5 +/- 36.5 minutes in controls; P less than 0.001), and a lower mean hematocrit than did the patients without splenomegaly (P less than 0.001). In the nine patients without splenomegaly, clearance was normal. After institution of antimalarial chemotherapy, however, the clearance in this group accelerated to supernormal rates similar to those in the patients with splenomegaly, but without the development of detectable splenomegaly. Clearance was not significantly altered by treatment in the group with splenomegaly. Six weeks later, normal clearance rates were reestablished in most patients in both groups. We conclude that splenic clearance of labeled erythrocytes is enhanced in patients with malaria if splenomegaly is present and is enhanced only after treatment if splenomegaly is absent. Whether this enhanced splenic function applies to parasite-infected erythrocytes in patients with malaria and has any clinical benefit will require further studies.
Margaret J Mackinnon
Full Text Available Mechanisms for differential regulation of gene expression may underlie much of the phenotypic variation and adaptability of malaria parasites. Here we describe transcriptional variation among culture-adapted field isolates of Plasmodium falciparum, the species responsible for most malarial disease. It was found that genes coding for parasite protein export into the red cell cytosol and onto its surface, and genes coding for sexual stage proteins involved in parasite transmission are up-regulated in field isolates compared with long-term laboratory isolates. Much of this variability was associated with the loss of small or large chromosomal segments, or other forms of gene copy number variation that are prevalent in the P. falciparum genome (copy number variants, CNVs. Expression levels of genes inside these segments were correlated to that of genes outside and adjacent to the segment boundaries, and this association declined with distance from the CNV boundary. This observation could not be explained by copy number variation in these adjacent genes. This suggests a local-acting regulatory role for CNVs in transcription of neighboring genes and helps explain the chromosomal clustering that we observed here. Transcriptional co-regulation of physical clusters of adaptive genes may provide a way for the parasite to readily adapt to its highly heterogeneous and strongly selective environment.
Miao, Jun; Li, Xiaolian; Cui, Liwang
Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038
Nicaise Tuikue Ndam
Full Text Available Cytoadherence of Plasmodium-falciparum-parasitized red blood cells (PRBCs to host receptors is the key phenomenon in the pathological process of the malaria disease. Some of these interactions can originate poor outcomes responsible for 1 to 3 million annual deaths mostly occurring among children in sub-Saharan Africa. Pregnancy-associated malaria (PAM represents an important exception of the disease occurring at adulthood in malaria endemic settings. Consequences of this are shared between the mother (maternal anemia and the baby (low birth weight and infant mortality. Demonstrating that parasites causing PAM express specific variant surface antigens (VSAPAM, including the P. falciparum erythrocyte membrane protein 1 (PfEMP1 variant VAR2CSA, that are targets for protective immunity has strengthened the possibility for the development of PAM-specific vaccine. In this paper, we review the molecular basis of malaria pathogenesis attributable to the erythrocyte stages of the parasites, and findings supporting potential anti-PAM vaccine components evidenced in PAM.
Jovel, Irina T; Mejía, Rosa E; Banegas, Engels; Piedade, Rita; Alger, Jackeline; Fontecha, Gustavo; Ferreira, Pedro E; Veiga, Maria I; Enamorado, Irma G; Bjorkman, Anders; Ursing, Johan
In Honduras, chloroquine and primaquine are recommended and still appear to be effective for treatment of Plasmodium falciparum and Plasmodium vivax malaria. The aim of this study was to determine the proportion of resistance associated genetic polymorphisms in P. falciparum and P. vivax collected in Honduras. Blood samples were collected from patients seeking medical attention at the Hospital Escuela in Tegucigalpa from 2004 to 2006 as well as three regional hospitals, two health centres and one regional laboratory during 2009. Single nucleotide polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes and in P. vivax multidrug resistance 1 (pvmdr1) and dihydrofolate reductase (pvdhfr) genes were detected using PCR based methods. Thirty seven P. falciparum and 64 P. vivax samples were collected. All P. falciparum infections acquired in Honduras carried pfcrt, pfmdr1, pfdhps and pfdhfr alleles associated with chloroquine, amodiaquine and sulphadoxine-pyrimethamine sensitivity only. One patient with parasites acquired on a Pacific Island had pfcrt 76 T and pfmdr1 86Y alleles. That patient and a patient infected in West Africa had pfdhfr 51I, 59 R and 108 N alleles. Pvmdr1 976 F was found in 7/37 and two copies of pvmdr1 were found in 1/37 samples. Pvdhfr 57 L + 58 R was observed in 2/57 samples. The results indicate that P. falciparum from Honduras remain sensitive to chloroquine and sulphadoxine-pyrimethamine. This suggests that chloroquine and sulphadoxine-pyrimethamine should be efficacious for treatment of uncomplicated P. falciparum malaria, supporting current national treatment guidelines. However, genetic polymorphisms associated with chloroquine and sulphadoxine-pyrimethamine tolerance were detected in local P. vivax and imported P. falciparum infections. Continuous monitoring of the prevalence of drug resistant/tolerant P
Full Text Available Genetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes. A gene homologous to a tyrosine site-specific recombinase has been identified in the genome of Plasmodium falciparum. The sequence is highly conserved among five other members of Plasmodia.The predicted open reading frame encodes for a ∼57 kDa protein containing a C-terminal domain including the putative tyrosine recombinase conserved active site residues R-H-R-(H/W-Y. The N-terminus has the typical alpha-helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. Pf-Int mRNA is expressed differentially during the P. falciparum erythrocytic life stages, peaking in the schizont stage. Recombinant Pf-Int and affinity chromatography of DNA from genomic or synthetic origin were used to identify potential DNA targets after sequencing or micro-array hybridization. Interestingly, the sequences captured also included highly variable subtelomeric genes such as var, rif, and stevor sequences. Electrophoretic mobility shift assays with DNA were carried out to verify Pf-Int/DNA binding. Finally, Pf-Int knock-out parasites were created in order to investigate the biological role of Pf-Int.Our data identify for the first time a malaria parasite gene with structural and functional features of recombinases. Pf-Int may bind to and alter DNA, either in a sequence specific or in a non-specific fashion, and may contribute to programmed or random DNA rearrangements. Pf-Int is the first molecular player identified with a potential role in genome plasticity in this pathogen. Finally, Pf-Int knock-out parasite is viable showing no detectable impact on blood stage development, which is compatible with such function.
Ángela Patricia Guerra; Eliana Patricia Calvo; Moisés Wasserman; Jacqueline Chaparro-Olaya
Introducción. La producción de proteínas recombinantes es fundamental para el estudio funcional de las proteínas de Plasmodium falciparum. Sin embargo, las proteínas recombinantes de P. falciparum están entre las más difíciles de expresar y, cuando lo hacen, usualmente se agregan dentro de cuerpos de inclusión insolubles. Objetivo. Evaluar la producción de cuatro proteínas de P. falciparum usando como sistema de expresión dos cepas de Escherichia coli genéticamente modificadas para favorec...
López, Mary Luz; Vommaro, Rossiane; Zalis, Mariano
-87 μM. However, their mode of action is unknown. Steroids regulate important cellular functions including cell growth, differentiation and death. Thus, the aim of this work was to determine the effects of S. nudum compounds on P. falciparum asexual blood stages and their association with cell death. We....... The Mitochondria presented no morphological alterations and the nuclei showed no abnormal chromatin condensation. By the use of S. nudum compounds, cell death in P. falciparum was evident by a decrease in mitochondrial membrane potential, DNA fragmentation and cytoplasmic acidification. The asexual blood stages...... of P. falciparum showed some apoptotic-like and autophagic-like cell death characteristics induced by SNs treatment....
Atroosh, Wahib M; Al-Mekhlafi, Hesham M; Al-Jasari, Adel; Sady, Hany; Al-Delaimy, Ahmed K; Nasr, Nabil A; Dawaki, Salwa; Abdulsalam, Awatif M; Ithoi, Init; Lau, Yee Ling; Fong, Mun Yik; Surin, Johari
The genetic variation in the Plasmodium falciparum histidine-rich protein 2 (pfhrp2) gene that may compromise the use of pfhrp2-based rapid diagnostic tests (RDTs) for the diagnosis of malaria was assessed in P. falciparum isolates from Yemen. This study was conducted in Hodeidah and Al-Mahwit governorates, Yemen. A total of 622 individuals with fever were examined for malaria by CareStart malaria HRP2-RDT and Giemsa-stained thin and thick blood films. The Pfhrp2 gene was amplified and sequenced from 180 isolates, and subjected to amino acid repeat types analysis. A total of 188 (30.2%) participants were found positive for P. falciparum by the RDT. Overall, 12 different amino acid repeat types were identified in Yemeni isolates. Six repeat types were detected in all the isolates (100%) namely types 1, 2, 6, 7, 10 and 12 while types 9 and 11 were not detected in any of the isolates. Moreover, the sensitivity and specificity of the used PfHRP2-based RDTs were high (90.5% and 96.1%, respectively). The present study provides data on the genetic variation within the pfhrp2 gene, and its potential impact on the PfHRP2-based RDTs commonly used in Yemen. CareStart Malaria HRP2-based RDT showed high sensitivity and specificity in endemic areas of Yemen.
Wanji, Samuel; Kengne-Ouafo, Arnaud J.; Joan Eyong, Ebanga E.; Kimbi, Helen K.; Tendongfor, Nicholas; Ndamukong-Nyanga, Judith L.; Nana-Djeunga, Hugues C.; Bourguinat, Catherine; Sofeu-Feugaing, David D.; Charvet, Claude L.
The present study analyzed the relationship between the genetic diversity of Plasmodium falciparum and parasitologic/entomologic indices in the Mount Cameroon region by using merozoite surface protein 1 as a genetic marker. Blood samples were collected from asymptomatic children from three altitude zones (high, intermediate, and low). Parasitologic and entomologic indices were determined by microscopy and landing catch mosquito collection/circumsporozoite protein–enzyme-linked immunosorbent assay, respectively. A total of 142 randomly selected P. falciparum-positive blood samples were genotyped by using a nested polymerase chain reaction–based technique. K-1 polymerase chain reaction products were also sequenced. As opposed to high altitude, the highest malaria prevalence (70.65%) and entomologic inoculation rate (2.43 infective/bites/night) were recorded at a low altitude site. Seven (18.91%), 22 (36.66%), and 19 (42.22%) samples from high, intermediate, and low altitudes, respectively, contained multiclonal infections. A new K-1 polymorphism was identified. This study shows a positive non-linear association between low/intermediate altitude (high malaria transmission) and an increase in P. falciparum merozoite surface protein 1 block 2 polymorphisms. PMID:22556072
Viana, Giselle Maria Rachid; Machado, Ricardo Luís Dantas; Calvosa, Vanja Sueli Pachiano; Póvoa, Marinete Marins
The objectives of this study were to investigate the molecular basis for Plasmodium falciparum resistance to chloroquine in isolates from the Brazilian Amazon and to identify polymorphisms in the pfmdr1 gene, codons 184, 1042, and 1246, the kappa and gamma regions of the cg2 gene, and the K76T mutation of the pfcrt gene, in order to calculate the distribution of polymorphism within each target gene, comparing samples from distinct geographic areas, using allele-specific polymerase chain reaction (PCR) for the pfmdr gene and PCR plus restriction fragment length polymorphism (RFLP) for the cg2 and pfcrt genes. The sample consisted of 40 human blood isolates, already collected and morphologically diagnosed as carriers of P. falciparum parasites, from four localities: Porto Velho in Rondonia State and Maraba, Itaituba, and Tailandia in Pará State. Distribution of P. falciparum in vitro chloroquine resistance in the isolates was 100% for pfmdr1, cg2 gamma region, and pfcrt, except for the polymorphism in the cg2 kappa region, which was not found.
Mendiola Martínez, Judith; Hernández, Hilda; Acuña, Deyanira; Esquivel, Macario; Scull Lizama, Ramón; Abreu Payrol, Juan
Several terpenoids have been identified in the genus Laurencia showing antimalarial activity; however it is a genus of great chemical complexity worldwide. The majority of these substances have been extracted from collections made in Philippines and Brazil. In order to know the presence of antimalarial activity in organisms of this genus from the North West Coast of Cuba, we prepared extracts in ethanol with the whole plant of specimens of L. obtusa and L. corallopsis and evaluated the inhibition produced by them against growth of Plasmodium falciparum, strain F32. The Minimum Inhibitory Concentrations against P. falciparum determined for Laurencia extracts were in the range of 44 and 162 microg/ml, and values of median inhibitory concentrations were between 14.82 and 51.3 microg/mL. The results are similar to those obtained for extracts from medicinal plants. The extracts did not improve chloroquine results. L. obtusa extracts gave strong reactions to the assays for terpenoids and for lactonic/coumarine compounds, but we also detected the presence of alkaloids, free reducing sugars, saponins and flavonoids. Several molecules could contribute to the observed antiplasmodial activity of the extracts, so research is in progress for the isolation and purification of new active principles.
Barry, Alyssa E.; Schultz, Lee; Senn, Nicholas; Nale, Joe; Kiniboro, Benson; Siba, Peter M.; Mueller, Ivo; Reeder, John C.
High levels of genetic diversity in Plasmodium falciparum populations are an obstacle to malaria control. Here, we investigate the relationship between local variation in malaria epidemiology and parasite genetic diversity in Papua New Guinea (PNG). Cross-sectional malaria surveys were performed in 14 villages spanning four distinct malaria-endemic areas on the north coast, including one area that was sampled during the dry season. High-resolution msp2 genotyping of 2,147 blood samples identified 761 P. falciparum infections containing a total of 1,392 clones whose genotypes were used to measure genetic diversity. Considerable variability in infection prevalence and mean multiplicity of infection was observed at all of the study sites, with the area sampled during the dry season showing particularly striking local variability. Genetic diversity was strongly associated with multiplicity of infection but not with infection prevalence. In highly endemic areas, differences in infection prevalence may not translate into a decrease in parasite population diversity. PMID:23400571
Full Text Available Summary: The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid, known as the apicoplast, that functions to produce essential metabolites, and drugs that target the apicoplast are clinically effective. Several prokaryotic caseinolytic protease (Clp genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss and was rescued by addition of the essential apicoplast-derived metabolite IPP. Using a double-conditional mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex. : Plasmodium falciparum contains a unique organelle, the apicoplast. Using genetic and phenotypic assays, Florentin et al. characterize the apicoplast Clp chaperone and protease. They find that the chaperone is essential for protease stability and that together they function to maintain organelle integrity and segregation into daughter cells. Keywords: malaria, Plasmodium, apicoplast, IPP, Clp, chaperone, caseinolytic protease
Weiner, Allon; Dahan-Pasternak, Noa; Shimoni, Eyal; Shinder, Vera; von Huth, Palle; Elbaum, Michael; Dzikowski, Ron
The deadliest form of human malaria is caused by the protozoan parasite Plasmodium falciparum. The complex life cycle of this parasite is associated with tight transcriptional regulation of gene expression. Nuclear positioning and chromatin dynamics may play an important role in regulating P. falciparum virulence genes. We have applied an emerging technique of electron microscopy to construct a 3D model of the parasite nucleus at distinct stages of development within the infected red blood cell. We have followed the distribution of nuclear pores and chromatin throughout the intra-erythrocytic cycle, and have found a striking coupling between the distributions of nuclear pores and chromatin organization. Pore dynamics involve clustering, biogenesis, and division among daughter cells, while chromatin undergoes stage-dependent changes in packaging. Dramatic changes in heterochromatin distribution coincide with a previously identified transition in gene expression and nucleosome positioning during the mid-to-late schizont phase. We also found a correlation between euchromatin positioning at the nuclear envelope and the local distribution of nuclear pores, as well as a dynamic nuclear polarity during schizogony. These results suggest that cyclic patterns in gene expression during parasite development correlate with gross changes in cellular and nuclear architecture. © 2011 Blackwell Publishing Ltd.
Full Text Available Introducción. Los estadios sexuales de Plasmodium falciparum han sido menos estudiados que los estadios asexuales. Al parecer, esto se debe a la carencia de cultivos estandarizados in vitro y a la dificultad de reconocer sus estadios de desarrollo. Estos hechos no permiten el estudio de aspectos biológicos, aspectos metabólicos, expresión de genes y síntesis de proteínas durante los estadios sexuales, temas de interés en la investigación de nuevos medicamentos antipalúdicos, principalmente los aislados de plantas, y la identificación de un potencial blanco contra Plasmodium. Objetivos. Establecer un cultivo in vitro de gametocitos, con la identificación de sus cinco estadios de desarrollo, y asegurar su continua producción. Materiales y métodos. El cultivo in vitro de gametocitos se realizó a partir de la cepa NF54 de P. falciparum en medio RPMI, con determinación de la parasitemia asexual y sexual, adición de glóbulos rojos A-Rh+ sólo el primer día de cultivo y cambio diario del medio con adición de mezcla de gases (90% N2, 5% O2; 5% CO2, asegurándose que el cultivo se mantuviera a 37 °C. Cuando la parasitemia asexual estuvo entre 3% y 5%, se comenzó a agregar el doble de volumen de medio. Resultados. Se obtuvieron gametocitos en estadios I, II y III a partir del día 11 de cultivo y estadios IV y V a partir del día 14 de cultivo. Conclusiones. Se estandarizó un cultivo in vitro para estadios sexuales de P. falciparum que puede usarse para futuros estudios de evaluación de compuestos, naturales o sintéticos, que actúen sobre los gametocitos, lo cual podría permitir el desarrollo de nuevas estrategias de control contra el paludismo.
White, John; Mascarenhas, Anjali; Pereira, Ligia; Dash, Rashmi; Walke, Jayashri T; Gawas, Pooja; Sharma, Ambika; Manoharan, Suresh Kumar; Guler, Jennifer L; Maki, Jennifer N; Kumar, Ashwani; Mahanta, Jagadish; Valecha, Neena; Dubhashi, Nagesh; Vaz, Marina; Gomes, Edwin; Chery, Laura; Rathod, Pradipsinh K
Culture-adapted Plasmodium falciparum parasites can offer deeper understanding of geographic variations in drug resistance, pathogenesis and immune evasion. To help ground population-based calculations and inferences from culture-adapted parasites, the complete range of parasites from a study area must be well represented in any collection. To this end, standardized adaptation methods and determinants of successful in vitro adaption were sought. Venous blood was collected from 33 P. falciparum-infected individuals at Goa Medical College and Hospital (Bambolim, Goa, India). Culture variables such as whole blood versus washed blood, heat-inactivated plasma versus Albumax, and different starting haematocrit levels were tested on fresh blood samples from patients. In vitro adaptation was considered successful when two four-fold or greater increases in parasitaemia were observed within, at most, 33 days of attempted culture. Subsequently, parasites from the same patients, which were originally cryopreserved following blood draw, were retested for adaptability for 45 days using identical host red blood cells (RBCs) and culture media. At a new endemic area research site, ~65% of tested patient samples, with varied patient history and clinical presentation, were successfully culture-adapted immediately after blood collection. Cultures set up at 1% haematocrit and 0.5% Albumax adapted most rapidly, but no single test condition was uniformly fatal to culture adaptation. Success was not limited by low patient parasitaemia nor by patient age. Some parasites emerged even after significant delays in sample processing and even after initiation of treatment with anti-malarials. When 'day 0' cryopreserved samples were retested in parallel many months later using identical host RBCs and media, speed to adaptation appeared to be an intrinsic property of the parasites collected from individual patients. Culture adaptation of P. falciparum in a field setting is formally shown to be
Chotivanich, K.; Udomsangpetch, R.; Dondorp, A.; Williams, T.; Angus, B.; Simpson, J. A.; Pukrittayakamee, S.; Looareesuwan, S.; Newbold, C. I.; White, N. J.
Studies were conducted to determine how malaria parasites are cleared from the blood after antimalarial treatment. Neither artesunate nor quinine decreased parasitized red cell deformability or increased antibody binding. In acute falciparum malaria, ring-infected erythrocyte surface antigen (RESA)
The purpose of this report is to present data on the evaluation of drugs and vaccines in the human malaria/Aotus lemurinus lemurinus monkey model experimentally infected with Plasmodium falciparum or vivax...
Khanh, N. X.; de Vries, P. J.; Ha, L. D.; van Boxtel, C. J.; Koopmans, R.; Kager, P. A.
Six patients with uncomplicated falciparum malaria received artesunate for 5 days. Plasma concentrations of artesunate and dihydroartemisinin were determined by high-performance liquid chromatography with electrochemical detection. The concentrations of dihydroartemisinin in plasma 2 h after a dose
Ting, L; Shi, W; Lewandowicz, A; Singh, V; Mwakingwe, A; Birck, M R; Taylor Ringia, E A; Bench, G; Madrid, D C; Tyler, P C; Evans, G B; Furneaux, R H; Schramm, V L; Kim, K.
Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of the polyamine pathway are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not previously been described. 5'-methylthio-Immucillin-H, a transition state analogue inhibitor that is selective for malarial over human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may have application as antimalarials
Buchholz, Ulrike; Kobbe, Robin; Danquah, Ina; Zanger, Philipp; Reither, Klaus; Abruquah, Harry H.; Grobusch, Martin P.; Ziniel, Peter; May, Jürgen; Mockenhaupt, Frank P.
Intermittent preventive treatment in infants with sulphadoxine-pyrimethamine (IPTi-SP) reduces malaria morbidity by 20% to 33%. Potentially, however, this intervention may compromise the acquisition of immunity, including the tolerance towards multiple infections with Plasmodium falciparum.
Venkatesan, Meera; Gadalla, Nahla B; Stepniewska, Kasia
Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated...... with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized...
Full Text Available Background: Cytoadherence of Plasmodium falciparum- infected erythrocytes to host cells is an important trait for parasite survival and has a major role in pathology of malaria disease. Infections with P. falciparum usually consist of several subpopulations of parasites with different adhesive properties. This study aimed to compare relative sizes of various binding subpopulations of different P. falciparum isolates. It also investigated the adhesive phenotype of a laboratory P. falciparum line, A4, using different binding techniques.Methods: Seven different P. falciparum isolates (ITG, A4, 3D7 and four field isolates were cultivated to late trophozoite and schizont and then cytoadherence to cell differentiation 36 (CD36, intercellular cell adhesion molecule-1 (ICAM-1, and vascular cell adhesion molecule (V-CAM and E-selectin were examined. The relative binding sizes of parasite subpopulations to human receptors were measured by mini-column cytoadherence method. The adhesion phenotype of P. falciparum-A4 line was evaluated by in vitro static, flow-based and mini-column binding assays.Results: The relative binding size of ITG, A4 and 3D7 clones to a column made with CHO/ICAM-1 was 68%, 54% and 0%, respectively. The relative binding sizes of these lines to CHO/CD36 were 59.7%, 28.7% and 0%, respectively. Different field isolates had variable sizes of respective CD36 and ICAM1-binding subpopulations. A4 line had five different subpopulations each with different binding sizes.Conclusion: This study provided further evidence that P. falciparum isolates have different binding subpopulations sizes in an infection. Furthermore, measurement of ICAM-1 or CD36 binding subpopulations may practical to study the cytoadherence phenotypes of P. falciparum field isolates at the molecular level.
Jakobsen, P H; Riley, E M; Allen, S J
A seroepidemiological and clinical study was performed in an area of West Africa (The Gambia) where Plasmodium falciparum is endemic with seasonal transmission. Plasma samples were tested by intermediate gel immunoelectrophoresis for antibodies against 7 soluble P. falciparum antigens. There were...... who had had a documented attack of clinical malaria or parasitaemia. There was no difference in antibody profiles to soluble antigens between children with sickle cell trait and children with normal haemoglobin....
Nag, Sidsel; Kofoed, Poul Erik; Ursing, Johan
Background: Plasmodium falciparum malaria remains a major health burden and genomic research represents one of the necessary approaches for continued progress towards malaria control and elimination. Sample acquisition for this purpose is troublesome, with the majority of malaria-infected individ......Background: Plasmodium falciparum malaria remains a major health burden and genomic research represents one of the necessary approaches for continued progress towards malaria control and elimination. Sample acquisition for this purpose is troublesome, with the majority of malaria...
clearance after artesunate- mefloquine treatment had been reported (7); and in Bandarban, Bangladesh, where artemisinins had been little used (Fig. S1...2007) Pfmdr1 and in vivo resistance to artesunate- mefloquine in falciparum malaria on the Cambodian-Thai border. Am J Trop Med Hyg 76(4): 641–647. 5...Denis MB, et al. (2006) Surveillance of the efficacy of artesunate and mefloquine combination for the treatment of uncomplicated falciparum malaria
Marschallek, Maria Rebekka
Background: Malaria remains one of the major causes of morbidity and mortality worldwide. Artemisinin-based combination therapies are used as first-line treatment in all endemic countries for uncomplicated P. falciparum malaria. artemether-lumefantrine was introduced as first-line treatment in Kenya in 2006. Since the first report of artemisinin-resistant P. falciparum malaria in 2009 on the Thai-Cambodian border, concerns about declining responsiveness to artemisinins have also been expresse...
John, Chandy C; Kutamba, Elizabeth; Mugarura, Keith; Opoka, Robert O
Severe malaria due to Plasmodium falciparum causes more than 800,000 deaths every year. Primary therapy with quinine or artesunate is generally effective in controlling P. falciparum parasitemia, but mortality from cerebral malaria and other forms of severe malaria remains unacceptably high. Long-term cognitive impairment is also common in children with cerebral malaria. Of the numerous adjunctive therapies for cerebral malaria and severe malaria studied over the past five decades, only one (...
Valenzuela Jesus G
Full Text Available Abstract Background The completion of the Plasmodium falciparum genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions. Results We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST, including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons versus 5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6% with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions. Conclusion Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively
Gerald, Noel; Mahajan, Babita; Kumar, Sanjai
Malaria is caused by intraerythrocytic protozoan parasites belonging to Plasmodium spp. (phylum Apicomplexa) that produce significant morbidity and mortality, mostly in developing countries. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. During the life cycle, the parasites undergo several cycles of extreme population growth within a brief span, and this is critical for their continued transmission and a contributing factor for their pathogenesis in the host. As with other eukaryotes, successful mitosis is an essential requirement for Plasmodium reproduction; however, some aspects of Plasmodium mitosis are quite distinct and not fully understood. In this review, we will discuss the current understanding of the architecture and key events of mitosis in Plasmodium falciparum and related parasites and compare them with the traditional mitotic events described for other eukaryotes. PMID:21317311
Brockelman, C R; Tan-Ariya, P
Three recently isolated Thai strains of Plasmodium falciparum were tested in vitro for their response to pyrimethamine, sulfadoxine, and both compounds in combination. One isolate, FCK, was moderately tolerant to pyrimethamine whereas FCM3 and FCM5 were not affected by this compound at serum level, being 100 times less responsive than a sensitive strain from the Gambia when tested similarly. This low degree of response is evidently due to drug resistance. Sensitivity to sulfadoxine was low in the Thai isolates, but was also lower than expected in the Gambian strain. Possible causes of the poor response are discussed. Pyrimethamine in combination with sulfadoxine showed a synergistic effect; the effective doses were reduced by ten and three times, respectively.
Rupp, Ingrid; Bosse, Rebecca; Schirmeister, Tanja; Pradel, Gabriele
Enzymes involved in sexual differentiation and fertilization of the human malaria parasite Plasmodium falciparum represent potential targets for transmission blocking strategies. Parasite proteases are putatively involved in several steps during fertilization, but the types of proteases, their targets and modes of action remain hitherto unknown. We investigated the involvement of proteases in gametogenesis via exflagellation and immunofluorescence assays, using a variety of commercially available as well as newly designed protease inhibitors. The assays revealed a blockade of microgamete formation by the cysteine/serine protease inhibitors TLCK and TPCK. The serine protease inhibitor PMSF, the falcipain-targeting inhibitor RV112D, and the aspartic protease inhibitor EPNP also significantly decreased formation of microgametes. The metalloprotease inhibitor 1,10-phenanthroline, on the other hand, inhibited exflagellation by interfering with microgamete motility. Furthermore, EPNP reduced the activation of male and female gametocytes. Our data point to a major involvement of serine proteases and a non-thermolysin-like zinc metalloprotease in microgametocyte exflagellation.
Ofori, Michael F; Lamptey, Helena; Dickson, Emmanuel K; Kyei-Baafour, Eric; Hviid, Lars
Plasmodium falciparum parasites causing placental malaria (PM) express the VAR2CSA type of the clonally variant antigen family PfEMP1. This enables evasion of pre-existing immunity and results in placental accumulation of infected erythrocytes (IEs). We present data on seasonal variation in levels of VAR2CSA-specific IgG and IgG specific for a PM-unrelated PfEMP1 protein among Ghanaian women at first antenatal visit. Our results indicate that PM does not require recent exposure to infected mosquitoes, in contrast to malaria in general. This has implications for the impact of insecticide-treated bed nets on PM incidence, and for antenatal care in woman with pre-existing immunity.
Miotto, Olivo; Almagro-Garcia, Jacob; Manske, Magnus; MacInnis, Bronwyn; Campino, Susana; Rockett, Kirk A; Amaratunga, Chanaki; Lim, Pharath; Suon, Seila; Sreng, Sokunthea; Anderson, Jennifer M; Duong, Socheat; Nguon, Chea; Chuor, Char Meng; Saunders, David; Se, Youry; Lon, Chantap; Fukuda, Mark M; Amenga-Etego, Lucas; Hodgson, Abraham VO; Asoala, Victor; Imwong, Mallika; Takala-Harrison, Shannon; Nosten, Francois; Su, Xin-zhuan; Ringwald, Pascal; Ariey, Frédéric; Dolecek, Christiane; Hien, Tran Tinh; Boni, Maciej F; Thai, Cao Quang; Amambua-Ngwa, Alfred; Conway, David J; Djimdé, Abdoulaye A; Doumbo, Ogobara K; Zongo, Issaka; Ouedraogo, Jean-Bosco; Alcock, Daniel; Drury, Eleanor; Auburn, Sarah; Koch, Oliver; Sanders, Mandy; Hubbart, Christina; Maslen, Gareth; Ruano-Rubio, Valentin; Jyothi, Dushyanth; Miles, Alistair; O’Brien, John; Gamble, Chris; Oyola, Samuel O; Rayner, Julian C; Newbold, Chris I; Berriman, Matthew; Spencer, Chris CA; McVean, Gilean; Day, Nicholas P; White, Nicholas J; Bethell, Delia; Dondorp, Arjen M; Plowe, Christopher V; Fairhurst, Rick M; Kwiatkowski, Dominic P
We describe an analysis of genome variation in 825 Plasmodium falciparum samples from Asia and Africa that reveals an unusual pattern of parasite population structure at the epicentre of artemisinin resistance in western Cambodia. Within this relatively small geographical area we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and remarkably high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalogue of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in various transporter proteins and DNA mismatch repair proteins. These data provide a population genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist its elimination. PMID:23624527
The emergence and spread of Plasmodium falciparum parasite resistant to sulfadoxine and pyrimethamine (SP) poses a serious public health problem. Resistance is caused by point mutations in dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps), the two key enzymes in the folate biosynthetic pathway. The use of microsatellite markers flanking pfdhfr has recently shown that the invasion of limited resistant lineages may explain the widespread SP resistance in many endemic regions. In Africa, however, multiple indigenous origins of pfdhfr triple mutants have been demonstrated. More new independent lineages and routes of geographical spread of resistance may be found by further molecular evolutionary analyses using samples from various endemic regions. Here, I review recent studies about the history of SP usage and the evolution and spread of resistant lineages while addressing the technical issue of microsatellite analysis. Copyright (c) 2009 Elsevier B.V. All rights reserved.
Jakobsen, P H; Morris-Jones, S D; Hviid, L
Plasma levels of antibodies against phosphatidylinositol (PI), phosphatidylcholine (PC) and cardiolipin (CL) were measured by enzyme-linked immunosorbent assay (ELISA) in patients from malaria endemic area of Sudan and The Gambia. Some Sudanese adults produced IgM antibodies against all three types...... of phospholipids (PL) during an acute Plasmodium falciparum infection. The anti-PL antibody titre returned to preinfection levels in most of the donors 30 days after the disease episode. IgG titres against PI, PC and CL were low. In Gambian children with malaria, IgM antibody titres against PI and PC were...... significantly higher in those with severe malaria than in those with mild malaria. These results show that a proportion of malaria patients produce anti-PL antibodies during infection and that titres of these antibodies are associated with the severity of disease....
Infection by Plasmodium falciparum parasites can lead to substantial protective immunity to malaria, and available evidence suggest that acquisition of protection against some severe malaria syndromes can be fairly rapid. Although these facts have raised hopes that the development of effective...... antigens on the surface of the infected erythrocytes, can explain some of the difficulties in relating particular immune responses with specificity for well-defined antigenic targets to clinical protection, and suggests a radically new approach to controlling malaria-related morbidity and mortality...... vaccines against this major cause of human misery is a realistic goal, the uncertainty regarding the antigenic targets of naturally acquired protective immunity and the immunological mechanisms involved remain major vaccine development obstacles. Nevertheless, a coherent theoretical framework of how...
Alyssa E Barry
Full Text Available Var genes encode the major surface antigen (PfEMP1 of the blood stages of the human malaria parasite Plasmodium falciparum. Differential expression of up to 60 diverse var genes in each parasite genome underlies immune evasion. We compared the diversity of the DBLalpha domain of var genes sampled from 30 parasite isolates from a malaria endemic area of Papua New Guinea (PNG and 59 from widespread geographic origins (global. Overall, we obtained over 8,000 quality-controlled DBLalpha sequences. Within our sampling frame, the global population had a total of 895 distinct DBLalpha "types" and negligible overlap among repertoires. This indicated that var gene diversity on a global scale is so immense that many genomes would need to be sequenced to capture its true extent. In contrast, we found a much lower diversity in PNG of 185 DBLalpha types, with an average of approximately 7% overlap among repertoires. While we identify marked geographic structuring, nearly 40% of types identified in PNG were also found in samples from different countries showing a cosmopolitan distribution for much of the diversity. We also present evidence to suggest that recombination plays a key role in maintaining the unprecedented levels of polymorphism found in these immune evasion genes. This population genomic framework provides a cost effective molecular epidemiological tool to rapidly explore the geographic diversity of var genes.
Jeeyapant, Atthanee; Kingston, Hugh W; Plewes, Katherine; Maude, Richard J; Hanson, Josh; Herdman, M Trent; Leopold, Stije J; Ngernseng, Thatsanun; Charunwatthana, Prakaykaew; Phu, Nguyen Hoan; Ghose, Aniruddha; Hasan, M Mahtab Uddin; Fanello, Caterina I; Faiz, Md Abul; Hien, Tran Tinh; Day, Nicholas P J; White, Nicholas J; Dondorp, Arjen M
Clinical trials in severe falciparum malaria require a large sample size to detect clinically meaningful differences in mortality. This means few interventions can be evaluated at any time. Using a validated surrogate endpoint for mortality would provide a useful alternative allowing a smaller sample size. Here we evaluate changes in coma score and plasma lactate as surrogate endpoints for mortality in severe falciparum malaria. Three datasets of clinical studies in severe malaria were re-evaluated: studies from Chittagong, Bangladesh (adults), the African 'AQUAMAT' trial comparing artesunate and quinine (children), and the Vietnamese 'AQ' study (adults) comparing artemether with quinine. The absolute change, relative change, slope of the normalization over time, and time to normalization were derived from sequential measurements of plasma lactate and coma score, and validated for their use as surrogate endpoint, including the proportion of treatment effect on mortality explained (PTE) by these surrogate measures. Improvements in lactate concentration or coma scores over the first 24 hours of admission, were strongly prognostic for survival in all datasets. In hyperlactataemic patients in the AQ study (n = 173), lower mortality with artemether compared to quinine closely correlated with faster reduction in plasma lactate concentration, with a high PTE of the relative change in plasma lactate at 8 and 12 hours of 0.81 and 0.75, respectively. In paediatric patients enrolled in the 'AQUAMAT' study with cerebral malaria (n = 785), mortality was lower with artesunate compared to quinine, but this was not associated with faster coma recovery. The relative changes in plasma lactate concentration assessed at 8 or 12 hours after admission are valid surrogate endpoints for severe malaria studies on antimalarial drugs or adjuvant treatments aiming at improving the microcirculation. Measures of coma recovery are not valid surrogate endpoints for mortality.
Full Text Available Abstract Background The increasing levels of Plasmodium falciparum resistance to chloroquine (CQ in Thailand have led to the use of alternative antimalarials, which are at present also becoming ineffective. In this context, any strategies that help improve the surveillance of drug resistance, become crucial in overcoming the problem. Methods In the present study, we have established the in vitro sensitivity to CQ, mefloquine (MF, quinine (QUIN and amodiaquine (AMQ of 52 P. falciparum isolates collected in Thailand, and assessed the prevalence of four putative genetic polymorphisms of drug resistance, pfcrt K76T, pfmdr1 N86Y, pfmdr1 D1042N and pfmdr1 Y1246D, by PCR-RFLP. Results The percentage of isolates resistant to CQ, MF, and AMQ was 96% (50/52, 62% (32/52, and 58% (18/31, respectively, while all parasites were found to be sensitive to QUIN. In addition, 41 (79% of the isolates assayed were resistant simultaneously to more than one drug; 25 to CQ and MF, 9 to CQ and AMQ, and 7 to all three drugs, CQ, MF and AMQ. There were two significant associations between drug sensitivity and presence of particular molecular markers, i CQ resistance / pfcrt 76T (P = 0.001, and ii MF resistance / pfmdr1 86N (P Conclusions i In Thailand, the high levels of CQ pressure have led to strong selection of the pfcrt 76T polymorphism and ii pfmdr1 86N appears to be a good predictor of in vitro MF resistance.
Zocher, Kathleen; Fritz-Wolf, Karin; Kehr, Sebastian; Fischer, Marina; Rahlfs, Stefan; Becker, Katja
Glutamate dehydrogenases (GDHs) play key roles in cellular redox, amino acid, and energy metabolism, thus representing potential targets for pharmacological interventions. Here we studied the functional network provided by the three known glutamate dehydrogenases of the malaria parasite Plasmodium falciparum. The recombinant production of the previously described PfGDH1 as hexahistidyl-tagged proteins was optimized. Additionally, PfGDH2 was cloned, recombinantly produced, and characterized. Like PfGDH1, PfGDH2 is an NADP(H)-dependent enzyme with a specific activity comparable to PfGDH1 but with slightly higher K(m) values for its substrates. The three-dimensional structure of hexameric PfGDH2 was solved to 3.1 Å resolution. The overall structure shows high similarity with PfGDH1 but with significant differences occurring at the subunit interface. As in mammalian GDH1, in PfGDH2 the subunit-subunit interactions are mainly assisted by hydrogen bonds and hydrophobic interactions, whereas in PfGDH1 these contacts are mediated by networks of salt bridges and hydrogen bonds. In accordance with this, the known bovine GDH inhibitors hexachlorophene, GW5074, and bithionol were more effective on PfGDH2 than on PfGDH1. Subcellular localization was determined for all three plasmodial GDHs by fusion with the green fluorescent protein. Based on our data, PfGDH1 and PfGDH3 are cytosolic proteins whereas PfGDH2 clearly localizes to the apicoplast, a plastid-like organelle specific for apicomplexan parasites. This study provides new insights into the structure and function of GDH isoenzymes of P. falciparum, which represent potential targets for the development of novel antimalarial drugs. Copyright Â© 2012 Elsevier B.V. All rights reserved.
Full Text Available Abstract Background Maps of malaria distribution are vital for optimal allocation of resources for anti-malarial activities. There is a lack of reliable contemporary malaria maps in endemic countries in sub-Saharan Africa. This problem is particularly acute in low malaria transmission countries such as those located in the horn of Africa. Methods Data from a national malaria cluster sample survey in 2005 and routine cluster surveys in 2007 were assembled for Somalia. Rapid diagnostic tests were used to examine the presence of Plasmodium falciparum parasites in finger-prick blood samples obtained from individuals across all age-groups. Bayesian geostatistical models, with environmental and survey covariates, were used to predict continuous maps of malaria prevalence across Somalia and to define the uncertainty associated with the predictions. Results For analyses the country was divided into north and south. In the north, the month of survey, distance to water, precipitation and temperature had no significant association with P. falciparum prevalence when spatial correlation was taken into account. In contrast, all the covariates, except distance to water, were significantly associated with parasite prevalence in the south. The inclusion of covariates improved model fit for the south but not for the north. Model precision was highest in the south. The majority of the country had a predicted prevalence of Conclusion The maps showed that malaria transmission in Somalia varied from hypo- to meso-endemic. However, even after including the selected covariates in the model, there still remained a considerable amount of unexplained spatial variation in parasite prevalence, indicating effects of other factors not captured in the study. Nonetheless the maps presented here provide the best contemporary information on malaria prevalence in Somalia.
Noor, Abdisalan M; Clements, Archie C A; Gething, Peter W; Moloney, Grainne; Borle, Mohammed; Shewchuk, Tanya; Hay, Simon I; Snow, Robert W
Maps of malaria distribution are vital for optimal allocation of resources for anti-malarial activities. There is a lack of reliable contemporary malaria maps in endemic countries in sub-Saharan Africa. This problem is particularly acute in low malaria transmission countries such as those located in the horn of Africa. Data from a national malaria cluster sample survey in 2005 and routine cluster surveys in 2007 were assembled for Somalia. Rapid diagnostic tests were used to examine the presence of Plasmodium falciparum parasites in finger-prick blood samples obtained from individuals across all age-groups. Bayesian geostatistical models, with environmental and survey covariates, were used to predict continuous maps of malaria prevalence across Somalia and to define the uncertainty associated with the predictions. For analyses the country was divided into north and south. In the north, the month of survey, distance to water, precipitation and temperature had no significant association with P. falciparum prevalence when spatial correlation was taken into account. In contrast, all the covariates, except distance to water, were significantly associated with parasite prevalence in the south. The inclusion of covariates improved model fit for the south but not for the north. Model precision was highest in the south. The majority of the country had a predicted prevalence of or = 5% prevalence were predominantly in the south. The maps showed that malaria transmission in Somalia varied from hypo- to meso-endemic. However, even after including the selected covariates in the model, there still remained a considerable amount of unexplained spatial variation in parasite prevalence, indicating effects of other factors not captured in the study. Nonetheless the maps presented here provide the best contemporary information on malaria prevalence in Somalia.
Tang Girdwood, Sonya C; Nenortas, Elizabeth; Shapiro, Theresa A
Drug-resistant malaria poses a major public health problem throughout the world and the need for new antimalarial drugs is growing. The apicoplast, a chloroplast-like organelle essential for malaria parasite survival and with no counterpart in humans, offers an attractive target for selectively toxic new therapies. The apicoplast genome (plDNA) is a 35 kb circular DNA that is served by gyrase, a prokaryotic type II topoisomerase. Gyrase is poisoned by fluoroquinolone antibacterials that stabilize a catalytically inert ternary complex of enzyme, its plDNA substrate, and inhibitor. We used fluoroquinolones to study the gyrase and plDNA of Plasmodium falciparum. New methods for isolating and separating plDNA reveal four topologically different forms and permit a quantitative exam of perturbations that result from gyrase poisoning. In keeping with its role in DNA replication, gyrase is most abundant in late stages of the parasite lifecycle, but several lines of evidence indicate that even in these cells the enzyme is present in relatively low abundance: about 1 enzyme for every two plDNAs or a ratio of 1 gyrase: 70 kb DNA. For a spectrum of quinolones, correlation was generally good between antimalarial activity and gyrase poisoning, the putative molecular mechanism of drug action. However, in P. falciparum there is evidence for off-target toxicity, particularly for ciprofloxacin. These studies highlight the utility of the new methods and of fluoroquinolones as a tool for studying the in situ workings of gyrase and its plDNA substrate. Copyright © 2015 Elsevier Inc. All rights reserved.
Full Text Available Abstract Background In areas where malaria is endemic, pregnancy is associated with increased susceptibility to malaria. It is generally agreed that this risk ends with delivery and decreases with the number of pregnancies. Our study aimed to demonstrate relationships between malarial parasitaemia and age, gravidity and anaemia in pregnant women in Libreville, the capital city of Gabon. Methods Peripheral blood was collected from 311 primigravidae and women in their second pregnancy. Thick blood smears were checked, as were the results of haemoglobin electrophoresis. We also looked for the presence of anaemia, fever, and checked whether the volunteers had had chemoprophylaxis. The study was performed in Gabon where malaria transmission is intense and perennial. Results A total of 177 women (57% had microscopic parasitaemia; 139 (64%of them were primigravidae, 38 (40% in their second pregnancy and 180 (64% were teenagers. The parasites densities were also higher in primigravidae and teenagers. The prevalence of anaemia was 71% and was associated with microscopic Plasmodium falciparum parasitaemia: women with moderate or severe anaemia had higher parasite prevalences and densities. However, the sickle cell trait, fever and the use of chemoprophylaxis did not have a significant association with the presence of P. falciparum. Conclusions These results suggest that the prevalence of malaria and the prevalence of anaemia, whether associated with malaria or not, are higher in pregnant women in Gabon. Primigravidae and young pregnant women are the most susceptible to infection. It is, therefore, urgent to design an effective regimen of malaria prophylaxis for this high risk population.
Ahmedou Salem, Mohamed Salem O; Ndiaye, Magatte; OuldAbdallahi, Mohamed; Lekweiry, Khadijetou M; Bogreau, Hervé; Konaté, Lassana; Faye, Babacar; Gaye, Oumar; Faye, Ousmane; Mohamed Salem O Boukhary, Ali O
The genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Mauritania. The present study examined and compared the genetic diversity of P. falciparum isolates in Mauritania. Plasmodium falciparum isolates blood samples were collected from 113 patients attending health facilities in Nouakchott and Hodh El Gharbi regions. K1, Mad20 and RO33 allelic family of msp-1 gene were determined by nested PCR amplification. K1 family was the predominant allelic type carried alone or in association with Ro33 and Mad20 types (90%; 102/113). Out of the 113 P. falciparum samples, 93(82.3%) harboured more than one parasite genotype. The overall multiplicity of infection was 3.2 genotypes per infection. There was no significant correlation between multiplicity of infection and age of patients. A significant increase of multiplicity of infection was correlated with parasite densities. The polymorphism of P. falciparum populations from Mauritania was high. Infection with multiple P. falciparum clones was observed, as well as a high multiplicity of infection reflecting both the high endemicity level and malaria transmission in Mauritania.
She, Guo-lin; Ma, Yu-Cai; Wang, Fu-biao
To analyze the current situation of the comprehensive prevention and control system for imported falciparum malaria in Hanjiang District and evaluate its effect. According to the Management Scheme on Control of Imported Falciparum Malaria in Yangzhou City, the comprehensive prevention and control system for imported falciparum malaria was implemented, and the relevant malaria data were collected and analyzed statistically. The data included plasmodium blood test ratio of fever patients among exported labors and those returned, the ratio of laboratory-confirmed cases among all reported cases of falciparum malaria, the ratio of falciparum malaria patients who received the standard treatment within 24 hours after onset, etc from 2010 to 2012. After the implementation of the comprehensive prevention and control system, the confirmation ratio of falciparum malaria cases within 24 hours following first visit has reached 60.47%, the average time from first visit to confirmation has shortened to 1.8 d, and the average time from onset to confirmation has shortened to 3.7 d. The health education coverage ratio was 100%, the health knowledge awareness ratio was 95.56%, the ratio of patients seeking treatment on own initiative was 100%, the laboratory-confirmed ratio was 100%, and the ratio of standard treatment after malaria diagnosis was 100%. The comprehensive prevention and control system carried out by Hanjiang District has made remarkable achievements.
Human recombinant antibodies against Plasmodium falciparum merozoite surface protein 3 cloned from peripheral blood leukocytes of individuals with immunity to malaria demonstrate antiparasitic properties
Lundquist, Rasmus; Nielsen, Leif Kofoed; Jafarshad, Ali
against MSP-3 residues 194 to 257 (MSP-3(194-257)) on the molecular level. mRNA from peripheral blood leukocytes from clinically immune individuals was used as a source of Fab (fragment antibody) genes. A Fab-phage display library was made, and three distinct antibodies designated RAM1, RAM2, and RAM3...... were isolated by panning. Immunoglobulin G1 (IgG1) and IgG3 full-length antibodies have been produced in CHO cells. Reactivity with the native parasite protein was demonstrated by immunofluorescence microscopy, flow cytometry, and immunoblotting. Furthermore, the antiparasitic effect of RAM1 has been...... tested in vitro in an antibody-dependent cellular inhibition (ADCI) assay. Both the IgG1 and the IgG3 versions of the antibody show an inhibitory effect on parasite growth....
Happi, Christian T; Gbotosho, Grace O; Folarin, Onikepe A; Milner, Danny; Sarr, Ousmane; Sowunmi, Akintunde; Kyle, Dennis E; Milhous, Wilbur K; Wirth, Dyann F; Oduola, Ayoade MJ
Abstract Background In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb) gene (Tyr268Ser and Tyr268Asn). However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa. Methods The prevalence of codon-268 mutations in the cytb gene of African P. falciparum...
Carol J. Palmer
Full Text Available We report on our investigation of a malaria outbreak in Honduras, Central America, in January 1997. We tested 202 patients with fever and chills using thin and thick blood film microscopy. Sixteen patients lived in the city and the rest lived in rural areas. A total of 95 samples (47% were positive for malaria parasites. Seventy-nine percent (63/80 of the rural patients were infected with Plasmodium vivax and 21% (17/80 were infected with P. falciparum. In the urban area, all 15 infected patients had P. vivax malaria and none showed evidence of P. falciparum. Since previous reports indicate that falciparum malaria accounts for only 2% of the overall malaria infections in Honduras, the results reported here suggest that there is a dramatic increase in falciparum malaria in the area of Honduras investigated in this study.Notificamos los resultados de un estudio de un brote de malaria que se produjo en Honduras, Centroamérica, en enero de 1997. Sometimos a examen microscópico frotis delgados y frotis gruesos de la sangre de 202 pacientes con fiebre y escalofríos. Dieciséis pacientes eran habitantes de la zona urbana y el resto de la zona rural. Un total de 95 especímenes (47% fueron positivos a parásitos de la malaria. Setenta y ocho por ciento (62/80 de los pacientes del área rural estaban infestados con Plasmodium vivax y 22% (17/80 con P. falciparum. En la zona urbana, todos los 15 pacientes que estaban infestados tenían P. vivax y en ninguno se detectó P. falciparum. Ya que según informes previos la malaria de tipo falciparum representa solamente 2% de todos los casos de malaria en Honduras, nuestros resultados sugieren que hay un gran incremento del número de casos de malaria falciparum en la zona de Honduras en que se llevó a cabo esta investigación.
Maja Verena Nielsen
Full Text Available Differentiation of infectious causes in severely ill children is essential but challenging in sub- Saharan Africa. The aim of the study was to determine clinical indicators that are able to identify bacterial co-infections in P. falciparum infected children in rural Ghana. In total, 1,915 severely ill children below the age of 15 years were recruited at Agogo Presbyterian Hospital in Ghana between May 2007 and February 2011. In 771 (40% of the children malaria parasites were detected. This group was analyzed for indicators of bacterial co-infections using bivariate and multivariate regression analyses with 24 socio-economic variables, 16 terms describing medical history and anthropometrical information and 68 variables describing clinical symptoms. The variables were tested for sensitivity, specificity, positive predictive value and negative predictive value. In 46 (6.0% of the children with malaria infection, bacterial co-infection was detected. The most frequent pathogens were non-typhoid salmonellae (45.7%, followed by Streptococcus spp. (13.0%. Coughing, dehydration, splenomegaly, severe anemia and leukocytosis were positively associated with bacteremia. Domestic hygiene and exclusive breastfeeding is negatively associated with bacteremia. In cases of high parasitemia (>10,000/μl, a significant association with bacteremia was found for splenomegaly (OR 8.8; CI 1.6-48.9, dehydration (OR 18.2; CI 2.0-166.0 and coughing (OR 9.0; CI 0.7-118.6. In children with low parasitemia, associations with bacteremia were found for vomiting (OR 4.7; CI 1.4-15.8, severe anemia (OR 3.3; CI 1.0-11.1 and leukocytosis (OR 6.8 CI 1.9-24.2. Clinical signs of impaired microcirculation were negatively associated with bacteremia. Ceftriaxone achieved best coverage of isolated pathogens. The results demonstrate the limitation of clinical symptoms to determine bacterial co-infections in P. falciparum infected children. Best clinical indicators are dependent on the
Full Text Available Abstract Background Understanding the geographical distribution of drug resistance of Plasmodium falciparum is important for the effective treatment of malaria. Drug resistance has previously been inferred mainly from records of clinical resistance. However, clinical resistance is not always consistent with the parasite's genetic resistance. Thus, molecular identification of the parasite's drug resistance is required. In Africa, clinical resistance to pyrimethamine (Pyr and chloroquine (CQ was evident before 1980 but few studies investigating the genetic resistance to these drugs were conducted before the late 1990s. In this study, genotyping of genes involved in resistance to Pyr and CQ was performed using archive blood samples from Africa between 1984 and 1998. Methods Parasite DNA was extracted from P. falciparum-infected blood smears collected from travellers returning to Japan from Africa between 1984 and 1998. Genotypes of the dihydrofolate reductase gene (dhfr and CQ-resistance transporter gene (pfcrt were determined by polymerase chain reaction amplification and sequencing. Results Genotyping of dhfr and pfcrt was successful in 59 and 80 samples, respectively. One wild-type and seven mutant dhfr genotypes were identified. Three dhfr genotypes lacking the S108N mutation (NRSI, ICSI, IRSI; amino acids at positions 51, 59, 108, and 164 with mutations underlined were highly prevalent before 1994 but reduced after 1995, accompanied by an increase in genotypes with the S108N mutation. The dhfr IRNI genotype was first identified in Nigeria in 1991 in the present samples, and its frequency gradually increased. However, two double mutants (ICNI and NRNI, the latter of which was exclusively found in West Africa, were more frequent than the IRNI genotype. Only two pfcrt genotypes were found, the wild-type and a Southeast Asian type (CVIET; amino acids at positions 72-76 with mutations underlined. The CVIET genotype was already present as early as
Shakya, Bikash; Penn, Wesley D.; Nakayasu, Ernesto S.; Lacount, Douglas J.
Plasmodium falciparum extensively modifies the infected red blood cell (RBC), resulting in changes in deformability, shape and surface properties. These alterations suggest that the RBC cytoskeleton is a major target for modification during infection. However, the molecular mechanisms leading to these changes are largely unknown. To begin to address this question, we screened for exported P. falciparum proteins that bound to the erythrocyte cytoskeleton proteins ankyrin 1 (ANK1) and band 4.1 (4.1R), which form critical interactions with other cytoskeletal proteins that contribute to the deformability and stability of RBCs. Yeast two-hybrid screens with ANK1 and 4.1R identified eight interactions with P. falciparum exported proteins, including an interaction between 4.1R and PF3D7_0402000 (PFD0090c). This interaction was first identified in a large-scale screen (Vignali et al., Malaria J, 7:211, 2008), which also reported an interaction between PF3D7_0402000 and ANK1. We confirmed the interactions of PF3D7_0402000 with 4.1R and ANK1 in pair-wise yeast two-hybrid and co-precipitation assays. In both cases, an intact PHIST domain in PF3D7_0402000 was required for binding. Complex purification followed by mass spectrometry analysis provided additional support for the interaction of PF3D7_0402000 with ANK1 and 4.1R. RBC ghost cells loaded with maltose-binding protein (MBP)-PF3D7_0402000 passed through a metal microsphere column less efficiently than mock- or MBP-loaded controls, consistent with an effect of PF3D7_0402000 on RBC rigidity or membrane stability. This study confirmed the interaction of PF3D7_0402000 with 4.1R in multiple independent assays, provided the first evidence that PF3D7_0402000 also binds to ANK1, and suggested that PF3D7_0402000 affects deformability or membrane stability of uninfected RBC ghosts.
Straimer, Judith; Gnädig, Nina F; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D; Urnov, Fyodor D; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M; Ménard, Didier; Fidock, David A
The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. Copyright © 2015, American Association for the Advancement of Science.
Full Text Available Abstract Background Accurate sampling of sub-microscopic gametocytes is necessary for epidemiological studies to identify the infectious reservoir of Plasmodium falciparum. Detection of gametocyte mRNA achieves sensitive detection, but requires careful handling of samples. Filter papers can be used for collecting RNA samples, but rigorous testing of their capacity to withstand adverse storage conditions has not been fully explored. Methods Three gametocyte dilutions: 10/μL, 1.0/μL and 0.1/μL were spotted onto Whatman™ 903 Protein Saver Cards, FTA Classic Cards and 3MM filter papers that were stored under frozen, cold chain or tropical conditions for up to 13 weeks . RNA was extracted, then detected by quantitative nucleic acid sequence-based amplification (QT-NASBA and reverse-transcriptase PCR (RT-PCR. Results Successful gametocyte detection was more frequently observed from the Whatman 903 Protein Saver Card compared to the Whatman FTA Classic Card, by both techniques (p Conclusions This study indicates the Whatman 903 Protein Saver Card is better for Pfs25 mRNA sampling compared to the Whatman FTA Classic Card, and that the Whatman 3MM filter paper may prove to be a satisfactory cheaper option for Pfs25 mRNA sampling. When appropriately dried, filter papers provide a useful approach to Pfs25 mRNA sampling, especially in settings where storage in RNA-protecting buffer is not possible.
Jones, Michael W.M., E-mail: firstname.lastname@example.org [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Dearnley, Megan K. [ARC Centre of Excellence for Coherent X-Ray Science, Department of Biochemistry and Molecular Biology, Bio21 Institute, The University of Melbourne, Victoria 3010 (Australia); Riessen, Grant A. van [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Abbey, Brian [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Melbourne Centre for Nanofabrication, Victoria 3168 (Australia); Putkunz, Corey T. [ARC Centre of Excellence for Coherent X-Ray Science, School of Physics, The University of Melbourne, Victoria 3010 (Australia); Junker, Mark D. [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Vine, David J. [Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 (United States); McNulty, Ian [Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 (United States); Centre for Nanoscale Materials, Argonne National Laboratory, Argonne, Illinois 60439 (United States); Nugent, Keith A. [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Peele, Andrew G. [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Australian Synchrotron, 800 Blackburn Road, Clayton 3168 (Australia); Tilley, Leann [ARC Centre of Excellence for Coherent X-Ray Science, Department of Biochemistry and Molecular Biology, Bio21 Institute, The University of Melbourne, Victoria 3010 (Australia)
Phase-diverse X-ray coherent diffractive imaging (CDI) provides a route to high sensitivity and spatial resolution with moderate radiation dose. It also provides a robust solution to the well-known phase-problem, making on-line image reconstruction feasible. Here we apply phase-diverse CDI to a cellular sample, obtaining images of an erythrocyte infected by the sexual stage of the malaria parasite, Plasmodium falciparum, with a radiation dose significantly lower than the lowest dose previously reported for cellular imaging using CDI. The high sensitivity and resolution allow key biological features to be identified within intact cells, providing complementary information to optical and electron microscopy. This high throughput method could be used for fast tomographic imaging, or to generate multiple replicates in two-dimensions of hydrated biological systems without freezing or fixing. This work demonstrates that phase-diverse CDI is a valuable complementary imaging method for the biological sciences and ready for immediate application. - Highlights: • Phase-diverse coherent X-ray diffraction microscopy provides high-resolution and high-contrast images of intact biological samples. • Rapid nanoscale resolution imaging is demonstrated at orders of magnitude lower dose than previously possible. • Phase-diverse coherent X-ray diffraction microscopy is a robust technique for rapid, quantitative, and correlative X-ray phase imaging.
Tran, Phuong N; Brown, Simon H J; Rug, Melanie; Ridgway, Melanie C; Mitchell, Todd W; Maier, Alexander G
The development of differentiated sexual stages (gametocytes) within human red blood cells is essential for the propagation of the malaria parasite, since only mature gametocytes will survive in the mosquito's midgut. Hence gametocytogenesis is a pre-requisite for transmission of the disease. Physiological changes involved in sexual differentiation are still enigmatic. In particular the lipid metabolism-despite being central to cellular regulation and development-is not well explored. Here the lipid profiles of red blood cells infected with the five different sexual stages of Plasmodium falciparum were analysed by mass spectrometry and compared to those from uninfected and asexual trophozoite infected erythrocytes. Fundamental differences between erythrocytes infected with the different parasite stages were revealed. In mature gametocytes many lipids that decrease in the trophozoite and early gametocyte infected red blood cells are regained. In particular, regulators of membrane fluidity, cholesterol and sphingomyelin, increased significantly during gametocyte maturation. Neutral lipids (serving mainly as caloriometric reserves) increased from 3 % of total lipids in uninfected to 27 % in stage V gametocyte infected red blood cells. The major membrane lipid class (phospholipids) decreased during gametocyte development. The lipid profiles of infected erythrocytes are characteristic for the particular parasite life cycle and maturity stages of gametocytes. The obtained lipid profiles are crucial in revealing the lipid metabolism of malaria parasites and identifying targets to interfere with this deadly disease.
Katherine T Andrews
Full Text Available Histone deacetylase (HDAC inhibitors are being intensively pursued as potential new drugs for a range of diseases, including malaria. HDAC inhibitors are also important tools for the study of epigenetic mechanisms, transcriptional control, and other important cellular processes. In this study the effects of three structurally related antimalarial HDAC inhibitors on P. falciparum malaria parasite gene expression were compared. The three hydroxamate-based compounds, trichostatin A (TSA, suberoylanilide hydroxamic acid (SAHA; Vorinostat® and a 2-aminosuberic acid derivative (2-ASA-9, all caused profound transcriptional effects, with ~2-21% of genes having >2-fold altered expression following 2 h exposure to the compounds. Only two genes, alpha tubulin II and a hydrolase, were up-regulated by all three compounds after 2 h exposure in all biological replicates examined. The transcriptional changes observed after 2 h exposure to HDAC inhibitors were found to be largely transitory, with only 1-5% of genes being regulated after removing the compounds and culturing for a further 2 h. Despite some structural similarity, the three inhibitors caused quite diverse transcriptional effects, possibly reflecting subtle differences in mode of action or cellular distribution. This dataset represents an important contribution to our understanding of how HDAC inhibitors act on malaria parasites and identifies alpha tubulin II as a potential transcriptional marker of HDAC inhibition in malaria parasites that may be able to be exploited for future development of HDAC inhibitors as new antimalarial agents.
Nilanju Pran Sarmah
Interpretation & conclusions: The P. falciparum population of NE India was diverse which might be responsible for higher plasticity leading to the survival of the parasite and in turn to the higher endemicity of falciparum malaria of this region.
Jessica T Lin
Full Text Available Mixed P. falciparum/P. vivax infections are common in southeast Asia. When patients with P. falciparum malaria are treated and followed for several weeks, a significant proportion will develop P. vivax malaria. In a combined analysis of 243 patients recruited to two malaria treatment trials in western Cambodia, 20/43 (47% of those with P. falciparum gametocytes on admission developed P. vivax malaria by Day 28 of follow-up. The presence of Pf gametocytes on an initial blood smear was associated with a 3.5-fold greater rate of vivax parasitemia post-treatment (IRR = 3.5, 95% CI 2.0-6.0, p<0.001. The increased rate of post-treatment P. vivax infection persisted when correlates of exposure and immunity such as a history of malaria, male gender, and age were controlled for (IRR = 3.0, 95% CI 1.9-4.7, p<0.001. Polymerase chain reaction (PCR confirmed that only a low proportion of subjects (5/55 or 9.1% who developed vivax during follow-up had detectable Pv parasites in the peripheral blood at baseline. Molecular detection of falciparum gametocytes by reverse transcriptase PCR in a subset of patients strengthened the observed association, while PCR detection of Pv parasitemia at follow-up was similar to microscopy results. These findings suggest that the majority of vivax infections arising after treatment of falciparum malaria originate from relapsing liver-stage parasites. In settings such as western Cambodia, the presence of both sexual and asexual forms of P. falciparum on blood smear at presentation with acute falciparum malaria serves as a marker for possible occult P. vivax coinfection and subsequent relapse. These patients may benefit from empiric treatment with an 8-aminoquinolone such as primaquine.
Full Text Available Malaria remains one of the world's most important infectious diseases and is responsible for enormous mortality and morbidity. Resistance to antimalarial drugs is a challenging problem in malaria control. Clinical malaria is associated with the proliferation and development of Plasmodium parasites in human erythrocytes. Especially, the development into the mature forms (trophozoite and schizont of Plasmodium falciparum (P. falciparum causes severe malaria symptoms due to a distinctive property, sequestration which is not shared by any other human malaria. Ca(2+ is well known to be a highly versatile intracellular messenger that regulates many different cellular processes. Cytosolic Ca(2+ increases evoked by extracellular stimuli are often observed in the form of oscillating Ca(2+ spikes (Ca(2+ oscillation in eukaryotic cells. However, in lower eukaryotic and plant cells the physiological roles and the molecular mechanisms of Ca(2+ oscillation are poorly understood. Here, we showed the observation of the inositol 1,4,5-trisphospate (IP(3-dependent spontaneous Ca(2+ oscillation in P. falciparum without any exogenous extracellular stimulation by using live cell fluorescence Ca(2+ imaging. Intraerythrocytic P. falciparum exhibited stage-specific Ca(2+ oscillations in ring form and trophozoite stages which were blocked by IP(3 receptor inhibitor, 2-aminoethyl diphenylborinate (2-APB. Analyses of parasitaemia and parasite size and electron micrograph of 2-APB-treated P. falciparum revealed that 2-APB severely obstructed the intraerythrocytic maturation, resulting in cell death of the parasites. Furthermore, we confirmed the similar lethal effect of 2-APB on the chloroquine-resistant strain of P. falciparum. To our best knowledge, we for the first time showed the existence of the spontaneous Ca(2+ oscillation in Plasmodium species and clearly demonstrated that IP(3-dependent spontaneous Ca(2+ oscillation in P. falciparum is critical for the development
Tibúrcio, Marta; Silvestrini, Francesco; Bertuccini, Lucia
endothelial cells. In conclusion, these results identify specific differences in molecular and cellular mechanisms of host cell remodelling and in adhesive properties, leading to clearly distinct host parasite interplays in the establishment of sequestration of stage I gametocytes and of asexual trophozoites.......In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains...... to ultrastructurally and biochemically analyse parasite-induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do...
Nixon, Christina E; Park, Sangshin; Pond-Tor, Sunthorn; Raj, Dipak; Lambert, Lynn E; Orr-Gonzalez, Sachy; Barnafo, Emma K; Rausch, Kelly M; Friedman, Jennifer F; Fried, Michal; Duffy, Patrick E; Kurtis, Jonathan D
Naturally acquired antibodies to Plasmodium falciparum schizont egress antigen 1 (PfSEA-1A) are associated with protection against severe malaria in children. Vaccination of mice with SEA-1A from Plasmodium berghei (PbSEA-1A) decreases parasitemia and prolongs survival following P. berghei ANKA challenge. To enhance the immunogenicity of PfSEA-1A, we identified five linear B-cell epitopes using peptide microarrays probed with antisera from nonhuman primates vaccinated with recombinant PfSEA-1A (rPfSEA-1A). We evaluated the relationship between epitope-specific antibody levels and protection from parasitemia in a longitudinal treatment-reinfection cohort in western Kenya. Antibodies to three epitopes were associated with 16 to 17% decreased parasitemia over an 18-week high transmission season. We are currently designing immunogens to enhance antibody responses to these three epitopes. Copyright © 2017 American Society for Microbiology.
Staines, Henry M; Powell, Trevor; Clive Ellory, J; Egée, Stéphane; Lapaix, Franck; Decherf, Gaëtan; Thomas, Serge L Y; Duranton, Christophe; Lang, Florian; Huber, Stephan M
Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum-infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole-cell configuration of the patch-clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole-cell recordings in malaria-infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4-fold and inward currents by approximately 2-fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail. PMID:12937282
Escobar, Carlos; Pateira, Sara; Lobo, Elsa; Lobo, Lis; Teodosio, Rosa; Dias, Fernanda; Fernandes, Natercia; Arez, Ana Paula; Varandas, Luis; Nogueira, Fatima
We report the presence of SNPs in Plasmodium falciparum K13-propeller gene in two African countries, Angola and Mozambique, where malaria is a serious public health problem. Samples were collected before and after ACT introduction as first-line treatment. In each country 50 samples collected before and 50 after ACT introduction were analysed. A total of three different mutations (R471R and R575R in Angola and V494I in Mozambique) were identified in five samples, all collected after the introduction of ACT. The R471R mutation detected in Angola has already been reported in Africa (DR-Congo and Gabon). However, the mutations R575R (Angola) and V494I (Mozambique), have never been reported. V494I is adjacent to the known K13 resistance-associated mutation Y493H, although functional analysis did not predict a deleterious effect on protein function.
Tanabe, Kazuyuki; Jombart, Thibaut; Horibe, Shun; Palacpac, Nirianne M Q; Honma, Hajime; Tachibana, Shin-Ichiro; Nakamura, Masatoshi; Horii, Toshihiro; Kishino, Hirohisa; Mita, Toshihiro
The geographical distribution of single nucleotide polymorphism (SNP) in the mitochondrial genome of the human malaria parasite Plasmodium falciparum was investigated. We identified 88 SNPs in 516 isolates from seven parasite populations in Africa, Southeast Asia and Oceania. Analysis of the SNPs postulated a sub-Saharan African origin and recovered a strong negative correlation between within-population SNP diversity and geographic distance from the putative African origin over Southeast Asia and Oceania. These results are consistent with those previously obtained for nuclear genome-encoded housekeeping genes, indicating that the pattern of inheritance does not substantially affect the geographical distribution of SNPs. Copyright © 2013 Elsevier B.V. All rights reserved.
Bagavan, Asokan; Rahuman, Abdul Abdul; Kamaraj, Chinnaperumal; Kaushik, Naveen Kumar; Mohanakrishnan, Dinesh; Sahal, Dinkar
The absence of a vaccine and the rampant resistance to almost all antimalarial drugs have accentuated the urgent need for new antimalarial drugs and drug targets for both prophylaxis and chemotherapy. The aim of the study was to discover effective plant extracts against Plasmodium falciparum. In the present study, the hexane, chloroform, ethyl acetate, acetone, and methanol extracts of Citrus sinensis (peel), Leucas aspera, Ocimum sanctum, Phyllanthus acidus (leaf), Terminalia chebula (seed) were tested for their antimalarial activity against chloroquine (CQ)-sensitive (3D7) strain of P. falciparum which was cultured following the candle-jar method. Antimalarial evaluations of daily replacement of culture medium containing CQ and different plant crude extracts were performed on 96-well plates at 37°C for 24 and 48 h. Parasitemia was determined microscopically on thin-film Giemsa-stained preparations. Plant extracts were tested for their cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on human laryngeal cancer cell line (HEp-2) and normal cell line (Vero). Out of the 25 extracts tested, six showed good (IC(50) 4.76-22.76 μg/mL), 15 exhibited moderate (IC(50) 31.42-88.03 μg/mL), while four displayed mild (IC(50) > 100 μg/mL) antiplasmodial activity. The leaf ethyl acetate and methanol extracts of L. aspera; ethyl acetate, acetone, and methanol extracts of P. acidus; and seed acetone extract of T. chebula had good antiplasmodial activity (IC(50) = 7.81, 22.76, 9.37, 14.65, 12.68, and 4.76 μg/mL) with selectivity indices 5.43, 2.04, 4.88, 3.35, 3.42, and 9.97 for HEp-2 and >5.79, >2.20, >11.75, >3.41, >3.94, and >7.38 for Vero cells, respectively. These analyses have revealed for the first time that the components present in the solvent extracts of L. aspera, P. acidus, and T. chebula have antiplasmodial activity. The high antiplasmodial activity observed make these plants good candidates for isolation of
Staalsoe, Trine; Jensen, Anja T R; Theander, Thor G
Malaria vaccine development has traditionally concentrated on careful molecular, biochemical, and immunological characterisation of candidate antigens. In contrast, evidence of the importance of identified antigens in immunity to human infection and disease has generally been limited......) in particular, to provide robust evidence of a causal link between the two in order to allow efficient and evidence-based identification of candidate antigens for malaria vaccine development....... to statistically significant co-variation with protection rather than on demonstration of causal relationships. We have studied the relationship between variant surface antigen-specific antibodies and clinical protection from Plasmodium falciparum malaria in general, and from pregnancy-associated malaria (PAM...
The influence of Maloprim chemoprophylaxis on cellular and humoral immune responses to Plasmodium falciparum asexual blood stage antigens in schoolchildren living in a malaria endemic area of Mozambique
Hogh, B; Thompson, R; Lobo, V
We examined the impact of chemoprophylaxis on the cellular and humoral immune responses to polypeptides of the asexual Plasmodium falciparum blood stage antigens, the glutamate rich protein GLURP and Pf155/RESA, both of which in previous field studies have been identified as potentially protective...... chemoprophylaxis successfully reduced the parasite rate during the rainy season from 43% to 4%, and during the dry season from 18% to 0%. Chemoprophylaxis may therefore have a useful role in combination with another partially effective malaria control measure such as insecticide-impregnated bed nets or a malaria...
Full Text Available The recent detection of clinical Artemisinin (ART resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca(2+-ATPase (PfSERCA or PfATP6 has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (~3.6 Kb in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs, including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average d(N/d(S ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance.
Kemp, Kåre; Akanmori, Bartholomew D; Kurtzhals, Jørgen A L
P. falciparum malaria is associated with increased activation among peripheral lymphocytes. In the present study, we investigated markers of susceptibility to apoptosis and expression of IFN-gamma and IL-4 by CD28-and CD28+T cells in West African children with acute P. falciparum malaria. The stu...... are sequestered away from the peripheral blood during P. falciparum malaria....
Hempel, Casper; Hoyer, Nils; Staalsø, Trine
. falciparum, making it a possible model for the effects of VEGF signalling in vivo during malaria. CONCLUSIONS: Inhibition of VEGFR-2 signalling reduces intra-erythrocytic growth of P. falciparum, likely due to tyrosine kinase inhibition. Internalisation of VEGF in P. falciparum-infected red blood cells does...
Full Text Available Abstract Background The population structure of the causative agents of human malaria, Plasmodium sp., including the most serious agent Plasmodium falciparum, depends on the local epidemiological and demographic situations, such as the incidence of infected people, the vector transmission intensity and migration of inhabitants (i.e. exchange between sites. Analysing the structure of P. falciparum populations at a large scale, such as continents, or with markers that are subject to non-neutral selection, can lead to a masking and misunderstanding of the effective process of transmission. Thus, knowledge of the genetic structure and organization of P. falciparum populations in a particular area with neutral genetic markers is needed to understand which epidemiological factors should be targeted for disease control. Limited reports are available on the population genetic diversity and structure of P. falciparum in Thailand, and this is of particular concern at the Thai-Myanmar and Thai-Cambodian borders, where there is a reported high resistance to anti-malarial drugs, for example mefloquine, with little understanding of its potential gene flow. Methods The diversity and genetic differentiation of P. falciparum populations were analysed using 12 polymorphic apparently neutral microsatellite loci distributed on eight of the 14 different chromosomes. Samples were collected from seven provinces in the western, eastern and southern parts of Thailand. Results A strong difference in the nuclear genetic structure was observed between most of the assayed populations. The genetic diversity was comparable to the intermediate level observed in low P. falciparum transmission areas (average HS = 0.65 ± 0.17, where the lowest is observed in South America and the highest in Africa. However, uniquely the Yala province, had only a single multilocus genotype present in all samples, leading to a strong geographic differentiation when compared to the other Thai
Branch, Donald R.; Hult, Annika K.; Olsson, Martin L.; Liles, W. Conrad; Cserti-Gazdewich, Christine M.; Kain, Kevin C.
Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A1, A2, and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria. PMID:23071435
del Portillo Hernando A
Full Text Available Abstract Background Heterologous promoter analysis in Plasmodium has revealed the existence of conserved cis regulatory elements as promoters from different species can drive expression of reporter genes in heterologous transfection assays. Here, the functional characterization of different Plasmodium vivax promoters in Plasmodium falciparum using luciferase as the reporter gene is presented. Methods Luciferase reporter plasmids harboring the upstream regions of the msp1, dhfr, and vir3 genes as well as the full-length intergenic regions of the vir23/24 and ef-1α genes of P. vivax were constructed and transiently transfected in P. falciparum. Results Only the constructs with the full-length intergenic regions of the vir23/24 and ef-1α genes were recognized by the P. falciparum transcription machinery albeit to values approximately two orders of magnitude lower than those reported by luc plasmids harbouring promoter regions from P. falciparum and Plasmodium berghei. A bioinformatics approach allowed the identification of a motif (GCATAT in the ef-1α intergenic region that is conserved in five Plasmodium species but is degenerate (GCANAN in P. vivax. Mutations of this motif in the P. berghei ef-1α promoter region decreased reporter expression indicating it is active in gene expression in Plasmodium. Conclusion Together, this data indicates that promoter regions of P. vivax are poorly or not recognized by the P. falciparum transcription machinery suggesting the existence of P. vivax-specific transcription regulatory elements.
Boechat, Núbia; Ferreira, Maria de Lourdes G; Pinheiro, Luiz C S; Jesus, Antônio M L; Leite, Milene M M; Júnior, Carlos C S; Aguiar, Anna C C; de Andrade, Isabel M; Krettli, Antoniana U
Malaria is one of the most prevalent parasitic diseases in the world. The global importance of this disease, current vector control limitations, and the absence of an effective vaccine make the use of therapeutic antimalarial drugs the main strategy to control malaria. Chloroquine is a cost-effective antimalarial drug with a relatively robust safety profile, or therapeutic index. However, chloroquine is no longer used alone to treat patients with Plasmodium falciparum due to the emergence and spread of chloroquine-resistant strains, which have also been reported for Plasmodium vivax. However, the activity of 1,2,3-triazole derivatives against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum has been reported in the literature. To enhance the anti-P. falciparum activity of quinoline derivatives, we synthesized 11 new quinoline-1H-1,2,3-triazole hybrids with different substituents in the 4-positions of the 1H-1,2,3-triazole ring, which were assayed against the W2-chloroquine-resistant P. falciparum clone. Six compounds exhibited activity against the P. falciparum W2 clone, chloroquine-resistant, with IC50 values ranging from 1.4 to 46 μm. None of these compounds was toxic to a normal monkey kidney cell line, thus exhibiting good selectivity indexes, as high 351 for one compound (11). © 2014 John Wiley & Sons A/S.
Yuan, Lili; Hao, Mingming; Wu, Lanou; Zhao, Zhen; Rosenthal, Benjamin M; Li, Xiaomei; He, Yongshu; Sun, Ling; Feng, Guohua; Xiang, Zheng; Cui, Liwang; Yang, Zhaoqing
Plasmodium falciparum is usually asynchronous during in vitro culture. Highly synchronized cultures of P. falciparum are routinely used in malaria research. Here, we describe a simple synchronization procedure for P. falciparum asexual erythrocytic culture, which involves storage at 4°C for 8-24 h followed by routine culture. When cultures with 27-60% of ring stage were synchronized using this procedure, 70-93% ring stages were obtained after 48 h of culture and relative growth synchrony remained for at least two erythrocytic cycles. To test the suitability of this procedure for subsequent work, drug sensitivity assays were performed using four laboratory strains and four freshly adapted clinical P. falciparum isolates. Parasites synchronized by sorbitol treatment or refrigeration showed similar dose-response curves and comparable IC50 values to four antimalarial drugs. The refrigeration synchronization method is simple, inexpensive, time-saving, and should be especially useful when large numbers of P. falciparum culture are handled. Copyright © 2014 Elsevier Inc. All rights reserved.
Agomo, Chimere Obiora; Oyibo, Wellington Aghoghovwia; Sutherland, Colin; Hallet, Rachael; Oguike, Mary
Background The use of antimalarial drugs for prevention and treatment is a major strategy in the prevention of malaria in pregnancy. Although sulphadoxine-pyrimethamine (SP) is currently recommended for intermittent preventive treatment of malaria during pregnancy in Nigeria, previously used drugs for prophylaxis such as chloroquine (CQ) and pyrimethamine are accessible as they are purchased over the counter. This study describes the markers of absence or presence of resistance to quinoline (Pfcrt and Pfmdr 1) and type 1 antifolate antimalarial medicines (Pfdhfr). Methods Plasmodium falciparum-positive dried blood spots from pregnant women attending antenatal clinics for the first time during current pregnancy were investigated for the presence of mutations at codons 72–76 of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene by real time polymerase chain reaction (PCR) using haplotype-specific probes. PCR followed by sequence analysis was used to identify mutations at codons 86, 184, 1034, 1042 and 1246 of P. falciparum multi-drug resistance-1 (Pfmdr1) gene; and codons 16, 50, 51, 59, 108, 140 and 164 of Pfdhfr gene. Results Two haplotypes of Pfcrt (n = 54) were observed: CVMNK 13(24.2%) and CVIET 41 (75.9%) of the samples. The SVMNT haplotype was absent in this population. The Pfmdr1 (n = 28) haplotypes were NYSND 15(53.6%), YYSND 5(17.9%), NFSND 6(21.4%) and YFSND 2(7.1%). The Pfdhfr (n = 15) were ACNCSVI 4(26.7%), and ACICNSVI 1(6.7%) and ACIRNVI 10 (66.7%). The rate of occurrence of Pfcrt 76T, Pfdhfr108N, Pfmdr186Yand184F were 75.9%, 73.3%, 25% and 28.1% respectively. The Pfmdr1 86Y was associated with low parasitaemia (median = 71 parasites/μl, P = 0.024) while Pfcrt 76T was associated with young maternal age (mean 24.1 ± 4.5 years; P = 0.006). The median parasitaemia were similar (P>0.05) in wild and mutant strains of Pfcrt 76, Pfmdr1 184 and Pfdhfr 108. There was no association between gravidity or gestational age of the women and
Jakobsen, P H; Jepsen, S; Riley, E M
The soluble amphiphilic glycoprotein, Ag1 (gp60), purified from supernatants of in vitro cultures of Plasmodium falciparum has a molecular mass of 60 kDa and did not exhibit size variation in the different P. falciparum isolates tested by immunoblotting. Ag1 was shown to interact with the lectin...
Jespersen, Jakob S.; Wang, Christian W.; Mkumbaye, Sixbert I.
Most severe Plasmodium falciparum infections are experienced by young children. Severe symptoms are precipitated by vascular sequestration of parasites expressing a particular subset of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion molecules. Parasites binding hum...
Jensen, A.; Magistrado, P.; Sharp, S.; Joergensen, L.; Lavstsen, T.; Chiucchiuini, A.; Salanti, A.; Vestergaard, L.S.; Lusingu, J.P.; Hermsen, R.; Sauerwein, R.W.; Christensen, J.; Nielsen, M.A.; Hviid, L.; Sutherland, C.J.; Staalsoe, T.; Theander, T.G.
Parasite-encoded variant surface antigens (VSAs) like the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in
Jakob M A Mauritz
Full Text Available The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15-32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before approximately 48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis. However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.
Shams-Eldin, Hosam; Santos de Macedo, Cristiana; Niehus, Sebastian; Dorn, Caroline; Kimmel, Juergen; Azzouz, Nahid; Schwarz, Ralph T.
Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker's yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups
Mrema, J E; Campbell, G H; Jaramillo, A L; Miranda, R; Rieckmann, K H
Spontaneously released merozoites were harvested from cultures in which 42-90% of the erythrocytes had been infected with mature forms of Plasmodium falciparum at the start of incubation. The mature forms had been extracted from asynchronous cultures by the use of Ficoll and Plasmagel gradients. As the mature forms consisted of both trophozoites and schizonts, merozoites were released into the culture medium over a long period of time. The synchrony of merozoite release did not appear to be improved by prior exposure of parasites to sorbitol. Over this prolonged period of incubation, the yield of merozoites was disappointingly low in cultures containing 2.5% of erythrocytes. At erythrocyte concentrations of 0.01-0.25%, 3-10 times more merozoites were released into the medium; 0.4-2.3 merozoites per initial mature form were harvested over a 15-19-hour period. In addition to merozoites, contents of the culture medium included intact erythrocytes, ghost cells, and other cellular fragments. Only intact erythrocytes were effectively removed from the medium by simple or Ficoll gradient centrifugation. Merozoite preparations that are free from host cellular material are important in the development of a human malaria vaccine.
Full Text Available Malaria in humans is caused by one of five species of obligate intracellular protozoan parasites of the genus Plasmodium. P. falciparum causes the most severe disease and is responsible for 600,000 deaths annually, primarily in Sub-Saharan Africa. It has long been suggested that during their development, malaria parasites are exposed to environmental and metabolic stresses. One strategy to drug discovery was to increase these stresses by interfering with the parasites’ antioxidant and redox systems, which may be a valuable approach to disease intervention. Plasmodium possesses two redox systems—the thioredoxin and the glutathione system—with overlapping but also distinct functions. Glutathione is the most abundant low molecular weight redox active thiol in the parasites existing primarily in its reduced form representing an excellent thiol redox buffer. This allows for an efficient maintenance of the intracellular reducing environment of the parasite cytoplasm and its organelles. This review will highlight the mechanisms that are responsible for sustaining an adequate concentration of glutathione and maintaining its redox state in Plasmodium. It will provide a summary of the functions of the tripeptide and will discuss the potential of glutathione metabolism for drug discovery against human malaria parasites.
Zenz, W; Trop, M; Kollaritsch, H; Reinthaler, F
Increasing tourism and growing numbers of immigrants from malaria-endemic countries are leading to a higher importation rate of rare tropical disorders in European countries. We describe, to the best of our knowledge, the first case of connatal malaria in Austria. The patient is the first child of a 24 year old mother who was born in Ghana and immigrated to Austria one and a half years before delivery. She did not stay in an endemic region during this period and did not show fever or any other signs of malaria. The boy was healthy for the first six weeks of his life. In the 8th week of life he was admitted to our hospital due to persistent fever of unknown origin. On physical examination he showed only mild splenomegaly. Routine laboratory testing revealed mild hemolytic anemia with a hemoglobin value of 8.3 g/l. In the blood smear Plasmodium falciparum and Plasmodium malariae were detected. Oral therapy with quinine hydrochloride was successful and blood smears became negative for Plasmodia within 6 days. This case shows that congenital malaria can occur in children of clinically healthy women who were born in malaria-endemic areas even one and a half year after they have immigrated to non-endemic regions.
Drinkwater, Nyssa; Sivaraman, Komagal Kannan; Bamert, Rebecca S; Rut, Wioletta; Mohamed, Khadija; Vinh, Natalie B; Scammells, Peter J; Drag, Marcin; McGowan, Sheena
Malaria is one of the world's most prevalent parasitic diseases, with over 200 million cases annually. Alarmingly, the spread of drug-resistant parasites threatens the effectiveness of current antimalarials and has made the development of novel therapeutic strategies a global health priority. Malaria parasites have a complicated lifecycle, involving an asymptomatic 'liver stage' and a symptomatic 'blood stage'. During the blood stage, the parasites utilise a proteolytic cascade to digest host hemoglobin, which produces free amino acids absolutely necessary for parasite growth and reproduction. The enzymes required for hemoglobin digestion are therefore attractive therapeutic targets. The final step of the cascade is catalyzed by several metalloaminopeptidases, including aminopeptidase P (APP). We developed a novel platform to examine the substrate fingerprint of APP from Plasmodium falciparum (PfAPP) and to show that it can catalyze the removal of any residue immediately prior to a proline. Further, we have determined the crystal structure of PfAPP and present the first examination of the 3D structure of this essential malarial enzyme. Together, these analyses provide insights into potential mechanisms of inhibition that could be used to develop novel antimalarial therapeutics. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.
Treeprasertsuk, Sombat; Krudsood, Srivicha; Tosukhowong, Thanawat; Maek-A-Nantawat, Wirach; Vannaphan, Suparp; Saengnetswang, Tosaporn; Looareesuwan, Sornchai; Kuhn, Walter F; Brittenham, Gary; Carroll, James
One hundred and eight patients with severe falciparum malaria underwent a placebo controlled trial with the antioxidant, N-acetylcysteine (NAC), as an adjunctive therapy along with standard intravenous artesunate therapy. Three NAC dosage regimens were used: an intravenous loading dose of 140 mg/kg followed by 70 mg/kg every four hours intravenously for up to 18 doses (Group 1); a single intravenous loading dose followed by oral NAC in the same amount as for Group 1 (Group 2); a regimen identical to Group 1 except that oral NAC was administered after the first 24 hours (Group 3). Fifty-four patients received placebo plus artesunate. Two critically ill patients died in Group 1. No patient sustained an adverse reaction to the NAC other than vomiting, and the deaths were attributed to severe disease with multiple organ involvement. The excellent results with NAC, the lack of adverse effects, and the rationale for NAC benefit supports the need for a large, double blind trial of NAC as an adjunctive therapy for severe malaria. PMID:12971512
Resistance to chloroquine of malaria strains is known to be associated with a parasite protein named PfCRT, the mutated form of which is able to reduce chloroquine accumulation in the digestive vacuole of the pathogen. Whether the protein mediates extrusion of the drug acting as a channel or as a carrier and which is the protonation state of its chloroquine substrate is the subject of a scientific debate. We present here an analytical approach that explores which combination of hypotheses on the mechanism of transport and the protonation state of chloroquine are consistent with available equilibrium experimental data. We show that the available experimental data are not, by themselves, sufficient to conclude whether the protein acts as a channel or as a transporter, which explains the origin of their different interpretation by different authors. Interestingly, though, each of the two models is only consistent with a subset of hypotheses on the protonation state of the transported molecule. The combination of these results with a sequence and structure analysis of PfCRT, which strongly suggests that the molecule is a carrier, indicates that the transported species is either or both the mono and di-protonated forms of chloroquine. We believe that our results, besides shedding light on the mechanism of chloroquine resistance in P. falciparum, have implications for the development of novel therapies against resistant malaria strains and demonstrate the usefulness of an approach combining systems biology strategies with structural bioinformatics and experimental data.
Ariey, Frédéric; Witkowski, Benoit; Amaratunga, Chanaki; Beghain, Johann; Langlois, Anne-Claire; Khim, Nimol; Kim, Saorin; Duru, Valentine; Bouchier, Christiane; Ma, Laurence; Lim, Pharath; Leang, Rithea; Duong, Socheat; Sreng, Sokunthea; Suon, Seila; Chuor, Char Meng; Bout, Denis Mey; Ménard, Sandie; Rogers, William O.; Genton, Blaise; Fandeur, Thierry; Miotto, Olivo; Ringwald, Pascal; Le Bras, Jacques; Berry, Antoine; Barale, Jean-Christophe; Fairhurst, Rick M.; Benoit-Vical, Françoise; Mercereau-Puijalon, Odile; Ménard, Didier
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. PMID:24352242
Pinkevych, Mykola; Petravic, Janka; Chelimo, Kiprotich; Kazura, James W.; Moormann, Ann M.; Davenport, Miles P.
Severe malaria occurs predominantly in young children and immunity to clinical disease is associated with cumulative exposure in holoendemic settings. The relative contribution of immunity against various stages of the parasite life cycle that results in controlling infection and limiting disease is not well understood. Here we analyse the dynamics of Plasmodium falciparum malaria infection after treatment in a cohort of 197 healthy study participants of different ages in order to model naturally acquired immunity. We find that both delayed time-to-infection and reductions in asymptomatic parasitaemias in older age groups can be explained by immunity that reduces the growth of blood stage as opposed to liver stage parasites. We found that this mechanism would require at least two components – a rapidly acting strain-specific component, as well as a slowly acquired cross-reactive or general immunity to all strains. Analysis and modelling of malaria infection dynamics and naturally acquired immunity with age provides important insights into what mechanisms of immune control may be harnessed by malaria vaccine strategists. PMID:23093922
Murray, Lee; Stewart, Lindsay B; Tarr, Sarah J; Ahouidi, Ambroise D; Diakite, Mahamadou; Amambua-Ngwa, Alfred; Conway, David J
It is important to understand intrinsic variation in asexual blood stage multiplication rates of the most virulent human malaria parasite, Plasmodium falciparum. Here, multiplication rates of long-term laboratory adapted parasite clones and new clinical isolates were measured, using a newly standardised assay of growth from low starting density in replicate parallel cultures with erythrocytes from multiple different donors, across multiple cycles. Multiplication rates of long-term established clones were between 7.6 and 10.5 fold per 48 hours, with clone Dd2 having a higher rate than others (clones 3D7, HB3 and D10). Parasite clone-specific growth was then analysed in co-culture assays with all possible heterologous pairwise combinations. This showed that co-culture of different parasites did not affect their replication rates, indicating that there were no suppressive interactions operating between parasites. Multiplication rates of eleven new clinical isolates were measured after a few weeks of culture, and showed a spectrum of replication rates between 2.3 and 6.0 fold per 48 hours, the entire range being lower than for the long-term laboratory adapted clones. Multiplication rate estimates remained stable over time for several isolates tested repeatedly up to three months after culture initiation, indicating considerable persistence of this important trait variation.
Susannah F. Calhoun
Full Text Available The human malaria parasite Plasmodium falciparum replicates within circulating red blood cells, where it is subjected to conditions that frequently cause DNA damage. The repair of DNA double-stranded breaks (DSBs is thought to rely almost exclusively on homologous recombination (HR, due to a lack of efficient nonhomologous end joining. However, given that the parasite is haploid during this stage of its life cycle, the mechanisms involved in maintaining genome stability are poorly understood. Of particular interest are the subtelomeric regions of the chromosomes, which contain the majority of the multicopy variant antigen-encoding genes responsible for virulence and disease severity. Here, we show that parasites utilize a competitive balance between de novo telomere addition, also called “telomere healing,” and HR to stabilize chromosome ends. Products of both repair pathways were observed in response to DSBs that occurred spontaneously during routine in vitro culture or resulted from experimentally induced DSBs, demonstrating that both pathways are active in repairing DSBs within subtelomeric regions and that the pathway utilized was determined by the DNA sequences immediately surrounding the break. In combination, these two repair pathways enable parasites to efficiently maintain chromosome stability while also contributing to the generation of genetic diversity.
Mboera, Leonard E G; Senkoro, Kesheni P; Rumisha, Susan F; Mayala, Benjamin K; Shayo, Elizabeth H; Mlozi, Malongo R S
In Sub-Saharan Africa, some individuals infected with malaria are also infected with helminths. However, the magnitude and distribution of such coinfections in relation to eco-systems remains poorly defined. This study was undertaken to determine the prevalence of Plasmodium falciparum and helminth coinfections among schoolchildren in relation to agro-ecosystems in Mvomero District, Tanzania. The agro-ecosystems were categorised as sugarcane, traditional flooding rice irrigation, improved non-flooding rice irrigation and savannah. Schoolchildren had their blood examined for P. falciparum and Wuchereria bancrofti; urine for Schistosoma haematobium and stool for intestinal helminths. Blood samples were also examined for haemoglobin concentration. A total of 578 schoolchildren (mean age = 7.96 years) were involved in the study. Overall, 60% of all schoolchildren had at least an infection of either P. falciparum, W. bancrofti, S. haematobium or hookworm. The highest prevalence of P. falciparum (75.3%), W. bancrofti (62.9%) and hookworm (24.7%) infections was observed among children in flooding rice irrigation ecosystem. P. falciparum+S. haematobium (10.9%) and P. falciparum+W. bancrofti (11.1%) were the most prevalent types of coinfection in the area. The highest prevalence of double parasitic infections was observed among children in the flooding rice irrigation ecosystems. The risk for acquiring coinfections of P. falciparum+W. bancrofti was significantly higher among children in the flooding rice irrigation ecosystem. Forty-five (7.8%) children were coinfected with three types of parasitic infections. The risk of acquiring triple infection among children from flooding rice irrigation was higher for P. falciparum+S. haematobium+W. bancrofti (p<0.001). Seven schoolchildren (1.2%) were found infected with four parasites and all were from the flooding rice irrigation ecosystem. Significantly high P. falciparum geometric parasite density was observed among children
Full Text Available Tanaman Ketepeng (Cassia alata L. dan Ketepeng Kecil (Cassia tora L. merupakan tanaman obat yang memiliki berbagaimacam kegunaan, diantaranya untuk mengobati malaria yang disebabkan oleh Plasmodium falciparum. Penelitian inibertujuan untuk mengetahui efektivitas ekstrak etanol daun ketepeng dan ketepeng kecil terhadap P. falcifarum secara invitro yang dihubungkan dengan periode waktu dengan pengenceran bertingkat dari larutan uji. Penelitian dilakukan dengantahapan: pengambilan sampel tanaman, pembuatan ekstrak dan uji aktivitas anti malaria secara in vitro. Kontrol positifmenggunakan klorokuin dan kontrol negatif menggunakan P. falciparum tanpa penambahan ekstrak uji. Ekstrak etanol daunketepeng (Cassia alata L menunjukkan penurunan jumlah pertumbuhan P. falciparum pada pengenceran 10-8. Ekstraketanol daun ketepeng dan ketepeng kecil tidak menunjukkan penghambatan pertumbuhan terhadap P. falciparum.Kata kunci: ekstrak, Cassia alata L., Cassia tora L., Plasmodium falciparum
Elizabeth H Aitken
Full Text Available Antibodies towards placental-binding P. falciparum are thought to protect against pregnancy malaria; however, environmental factors may affect antibody development.Using plasma from pregnant Malawian women, we measured IgG against placental-binding P. falciparum parasites by flow cytometry, and related results to intermittent preventive treatment (IPTp regime, and bed net use. Bed net use was associated with decreased antibody levels at mid-pregnancy but not at 1 month post partum (1 mpp. At 1 mpp a more intensive IPTp regime was associated with decreased antibody levels in primigravidae, but not multigravidae.Results suggest bed nets and IPTp regime influence acquisition of pregnancy-specific P. falciparum immunity.
Dziegiel, Morten Hanefeld; Borre, M B; Jepsen, S
A method for purification of a recombinant Plasmodium falciparum protein produced in E. coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described. The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P....... falciparum glutamate rich protein (GLURP), with a molecular weight of 220 kilodalton. The protein is associated with all parasite stages in the human host. Examination of sera from 105 adult Liberians living in a malaria endemic area revealed anti-GLURP IgG antibodies in 98% of the sera. The recombinant...... New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P. falciparum. Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control...
Hazelton, Keith Z. [Yeshiva Univ., New York, NY (United States); Ho, Meng-Chaio [Yeshiva Univ., New York, NY (United States); Cassera, Maria B. [Yeshiva Univ., New York, NY (United States); Clinch, Keith [Industrial Research Ltd., Lower Hutt (New Zealand); Crump, Douglas R. [Industrial Research Ltd., Lower Hutt (New Zealand); Rosario Jr., Irving [Yeshiva Univ., New York, NY (United States); Merino, Emilio F. [Yeshiva Univ., New York, NY (United States); Almo, Steve C. [Yeshiva Univ., New York, NY (United States); Tyler, Peter C. [Industrial Research Ltd., Lower Hutt (New Zealand); Schramm, Vern L. [Yeshiva Univ., New York, NY (United States)
We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.
Full Text Available An in vitro sensitivity test was conducted to study the sensitivity of Plasmodium falciparum against chloroquine and pyrimethamine/sulphadoxine combination. The relationship between sensitivity of the parasite to the two drugs was also studied. A total of 72 patients from five localities were examined during 1984-1985. Test against chloroquine was conducted according to WHO method, while against pyrimethamine/sulphadoxine combination, a modified method of Nguyen Dinh and Payne and Eastham and Rieckmann was used. The results showed that there is no relationship between the sensitivity of P. falciparum against pyrimethamine/ sulphadoxine combination and chloroquine. It can be concluded that in case of chloroquine resistant P. falciparum, pyrimethamine/sulphadoxine combination could be applied as an alternative chemotherapy.
Lensen, A H; Bolmer-Van de Vegte, M; van Gemert, G J; Eling, W M; Sauerwein, R W
Mosquitoes are infected with Plasmodium falciparum by taking a blood meal from a gametocyte carrier. Since a mosquito takes a volume of 1 to 2 microl, a blood meal may contain 1 x 10(4) to 3 x 10(4) leukocytes (WBC). The majority of WBC are composed of neutrophils which may phagocytose and kill developing gametes inside the mosquito midgut. Phagocytosis was measured in vitro by a luminol-dependent chemiluminescence (CL) assay. In the presence of P. falciparum gametes, sera from areas of endemicity had an increased CL response compared to controls. In mosquito membrane feeding experiments some such sera showed a transmission reduction which was related to the presence of viable WBC. The results of this study suggest that phagocytosis of opsonized gametes inside the mosquito midgut occurs and can contribute to a reduction in the transmission of P. falciparum parasites. PMID:9284160
Manser, Monika; Olufsen, Catherine; Andrews, Nick; Chiodini, Peter L
To examine performance of the identification and estimation of percentage parasitaemia of Plasmodium falciparum in stained blood films distributed in the UK National External Quality Assessment Scheme (UKNEQAS) Blood Parasitology Scheme. Analysis of performance for the diagnosis and estimation of the percentage parasitaemia of P. falciparum in Giemsa-stained thin blood films was made over a 15-year period to look for trends in performance. An average of 25% of participants failed to estimate the percentage parasitaemia, 17% overestimated and 8% underestimated, whilst 5% misidentified the malaria species present. Although the results achieved by participants for other blood parasites have shown an overall improvement, the level of performance for estimation of the parasitaemia of P. falciparum remains unchanged over 15 years. Possible reasons include incorrect calculation, not examining the correct part of the film and not examining an adequate number of microscope fields.
Moura, Ivan Cruz; Wunderlich, Gerhard; Uhrig, Maria L.; Couto, Alicia S.; Peres, Valnice J.; Katzin, Alejandro M.; Kimura, Emília A.
Isoprenylation is an essential protein modification in eukaryotic cells. Herein, we report that in Plasmodium falciparum, a number of proteins were labeled upon incubation of intraerythrocytic forms with either [3H]farnesyl pyrophosphate or [3H]geranylgeranyl pyrophosphate. By thin-layer chromatography, we showed that attached isoprenoids are partially modified to dolichol and other, uncharacterized, residues, confirming active isoprenoid metabolism in this parasite. Incubation of blood-stage P. falciparum treated with the isoprenylation inhibitor limonene significantly decreased the parasites' progression from the ring stage to the trophozoite stage and at 1.22 mM, 50% of the parasites died after the first cycle. Using Ras- and Rap-specific monoclonal antibodies, putative Rap and Ras proteins of P. falciparum were immunoprecipitated. Upon treatment with 0.5 mM limonene, isoprenylation of these proteins was significantly decreased, possibly explaining the observed arrest of parasite development. PMID:11502528
Mkumbaye, Sixbert I; Minja, Daniel T R; Jespersen, Jakob S
BACKGROUND: Establishing in vitro Plasmodium falciparum culture lines from patient parasite isolates can offer deeper understanding of geographic variations of drug sensitivity and mechanisms of malaria pathogenesis and immunity. Cellulose column filtration of blood is an inexpensive, rapid...... and effective method for the removal of host factors, such as leucocytes and platelets, significantly improving the purification of parasite DNA in a blood sample. METHODS: In this study, the effect of cellulose column filtration of venous blood on the initial in vitro growth of P. falciparum parasite isolates....... falciparum merozoite surface protein 2 genotyping was performed using nested PCR on extracted genomic DNA, and the var gene transcript levels were investigated, using quantitative PCR on extracted RNA, at admission and 4 days of culture. RESULTS: The cellulose-filtered parasites grew to higher parasitaemia...
Guitard, Juliette; Andersen, Pernille; Ermont, Caroline
Background: Pregnant women acquire protective antibodies that cross-react with geographically diverse placental Plasmodium falciparum isolates, suggesting that surface molecules expressed on infected erythrocytes by pregnancy-associated malaria (PAM) parasites have conserved epitopes and, that de......Background: Pregnant women acquire protective antibodies that cross-react with geographically diverse placental Plasmodium falciparum isolates, suggesting that surface molecules expressed on infected erythrocytes by pregnancy-associated malaria (PAM) parasites have conserved epitopes and......, that designing a PAM vaccine may be envisaged. VAR2CSA is the main candidate for a pregnancy malaria vaccine, but vaccine development may be complicated by its sequence polymorphism. Methods: The dynamics of P. falciparum genotypes during pregnancy in 32 women in relation to VAR2CSA polymorphism and immunity...
Valente, Bianor; Campos, Paulo A; do Rosário, Virgílio E; Varandas, Luis; Silveira, Henrique
Pregnant women are at increased risk of malaria, but in Angola, epidemiologic data from this group is almost inexistent. We conducted a cross-sectional study to determine the prevalence and risk factors of Plasmodium falciparum infections in 567 pregnant Angolan women living in Luanda province. One in five women had P. falciparum at delivery, diagnosed by PCR assay. Age, residence and history of malaria during pregnancy were significantly associated with P. falciparum infection, but gravidity and use of anti-malarial drugs were not. Placental infections were significantly more common in women ≤18 years old and in primigravidae, but we could not correlate placental infections with poor pregnancy outcomes. These findings are relevant to malaria control policies in Luanda, Angola. © 2011 Blackwell Publishing Ltd.
Ali, H.; Mahmood, T.; Ahmed, N.
To determine the impact of percentage parasitemia and clinical features on morbidity and mortality in patients with P. falciparum malaria. Seventy-six adult patients of smear positive P. falciparum malaria were selected for the study. Parasite density was estimated on thin blood film and expressed as percentage of red blood cells parasitized. Patients were divided into three groups on the basis of parasite density. The data was analyzed on SPSS version 12. Results were expressed as percentages, mean and standard deviations. P-value 10%. Comparative analysis of the groups showed that pallor, impaired consciousness, jaundice or malarial hepatitis, thrombocytopenia, acute renal failure, DIC, and mortality were all strongly associated with the density of Plasmodium falciparum malaria (p=0.001). Parasite density was not related to age, gender and hepatosplenomegaly. High parasite density was associated with severe clinical illness, complications and mortality. Parasite counts of > 5% may be considered as hyperparasitaemia in this population of the world. (author)
Jakobsen, P H; McKay, V; N'Jie, R
with levels in children with asymptomatic P. falciparum infections and in healthy children. Concentrations of IL-10 and IL-1Ra were correlated with levels of parasitaemia, but the association of cytokine levels with disease was independent of the association with parasitaemia. Children may tolerate a high......A proportion of children with Plasmodium falciparum infection have a high parasitaemia without accompanying fever, indicative of different clinical thresholds of parasitaemia. Higher levels of IL-10, IL-1Ra and sIL-4R but not sIL-2R were found in children with P. falciparum malaria, compared...... parasitaemia by neutralizing the parasite-derived toxins. When studying potential anti-toxic molecules we found that children with symptomatic infections had lower concentrations of a phospholipid-binding molecule, beta 2-glycoprotein I (beta 2-GPI), compared with children with asymptomatic infections...
Theander, T G; Andersen, B J; Pedersen, B K
by either SPag or PPD in the presence of immune serum. Studies on subpopulations of PBMC indicated that the inhibitory cells resided among the adherent cell fraction. Furthermore we tested PBMC for cytotoxic activity against P. falciparum-infected autologous or heterologous erythrocytes. Experiments were......Blood mononuclear cells (PBMC) recognizing soluble malaria antigens (SPag) are present in the peripheral blood of individuals clinically immune to malaria, and they proliferate after exposure to such antigens. To test whether these cells have effector activity against Plasmodium falciparum, we...... stimulated PBMC from malaria-immune donors by SPag and purified protein derivative (PPD) in culture for 7 days. The PBMC were then co-incubated with P. falciparum for 48 h, and parasitaemia was determined by microscopy. Parasite growth was only significantly impaired after incubation with PBMC stimulated...
Björkman, Anders; Cook, Jackie; Sturrock, Hugh; Msellem, Mwinyi; Ali, Abdullah; Xu, Weiping; Molteni, Fabrizio; Gosling, Roly; Drakeley, Chris; Mårtensson, Andreas
Optimal use of mass/targeted screen-and-treat or mass or focal drug administration as malaria elimination strategies remains unclear. We therefore studied spatial distribution of Plasmodium falciparum infections to compare simulated effects of these strategies on reducing the parasite reservoir in a pre-elimination setting. P. falciparum rapid diagnostic tests (RDTs) and molecular (polymerase chain reaction [PCR]) and serological (enzyme-linked immunosorbent assay) analyses were performed on finger-prick blood samples from a population-based survey in 3 adjacent communities. Among 5278 persons screened, 13 (0.2%) were positive by RDT and 123 (2.3%) by PCR. PCR-positive individuals were scattered over the study area, but logistic regression analysis suggested a propensity of these infections to cluster around RDT-positive individuals. The odds ratios for being PCR positive was 7.4 (95% confidence interval, 2.8-19.9) for those living in the household of an RDT-positive individual and 1.64 (1.0-2.8; P = .06) for those living within 1000 m. Treating everyone within households of RDT-positive individuals (1% population) would target 13% of those who are PCR positive. Treating all living within a radius of <300 or <1000 m (14% or 58% population) would target 30% or 66% of infections, respectively. Among 4431 serologically screened individuals, 26% were seropositive. Treating everyone within seropositive households (63% population) would target 77% of PCR-positive individuals. Presumptive malaria treatment seemed justified within RDT-positive households and potentially worth considering within, for example, a radius of <300 m. Serology was not discriminative enough in identifying ongoing infections for improving focal interventions in this setting but may rather be useful to detect larger transmission foci. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.
Full Text Available Novel antimalarial therapies are urgently needed for the fight against drug-resistant parasites. The metabolism of malaria parasites in infected cells is an attractive source of drug targets but is rather complex. Computational methods can handle this complexity and allow integrative analyses of cell metabolism. In this study, we present a genome-scale metabolic model (iPfa of the deadliest malaria parasite, Plasmodium falciparum, and its thermodynamics-based flux analysis (TFA. Using previous absolute concentration data of the intraerythrocytic parasite, we applied TFA to iPfa and predicted up to 63 essential genes and 26 essential pairs of genes. Of the 63 genes, 35 have been experimentally validated and reported in the literature, and 28 have not been experimentally tested and include previously hypothesized or novel predictions of essential metabolic capabilities. Without metabolomics data, four of the genes would have been incorrectly predicted to be non-essential. TFA also indicated that substrate channeling should exist in two metabolic pathways to ensure the thermodynamic feasibility of the flux. Finally, analysis of the metabolic capabilities of P. falciparum led to the identification of both the minimal nutritional requirements and the genes that can become indispensable upon substrate inaccessibility. This model provides novel insight into the metabolic needs and capabilities of the malaria parasite and highlights metabolites and pathways that should be measured and characterized to identify potential thermodynamic bottlenecks and substrate channeling. The hypotheses presented seek to guide experimental studies to facilitate a better understanding of the parasite metabolism and the identification of targets for more efficient intervention.
Full Text Available Malaria represents a major public health problem in Africa. In the East African highlands, the high-altitude areas were previously considered too cold to support vector population and parasite transmission, rendering the region particularly prone to epidemic malaria due to the lack of protective immunity of the population. Since the 1980's, frequent malaria epidemics have been reported and these successive outbreaks may have generated some immunity against Plasmodium falciparum amongst the highland residents. Serological studies reveal indirect evidence of human exposure to the parasite, and can reliably assess prevalence of exposure and transmission intensity in an endemic area. However, the vast majority of serological studies of malaria have been, hereto, limited to a small number of the parasite's antigens. We surveyed and compared the antibody response profiles of age-stratified sera from residents of two endemic areas in the western Kenyan highlands with differing malaria transmission intensities, during two distinct seasons, against 854 polypeptides of P. falciparum using high-throughput proteomic microarray technology. We identified 107 proteins as serum antibody targets, which were then characterized for their gene ontology biological process and cellular component of the parasite, and showed significant enrichment for categories related to immune evasion, pathogenesis and expression on the host's cell and parasite's surface. Additionally, we calculated age-fitted annual seroconversion rates for the immunogenic proteins, and contrasted the age-dependent antibody acquisition for those antigens between the two sampling sites. We observed highly immunogenic antigens that produce stable antibody responses from early age in both sites, as well as less immunogenic proteins that require repeated exposure for stable responses to develop and produce different seroconversion rates between sites. We propose that a combination of highly and less
Full Text Available INTRODUCTION: Microscopy is the gold standard for diagnosis of malaria, however, manual evaluation of blood films is highly dependent on skilled personnel in a time-consuming, error-prone and repetitive process. In this study we propose a method using computer vision detection and visualization of only the diagnostically most relevant sample regions in digitized blood smears. METHODS: Giemsa-stained thin blood films with P. falciparum ring-stage trophozoites (n = 27 and uninfected controls (n = 20 were digitally scanned with an oil immersion objective (0.1 µm/pixel to capture approximately 50,000 erythrocytes per sample. Parasite candidate regions were identified based on color and object size, followed by extraction of image features (local binary patterns, local contrast and Scale-invariant feature transform descriptors used as input to a support vector machine classifier. The classifier was trained on digital slides from ten patients and validated on six samples. RESULTS: The diagnostic accuracy was tested on 31 samples (19 infected and 12 controls. From each digitized area of a blood smear, a panel with the 128 most probable parasite candidate regions was generated. Two expert microscopists were asked to visually inspect the panel on a tablet computer and to judge whether the patient was infected with P. falciparum. The method achieved a diagnostic sensitivity and specificity of 95% and 100% as well as 90% and 100% for the two readers respectively using the diagnostic tool. Parasitemia was separately calculated by the automated system and the correlation coefficient between manual and automated parasitemia counts was 0.97. CONCLUSION: We developed a decision support system for detecting malaria parasites using a computer vision algorithm combined with visualization of sample areas with the highest probability of malaria infection. The system provides a novel method for blood smear screening with a significantly reduced need for
Chiappino-Pepe, Anush; Ataman, Meriç
Novel antimalarial therapies are urgently needed for the fight against drug-resistant parasites. The metabolism of malaria parasites in infected cells is an attractive source of drug targets but is rather complex. Computational methods can handle this complexity and allow integrative analyses of cell metabolism. In this study, we present a genome-scale metabolic model (iPfa) of the deadliest malaria parasite, Plasmodium falciparum, and its thermodynamics-based flux analysis (TFA). Using previous absolute concentration data of the intraerythrocytic parasite, we applied TFA to iPfa and predicted up to 63 essential genes and 26 essential pairs of genes. Of the 63 genes, 35 have been experimentally validated and reported in the literature, and 28 have not been experimentally tested and include previously hypothesized or novel predictions of essential metabolic capabilities. Without metabolomics data, four of the genes would have been incorrectly predicted to be non-essential. TFA also indicated that substrate channeling should exist in two metabolic pathways to ensure the thermodynamic feasibility of the flux. Finally, analysis of the metabolic capabilities of P. falciparum led to the identification of both the minimal nutritional requirements and the genes that can become indispensable upon substrate inaccessibility. This model provides novel insight into the metabolic needs and capabilities of the malaria parasite and highlights metabolites and pathways that should be measured and characterized to identify potential thermodynamic bottlenecks and substrate channeling. The hypotheses presented seek to guide experimental studies to facilitate a better understanding of the parasite metabolism and the identification of targets for more efficient intervention. PMID:28333921
Full Text Available BACKGROUND: Large studies on severe imported malaria in non-endemic industrialized countries are lacking. We sought to describe the clinical spectrum of severe imported malaria in French adults and to identify risk factors for mortality at admission to the intensive care unit. METHODOLOGY AND PRINCIPAL FINDINGS: Retrospective review of severe Plasmodium falciparum malaria episodes according to the 2000 World Health Organization definition and requiring admission to the intensive care unit. Data were collected from medical charts using standardised case-report forms, in 45 French intensive care units in 2000-2006. Risk factors for in-hospital mortality were identified by univariate and multivariate analyses. Data from 400 adults admitted to the intensive care unit were analysed, representing the largest series of severe imported malaria to date. Median age was 45 years; 60% of patients were white, 96% acquired the disease in sub-Saharan Africa, and 65% had not taken antimalarial chemoprophylaxis. Curative quinine treatment was used in 97% of patients. Intensive care unit mortality was 10.5% (42 deaths. By multivariate analysis, three variables at intensive care unit admission were independently associated with hospital death: older age (per 10-year increment, odds ratio [OR], 1.72; 95% confidence interval [95%CI], 1.28-2.32; P = 0.0004, Glasgow Coma Scale score (per 1-point decrease, OR, 1.32; 95%CI, 1.20-1.45; P<0.0001, and higher parasitemia (per 5% increment, OR, 1.41; 95%CI, 1.22-1.62; P<0.0001. CONCLUSIONS AND SIGNIFICANCE: In a large population of adults treated in a non-endemic industrialized country, severe malaria still carried a high mortality rate. Our data, including predictors of death, can probably be generalized to other non-endemic countries where high-quality healthcare is available.
Edgar Leonardo Martínez-Salazar
Full Text Available Introduction Thrombocytopenia is a common complication in malaria patients. The relationship between abnormal platelet profile and clinical status in malaria patients is unclear. In low and unstable endemic regions where vivax malaria predominates, the hematologic profiles of malaria patients and their clinical utility are poorly understood. The aim of this study was to characterize the thrombograms of malaria patients from Colombia, where Plasmodium vivax infection is common, and to explore the relationship between thrombograms and clinical status. Methods Eight hundred sixty-two malaria patients were enrolled, including 533 (61.8% patients infected with Plasmodium falciparum, 311 (36.1% patients infected with Plasmodium vivax and 18 (2.1% patients with mixed infections. Results The most frequently observed changes were low platelet count (PC and high platelet distribution width (PDW, which were observed in 65% of patients; thrombocytopenia with <50,000 platelets/µL was identified in 11% of patients. Patients with complications had lower PC and plateletcrit (PT and higher PDW values. A higher risk of thrombocytopenia was identified in patients with severe anemia, neurologic complications, pulmonary complications, liver dysfunction, renal impairment and severe hypoglycemia. The presence of thrombocytopenia (<150,000 platelets/µL was associated with a higher probability of liver dysfunction. Conclusions Young age, longer duration of illness and higher parasitemia are associated with severe thrombocytopenia. Our study showed that thrombocytopenia is related to malaria complications, especially liver dysfunction. High PDW in patients with severe malaria may explain the mechanisms of thrombocytopenia that is common in this group of patients.
Full Text Available Abstract Background Elevated parasite biomass in the human red blood cells can lead to increased malaria morbidity. The genes and mechanisms regulating growth and development of Plasmodium falciparum through its erythrocytic cycle are not well understood. We previously showed that strains HB3 and Dd2 diverge in their proliferation rates, and here use quantitative trait loci mapping in 34 progeny from a cross between these parent clones along with integrative bioinformatics to identify genetic loci and candidate genes that control divergences in cell cycle duration. Results Genetic mapping of cell cycle duration revealed a four-locus genetic model, including a major genetic effect on chromosome 12, which accounts for 75% of the inherited phenotype variation. These QTL span 165 genes, the majority of which have no predicted function based on homology. We present a method to systematically prioritize candidate genes using the extensive sequence and transcriptional information available for the parent lines. Putative functions were assigned to the prioritized genes based on protein interaction networks and expression eQTL from our earlier study. DNA metabolism or antigenic variation functional categories were enriched among our prioritized candidate genes. Genes were then analyzed to determine if they interact with cyclins or other proteins known to be involved in the regulation of cell cycle. Conclusions We show that the divergent proliferation rate between a drug resistant and drug sensitive parent clone is under genetic regulation and is segregating as a complex trait in 34 progeny. We map a major locus along with additional secondary effects, and use the wealth of genome data to identify key candidate genes. Of particular interest are a nucleosome assembly protein (PFL0185c, a Zinc finger transcription factor (PFL0465c both on chromosome 12 and a ribosomal protein L7Ae-related on chromosome 4 (PFD0960c.
Peter Klein Klouwenberg
Full Text Available BACKGROUND: In sub-Saharan Africa, Plasmodium falciparum and hepatitis A (HAV infections are common, especially in children. Co-infections with these two pathogens may therefore occur, but it is unknown if temporal clustering exists. MATERIALS AND METHODS: We studied the pattern of co-infection of P. falciparum malaria and acute HAV in Kenyan children under the age of 5 years in a cohort of children presenting with uncomplicated P. falciparum malaria. HAV status was determined during a 3-month follow-up period. DISCUSSION: Among 222 cases of uncomplicated malaria, 10 patients were anti-HAV IgM positive. The incidence of HAV infections during P. falciparum malaria was 1.7 (95% CI 0.81-3.1 infections/person-year while the cumulative incidence of HAV over the 3-month follow-up period was 0.27 (95% CI 0.14-0.50 infections/person-year. Children with or without HAV co-infections had similar mean P. falciparum asexual parasite densities at presentation (31,000/µL vs. 34,000/µL, respectively, largely exceeding the pyrogenic threshold of 2,500 parasites/µL in this population and minimizing risk of over-diagnosis of malaria as an explanation. CONCLUSION: The observed temporal association between acute HAV and P. falciparum malaria suggests that co-infections of these two hepatotrophic human pathogens may result from changes in host susceptibility. Testing this hypothesis will require larger prospective studies.
Full Text Available Abstract Background Chloroquine was used as first-line treatment for Plasmodium falciparum or Plasmodium vivax in Indonesia before the initial launch of artemisinin combination therapy in 2004. A study to evaluate efficacies of chloroquine against P. falciparum and P. vivax was undertaken at Lampung in southern Sumatra, western Indonesia in 2002. Methods Patients infected by P. falciparum or P. vivax were treated with 25 mg/kg chloroquine base in three daily doses over 48 hr. Finger prick blood was collected on Days 0, 2, 3, 7, 14, 21 and 28 after starting drug administration. Whole blood chloroquine and its desethyl metabolite were measured on Days-0, -3 and -28, or on the day of recurrent parasitaemia. Results 42 patients infected by P. falciparum were enrolled, and 38 fullfilled criteria for per protocol analysis. Only six of 38 (16% showed a response consistent with senstivity to chloroquine. 25 of 32 failures were confirmed resistant by demonstrating chloroquine levels on day of recurrence exceeding the minimally effective concentration (200 ng/mL whole blood. The 28-day cumulative incidence of resistance in P. falciparum was 68% (95% CI: 0.5260 - 0.8306. Thirty one patients infected by P. vivax were enrolled, and 23 were evaluable for per protocol analysis. 15 out of 23 (65% subjects had persistent or recurrent parasitaemia. Measurement of chloroquine levels confirmed all treatment failures prior to Day-15 as resistant. Beyond Day-15, 4 of 7 recurrences also had drug levels above 100 ng/mL and were classified as resistant. The 28-day cumulative incidence of chloroquine resistance in P. vivax was 43% (95% CI: 0.2715 - 0.6384. Conclusion These findings confirm persistantly high levels of resistance to chloroquine by P. falciparum in southern Sumatra, and suggest that high-grade and frequent resistance to chloroquine by P. vivax may be spreading westward in the Indonesia archipelago.
Oyola, Samuel O; Ariani, Cristina V; Hamilton, William L; Kekre, Mihir; Amenga-Etego, Lucas N; Ghansah, Anita; Rutledge, Gavin G; Redmond, Seth; Manske, Magnus; Jyothi, Dushyanth; Jacob, Chris G; Otto, Thomas D; Rockett, Kirk; Newbold, Chris I; Berriman, Matthew; Kwiatkowski, Dominic P
Translating genomic technologies into healthcare applications for the malaria parasite Plasmodium falciparum has been limited by the technical and logistical difficulties of obtaining high quality clinical samples from the field. Sampling by dried blood spot (DBS) finger-pricks can be performed safely and efficiently with minimal resource and storage requirements compared with venous blood (VB). Here, the use of selective whole genome amplification (sWGA) to sequence the P. falciparum genome from clinical DBS samples was evaluated, and the results compared with current methods that use leucodepleted VB. Parasite DNA with high (>95%) human DNA contamination was selectively amplified by Phi29 polymerase using short oligonucleotide probes of 8-12 mers as primers. These primers were selected on the basis of their differential frequency of binding the desired (P. falciparum DNA) and contaminating (human) genomes. Using sWGA method, clinical samples from 156 malaria patients, including 120 paired samples for head-to-head comparison of DBS and leucodepleted VB were sequenced. Greater than 18-fold enrichment of P. falciparum DNA was achieved from DBS extracts. The parasitaemia threshold to achieve >5× coverage for 50% of the genome was 0.03% (40 parasites per 200 white blood cells). Over 99% SNP concordance between VB and DBS samples was achieved after excluding missing calls. The sWGA methods described here provide a reliable and scalable way of generating P. falciparum genome sequence data from DBS samples. The current data indicate that it will be possible to get good quality sequence on most if not all drug resistance loci from the majority of symptomatic malaria patients. This technique overcomes a major limiting factor in P. falciparum genome sequencing from field samples, and paves the way for large-scale epidemiological applications.
Abellan van Kan, Gabor; Houles, Mathieu; Vellas, Bruno
The present review describes and discusses the currently available definitions for sarcopenia from consensus studies. Different sarcopenia definitions have been proposed in these last years. Six main approaches to an operative definition of sarcopenia have been identified. Although the first definitions were solely based on the assessment of the amount of muscle mass, current definitions seem to consistently recognize a bi-dimensional nature of sarcopenia. So, these approaches imply the need of simultaneously assessing both age-related quantitative (i.e. amount of muscle mass) and qualitative (i.e. muscle strength and function) declines of skeletal muscle. Although current consensus exists about a bi-dimensional nature, the proposed approaches to measure sarcopenia are characterized by methodological differences. The majority of the operative definitions proposes to assess muscle mass as an index of appendicular muscle mass divided by squared height (evaluated by dual energy X-ray absorptiometry), assess strength using hand-held dynamometers, and assess function by evaluating gait speed at habitual pace over a short distance. Nevertheless, the clinically relevant thresholds and how to combine the three aspects in an operative definition in order to identify sarcopenia are heterogeneous. A main drawback is that supportive empirical data are missing for these conceptual definitions regarding the risk-assessment of different clinically significant adverse outcomes.
Duffy, Michael F; Reeder, John C; Brown, Graham V
Plasmodium falciparum employs a strategy of clonal antigenic variation to evade the host immune response during the intraerythrocytic stage of its life cycle. The major variant parasite molecule is the P. falciparum erythrocyte membrane protein (PfEMP)1, which is encoded by the var multigene family. The parasite switches between different PfEMP1 molecules through regulation of var transcription. Recent studies have shed considerable light on this process, but much remains unknown. However, striking parallels between transcriptional control of var and genes in other organisms provide direction for future studies.
Dinio, Theresa; Gorka, Alexander P.; McGinniss, Andrew; Roepe, Paul D.; Morgan, Jeremy B.
Plasmodium falciparum, the deadliest malarial parasite species, has developed resistance against nearly all man-made antimalarial drugs within the past century. However, quinine (QN), the first antimalarial drug, remains efficacious worldwide. Some chloroquine resistant (CQR) P. falciparum strains or isolates show mild cross resistance to QN, but many do not. Further optimization of QN may provide well-tolerated therapy with improved activity vs. CQR malaria. Thus, using the Heck reaction, we have pursued a structure-activity relationship study, including vinyl group modifications of QN. Certain derivatives show good antiplasmodial activity in QN-resistant and QN-sensitive strains, with lower IC50 values relative to QN. PMID:22512909
Dziegiel, Morten Hanefeld; Borre, M B; Jepsen, S
New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P. falciparum. Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control...... GLURP489-1271 was expressed as a chimeric protein, fused with E. coli beta-galactosidase. However, antibodies in sera were directed only against the malaria part of the fusion protein and not against beta-galactosidase. Antigen from in vitro P. falciparum cultures of isolates from Tanzania (F32), Papua...
Abu-Zeid, Y A; Theander, T G; Abdulhadi, N H
Plasma and peripheral blood mononuclear cells (PBMC) were obtained from P. falciparum-infected individuals with and without the sickle cell trait at diagnosis and 7 days after treatment. HbAA and HbAS patients were compared for levels of plasma soluble IL-2 receptors (IL-2R) and the in vitro...... cellular reactivity to affinity-purified soluble P. falciparum antigens (SPAg), PPD and phytohaemagglutinin (PHA). At diagnosis, HbAS patients with clinical disease had lower plasma-soluble IL-2R levels and parasite counts than the corresponding HbAA patients, whereas HbAS and HbAA patients...
Foumane Ngane, Vincent; Allico Djaman, Joseph; Culeux, Cécile; Piette, Nathalie; Carnevale, Pierre; Besnard, Patrick; Fortes, Filomeno; Basco, Leonardo K; Tahar, Rachida
The malaria situation has been worsening in Angola, partly due to armed conflict until the recent past and drug-resistant Plasmodium falciparum. Malaria transmission is heterogeneous within the country, and data on drug-resistant malaria in different parts of the country are incomplete. The aim of the present study was to evaluate resistance to 4-aminoquinolines and antifolate drugs in P. falciparum isolates collected in Benguela province, central Angola, using molecular markers. Fingerprick capillary blood was collected from asymptomatic children aged less than 15 years old during a household survey in and around Balombo town in 2010-2011. Samples were screened for P. falciparum by nested PCR. Molecular markers (P. falciparum dihydrofolate reductase [pfdhfr], P. falciparum dihydropteroate synthase [pfdhps], P. falciparum chloroquine resistance transporter [pfcrt], and P. falciparum multidrug-resistance gene 1 [pfmdr1]) were sequenced to determine the key codons associated with drug resistance. A total of 60 blood samples were positive for P. falciparum. Most isolates with successful PCR amplification had mutant pfdhfr alleles, with either double mutant AICNI (69%) or triple mutant AIRNI (21%) haplotypes. A16V, S108T, and I164L substitutions were not found. Many of the isolates were carriers of either SGKAA (60%) or AGKAA (27%) pfdhps haplotype. K540E substitution was absent. There were only two pfcrt haplotypes: wild-type CVMNK (11%) and mutant CVIET (89%). Wild-type pfmdr1 NYSND haplotype was found in 19% of the isolates, whereas single mutant pfmdr1 YYSND and NFSND haplotypes occurred in 48% and 11%, respectively. Double mutant pfmdr1 haplotypes (YFSND and YYSNY) occurred rarely. The results suggest that the high prevalence of mutant pfcrt CVIET haplotype is in agreement with low clinical efficacy of chloroquine observed in earlier studies and that the double pfdhfr mutant AICNI and single pfdhps mutant SGKAA are currently the predominant haplotypes associated
Molyneux Malcolm E
Full Text Available Abstract Background Described here is the first population genetic study of Plasmodium malariae, the causative agent of quartan malaria. Although not as deadly as Plasmodium falciparum, P. malariae is more common than previously thought, and is frequently in sympatry and co-infection with P. falciparum, making its study increasingly important. This study compares the population parameters of the two species in two districts of Malawi with different malaria transmission patterns - one seasonal, one perennial - to explore the effects of transmission on population structures. Methods Six species-specific microsatellite markers were used to analyse 257 P. malariae samples and 257 P. falciparum samples matched for age, gender and village of residence. Allele sizes were scored to within 2 bp for each locus and haplotypes were constructed from dominant alleles in multiple infections. Analysis of multiplicity of infection (MOI, population differentiation, clustering of haplotypes and linkage disequilibrium was performed for both species. Regression analyses were used to determine association of MOI measurements with clinical malaria parameters. Results Multiple-genotype infections within each species were common in both districts, accounting for 86.0% of P. falciparum and 73.2% of P. malariae infections and did not differ significantly with transmission setting. Mean MOI of P. falciparum was increased under perennial transmission compared with seasonal (3.14 vs 2.59, p = 0.008 and was greater in children compared with adults. In contrast, P. malariae mean MOI was similar between transmission settings (2.12 vs 2.11 and there was no difference between children and adults. Population differentiation showed no significant differences between villages or districts for either species. There was no evidence of geographical clustering of haplotypes. Linkage disequilibrium amongst loci was found only for P. falciparum samples from the seasonal transmission
Jakobsen, P H; Bygbjerg, I C; Theander, T G
. falciparum malaria compared to the levels during acute disease. Thus, both soluble Hb and haptoglobin appear to be markers of recent P. falciparum infections. Very high levels of CRP protein were measured in some of the malaria patients at the day of treatment while lower levels were recorded 7 and 30 days...... after treatment. Soluble Hb levels were associated with malariometric parameters in a similar fashion to haptoglobin. The new Mab-based assay for measuring soluble Hb in the peripheral blood of malaria patients may be useful for future epidemiological studies of malaria....
Alistair R D McLean
Full Text Available During pregnancy, immunoglobulin G (IgG is transferred from the mother to the fetus, providing protection from disease in early infancy. Plasmodium falciparum infections may reduce maternofetal antibody transfer efficiency, but mechanisms remain unclear.Mother-cord paired serum samples collected at delivery from Papua New Guinea (PNG and the Thailand-Myanmar Border Area (TMBA were tested for IgG1 and IgG3 to four P. falciparum antigens and measles antigen, as well as total serum IgG. Multivariable linear regression was conducted to assess the association of peripheral P. falciparum infection during pregnancy or placental P. falciparum infection assessed at delivery with maternofetal antibody transfer efficiency. Path analysis assessed the extent to which associations between P. falciparum infection and antibody transfer were mediated by gestational age at delivery or levels of maternal total serum IgG.Maternofetal antibody transfer efficiency of IgG1 and IgG3 was lower in PNG compared to TMBA (mean difference in cord antibody levels (controlling for maternal antibody levels ranged from -0.88 to 0.09, median of -0.20 log2 units. Placental P. falciparum infections were associated with substantially lower maternofetal antibody transfer efficiency in PNG primigravid women (mean difference in cord antibody levels (controlling for maternal antibody levels ranged from -0.62 to -0.10, median of -0.36 log2 units, but not multigravid women. The lower antibody transfer efficiency amongst primigravid women with placental infection was only partially mediated by gestational age at delivery (proportion indirect effect ranged from 0% to 18%, whereas no mediation effects of maternal total serum IgG were observed.Primigravid women may be at risk of impaired maternofetal antibody transport with placental P. falciparum infection. Direct effects of P. falciparum on the placenta, rather than earlier gestational age and elevated serum IgG, are likely responsible for
Ogouyèmi-Hounto, Aurore; Gazard, Dorothée Kinde; Ndam, Nicaise; Topanou, Elsa; Garba, Olivia; Elegbe, Pancras; Hountohotegbe, Tatiana; Massougbodji, Achille
The aim of this study was to determine the genetic diversity of Plasmodium falciparum by analyzing the polymorphism of the msp-1 and msp-2 genes and the multiplicity of infection in children with uncomplicated malaria in southern Benin. Blood samples of children with fever or history of fever with thick smear positive P. falciparum were collected on filter paper. After extraction of DNA by Chelex®, the samples underwent nested PCR. 93 isolates from children were genotyped. For the msp-1 gene, the K1 and R033 sequences were the most represented in the study population with 85.2% and 83% prevalence, respectively. Regarding the msp-2 gene, the FC27 family was more highly represented with 99% prevalence against 81.5% for 3D7. Mixed infections accounted for 80.4% of the samples. Twenty-five alleles were identified for msp-1 and 28 for msp-2. Fourteen and ten alleles belonged to the K1 (100-500 bp) and MAD20 (100-500 bp) families, respectively. The RO33 sequence did not show any polymorphism, with only one variant (160 bp) detected. The msp-2 gene was present as 16 FC27 family fragments (250-800 bp) and 12 of the 3D7 family (350-700 bp). The multiplicity of infection was estimated at 3.8 for msp-1 and 3.9 for msp-2 with 77 (87.5%) and 84 (91.3%) samples harboring more than one parasite genotype for msp-1 and msp-2, respectively. The multiplicity of infection (MOI) was influenced neither by age nor by parasite density. This study shows a significant diversity of P. falciparum in southern Benin with an MOI unaffected by age or by parasite density. © A. Ogouyèmi-Hounto et al., published by EDP Sciences, 2013.
Identification of resistance of Plasmodium falciparum to artesunate-mefloquine combination in an area along the Thai-Myanmar border: integration of clinico-parasitological response, systemic drug exposure, and in vitro parasite sensitivity.
Na-Bangchang, Kesara; Muhamad, Phunuch; Ruaengweerayut, Ronnatrai; Chaijaroenkul, Wanna; Karbwang, Juntra
A markedly high failure rate of three-day artesunate-mefloquine was observed in the area along the Thai-Myanmar border. Identification of Plasmodium falciparum isolates with intrinsic resistance to each component of the artesunate-mefloquine combination was analysed with integrated information on clinico-parasitological response, together with systemic drug exposure (area under blood/plasma concentration-time curves (AUC)) of dihydroartemisinin and mefloquine, and in vitro sensitivity of P. falciparum in a total of 17 out of 29 P. falciparum isolates from patients with acute uncomplicated falciparum malaria. Analysis of the contribution of in vitro parasite sensitivity and systemic drug exposure and relationship with pfmdr1 copy number in the group with sensitive response was performed in 21 of 69 cases. Identification of resistance and/or reduced intrinsic parasitocidal activity of artesunate and/or mefloquine without pharmacokinetic or other host-related factors were confirmed in six cases: one with reduced sensitivity to artesunate alone, two with resistance to mefloquine alone, and three with reduced sensitivity to artesunate combined with resistance to mefloquine. Resistance and/or reduced intrinsic parasitocidal activity of mefloquine/artesunate, together with contribution of pharmacokinetic factor of mefloquine and/or artesunate were identified in seven cases: two with resistance to mefloquine alone, and five with resistance to mefloquine combined with reduced sensitivity to artesunate. Pharmacokinetic factor alone contributed to recrudescence in three cases, all of which had inadequate whole blood mefloquine levels (AUC0-7days). Other host-related factors contributed to recrudescence in one case. Amplification of pfmdr1 (increasing of pfmdr1 copy number) is a related molecular marker of artesunate-mefloquine resistance and seems to be a suitable molecular marker to predict occurrence of recrudescence. Despite the evidence of a low level of a decline in
Full Text Available Clinical trials of interventions designed to prevent severe falciparum malaria in children require a clear endpoint. The internationally accepted definition of severe malaria is sensitive, and appropriate for clinical purposes. However, this definition includes individuals with severe nonmalarial disease and coincident parasitaemia, so may lack specificity in vaccine trials. Although there is no "gold standard" individual test for severe malaria, malaria-attributable fractions (MAFs can be estimated among groups of children using a logistic model, which we use to test the suitability of various case definitions as trial endpoints.A total of 4,583 blood samples were taken from well children in cross-sectional surveys and from 1,361 children admitted to a Kenyan District hospital with severe disease. Among children under 2 y old with severe disease and over 2,500 parasites per microliter of blood, the MAFs were above 85% in moderate- and low-transmission areas, but only 61% in a high-transmission area. HIV and malnutrition were not associated with reduced MAFs, but gastroenteritis with severe dehydration (defined by reduced skin turgor, lower respiratory tract infection (clinician's final diagnosis, meningitis (on cerebrospinal fluid [CSF] examination, and bacteraemia were associated with reduced MAFs. The overall MAF was 85% (95% confidence interval [CI] 83.8%-86.1% without excluding these conditions, 89% (95% CI 88.4%-90.2% after exclusions, and 95% (95% CI 94.0%-95.5% when a threshold of 2,500 parasites/mul was also applied. Applying a threshold and exclusion criteria reduced sensitivity to 80% (95% CI 77%-83%.The specificity of a case definition for severe malaria is improved by applying a parasite density threshold and by excluding children with meningitis, lower respiratory tract infection (clinician's diagnosis, bacteraemia, and gastroenteritis with severe dehydration, but not by excluding children with HIV or malnutrition.
Lunev, Sergey; Bosch, Soraya S; Batista, Fernando de Assis; Wrenger, Carsten; Groves, Matthew R
The de novo pyrimidine-biosynthesis pathway of Plasmodium falciparum is a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is unable to take up pyrimidines from the host. Direct (or indirect) inhibition of de novo pyrimidine biosynthesis via dihydroorotate dehydrogenase (PfDHODH), the fourth enzyme of the pathway, has already been shown to be lethal to the parasite. In the second step of the plasmodial pyrimidine-synthesis pathway, aspartate and carbamoyl phosphate are condensed to N-carbamoyl-L-aspartate and inorganic phosphate by aspartate transcarbamoylase (PfATC). In this paper, the 2.5 Å resolution crystal structure of PfATC is reported. The space group of the PfATC crystals was determined to be monoclinic P21, with unit-cell parameters a = 87.0, b = 103.8, c = 87.1 Å, α = 90.0, β = 117.7, γ = 90.0°. The presented PfATC model shares a high degree of homology with the catalytic domain of Escherichia coli ATC. There is as yet no evidence of the existence of a regulatory domain in PfATC. Similarly to E. coli ATC, PfATC was modelled as a homotrimer in which each of the three active sites is formed at the oligomeric interface. Each active site comprises residues from two adjacent subunits in the trimer with a high degree of evolutional conservation. Here, the activity loss owing to mutagenesis of the key active-site residues is also described.
Hasang, Wina; Dembo, Edson G; Wijesinghe, Rushika; Molyneux, Malcolm E; Kublin, James G; Rogerson, Stephen
Coinfection with human immunodeficiency virus (HIV) may increase susceptibility to malaria by compromising naturally acquired immunity. In 339 adults (64% HIV infected), we measured antibodies to Plasmodium falciparum variant surface antigens (VSA) and antibodies that opsonise infected erythrocytes using parasite lines FCR3, E8B, and R29, and antibodies to merozoite antigens AMA-1 and MSP2. We determined the relationship between malaria antibodies, HIV infection, markers of immune compromise, and risk of incident parasitemia. HIV-infected adults had significantly lower mean levels of opsonizing antibody to all parasite lines (P < .0001), and lower levels of antibody to AMA-1 (P = .01) and MSP2 (P < .0001). Levels of immunoglobulin G (IgG) to VSA were not affected by HIV status. Opsonising antibody titres against some isolates were positively correlated with CD4 count. There were negative associations between human immunodeficiency virus type 1 (HIV-1) viral load and opsonizing antibodies to FCR3 (P = .04), and levels of IgG to AMA-1 (P ≤ .03) and MSP2-3D7 (P = .05). Lower opsonizing antibody levels on enrollment were seen in those who became parasitemic during follow-up, independent of HIV infection (P ≤ .04 for each line). HIV-1 infection decreases opsonizing antibodies to VSA, and antibody to merozoite antigens. Opsonizing antibodies were associated with lack of parasitemia during follow up, suggesting a role in protection. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: email@example.com.
Mutations in the pfmdr1, cg2, and pfcrt genes in Plasmodium falciparum samples from endemic malaria areas in Rondonia and Pará State, Brazilian Amazon Region Mutações nos genes pfmdr1, cg2 e pfcrt em isolados de Plasmodium falciparum provenientes de localidades malarígenas dos Estados de Rondônia e Pará, Amazônia Legal Brasileira
Giselle Maria Rachid Viana
Full Text Available The objectives of this study were to investigate the molecular basis for Plasmodium falciparum resistance to chloroquine in isolates from the Brazilian Amazon and to identify polymorphisms in the pfmdr1 gene, codons 184, 1042, and 1246, the kappa and gamma regions of the cg2 gene, and the K76T mutation of the pfcrt gene, in order to calculate the distribution of polymorphism within each target gene, comparing samples from distinct geographic areas, using allele-specific polymerase chain reaction (PCR for the pfmdr gene and PCR plus restriction fragment length polymorphism (RFLP for the cg2 and pfcrt genes. The sample consisted of 40 human blood isolates, already collected and morphologically diagnosed as carriers of P. falciparum parasites, from four localities: Porto Velho in Rondonia State and Maraba, Itaituba, and Tailandia in Pará State. Distribution of P. falciparum in vitro chloroquine resistance in the isolates was 100% for pfmdr1, cg2 gamma region, and pfcrt, except for the polymorphism in the cg2 kappa region, which was not found.O estudo foi desenvolvido para investigar a base molecular da resistência do Plasmodium falciparum à cloroquina em isolados da região Amazônica brasileira e identificar os polimorfismos nos códons TYR184PHE, ASN1042ASP e ASP1246TYR do gene pfmdr1, as regiões kappa e gamma do gene cg2 e a mutação K76T do gene pfcrt, a fim de determinar a distribuição percentual dos alelos de cada gene estudado, comparando amostras de áreas geográficas distintas, utilizando a reação em cadeia da polimerase (PCR alelo-específica para o pfmdr1 e a PCR e o polimorfismo do comprimento do fragmento de restrição (RFLP para os genes cg2 e pfcrt. A amostra foi constituída de quarenta isolados de sangue humano já coletados e microscopicamente diagnosticados com malária por P. falciparum das localidades de Porto Velho (Rondônia e Marabá, Itaituba e Tailândia (Pará. A distribuição percentual da resistência in
Full Text Available Epstein–Barr virus (EBV is a necessary cause of endemic Burkitt lymphoma (eBL, while the role of Plasmodium falciparum in eBL remains uncertain. This study aimed to generate new hypotheses on the interplay between both infections in the development of eBL by investigating the IgG and IgM profiles against several EBV and P. falciparum antigens. Serum samples collected in a childhood study in Malawi (2005–2006 from 442 HIV-seronegative children (271 eBL cases and 171 controls between 1.4 and 15 years old were tested by quantitative suspension array technology against a newly developed multiplex panel combining 4 EBV antigens [Z Epstein–Barr replication activator protein (ZEBRA, early antigen-diffuse component (EA-D, EBV nuclear antigen 1, and viral capsid antigen p18 subunit (VCA-p18] and 15 P. falciparum antigens selected for their immunogenicity, role in malaria pathogenesis, and presence in different parasite stages. Principal component analyses, multivariate logistic models, and elastic-net regressions were used. As expected, elevated levels of EBV IgG (especially against the lytic antigens ZEBRA, EA-D, and VCA-p18 were strongly associated with eBL [high vs low tertile odds ratio (OR = 8.67, 95% confidence interval (CI = 4.81–15.64]. Higher IgG responses to the merozoite surface protein 3 were observed in children with eBL compared with controls (OR = 1.29, 95% CI = 1.02–1.64, showing an additive interaction with EBV IgGs (OR = 10.6, 95% CI = 5.1–22.2, P = 0.05. Using elastic-net regression models, eBL serological profile was further characterized by lower IgM levels against P. falciparum preerythrocytic-stage antigen CelTOS and EBV lytic antigen VCA-p18 compared with controls. In a secondary analysis, abdominal Burkitt lymphoma had lower IgM to EBV and higher IgG to EA-D levels than cases with head involvement. Overall, this exploratory study confirmed the strong role of EBV in eBL and identified
Odaro Stanley Imade
Full Text Available Our study aimed at substantiating the recent claim of myocardial complications in severe malaria by experimentally inducing severe Plasmodium falciparum infection in a humanized mouse model employed as human surrogate.Twenty five humanized mice were inoculated with standard in vitro cultured P. falciparum and blood extracts collected from the inner cardiac muscles of infected mice that died were examined for the presence of the infectious cause of death. The therapeutic effect of quinine on 7 mice severely infected with P. falciparum was also evaluated.All the 25 humanized mice inoculated with the in vitro cultured P. falciparum revealed peripheral parasitemia with a total of 10 deaths recorded. Postmortem examination of the inner cardiac muscles of the dead mice also revealed massive sequestration of mature P. falciparum as well as significant infiltration of inflammatory cells such as lymphocytes and monocytes. Postmortem evaluation of the inner cardiac muscles of the P. falciparum-infected mice after quinine therapy showed significant decline in parasite density with no death of mice recorded.Data obtained from our study significantly corroborated the findings of myocardial dysfunction as the primary cause of death in recent case reports of humans infected with P. falciparum.
Full Text Available El objetivo de este trabajo es presentar los casos de paludismo por Plasmodium falciparum ocurridos en viajeros provenientes del África tropical, atendidos en el Hospital Alemán. Se definió paludismo de origen africano como la infección adquirida en un país del África subsahariana, diagnosticado y tratado en la Argentina. El diagnóstico se realizó por la clínica y la microscopía óptica en frotis de sangre periférica coloreados con Giemsa. Se revieron las historias clínicas de 11 pacientes adultos -cinco turistas y seis marineros mercantes- no oriundos de área endémica, sin condición inmunosupresora, ni morbilidad asociada, internados entre 1993 y 2007. El rango de edad fue de 21 a 48 años; nueve hombres y dos mujeres. Los pacientes fueron clasificados retrospectivamente en malaria grave (seis o no grave (cinco según cumplieran con uno o más de los criterios de gravedad de la Organización Mundial de la Salud. Todos presentaron fiebre como signo más significativo. Como complicaciones graves se observaron casos de insuficiencia renal, epistaxis, hemoglobinuria, hipoglucemia, edema pulmonar, acidosis y coma. Tres pacientes requirieron internación en la unidad de terapia intensiva. Todos sobrevivieron y solamente tres habían recibido la quimioprofilaxis correcta antes de viajar. El tratamiento se realizó con una o más de las siguientes drogas: mefloquina, quinidina, clindamicina y cotrimoxazol.The purpose of this paper is to present the cases of malaria caused by Plasmodium falciparum in travelers coming from tropical Africa, who were treated at the Hospital Alemán (Buenos Aires. African malaria was defined as an infection acquired in any country within Africa, diagnosed and treated in Argentina. Diagnostic tools included clinical features and optic microscopy with Giemsa stained peripheral blood films. We reviewed the medical records of 11 adult patients -five tourists and six sailors- with no history of malaria
Subramanian, Gowtham; Belekar, Meenakshi A; Shukla, Anurag; Tong, Jie Xin; Sinha, Ameya; Chu, Trang T T; Kulkarni, Akshay S; Preiser, Peter R; Reddy, D Srinivasa; Tan, Kevin S W; Shanmugam, Dhanasekaran; Chandramohanadas, Rajesh
The Malaria Box collection includes 400 chemically diverse small molecules with documented potency against malaria parasite growth, but the underlying modes of action are largely unknown. Using complementary phenotypic screens against Plasmodium falciparum and Toxoplasma gondii , we report phenotype-specific hits based on inhibition of overall parasite growth, apicoplast segregation, and egress or host invasion, providing hitherto unavailable insights into the possible mechanisms affected. First, the Malaria Box library was screened against tachyzoite stage T. gondii and the half-maximal effective concentrations (EC 50 s) of molecules showing ≥80% growth inhibition at 10 µM were determined. Comparison of the EC 50 s for T. gondii and P. falciparum identified a subset of 24 molecules with nanomolar potency against both parasites. Thirty molecules that failed to induce acute growth inhibition in T. gondii tachyzoites in a 2-day assay caused delayed parasite death upon extended exposure, with at least three molecules interfering with apicoplast segregation during daughter cell formation. Using flow cytometry and microscopy-based examinations, we prioritized 26 molecules with the potential to inhibit host cell egress/invasion during asexual developmental stages of P. falciparum . None of the inhibitors affected digestive vacuole integrity, ruling out a mechanism mediated by broadly specific protease inhibitor activity. Interestingly, five of the plasmodial egress inhibitors inhibited ionophore-induced egress of T. gondii tachyzoites. These findings highlight the advantage of comparative and targeted phenotypic screens in related species as a means to identify lead molecules with a conserved mode of action. Further work on target identification and mechanism analysis will facilitate the development of antiparasitic compounds with cross-species efficacy. IMPORTANCE The phylum Apicomplexa includes many human and animal pathogens, such as Plasmodium falciparum
Bhaumik, Prasenjit; Horimoto, Yasumi; Xiao, Huogen; Miura, Takuya; Hidaka, Koushi; Kiso, Yoshiaki; Wlodawer, Alexander; Yada, Rickey Y; Gustchina, Alla
Plasmepsin I (PMI) is one of the four vacuolar pepsin-like proteases responsible for hemoglobin degradation by the malarial parasite Plasmodium falciparum, and the only one with no crystal structure reported to date. Due to substantial functional redundancy of these enzymes, lack of inhibition of even a single plasmepsin can defeat efforts in creating effective antiparasitic agents. We have now solved crystal structures of the recombinant PMI as apoenzyme and in complex with the potent peptidic inhibitor, KNI-10006, at the resolution of 2.4 and 3.1Å, respectively. The apoenzyme crystallized in the orthorhombic space group P2(1)2(1)2(1) with two molecules in the asymmetric unit and the structure has been refined to the final R-factor of 20.7%. The KNI-10006 bound enzyme crystallized in the tetragonal space group P4(3) with four molecules in the asymmetric unit and the structure has been refined to the final R-factor of 21.1%. In the PMI-KNI-10006 complex, the inhibitors were bound identically to all four enzyme molecules, with the opposite directionality of the main chain of KNI-10006 relative to the direction of the enzyme substrates. Such a mode of binding of inhibitors containing an allophenylnorstatine-dimethylthioproline insert in the P1-P1' positions, previously reported in a complex with PMIV, demonstrates the importance of satisfying the requirements for the proper positioning of the functional groups in the mechanism-based inhibitors towards the catalytic machinery of aspartic proteases, as opposed to binding driven solely by the specificity of the individual enzymes. A comparison of the structure of the PMI-KNI-10006 complex with the structures of other vacuolar plasmepsins identified the important differences between them and may help in the design of specific inhibitors targeting the individual enzymes. Published by Elsevier Inc.
Tan John C
Full Text Available Abstract Background Knowledge of the origins, distribution, and inheritance of variation in the malaria parasite (Plasmodium falciparum genome is crucial for understanding its evolution; however the 81% (A+T genome poses challenges to high-throughput sequencing technologies. We explore the viability of the Roche 454 Genome Sequencer FLX (GS FLX high throughput sequencing technology for both whole genome sequencing and fine-resolution characterization of genetic exchange in malaria parasites. Results We present a scheme to survey recombination in the haploid stage genomes of two sibling parasite clones, using whole genome pyrosequencing that includes a sliding window approach to predict recombination breakpoints. Whole genome shotgun (WGS sequencing generated approximately 2 million reads, with an average read length of approximately 300 bp. De novo assembly using a combination of WGS and 3 kb paired end libraries resulted in contigs ≤ 34 kb. More than 8,000 of the 24,599 SNP markers identified between parents were genotyped in the progeny, resulting in a marker density of approximately 1 marker/3.3 kb and allowing for the detection of previously unrecognized crossovers (COs and many non crossover (NCO gene conversions throughout the genome. Conclusions By sequencing the 23 Mb genomes of two haploid progeny clones derived from a genetic cross at more than 30× coverage, we captured high resolution information on COs, NCOs and genetic variation within the progeny genomes. This study is the first to resequence progeny clones to examine fine structure of COs and NCOs in malaria parasites.
Susanne H Sheehy
Full Text Available Controlled human malaria infection (CHMI studies have become a routine tool to evaluate efficacy of candidate anti-malarial drugs and vaccines. To date, CHMI trials have mostly been conducted using the bite of infected mosquitoes, restricting the number of trial sites that can perform CHMI studies. Aseptic, cryopreserved P. falciparum sporozoites (PfSPZ Challenge provide a potentially more accurate, reproducible and practical alternative, allowing a known number of sporozoites to be administered simply by injection.We sought to assess the infectivity of PfSPZ Challenge administered in different dosing regimens to malaria-naive healthy adults (n = 18. Six participants received 2,500 sporozoites intradermally (ID, six received 2,500 sporozoites intramuscularly (IM and six received 25,000 sporozoites IM.Five out of six participants receiving 2,500 sporozoites ID, 3/6 participants receiving 2,500 sporozoites IM and 6/6 participants receiving 25,000 sporozoites IM were successfully infected. The median time to diagnosis was 13.2, 17.8 and 12.7 days for 2,500 sporozoites ID, 2,500 sporozoites IM and 25,000 sporozoites IM respectively (Kaplan Meier method; p = 0.024 log rank test.2,500 sporozoites ID and 25,000 sporozoites IM have similar infectivities. Given the dose response in infectivity seen with IM administration, further work should evaluate increasing doses of PfSPZ Challenge IM to identify a dosing regimen that reliably infects 100% of participants.ClinicalTrials.gov NCT01465048.
Full Text Available Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8 employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64 and aspartic (pepstatin protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine, indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in
Lanneaux, Justine; Dauger, Stéphane; Pham, Luu-Ly; Naudin, Jérôme; Faye, Albert; Gillet, Yves; Bosdure, Emmanuelle; Carbajal, Ricardo; Dubos, François; Vialet, Renaud; Chéron, Gérard; Angoulvant, François
The World Health Organization (WHO) severity criteria for paediatric Plasmodium falciparum (Pf) malaria are based on studies in countries of endemic malaria. The relevance of these criteria for other countries remains unclear. We assessed the relevance of these criteria in an industrialised country. Retrospective case-control study. Eight French university hospitals, from 2006 to 2012. Children with Pf malaria admitted to paediatric intensive care units (cases: n=55) or paediatric emergency departments (controls: n=110). Descriptive analysis of WHO severity criteria and major interventions (mechanical ventilation, blood transfusion, fluid challenge, treatment of cerebral oedema, renal replacement therapy). Thresholds were set by receiver operating characteristics curve analysis. Altered consciousness (71% vs 5%), shock (24% vs 1%), renal failure (20% vs 1%), anaemia 50 µmol/L (25% vs 8%) and parasitaemia >10% (30% vs 8%) were more frequent in cases (p<0.01). All these criteria were associated with major interventions (p<0.001). Respiratory distress (six cases), and hypoglycaemia (two cases) were infrequent. Thrombocytopenia <50 000/mm 3 (46% vs 7%) and anaemia (haemoglobin concentration <70 g/L (41% vs 13%)) were more frequent in cases (p<0.0001). The WHO severity criteria for paediatric Pf malaria are relevant for countries without endemic malaria. The infrequent but severe complications also provide a timely reminder of the morbidity and mortality associated with this condition worldwide. In non-endemic countries haemoglobin <70 g/L and platelet count <50 000/mm 3 could be used as additional criteria to identify children needing high level of care. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.
Full Text Available BACKGROUND: Care for malaria patients in endemic areas has been improved through the increasing use of Rapid Diagnostic Tests (RDTs. Most RDTs target the histidine-rich protein-2 antigen (PfHRP2 to detect P. falciparum, as it is abundant and shows great heat stability. However, their use in South America has been widely questioned following a recent publication that pinpoints the high prevalence of Peruvian field isolates lacking the gene encoding this protein. In the remote rural health centers of French Guiana, RDTs are the main diagnosis tools. Therefore, a study of PfHRP2 RDT performances and pfhrp2 genotyping was conducted to determine whether a replacement of the current pLDH-based kit could be considered. METHODS: The performance study compared the SD Malaria Ag test P.f/Pan® kit with the current gold standard diagnosis by microscopy. The prevalence of pfhrp2 and pfhrp3 deletions were evaluated from 221 P. falciparum isolates collected between 2009 and 2011 in French Guiana. RESULTS: Between January 2010 and August 2011, 960 suspected cases of malaria were analyzed using microscopy and RDTs. The sensitivity of the SD Malaria Ag test P.f/Pan® for detection of P. falciparum was 96.8% (95% CI: 90.9-99.3, and 86.0% (95% CI: 78.9-91.5 for the detection of P. vivax. No isolates (95% CI: 0-4.5 lacking either exon of the pfhrp2 gene were identified among the 221 P. falciparum isolates analyzed, but 7.4% (95% CI: 2.8-15.4 lacked the exon 2 part of the pfhrp3 gene. CONCLUSIONS: Field isolates lacking either exon of the pfhrp2 gene are absent in this western part of South America. Despite its sensibility to detect P. vivax, the SD Malaria Ag test P.f/Pan® kit is a satisfying alternative to microscopy in remote health centers, where it is difficult to provide highly skilled microscopists and to maintain the necessary equipment.
Kyle Dennis E
Full Text Available Abstract Background In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb gene (Tyr268Ser and Tyr268Asn. However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa. Methods The prevalence of codon-268 mutations in the cytb gene of African P. falciparum isolates from Nigeria, Malawi and Senegal, where atovaquone-proguanil has not been introduced for treatment of malaria was assessed. Genotyping of the cytb gene in isolates of P. falciparum was performed by PCR-restriction fragment length polymorphism and confirmed by sequencing. Results 295 samples from Nigeria (111, Malawi (91 and Senegal (93 were successfully analyzed for detection of either mutant Tyr268Ser or Tyr268Asn. No case of Ser268 or Asn268 was detected in cytb gene of parasites from Malawi or Senegal. However, Asn268 was detected in five out of 111 (4.5% unexposed P. falciparum isolates from Nigeria. In addition, one out of these five mutant Asn268 isolates showed an additional cytb mutation leading to a Pro266Thr substitution inside the ubiquinone reduction site. Conclusion No Tyr268Ser mutation is found in cytb of P. falciparum isolates from Nigeria, Malawi or Senegal. This study reports for the first time cytb Tyr268Asn mutation in unexposed P. falciparum isolates from Nigeria. The emergence in Africa of P. falciparum isolates with cytb Tyr268Asn mutation is a matter of serious concern. Continuous monitoring of atovaquone-proguanil resistant P. falciparum in Africa is warranted for the rational use of this new antimalarial drug, especially in non-immune travelers.
Happi, Christian T; Gbotosho, Grace O; Folarin, Onikepe A; Milner, Danny; Sarr, Ousmane; Sowunmi, Akintunde; Kyle, Dennis E; Milhous, Wilbur K; Wirth, Dyann F; Oduola, Ayoade M J
In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb) gene (Tyr268Ser and Tyr268Asn). However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa. The prevalence of codon-268 mutations in the cytb gene of African P. falciparum isolates from Nigeria, Malawi and Senegal, where atovaquone-proguanil has not been introduced for treatment of malaria was assessed. Genotyping of the cytb gene in isolates of P. falciparum was performed by PCR-restriction fragment length polymorphism and confirmed by sequencing. 295 samples from Nigeria (111), Malawi (91) and Senegal (93) were successfully analyzed for detection of either mutant Tyr268Ser or Tyr268Asn. No case of Ser268 or Asn268 was detected in cytb gene of parasites from Malawi or Senegal. However, Asn268 was detected in five out of 111 (4.5%) unexposed P. falciparum isolates from Nigeria. In addition, one out of these five mutant Asn268 isolates showed an additional cytb mutation leading to a Pro266Thr substitution inside the ubiquinone reduction site. No Tyr268Ser mutation is found in cytb of P. falciparum isolates from Nigeria, Malawi or Senegal. This study reports for the first time cytb Tyr268Asn mutation in unexposed P. falciparum isolates from Nigeria. The emergence in Africa of P. falciparum isolates with cytb Tyr268Asn mutation is a matter of serious concern. Continuous monitoring of atovaquone-proguanil resistant P. falciparum in Africa is warranted for the rational use of this new antimalarial drug, especially in non-immune travelers.
Afoakwah, Richmond; Aubyn, Edmond; Prah, James; Nwaefuna, Ekene Kwabena; Boampong, Johnson N
The clinical outcome of falciparum malaria in endemic areas is influenced by erythrocyte polymorphisms including the ABO blood groups. Studies have reported association of ABO blood group to resistance, susceptibility, and severity of P. falciparum malaria infection. Individuals with blood group "A" have been found to be highly susceptible to falciparum malaria whereas blood group "O" is said to confer protection against complicated cases. We analyzed samples from 293 young children less than six years old with malaria in the Korle-Bu Teaching Hospital in Accra, Ghana. It was observed that group O was present in about 16.1% of complicated cases weighed against 40.9% of uncomplicated controls. Individuals with complicated malaria were about twice likely to be of blood groups A and B compared to group O (A versus O, OR = 1.90, 95% CI = 1.59-2.26, P Blood group O participants with complicated diseases had low parasitaemia compared to the other blood groups (P blood group O individuals a survival advantage over the other groups in complicated malaria as suggested. Participants with complicated falciparum malaria were generally anaemic and younger than those with uncomplicated disease.
Blood samples were collected from 100 children presenting with microscopically confirmed P. falciparum. Parasite DNA extracted from dried blood spots by Chelex method was analysed with a primary Polymerase Chain Reaction (PCR) and nested PCR for specific DHFR codons; 108, 51 and 59. Overall, 83% had resistant ...
Abu-Zeid, Y A; Abdulhadi, N H; Hviid, L
Blood mononuclear cells (BMNC) were isolated from sickle cell trait (HbAS) healthy donors and normal haemoglobin (HbAA) healthy donors resident in a P. falciparum endemic area of eastern Sudan. Blood samples were collected during the malaria season. BMNC were tested for their proliferative...
Masseno, Victor; Muriithi, Steven; Nzila, Alexis
We tested the effect of probenecid and verapamil in chemosensitizing Plasmodium falciparum to 14 antimalarials using the multidrug-resistant strain V1S and the drug-sensitive 3D7. Verapamil chemosensitizes V1S to quinine and chloroquine. Interestingly, probenecid profoundly chemosensitizes V1S to piperaquine. Thus, probenecid could be used to increase piperaquine efficacy in vivo.
Masseno, Victor; Muriithi, Steven; Nzila, Alexis
We tested the effect of probenecid and verapamil in chemosensitizing Plasmodium falciparum to 14 antimalarials using the multidrug-resistant strain V1S and the drug-sensitive 3D7. Verapamil chemosensitizes V1S to quinine and chloroquine. Interestingly, probenecid profoundly chemosensitizes V1S to piperaquine. Thus, probenecid could be used to increase piperaquine efficacy in vivo.
Dziegiel, Morten Hanefeld; Rowe, P; Bennett, S
The aims of the present study were to describe the age-related immunoglobulin M (IgM) and IgG response to part of a 220-kDa glutamate-rich protein (GLURP) from Plasmodium falciparum and to determine possible correlations of possession of these antibodies with malaria morbidity. IgM and IgG levels...
Beards, S. C.; Joynt, G. M.; Lipman, J.
We describe the haemodynamic and oxygen transport response in a patient undergoing exchange transfusion for severe falciparum malaria. We found that exchange transfusion produced a significant increase in left ventricular stroke work index, systemic oxygen delivery and oxygen consumption. This potentially beneficial effect of exchange transfusion has not been reported previously.
Dekker, E.; Romijn, J. A.; Waruiru, C.; Ackermans, M. T.; Weverling, G. J.; Sauerwein, R. W.; Endert, E.; Peshu, N.; Marsh, K.; Sauerwein, H. P.
The pathophysiology of hypoglycaemia in children with acute falciparum malaria, a frequent and serious complication, is unknown due to absence of data on glucose kinetics. We investigated the correlation between basal glucose production and plasma glucose concentration in 20 children (8 girls) with
de Vries, P. J.; Bich, N. N.; van Thien, H.; Hung, L. N.; Anh, T. K.; Kager, P. A.; Heisterkamp, S. H.
Combinations of artemisinin and quinine for uncomplicated falciparum malaria were studied. A total of 268 patients were randomized to 7 days of quinine at 10 mg/kg of body weight three times a day (Q) or to artemisinin at 20 mg/kg of body weight followed by 3 (AQ3) or 5 (AQ5) days of quinine.
Tange, R. A.; Dreschler, W. A.; Claessen, F. A.; Perenboom, R. M.
Audiometric changes following quinine administration were studied in healthy Caucasian subjects and patients suffering from falciparum malaria disease. Quinine-dihydrochloride was administered intravenously as a single dose of 300 mg to 12 healthy subjects and as multiple doses of 600 mg in 4 h
Claessen, F. A.; van Boxtel, C. J.; Perenboom, R. M.; Tange, R. A.; Wetsteijn, J. C.; Kager, P. A.
To study the pharmacokinetic behaviour of quinine in Caucasians with and without malaria. Quinine-dihydrochloride was administered intravenously as a single dose of 300 mg to 12 healthy subjects and as multiple doses of 600 mg in 4 h every 8 h in 10 patients with falciparum malaria. Plasma quinine
Interpretation & conclusions: As the increase in IC50 and IC90 values of DHA against field isolates of P. falciparum was not significant, the tolerance to DHA-piperaquine (PPQ combination might be because of PPQ only. Further study is required on more number of such isolates to generate data for a meaningful conclusion.
Baswin, A.; Siregar, M. L.; Jamil, K. F.
P. falciparum-induced severe malaria with life-threatening complications like acute kidney injury (AKI), jaundice, cerebral malaria, severe anemia, acidosis, and acute respiratory distress syndrome (ARDS). A 31-year-old soldier man who works in Aceh Singkil, Indonesia which is an endemic malaria area presented with a paroxysm of fever, shaking chills and sweats over four days, headache, arthralgia, abdominal pain, pale, jaundice, and oliguria. Urinalysis showed hemoglobinuria. Blood examination showed hemolytic anemia, thrombocytopenia, and hyperbilirubinemia. Falciparum malaria was then confirmed by peripheral blood smear, antimalarial medications were initiated, and hemodialysis was performed for eight times. The patient’s condition and laboratory results were quickly normalized. We report a case of P. falciparum-induced severe malaria with AKI and jaundice. The present case suggests that P. falciparum may induce severe malaria with life-threatening complications, early diagnosis and treatment is important to improve the quality of life of patients. Physicians must be alert for correct diagnosis and proper management of imported tropical malaria when patients have travel history in endemic areas.
Full Text Available The presence of the P. falciparum resistance and decreased of efficacy against artemisinin and its derivatives result in increasingly complex malaria issues. Malaria has become one of the currently unresolved world’s health problems due to the lack of new artemisinin replacement drugs. This study aimed to provide evidence that the repeated exposure of in vitro artemisinin may cause a change in P. falciparum Papua 2300 strain phenotypic. This study was conducted during the period of February to November 2013 in Biomedics Brawijaya University and the Faculty of Veterinary Medicine, Airlangga University. A post-test control only experimental design was used. In vitro cultures of P. falciparum Papua 2300 strain were treated by repeated artemisin in IC50 concentration and were observed for their viability and IC50 using probit analysis. The control group did not show any changes after IC50value and PO1 treatment. An increase in IC50 value was occurred after PO2. Repeated exposures of artemisinin in PO2, PO3 and PO4 had shorter viability periods than PO1. The viability of was stable after PO3 in this group. In conclusion, repeated exposures of artemisinin influence changes in IC50 value and viability period of P. falciparum Papua 2300 strain.
Hamad, A A; El Hassan, I M; El Khalifa, A A
the October-December malaria season of 1996, 51 individuals out of a population of 420 had confirmed and treated P. falciparum malaria in the village of Daraweesh in eastern Sudan. In a cross-sectional survey carried out in December 1996, an additional 6 individuals were found to harbour a microscopically...
Elhassan, I M; Satti, G H; Ali, A E
The efficacy of artemether (a qinghaosu derivative) administered intramuscularly for the treatment of Plasmodium falciparum malaria was compared to quinine in an open randomized trial including 54 patients in eastern Sudan, where chloroquine resistance is common. The artemether treatment (5 d...
ORJUELA-SÁNCHEZ, P.; SILVA-NUNES, M. DA; DA SILVA, N. S.; SCOPEL, K.K.G.; GONÇALVES, R. M.; MALAFRONTE, R. S.; FERREIRA, M. U.
SUMMARY Temporal changes in the prevalence of antigenic variants in Plasmodium falciparum populations have been interpreted as evidence of immune-mediated frequency-dependent selection, but evolutively neutral processes may generate similar patterns of serotype replacement. Over 4 years, we investigated the population dynamics of P. falciparum polymorphisms at the community level by using 11 putatively neutral microsatellite markers. Plasmodium falciparum populations were less diverse than sympatric P. vivax isolates, with less multiple-clone infections, lower number of alleles per locus and lower virtual heterozygosity, but both species showed significant multilocus linkage disequilibrium. Evolutively neutral P. falciparum polymorphisms showed a high turnover rate, with few lineages persisting for several months in the population. Similar results had previously been obtained, in the same community, for sympatric P. vivax isolates. In contrast, the prevalence of the 2 dimorphic types of a major antigen, MSP-2, remained remarkably stable throughout the study period. We suggest that the relatively fast turnover of parasite lineages represents the typical population dynamics of neutral polymorphisms in small populations, with clear implications for the detection of frequency-dependent selection of polymorphisms. PMID:19631016
Increased lung uptake of /sup 99m/Tc-sulfur colloid was seen during liver scanning in a patient with falciparum malaria. This finding was due to the enhanced activity of the phagocytic cells of the reticuloendothelial system in the liver, spleen, and lung found in human and experimental malaria. Similar findings in other clinical situations and the relevant literature are reviewed.
Villasis, Elizabeth; Lopez-Perez, Mary; Torres, Katherine
Background: Erythrocyte invasion by Plasmodium falciparum is a complex process that involves two families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins. Antibodies that inhibit merozoite attachment and invasion are believed to be important in mediating naturall...
Availability of complete genome sequence for Plasmodium falciparum has been useful in drawing a comprehensive metabolic map of the parasite. Distinct and unique metabolic characteristics of the parasite may be exploited as potential targets for new antimalarial drug discovery research. Reversible ph...
Tuikue Ndam, Nicaise G; Salanti, Ali; Bertin, Gwladys
Plasmodium falciparum parasites that bind to chondroitin sulphate A (CSA) express unique variant surface antigens that are involved in the placental sequestration that precipitates pregnancy-associated malaria (PAM). Two var gene subfamilies, var1csa and var2csa, have been associated with CSA bin...
Wang, C.W.; Mwakalinga, S.B.; Sutherland, C.J.; Schwank, S.; Sharp, S.; Hermsen, C.C.; Sauerwein, R.W.; Theander, T.G.; Lavstsen, T.
BACKGROUND: The Plasmodium falciparum parasite is transmitted in its sexual gametocyte stage from man to mosquito and as asexual sporozoites from mosquito to man. Developing gametocytes sequester preferentially in the bone marrow, but mature stage gametocytes are released to the bloodstream. Sexual
There is substantial immuno-epidemiological evidence that the parasite-encoded, so-called variant surface antigens (VSAs) such as PfEMP1 on the surface of infected erythrocytes (IEs) are important-in some cases probably decisive-determinants of clinical outcome of P. falciparum malaria. The evide...
Thien, Huynh V.; Kager, Piet A.; Sauerwein, Hans P.
Hypoglycemia is a frequently encountered complication in falciparum malaria that is usually ascribed to increased glucose use and impaired glucose production caused by the inhibition of gluconeogenesis. Here, in light of recent data showing that glucose production and gluconeogenesis are often
The acquisition of substantial anti-malarial protection in people naturally exposed to P. falciparum is often cited as evidence that malaria vaccines can be developed, but is rarely used to guide the development. We are pursuing the development of vaccines based on antigens and immune responses...
Anabwani, G; Canfield, C J; Hutchinson, D B
Malaria is a major cause of pediatric mortality in sub-Saharan Africa. Worldwide estimates of mortality among children with Plasmodium falciparum malaria range from 1 to 2 million deaths per year. Management of malaria is increasingly difficult because of the global spread of drug-resistant strains of P. falciparum. There is an urgent need for safe and effective new therapies to treat multidrug-resistant malaria. This open label, randomized trial compared atovaquone and proguanil hydrochloride with halofantrine for treatment of acute, uncomplicated P. falciparum malaria in children age 3 to 12 years (84 patients per group). Study drug dosages were adjusted by weight (approximately 20 and 8 mg/kg daily for three doses for atovaquone and proguanil hydrochloride and 8 mg/kg every 6 h for three doses for halofantrine). Patients were monitored by serial clinical and laboratory assessments for 28 days after starting treatment. Both regimens were effective (cure rate, 93.8% for atovaquone and proguanil hydrochloride and 90.4% for halofantrine) and produced prompt defervescence. Mean parasite clearance times were 50.2 h for halofantrine and 64.9 h for atovaquone and proguanil hydrochloride. More adverse experiences were reported in children treated with halofantrine (119) than with atovaquone and proguanil hydrochloride (73). In Kenyan children the combination of atovaquone and proguanil hydrochloride has efficacy comparable with that of halofantrine for treatment of acute uncomplicated multidrug-resistant falciparum malaria and is associated with a lower rate of adverse events.
Gallo, Valentina; Schwarzer, Evelin; Rahlfs, Stefan; Schirmer, R. Heiner; van Zwieten, Rob; Roos, Dirk; Arese, Paolo; Becker, Katja
In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions
Loscertales, María-Paz; Owens, Stephen; O'Donnell, James; Bunn, James; Bosch-Capblanch, Xavier; Brabin, Bernard J.
Host susceptibility to Plasmodium falciparum infection is central for improved understanding of malaria in human populations. Red blood cell (RBC) polymorphisms have been proposed as factors associated with malaria infection or its severity, although no systematic appraisal of ABO phenotypes and
Pribadi, W; Dakung, L S; Gandahusada, S; Daldyono
A report was made of 4 cases of chloroquine resistant Plasmodium falciparum infections. The infections, detected in Jakarta, were imported from Kotabumi, Tanjung Karang, the Island of Pidada in the Lampung Province and from Pangkalpinang on the Island Bangka in the Province of South Sumatra. Treatment with courses of 1500 mg chloroquine base and with increased dosages up to 2250 mg base failed to cure the patients. The chloroquine sensitivity test in vitro was carried out in 3 patients, which showed that the Plasmodium falciparum strains were resistant to chloroquine at the R I level. The strains appeared to be similar to the Malaya Camp strain. In vivo observations revealed that the parasites were resistant at the R I level with a delayed recrudescence. The chloroquine resistant falciparum malaria cases, acquired in South Sumatra, may therefore be regarded as the first reported cases from a focus outside the already known two foci in Indonesia, namely East Kalimantan and Irian Jaya. It may be expected that chloroquine resistant Plasmodium falciparum will be encountered in other parts of Indonesia in the near future. The use of a combination of sulfadoxine and pyrimethamine should not be recommended in Indonesia because chloroquine is still considered the drug of choice against all malaria infections in Indonesia.
Zhang , Y., Miles, A.P., Chitsaz, F., Saul, A., Long, C.A., Miller, L.H., Stowers, A.W., 2002. In vitro studies with recombinant Plasmodium falciparum...Otsyula, N., Angov, E., Bergmann-Leitner, E., Koech, M., Khan, F., Bennett, J., Otieno, L., Cummings, J., Andagalu, B., Tosh , D., et al., 2013. Results from
Ursing, Johan; Rombo, Lars; Bergqvist, Yngve
to determine the in vivo efficacy of higher chloroquine concentrations against P. falciparum with resistance-conferring genotypes. METHODS: Standard or double-dose chloroquine was given to 892 children aged malaria during 3 clinical trials (2001-2008) with ≥35 days follow...
Charnaud, Sarah C; Dixon, Matthew W A; Nie, Catherine Q; Chappell, Lia; Sanders, Paul R; Nebl, Thomas; Hanssen, Eric; Berriman, Matthew; Chan, Jo-Anne; Blanch, Adam J; Beeson, James G; Rayner, Julian C; Przyborski, Jude M; Tilley, Leann; Crabb, Brendan S; Gilson, Paul R
Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.
Sarah C Charnaud
Full Text Available Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.
Resende, Mafalda; Nielsen, Morten A.; Dahlbaeck, Madeleine
Background: Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistan...
Kemp, Kåre; Kurtzhals, Jørgen; Akanmori, Bartholomew D
Levels of soluble CD30 (sCD30) in serum were elevated in patients with Plasmodium falciparum malaria but showed decline following treatment. The levels of sCD30 in serum were correlated significantly with the expression of gamma interferon by peripheral T cells. These data suggest that CD30...
Larrañaga, Nerea; Mejía, Rosa E; Hormaza, José I; Montoya, Alberto; Soto, Aida; Fontecha, Gustavo A
The Caribbean coast of Central America remains an area of malaria transmission caused by Plasmodium falciparum despite the fact that morbidity has been reduced in recent years. Parasite populations in that region show interesting characteristics such as chloroquine susceptibility and low mortality rates. Genetic structure and diversity of P. falciparum populations in the Honduras-Nicaragua border were analysed in this study. Seven neutral microsatellite loci were analysed in 110 P. falciparum isolates from endemic areas of Honduras (n = 77) and Nicaragua (n = 33), mostly from the border region called the Moskitia. Several analyses concerning the genetic diversity, linkage disequilibrium, population structure, molecular variance, and haplotype clustering were conducted. There was a low level of genetic diversity in P. falciparum populations from Honduras and Nicaragua. Expected heterozigosity (H(e)) results were similarly low for both populations. A moderate differentiation was revealed by the F(ST) index between both populations, and two putative clusters were defined through a structure analysis. The main cluster grouped most of samples from Honduras and Nicaragua, while the second cluster was smaller and included all the samples from the Siuna community in Nicaragua. This result could partially explain the stronger linkage disequilibrium (LD) in the parasite population from that country. These findings are congruent with the decreasing rates of malaria endemicity in Central America.
Full Text Available The clinical outcome of falciparum malaria in endemic areas is influenced by erythrocyte polymorphisms including the ABO blood groups. Studies have reported association of ABO blood group to resistance, susceptibility, and severity of P. falciparum malaria infection. Individuals with blood group “A” have been found to be highly susceptible to falciparum malaria whereas blood group “O” is said to confer protection against complicated cases. We analyzed samples from 293 young children less than six years old with malaria in the Korle-Bu Teaching Hospital in Accra, Ghana. It was observed that group O was present in about 16.1% of complicated cases weighed against 40.9% of uncomplicated controls. Individuals with complicated malaria were about twice likely to be of blood groups A and B compared to group O (A versus O, OR = 1.90, 95% CI = 1.59–2.26, P<0.0001; B versus O, OR = 1.82. 95% CI = 1.57–2.23, P<0.0001. Blood group O participants with complicated diseases had low parasitaemia compared to the other blood groups (P<0.0001. This may give blood group O individuals a survival advantage over the other groups in complicated malaria as suggested. Participants with complicated falciparum malaria were generally anaemic and younger than those with uncomplicated disease.
Carvalho Leonardo JM
Full Text Available Aotus is one of the WHO-recommended primate models for studies in malaria, and several species can be infected with Plasmodium falciparum or P. vivax. Here we describe the successful infection of the species A. infulatus from eastern Amazon with blood stages of P. falciparum. Both intact and splenectomized animals were susceptible to infection; the intact ones were able to keep parasitemias at lower levels for several days, but developed complications such as severe anemia; splenectomized monkeys developed higher parasitemias but no major complications. We conclude that A. infulatus is susceptible to P. falciparum infection and may represent an alternative model for studies in malaria.
Wang, Christian W; Hermsen, Cornelus C; Sauerwein, Robert W
transcript distribution of var genes in a P. falciparum-infected non-immune individual and show that the initial expression of PfEMP1 is based on a strategy that allows all or most variants of PfEMP1s to be expressed by the parasite population at the onset of the blood stage infection.......The var genes encode a family of adhesion receptor proteins, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which profoundly influence malaria pathogenesis. Only a single var gene is transcribed and one PfEMP1 expressed per P.falciparum parasite. Here we present the in vivo...
Full Text Available Abstract Background Erythrocyte invasion by Plasmodium falciparum is a complex process that involves two families; Erythrocyte Binding-Like (EBL and the Reticulocyte Binding-Like (PfRh proteins. Antibodies that inhibit merozoite attachment and invasion are believed to be important in mediating naturally acquired immunity and immunity generated by parasite blood stage vaccine candidates. The hypotheses tested in this study were 1 that antibody responses against specific P. falciparum invasion ligands (EBL and PfRh differ between symptomatic and asymptomatic individuals living in the low-transmission region of the Peruvian Amazon and 2, such antibody responses might have an association, either direct or indirect, with clinical immunity observed in asymptomatically parasitaemic individuals. Methods ELISA was used to assess antibody responses (IgG, IgG1 and IgG3 against recombinant P. falciparum invasion ligands of the EBL (EBA-175, EBA-181, EBA-140 and PfRh families (PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5 in 45 individuals infected with P. falciparum from Peruvian Amazon. Individuals were classified as having symptomatic malaria (N=37 or asymptomatic infection (N=8. Results Antibody responses against both EBL and PfRh family proteins were significantly higher in asymptomatic compared to symptomatic individuals, demonstrating an association with clinical immunity. Significant differences in the total IgG responses were observed with EBA-175, EBA-181, PfRh2b, and MSP119 (as a control. IgG1 responses against EBA-181, PfRh2a and PfRh2b were significantly higher in the asymptomatic individuals. Total IgG antibody responses against PfRh1, PfRh2a, PfRh2b, PfRh5, EBA-175, EBA-181 and MSP119 proteins were negatively correlated with level of parasitaemia. IgG1 responses against EBA-181, PfRh2a and PfRh2b and IgG3 response for PfRh2a were also negatively correlated with parasitaemia. Conclusions These data suggest that falciparum malaria patients who develop
Zani, Babalwa; Gathu, Michael; Donegan, Sarah; Olliaro, Piero L; Sinclair, David
Background The World Health Organization (WHO) recommends Artemisinin-based Combination Therapy (ACT) for treating uncomplicated Plasmodium falciparum malaria. This review aims to assist the decision-making of malaria control programmes by providing an overview of the relative effects of dihydroartemisinin-piperaquine (DHA-P) versus other recommended ACTs. Objectives To evaluate the effectiveness and safety of DHA-P compared to other ACTs for treating uncomplicated P. falciparum malaria in adults and children. Search methods We searched the Cochrane Infectious Diseases Group Specialized Register; the Cochrane Central Register of Controlled Trials (CENTRAL) published in The Cochrane Library; MEDLINE; EMBASE; LILACS, and the metaRegister of Controlled Trials (mRCT) up to July 2013. Selection criteria Randomized controlled trials comparing a three-day course of DHA-P to a three-day course of an alternative WHO recommended ACT in uncomplicated P. falciparum malaria. Data collection and analysis Two authors independently assessed trials for eligibility and risk of bias, and extracted data. We analysed primary outcomes in line with the WHO 'Protocol for assessing and monitoring antimalarial drug efficacy’ and compared drugs using risk ratios (RR) and 95% confidence intervals (CI). Secondary outcomes were effects on gametocytes, haemoglobin, and adverse events. We assessed the quality of evidence using the GRADE approach. Main results We included 27 trials, enrolling 16,382 adults and children, and conducted between 2002 and 2010. Most trials excluded infants aged less than six months and pregnant women. DHA-P versus artemether-lumefantrine In Africa, over 28 days follow-up, DHA-P is superior to artemether-lumefantrine at preventing further parasitaemia (PCR-unadjusted treatment failure: RR 0.34, 95% CI 0.30 to 0.39, nine trials, 6200 participants, high quality evidence), and although PCR-adjusted treatment failure was below 5% for both ACTs, it was consistently lower
Molecular epidemiology of malaria in Cameroon. XXX. sequence analysis of Plasmodium falciparum ATPase 6, dihydrofolate reductase, and dihydropteroate synthase resistance markers in clinical isolates from children treated with an artesunate-sulfadoxine-pyrimethamine combination.
Menemedengue, Virginie; Sahnouni, Khalifa; Basco, Leonardo; Tahar, Rachida
Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are reliable molecular markers for antifolate resistance. The P. falciparum ATPase 6 (pfatp6) gene has been proposed to be a potential marker for artemisinin resistance. In our previous clinical study, we showed that artesunate-sulfadoxine-pyrimethamine is highly effective against uncomplicated malaria in Yaoundé, Cameroon. In the present study, dhfr, dhps, and pfatp6 mutations in P. falciparum isolates obtained from children treated with artesunate-sulfadoxine-pyrimethamine were determined. All 61 isolates had wild-type Pfatp6 263, 623, and 769 alleles, and 11 (18%) had a single E431K substitution. Three additional mutations, E643Q, E432K, and E641Q, were detected. The results did not indicate any warning signal of serious concern (i.e., no parasites were seen with quintuple dhfr-dhps, DHFR Ile164Leu, or pfatp6 mutations), as confirmed by the high clinical efficacy of artesunate-sulfadoxine-pyrimethamine. Further studies are required to identify a molecular marker that reliably predicts artemisinin resistance.
Michelle R. Sanford
Full Text Available Presence of Plasmodium falciparum circumsporozoite protein (CSP was detected by enzyme linked immunosorbent assay (ELISA in a sample of Anopheles gambiae s.s., A. melas and A. pharoensis collected in Guinea-Bissau during October and November 2009. The percentage of P. falciparum infected samples (10.2% overall; confidence interval (CI: 7.45-13.6% was comparable to earlier studies from other sites in Guinea-Bissau (9.6-12.4%. The majority of the specimens collected were identified as A. gambiae which had an individual infection rate of 12.6 % (CI: 8.88-17.6 across collection sites. A small number of specimens of A. coluzzii, A. coluzzii x A. gambiae hybrids, A. melas and A. pharoensis were collected and had infection rates of 4.3% (CI:0.98-12.4, 4.1% (CI:0.35-14.5, 11.1% (CI:1.86-34.1 and 33.3% (CI:9.25-70.4 respectively. Despite being present in low numbers in indoor collections, the exophilic feeding behaviors of A. melas (N=18 and A. pharoensis (N=6 and high infection rates observed in this survey suggest falciparum-malaria transmission potential outside of the protection of bed nets.
Full Text Available Maternal parasitoses modulate fetal immune development, manifesting as altered cellular immunological activity in cord blood that may be linked to enhanced susceptibility to infections in early life. Plasmodium falciparum typifies such infections, with distinct placental infection-related changes in cord blood exemplified by expanded populations of parasite antigen-specific regulatory T cells. Here we addressed whether such early-onset cellular immunological alterations persist through infancy. Specifically, in order to assess the potential impacts of P. falciparum infections either during pregnancy or during infancy, we quantified lymphocyte subsets in cord blood and in infants' peripheral blood during the first year of life. The principal age-related changes observed, independent of infection status, concerned decreases in the frequencies of CD4+, NKdim and NKT cells, whilst CD8+, Treg and Teff cells' frequencies increased from birth to 12 months of age. P. falciparum infections present at delivery, but not those earlier in gestation, were associated with increased frequencies of Treg and CD8+ T cells but fewer CD4+ and NKT cells during infancy, thus accentuating the observed age-related patterns. Overall, P. falciparum infections arising during infancy were associated with a reversal of the trends associated with maternal infection i.e. with more CD4+ cells, with fewer Treg and CD8+ cells. We conclude that maternal P. falciparum infection at delivery has significant and, in some cases, year-long effects on the composition of infants' peripheral blood lymphocyte populations. Those effects are superimposed on separate and independent age- as well as infant infection-related alterations that, respectively, either match or run counter to them.
Raquel M Gonçalves
Full Text Available BACKGROUND: The mechanisms by which humans regulate pro- and anti-inflammatory responses on exposure to different malaria parasites remains unclear. Although Plasmodium vivax usually causes a relatively benign disease, this parasite has been suggested to elicit more host inflammation per parasitized red blood cell than P. falciparum. METHODOLOGY/PRINCIPAL FINDINGS: We measured plasma concentrations of seven cytokines and two soluble tumor necrosis factor (TNF-α receptors, and evaluated clinical and laboratory outcomes, in Brazilians with acute uncomplicated infections with P. vivax (n = 85, P. falciparum (n = 30, or both species (n = 12, and in 45 asymptomatic carriers of low-density P. vivax infection. Symptomatic vivax malaria patients, compared to those infected with P. falciparum or both species, had more intense paroxysms, but they had no clear association with a pro-inflammatory imbalance. To the contrary, these patients had higher levels of the regulatory cytokine interleukin (IL-10, which correlated positively with parasite density, and elevated IL-10/TNF-α, IL-10/interferon (IFN-γ, IL-10/IL-6 and sTNFRII/TNF-α ratios, compared to falciparum or mixed-species malaria patient groups. Vivax malaria patients had the highest levels of circulating soluble TNF-α receptor sTNFRII. Levels of regulatory cytokines returned to normal values 28 days after P. vivax clearance following chemotherapy. Finally, asymptomatic carriers of low P. vivax parasitemias had substantially lower levels of both inflammatory and regulatory cytokines than did patients with clinical malaria due to either species. CONCLUSIONS: Controlling fast-multiplying P. falciparum blood stages requires a strong inflammatory response to prevent fulminant infections, while reducing inflammation-related tissue damage with early regulatory cytokine responses may be a more cost-effective strategy in infections with the less virulent P. vivax parasite. The early induction
Full Text Available We assessed the prevalence of Plasmodium falciparum and the frequency of the dhfr triple mutation that is associated with antifolate drug resistance among P. falciparumisolates obtained from pregnant women in Ilorin, Nigeria. The study included 179 women in the second and third trimester of pregnancy who have been exposed to intermittent preventive treatment in pregnancy (IPTp with sulfadoxinepyrimethamine. Thick and thin blood films and PCR were used for malaria parasite detection. Blood group and hemoglobin concentration were also determined. Mutations in P. falciparum dhfr were analyzed by sequencing DNA obtained from blood spots on filter paper. Prevalence of P. falciparum in the population (PCR corrected was 44.1% (79/179 with 66.7% and 33.3% in the second and third trimester, respectively. Primigravide (51.3% were more infected than multigravide (48.7% but the difference was not statistically significant. Women in blood group A had the highest P. falciparum malaria infection (30.8%. The mean hemoglobin concentration was lower among those infected with malaria parasite. Also, more women with the malaria parasite (38.4% had anemia compare to those without (21.4%. The prevalence of the P. falciparum dhfr mutant alleles was 64.1%, 61.5%, 38.5%, and 12.8% for I51, R59, N108 and T108, respectively. None of the samples had the L164 mutation. The combined triple dhfr mutation (51 + 59 + 108 in the population was 17.9% (7 of 39. Also, the prevalence of the triple mutant alleles was not significantly associated to the number of doses of SP taken by the women. These findings highlight the need for a regular assessment of IPTp/SP efficacy, and evaluation of possible alternative drugs.
Tanabe, Kazuyuki; Zollner, Gabriela; Vaughan, Jefferson A; Sattabongkot, Jetsumon; Khuntirat, Benjawan; Honma, Hajime; Mita, Toshihiro; Tsuboi, Takafumi; Coleman, Russell
Genetic diversity of Plasmodium falciparum is intimately associated with morbidity, mortality and malaria control strategies. It is therefore imperative to study genetic makeup and population structure of this parasite in endemic areas. In Kong Mong Tha, an isolated village in western Thailand, the majority of P. falciparum infections are asymptomatic. In this study we investigated complexity of infections and single nucleotide polymorphisms (SNPs) in the P. falciparum population of Kong Mong Tha, and compared results with those previously obtained from Mae Sod, in northwestern Thailand, where the majority of infections were symptomatic. Using PCR-based determination of the 5' merozoite surface protein 1 gene (msp1) recombinant types, we found that 39% of 59 P. falciparum isolates from Kong Mong Tha had multiple 5' recombinant types with a mean number of 1.54. These values were much lower than those obtained from Mae Sod: 96% for multiple infections and with a mean number of 3.61. Analysis of full-length sequences of two housekeeping genes, the P-type Ca(2+)-transporting ATPase gene (n=33) plus adenylosuccinate lyase gene (n=33), and three vaccine candidate antigen genes, msp1 (n=26), the circumsporozoite protein gene, csp (n=30) and the apical membrane antigen 1 gene, ama 1 (n=32), revealed that in all of these genes within-population SNP diversity was at similar levels between Kong Mong Tha and Mae Sod, suggesting that the extent of MOI and clinical manifestations of malaria are not strongly associated with genetic diversity. Additionally, we did not detect significant genetic differentiation between the two parasite populations, as estimated by the Wright's fixation index of inter-population variance in allele frequencies, suggesting that gene flow prevented the formation of population structuring. Thus, this study highlights unique features of P. falciparum populations in Thailand. The implications of these finding are discussed. © 2013.
Forni, Diego; Pontremoli, Chiara; Cagliani, Rachele; Pozzoli, Uberto; Clerici, Mario; Sironi, Manuela
Plasmodium falciparum, the causative agent of the deadliest form of malaria, is a member of the Laverania subgenus, which includes ape-infecting parasites. P. falciparum is thought to have originated in gorillas, although infection is now restricted to humans. Laverania parasites display remarkable host-specificity, which is partially mediated by the interaction between parasite ligands and host receptors. We analyse the evolution of BSG (basigin) and GYPA (glycophorin A) in primates/hominins, as well as of their Plasmodium-encoded ligands, PfRH5 and PfEBA175. We show that, in primates, positive selection targeted two sites in BSG (F27 and H102), both involved in PfRH5 binding. A population genetics-phylogenetics approach detected the strongest selection for the gorilla lineage: one of the positively selected sites (K191) is a major determinant of PfRH5 binding affinity. Analysis of RH5 genes indicated episodic selection on the P. falciparum branch; the positively selected W447 site is known to stabilize the interaction with human basigin. Conversely, we detect no selection in the receptor-binding region of EBA175 in the P. falciparum lineage. Its host receptor, GYPA, shows evidence of positive selection in all hominid lineages; selected codons include glycosylation sites that modulate PfEBA175 binding affinity. Data herein provide an evolutionary explanation for species-specific binding of the PfRH5-BSG ligand-receptor pair and support the hypothesis that positive selection at these genes drove the host shift leading to the emergence of P. falciparum as a human pathogen. © 2015 John Wiley & Sons Ltd.
Potipitak, Tiparat; Ngrenngarmlert, Warunee; Promptmas, Chamras; Chomean, Sirinart; Ittarat, Wanida
Malaria infection with Plasmodium falciparum is an important basic health problem in the tropical and sub-tropical countries. The standard diagnostic method is blood film examination to visualize parasite morphology. However, in cases of low parasitemia or mixed infection with very low cryptic species, microscopy is not sensitive enough. Therefore, molecular techniques have been widely employed. A label-free DNA biosensor based on quartz crystal microbalance (QCM) to diagnose and genotype P. falciparum was developed. Avidin-biotin interaction was used to coat the specific biotinylated probe on the gold surface of QCM. The gene encoding merozoite surface protein 2 (msp2) was amplified and the PCR products were then cut with restriction enzyme (MwoI). Enzymatic cutting made the PCR products suitable for QCM development. Hybridization between probe and enzymatic cutting DNA fragments resulted in frequency changes of the QCM. The newly developed QCM was tested for its diagnosis ability using both malaria laboratory strains and clinical isolates. The biosensor was sensitive at the sub-nanogram level, specific for only P. falciparum detection, no cross-reaction with P. vivax, and stable at room temperature for up to 6 months. Selection of msp2 as a target gene and a geno-typing marker made the QCM potentially useful for falciparum diagnosis simultaneously with genotyping. Potency was tested by genotyping two allelic families of P. falciparum, FC27 and IC1, using malaria laboratory strains, K1 and 3D7, respectively. The dual function QCM was successfully developed with high sensitivity and specificity, and was cost-effective, stable and field adaptable.
Lasonder, Edwin; Janse, Chris J; van Gemert, Geert-Jan
Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature...... whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed...... three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may...
Full Text Available Zanthoxylum chiloperone var. angustifolium Engl., Rutaceae, is used in traditional medicine to treat fungal and protozoal infections in the central area of South America. Considering the increasing resistance of Plasmodium falciparum in malarial ridden areas, we explored the anti-plasmodial effects of three compounds isolated from Z. chiloperone. The pyranocoumarin transavicennol and the canthinone alkaloids, canthin-6-one and 5-methoxycanthin-6-one, were found to have IC50 on chloroquine/mefloquine resistant and sensitive strains of P. falciparum of 0.5-2.7, 2.0-5.3 and 5.1-10.4 ƒÊg/mL, respectively. Moreover, the formation of heme adducts by these compounds is described by a novel alternative method based on MS-CID methods. The alkylamide sanshool was also identified, for first time in this plant, in the dichloromethanic and ethanolic extracts and the extracts were found to be notably non-toxic and displayed good anti-plasmodial effects.
Serge Brice Assi
Full Text Available The objective of this study was to monitor the effectiveness of artesunate-amodiaquine fixed-dose combination tablets (ASAQ Winthrop® in the treatment of uncomplicated Plasmodium falciparum malaria in Côte d’Ivoire. Two enrolment periods (November 2009 to May 2010 and March to October 2013 were compared using an identical design. Subjects with proven monospecific P. falciparum infection according to the WHO diagnostic criteria were eligible. 290 patients during each period received a dose of ASAQ Winthrop tablets appropriate for their age. The primary outcome measure was PCR-corrected adequate clinical and parasitological response at Day 28 in the per protocol population (255 in Period 1 and 240 in Period 2. This was achieved by 95.7% of patients during Period 1 and 96.3% during Period 2. Over 95% of patients were afebrile at Day 3 and complete parasite clearance was achieved at Day 3 in >99% of patients. Nineteen adverse events in nineteen patients were considered as possibly related to treatment, principally vomiting, abnormal liver function tests, and pruritus. There was no evidence for loss of effectiveness over the three-year period in spite of strong drug pressure. This trial was registered in the US Clinical Trials Registry (clinical.trials.gov under the identifier number NCT01023399.
Llanos-Chea, Fiorella; Martínez, Dalila; Rosas, Angel; Samalvides, Frine; Vinetz, Joseph M; Llanos-Cuentas, Alejandro
Severe Plasmodium falciparum malaria is uncommon in South America. Lima, Peru, while not endemic for malaria, is home to specialized centers for infectious diseases that admit and manage patients with severe malaria (SM), all of whom contracted infection during travel. This retrospective study describes severe travel-related malaria in individuals admitted to one tertiary care referral hospital in Lima, Peru; severity was classified based on criteria published by the World Health Organization in 2000. Data were abstracted from medical records of patients with SM admitted to Hospital Nacional Cayetano Heredia from 2006 to 2011. Of 33 SM cases with complete clinical data, the mean age was 39 years and the male/female ratio was 2.8. Most cases were contracted in known endemic regions within Peru: Amazonia (47%), the central jungle (18%), and the northern coast (12%); cases were also found in five (15%) travelers returning from Africa. Plasmodium vivax was most commonly identified (71%) among the severe infections, followed by P. falciparum (18%); mixed infections composed 11% of the group. Among the criteria of severity, jaundice was most common (58%), followed by severe thrombocytopenia (47%), hyperpyrexia (32%), and shock (15%). Plasmodium vivax mono-infection predominated as the etiology of SM in cases acquired in Peru. © The American Society of Tropical Medicine and Hygiene.
Jakobsen, P H; Rasheed, F N; Bulmer, J N
falciparum protein, glutamate-rich protein (GLURP) and antibodies to P. falciparum rhoptry-associated protein-1 were measured among 105 Gambian women and their neonates. Peripheral blood concentrations of IL-10, soluble cytokine receptors and soluble adhesion molecules were found to be different from those...... as an immunoprivileged site. Concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intracellular adhesion molecule-1 (sICAM-1), potential adhesion receptors for malaria parasites, were associated with an active P. falciparum infection in the placenta although the associations did not reach...... significance. P. falciparum exoantigen, GLURP, was detected in cord blood indicating transplacental passage of malarial antigens. Concentrations of E-selectin were higher in cord blood samples compared with peripheral blood samples. This appeared to be associated with development of cord endothelial cells...
Full Text Available Plasmodium falciparum secretes a homologue of the translationally controlled tumor protein (TCTP into serum of infected individuals, although its role in pathogenesis or virulence is unknown. To determine the effect of P. falciparum TCTP on B cells as compared to human TCTP, fluorescently labeled proteins were incubated on primary cultures of mouse splenic B cells and analyzed by flow cytometry and confocal microscopy. Our results indicate that both recombinant proteins are incorporated into B cells, but differ significantly in their rate and percentage of incorporation, being significantly higher for P. falciparum TCTP. Furthermore, P. falciparum TCTP showed a lower B cell proliferative effect than human TCTP, suggesting a mechanism through which the former could interfere in the host's immune response.
Murungi, Linda M; Sondén, Klara; Llewellyn, David
Severe malaria (SM) is a life-threatening complication of infection with Plasmodium falciparum Epidemiological observations have long indicated that immunity against SM is acquired relatively rapidly, but prospective studies to investigate its immunological basis are logistically challenging and ...
Staalsoe, T; Giha, H A; Dodoo, D
BACKGROUND: Naturally induced antibodies binding to surface antigens of Plasmodium falciparum-infected erythrocytes can be detected by direct agglutination of infected erythrocytes or by indirect immunofluorescence on intact, unfixed, infected erythrocytes. Agglutinating antibodies have previously...
Mekonnen, Seleshi Kebede; Aseffa, Abraham; Berhe, Nega; Teklehaymanot, Tilahun; Clouse, Ronald M; Gebru, Tamirat; Medhin, Girmay; Velavan, Thirumalaisamy P
Increased resistance by Plasmodium falciparum parasites led to the withdrawal of the antimalarial drugs chloroquine and sulphadoxine-pyrimethamine in Ethiopia. Since 2004 artemether-lumefantrine has served to treat uncomplicated P. falciparum malaria. However, increasing reports on delayed parasite clearance to artemisinin opens up a new challenge in anti-malarial therapy. With the complete withdrawal of CQ for the treatment of Plasmodium falciparum malaria, this study assessed the evolution of CQ resistance by investigating the prevalence of mutant alleles in the pfmdr1 and pfcrt genes in P. falciparum and pvmdr1 gene in Plasmodium vivax in Southern and Eastern Ethiopia. Of the 1,416 febrile patients attending primary health facilities in Southern Ethiopia, 329 febrile patients positive for P. falciparum or P. vivax were recruited. Similarly of the 1,304 febrile patients from Eastern Ethiopia, 81 febrile patients positive for P. falciparum or P. vivax were included in the study. Of the 410 finger prick blood samples collected from malaria patients, we used direct sequencing to investigate the prevalence of mutations in pfcrt and pfmdr1. This included determining the gene copy number in pfmdr1 in 195 P. falciparum clinical isolates, and mutations in the pvmdr1 locus in 215 P. vivax clinical isolates. The pfcrt K76 CQ-sensitive allele was observed in 84.1% of the investigated P.falciparum clinical isolates. The pfcrt double mutations (K76T and C72S) were observed less than 3%. The pfcrt SVMNT haplotype was also found to be present in clinical isolates from Ethiopia. The pfcrt CVMNK-sensitive haplotypes were frequently observed (95.9%). The pfmdr1 mutation N86Y was observed only in 14.9% compared to 85.1% of the clinical isolates that carried sensitive alleles. Also, the sensitive pfmdr1 Y184 allele was more common, in 94.9% of clinical isolates. None of the investigated P. falciparum clinical isolates carried S1034C, N1042D and D1246Y pfmdr1 polymorphisms. All
Die vorliegende Arbeit beschreibt die Aktivität von Chloroquin und seinen Derivaten Amodiaquin und Ferroquin, sowie Artesunat, Chinin und Mefloquin gegen Plasmodium falciparum Isolate in Lambaréné (Stand 2010). Ein besonderer Augenmerk wurde auf das Chloroquinderivat Ferroquin gelegt, welches sich – in Kombination mit Artesunat – in der Phase 2 der klinischen Forschung befindet. Ziel dieser Arbeit ist die Erhebung des aktuellen Resistenzstatus der genannten Wirkstoffe bei P. falciparum...
Noviyanti, Rintis; Coutrier, Farah; Utami, Retno A S; Trimarsanto, Hidayat; Tirta, Yusrifar K; Trianty, Leily; Kusuma, Andreas; Sutanto, Inge; Kosasih, Ayleen; Kusriastuti, Rita; Hawley, William A; Laihad, Ferdinand; Lobo, Neil; Marfurt, Jutta; Clark, Taane G; Price, Ric N; Auburn, Sarah
Outside of Africa, P. falciparum and P. vivax usually coexist. In such co-endemic regions, successful malaria control programs have a greater impact on reducing falciparum malaria, resulting in P. vivax becoming the predominant species of infection. Adding to the challenges of elimination, the dormant liver stage complicates efforts to monitor the impact of ongoing interventions against P. vivax. We investigated molecular approaches to inform the respective transmission dynamics of P. falciparum and P. vivax and how these could help to prioritize public health interventions. Genotype data generated at 8 and 9 microsatellite loci were analysed in 168 P. falciparum and 166 P. vivax isolates, respectively, from four co-endemic sites in Indonesia (Bangka, Kalimantan, Sumba and West Timor). Measures of diversity, linkage disequilibrium (LD) and population structure were used to gauge the transmission dynamics of each species in each setting. Marked differences were observed in the diversity and population structure of P. vivax versus P. falciparum. In Bangka, Kalimantan and Timor, P. falciparum diversity was low, and LD patterns were consistent with unstable, epidemic transmission, amenable to targeted intervention. In contrast, P. vivax diversity was higher and transmission appeared more stable. Population differentiation was lower in P. vivax versus P. falciparum, suggesting that the hypnozoite reservoir might play an important role in sustaining local transmission and facilitating the spread of P. vivax infections in different endemic settings. P. vivax polyclonality varied with local endemicity, demonstrating potential utility in informing on transmission intensity in this species. Molecular approaches can provide important information on malaria transmission that is not readily available from traditional epidemiological measures. Elucidation of the transmission dynamics circulating in a given setting will have a major role in prioritising malaria control strategies
Full Text Available Outside of Africa, P. falciparum and P. vivax usually coexist. In such co-endemic regions, successful malaria control programs have a greater impact on reducing falciparum malaria, resulting in P. vivax becoming the predominant species of infection. Adding to the challenges of elimination, the dormant liver stage complicates efforts to monitor the impact of ongoing interventions against P. vivax. We investigated molecular approaches to inform the respective transmission dynamics of P. falciparum and P. vivax and how these could help to prioritize public health interventions.Genotype data generated at 8 and 9 microsatellite loci were analysed in 168 P. falciparum and 166 P. vivax isolates, respectively, from four co-endemic sites in Indonesia (Bangka, Kalimantan, Sumba and West Timor. Measures of diversity, linkage disequilibrium (LD and population structure were used to gauge the transmission dynamics of each species in each setting. Marked differences were observed in the diversity and population structure of P. vivax versus P. falciparum. In Bangka, Kalimantan and Timor, P. falciparum diversity was low, and LD patterns were consistent with unstable, epidemic transmission, amenable to targeted intervention. In contrast, P. vivax diversity was higher and transmission appeared more stable. Population differentiation was lower in P. vivax versus P. falciparum, suggesting that the hypnozoite reservoir might play an important role in sustaining local transmission and facilitating the spread of P. vivax infections in different endemic settings. P. vivax polyclonality varied with local endemicity, demonstrating potential utility in informing on transmission intensity in this species.Molecular approaches can provide important information on malaria transmission that is not readily available from traditional epidemiological measures. Elucidation of the transmission dynamics circulating in a given setting will have a major role in prioritising malaria
Jakobsen, P H; Jepsen, S; Riley, E M
The soluble amphiphilic glycoprotein, Ag1 (gp60), purified from supernatants of in vitro cultures of Plasmodium falciparum has a molecular mass of 60 kDa and did not exhibit size variation in the different P. falciparum isolates tested by immunoblotting. Ag1 was shown to interact with the lectin...... from six different malaria-endemic regions. Ag1 induces in vitro proliferation of lymphocytes from malaria-immune individuals in an antigen-specific manner....
Kerkhof, Karen; Sluydts, Vincent; Heng, Somony; Kim, Saorin; Pareyn, Myrthe; Willen, Laura; Canier, Lydie; Sovannaroth, Siv; Ménard, Didier; Sochantha, Tho; Coosemans, Marc; Durnez, Lies
Malaria transmission is highly heterogeneous, especially in low endemic countries, such as Cambodia. This results in geographical clusters of residual transmission in the dry, low transmission season, which can fuel the transmission to wider areas or populations during the wet season. A better understanding of spatial clustering of malaria can lead to a more efficient, targeted strategy to reduce malaria transmission. This study aims to evaluate the potential of the use of serological markers to define spatial patterns in malaria exposure. Blood samples collected in a community-based randomized trial performed in 98 high endemic communities in Ratanakiri province, north-eastern Cambodia, were screened with a multiplex serological assay for five serological markers (three Plasmodium falciparum and two Plasmodium vivax). The antibody half-lives range from approximately six months until more than two years. Geographical heterogeneity in malaria transmission was examined using a spatial scan statistic on serology, PCR prevalence and malaria incidence rate data. Furthermore, to identify behavioural patterns or intrinsic factors associated with malaria exposure (antibody levels), risk factor analyses were performed by using multivariable random effect logistic regression models. The serological outcomes were then compared to PCR prevalence and malaria incidence data. A total of 6502 samples from two surveys were screened in an area where the average parasite prevalence estimated by PCR among the selected villages is 3.4 %. High-risk malaria pockets were observed adjacent to the 'Tonle San River' and neighbouring Vietnam for all three sets of data (serology, PCR prevalence and malaria incidence rates). The main risk factors for all P. falciparum antigens and P. vivax MSP1.19 are age, ethnicity and staying overnight at the plot hut. It is possible to identify similar malaria pockets of higher malaria transmission together with the potential risk factors by using serology
Munigunti, Ranjith; Nelson, Nicholas; Mulabagal, Vanisree; Gupta, Mahabir P; Brun, Reto; Calderón, Angela I
Our current research on applications of mass spectrometry to natural product drug discovery against malaria aims to screen plant extracts for new ligands to Plasmodium falciparum thioredoxin reductase (PfTrxR) followed by their identification and structure elucidation. PfTrxR is involved in the antioxidant defense and redox regulation of the parasite and is validated as a promising target for therapeutic intervention against malaria. In the present study, detannified methanol extracts from Guatteria recurvisepala, Licania kallunkiae, and Topobea watsonii were screened for ligands to PfTrxR using ultrafiltration and liquid chromatography/mass spectrometry-based binding experiments. The PfTrxR ligand identified in the extract of Guatteria recurvisepala displayed a relative binding affinity of 3.5-fold when incubated with 1 μM PfTrxR. The ligand corresponding to the protonated molecule m/z 282.2792 [M+ H]+ was eluted at a retention time of 17.95 min in a 20-min gradient of 95% B consisting of (A) 0.1%formic acid in 95% H₂O-5% ACN, and (B) 0.1% formic acid in 95% ACN-5% H₂O in an LC-QTOF-MS.Tandem MS of the protonated molecule m/z 282.2792 [M + H]+, C₁₈H₃₆NO (DBE: 2; error: 1.13 ppm) resulted in two daughter ions m/z 265.2516[M + H-NH₃]+ (DBE: 3; error: 0.35 ppm) and m/z 247.2405 [M + H-NH₃-H₂O] +, (DBE: 4; error:2.26 ppm). The PfTrxR ligand was identified as oleamide and confirmed by comparison of the retention time, molecular formula, accurate mass,and double bond equivalence with the standard oleamide. This is the first report on the identification of oleamide as a PfTrxR ligand from Guatteria recurvisepala R. E. Fr. and the corresponding in vitro activity against P. falciparum strain K1 (IC₅₀ 4.29 μg/mL). © Georg Thieme Verlag KG Stuttgart · New York.
Dieudonné Makaba Mvumbi
Full Text Available Malaria is a major public health problem in the Democratic Republic of Congo. Despite progress achieved over the past decade in the fight against malaria, further efforts have to be done such as in the surveillance and the containment of Plasmodium falciparum resistant strains. We investigated resistance to artemisinin-based combination therapies currently in use in Democratic Republic of Congo by surveying molecular polymorphisms in three genes: pfcrt, pfmdr1 and pfk13 to explore possible emergence of amodiaquine, lumefantrine or artemisinin resistance in Democratic Republic of Congo. This study essentially revealed that resistance to chloroquine is still decreasing while polymorphism related to amodiaquine resistance seems to be not present in Democratic Republic of Congo, that three samples, located in the east of the country, harbor Pfmdr1 amplification and that none of the mutations found in South-East Asia correlated with artemisinine resistance have been found in Democratic Republic of Congo. But new mutations have been identified, especially the M476K, occurred in the same position that the M476I previously identified in the F32-ART strain, strongly resistant to artemisinine. Antimalarial first-line treatments currently in use in Democratic Republic of Congo are not associated with emergence of molecular markers of resistance.
Ranjitkar, Samir; Schousboe, Mette L; Thomsen, Thomas
endemic areas. However, SP is still used against P. falciparum infections in low endemic areas while CQ is used in suspected cases in areas with lack of diagnostic facilities. This study examines the prevalence of molecular markers of P. falciparum and Plasmodium vivax CQ and SP resistance to determine...... if high levels of in vivo resistance are reflected at molecular level as well. METHODS: Finger prick blood samples (n=189) were collected from malaria positive patients from two high endemic districts and analysed for single nucleotide polymorphisms (SNPs) in the resistance related genes of P. falciparum...... on a few P. falciparum samples, the molecular level of CQ resistance in P. falciparum was high since nearly all parasites had the Pfcrt mutant haplotypes CVIET (55%) or SVMNT (42%), though frequency of Pfmdr1 wild type haplotype was relatively low (35%). Molecular levels of SP resistance in P. falciparum...
Ana Maria Fonseca
Full Text Available Pregnant women exposed to Plasmodium falciparum generate antibodies against VAR2CSA, the parasite protein that mediates adhesion of infected erythrocytes to the placenta. There is a need of high-throughput tools to determine the fine specificity of these antibodies that can be used to identify immune correlates of protection and exposure. Here we aimed at developing a multiplex-immunoassay to detect antibodies against VAR2CSA antigens.We constructed two multiplex-bead arrays, one composed of 3 VAR2CSA recombinant-domains (DBL3X, DBL5Ɛ and DBL6Ɛ and another composed of 46 new peptides covering VAR2CSA conserved and semi-conserved regions. IgG reactivity was similar in multiplexed and singleplexed determinations (Pearson correlation, protein array: R2 = 0.99 and peptide array: R2 = 0.87. IgG recognition of 25 out of 46 peptides and all recombinant-domains was higher in pregnant Mozambican women (n = 106 than in Mozambican men (n = 102 and Spanish individuals (n = 101; p<0.05. Agreement of IgG levels detected in cryopreserved plasma and in elutions from dried blood spots was good after exclusion of inappropriate filter papers. Under heterogeneous levels of exposure to malaria, similar seropositivity cutoffs were obtained using finite mixture models applied to antibodies measured on pregnant Mozambican women and average of antibodies measured on pregnant Spanish women never exposed to malaria. The application of the multiplex-bead array developed here, allowed the assessment of higher IgG levels and seroprevalences against VAR2CSA-derived antigens in women pregnant during 2003-2005 than during 2010-2012, in accordance with the levels of malaria transmission reported for these years in Mozambique.The multiplex bead-based immunoassay to detect antibodies against selected 25 VAR2CSA new-peptides and recombinant-domains was successfully implemented. Analysis of field samples showed that responses were specific among pregnant women and dependent on the
Adam Mayyada B
Full Text Available Abstract Background Blood platelet levels are being evaluated as predictive and prognostic indicators of the severity of malaria infections in humans. However, there are few studies on platelets and Plasmodium falciparum malaria during pregnancy. Methods A case–control study was conducted at Gadarif Hospital in Eastern Sudan, an area characterized by unstable malaria transmission. The aim of the study was to investigate thrombocytopenia in pregnant women with P. falciparum malaria (cases and healthy pregnant women (controls. Results The median (interquartile platelet counts were significantly lower in patients with malaria (N = 60 than in the controls (N = 60, 61, 000 (43,000–85,000 vs. 249,000 (204,000–300,000/μL, respectively, p P. falciparum malaria (N = 12 compared with those patients with uncomplicated P. falciparum malaria (N = 48, 68, 000 (33,000-88,000/μL vs. 61, 000 (45,000–85,000/μL, respectively, p = 0.8. While none of the control group had thrombocytopenia (platelet count p P. falciparum malaria, compared with the pregnant healthy control group, were at higher risk (OR = 10.1, 95% CI = 4.1–25.18; p Conclusion P. falciparum malaria is associated with thrombocytopenia in pregnant women in this setting. More research is needed.
Sandra L.M. AVILA
Full Text Available The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20oC up to 10 days or at -70oC for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.O objetivo deste estudo foi padronizar variáveis técnicas para o armazenamento de Plasmodium falciparum e de seus componentes antigênicos. Sedimentos de parasitas foram obtidos do cultivo in vitro de P.falciparum e estocados em diferentes temperaturas por diferentes períodos de tempo. De cada variável, foram extraídos os componentes antigênicos com detergente anfótero Zwittergent na presença e na ausência de inibidores de proteases e submetidos ou não a posterior diálise. Os produtos foram estocados por 15, 30 e 60 dias em diferentes temperaturas e caracterizados por SDS-PAGE. A atividade antigênica de cada extrato foi determinada por ELISA e Western blotting usando soros positivos e negativos para anticorpos IgG anti-formas eritrocitárias de P.falciparum. Os extratos antigênicos obtidos de parasitas estocados até 10 dias a _20ºC ou por 2 meses a _70ºC e tratados com inibidores de proteases, sob as
Elbadry, Maha A; Existe, Alexandre; Victor, Yves S; Memnon, Gladys; Fukuda, Mark; Dame, John B; Yowell, Charles A; Okech, Bernard A
In Haiti where chloroquine (CQ) is widely used for malaria treatment, reports of resistance are scarce. However, recent identification of CQ resistance genotypes in one site is suggestive of an emerging problem. Additional studies are needed to evaluate genetic mutations associated with CQ resistance, especially in the Plasmodium falciparum multi-drug resistance-1 gene (pfmdr1) while expanding the already available information on P. falciparum CQ transporter gene (pfcrt) in Haiti. Blood samples were collected on Whatman filter cards (FTA) from eight clinics spread across Haiti. Following the confirmation of P. falciparum in the samples, PCR protocols were used to amplify regions of pfmdr1and pfcrt codons of interest, (86, 184, 1034, 1042, and 1246) and (72-76), respectively. Sequencing and site-specific restriction enzyme digestions were used to analyse these DNA fragments for the presence of single nucleotide polymorphisms (SNPs) known to confer resistance to anti-malarial drugs. P. falciparum infection was confirmed in160 samples by amplifying a segment of the P. falciparum 18S small subunit ribosomal RNA gene (pfssurrna). The sequence of pfmdr1 in 54 of these samples was determined between codons 86,184 codons 1034, 1042 and 1246. No sequence differences from that of the NF54 clone 3D7 were found among the 54 samples except at codon 184, where a non-silent mutation was found in all samples predicted to alter the amino acid sequence replacing tyrosine with phenylalanine (Y184F). This altered sequence was also confirmed by restriction enzyme digestion. The sequence of pfmdr1 at codons 86, 184, 1034 and 1042 encoded the NFSN haplotype. The sequence of pfcrt codons 72-76 from 79 samples was determined and found to encode CVMNK, consistent with a CQ sensitive genotype. The presence of the Y184F mutation in pfmdr1 of P. falciparum parasites in Haiti may have implications for resistance to antimalarial drugs. The absence of mutation in pfcrt at codon 76 among 79
Kang, Jung-Mi; Ju, Hye-Lim; Cho, Pyo Yun; Moon, Sung-Ung; Ahn, Seong Kyu; Sohn, Woon-Mok; Lee, Hyeong-Woo; Kim, Tong-Soo; Na, Byoung-Kuk
The merozoite surface protein-3β of Plasmodium vivax (PvMSP-3β) is one of the candidate antigens for blood stage malaria vaccine development. The polymorphisms in PvMSP-3β have been reported in certain P. vivax isolates. However, the diversity of PvMSP-3β throughout its global distribution has not been well understood. In this study, the genetic diversity and the effects of natural selection in PvMSP-3β among P. vivax Korean isolates were analysed. Blood samples were collected from 95 patients with vivax malaria in Korea. The region flanking full-length PvMSP-3β was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvMSP-3β sequence of each isolate was determined and the polymorphic characteristics and effects of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs. Five different subtypes of PvMSP-3β were identified based on single nucleotide polymorphisms (SNPs), insertions, and deletions. Although a high level of sequence diversity was observed in the PvMSP-3β gene, the coiled-coil tertiary structure of the PvMSP-3β protein was well conserved in all of the sequences. The PvMSP-3β of Korean isolates is under natural selection. DNA polymerase slippage and intragenic recombination likely contributed to PvMSP-3β diversity in Korean P. vivax isolates. The PvMSP-3β of Korean P. vivax isolates displayed polymorphisms, with SNPs, insertions and deletions scattered throughout of the gene. These results of parasite heterogeneity are relevant to the development of a PvMSP-3β based vaccine against P. vivax and the implementation of malaria control programmes in Korea.
Hrycyna, Christine A; Summers, Robert L; Lehane, Adele M; Pires, Marcos M; Namanja, Hilda; Bohn, Kelsey; Kuriakose, Jerrin; Ferdig, Michael; Henrich, Philipp P; Fidock, David A; Kirk, Kiaran; Chmielewski, Jean; Martin, Rowena E
Chloroquine (CQ) resistance in the human malaria parasite Plasmodium falciparum is primarily conferred by mutations in the "chloroquine resistance transporter" (PfCRT). The resistance-conferring form of PfCRT (PfCRT(CQR)) mediates CQ resistance by effluxing the drug from the parasite's digestive vacuole, the acidic compartment in which CQ exerts its antiplasmodial effect. PfCRT(CQR) can also decrease the parasite's susceptibility to other quinoline drugs, including the current antimalarials quinine and amodiaquine. Here we describe interactions between PfCRT(CQR) and a series of dimeric quinine molecules using a Xenopus laevis oocyte system for the heterologous expression of PfCRT and using an assay that detects the drug-associated efflux of H(+) ions from the digestive vacuole in parasites that harbor different forms of PfCRT. The antiplasmodial activities of dimers 1 and 6 were also examined in vitro (against drug-sensitive and drug-resistant strains of P. falciparum) and in vivo (against drug-sensitive P. berghei). Our data reveal that the quinine dimers are the most potent inhibitors of PfCRT(CQR) reported to date. Furthermore, the lead compounds (1 and 6) were not effluxed by PfCRT(CQR) from the digestive vacuole but instead accumulated to very high levels within this organelle. Both 1 and 6 exhibited in vitro antiplasmodial activities that were inversely correlated with CQ. Moreover, the additional parasiticidal effect exerted by 1 and 6 in the drug-resistant parasites was attributable, at least in part, to their ability to inhibit PfCRT(CQR). This highlights the potential for devising new antimalarial therapies that exploit inherent weaknesses in a key resistance mechanism of P. falciparum.
Hrycyna, Christine A.; Summers, Robert L.; Lehane, Adele M.; Pires, Marcos M.; Namanja, Hilda; Bohn, Kelsey; Kuriakose, Jerrin; Ferdig, Michael; Henrich, Philipp P.; Fidock, David A.; Kirk, Kiaran; Chmielewski, Jean; Martin, Rowena E.
Chloroquine (CQ) resistance in the human malaria parasite Plasmodium falciparum is primarily conferred by mutations in the “chloroquine resistance transporter” (PfCRT). The resistance-conferring form of PfCRT (PfCRTCQR) mediates CQ resistance by effluxing the drug from the parasite’s digestive vacuole, the acidic compartment in which CQ exerts its antiplasmodial effect. PfCRTCQR can also decrease the parasite’s susceptibility to other quinoline drugs, including the current antimalarials quinine and amodiaquine. Here we describe interactions between PfCRTCQR and a series of dimeric quinine molecules using a Xenopus laevis oocyte system for the heterologous expression of PfCRT and using an assay that detects the drug-associated efflux of H+ ions from the digestive vacuole in parasites that harbor different forms of PfCRT. The antiplasmodial activities of dimers 1 and 6 were also examined in vitro (against drug-sensitive and drug-resistant strains of P. falciparum) and in vivo (against drug-sensitive P. berghei). Our data reveal that the quinine dimers are the most potent inhibitors of PfCRTCQR reported to date. Furthermore, the lead compounds (1 and 6) were not effluxed by PfCRTCQR from the digestive vacuole but instead accumulated to very high levels within this organelle. Both 1 and 6 exhibited in vitro antiplasmodial activities that were inversely correlated with CQ. Moreover, the additional parasiticidal effect exerted by 1 and 6 in the drug-resistant parasites was attributable, at least in part, to their ability to inhibit PfCRTCQR. This highlights the potential for devising new antimalarial therapies that exploit inherent weaknesses in a key resistance mechanism of P. falciparum. PMID:24369685
Harbarth; Meyer; Grau; Loutan; Ricou
Incidence of falciparum malaria in developed countries has increased in recent years due to tourism to tropical countries and immigration from Asia and Africa. In Switzerland, about 250 cases of malaria were reported in 1994 to the Federal Office of Health, including three cases with fatal outcome.1 The most commonly described complications of plasmodia infection are cerebral malaria, acute renal failure, and severe anemia with disseminated intravascular coagulation. However, pulmonary involvement occurs in 3 to 10% of cases and represents the most serious complication of this infection, with a lethality of 70%.2,3 Furthermore, a pronounced general immunosuppression has been reported in malaria patients, which may predispose them to opportunistic infections.4 We report a case of Plasmodium falciparum infection complicated by severe acute respiratory distress syndrome (ARDS) with development of systemic cytomegalovirus (CMV) infection leading to death. This evolution implies a severe immune deficiency associated with malaria, as previously suggested in the literature.
Lorena M Coronado
Full Text Available The development of resistance to insecticides by the vector of malaria and the increasingly faster appearance of resistance to antimalarial drugs by the parasite can dangerously hamper efforts to control and eradicate the disease. Alternative ways to treat this disease are urgently needed. Here we evaluate the in vitro effect of direct current (DC capacitive coupling electrical stimulation on the biology and viability of Plasmodium falciparum. We designed a system that exposes infected erythrocytes to different capacitively coupled electric fields in order to evaluate their effect on P. falciparum. The effect on growth of the parasite, replication of DNA, mitochondrial membrane potential and level of reactive oxygen species after exposure to electric fields demonstrate that the parasite is biologically able to respond to stimuli from DC electric fields involving calcium signaling pathways.
Full Text Available From which host did the most malignant human malaria come: birds, primates, or rodents? When did the transfer occur? Over the last half century, these have been some of the questions up for debate about the origin of Plasmodium falciparum, the most common and deadliest human malaria parasite, which is responsible for at least one million deaths every year. Recent findings bring elements in favor of a transfer from great apes, but are these evidences really solid? What are the grey areas that remain to be clarified? Here, we examine in depth these new elements and discuss how they modify our perception of the origin and evolution of P. falciparum. We also discuss the perspectives these new discoveries open.
Eltahir, Hatim G; Omer, Abubaker A; Mohamed, Ayoub A; Adam, Ishag
Sixty-six children presenting to Singa hospital, Sudan with different manifestations of severe Plasmodium falciparum malaria were randomly divided into two well-matched groups (33 in each arm) to receive either intravenous artesunate 2·4 mg/kg at 0, 12, and 24 hours, then daily, or intravenous quinine 20mg/kg initially then 10mg/kg three times a day. There was no significant difference in the fever, parasite clearance, and coma resolution times. Three patients died, one in the artesunate and two in the quinine groups. One patient developed hypoglycaemia following quinine infusion. Thus, artesunate can be used for the treatment of severe falciparum malaria. Copyright © 2010 Royal Society of Tropical Medicine and Hygiene.
Chulay, J.D.; Haynes, J.D.; Diggs, C.L.
To evaluate rapidly Plasmodium falciparum growth in Vitro, [ 3 H]hypoxanthine was added to parasite microcultures and radioisotope incorporation was measured. When culture parameters were carefully controlled, [ 3 H]hypoxanthine incorporation was proportional to the number of parasitized erythrocytes present. Factors affecting [ 3 H]hypoxanthine incorporation included initial parasitemia, duration of culture, duration of radioisotope pulse, parasite stage, concentration of uninfected erythrocytes, the use of serum or plasma to supplement growth, and the concentration of a variety of purines in the culture medium. The method described can be used to measure inhibition of P. falciparum growth by immune serum and has previously been used to study antimalarial drug activity in vitro
Ndam, Nicaise Tuikue; Mbuba, Emmanuel; González, Raquel; Cisteró, Pau; Kariuki, Simon; Sevene, Esperança; Rupérez, María; Fonseca, Ana Maria; Vala, Anifa; Maculuve, Sonia; Jiménez, Alfons; Quintó, Llorenç; Ouma, Peter; Ramharter, Michael; Aponte, John J; Nhacolo, Arsenio; Massougbodji, Achille; Briand, Valerie; Kremsner, Peter G; Mombo-Ngoma, Ghyslain; Desai, Meghna; Macete, Eusebio; Cot, Michel; Menéndez, Clara; Mayor, Alfredo
Resistance and tolerance to Plasmodium falciparum can determine the progression of malaria disease. However, quantitative evidence of tolerance is still limited. We investigated variations in the adverse impact of P. falciparum infections among African pregnant women under different intensities of malaria transmission. P. falciparum at delivery was assessed by microscopy, quantitative PCR (qPCR) and placental histology in 946 HIV-uninfected and 768 HIV-infected pregnant women from Benin, Gabon, Kenya and Mozambique. Resistance was defined by the proportion of submicroscopic infectio