Antibodies among men and children to placental-binding Plasmodium falciparum-infected erythrocytes that express var2csa.

PubMed

Beeson, James G; Ndungu, Francis; Persson, Kristina E M; Chesson, Joanne M; Kelly, Greg L; Uyoga, Sophie; Hallamore, Sandra L; Williams, Thomas N; Reeder, John C; Brown, Graham V; Marsh, Kevin

2007-07-01

During pregnancy, specific variants of Plasmodium falciparum-infected erythrocytes (IEs) can accumulate in the placenta through adhesion to chondroitin sulfate A (CSA) mediated by expression of PfEMP1 encoded by var2csa-type genes. Antibodies against these variants are associated with protection from maternal malaria. We evaluated antibodies among Kenyan, Papua New Guinean, and Malawian men and Kenyan children against two different CSA-binding P. falciparum isolates expressing var2csa variants. Specific IgG was present at significant levels among some men and children from each population, suggesting exposure to these variants is not exclusive to pregnancy. However, the level and prevalence of antibodies was substantially lower overall than exposed multigravidas. IgG-binding was specific and did not represent antibodies to subpopulations of non-CSA-binding IEs, and some sera inhibited IE adhesion to CSA. These findings have significant implications for understanding malaria pathogenesis and immunity and may be significant for understanding the acquisition of immunity to maternal malaria.

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

PubMed Central

Ostera, Graciela; Tokumasu, Fuyuki; Oliveira, Fabiano; Sa, Juliana; Furuya, Tetsuya; Teixeira, Clarissa; Dvorak, James

2008-01-01

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P.falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite, PMID:18504040

Population genetic structure and natural selection of Plasmodium falciparum apical membrane antigen-1 in Myanmar isolates.

PubMed

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

2018-02-07

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 strong selective pressure of host immunity on the PfAMA-1 gene. These results have significant implications in understanding the nature of Myanmar PfAMA-1 along with global PfAMA-1. They also provide useful information for the development of effective malaria vaccine based on this antigen.

A Large Proportion of P. falciparum Isolates in the Amazon Region of Peru Lack pfhrp2 and pfhrp3: Implications for Malaria Rapid Diagnostic Tests

PubMed Central

Gamboa, Dionicia; Ho, Mei-Fong; Bendezu, Jorge; Torres, Katherine; Chiodini, Peter L.; Barnwell, John W.; Incardona, Sandra; Perkins, Mark; Bell, David; McCarthy, James; Cheng, Qin

2010-01-01

Background Malaria rapid diagnostic tests (RDTs) offer significant potential to improve the diagnosis of malaria, and are playing an increasing role in malaria case management, control and elimination. Peru, along with other South American countries, is moving to introduce malaria RDTs as components of malaria control programmes supported by the Global Fund for AIDS, TB and malaria. The selection of the most suitable malaria RDTs is critical to the success of the programmes. Methods Eight of nine microscopy positive P. falciparum samples collected in Iquitos, Peru tested negative or weak positive using HRP2-detecting RDTs. These samples were tested for the presence of pfhrp2 and pfhrp3 and their flanking genes by PCR, as well as the presence of HRP proteins by ELISA. To investigate for geographic extent of HRP-deleted parasites and their temporal occurrence a retrospective study was undertaken on 148 microscopy positive P. falciparum samples collected in different areas of the Amazon region of Peru. Findings Eight of the nine isolates lacked the pfhrp2 and/or pfhrp3 genes and one or both flanking genes, and the absence of HRP was confirmed by ELISA. The retrospective study showed that 61 (41%) and 103 (70%) of the 148 samples lacked the pfhrp2 or pfhrp3 genes respectively, with 32 (21.6%) samples lacking both hrp genes. Conclusions This is the first documentation of P. falciparum field isolates lacking pfhrp2 and/or pfhrp3. The high frequency and wide distribution of different parasites lacking pfhrp2 and/or pfhrp3 in widely dispersed areas in the Peruvian Amazon implies that malaria RDTs targeting HRP2 will fail to detect a high proportion of P. falciparum in malaria-endemic areas of Peru and should not be used. RDTs detecting parasite LDH or aldolase and quality microscopy should be use for malaria diagnosis in this region. There is an urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighbouring countries. PMID:20111602

Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum*

PubMed Central

Yam, Xue Yan; Birago, Cecilia; Fratini, Federica; Di Girolamo, Francesco; Raggi, Carla; Sargiacomo, Massimo; Bachi, Angela; Berry, Laurence; Fall, Gamou; Currà, Chiara; Pizzi, Elisabetta; Breton, Catherine Braun; Ponzi, Marta

2013-01-01

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum. PMID:24045696

Contrasting Transmission Dynamics of Co-endemic Plasmodium vivax and P. falciparum: Implications for Malaria Control and Elimination

PubMed Central

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

2015-01-01

Background 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. Methodology/ Principal Findings 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. Conclusions/ Significance 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, particularly against the relatively neglected non-falciparum species. PMID:25951184

Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar

PubMed Central

Juliano, Jonathan J; Randrianarivelojosia, Milijaona; Ramarosandratana, Benjamin; Ariey, Frédéric; Mwapasa, Victor; Meshnick, Steven R

2009-01-01

Background Strains of Plasmodium falciparum genetically resistant to chloroquine (CQ) due to the presence of pfcrt 76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant P. falciparum isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect pfcrt 76T-bearing P. falciparum minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings. Methods This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect pfcrt 76T-bearing minority variant P. falciparum. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples. Results Thirty one clinical P. falciparum isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the pfcrt K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant P. falciparum strains after therapy as detected by HTA. RFLP analysis failed to detect any pfcrt K76T-bearing isolates. Conclusion These findings indicate that genetically CQ-resistant P. falciparum are more common than previously thought in Madagascar even though the fitness of the minority variant pfcrt 76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the surveillance of anti-malarial resistance. The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries. PMID:19291288

Genetic diversity of Plasmodium falciparum isolates from naturally infected children in north-central Nigeria using the merozoite surface protein-2 as molecular marker.

PubMed

Oyedeji, Segun Isaac; Awobode, Henrietta Oluwatoyin; Anumudu, Chiaka; Kun, Jürgen

2013-08-01

To characterize the genetic diversity of Plasmodium falciparum (P. falciparum) field isolates in children from Lafia, North-central Nigeria, using the highly polymorphic P. falciparum merozoite surface protein 2 (MSP-2) gene as molecular marker. Three hundred and twenty children were enrolled into the study between 2005 and 2006. These included 140 children who presented with uncomplicated malaria at the Dalhatu Araf Specialist Hospital, Lafia and another 180 children from the study area with asymptomatic infection. DNA was extracted from blood spot on filter paper and MSP-2 genes were genotyped using allele-specific nested PCR in order to analyze the genetic diversity of parasite isolates. A total of 31 and 34 distinct MSP-2 alleles were identified in the asymptomatic and uncomplicated malaria groups respectively. No difference was found between the multiplicity of infection in the asymptomatic group and that of the uncomplicated malaria group (P>0.05). However, isolates of the FC27 allele type were dominant in the asymptomatic group whereas isolates of the 3D7 allele type were dominant in the uncomplicated malaria group. This study showed a high genetic diversity of P. falciparum isolates in North-central Nigeria and is comparable to reports from similar areas with high malaria transmission intensity. Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

Ex Vivo Drug Susceptibility of Ferroquine against Chloroquine-Resistant Isolates of Plasmodium falciparum and P. vivax▿

PubMed Central

Marfurt, Jutta; Chalfein, Ferryanto; Prayoga, Pak; Wabiser, Frans; Kenangalem, Enny; Piera, Kim A.; MacHunter, Barbara; Tjitra, Emiliana; Anstey, Nicholas M.; Price, Ric N.

2011-01-01

Ferroquine (FQ; SSR97193), a ferrocene-containing 4-aminoquinoline derivate, has potent in vitro efficacy against chloroquine (CQ)-resistant Plasmodium falciparum and CQ-sensitive P. vivax. In the current study, ex vivo FQ activity was tested in multidrug-resistant P. falciparum and P. vivax field isolates using a schizont maturation assay. Although FQ showed excellent activity against CQ-sensitive and -resistant P. falciparum and P. vivax (median 50% inhibitory concentrations [IC50s], 9.6 nM and 18.8 nM, respectively), there was significant cross-susceptibility with the quinoline-based drugs chloroquine, amodiaquine, and piperaquine (for P. falciparum, r = 0.546 to 0.700, P < 0.001; for P. vivax, r = 0.677 to 0.821, P < 0.001). The observed ex vivo cross-susceptibility is likely to reflect similar mechanisms of drug uptake/efflux and modes of drug action of this drug class. However, the potent activity of FQ against resistant isolates of both P. falciparum and P. vivax highlights a promising role for FQ as a lead antimalarial against CQ-resistant Plasmodium and a useful partner drug for artemisinin-based combination therapy. PMID:21730116

Plasmodium diversity in non-malaria individuals from the Bioko Island in Equatorial Guinea (West Central-Africa)

PubMed Central

Guerra-Neira, Ana; Rubio, José M; Royo, Jesús Roche; Ortega, Jorge Cano; Auñón, Antonio Sarrión; Diaz, Pedro Berzosa; LLanes, Agustín Benito

2006-01-01

Background In this paper we analyse the Plasmodium sp. prevalence in three villages with different isolation status on the island of Bioko (Equatorial Guinea) where malaria is a hyper-endemic disease. We also describe the genetic diversity of P. falciparum, using several plasmodia proteins as markers which show a high degree of polymorphism (MSP-1 and MSP-2). The results obtained from three different populations are compared in order to establish the impact of human movements and interventions. Methods Plasmodium sp. were analysed in three villages on Bioko Island (Equatorial Guinea), one of which (Southern) is isolated by geographical barriers. The semi-nested multiplex polymerase chain reaction (PCR) technique was used to determine the prevalence of the four human plasmodia species. The genotyping and frequency of P. falciparum populations were determined by PCR assay target polymorphism regions of the merozoite surface proteins 1 and 2 genes (MSP-1 and MSP-2). Results The data obtained show that there are no differences in plasmodia population flow between the Northwest and Eastern regions as regards the prevalence of the different Plasmodium species. The Southern population, on the other hand, shows a minor presence of P. malariae and a higher prevalence of P. ovale, suggesting some kind of transmission isolated from the other two. The P. falciparum genotyping in the different regions points to a considerable allelic diversity in the parasite population on Bioko Island, although this is somewhat higher in the Southern region than the others. There was a correlation between parasitaemia levels and the age of the individual with the multiplicity of infection (MOI). Conclusion Results could be explained by the selection of particular MSP alleles. This would tend to limit diversity in the parasite population and leading up to the extinction of rare alleles. On the other hand, the parasite population in the isolated village has less outside influence and the diversity of P. falciparum is maintained higher. The knowledge of parasite populations and their relationships is necessary to study their implications for control intervention. PMID:16784527

Evidence of triple mutant Pfdhps ISGNGA haplotype in Plasmodium falciparum isolates from North-east India: An analysis of sulfadoxine resistant haplotype selection.

PubMed

Das, Manuj K; Chetry, Sumi; Kalita, Mohan C; Dutta, Prafulla

2016-12-01

North-east region of India has consistent role in the spread of multi drug resistant Plasmodium (P.) falciparum to other parts of Southeast Asia. After rapid clinical treatment failure of Artemisinin based combination therapy-Sulphadoxine/Pyrimethamine (ACT-SP) chemoprophylaxis, Artemether-Lumefantrine (ACT-AL) combination therapy was introduced in the year 2012 in this region for the treatment of uncomplicated P. falciparum malaria. In a DNA sequencing based polymorphism analysis, seven codons of P. falciparum dihydropteroate synthetase ( Pf dhps) gene were screened in a total of 127 P. falciparum isolates collected from Assam, Arunachal Pradesh and Tripura of North-east India during the year 2014 and 2015 to document current sulfadoxine resistant haplotypes. Sequences were analyzed to rearrange both nucleotide and protein haplotypes. Molecular diversity indices were analyzed in DNA Sequence Polymorphism software (DnaSP) on the basis of Pf dhps gene sequences. Disappearance from selective neutrality was assessed based on the ratio of non-synonomous to synonomous nucleotide substitutions [dN/dS ratio]. Moreover, two-tailed Z test was performed in search of the significance for probability of rejecting null hypothesis of strict neutrality [dN = dS]. Presence of mutant P. falciparum multidrug resistance protein1 ( Pf mdr1) was also checked in those isolates that were present with new Pf dhps haplotypes. Phylogenetic relationship based on Pf dhps gene was reconstructed in Molecular Evolutionary Genetics Analysis (MEGA). Among eight different sulfadoxine resistant haplotypes found, IS GNG A haplotype was documented in a total of five isolates from Tripura with association of a new mutant M538 R allele. Sequence analysis of Pf mdr1 gene in these five isolates came to notice that not all but only one isolate was mutant at codon 86 (N86 Y ; Y YSND) in the multidrug resistance protein. Molecular diversity based on Pf dhps haplotypes revealed that P. falciparum populations in Assam and Tripura were under balancing selection for sulfadoxine resistant haplotypes but population from Arunachal Pradesh was under positive selection with comparatively high haplotype diversity ( h  = 0.870). In reconstructed phylogenetic analysis, isolates having IS GNG A haplotype were grouped into two separate sub-clusters from the other isolates based on their genetic distances and diversities. This study suggests that sulfadoxine resistant isolates are still migrating from its epicenter to the other parts of Southeast Asia and hence control and elimination of the drug resistant isolates have become impedimental. Moreover, P. falciparum populations in different areas may undergo selection of particular sulfadoxine resistant haplotypes either in the presence of drug or after its removal to maintain their plasticity.

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

PubMed Central

Gentz, R; Certa, U; Takacs, B; Matile, H; Döbeli, H; Pink, R; Mackay, M; Bone, N; Scaife, J G

1988-01-01

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene. Images PMID:2452082

DNA Sequence Polymorphism of the Lactate Dehydrogenase Genefrom Iranian Plasmodium vivax and Plasmodium falciparum Isolates.

PubMed

Getacher Feleke, Daniel; Nateghpour, Mehdi; Motevalli Haghi, Afsaneh; Hajjaran, Homa; Farivar, Leila; Mohebali, Mehdi; Raoofian, Reza

2015-01-01

Parasite lactate dehydrogenase (pLDH) is extensively employed as malaria rapid diagnostic tests (RDTs). Moreover, it is a well-known drug target candidate. However, the genetic diversity of this gene might influence performance of RDT kits and its drug target candidacy. This study aimed to determine polymorphism of pLDH gene from Iranian isolates of P. vivax and P. falciparum. Genomic DNA was extracted from whole blood of microscopically confirmed P. vivax and P. falciparum infected patients. pLDH gene of P. falciparum and P. vivax was amplified using conventional PCR from 43 symptomatic malaria patients from Sistan and Baluchistan Province, Southeast Iran from 2012 to 2013. Sequence analysis of 15 P. vivax LDH showed fourteen had 100% identity with P. vivax Sal-1 and Belem strains. Two nucleotide substitutions were detected with only one resulted in amino acid change. Analysis of P. falciparum LDH sequences showed six of the seven sequences had 100% homology with P. falciparum 3D7 and Mzr-1. Moreover, PfLDH displayed three nucleotide changes that resulted in changing only one amino acid. PvLDH and PfLDH showed 75%-76% nucleotide and 90.4%-90.76% amino acid homology. pLDH gene from Iranian P. falciparum and P. vivax isolates displayed 98.8-100% homology with 1-3 nucleotide substitutions. This indicated this gene was relatively conserved. Additional studies can be done weather this genetic variation can influence the performance of pLDH based RDTs or not.

Genetic characterization of an epidemic of Plasmodium falciparum malaria among Yanomami Amerindians.

PubMed

Laserson, K F; Petralanda, I; Almera, R; Barker, R H; Spielman, A; Maguire, J H; Wirth, D F

1999-12-01

Malaria parasites are genetically diverse at all levels of endemicity. In contrast, the merozoite surface protein (MSP) alleles in samples from 2 isolated populations of Yanomami Amerindians during an epidemic of Plasmodium falciparum were identical. The nonvariable restriction fragment length polymorphism patterns further suggested that the sequential outbreak comprised only a single P. falciparum genotype. By examination of serial samples from single human infections, the MSP characteristics were found to remain constant throughout the course of infection. An apparent clonal population structure of parasites seemed to cause outbreaks in small isolated villages. The use of standard molecular epidemiologic methods to measure genetic diversity in malaria revealed the occurrence of a genetically monomorphic population of P. falciparum within a human community.

MOLECULAR SURVEILLANCE OF Plasmodium vivax AND Plasmodium falciparum DHFR MUTATIONS IN ISOLATES FROM SOUTHERN IRAN

PubMed Central

SHARIFI-SARASIABI, Khojasteh; HAGHIGHI, Ali; KAZEMI, Bahram; TAGHIPOUR, Niloofar; MOJARAD, Ehsan Nazemalhosseini; GACHKAR, Latif

2016-01-01

In Iran, both Plasmodium vivax and P. falciparum malaria have been detected, but P. vivax is the predominant species. Point mutations in dihydrofolate reductase (dhfr) gene in both Plasmodia are the major mechanisms of pyrimethamine resistance. From April 2007 to June 2009, a total of 134 blood samples in two endemic areas of southern Iran were collected from patients infected with P. vivax and P. falciparum. The isolates were analyzed for P. vivax dihydrofolate reductase (pvdhfr) and P. falciparum dihydrofolate reductase (pfdhfr) point mutations using various PCR-based methods. The majority of the isolates (72.9%) had wild type amino acids at five codons of pvdhfr. Amongst mutant isolates, the most common pvdhfr alleles were double mutant in 58 and 117 amino acids (58R-117N). Triple mutation in 57, 58, and 117 amino acids (57L/58R/117N) was identified for the first time in the pvdhfr gene of Iranian P. vivax isolates. All the P. falciparumsamples analyzed (n = 16) possessed a double mutant pfdhfrallele (59R/108N) and retained a wild-type mutation at position 51. This may be attributed to the fact that the falciparum malaria patients were treated using sulfadoxine-pyrimethamine (SP) in Iran. The presence of mutant haplotypes in P. vivax is worrying, but has not yet reached an alarming threshold regarding drugs such as SP. The results of this study reinforce the importance of performing a molecular surveillance by means of a continuous chemoresistance assessment. PMID:27007559

Polymorphisms of the artemisinin resistant marker (K13) in Plasmodium falciparum parasite populations of Grande Comore Island 10 years after artemisinin combination therapy.

PubMed

Huang, Bo; Deng, Changsheng; Yang, Tao; Xue, Linlu; Wang, Qi; Huang, Shiguang; Su, Xin-zhuan; Liu, Yajun; Zheng, Shaoqin; Guan, Yezhi; Xu, Qin; Zhou, Jiuyao; Yuan, Jie; Bacar, Afane; Abdallah, Kamal Said; Attoumane, Rachad; Mliva, Ahamada M S A; Zhong, Yanchun; Lu, Fangli; Song, Jianping

2015-12-15

Plasmodium falciparum malaria is a significant public health problem in Comoros, and artemisinin combination therapy (ACT) remains the first choice for treating acute uncomplicated P. falciparum. The emergence and spread of artemisinin-resistant P. falciparum in Southeast Asia, associated with mutations in K13-propeller gene, poses a potential threat to ACT efficacy. Detection of mutations in the P. falciparum K13-propeller gene may provide the first-hand information on changes in parasite susceptibility to artemisinin. The objective of this study is to determinate the prevalence of mutant K13-propeller gene among the P. falciparum isolates collected from Grande Comore Island, Union of Comoros, where ACT has been in use since 2004. A total of 207 P. falciparum clinical isolates were collected from the island during March 2006 and October 2007 (n = 118) and March 2013 and December 2014 (n = 89). All isolates were analysed for single nucleotide polymorphisms (SNPs) and haplotypes in the K13-propeller gene using nested PCR and DNA sequencing. Only three 2006-2007 samples carried SNPs in the K13-propeller gene, one having a synonymous (G538G) and the other having two non-synonymous (S477Y and D584E) substitutions leading to two mutated haplotypes (2.2%, 2/95). Three synonymous mutations (R471R, Y500Y, and G538G) (5.9%, 5/85) and 7 non-synonymous substitutions (21.2%, 18/85) with nine mutated haplotypes (18.8%, 16/85) were found in isolates from 2013 to 2014. However, none of the polymorphisms associated with artemisinin-resistance in Southeast Asia was detected from any of the parasites examined. This study showed increased K13-propeller gene diversity among P. falciparum populations on the Island over the course of 8 years (2006-2014). Nevertheless, none of the polymorphisms known to be associated with artemisinin resistance in Asia was detected in the parasite populations examined. Our data suggest that P. falciparum populations in Grande Comore are still effectively susceptible to artemisinin. Our results provide insights into P. falciparum populations regarding mutations in the gene associated with artemisinin resistance and will be useful for developing and updating anti-malarial guidance in Comoros.

Cellular mechanisms of action and resistance of Plasmodium falciparum to artemisinin.

PubMed

Phompradit, Papichaya; Chaijaroenkul, Wanna; Na-Bangchang, Kesara

2017-12-01

The recent reports of high failure rates and decline in in vitro sensitivity of Plasmodium falciparum to artemisinin-based combination therapies (ACTs) suggest the possibility of clinical artemisinin resistance along the Thai-Cambodian and Thai-Myanmar borders. The study investigated cellular mechanisms of action and resistance of P. falciparum to artesunate (stage specific activity, interaction with hemozoin, and anti-oxidant levels) in the two paired P. falciparum isolates (MSF046 and MSF060) collected before treatment with a 3-day artesunate-mefloquine and at the time of recrudescence. In addition, the link of these cellular mechanisms to the polymorphisms of the candidate artemisinin-resistant genes (pfatp6, pfcrt, pfmdr1, pfmrp1, and K13 propeller) was also investigated. Morphological change was observed in both pairs of the primary and recrudesced P. falciparum isolates during 12-48 h of exposure to artesunate (at IC 90 ). A marked decrease in parasite viability was found in the recrudesced isolates of both MSF046 and MSD060. The extent of the reduction (% change of baseline) in total glutathione concentrations was significantly lower in recrudesced (32.1 and 1.7%) compared with primary (45.5 and 53.7%) isolates of both MSF046 and MSF060. The extent of reduction of hemozoin content in MSF046 was significantly higher in the recrudesced (76.8%) isolate compared with the primary isolate (99.5%). For MSF060 on the other hand, increase in hemozoin content was found in the recrudesced isolate and the extent of such increase was significantly higher in recrudesced (93.1%) than the primary isolate (87.5%). Polymorphism of K13 (N458Y) together with pfmdr1 copy number correlated well with sensitivity of both isolates to artesunate. Results of this preliminary study suggests possible role of glutathione-dependent detoxification system as well as heme degradation as cellular mechanisms of action and resistance of artemisinins.

Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes is associated with high parasitemia but not severe clinical manifestations of malaria in African children

PubMed Central

Arman, Mònica; Raza, Ahmed; Tempest, Louisa J.; Lyke, Kirsten E.; Thera, Mahamadou A.; Koné, Abdoulaye; Plowe, Christopher V.; Doumbo, Ogobara K.; Rowe, J. Alexandra

2009-01-01

Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes is an adhesive phenotype commonly found in field isolates that has previously been associated with severe malaria. Here, clumping was assessed in 131 isolates from Malian children. The clumping phenotype was seen in 6% (n=51) of uncomplicated malaria, 24% (n=51) of severe malaria, and 45% (n=29) of high parasitemia non-severe malaria isolates. Multivariate analysis indicated that clumping was strongly positively associated with parasitemia (F1,122=24.1, p<0.001) but not with disease category (F2,122=1.8, p=0.17). Therefore platelet-mediated clumping in Malian P. falciparum isolates is primarily associated with high parasitemia and not with severe clinical manifestations of malaria. PMID:17984358

Influence of the pfmdr1 Gene on In Vitro Sensitivities of Piperaquine in Thai Isolates of Plasmodium falciparum

PubMed Central

Mungthin, Mathirut; Watanatanasup, Ekularn; Sitthichot, Naruemon; Suwandittakul, Nantana; Khositnithikul, Rommanee; Ward, Stephen A.

2017-01-01

Piperaquine combined with dihydroartemisinin is one of the artemisinin derivative combination therapies, which can replace artesunate–mefloquine in treating uncomplicated falciparum malaria in Thailand. The aim of this study was to determine the in vitro sensitivity of Thai Plasmodium falciparum isolates against piperaquine and the influence of the pfmdr1 gene on in vitro response. One hundred and thirty-seven standard laboratory and adapted Thai isolates of P. falciparum were assessed for in vitro piperaquine sensitivity. Polymorphisms of the pfmdr1 gene were determined by polymerase chain reaction methods. The mean and standard deviation of the piperaquine IC50 in Thai isolates of P. falciparum were 16.7 ± 6.3 nM. The parasites exhibiting chloroquine IC50 of ≥ 100 nM were significantly less sensitive to piperaquine compared with the parasite with chloroquine IC50 of < 100 nM. No significant association between the pfmdr1 copy number and piperaquine IC50 values was found. In contrast, the parasites containing the pfmdr1 86Y allele exhibited significantly reduced piperaquine sensitivity. Before nationwide implementation of dihydroartemisinin–piperaquine as the first-line treatment in Thailand, in vitro and in vivo evaluations of this combination should be performed especially in areas where parasites containing the pfmdr1 86Y allele are predominant such as the Thai–Malaysian border. PMID:28044042

Patterns of protective associations differ for antibodies to P. falciparum-infected erythrocytes and merozoites in immunity against malaria in children.

PubMed

Chan, Jo-Anne; Stanisic, Danielle I; Duffy, Michael F; Robinson, Leanne J; Lin, Enmoore; Kazura, James W; King, Christopher L; Siba, Peter M; Fowkes, Freya Ji; Mueller, Ivo; Beeson, James G

2017-12-01

Acquired antibodies play an important role in immunity to P. falciparum malaria and are typically directed towards surface antigens expressed by merozoites and infected erythrocytes (IEs). The importance of specific IE surface antigens as immune targets remains unclear. We evaluated antibodies and protective associations in two cohorts of children in Papua New Guinea. We used genetically-modified P. falciparum to evaluate the importance of PfEMP1 and a P. falciparum isolate with a virulent phenotype. Our findings suggested that PfEMP1 was the dominant target of antibodies to the IE surface, including functional antibodies that promoted opsonic phagocytosis by monocytes. Antibodies were associated with increasing age and concurrent parasitemia, and were higher among children exposed to a higher force-of-infection as determined using molecular detection. Antibodies to IE surface antigens were consistently associated with reduced risk of malaria in both younger and older children. However, protective associations for antibodies to merozoite surface antigens were only observed in older children. This suggests that antibodies to IE surface antigens, particularly PfEMP1, play an earlier role in acquired immunity to malaria, whereas greater exposure is required for protective antibodies to merozoite antigens. These findings have implications for vaccine design and serosurveillance of malaria transmission and immunity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetic deletion of HRP2 and HRP3 in Indian Plasmodium falciparum population and false negative malaria rapid diagnostic test.

PubMed

Kumar, Navin; Pande, Veena; Bhatt, R M; Shah, Naman K; Mishra, Neelima; Srivastava, Bina; Valecha, Neena; Anvikar, Anupkumar R

2013-01-01

Genetic polymorphisms in diagnostic antigens are important factors responsible for variable performance of rapid diagnostic tests. Additionally, the failure of antigen expression due to gene deletion may also contribute to variable performance. We report Indian Plasmodium falciparum field isolates lacking both Pfhrp2 and Pfhrp3 genes leading to false negative results of rapid diagnostic tests. The study highlights need to determine the prevalence of P. falciparum isolates lacking these genes in larger field populations in India. Copyright © 2012 Elsevier B.V. All rights reserved.

Genetic variation of pfhrp2 in Plasmodium falciparum isolates from Yemen and the performance of HRP2-based malaria rapid diagnostic test.

PubMed

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

2015-07-22

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.

Historical review: Does falciparum malaria destroy isolated tribal populations?

PubMed

Shanks, G Dennis

Many isolated populations of tribal peoples were nearly destroyed when they first contacted infectious diseases particularly respiratory pathogens such as measles and smallpox. Surviving groups have often been found to have declining populations in the face of multiple social and infectious threats. Malaria, especially Plasmodium falciparum, was thought to be a major cause of depopulation in some tribal peoples isolated in tropical jungles. The dynamics of such host parasite interactions is unclear especially since most such populations would have had long histories of exposure to malaria. Three groups are individually reviewed: Meruts of Borneo, Yanomami of Amazonia, Jarawas of the Andaman Islands. The purpose of this review is to examine the role of falciparum malaria in the depopulation of some isolated tribal groups in order to understand what measures, if any, would be likely to prevent such losses. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular Surveillance for Multidrug-Resistant Plasmodium falciparum, Cambodia

PubMed Central

Shah, Naman K.; Alker, Alisa P.; Sem, Rithy; Susanti, Agustina Ika; Muth, Sinuon; Maguire, Jason D.; Duong, Socheat; Ariey, Frederic; Meshnick, Steven R.

2008-01-01

We conducted surveillance for multidrug-resistant Plasmodium falciparum in Cambodia during 2004–2006 by assessing molecular changes in pfmdr1. The high prevalence of isolates with multiple pfmdr1 copies found in western Cambodia near the Thai border, where artesunate–mefloquine therapy failures occur, contrasts with isolates from eastern Cambodia, where this combination therapy remains highly effective. PMID:18826834

An Adjustable Gas-Mixing Device to Increase Feasibility of In Vitro Culture of Plasmodium falciparum Parasites in the Field

PubMed Central

Volkman, Sarah K.; Ahouidi, Ambroise D.; Ndiaye, Daouda; Mboup, Souleymane; Wirth, Dyann F.

2014-01-01

A challenge to conducting high-impact and reproducible studies of the mechanisms of P. falciparum drug resistance, invasion, virulence, and immunity is the lack of robust and sustainable in vitro culture in the field. While the technology exists and is routinely utilized in developed countries, various factors–from cost, to supply, to quality–make it hard to implement in malaria endemic countries. Here, we design and rigorously evaluate an adjustable gas-mixing device for the in vitro culture of P. falciparum parasites in the field to circumvent this challenge. The device accurately replicates the gas concentrations needed to culture laboratory isolates, short-term adapted field isolates, cryopreserved previously non-adapted isolates, as well as to adapt ex vivo isolates to in vitro culture in the field. We also show an advantage over existing alternatives both in cost and in supply. Furthermore, the adjustable nature of the device makes it an ideal tool for many applications in which varied gas concentrations could be critical to culture success. This adjustable gas-mixing device will dramatically improve the feasibility of in vitro culture of Plasmodium falciparum parasites in malaria endemic countries given its numerous advantages. PMID:24603696

Plasmodium falciparum merozoite protein-1 genetic diversity and multiplicity of infection in isolates from Congolese children consulting in a pediatric hospital in Brazzaville.

PubMed

Gueye, Nerly Shirère Gampio; Ntoumi, Francine; Vouvoungui, Christevy; Kobawila, Simon Charles; NKombo, Michael; Mouanga, Alain M; Deibert, Julia; Koukouikila-Koussounda, Felix

2018-07-01

As in many sub-Saharan African countries, the burden of malaria has been reduced in the Republic of Congo as a result of massive deployment of insecticide treated nets and availability of artemisinin-combinations therapies (ACTs). High to moderate genetic diversity of msp-1 gene of Plasmodium falciparum (P. falciparum) has been reported from different parts of the world but limited data are available from Central Africa including the Republic of Congo. For this reason, the aim of study was to investigate the P. falciparum genetic diversity and to determine the multiplicity of infection in P. falciparum isolates from Congolese children in order to dispose of an additional parameter to measure the impact malaria control intervention. A total of 229 blood samples were collected from September 2014 to February 2015 in children aged from one to ten years presenting a paediatric hospital Marien NGOUABI located in Northern part of Brazzaville. Inclusion criterion was fever (axillary temperature ≥ 37.5 °C) or history of fever in the preceding 48 h before inclusion in this study. Then thick and thin blood smears were done to detect malaria parasites, to determine parasite density and to identify plasmodial species. Sub-microscopic infection was detected by PCR using the P. falciparum msp-1 gene as molecular marker. The prevalence of microscopic and sub-microscopic infection in this cohort was 10% and 27.5%, respectively. The K1 allelic family was predominant (45% of isolates) whereas the RO33 and MAD20 represented 35% and 20%, respectively of isolates. In this study 48% (38/79) of isolates harbored more than one parasite clone. Overall the multiplicity of infection (MOI) was 1.7. According to type of infection, the MOI was significantly higher in children with microscopic infection (2.5 vs 1.4 for submicroscopic infection, P = .001). When considering age, hemoglobin genotype (AA or AS) and level and parasite density, no association was observed with the MOI. This study reveals that the P. falciparum genetic diversity in isolates from Congolese children is high but with low multiplicity of infection. Copyright © 2018. Published by Elsevier B.V.

The Plasmodium falciparum chloroquine resistance transporter is associated with the ex vivo P. falciparum African parasite response to pyronaridine.

PubMed

Madamet, Marylin; Briolant, Sébastien; Amalvict, Rémy; Benoit, Nicolas; Bouchiba, Housem; Cren, Julien; Pradines, Bruno

2016-02-09

The pyronaridine-artesunate combination is one of the most recent oral artemisinin-based therapeutic combinations (ACTs) recommended for the treatment of uncomplicated P. falciparum malaria. The emergence of P. falciparum resistance to artemisinin has recently developed in Southeast Asia. Little data are available on the association between pyronaridine susceptibility and polymorphisms in genes involved in antimalarial drug resistance. The objective of the present study was to investigate the association between ex vivo responses to pyronaridine and the K76T mutation in the pfcrt gene in P. falciparum isolates. The assessment of ex vivo susceptibility to pyronaridine was performed on 296 P. falciparum isolates using a standard 42-h 3H-hypoxanthine uptake inhibition method. The K76T mutation was also investigated. The pyronaridine IC50 (inhibitory concentration 50 %) ranged from 0.55 to 80.0 nM. Ex vivo responses to pyronaridine were significantly associated with the K76T mutation (p-value = 0.020). The reduced susceptibility to pyronaridine, defined as IC50 > 60 nM, was significantly associated with the K76T mutation (p-value = 0.004). Using a Bayesian mixture modelling approach, the pyronaridine IC50 were classified into three components: component A (IC50 median 15.9 nM), component B (IC50 median 34.2 nM) and component C (IC50 median 63.3 nM). The K76T mutation was represented in 46.3% of the isolates in component A, 47.2% of the isolates in component B and 73.3% of the isolates in component C (p-value = 0.021). These results showed the ex vivo reduced susceptibility to pyronaridine, i.e., IC50 > 60 nM, associated with the K76T mutation.

Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance

PubMed Central

Chaorattanakawee, Suwanna; Saunders, David L.; Sea, Darapiseth; Chanarat, Nitima; Yingyuen, Kritsanai; Sundrakes, Siratchana; Saingam, Piyaporn; Buathong, Nillawan; Sriwichai, Sabaithip; Chann, Soklyda; Se, Youry; Yom, You; Heng, Thay Kheng; Kong, Nareth; Kuntawunginn, Worachet; Tangthongchaiwiriya, Kuntida; Jacob, Christopher; Takala-Harrison, Shannon; Plowe, Christopher; Lin, Jessica T.; Chuor, Char Meng; Prom, Satharath; Tyner, Stuart D.; Gosi, Panita; Teja-Isavadharm, Paktiya; Lon, Chanthap

2015-01-01

Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure. PMID:26014942

High prevalence of sulphadoxine-pyrimethamine resistance-associated mutations in Plasmodium falciparum field isolates from pregnant women in Brazzaville, Republic of Congo.

PubMed

Koukouikila-Koussounda, Felix; Bakoua, Damien; Fesser, Anna; Nkombo, Michael; Vouvoungui, Christevy; Ntoumi, Francine

2015-07-01

Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) has not been evaluated in the Republic of Congo since its implementation in 2006 and there is no published data on molecular markers of SP resistance among Plasmodium falciparum isolates from pregnant women. This first study in this country aimed to describe the prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) point mutations and haplotypes in P. falciparum isolates collected from pregnant women with asymptomatic infection. From March 2012 to December 2013, pregnant women attending Madibou health centre (in Southern Brazzaville) for antenatal visits were enrolled in this study after obtaining their written informed consent. Blood samples were collected and P. falciparum infections were characterized using PCR. A total of 363 pregnant women were enrolled. P. falciparum infection was detected in 67 (18.4%) samples as their PCR amplification of dhfr and dhps genes yielded bands and all the PCR products were successfully digested. Out of these 67 isolates, 59 (88%), 57 (85%) and 53 (79.1%) carried 51I, 59R and 108N dhfr mutant alleles, respectively. The prevalence of dhps 436A, 437G and 540E mutations were 67.1% (45/67), 98.5% (66/67) and 55.2% (37/67), respectively. More than one-half of the isolates carried quintuple mutations, with highly resistant haplotype dhfr51I/59R/108N + dhps437G/540E detected in 33% (22/67) whereas 25% (17/67) were found to carry sextuple mutations. We observed significantly higher frequencies of triple dhps mutations 436A/437G/540E and quintuple mutations dhfr51I/59R/108N+dhps437G/540E in isolates from women who received IPTp-SP than those who did not. Overall, this study shows high prevalence rates of SP-associated resistance mutations in P. falciparum isolates collected from pregnant women. The presence of the dhps mutant allele 540E and the high prevalence of isolates carrying quintuple dhfr/dhps mutations are here reported for the first time in the Republic of Congo. The increasing prevalence of multiple mutant alleles observed in this study is alarming and may present a challenge for the future interventions including IPTp-SP in the country. Copyright © 2015 Elsevier B.V. All rights reserved.

Culture adaptation of malaria parasites selects for convergent loss-of-function mutants.

PubMed

Claessens, Antoine; Affara, Muna; Assefa, Samuel A; Kwiatkowski, Dominic P; Conway, David J

2017-01-24

Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.

Lack of artemisinin resistance in Plasmodium falciparum in northwest Benin after 10 years of use of artemisinin-based combination therapy.

PubMed

Ogouyèmi-Hounto, Aurore; Damien, Georgia; Deme, Awa Bineta; Ndam, Nicaise T; Assohou, Constance; Tchonlin, Didier; Mama, Atika; Hounkpe, Virgile Olivier; Moutouama, Jules Doumitou; Remoué, Franck; Ndiaye, Daouda; Gazard, Dorothée Kinde

2016-01-01

In Benin, artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment for uncomplicated Plasmodium falciparum malaria since 2004. The emergence in Southeast Asia of parasites that are resistant to artemisinins poses a serious threat to global control of this disease. The presence of artemisinin resistance genotypes in parasite populations in Benin is currently unknown. The present study investigated the prevalence of relevant K13-propeller gene polymorphisms in parasite isolates from the north-western region of Benin. Plasmodium falciparum isolates were collected from children with a confirmed diagnosis of malaria aged 6 months to 5 years in two towns, Cobly and Djougou, in the north-western part of Benin. The study was conducted during the rainy season from July to November 2014 in local health facilities. The K13-propeller gene was amplified in parasite isolates using nested PCR and subsequently sequenced. A total of 108 children were recruited into the study. The efficiency of amplification reactions was 72% (78/108). The propeller domain of the K13 gene was successfully sequenced in 78 P. falciparum isolates; all of them were wild type with no polymorphisms detectable. The absence of mutations in the K13 gene indicates that P. falciparum parasite populations in the study area are still fully susceptible to artemisinins. © A. Ogouyèmi-Hounto et al., published by EDP Sciences, 2016.

Prevalence of mutation and phenotypic expression associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum and Plasmodium vivax.

PubMed

Zakai, Haytham A; Khan, Wajihullah; Asma, Umme

2013-09-01

Therapeutic efficacy of sulfadoxine-pyrimethamine (SP), which is commonly used to treat falciparum malaria, was assessed in isolates of Plasmodium falciparum (Welch, 1897) and Plasmodium vivax (Grassi et Feletti, 1890) ofAligarh, Uttar Pradesh, North India and Taif, Saudi Arabia during 2011-2012. Both the species showed mutations in dihydrofolate reductase (DHFR) enzyme as they have common biochemical drug targets. Mutation rate for pfdhfr was higher compared to pvdhfr because the drug was mainly given to treat falciparum malaria. Since both the species coexist, P. vivax was also exposed to SP due to faulty species diagnosis or medication without specific diagnosis. Low level of mutations against SP in P. falciparum of Saudi isolates indicates that the SP combination is still effective for the treatment of falciparum malaria. Since SP is used as first-line of treatment because of high level of resistance against chloroquine (CQ), it may result in spread of higher level of mutations resulting in drug resistance and treatment failure in near future. Therefore, to avoid further higher mutations in the parasite, use of better treatment regimens such as artesunate combination therapy must be introduced against SP combination.

Population genetics of GYPB and association study between GYPB*S/s polymorphism and susceptibility to P. falciparum infection in the Brazilian Amazon.

PubMed

Tarazona-Santos, Eduardo; Castilho, Lilian; Amaral, Daphne R T; Costa, Daiane C; Furlani, Natália G; Zuccherato, Luciana W; Machado, Moara; Reid, Marion E; Zalis, Mariano G; Rossit, Andréa R; Santos, Sidney E B; Machado, Ricardo L; Lustigman, Sara

2011-01-24

Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil. Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection. GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity. Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population.

Multinormal In Vitro Distribution Model Suitable for the Distribution of Plasmodium falciparum Chemosusceptibility to Doxycycline▿

PubMed Central

Briolant, Sébastien; Baragatti, Meili; Parola, Philippe; Simon, Fabrice; Tall, Adama; Sokhna, Cheikh; Hovette, Philippe; Mamfoumbi, Modeste Mabika; Koeck, Jean-Louis; Delmont, Jean; Spiegel, André; Castello, Jacky; Gardair, Jean Pierre; Trape, Jean Francois; Kombila, Maryvonne; Minodier, Philippe; Fusai, Thierry; Rogier, Christophe; Pradines, Bruno

2009-01-01

The distribution and range of 50% inhibitory concentrations (IC50s) of doxycycline were determined for 747 isolates obtained between 1997 and 2006 from patients living in Senegal, Republic of the Congo, and Gabon and patients hospitalized in France for imported malaria. The statistical analysis was designed to answer the specific question of whether Plasmodium falciparum has different phenotypes of susceptibility to doxycycline. A triple normal distribution was fitted to the data using a Bayesian mixture modeling approach. The IC50 geometric mean ranged from 6.2 μM to 11.1 μM according to the geographical origin, with a mean of 9.3 μM for all 747 parasites. The values for all 747 isolates were classified into three components: component A, with an IC50 mean of 4.9 μM (±2.1 μM [standard deviation]); component B, with an IC50 mean of 7.7 μM (±1.2 μM); and component C, with an IC50 mean of 17.9 μM (±1.4 μM). According to the origin of the P. falciparum isolates, the triple normal distribution was found in each subgroup. However, the proportion of isolates predicted to belong to component B was most important in isolates from Gabon and Congo and in isolates imported from Africa (from 46 to 56%). In Senegal, 55% of the P. falciparum isolates were predicted to be classified as component C. The cutoff of reduced susceptibility to doxycycline in vitro was estimated to be 35 μM. PMID:19047651

Plasmodium falciparum In Vitro Resistance to Monodesethylamodiaquine, Dakar, Senegal, 2014

PubMed Central

Fall, Bécaye; Madamet, Marylin; Camara, Cheikhou; Amalvict, Rémy; Fall, Mansour; Nakoulima, Aminata; Diatta, Bakary; Diémé, Yaya; Wade, Boubacar

2016-01-01

We successfully cultured 36 Plasmodium falciparum isolates from blood samples of 44 malaria patients admitted to the Hôpital Principal de Dakar (Dakar, Senegal) during August–December 2014. The prevalence of isolates with in vitro reduced susceptibility was 30.6% for monodesethylamodiaquine, 52.8% for chloroquine, 44.1% for mefloquine, 16.7% for doxycycline, 11.8% for piperaquine, 8.3% for artesunate, 5.9% for pyronaridine, 2.8% for quinine and dihydroartemisinin, and 0.0% for lumefantrine. The prevalence of isolates with reduced in vitro susceptibility to the artemisinin-based combination therapy partner monodesethylamodiaquine increased from 5.6% in 2013 to 30.6% in 2014. Because of the increased prevalence of P. falciparum parasites with impaired in vitro susceptibility to monodesethylamodiaquine, the implementation of in vitro and in vivo surveillance of all artemisinin-based combination therapy partners is warranted. PMID:27088703

Longitudinal genomic surveillance of Plasmodium falciparum malaria parasites reveals complex genomic architecture of emerging artemisinin resistance.

PubMed

Cerqueira, Gustavo C; Cheeseman, Ian H; Schaffner, Steve F; Nair, Shalini; McDew-White, Marina; Phyo, Aung Pyae; Ashley, Elizabeth A; Melnikov, Alexandre; Rogov, Peter; Birren, Bruce W; Nosten, François; Anderson, Timothy J C; Neafsey, Daniel E

2017-04-28

Artemisinin-based combination therapies are the first line of treatment for Plasmodium falciparum infections worldwide, but artemisinin resistance has risen rapidly in Southeast Asia over the past decade. Mutations in the kelch13 gene have been implicated in this resistance. We used longitudinal genomic surveillance to detect signals in kelch13 and other loci that contribute to artemisinin or partner drug resistance. We retrospectively sequenced the genomes of 194 P. falciparum isolates from five sites in Northwest Thailand, over the period of a rapid increase in the emergence of artemisinin resistance (2001-2014). We evaluate statistical metrics for temporal change in the frequency of individual SNPs, assuming that SNPs associated with resistance increase in frequency over this period. After Kelch13-C580Y, the strongest temporal change is seen at a SNP in phosphatidylinositol 4-kinase, which is involved in a pathway recently implicated in artemisinin resistance. Furthermore, other loci exhibit strong temporal signatures which warrant further investigation for involvement in artemisinin resistance evolution. Through genome-wide association analysis we identify a variant in a kelch domain-containing gene on chromosome 10 that may epistatically modulate artemisinin resistance. This analysis demonstrates the potential of a longitudinal genomic surveillance approach to detect resistance-associated gene loci to improve our mechanistic understanding of how resistance develops. Evidence for additional genomic regions outside of the kelch13 locus associated with artemisinin-resistant parasites may yield new molecular markers for resistance surveillance, which may be useful in efforts to reduce the emergence or spread of artemisinin resistance in African parasite populations.

Artemisinin Resistance-Associated Polymorphisms at the K13-Propeller Locus Are Absent in Plasmodium falciparum Isolates from Haiti

PubMed Central

Carter, Tamar E.; Boulter, Alexis; Existe, Alexandre; Romain, Jean R.; St. Victor, Jean Yves; Mulligan, Connie J.; Okech, Bernard A.

2015-01-01

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

Decreasing pfmdr1 Copy Number Suggests that Plasmodium falciparum in Western Cambodia Is Regaining In Vitro Susceptibility to Mefloquine

PubMed Central

Lim, Pharath; Dek, Dalin; Try, Vorleak; Sreng, Sokunthea; Suon, Seila

2015-01-01

Dihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) for Plasmodium falciparum malaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS+MQ) as a replacement ACT, we measured the prevalence of multiple pfmdr1 copies—a molecular marker for MQ resistance—in 844 P. falciparum clinical isolates collected in 2008 to 2013. The pfmdr1 copy number is decreasing in Western Cambodia, suggesting that P. falciparum is regaining in vitro susceptibility to MQ. PMID:25712365

[Presence of the double pfmdr1 mutation 86Tyr and 1246 Tyr in clones of a chloroquine-resistant west African isolate of Plasmodium falciparum].

PubMed

Pinheiro, L; Franco, S; Adagu, I S; Rosa, R; Rosário, V E; Warhurst, D C

2003-01-01

Isolates of Plasmodium falciparum from three areas of West Africa were recovered from cryopreservation and their chloroquine-sensitivity were determined in vitro. Of the 90 samples studied, 60 were from Guinea-Bissau (30Resistant/30Sensitive), 15 were from S. Tomé and Príncipe (11Resistant/4Sensitive) and 15 were from Angola (11Resistant/4Sensitive). All the isolates were sensitive to mefloquine. Using the polymerase chain reaction/restriction fragment length polymorphism technique (PCR/RFLP) it was possible to detect two mutations in the pfmdr1 gene, often associated with chloroquine-resistance. 66% of the samples from Guiné-Bissau showed a correlation with chloroquine-resistance while 73% of the samples from São Tomé and Angola altogether had the 86Tyr mutation. The present study on West African isolates and clones showed, for the first time, the presence of a double point mutation in the pfmdr1 gene one being found, up to now, only in South America isolates of Plasmodium falciparum.

Molecular analysis demonstrates high prevalence of chloroquine resistance but no evidence of artemisinin resistance in Plasmodium falciparum in the Chittagong Hill Tracts of Bangladesh.

PubMed

Alam, Mohammad Shafiul; Ley, Benedikt; Nima, Maisha Khair; Johora, Fatema Tuj; Hossain, Mohammad Enayet; Thriemer, Kamala; Auburn, Sarah; Marfurt, Jutta; Price, Ric N; Khan, Wasif A

2017-08-15

Artemisinin resistance is present in the Greater Mekong region and poses a significant threat for current anti-malarial treatment guidelines in Bangladesh. The aim of this molecular study was to assess the current status of drug resistance in the Chittagong Hill Tracts of Bangladesh near the Myanmar border. Samples were obtained from patients enrolled into a Clinical Trial (NCT02389374) conducted in Alikadam, Bandarban between August 2014 and January 2015. Plasmodium falciparum infections were confirmed by PCR and all P. falciparum positive isolates genotyped for the pfcrt K76T and pfmdr1 N86Y markers. The propeller region of the kelch 13 (k13) gene was sequenced from isolates from patients with delayed parasite clearance. In total, 130 P. falciparum isolates were available for analysis. The pfcrt mutation K76T, associated with chloroquine resistance was found in 81.5% (106/130) of cases and the pfmdr1 mutation N86Y in 13.9% (18/130) cases. No single nucleotide polymorphisms were observed in the k13 propeller region. This study provides molecular evidence for the ongoing presence of chloroquine resistant P. falciparum in Bangladesh, but no evidence of mutations in the k13 propeller domain associated with artemisinin resistance. Monitoring for artemisinin susceptibility in Bangladesh is needed to ensure early detection and containment emerging anti-malarial resistance.

K13 Propeller Alleles, mdr1 Polymorphism, and Drug Effectiveness at Day 3 after Artemether-Lumefantrine Treatment for Plasmodium falciparum Malaria in Colombia, 2014-2015.

PubMed

Montenegro, Madeline; Neal, Aaron T; Posada, Maritza; De Las Salas, Briegel; Lopera-Mesa, Tatiana M; Fairhurst, Rick M; Tobon-Castaño, Alberto

2017-12-01

High treatment failure rates for Plasmodium falciparum malaria have been reported in Colombia for chloroquine, amodiaquine, and sulfadoxine-pyrimethamine. Artemisinin combination therapies were introduced in 2006 in Colombia, where artemether-lumefantrine (AL) is currently used to treat uncomplicated P. falciparum malaria. Artemisinin (ART) resistance was initially observed in Southeast Asia as an increased parasite clearance time, manifesting as a positive thick-blood smear on day 3 after treatment (D3 positivity). Recently, mutations in the propeller domain of the P. falciparum kelch13 gene ( K13 propeller) have been associated with ART resistance. In this study, we surveyed AL effectiveness at D3 and molecular markers of drug resistance among 187 uncomplicated P. falciparum cases in 4 regions of Colombia from June 2014 to July 2015. We found that 3.2% (4/125) of patients showed D3 positivity, 100% (163/163) of isolates carried wild-type K13 propeller alleles, 12.9% (23/178) of isolates had multiple copies of the multidrug resistance 1 gene ( mdr1 ), and 75.8% (113/149) of isolates harbored the double mutant N F S D D mdr1 haplotype (the underlining indicates mutant alleles). These data suggest that ART resistance is not currently suspected in Colombia but that monitoring for lumefantrine resistance and AL failures should continue. Copyright © 2017 American Society for Microbiology.

Genetic diversity of merozoite surface protein-2 in Plasmodium falciparum isolates from Aceh province, Indonesia

NASA Astrophysics Data System (ADS)

Jamil, K. F.; Supargiyono, S.; Syafruddin, D.; Pratama, N.; Silvy, S.

2018-03-01

Estimated 3.3 million Indonesian population were infected with malaria. However, extensive genetic polymorphism of the field isolates MSP-2 of P. falciparum represents a major obstacle for the development of malaria treatment. The aim of this study to investigate the genetic diversity of MSP-2 genotype in field isolates of P. falciparum collected in Aceh Province. A total of 90 patients enrolled in this study who were selected from positive malaria from eleven district Hospitals in Aceh from 2013-2015. Data was collected by anamnesis, complete physical examination and laboratory tests for MSP-2. All protocol to diagnose malaria assigned following the WHO 2010 guideline. All samples were stored in Eijkman Biology Molecular Institute, Jakarta.Among 90 samples were 57.7% male and 42.3% female with the most cases ages between 21-30 years old. Allele typing analysis displayed the polymorphic nature of P. falciparum. The MSP-2 have two alleles, 62.2% (56/90) for FC27 type and 58.9% (53/90) for 3D7 type and 21.2% (19/90) for mixed FC27 and 3D7 infection were identified. Diverse allele types from Aceh Province was identified in MSP-2 P. falciparum patients; there is the almost similar number of patients infected with both allele. A moderate level of the mixed allele was also observed.

K13 Propeller Alleles, mdr1 Polymorphism, and Drug Effectiveness at Day 3 after Artemether-Lumefantrine Treatment for Plasmodium falciparum Malaria in Colombia, 2014-2015

PubMed Central

Montenegro, Madeline; Neal, Aaron T.; Posada, Maritza; De las Salas, Briegel; Lopera-Mesa, Tatiana M.

2017-01-01

ABSTRACT High treatment failure rates for Plasmodium falciparum malaria have been reported in Colombia for chloroquine, amodiaquine, and sulfadoxine-pyrimethamine. Artemisinin combination therapies were introduced in 2006 in Colombia, where artemether-lumefantrine (AL) is currently used to treat uncomplicated P. falciparum malaria. Artemisinin (ART) resistance was initially observed in Southeast Asia as an increased parasite clearance time, manifesting as a positive thick-blood smear on day 3 after treatment (D3 positivity). Recently, mutations in the propeller domain of the P. falciparum kelch13 gene (K13 propeller) have been associated with ART resistance. In this study, we surveyed AL effectiveness at D3 and molecular markers of drug resistance among 187 uncomplicated P. falciparum cases in 4 regions of Colombia from June 2014 to July 2015. We found that 3.2% (4/125) of patients showed D3 positivity, 100% (163/163) of isolates carried wild-type K13 propeller alleles, 12.9% (23/178) of isolates had multiple copies of the multidrug resistance 1 gene (mdr1), and 75.8% (113/149) of isolates harbored the double mutant NFSDD mdr1 haplotype (the underlining indicates mutant alleles). These data suggest that ART resistance is not currently suspected in Colombia but that monitoring for lumefantrine resistance and AL failures should continue. PMID:28947476

Contrasting ex vivo efficacies of "reversed chloroquine" compounds in chloroquine-resistant Plasmodium falciparum and P. vivax isolates.

PubMed

Wirjanata, Grennady; Sebayang, Boni F; Chalfein, Ferryanto; Prayoga; Handayuni, Irene; Noviyanti, Rintis; Kenangalem, Enny; Poespoprodjo, Jeanne Rini; Burgess, Steven J; Peyton, David H; Price, Ric N; Marfurt, Jutta

2015-09-01

Chloroquine (CQ) has been the mainstay of malaria treatment for more than 60 years. However, the emergence and spread of CQ resistance now restrict its use to only a few areas where malaria is endemic. The aim of the present study was to investigate whether a novel combination of a CQ-like moiety and an imipramine-like pharmacophore can reverse CQ resistance ex vivo. Between March to October 2011 and January to September 2013, two "reversed chloroquine" (RCQ) compounds (PL69 and PL106) were tested against multidrug-resistant field isolates of Plasmodium falciparum (n = 41) and Plasmodium vivax (n = 45) in Papua, Indonesia, using a modified ex vivo schizont maturation assay. The RCQ compounds showed high efficacy against both CQ-resistant P. falciparum and P. vivax field isolates. For P. falciparum, the median 50% inhibitory concentrations (IC50s) were 23.2 nM for PL69 and 26.6 nM for PL106, compared to 79.4 nM for unmodified CQ (P < 0.001 and P = 0.036, respectively). The corresponding values for P. vivax were 19.0, 60.0, and 60.9 nM (P < 0.001 and P = 0.018, respectively). There was a significant correlation between IC50s of CQ and PL69 (Spearman's rank correlation coefficient [r s] = 0.727, P < 0.001) and PL106 (rs = 0.830, P < 0.001) in P. vivax but not in P. falciparum. Both RCQs were equally active against the ring and trophozoite stages of P. falciparum, but in P. vivax, PL69 and PL106 showed less potent activity against trophozoite stages (median IC50s, 130.2 and 172.5 nM) compared to ring stages (median IC50s, 17.6 and 91.3 nM). RCQ compounds have enhanced ex vivo activity against CQ-resistant clinical isolates of P. falciparum and P. vivax, suggesting the potential use of reversal agents in antimalarial drug development. Interspecies differences in RCQ compound activity may indicate differences in CQ pharmacokinetics between the two Plasmodium species. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Genetic micro-epidemiology of malaria in Papua Indonesia: Extensive P. vivax diversity and a distinct subpopulation of asymptomatic P. falciparum infections.

PubMed

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

2017-01-01

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.

Higher microsatellite diversity in Plasmodium vivax than in sympatric Plasmodium falciparum populations in Pursat, Western Cambodia.

PubMed

Orjuela-Sánchez, Pamela; Sá, Juliana M; Brandi, Michelle C C; Rodrigues, Priscila T; Bastos, Melissa S; Amaratunga, Chanaki; Duong, Socheat; Fairhurst, Rick M; Ferreira, Marcelo U

2013-07-01

Previous microsatellite analyses of sympatric populations of Plasmodium vivax and Plasmodium falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax (n=87) and P. falciparum (n=164) parasites from Pursat province, Western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax, we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [HE], 0.87 vs. 0.66, P=0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P=0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax. Copyright © 2013 Elsevier Inc. All rights reserved.

Cloning of Plasmodium falciparum by single-cell sorting

PubMed Central

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-01-01

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

A polyvalent hybrid protein elicits antibodies against the diverse allelic types of block 2 in Plasmodium falciparum merozoite surface protein 1.

PubMed

Tetteh, Kevin K A; Conway, David J

2011-10-13

Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice. The full hybrid protein induced high titre antibodies against each of the major block 2 allelic types expressed as separate recombinant proteins and against a wide range of allelic types naturally expressed by a panel of diverse P. falciparum isolates, while the sub-component modules had partial antigenic coverage as expected. This encourages further development and evaluation of the full MSP1 block 2 polyvalent hybrid protein as a candidate blood-stage component of a malaria vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

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.

PubMed

Menemedengue, Virginie; Sahnouni, Khalifa; Basco, Leonardo; Tahar, Rachida

2011-07-01

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.

The genetic polymorphism of merozoite surface protein-1 in Plasmodium falciparum isolates from Aceh province, Indonesia

NASA Astrophysics Data System (ADS)

Jamil, K. F.; Supargiyono, S.; Syafruddin, D.; Pratama, N.; Silvy, S.

2018-03-01

An estimated of 3.3 million Indonesian population were infected with malaria. However, extensive genetic polymorphism of the field isolates msp-1 of P. falciparum represents a major obstacle for the development of malaria treatment. The aim of this study was to investigate the genetic diversity of msp-1 genotype in field isolates of P. falciparum collected in Aceh Province. A total of 90 patients with malaria (+) were selected from eleven district hospitals in Aceh from 2013-2015. Data were collected by anamnesis, complete physical examination and laboratory tests for msp-1. All protocols to diagnose malaria followed the WHO 2010 guideline. All samples were stored in Eijkman Biology Molecular Institute, Jakarta. Among 90 samples, 57.7% were male, and 42.3% were female with the most cases found between 21-30 years old. From the allele typing analysis of P. falciparum from Aceh; K1, MAD20, and RO33 allele types were identified. MAD20 type was the highest allele found in this study (57.9%). It was found in single and mixed infection. A moderate level of the mixed allele was also observed.

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

PubMed Central

Menemedengue, Virginie; Sahnouni, Khalifa; Basco, Leonardo; Tahar, Rachida

2011-01-01

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. PMID:21734119

Plasmodium falciparum and Plasmodium vivax Demonstrate Contrasting Chloroquine Resistance Reversal Phenotypes.

PubMed

Wirjanata, Grennady; Handayuni, Irene; Prayoga, Pak; Leonardo, Leo; Apriyanti, Dwi; Trianty, Leily; Wandosa, Ruland; Gobay, Basbak; Kenangalem, Enny; Poespoprodjo, Jeanne Rini; Noviyanti, Rintis; Kyle, Dennis E; Cheng, Qin; Price, Ric N; Marfurt, Jutta

2017-08-01

High-grade chloroquine (CQ) resistance has emerged in both Plasmodium falciparum and P. vivax The aim of the present study was to investigate the phenotypic differences of CQ resistance in both of these species and the ability of known CQ resistance reversal agents (CQRRAs) to alter CQ susceptibility. Between April 2015 and April 2016, the potential of verapamil (VP), mibefradil (MF), L703,606 (L7), and primaquine (PQ) to reverse CQ resistance was assessed in 46 P. falciparum and 34 P. vivax clinical isolates in Papua, Indonesia, where CQ resistance is present in both species, using a modified schizont maturation assay. In P. falciparum , CQ 50% inhibitory concentrations (IC 50 s) were reduced when CQ was combined with VP (1.4-fold), MF (1.2-fold), L7 (4.2-fold), or PQ (1.8-fold). The degree of CQ resistance reversal in P. falciparum was highly correlated with CQ susceptibility for all CQRRAs ( R 2 = 0.951, 0.852, 0.962, and 0.901 for VP, MF, L7, and PQ, respectively), in line with observations in P. falciparum laboratory strains. In contrast, no reduction in the CQ IC 50 s was observed with any of the CQRRAs in P. vivax , even in those isolates with high chloroquine IC 50 s. The differential effect of CQRRAs in P. falciparum and P. vivax suggests significant differences in CQ kinetics and, potentially, the likely mechanism of CQ resistance between these two species. © Crown copyright 2017.

Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

DTIC Science & Technology

2010-06-17

Pennsylvania State University College of Medicine, Hershey , Pennsylvania, United States of America Abstract Plasmodium falciparum is a highly lethal malaria...www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000968 Zimmerli S, Edwards S, Ernst JD ( 1996 ) Selective receptor blockade...in field isolates. J Immunol 165: 6341–6346. 22. Baruch DI, Gormely JA, Ma C, Howard RJ, Pasloske BL ( 1996 ) Plasmodium falciparum erythrocyte

Antimalarial Activities of Peptide Antibiotics Isolated from Fungi

PubMed Central

Nagaraj, G.; Uma, M. V.; Shivayogi, M. S.; Balaram, Hemalatha

2001-01-01

Malaria caused by Plasmodium falciparum is a major public health problem in the developing countries of the world. Clinical treatment of malaria has become complicated due to the occurrence of infections caused by drug resistant parasites. Secondary metabolites from fungi are an attractive source of chemotherapeutic agents. This work reports the isolation and in vitro antiplasmodial activities of peptide antibiotics of fungal origin. The three peptide antibiotics used in this study were efrapeptins, zervamicins, and antiamoebin. The high-performance liquid chromatography-purified peptides were characterized by nuclear magnetic resonance and mass spectral analysis. All three fungal peptides kill P. falciparum in culture with 50% inhibitory concentrations in the micromolar range. A possible mode of action of these peptide antibiotics on P. falciparum is presented. PMID:11120957

Genetic Diversity of Plasmodium falciparum Field Isolates in Central Sudan Inferred by PCR Genotyping of Merozoite Surface Protein 1 and 2.

PubMed

Hamid, Muzamil M Abdel; Mohammed, Sara B; El Hassan, Ibrahim M

2013-02-01

Characterization of Plasmodium falciparum diversity is commonly achieved by amplification of the polymorphic regions of the merozoite surface proteins 1 (MSP1) and 2 (MSP2) genes. The present study aimed to determine the allelic variants distribution of MSP1 and MSP2 and multiplicity of infection in P. falciparum field isolates from Kosti, central Sudan, an area characterized by seasonal malaria transmission. Total 121 samples (N = 121) were collected during a cross-sectional survey between March and April 2003. DNA was extracted and MSP1 and MSP2 polymorphic loci were genotyped. The total number of alleles identified in MSP1 block 2 was 11, while 16 alleles were observed in MSP2 block 3. In MSP1, RO33 was found to be the predominant allelic type, carried alone or in combination with MAD20 and K1 types, whereas FC27 family was the most prevalent in MSP2. Sixty two percent of isolates had multiple genotypes and the overall mean multiplicity of infection was 1.93 (CI 95% 1.66-2.20). Age correlated with parasite density (P = 0.017). In addition, a positive correlation was observed between parasite densities and the number of alleles (P = 0.022). Genetic diversity in P. falciparum field isolates in central Sudan was high and consisted of multiple clones.

Artemisinin resistance-associated polymorphisms at the K13-propeller locus are absent in Plasmodium falciparum isolates from Haiti.

PubMed

Carter, Tamar E; Boulter, Alexis; Existe, Alexandre; Romain, Jean R; St Victor, Jean Yves; Mulligan, Connie J; Okech, Bernard A

2015-03-01

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. © The American Society of Tropical Medicine and Hygiene.

Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Honduras

PubMed Central

2012-01-01

Background 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. Methods 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. Results and conclusion 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. PMID:23181845

Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Honduras.

PubMed

Lopez, Ana Cecilia; Ortiz, Andres; Coello, Jorge; Sosa-Ochoa, Wilfredo; Torres, Rosa E Mejia; Banegas, Engels I; Jovel, Irina; Fontecha, Gustavo A

2012-11-26

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.

Genetic diversity of Plasmodium falciparum histidine-rich protein 2 in the China-Myanmar border area

PubMed Central

Li, Peipei; Xing, Hua; Zhao, Zhenjun; Yang, Zhaoqing; Cao, Yaming; Yan, Guiyun; Sattabongkot, Jetsumon; Cui, Liwang; Fan, Qi

2016-01-01

Deletion of the Plasmodium falciparum histidine-rich protein 2 (pfhrp2) gene may affect the performance of PfHRP2-based rapid diagnostic tests (RDTs). Here we investigated the genetic diversity of the pfhrp2 gene in clinical parasite isolates collected in recent years from the China-Myanmar border area. Deletion of pfhrp2 has been identified in 4 out of 97 parasite isolates. Sequencing of the pfhrp2 exon 2 from 67 isolates revealed a high level of genetic diversity in pfhrp2, which is reflected in the presence of many repeat types and their variants, as well as variable copy numbers and different arrangements of these repeats in parasite isolates. In addition, we observed pfhrp3 deletion in three of the four parasites harboring pfhrp2 deletion, suggesting of double deletions of both genes in these three isolates. Analysis of two cases, which were P. falciparum-positive by microscopy and PCR but failed by two PfHRP2-based RDTs, did not find pfhrp2 deletion. Further correlational studies of pfhrp2 polymorphisms with detection sensitivity are needed to identify factors influencing the performance of RDTs in malaria-endemic areas. PMID:26297799

Effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum infected erythrocytes

PubMed Central

Saiwaew, Somporn; Sritabal, Juntima; Piaraksa, Nattaporn; Keayarsa, Srisuda; Ruengweerayut, Ronnatrai; Utaisin, Chirapong; Sila, Patima; Niramis, Rangsan; Udomsangpetch, Rachanee; Charunwatthana, Prakaykaew; Pongponratn, Emsri; Pukrittayakamee, Sasithon; Leitgeb, Anna M.; Wahlgren, Mats; Lee, Sue J.; Day, Nicholas P. J.; White, Nicholas J.; Dondorp, Arjen M.; Chotivanich, Kesinee

2017-01-01

In severe falciparum malaria cytoadherence of parasitised red blood cells (PRBCs) to vascular endothelium (causing sequestration) and to uninfected red cells (causing rosette formation) contribute to microcirculatory flow obstruction in vital organs. Heparin can reverse the underlying ligand-receptor interactions, but may increase the bleeding risks. As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Sevuparin has been evaluated recently in patients with uncomplicated falciparum malaria, and is currently investigated in a clinical trial for sickle cell disease. The effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum isolates from Thailand were investigated. Trophozoite stages of P. falciparum-infected RBCs (Pf-iRBCs) were cultured from 49 patients with malaria. Pf-iRBCs were treated with sevuparin at 37°C and assessed in rosetting and in cytoadhesion assays with human dermal microvascular endothelial cells (HDMECs) under static and flow conditions. The proportion of Pf-iRBCs forming rosettes ranged from 6.5% to 26.0% (median = 12.2%). Rosetting was dose dependently disrupted by sevuparin (50% disruption by 250 μg/mL). Overall 57% of P. falciparum isolates bound to HDMECs under static conditions; median (interquartile range) Pf-iRBC binding was 8.5 (3.0–38.0) Pf-iRBCs/1000 HDMECs. Sevuparin in concentrations ≥ 100 μg/mL inhibited cytoadherence. Sevuparin disrupts P. falciparum rosette formation in a dose dependent manner and inhibits cytoadherence to endothelial cells. The data support assessment of sevuparin as an adjunctive treatment to the standard therapy in severe falciparum malaria. PMID:28249043

Ferrocene-chloroquine analogues as antimalarial agents: in vitro activity of ferrochloroquine against 103 Gabonese isolates of Plasmodium falciparum.

PubMed

Pradines, B; Fusai, T; Daries, W; Laloge, V; Rogier, C; Millet, P; Panconi, E; Kombila, M; Parzy, D

2001-08-01

The in vitro activities of ferrochloroquine, chloroquine, quinine, mefloquine, halofantrine, amodiaquine, primaquine, atovaquone and artesunate were evaluated against Plasmodium falciparum isolates from children with uncomplicated malaria from Libreville (Gabon), using an isotopic, micro, drug susceptibility test. The IC(50) values for ferrochloroquine were in the range 0.43-30.9 nM and the geometric mean IC(50) for the 103 isolates was 10.8 nM (95% CI 8.6-13.5 nM), while the geometric means for chloroquine, quinine, mefloquine, amodiaquine and primaquine were 370 nM, 341 nM, 8.3 nM, 18.1 nM and 7.6 microM, respectively. Ferrochloroquine was active against P. falciparum isolates, 95% of which showed in vitro resistance to chloroquine. Weak positive significant correlations were observed between the responses to ferrochloroquine and that to chloroquine, amodiaquine and quinine, but too low to suggest cross-resistance. There was no significant correlation between the response to ferrochloroquine and those to mefloquine, halofantrine, primaquine, atovaquone or artesunate. Ferrochloroquine may be an important alternative drug for the treatment of chloroquine-resistant malaria.

Genetic micro-epidemiology of malaria in Papua Indonesia: Extensive P. vivax diversity and a distinct subpopulation of asymptomatic P. falciparum infections

PubMed Central

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

2017-01-01

Background 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. Methods 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. Results 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. Conclusions 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. PMID:28498860

Assessment of copy number variation in genes related to drug resistance in Plasmodium vivax and Plasmodium falciparum isolates from the Brazilian Amazon and a systematic review of the literature.

PubMed

Costa, Gabriel Luíz; Amaral, Lara Cotta; Fontes, Cor Jesus Fernandes; Carvalho, Luzia Helena; de Brito, Cristiana Ferreira Alves; de Sousa, Taís Nóbrega

2017-04-19

Parasite resistance to anti-malarials represents a great obstacle for malaria elimination. The majority of studies have investigated the association between single-nucleotide polymorphisms (SNPs) and drug resistance; however, it is becoming clear that the copy number variation (CNV) is also associated with this parasite phenotype. To provide a baseline for molecular surveillance of anti-malarial drug resistance in the Brazilian Amazon, the present study characterized the genetic profile of both markers in the most common genes associated with drug resistance in Plasmodium falciparum and Plasmodium vivax isolates. Additionally, these data were compared to data published elsewhere applying a systematic review of the literature published over a 20-year time period. The genomic DNA of 67 patients infected by P. falciparum and P. vivax from three Brazilian States was obtained between 2002 and 2012. CNV in P. falciparum multidrug resistance gene-1 (pfmdr1), GTP cyclohydrolase 1 (pfgch1) and P. vivax multidrug resistance gene-1 (pvmdr1) were assessed by real-time PCR assays. SNPs in the pfmdr1 and pfcrt genes were assessed by PCR-RFLP. A literature search for studies that analysed CNP in the same genes of P. falciparum and P. vivax was conducted between May 2014 and March 2017 across four databases. All analysed samples of P. falciparum carried only one copy of pfmdr1 or pfgch1. Although the pfcrt K76T polymorphism, a determinant of CQ resistance, was present in all samples genotyped, the pfmdr1 N86Y was absent. For P. vivax isolates, an amplification rate of 20% was found for the pvmdr1 gene. The results of the study are in agreement with the low amplification rates for pfmdr1 gene evidenced in the Americas and Africa, while higher rates have been described in Southeast Asia. For P. vivax, very low rates of amplification for pvmdr1 have been described worldwide, with exceptions in French Guiana, Cambodia, Thailand and Brazil. The present study was the first to evaluate gch1 CNV in P. falciparum isolates from Brazil, showing an absence of amplification of this gene more than 20 years after the withdrawal of the Brazilian antifolates therapeutic scheme. Furthermore, the rate of pvmdr1 amplification was significantly higher than that previously reported for isolates circulating in Northern Brazil.

DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

NASA Astrophysics Data System (ADS)

Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

1984-08-01

A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

Genetic mutations in Plasmodium falciparum and Plasmodium vivax dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) in Vanuatu and Solomon Islands prior to the introduction of artemisinin combination therapy.

PubMed

Gresty, Karryn J; Gray, Karen-Ann; Bobogare, Albino; Wini, Lyndes; Taleo, George; Hii, Jeffrey; Cheng, Qin; Waters, Norman C

2014-10-14

Plasmodium falciparum and Plasmodium vivax are endemic in Vanuatu and the Solomon Islands. While both countries have introduced artemether-lumefantrine (AL) as first-line therapy for both P. falciparum and P. vivax since 2008, chloroquine and sulphadoxine-pyrimethamine (SP) were used as first-line therapy for many years prior to the introduction of AL. Limited data are available on the extent of SP resistance at the time of policy change. Blood spots were obtained from epidemiological surveys conducted on Tanna Island, Tafea Province, Vanuatu and Temotu Province, Solomon Islands in 2008. Additional samples from Malaita Province, Solomon Islands were collected as part of an AL therapeutic efficacy study conducted in 2008. Plasmodium vivax and P. falciparum dhfr and dhps genes were sequenced to detect nucleotide polymorphisms. All P. falciparum samples analysed (n=114) possessed a double mutant pfdhfr allele (C59R/S108N). Additionally, mutation A437G in pfhdps was detected in a small number of samples 2/13, 1/17 and 3/26 from Tanna Island, Vanuatu and Temotu and Malaita Provinces Solomon Islands respectively. Mutations were also common in pvdhfr from Tanna Island, Vanuatu, where 33/51 parasites carried the double amino acid substitution S58R/S117N, while in Temotu and Malaita Provinces, Solomon Islands 32/40 and 39/46 isolates carried the quadruple amino acid substitution F57L/S58R/T61M/S117T in DHFR respectively. No mutations in pvdhps (n=108) were detected in these three island groups. Prior to the introduction of AL, there was a moderate level of SP resistance in the P. falciparum population that may cause SP treatment failure in young children. Of the P. vivax isolates, a majority of Solomon Islands isolates carried quadruple mutant pvdhfr alleles while a majority of Vanuatu isolates carried double mutant pvdhfr alleles. This suggests a higher level of SP resistance in the P. vivax population in Solomon Islands compared to the sympatric P. falciparum population and there is a higher level of SP resistance in P. vivax parasites from Solomon Islands than Vanuatu. This study demonstrates that the change of treatment policy in these countries from SP to ACT was timely. The information also provides a baseline for future monitoring.

Cloning of Plasmodium falciparum by single-cell sorting.

PubMed

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-10-01

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. Copyright 2010 Elsevier Inc. All rights reserved.

A simple field kit for the determination of drug susceptibility in Plasmodium falciparum.

PubMed

Nguyen-Dinh, P; Magloire, R; Chin, W

1983-05-01

A field kit has been developed which greatly simplifies the performance of the 48-hour in vitro test for drug resistance in Plasmodium falciparum. The kit uses an easily reconstituted lyophilized culture medium, and requires only a fingerprick blood sample. In parallel tests with 13 isolates of P. falciparum in Haiti, the new technique had a success rate equal to that of the previously described method, with comparable results in terms of parasite susceptibility in vitro to chloroquine and pyrimethamine.

Cytoadhesion to gC1qR through Plasmodium falciparum Erythrocyte Membrane Protein 1 in Severe Malaria

PubMed Central

Magallón-Tejada, Ariel; Machevo, Sónia; Cisteró, Pau; Lavstsen, Thomas; Aide, Pedro; Jiménez, Alfons; Turner, Louise; Gupta, Himanshu; De Las Salas, Briegel; Mandomando, Inacio; Wang, Christian W.; Petersen, Jens E. V.; Muñoz, Jose; Gascón, Joaquim; Macete, Eusebio; Alonso, Pedro L.; Chitnis, Chetan E.

2016-01-01

Cytoadhesion of Plasmodium falciparum infected erythrocytes to gC1qR has been associated with severe malaria, but the parasite ligand involved is currently unknown. To assess if binding to gC1qR is mediated through the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, we analyzed by static binding assays and qPCR the cytoadhesion and var gene transcriptional profile of 86 P. falciparum isolates from Mozambican children with severe and uncomplicated malaria, as well as of a P. falciparum 3D7 line selected for binding to gC1qR (Pf3D7gC1qR). Transcript levels of DC8 correlated positively with cytoadhesion to gC1qR (rho = 0.287, P = 0.007), were higher in isolates from children with severe anemia than with uncomplicated malaria, as well as in isolates from Europeans presenting a first episode of malaria (n = 21) than Mozambican adults (n = 25), and were associated with an increased IgG recognition of infected erythrocytes by flow cytometry. Pf3D7gC1qR overexpressed the DC8 type PFD0020c (5.3-fold transcript levels relative to Seryl-tRNA-synthetase gene) compared to the unselected line (0.001-fold). DBLβ12 from PFD0020c bound to gC1qR in ELISA-based binding assays and polyclonal antibodies against this domain were able to inhibit binding to gC1qR of Pf3D7gC1qR and four Mozambican P. falciparum isolates by 50%. Our results show that DC8-type PfEMP1s mediate binding to gC1qR through conserved surface epitopes in DBLβ12 domain which can be inhibited by strain-transcending functional antibodies. This study supports a key role for gC1qR in malaria-associated endovascular pathogenesis and suggests the feasibility of designing interventions against severe malaria targeting this specific interaction. PMID:27835682

Microsatellite Genotyping of Plasmodium vivax Isolates from Pregnant Women in Four Malaria Endemic Countries

PubMed Central

Menegon, Michela; Bardají, Azucena; Martínez-Espinosa, Flor; Bôtto-Menezes, Camila; Ome-Kaius, Maria; Mueller, Ivo; Betuela, Inoni; Arévalo-Herrera, Myriam; Kochar, Swati; Kochar, Sanjay K.; Jaju, Puneet; Hans, Dhiraj; Chitnis, Chetan; Padilla, Norma; Castellanos, María Eugenia; Ortiz, Lucía; Sanz, Sergi; Piqueras, Mireia; Desai, Meghna; Mayor, Alfredo; del Portillo, Hernando; Menéndez, Clara; Severini, Carlo

2016-01-01

Plasmodium vivax is the most widely distributed human parasite and the main cause of human malaria outside the African continent. However, the knowledge about the genetic variability of P. vivax is limited when compared to the information available for P. falciparum. We present the results of a study aimed at characterizing the genetic structure of P. vivax populations obtained from pregnant women from different malaria endemic settings. Between June 2008 and October 2011 nearly 2000 pregnant women were recruited during routine antenatal care at each site and followed up until delivery. A capillary blood sample from the study participants was collected for genotyping at different time points. Seven P. vivax microsatellite markers were used for genotypic characterization on a total of 229 P. vivax isolates obtained from Brazil, Colombia, India and Papua New Guinea. In each population, the number of alleles per locus, the expected heterozygosity and the levels of multilocus linkage disequilibrium were assessed. The extent of genetic differentiation among populations was also estimated. Six microsatellite loci on 137 P. falciparum isolates from three countries were screened for comparison. The mean value of expected heterozygosity per country ranged from 0.839 to 0.874 for P. vivax and from 0.578 to 0.758 for P. falciparum. P. vivax populations were more diverse than those of P. falciparum. In some of the studied countries, the diversity of P. vivax population was very high compared to the respective level of endemicity. The level of inter-population differentiation was moderate to high in all P. vivax and P. falciparum populations studied. PMID:27011010

Genetic diversity of the DBLalpha region in Plasmodium falciparum var genes among Asia-Pacific isolates.

PubMed

Fowler, Elizabeth V; Peters, Jennifer M; Gatton, Michelle L; Chen, Nanhua; Cheng, Qin

2002-03-01

In Plasmodium falciparum a highly polymorphic multi-copy gene family, var, encodes the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1), which has an important role in cytoadherence and immune evasion. Using previously described universal PCR primers for the first Duffy binding-like domain (DBLalpha) of var we analysed the DBLalpha repertoires of Dd2 (originally from Thailand) and eight isolates from the Solomon Islands (n=4), Philippines (n=2), Papua New Guinea (n=1) and Africa (n=1). We found 15-32 unique DBLalpha sequence types among these isolates and estimated detectable DBLalpha repertoire sizes ranging from 33-38 to 52-57 copies per genome. Our data suggest that var gene repertoires generally consist of 40-50 copies per genome. Eighteen DBLalpha sequences appeared in more than one Asia-Pacific isolate with the number of sequences shared between any two isolates ranging from 0 to 6 (mean=2.0 +/-1.6). At the amino acid level DBLalpha sequence similarity within isolates ranged from 45.2 +/- 7.1 to 50.2 +/- 6.9%, and was not significantly different from the DBLalpha amino acid sequence similarity among isolates (P>0.1). Comparisons with published sequences also revealed little overlap among DBLalpha sequences from different regions. High DBLalpha sequence diversity and minimal overlap among these isolates suggest that the global var gene repertoire is immense, and may potentially be selected for by the host's protective immune response to the var gene products, PfEMP1.

In vitro activity of tafenoquine alone and in combination with artemisinin against Plasmodium falciparum.

PubMed

Ramharter, Michael; Noedl, Harald; Thimasarn, Krongthong; Wiedermann, Gerhard; Wernsdorfer, Gunther; Wernsdorfer, Walther H

2002-07-01

Emergence and spread of drug-resistant falciparum malaria has created an urgent demand for alternative therapeutic agents. This study was conducted to assess the in vitro blood schizontocidal activity of tafenoquine, the most advanced candidate drug of the 8-aminoquinolines, and of its 1:1 combination with artemisinin in fresh isolates of Plasmodium falciparum in an area with multi-drug resistance, measuring the inhibition of schizont maturation. In 43 successfully tested parasite isolates, the mean effective concentrations (ECs) of tafenoquine were 209 nmol/L for the EC50, and 1,414 nmol/L for the EC90. Tafenoquine showed no significant activity relationships with mefloquine, artemisinin, and chloroquine. With quinine, a highly significant activity relationship was observed at the EC50, but not at the EC90. The EC50, and EC90 of the tafenoquine-artemisinin combination were 15.9 nmol/L and 84.3 nmol/L. The combination was synergistic. Tafenoquine appears to be a promising candidate for treating multidrug-resistant falciparum malaria, especially in combination with artemisinin derivatives.

Acute Malaria Induces PD1+CTLA4+ Effector T Cells with Cell-Extrinsic Suppressor Function

PubMed Central

Mackroth, Maria Sophia; Abel, Annemieke; Steeg, Christiane; Schulze zur Wiesch, Julian; Jacobs, Thomas

2016-01-01

In acute Plasmodium falciparum (P. falciparum) malaria, the pro- and anti-inflammatory immune pathways must be delicately balanced so that the parasitemia is controlled without inducing immunopathology. An important mechanism to fine-tune T cell responses in the periphery is the induction of coinhibitory receptors such as CTLA4 and PD1. However, their role in acute infections such as P. falciparum malaria remains poorly understood. To test whether coinhibitory receptors modulate CD4+ T cell functions in malaria, blood samples were obtained from patients with acute P. falciparum malaria treated in Germany. Flow cytometric analysis showed a more frequent expression of CTLA4 and PD1 on CD4+ T cells of malaria patients than of healthy control subjects. In vitro stimulation with P. falciparum-infected red blood cells revealed a distinct population of PD1+CTLA4+CD4+ T cells that simultaneously produced IFNγ and IL10. This antigen-specific cytokine production was enhanced by blocking PD1/PDL1 and CTLA4. PD1+CTLA4+CD4+ T cells were further isolated based on surface expression of PD1 and their inhibitory function investigated in-vitro. Isolated PD1+CTLA4+CD4+ T cells suppressed the proliferation of the total CD4+ population in response to anti-CD3/28 and plasmodial antigens in a cell-extrinsic manner. The response to other specific antigens was not suppressed. Thus, acute P. falciparum malaria induces P. falciparum-specific PD1+CTLA4+CD4+ Teffector cells that coproduce IFNγ and IL10, and inhibit other CD4+ T cells. Transient induction of regulatory Teffector cells may be an important mechanism that controls T cell responses and might prevent severe inflammation in patients with malaria and potentially other acute infections. PMID:27802341

A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface

PubMed Central

Oberli, Alexander; Slater, Leanne M.; Cutts, Erin; Brand, Françoise; Mundwiler-Pachlatko, Esther; Rusch, Sebastian; Masik, Martin F. G.; Erat, Michèle C.; Beck, Hans-Peter; Vakonakis, Ioannis

2014-01-01

Uniquely among malaria parasites, Plasmodium falciparum-infected erythrocytes (iRBCs) develop membrane protrusions, known as knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed subtelomeric (PHIST) domains has attracted attention, with members being implicated in host-parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 μM, comigrates with PfEMP1 during export, and locates in knobs. PHIST variants that do not locate in knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1-cytoskeletal connection in knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome.—Oberli, A., Slater, L. M., Cutts, E., Brand, F., Mundwiler-Pachlatko, E., Rusch, S., Masik, M. F. G., Erat, M. C., Beck, H.-P., Vakonakis, I. A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface. PMID:24983468

Trans-kingdom small RNA transfer during host-pathogen interactions: The case of P. falciparum and erythrocytes.

PubMed

Walzer, Katelyn A; Chi, Jen-Tsan

2017-04-03

This review focuses on the role of trans-kingdom movement of small RNA (sRNA) molecules between parasites, particularly Plasmodium falciparum, and their respective host cells. While the intercellular transfer of sRNAs within organisms is well recognized, recent studies illustrate many examples of trans-kingdom sRNA exchange within the context of host-parasite interactions. These interactions are predominantly found in the transfer of host sRNAs between erythrocytes and the invading P. falciparum, as well as other host cell types. In addition, parasite-encoded sRNAs can also be transferred to host cells to evade the immune system. The transport of these parasite sRNAs in the body fluids of the host may also offer means to detect and monitor the parasite infection. These isolated examples may only represent the tip of the iceberg in which the transfer of sRNA between host and parasites is a critical aspect of host-pathogen interactions. In addition, the levels of these sRNAs and their speed of transfer may vary dramatically under different contexts to push the biologic equilibrium toward the benefit of hosts vs. parasites. Therefore, these sRNA transfers may offer potential strategies to detect, prevent or treat parasite infections. Here, we review a brief history of the discovery of host erythrocyte sRNAs, their transfers and interactions in the context of P. falciparum infection. We also provide examples and discuss the functional significance of the reciprocal transfer of parasite-encoded sRNAs into hosts. These understandings of sRNA exchanges are put in the context of their implications for parasite pathogenesis, host defenses and the evolution of host polymorphisms driven by host interactions with these parasites.

Pro-inflammatory Cytokine Response and Genetic Diversity in Merozoite Surface Protein 2 of Plasmodium falciparum Isolates from Nigeria.

PubMed

Ajibaye, Olusola; Osuntoki, Akinniyi A; Ebuehi, Albert Ot; Iwalokun, Bamidele A; Balogun, Emmanuel O; Egbuna, Kathleen N

2017-01-01

Polymorphisms in Plasmodium falciparum merozoite surface protein-2 ( msp -2) and associated parasite genetic diversity which varies between malaria-endemic regions remain a limitation in malaria vaccine development. Pro-inflammatory cytokines are important in immunity against malaria, understanding the influence of genetic diversity on cytokine response is important for effective vaccine design. P. falciparum isolates obtained from 300 Nigerians with uncomplicated falciparum malaria at Ijede General Hospital, Ijede (IJE), General Hospital Ajeromi, Ajeromi (AJE) and Saint Kizito Mission Hospital, Lekki, were genotyped by nested polymerase chain reaction of msp -2 block 3 while ELISA was used to determine the pro-inflammatory cytokine response to describe the genetic diversity of P. falciparum . Eighteen alleles were observed for msp -2 loci. Of the 195 isolates, 61 (31.0%) had only FC27-type alleles, 38 (19.7%) had only 3D7-type alleles, and 49.3% had multiple parasite lines with both alleles. Band sizes were 275-625 bp for FC27 and 150-425 bp for 3D7. Four alleles were observed from LEK, 2 (375-425 bp) and 2 (275-325 bp) of FC27-and 3D7-types, respectively; 12 alleles from AJE, 9 (275-625 bp) and 3 (325-425 bp) of FC27-types and 3D7-types, respectively; while IJE had a total of 12 alleles, 9 (275-625 bp) and 3 (325-425 bp) of FC27-types and 3D7-types, respectively. Mean multiplicity of infection (MOI) was 1.54. Heterozygosity ( H E ) ranged from 0.77 to 0.87 and was highest for IJE (0.87). Cytokine response was higher among <5 years and was significantly associated with MOI ( P > 0.05) but with neither parasite density nor infection type. P. falciparum genetic diversity is extensive in Nigeria, protection via pro-inflammatory cytokines have little or no interplay with infection multiplicity.

The D113N mutation in the RING E3 ubiquitin protein ligase gene is not associated with ex vivo susceptibility to common anti-malarial drugs in African Plasmodium falciparum isolates.

PubMed

Gendrot, Mathieu; Foguim, Francis Tsombeng; Robert, Marie Gladys; Amalvict, Rémy; Mosnier, Joel; Benoit, Nicolas; Madamet, Marylin; Pradines, Bruno

2018-03-12

Plasmodium falciparum resistance to artemisinin-based combination therapy has emerged and spread in Southeast Asia. In areas where artemisinin resistance is emerging, the efficacy of combination is now based on partner drugs. In this context, the identification of novel markers of resistance is essential to monitor the emergence and spread of resistance to these partner drugs. The ubiquitylation pathway could be a possible target for anti-malarial compounds and might be involved in resistance. Polymorphisms in the E3 ubiquitin-protein ligase (PF3D7_0627300) gene could be associated with decreased in vitro susceptibility to anti-malarial drugs. Plasmodium falciparum isolates were collected from patients hospitalized in France with imported malaria from a malaria-endemic country from January 2015 to December 2016 and, more particularly, from African French-speaking countries. In total, 215 isolates were successfully sequenced for the E3 ubiquitin-protein ligase gene and assessed for ex vivo susceptibility to anti-malarial drugs. The D113N mutation in the RING E3 ubiquitin-protein ligase gene was present in 147 out of the 215 samples (68.4%). The IC 50 values for the ten anti-malarial drugs were not significantly different between the wild-type and mutant parasites (p values between 0.225 and 0.933). There was no significant difference in terms of the percentage of parasites with decreased susceptibility between the D113 wild-type and the 133N mutated P. falciparum strains (p values between 0.541 and 1). The present data confirmed the absence of the association between polymorphisms in the RING E3 ubiquitin-protein ligase gene and the ex vivo susceptibility to common anti-malarial drugs in African P. falciparum isolates.

Prevalence of Plasmodium falciparum Molecular Markers of Antimalarial Drug Resistance in a Residual Malaria Focus Area in Sabah, Malaysia

PubMed Central

Mohd Abd Razak, Mohd Ridzuan; Abdullah, Noor Rain; Sastu, Umi Rubiah; Imwong, Mallika; Muniandy, Prem Kumar; Saat, Muhammad Nor Farhan; Muhammad, Amirrudin; Jelip, Jenarun; Tikuson, Moizin; Yusof, Norsalleh; Rundi, Christina; Mudin, Rose Nani; Syed Mohamed, Ami Fazlin

2016-01-01

Chloroquine (CQ) and fansidar (sulphadoxine-pyrimethamine, SP) were widely used for treatment of Plasmodium falciparum for several decades in Malaysia prior to the introduction of Artemisinin-based Combination Therapy (ACT) in 2008. Our previous study in Kalabakan, located in south-east coast of Sabah showed a high prevalence of resistance to CQ and SP, suggesting the use of the treatment may no longer be effective in the area. This study aimed to provide a baseline data of antimalarial drug resistant markers on P. falciparum isolates in Kota Marudu located in the north-east coast of Sabah. Mutations on genes associated with CQ (pfcrt and pfmdr1) and SP (pfdhps and pfdhfr) were assessed by PCR amplification and restriction fragment length polymorphism. Mutations on the kelch13 marker (K13) associated with artemisinin resistance were determined by DNA sequencing technique. The assessment of pfmdr1 copy number variation associated with mefloquine resistant was done by real-time PCR technique. A low prevalence (6.9%) was indicated for both pfcrt K76T and pfmdr1 N86Y mutations. All P. falciparum isolates harboured the pfdhps A437G mutation. Prevalence of pfdhfr gene mutations, S108N and I164L, were 100% and 10.3%, respectively. Combining the different resistant markers, only two isolates were conferred to have CQ and SP treatment failure markers as they contained mutant alleles of pfcrt and pfmdr1 together with quintuple pfdhps/pfdhfr mutation (combination of pfdhps A437G+A581G and pfdhfr C59R+S108N+I164L). All P. falciparum isolates carried single copy number of pfmdr1 and wild type K13 marker. This study has demonstrated a low prevalence of CQ and SP resistance alleles in the study area. Continuous monitoring of antimalarial drug efficacy is warranted and the findings provide information for policy makers in ensuring a proper malaria control. PMID:27788228

Impact of Human Immunodeficiency Virus Infection in Pregnant Women on Variant-Specific Immunity to Malaria▿

PubMed Central

Dembo, Edson G.; Mwapasa, Victor; Montgomery, Jacqui; Craig, Alister G.; Porter, Kimberly A.; Meshnick, Steven R.; Molyneux, Malcolm E.; Rogerson, Stephen J.

2008-01-01

Human immunodeficiency virus (HIV) increases susceptibility to Plasmodium falciparum infection, and this has most clearly been demonstrated in pregnant women. Variant surface antigens on the surfaces of erythrocytes infected with P. falciparum are major targets of protective immunity. We studied the impact of HIV infection on pregnant women's humoral immunity to variant surface antigens expressed by placental and pediatric isolates of P. falciparum. By flow cytometry, sera from HIV-infected women more frequently lacked antibodies to these antigens than sera from HIV-uninfected women. This difference was similar in magnitude for pediatric isolates (unadjusted odds ratio [OR] = 6.36; 95% confidence interval [CI] = 1.14, 35.32; P < 0.05) and placental isolates (unadjusted OR = 6.47; 95% CI = 0.75, 55.64; P < 0.10). We divided women into high and low responders on the basis of their antibody levels. After adjustment for CD4 count, maternal age, and gravidity, we found that HIV-infected women more frequently had low responses to both pediatric isolates (OR = 5.34; 95% CI = 1.23, 23.16; P = 0.025) and placental isolates (OR = 4.14; 95% CI = 1.71, 10.02; P = 0.002). The relative quantity of antibodies to both pediatric isolates (P = 0.035) and placental isolates (P = 0.005) was lower in HIV-infected women than in HIV-uninfected women. HIV infection has a broad impact on variant-specific immunity, which may explain the susceptibility of infected individuals to clinical malaria episodes. PMID:18199738

Refrigeration provides a simple means to synchronize in vitro cultures of Plasmodium falciparum.

PubMed

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

2014-05-01

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.

Refrigeration provides a simple means to synchronize in vitro cultures of Plasmodium falciparum

PubMed Central

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

2014-01-01

Plasmodium falciparum is usually asynchronous during in vitro culture. Highly synchronized cultures of Plasmodium 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. PMID:24632190

High performance of histidine-rich protein 2 based rapid diagnostic tests in French Guiana are explained by the absence of pfhrp2 gene deletion in P. falciparum.

PubMed

Trouvay, Mélanie; Palazon, Georges; Berger, Franck; Volney, Béatrice; Blanchet, Denis; Faway, Emilie; Donato, Damien; Legrand, Eric; Carme, Bernard; Musset, Lise

2013-01-01

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. 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. 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. 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.

False-negative malaria rapid diagnostic tests in Rwanda: impact of Plasmodium falciparum isolates lacking hrp2 and declining malaria transmission.

PubMed

Kozycki, Christina T; Umulisa, Noella; Rulisa, Stephen; Mwikarago, Emil I; Musabyimana, Jean Pierre; Habimana, Jean Pierre; Karema, Corine; Krogstad, Donald J

2017-03-20

Rapid diagnostic tests (RDTs) for histidine rich protein 2 (HRP2) are often used to determine whether persons with fever should be treated with anti-malarials. However, Plasmodium falciparum parasites with a deletion of the hrp2 gene yield false-negative RDTs and there are concerns the sensitivity of HRP2-based RDTs may fall when the intensity of transmission decreases. This observational study enrolled 9226 patients at three health centres in Rwanda from April 2014 to April 2015. It then compared the sensitivity of RDTs based on HRP2 and the Plasmodium lactate dehydrogenase (pLDH) to microscopy (thick smears) for the diagnosis of malaria. PCR was used to determine whether deletions of the histidine-rich central repeat region of the hrp2 gene (exon 2) were associated with false-negative HRP2-based RDTs. In comparison to microscopy, the sensitivity and specificity of HRP2- and pLDH-based RDTs were 89.5 and 86.2% and 80.2 and 94.3%, respectively. When the results for both RDTs were combined, sensitivity rose to 91.8% and specificity was 85.7%. Additionally, when smear positivity fell from 46 to 3%, the sensitivity of the HRP2-based RDT fell from 88 to 67%. Of 370 samples with false-negative HRP2 RDT results for which PCR was performed, 140 (38%) were identified as P. falciparum by PCR. Of the isolates identified as P. falciparum by PCR, 32 (23%) were negative for the hrp2 gene based on PCR. Of the 32 P. falciparum isolates negative for hrp2 by PCR, 17 (53%) were positive based on the pLDH RDT. This prospective study of RDT performance coincided with a decline in the intensity of malaria transmission in Kibirizi (fall in slide positivity from 46 to 3%). This decline was associated with a decrease in HRP2 RDT sensitivity (from 88 to 67%). While P. falciparum isolates without the hrp2 gene were an important cause of false-negative HRP2-based RDTs, most were identified by the pLDH-based RDT. Although WHO does not recommend the use of combined HRP2/pLDH testing in sub-Saharan Africa, these results suggest that combination HRP2/pLDH-based RDTs could reduce the impact of false-negative HRP2-based RDTs for detection of symptomatic P. falciparum malaria.

Mirincamycin, an old candidate for malaria combination treatment and prophylaxis in the 21st century: in vitro interaction profiles with potential partner drugs in continuous culture and field isolates.

PubMed

Starzengruber, Peter; Fuehrer, Hans-Peter; Swoboda, Paul; Ganesh, Deepa; Haque, Rashidul; Khan, Wasif A; Graninger, Wolfgang; Noedl, Harald

2014-06-10

Spreading resistance of Plasmodium falciparum to existing drugs calls for the search for novel anti-malarial drugs and combinations for the treatment of falciparum malaria. In vitro and ex vivo investigations were conducted with fresh P. falciparum field isolates and culture-adapted P. falciparum clones to evaluate the anti-malarial potential of mirincamycin, a lincosamide, alone and in combination with tafenoquine (TQ), dihydroartemisinin (DHA), and chloroquine (CQ). All samples were tested in a histidine-rich protein 2 (HRP2) drug susceptibility assay. Interaction analysis showed additive to synergistic interaction profiles with these potential partner drugs, with an overall geometric mean fractional inhibitory concentration at 50% inhibition (FIC₅₀) of 0.78, 0.80 and 0.80 for mirincamycin with TQ, DHA, and CQ, respectively. Antagonism was not found in any of the tested field isolates or clones. The strongest tendency toward synergy (i.e. the lowest FIC) was seen with a combination ratio of 1:0.27 to 1:7.2 (mean 1:2.7) for the combination with tafenoquine. The optimal combination ratios for DHA and CQ were 1:444.4 to 1:36,000 (mean 1:10,755.5) and 1:2.7 to 1:216 (mean 1:64.5), respectively. No evidence of an activity correlation (i.e. potential cross-resistance) with DHA, mefloquine, quinine or chloroquine was seen whereas a significant correlation with the activity of clindamycin and azithromycin was detected. Mirincamycin combinations may be promising candidates for further clinical investigations in the therapy and prophylaxis of multidrug-resistant falciparum malaria or in combination with 4 or 8-aminoquinolines for the treatment and relapse prevention of vivax malaria.

Mirincamycin, an old candidate for malaria combination treatment and prophylaxis in the 21st century: in vitro interaction profiles with potential partner drugs in continuous culture and field isolates

PubMed Central

2014-01-01

Background Spreading resistance of Plasmodium falciparum to existing drugs calls for the search for novel anti-malarial drugs and combinations for the treatment of falciparum malaria. Methods In vitro and ex vivo investigations were conducted with fresh P. falciparum field isolates and culture-adapted P. falciparum clones to evaluate the anti-malarial potential of mirincamycin, a lincosamide, alone and in combination with tafenoquine (TQ), dihydroartemisinin (DHA), and chloroquine (CQ). All samples were tested in a histidine-rich protein 2 (HRP2) drug susceptibility assay. Results Interaction analysis showed additive to synergistic interaction profiles with these potential partner drugs, with an overall geometric mean fractional inhibitory concentration at 50% inhibition (FIC50) of 0.78, 0.80 and 0.80 for mirincamycin with TQ, DHA, and CQ, respectively. Antagonism was not found in any of the tested field isolates or clones. The strongest tendency toward synergy (i.e. the lowest FIC) was seen with a combination ratio of 1:0.27 to 1:7.2 (mean 1:2.7) for the combination with tafenoquine. The optimal combination ratios for DHA and CQ were 1:444.4 to 1:36,000 (mean 1:10,755.5) and 1:2.7 to 1:216 (mean 1:64.5), respectively. No evidence of an activity correlation (i.e. potential cross-resistance) with DHA, mefloquine, quinine or chloroquine was seen whereas a significant correlation with the activity of clindamycin and azithromycin was detected. Conclusions Mirincamycin combinations may be promising candidates for further clinical investigations in the therapy and prophylaxis of multidrug-resistant falciparum malaria or in combination with 4 or 8-aminoquinolines for the treatment and relapse prevention of vivax malaria. PMID:24916383

Genetic evidence for contribution of human dispersal to the genetic diversity of EBA-175 in Plasmodium falciparum.

PubMed

Yasukochi, Yoshiki; Naka, Izumi; Patarapotikul, Jintana; Hananantachai, Hathairad; Ohashi, Jun

2015-08-01

The 175-kDa erythrocyte binding antigen (EBA-175) of Plasmodium falciparum plays a crucial role in merozoite invasion into human erythrocytes. EBA-175 is believed to have been under diversifying selection; however, there have been no studies investigating the effect of dispersal of humans out of Africa on the genetic variation of EBA-175 in P. falciparum. The PCR-direct sequencing was performed for a part of the eba-175 gene (regions II and III) using DNA samples obtained from Thai patients infected with P. falciparum. The divergence times for the P. falciparum eba-175 alleles were estimated assuming that P. falciparum/Plasmodium reichenowi divergence occurred 6 million years ago (MYA). To examine the possibility of diversifying selection, nonsynonymous and synonymous substitution rates for Plasmodium species were also estimated. A total of 32 eba-175 alleles were identified from 131 Thai P. falciparum isolates. Their estimated divergence time was 0.13-0.14 MYA, before the exodus of humans from Africa. A phylogenetic tree for a large sequence dataset of P. falciparum eba-175 alleles from across the world showed the presence of a basal Asian-specific cluster for all P. falciparum sequences. A markedly more nonsynonymous substitutions than synonymous substitutions in region II in P. falciparum was also detected, but not within Plasmodium species parasitizing African apes, suggesting that diversifying selection has acted specifically on P. falciparum eba-175. Plasmodium falciparum eba-175 genetic diversity appeared to increase following the exodus of Asian ancestors from Africa. Diversifying selection may have played an important role in the diversification of eba-175 allelic lineages. The present results suggest that the dispersals of humans out of Africa influenced significantly the molecular evolution of P. falciparum EBA-175.

Molecular identification of sulfadoxine-pyrimethamine resistance in malaria infected women who received intermittent preventive treatment in the Democratic Republic of Congo.

PubMed

Ruh, Emrah; Bateko, Jean Paul; Imir, Turgut; Taylan-Ozkan, Aysegul

2018-01-09

Point mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes which confer resistance to sulfadoxine-pyrimethamine (SP) occur at increasing rates. The present study aimed to identify Pfdhfr and Pfdhps mutations in P. falciparum isolates recovered from women who received two doses of SP during pregnancy in Bandundu, the Democratic Republic of Congo (DRC). A total of 48 women with confirmed P. falciparum infection were enrolled in the study. Finger-prick blood samples that were collected on filter paper at the time of delivery were used for DNA isolation. Pfdhfr and Pfdhps genes were amplified by a nested PCR protocol. DNA sequencing was performed on both strands, and the point mutations were analysed. All of the 48 (100.0%) P. falciparum isolates carried at least one polymorphism in both genes. The wild-type haplotypes of Pfdhfr (CNCSI [C50, N51, C59, S108, I164]) and Pfdhps (SAKAA [S436, A437, K540, A581, A613]) were not observed in the study. In Pfdhfr, N51I (85.4%), C59R (60.4%), and S108N (100.0%) polymorphisms were detected. Triple mutation (CIRNI) (mutant amino acids are underlined) was the most prevalent (47.9%) Pfdhfr haplotype. In the study, all P. falciparum isolates (100.0%) harboured the A437G allele in Pfdhps gene. Also, K540E and A581G polymorphisms were observed in one (2.1%) isolate. Single mutant haplotype (SGKAA) was detected in 97.9% of the isolates. Mutant Pfdhfr and Pfdhps allele combinations revealed quintuple (CICNI-SGEGA; 2.1%), quadruple (CIRNI-SGKAA; 47.9%), triple (CICNI-SGKAA; 35.4%, CNRNI-SGKAA; 12.5%), and double (CNCNI-SGKAA; 2.1%) haplotypes. In the study, the rate of SGEGA haplotype was low (2.1%). Although K540E and A581G alleles are more common in Eastern Africa, a distinct lineage of SGEGA is also present in the DRC, which is located in Central Africa. This haplotype is associated with decreased efficacy of SP in pregnant women and infants, therefore, it should be carefully considered in the DRC and SP resistance should be routinely monitored.

In vitro inhibition of Plasmodium falciparum by substances isolated from Amazonian antimalarial plants.

PubMed

de Andrade-Neto, Valter F; Pohlit, Adrian M; Pinto, Ana Cristina S; Silva, Ellen Cristina C; Nogueira, Karla L; Melo, Márcia R S; Henrique, Marycleuma C; Amorim, Rodrigo C N; Silva, Luis Francisco R; Costa, Mônica R F; Nunomura, Rita C S; Nunomura, Sergio M; Alecrim, Wilson D; Alecrim, M das Graças C; Chaves, F Célio M; Vieira, Pedro Paulo R

2007-06-01

In the present study, a quassinoid, neosergeolide, isolated from the roots and stems of Picrolemma sprucei (Simaroubaceae), the indole alkaloids ellipticine and aspidocarpine, isolated from the bark of Aspidosperma vargasii and A. desmanthum (Apocynaceae), respectively, and 4-nerolidylcatechol, isolated from the roots of Pothomorphe peltata (Piperaceae), all presented significant in vitro inhibition (more active than quinine and chloroquine) of the multi-drug resistant K1 strain of Plasmodium falciparum. Neosergeolide presented activity in the nanomolar range. This is the first report on the antimalarial activity of these known, natural compounds. This is also the first report on the isolation of aspidocarpine from A. desmanthum. These compounds are good candidates for pre-clinical tests as novel lead structures with the aim of finding new antimalarial prototypes and lend support to the traditional use of the plants from which these compounds are derived.

Case Report: A Case of Plasmodium falciparum hrp2 and hrp3 Gene Mutation in Bangladesh.

PubMed

Nima, Maisha Khair; Hougard, Thomas; Hossain, Mohammad Enayet; Kibria, Mohammad Golam; Mohon, Abu Naser; Johora, Fatema Tuj; Rahman, Rajibur; Haque, Rashidul; Alam, Mohammad Shafiul

2017-10-01

Several species of Plasmodium are responsible for causing malaria in humans. Proper diagnoses are crucial to case management, because severity and treatment varies between species. Diagnoses can be made using rapid diagnostic tests (RDTs), which detect Plasmodium proteins. Plasmodium falciparum causes the most virulent cases of malaria, and P. falciparum histidine-rich protein 2 (PfHRP2) is a common target of falciparum malaria RDTs. Here we report a case in which a falciparum malaria patient in Bangladesh tested negative on PfHRP2-based RDTs. The negative results can be attributed to a deletion of part of the pfhrp2 gene and frameshift mutations in both pfhrp2 and pfhrp3 gene. This finding may have implications for malaria diagnostics and case management in Bangladesh and other regions of South Asia.

Deletion of Plasmodium falciparum Histidine-Rich Protein 2 (pfhrp2) and Histidine-Rich Protein 3 (pfhrp3) Genes in Colombian Parasites.

PubMed

Murillo Solano, Claribel; Akinyi Okoth, Sheila; Abdallah, Joseph F; Pava, Zuleima; Dorado, Erika; Incardona, Sandra; Huber, Curtis S; Macedo de Oliveira, Alexandre; Bell, David; Udhayakumar, Venkatachalam; Barnwell, John W

2015-01-01

A number of studies have analyzed the performance of malaria rapid diagnostic tests (RDTs) in Colombia with discrepancies in performance being attributed to a combination of factors such as parasite levels, interpretation of RDT results and/or the handling and storage of RDT kits. However, some of the inconsistencies observed with results from Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs could also be explained by the deletion of the gene that encodes the protein, pfhrp2, and its structural homolog, pfhrp3, in some parasite isolates. Given that pfhrp2- and pfhrp3-negative P. falciparum isolates have been detected in the neighboring Peruvian and Brazilian Amazon regions, we hypothesized that parasites with deletions of pfhrp2 and pfhrp3 may also be present in Colombia. In this study we tested 100 historical samples collected between 1999 and 2009 from six Departments in Colombia for the presence of pfhrp2, pfhrp3 and their flanking genes. Seven neutral microsatellites were also used to determine the genetic background of these parasites. In total 18 of 100 parasite isolates were found to have deleted pfhrp2, a majority of which (14 of 18) were collected from Amazonas Department, which borders Peru and Brazil. pfhrp3 deletions were found in 52 of the 100 samples collected from all regions of the country. pfhrp2 flanking genes PF3D7_0831900 and PF3D7_0831700 were deleted in 22 of 100 and in 1 of 100 samples, respectively. pfhrp3 flanking genes PF3D7_1372100 and PF3D7_1372400 were missing in 55 of 100 and in 57 of 100 samples. Structure analysis of microsatellite data indicated that Colombian samples tested in this study belonged to four clusters and they segregated mostly based on their geographic region. Most of the pfhrp2-deleted parasites were assigned to a single cluster and originated from Amazonas Department although a few pfhrp2-negative parasites originated from the other three clusters. The presence of a high proportion of pfhrp2-negative isolates in the Colombian Amazon may have implications for the use of PfHRP2-based RDTs in the region and may explain inconsistencies observed when PfHRP2-based tests and assays are performed.

Deletion of Plasmodium falciparum Histidine-Rich Protein 2 (pfhrp2) and Histidine-Rich Protein 3 (pfhrp3) Genes in Colombian Parasites

PubMed Central

Murillo Solano, Claribel; Akinyi Okoth, Sheila; Abdallah, Joseph F.; Pava, Zuleima; Dorado, Erika; Incardona, Sandra; Huber, Curtis S.; Macedo de Oliveira, Alexandre; Bell, David; Udhayakumar, Venkatachalam; Barnwell, John W.

2015-01-01

A number of studies have analyzed the performance of malaria rapid diagnostic tests (RDTs) in Colombia with discrepancies in performance being attributed to a combination of factors such as parasite levels, interpretation of RDT results and/or the handling and storage of RDT kits. However, some of the inconsistencies observed with results from Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs could also be explained by the deletion of the gene that encodes the protein, pfhrp2, and its structural homolog, pfhrp3, in some parasite isolates. Given that pfhrp2- and pfhrp3-negative P. falciparum isolates have been detected in the neighboring Peruvian and Brazilian Amazon regions, we hypothesized that parasites with deletions of pfhrp2 and pfhrp3 may also be present in Colombia. In this study we tested 100 historical samples collected between 1999 and 2009 from six Departments in Colombia for the presence of pfhrp2, pfhrp3 and their flanking genes. Seven neutral microsatellites were also used to determine the genetic background of these parasites. In total 18 of 100 parasite isolates were found to have deleted pfhrp2, a majority of which (14 of 18) were collected from Amazonas Department, which borders Peru and Brazil. pfhrp3 deletions were found in 52 of the100 samples collected from all regions of the country. pfhrp2 flanking genes PF3D7_0831900 and PF3D7_0831700 were deleted in 22 of 100 and in 1 of 100 samples, respectively. pfhrp3 flanking genes PF3D7_1372100 and PF3D7_1372400 were missing in 55 of 100 and in 57 of 100 samples. Structure analysis of microsatellite data indicated that Colombian samples tested in this study belonged to four clusters and they segregated mostly based on their geographic region. Most of the pfhrp2-deleted parasites were assigned to a single cluster and originated from Amazonas Department although a few pfhrp2-negative parasites originated from the other three clusters. The presence of a high proportion of pfhrp2-negative isolates in the Colombian Amazon may have implications for the use of PfHRP2-based RDTs in the region and may explain inconsistencies observed when PfHRP2-based tests and assays are performed. PMID:26151448

Infectivity of Plasmodium falciparum sporozoites determines emerging parasitemia in infected volunteers.

PubMed

McCall, Matthew B B; Wammes, Linda J; Langenberg, Marijke C C; van Gemert, Geert-Jan; Walk, Jona; Hermsen, Cornelus C; Graumans, Wouter; Koelewijn, Rob; Franetich, Jean-François; Chishimba, Sandra; Gerdsen, Max; Lorthiois, Audrey; van de Vegte, Marga; Mazier, Dominique; Bijker, Else M; van Hellemond, Jaap J; van Genderen, Perry J J; Sauerwein, Robert W

2017-06-21

Malaria sporozoites must first undergo intrahepatic development before a pathogenic blood-stage infection is established. The success of infection depends on host and parasite factors. In healthy human volunteers undergoing controlled human malaria infection (CHMI), we directly compared three clinical Plasmodium falciparum isolates for their ability to infect primary human hepatocytes in vitro and to drive the production of blood-stage parasites in vivo. Our data show a correlation between the efficiency of strain-specific sporozoite invasion of human hepatocytes and the dynamics of patent parasitemia in study subjects, highlighting intrinsic differences in infectivity among P. falciparum isolates from distinct geographical locales. The observed heterogeneity in infectivity among strains underscores the value of assessing the protective efficacy of candidate malaria vaccines against heterologous strains in the CHMI model. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9.

PubMed Central

Day, K P; Karamalis, F; Thompson, J; Barnes, D A; Peterson, C; Brown, H; Brown, G V; Kemp, D J

1993-01-01

Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria. Images Fig. 1 Fig. 2 Fig. 3 PMID:8367496

Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and clindamycin-resistant parasites

PubMed Central

Dharia, Neekesh V.; Plouffe, David; Bopp, Selina E.R.; González-Páez, Gonzalo E.; Lucas, Carmen; Salas, Carola; Soberon, Valeria; Bursulaya, Badry; Kochel, Tadeusz J.; Bacon, David J.; Winzeler, Elizabeth A.

2010-01-01

Here, we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos region using genome scanning, a microarray-based technique that delineates the majority of single-base changes, indels, and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon bears a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes, indicating mutations arising during self-mating or mitotic replication. The data also reveal that one or two meioses separate different isolates, showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pairwise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species, but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase of clindamycin EC50 in strains harboring one of these mutations. This evidence of clindamycin-resistant parasites in the Amazon suggests that a shift should be made in health policy away from quinine + clindamycin therapy for malaria in pregnant women and infants, and that the development of new lincosamide antibiotics for malaria should be reconsidered. PMID:20829224

In vitro sensitivity of Plasmodium falciparum to artesunate in Thailand.

PubMed Central

Wongsrichanalai, C.; Wimonwattrawatee, T.; Sookto, P.; Laoboonchai, A.; Heppner, D. G.; Kyle, D. E.; Wernsdorfer, W. H.

1999-01-01

Reported are the in vitro susceptibilities of Plasmodium falciparum to artesunate, mefloquine, quinine and chloroquine of 86 isolates and to dihydroartemisinin of 45 isolates collected from areas of high resistance to mefloquine within Thailand near the borders with Myanmar and Cambodia, and from southern Thailand where P. falciparum is generally still sensitive to mefloquine. All the isolates were highly sensitive to artesunate, but the geometric mean IC50S were higher in isolates from the Thai-Myanmar and Thai-Cambodian borders than in those from southern Thailand. The IC50S for mefloquine and artesunate were strongly correlated (Pearson r = 0.605; n = 86; P < 0.00001). As expected, the in vitro sensitivities to dihydroartemisinin and artesunate were similar and strongly correlated (at IC50, Pearson r = 0.695; n = 45; P < 0.00002). The correlation between the activity of mefloquine and artesunate requires further investigation in order to determine the potential for development of cross-resistance in nature. Our results suggest that combination with mefloquine is not the ideal way of protecting the usefulness of artemisinin and its derivatives. A search for more suitable partner drugs to these compounds and careful regulation of their use are necessary in the interest of ensuring their long therapeutic life span. PMID:10361756

Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia.

PubMed

Rachid Viana, Giselle Maria; Akinyi Okoth, Sheila; Silva-Flannery, Luciana; Lima Barbosa, Danielle Regina; Macedo de Oliveira, Alexandre; Goldman, Ira F; Morton, Lindsay C; Huber, Curtis; Anez, Arletta; Dantas Machado, Ricardo Luiz; Aranha Camargo, Luís Marcelo; Costa Negreiros do Valle, Suiane; Marins Póvoa, Marinete; Udhayakumar, Venkatachalam; Barnwell, John W

2017-01-01

More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region.

Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia

PubMed Central

Rachid Viana, Giselle Maria; Akinyi Okoth, Sheila; Silva-Flannery, Luciana; Lima Barbosa, Danielle Regina; Macedo de Oliveira, Alexandre; Goldman, Ira F.; Morton, Lindsay C.; Huber, Curtis; Anez, Arletta; Dantas Machado, Ricardo Luiz; Aranha Camargo, Luís Marcelo; Costa Negreiros do Valle, Suiane; Marins Póvoa, Marinete; Barnwell, John W.

2017-01-01

More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region. PMID:28301474

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

PubMed Central

2013-01-01

Background A markedly high failure rate of three-day artesunate-mefloquine was observed in the area along the Thai-Myanmar border. Methods 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. Results 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. Conclusions Despite the evidence of a low level of a decline in sensitivity of P. falciparum isolates to artemisinins in areas along the Thai-Myanmar border, artemisinin-based combination therapy (ACT) would be expected to remain the key anti-malarial drug for treatment of multidrug resistance P. falciparum. Continued monitoring and active surveillance of clinical efficacy of ACT, including identification of true artemisinin resistant parasites, is required for appropriate implementation of malaria control policy in this area. PMID:23898808

PFMDR1 POLYMORPHISMS INFLUENCE ON IN VITRO SENSITIVITY OF THAI PLASMODIUM FALCIPARUM ISOLATES TO PRIMAQUINE, SITAMAQUINE AND TAFENOQUINE.

PubMed

Kaewpruk, Napaporn; Tan-ariya, Peerapan; Ward, Stephen A; Sitthichot, Naruemon; Suwandittakul, Nantana; Mungthin, Mathirut

2016-05-01

Primaquine (PQ), an 8-aminoquinoline, is considered a tissue schizonticide drug for radical cure in vivax and ovale malaria, with minimal impact on asexual erythrocytic stages at therapeutic concentrations. Tafenoquine (TQ), a new 8-aminoquinoline analog of PQ, is active against both malaria parasite tissue and blood stages and is being promoted as a drug candidate for antimalarial chemotherapy and chemoprophylaxis and potential transmission blocking against Plasmodium vivax and P. falciparum. This study compared in vitro sensitivity of Thai P. falciparum isolates against three 8-aminoquinolines, PQ, TQ and sitamaquine (SQ), a related 8-aminoquinoline and assessed the importance of pfmdr1 polymorphism on the in vitro response. Seventy-eight laboratory adapted Thai P. falciparum isolates were evaluated for in vitro sensitivity to the three 8-aminoquinolines using a radioisotopic assay, and pfmdr1 polymorphisms were determined using PCR-based methods. All three drugs have weak antiplasmodial activity against asexual erythrocytic stage with SQ being the most potent by almost 10 folds. Cross susceptibility was observed in all three 8-aminoquinolines. Parasites containing pfmdr1 86Y, 184Y or 1034S allele exhibit significantly higher PQ IC₅₀. TQ sensitivity was reduced in those parasites containing pfmdr1 86Y, 1034S or 1042N allele. However, there was no significant influence of pfmdr1 alleles on SQ sensitivity. The data highlight unique differences among three representative 8-aminoquinoline drugs that may be useful in understanding their potential utility in antimalarial development.

Microsatellite analysis of chloroquine resistance associated alleles and neutral loci reveal genetic structure of Indian Plasmodium falciparum

PubMed Central

Mallick, Prashant K.; Sutton, Patrick L.; Singh, Ruchi; Singh, Om P.; Dash, Aditya P.; Singh, Ashok K.; Carlton, Jane M.; Bhasin, Virendra K.

2013-01-01

Efforts to control malignant malaria caused by Plasmodium falciparum are hampered by the parasite’s acquisition of resistance to antimalarial drugs, e.g., chloroquine. This necessitates evaluating the spread of chloroquine resistance in any malaria-endemic area. India displays highly variable malaria epidemiology and also shares porous international borders with malaria-endemic Southeast Asian countries having multi-drug resistant malaria. Malaria epidemiology in India is believed to be affected by two major factors: high genetic diversity and evolving drug resistance in P. falciparum. How transmission intensity of malaria can influence the genetic structure of chloroquine-resistant P. falciparum population in India is unknown. Here, genetic diversity within and among P. falciparum populations is analyzed with respect to their prevalence and chloroquine resistance observed in 13 different locations in India. Microsatellites developed for P. falciparum, including three putatively neutral and seven microsatellites thought to be under a hitchhiking effect due to chloroquine selection were used. Genetic hitchhiking is observed in five of seven microsatellites flanking the gene responsible for chloroquine resistance. Genetic admixture analysis and F-statistics detected genetically distinct groups in accordance with transmission intensity of different locations and the probable use of chloroquine. A large genetic break between the chloroquine-resistant parasite of the Northeast-East-Island group and Southwest group (FST = 0.253, P<0.001) suggests a long period of isolation or a possibility of different origin between them. A pattern of significant isolation by distance was observed in low transmission areas (r = 0.49, P=0.003, N = 83, Mantel test). An unanticipated pattern of spread of hitchhiking suggests genetic structure for Indian P. falciparum population. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation at both neutral and adaptive loci across India. PMID:23871774

Microsatellite analysis of chloroquine resistance associated alleles and neutral loci reveal genetic structure of Indian Plasmodium falciparum.

PubMed

Mallick, Prashant K; Sutton, Patrick L; Singh, Ruchi; Singh, Om P; Dash, Aditya P; Singh, Ashok K; Carlton, Jane M; Bhasin, Virendra K

2013-10-01

Efforts to control malignant malaria caused by Plasmodium falciparum are hampered by the parasite's acquisition of resistance to antimalarial drugs, e.g., chloroquine. This necessitates evaluating the spread of chloroquine resistance in any malaria-endemic area. India displays highly variable malaria epidemiology and also shares porous international borders with malaria-endemic Southeast Asian countries having multi-drug resistant malaria. Malaria epidemiology in India is believed to be affected by two major factors: high genetic diversity and evolving drug resistance in P. falciparum. How transmission intensity of malaria can influence the genetic structure of chloroquine-resistant P. falciparum population in India is unknown. Here, genetic diversity within and among P. falciparum populations is analyzed with respect to their prevalence and chloroquine resistance observed in 13 different locations in India. Microsatellites developed for P. falciparum, including three putatively neutral and seven microsatellites thought to be under a hitchhiking effect due to chloroquine selection were used. Genetic hitchhiking is observed in five of seven microsatellites flanking the gene responsible for chloroquine resistance. Genetic admixture analysis and F-statistics detected genetically distinct groups in accordance with transmission intensity of different locations and the probable use of chloroquine. A large genetic break between the chloroquine-resistant parasite of the Northeast-East-Island group and Southwest group (FST=0.253, P<0.001) suggests a long period of isolation or a possibility of different origin between them. A pattern of significant isolation by distance was observed in low transmission areas (r=0.49, P=0.003, N=83, Mantel test). An unanticipated pattern of spread of hitchhiking suggests genetic structure for Indian P. falciparum population. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation at both neutral and adaptive loci across India. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular mutation profile of pfcrt in Plasmodium falciparum isolates imported from Africa in Henan province.

PubMed

Zhou, Rui-Min; Zhang, Hong-Wei; Yang, Cheng-Yun; Liu, Ying; Zhao, Yu-Ling; Li, Su-Hua; Qian, Dan; Xu, Bian-Li

2016-05-10

Anti-malarial drug resistance is a primary public health problem. Haplotypes of pfcrt gene have been implicated to be molecular markers of chloroquine (CQ) resistance. This study aims to explore the prevalence of polymorphisms in pfcrt in Plasmodium falciparum-infected patients imported from Africa in Henan province. Blood samples were collected from 502 patients who were infected with P. falciparum returning from Africa in Henan province during 2012-2015. The single nucleotide polymorphisms in pfcrt (codons 72-76) were assessed by nested PCR with DNA sequencing and restriction digestion, the haplotype prevalences were also determined. Four haplotypes coding 72-76 of pfcrt were found including CVMNK (wild type), CVIET (mutation type), CVIEK (mutation type), and CV M/I N/E/D/K K/T (mixed type), with 61.95 % (311/502), 33.07 % (166/502), 0.20 % (1/502), and 4.78 % (24/502) prevalence, respectively. Except mixed type, CVIET and CVIEK were the largest proportion of the mutant type in West Africa, accounting for 44.83 % (91/203), followed by East Africa (8/21, 38.10 %), North Africa (4/11, 36.36 %), Central Africa (36/135, 26.67 %), and South Africa (28/132, 21.21 %). There was significant difference among the groups (χ(2) = 23.78, P < 0.05). Mixed type was the largest proportion in North Africa (9.09 %), followed by Central Africa (6.67 %), East Africa (4.76 %), South Africa (4.55 %), and West Africa (3.45 %). There was no significant difference among the groups (χ(2) = 2.31, P > 0.05). The position 72 and 73 of pfcrt showed predominance for the wild type with rates of 100 % (502/502). This study identified four haplotypes of pfcrt in P. falciparum-infected patients imported from Africa in Henan province. The prevalence of mutations in the pfcrt was dropped comparing with other people's researches. It establishes fundamental data for detection of P. falciparum CQR with molecular markers for the imported P. falciparum in China, and it also provides complementary information of CQR for the malaria endemic countries and assesses the evolution of anti-malarial drug resistance.

Genotype comparison of Plasmodium vivax and Plasmodium falciparum clones from pregnant and non-pregnant populations in North-west Colombia

PubMed Central

2012-01-01

Background Placental malaria is the predominant pathology secondary to malaria in pregnancy, causing substantial maternal and infant morbidity and mortality in tropical areas. While it is clear that placental parasites are phenotypically different from those in the peripheral circulation, it is not known whether unique genotypes are associated specifically with placental infection or perhaps more generally with pregnancy. In this study, genetic analysis was performed on Plasmodium vivax and Plasmodium falciparum parasites isolated from peripheral and placental blood in pregnant women living in North-west Colombia, and compared with parasites causing acute malaria in non-pregnant populations. Methods A total of 57 pregnant women at delivery with malaria infection confirmed by real-time PCR in peripheral or placental blood were included, as well as 50 pregnant women in antenatal care and 80 men or non-pregnant women with acute malaria confirmed by a positive thick smear for P. vivax or P. falciparum. Five molecular markers per species were genotyped by nested PCR and capillary electrophoresis. Genetic diversity and the fixation index FST per species and study group were calculated and compared. Results Almost all infections at delivery were asymptomatic with significantly lower levels of infection compared with the groups with acute malaria. Expected heterozygosity for P. vivax molecular markers ranged from 0.765 to 0.928 and for P. falciparum markers ranged from 0.331 to 0.604. For P. vivax infections, the genetic diversity was similar amongst the four study groups and the fixation index from each pairwise comparison failed to show significant genetic differentiation. For P. falciparum, no genetic differentiation was observed between placental and peripheral parasites from the same woman at delivery, but the parasites isolated at delivery showed significant genetic differentiation compared with parasites isolated from subjects with acute malaria. Conclusions In North-west Colombia, P. vivax parasites have high genetic diversity that is equivalent in pregnant and non-pregnant populations as well as in symptomatic and asymptomatic infections. For P. falciparum, the overall genetic diversity is lower, with specific genotypes associated with asymptomatic infections at delivery. PMID:23181896

Differences in Gene Transcriptomic Pattern of Plasmodium falciparum in Children with Cerebral Malaria and Asymptomatic Carriers

PubMed Central

Almelli, Talleh; Nuel, Grégory; Bischoff, Emmanuel; Aubouy, Agnès; Elati, Mohamed; Wang, Christian William; Dillies, Marie-Agnès; Coppée, Jean-Yves; Ayissi, Georges Nko; Basco, Leonardo Kishi; Rogier, Christophe; Ndam, Nicaise Tuikue; Deloron, Philippe; Tahar, Rachida

2014-01-01

The mechanisms underlying the heterogeneity of clinical malaria remain largely unknown. We hypothesized that differential gene expression contributes to phenotypic variation of parasites which results in a specific interaction with the host, leading to different clinical features of malaria. In this study, we analyzed the transcriptomes of isolates obtained from asymptomatic carriers and patients with uncomplicated or cerebral malaria. We also investigated the transcriptomes of 3D7 clone and 3D7-Lib that expresses severe malaria associated-variant surface antigen. Our findings revealed a specific up-regulation of genes involved in pathogenesis, adhesion to host cell, and erythrocyte aggregation in parasites from patients with cerebral malaria and 3D7-Lib, compared to parasites from asymptomatic carriers and 3D7, respectively. However, we did not find any significant difference between the transcriptomes of parasites from cerebral malaria and uncomplicated malaria, suggesting similar transcriptomic pattern in these two parasite populations. The difference between isolates from asymptomatic children and cerebral malaria concerned genes coding for exported proteins, Maurer's cleft proteins, transcriptional factor proteins, proteins implicated in protein transport, as well as Plasmodium conserved and hypothetical proteins. Interestingly, UPs A1, A2, A3 and UPs B1 of var genes were predominantly found in cerebral malaria-associated isolates and those containing architectural domains of DC4, DC5, DC13 and their neighboring rif genes in 3D7-lib. Therefore, more investigations are needed to analyze the effective role of these genes during malaria infection to provide with new knowledge on malaria pathology. In addition, concomitant regulation of genes within the chromosomal neighborhood suggests a common mechanism of gene regulation in P. falciparum. PMID:25479608

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum.

PubMed

El Bissati, Kamal; Downie, Megan J; Kim, Seong-Kyoun; Horowitz, Michael; Carter, Nicola; Ullman, Buddy; Ben Mamoun, Choukri

2008-10-01

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine.

Direct Comparison of the Histidine-rich Protein-2 Enzyme-linked Immunosorbent Assay (HRP-2 ELISA) and Malaria SYBR Green I Fluorescence (MSF) Drug Sensitivity Tests in Plasmodium falciparum Reference Clones and Fresh ex vivo Field Isolates from Cambodia

DTIC Science & Technology

2013-07-12

assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 1979, 16:710–718. 3. Noedl H, Attlmayr B...40:685–691. 32. Hawley SR, Bray PG, Mungthin M, Atkinson JD, O’Neill PM, Ward SA: Relationship between antimalarial drug activity , accumulation, and...success rate when testing DHA, AS, MQ, QN, CQ, and PPQ activities . A “successful” IC50 assay result for each P. falciparum clinical isolate was defined as

Prevalence of mutations associated with antimalarial drugs in Plasmodium falciparum isolates prior to the introduction of sulphadoxine-pyrimethamine as first-line treatment in Iran

PubMed Central

Zakeri, Sedigheh; Afsharpad, Mandana; Raeisi, Ahmad; Djadid, Navid Dinparast

2007-01-01

Background This work was carried out to assess the patterns and prevalence of resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) in Iran. Methods The prevalence of pfcrt K76T, pfmdr1 N86Y, pfdhfr N51I, C59R, S108N/T and I164L and codons S436F/A, A437G, K540E, A581E, and A613S/T in pfdhps genes were genotyped by PCR/RFLP methods in 206 Plasmodium falciparum isolates from Chabahar and Sarbaz districts in Sistan and Baluchistan province, Iran, during 2003–2005. Results All P. falciparum isolates carried the 108N, while 98.5% parasite isolates carried the 59R mutation. 98.5% of patients carried both 108N and 59R. The prevalence of pfdhps 437G mutation was 17% (Chabahar) and 33% (Sarbaz) isolates. 20.4% of samples presented the pfdhfr 108N, 59R with pfdhps 437G mutations. The frequency of allele pfcrt 76T was 98%, while 41.4% (Chabahar) and 27.7% (Sarbaz) isolates carried pfmdr1 86Y allele. Eight distinct haplotypes were identified in all 206 samples, while the most prevalent haplotype was T76/N86/N51R59N108/A437 among both study areas. Conclusion Finding the fixed level of CQ resistance polymorphisms (pfcrt 76T) suggests that CQ must be withdrawn from the current treatment strategy in Iran, while SP may remain the treatment of choice for uncomplicated malaria. PMID:17999755

Alisiaquinones and alisiaquinol, dual inhibitors of Plasmodium falciparum enzyme targets from a New Caledonian deep water sponge.

PubMed

Desoubzdanne, Denis; Marcourt, Laurence; Raux, Roselyne; Chevalley, Séverine; Dorin, Dominique; Doerig, Christian; Valentin, Alexis; Ausseil, Frédéric; Debitus, Cécile

2008-07-01

Four new meroterpenes, alisiaquinones A-C (1-3) and alisiaquinol (4), were isolated from a New Caledonian deep water sponge. Their structures and relative stereochemistry were elucidated by spectroscopic data analysis. They are related to xestoquinone, but showed unusual substitution on a tetrahydrofuran junction. They displayed micromolar range activity on two enzymatic targets of importance for the control of malaria, the plasmodial kinase Pfnek-1 and a protein farnesyl transferase, as well as on different chloroquine-sensitive and -resistant strains of Plasmodium falciparum. Alisiaquinone C displayed a submicromolar activity on P. falciparum and a competitive selectivity index on the different plasmodial strains.

Sequence diversity of the C-terminal region of Plasmodium falciparum merozoite surface protein 1 in southern Iran.

PubMed

Zamani, Zahra; Razavi, Mohammad Reza; Sadeghi, Sedigheh; Naddaf, Saeed; Pourfallah, Fatemeh; Mirkhani, Fatemeh; Arjmand, Mohammad; Feizhaddad, Hossein; Rad, Mina Ebrahimi; Ebrahimi Rad, Mina; Tameemi, Marzieh; Assmar, Mehdi

2009-01-01

The C-terminal region of the merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is a strong vaccine candidate as it is associated with immunity to the parasite. This corresponds approximately to the conserved 17th block of the gene and is composed of two EGF- like domains. These domains exhibit only four single amino acid substitutions which show several potential variants in this region of the gene. As the variations might be important for a regional vaccine design, a study was carried out to determine the variations present in P. falciparum isolates from southern Iran. Besides the usual E-T-S-R-L and the Q-K-N-G-F types, we found Q-T-S-R-L, E-K-N-G-F, E-T-S-G-L, Z-T-S-G-L and Z-T-S-R-L types, where Z was E or Q signifying the presence of mixed clones in single isolates.

Adherence to human lung microvascular endothelial cells (HMVEC-L) of Plasmodium vivax isolates from Colombia

PubMed Central

2013-01-01

Background For years Plasmodium vivax has been considered the cause of benign malaria. Nevertheless, it has been observed that this parasite can produce a severe disease comparable to Plasmodium falciparum. It has been suggested that some physiopathogenic processes might be shared by these two species, such as cytoadherence. Recently, it has been demonstrated that P. vivax-infected erythrocytes (Pv-iEs) have the capacity to adhere to endothelial cells, in which intercellular adhesion molecule-1 (ICAM-1) seems to be involved in this process. Methods Adherence capacity of 21 Colombian isolates, from patients with P. vivax mono-infection to a microvascular line of human lung endothelium (HMVEC-L) was assessed in static conditions and binding was evaluated at basal levels or in tumor necrosis factor (TNF) stimulated cells. The adherence specificity for the ICAM-1 receptor was determined through inhibition with an anti-CD54 monoclonal antibody. Results The majority of P. vivax isolates, 13 out of 21 (61.9%), adhered to the HMVEC-L cells, but P. vivax adherence was at least seven times lower when compared to the four P. falciparum isolates. Moreover, HMVEC-L stimulation with TNF led to an increase of 1.6-fold in P. vivax cytoadhesion, similar to P. falciparum isolates (1.8-fold) at comparable conditions. Also, blockage of ICAM-1 receptor with specific antibodies showed a significant 50% adherence reduction. Conclusions Plasmodium vivax isolates found in Colombia are also capable of adhering specifically in vitro to lung endothelial cells, via ICAM-1 cell receptor, both at basal state and after cell stimulation with TNF. Collectively, these findings reinforce the concept of cytoadherence for P. vivax, but here, to a different endothelial cell line and using geographical distinct isolates, thus contributing to understanding P. vivax biology. PMID:24080027

Multiple independent introductions of Plasmodium falciparum in South America

PubMed Central

Yalcindag, Erhan; Elguero, Eric; Arnathau, Céline; Durand, Patrick; Akiana, Jean; Anderson, Timothy J.; Aubouy, Agnes; Balloux, François; Besnard, Patrick; Bogreau, Hervé; Carnevale, Pierre; D'Alessandro, Umberto; Fontenille, Didier; Gamboa, Dionicia; Jombart, Thibaut; Le Mire, Jacques; Leroy, Eric; Maestre, Amanda; Mayxay, Mayfong; Ménard, Didier; Musset, Lise; Newton, Paul N.; Nkoghé, Dieudonné; Noya, Oscar; Ollomo, Benjamin; Rogier, Christophe; Veron, Vincent; Wide, Albina; Zakeri, Sedigheh; Carme, Bernard; Legrand, Eric; Chevillon, Christine; Ayala, Francisco J.; Renaud, François; Prugnolle, Franck

2012-01-01

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence—archeological and genetic—suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade. PMID:22203975

Antiplasmodial activities and cytotoxic effects of aqueous extracts and sesquiterpene lactones from Neurolaena lobata.

PubMed

François, G; Passreiter, C M; Woerdenbag, H J; Van Looveren, M

1996-04-01

Aqueous and lipophilic extracts of Neurolaena lobata (Asteraceae), obtained from Guatemala, were tested against Plasmodium falciparum in vitro. Moreover, sesquiterpene lactones, of the germacranolide and furanoheliangolide type, isolated from N. lobata, were shown to be active against P. falciparum in vitro. In addition to their antiplasmodial activity, their cytotoxic effects on human carcinoma cell lines were evaluated. Structure-activity relationships are discussed.

Detection of the Plasmodium falciparum Kelch-13 gene P553L mutation in sporozoites isolated from mosquito salivary glands in South-Central Vietnam.

PubMed

Maeno, Yoshimasa; Quang, Nguyen Tuyen; Culleton, Richard; Kawai, Satoru; Masuda, Gaku; Hori, Kaoru; Nakazawa, Shusuke; Marchand, Ron P

2017-06-24

Plasmodium falciparum has developed resistance against artemisinin in Southeast Asia. Mutations in the P. falciparum Kelch-13 (Pfk13) gene are associated with artemisinin resistance in vitro and in vivo. We investigated the prevalence of mutations in PfK13 from sporozoite-stage parasites isolated from the salivary glands of Anopheles dirus mosquitoes. Mosquitoes were caught by human-landing catches at two locations within the Khanh Phu commune, South-Central Vietnam. Identification of Anopheles species was performed based on morphological features and nucleotide sequence analysis. Sporozoite-infected salivary glands were stored on filter paper and at 4-6 °C. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification. Pfk13 was amplified by nested PCR, and subjected to nucleotide sequencing. Five of 33 P. falciparum sporozoite samples carried the P553L mutation at the PfK13 locus. This mutation has been recorded previously in Vietnam, but not in Khanh Hoa province, were surveys of K13 polymorphism have not previously been carried out. These results demonstrate the utility of mosquito-stage malaria parasite samples for studies on the molecular epidemiology of drug resistance.

Ex vivo activity of Proveblue, a methylene blue, against field isolates of Plasmodium falciparum in Dakar, Senegal from 2013-2015.

PubMed

Fall, Bécaye; Madamet, Marylin; Diawara, Silman; Briolant, Sébastien; Wade, Khalifa Ababacar; Lo, Gora; Nakoulima, Aminata; Fall, Mansour; Bercion, Raymond; Kounta, Mame Bou; Amalvict, Rémi; Benoit, Nicolas; Gueye, Mamadou Wague; Diatta, Bakary; Wade, Boubacar; Pradines, Bruno

2017-08-01

Resistance to most antimalarial drugs has spread from Southeast Asia to Africa. Accordingly, new therapies to use with artemisinin-based combination therapy (triple ACT) are urgently needed. Proveblue, a methylene blue preparation, was found to exhibit antimalarial activity against Plasmodium falciparum strains in vitro. Proveblue has synergistic effects when used in combination with dihydroartemisinin, and has been shown to significantly reduce or prevent cerebral malaria in mice. The objectives of the current study were to evaluate the in vitro baseline susceptibility of clinical field isolates to Proveblue, compare its activity with that of other standard antimalarial drugs and define the patterns of cross-susceptibility between Proveblue and conventional antimalarial drugs. The Proveblue IC 50 of 76 P. falciparum isolates ranged from 0.5 nM to 135.1 nM, with a mean of 8.1 nM [95% confidence interval, 6.4-10.3]. Proveblue was found to be more active against P. falciparum parasites than chloroquine, quinine, monodesethylamodiaquine, mefloquine, piperaquine, doxycycline (P <0.001) and lumefantrine (P = 0.014). Proveblue was as active as pyronaridine (P = 0.927), but was less active than dihydroartemisinin and artesunate (P <0.001). The only significant cross-susceptibilities found were between Proveblue and dihydroartemisinin (r 2  = 0.195, P = 0.0001), artesunate (r 2  = 0.187, P = 0.0002) and piperaquine (r 2  = 0.063, P = 0.029). The present study clearly demonstrates the potential of Proveblue as an effective therapeutic agent against P. falciparum. In this context, the use of Proveblue as part of the triple ACT treatment for multidrug-resistant malaria warrants further investigation. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

K13 Propeller Mutations in Plasmodium falciparum Populations in Regions of Malaria Endemicity in Vietnam from 2009 to 2016.

PubMed

Thuy-Nhien, Nguyen; Tuyen, Nguyen Kim; Tong, Nguyen Thanh; Vy, Nguyen Tuong; Thanh, Ngo Viet; Van, Huynh Thuy; Huong-Thu, Pham; Quang, Huynh Hong; Boni, Maciej F; Dolecek, Christiane; Farrar, Jeremy; Thwaites, Guy E; Miotto, Olivo; White, Nicholas J; Hien, Tran Tinh

2017-04-01

The spread of artemisinin-resistant Plasmodium falciparum compromises the therapeutic efficacy of artemisinin combination therapies (ACTs) and is considered the greatest threat to current global initiatives to control and eliminate malaria. This is particularly relevant in Vietnam, where dihydroartemisinin-piperaquine (DP) is the recommended ACT for P. falciparum infection. The propeller domain gene of K13, a molecular marker of artemisinin resistance, was successfully sequenced in 1,060 P. falciparum isolates collected at 3 malaria hot spots in Vietnam between 2009 and 2016. Eight K13 propeller mutations (Thr474Ile, Tyr493His, Arg539Thr, Ile543Thr, Pro553Leu, Val568Gly, Pro574Leu, and Cys580Tyr), including several that have been validated to be artemisinin resistance markers, were found. The prevalences of K13 mutations were 29% (222/767), 6% (11/188), and 43% (45/105) in the Binh Phuoc, Ninh Thuan, and Gia Lai Provinces of Vietnam, respectively. Cys580Tyr became the dominant genotype in recent years, with 79.1% (34/43) of isolates in Binh Phuoc Province and 63% (17/27) of isolates in Gia Lai Province carrying this mutation. K13 mutations were associated with reduced ring-stage susceptibility to dihydroartemisinin (DHA) in vitro and prolonged parasite clearance in vivo An analysis of haplotypes flanking K13 suggested the presence of multiple strains with the Cys580Tyr mutation rather than a single strain expanding across the three sites. Copyright © 2017 Thuy-Nhien et al.

Genetic Analysis and Species Specific Amplification of the Artemisinin Resistance-Associated Kelch Propeller Domain in P. falciparum and P. vivax

PubMed Central

Talundzic, Eldin; Chenet, Stella M.; Goldman, Ira F.; Patel, Dhruviben S.; Nelson, Julia A.; Plucinski, Mateusz M.; Barnwell, John W.; Udhayakumar, Venkatachalam

2015-01-01

Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species. PMID:26292024

Genetic Analysis and Species Specific Amplification of the Artemisinin Resistance-Associated Kelch Propeller Domain in P. falciparum and P. vivax.

PubMed

Talundzic, Eldin; Chenet, Stella M; Goldman, Ira F; Patel, Dhruviben S; Nelson, Julia A; Plucinski, Mateusz M; Barnwell, John W; Udhayakumar, Venkatachalam

2015-01-01

Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species.

Magnetic isolation of Plasmodium falciparum schizonts iRBCs to generate a high parasitaemia and synchronized in vitro culture.

PubMed

Mata-Cantero, Lydia; Lafuente, Maria J; Sanz, Laura; Rodriguez, Manuel S

2014-03-21

The establishment of methods for an in vitro continuous culture of Plasmodium falciparum is essential for gaining knowledge into its biology and for the development of new treatments. Previously, several techniques have been used to synchronize, enrich and concentrate P. falciparum, although obtaining cultures with high parasitaemia continues being a challenging process. Current methods produce high parasitaemia levels of synchronized P. falciparum cultures by frequent changes of culture medium or reducing the haematocrit. However, these methods are time consuming and sometimes lead to the loss of synchrony. A procedure that combines Percoll and sorbitol treatments, the use of magnetic columns, and the optimization of the in vitro culture conditions to reach high parasitaemia levels for synchronized Plasmodium falciparum cultures is described. A new procedure has been established using P. falciparum 3D7, combining previous reported methodologies to achieve in vitro parasite cultures that reach parasitaemia up to 40% at any intra-erythrocytic stage. High parasitaemia levels are obtained only one day after magnetic column purification without compromising the parasite viability and synchrony. The described procedure allows obtaining a large scale synchronized parasite culture at a high parasitaemia with less manipulations than other methods previously described.

Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area.

PubMed

Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

2016-04-01

To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs. Copyright © 2016 Elsevier B.V. All rights reserved.

Investigating the activity of quinine analogues versus chloroquine resistant Plasmodium falciparum.

PubMed

Dinio, Theresa; Gorka, Alexander P; McGinniss, Andrew; Roepe, Paul D; Morgan, Jeremy B

2012-05-15

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 a well-tolerated therapy with improved activity versus 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 IC(50) values relative to QN. Copyright © 2012 Elsevier Ltd. All rights reserved.

In vitro activity of anti-malarial ozonides against an artemisinin-resistant isolate.

PubMed

Baumgärtner, Fabian; Jourdan, Joëlle; Scheurer, Christian; Blasco, Benjamin; Campo, Brice; Mäser, Pascal; Wittlin, Sergio

2017-01-25

Recently published data suggest that artemisinin derivatives and synthetic peroxides, such as the ozonides OZ277 and OZ439, have a similar mode of action. Here the cross-resistance of OZ277 and OZ439 and four additional next-generation ozonides was probed against the artemisinin-resistant clinical isolate Plasmodium falciparum Cam3.I, which carries the K13-propeller mutation R539T (Cam3.I R539T ). The previously described in vitro ring-stage survival assay (RSA 0-3h ) was employed and a simplified variation of the original protocol was developed. At the pharmacologically relevant concentration of 700 nM, all six ozonides were highly effective against the dihydroartemisinin-resistant P. falciparum Cam3.I R539T parasites, showing a per cent survival ranging from <0.01 to 1.83%. A simplified version of the original RSA 0-3h method was developed and gave similar results, thus providing a practical drug discovery tool for further optimization of next-generation anti-malarial peroxides. The absence of in vitro cross-resistance against the artemisinin-resistant clinical isolate Cam3.I R539T suggests that ozonides could be effective against artemisinin-resistant P. falciparum. How this will translate to the human situation in clinical settings remains to be investigated.

Global sequence diversity of the lactate dehydrogenase gene in Plasmodium falciparum.

PubMed

Simpalipan, Phumin; Pattaradilokrat, Sittiporn; Harnyuttanakorn, Pongchai

2018-01-09

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 LDH as a therapeutic drug target.

Genetic Characterisation of Plasmodium falciparum Isolates with Deletion of the pfhrp2 and/or pfhrp3 Genes in Colombia: The Amazon Region, a Challenge for Malaria Diagnosis and Control

PubMed Central

Dorado, Erika Jimena; Okoth, Sheila Akinyi; Montenegro, Lidia Madeline; Diaz, Gustavo; Barnwell, John W.; Udhayakumar, Venkatachalam; Murillo Solano, Claribel

2016-01-01

Most Plasmodium falciparum-detecting rapid diagnostic tests (RDTs) target histidine-rich protein 2 (PfHRP2). However, P. falciparum isolates with deletion of the pfhrp2 gene and its homolog gene, pfhrp3, have been detected. We carried out an extensive investigation on 365 P. falciparum dried blood samples collected from seven P. falciparum endemic sites in Colombia between 2003 and 2012 to genetically characterise and geographically map pfhrp2- and/or pfhrp3-negative P. falciparum parasites in the country. We found a high proportion of pfhrp2-negative parasites only in Amazonas (15/39; 38.5%), and these parasites were also pfhrp3-negative. These parasites were collected between 2008 and 2009 in Amazonas, while pfhrp3-negative parasites (157/365, 43%) were found in all the sites and from each of the sample collection years evaluated (2003 to 2012). We also found that all pfhrp2- and/or pfhrp3-negative parasites were also negative for one or both flanking genes. Six sub-population clusters were established with 93.3% (14/15) of the pfhrp2-negative parasites grouped in the same cluster and sharing the same haplotype. This haplotype corresponded with the genetic lineage BV1, a multidrug resistant strain that caused two outbreaks reported in Peru between 2010 and 2013. We found this BV1 lineage in the Colombian Amazon as early as 2006. Two new clonal lineages were identified in these parasites from Colombia: the genetic lineages EV1 and F. PfHRP2 sequence analysis revealed high genetic diversity at the amino acid level, with 17 unique sequences identified among 53 PfHRP2 sequences analysed. The use of PfHRP2-based RDTs is not recommended in Amazonas because of the high proportion of parasites with pfhrp2 deletion (38.5%), and implementation of new strategies for malaria diagnosis and control in Amazonas must be prioritised. Moreover, studies to monitor and genetically characterise pfhrp2-negative P. falciparum parasites in the Americas are warranted, given the extensive human migration occurring in the region. PMID:27636709

K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates

PubMed Central

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.

2015-01-01

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. PMID:25502314

Artemisinin-resistant Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa

PubMed Central

St. Laurent, Brandyce; Miller, Becky; Burton, Timothy A.; Amaratunga, Chanaki; Men, Sary; Sovannaroth, Siv; Fay, Michael P.; Miotto, Olivo; Gwadz, Robert W.; Anderson, Jennifer M.; Fairhurst, Rick M.

2015-01-01

Artemisinin-resistant Plasmodium falciparum parasites are rapidly spreading in Southeast Asia, yet nothing is known about their transmission. This knowledge gap and the possibility that these parasites will spread to Africa endanger global efforts to eliminate malaria. Here we produce gametocytes from parasite clinical isolates that displayed artemisinin resistance in patients and in vitro, and use them to infect native and non-native mosquito vectors. We show that contemporary artemisinin-resistant isolates from Cambodia develop and produce sporozoites in two Southeast Asian vectors, Anopheles dirus and Anopheles minimus, and the major African vector, Anopheles coluzzii (formerly Anopheles gambiae M). The ability of artemisinin-resistant parasites to infect such highly diverse Anopheles species, combined with their higher gametocyte prevalence in patients, may explain the rapid expansion of these parasites in Cambodia and neighbouring countries, and further compromise efforts to prevent their global spread. PMID:26485448

Artemisinin-resistant Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa.

PubMed

St Laurent, Brandyce; Miller, Becky; Burton, Timothy A; Amaratunga, Chanaki; Men, Sary; Sovannaroth, Siv; Fay, Michael P; Miotto, Olivo; Gwadz, Robert W; Anderson, Jennifer M; Fairhurst, Rick M

2015-10-20

Artemisinin-resistant Plasmodium falciparum parasites are rapidly spreading in Southeast Asia, yet nothing is known about their transmission. This knowledge gap and the possibility that these parasites will spread to Africa endanger global efforts to eliminate malaria. Here we produce gametocytes from parasite clinical isolates that displayed artemisinin resistance in patients and in vitro, and use them to infect native and non-native mosquito vectors. We show that contemporary artemisinin-resistant isolates from Cambodia develop and produce sporozoites in two Southeast Asian vectors, Anopheles dirus and Anopheles minimus, and the major African vector, Anopheles coluzzii (formerly Anopheles gambiae M). The ability of artemisinin-resistant parasites to infect such highly diverse Anopheles species, combined with their higher gametocyte prevalence in patients, may explain the rapid expansion of these parasites in Cambodia and neighbouring countries, and further compromise efforts to prevent their global spread.

Novel pfdhps Haplotypes among Imported Cases of Plasmodium falciparum Malaria in the United Kingdom ▿

PubMed Central

Sutherland, Colin J.; Fifer, Helen; Pearce, Richard J.; bin Reza, Faisal; Nicholas, Meredydd; Haustein, Thomas; Njimgye-Tekumafor, Njah E.; Doherty, Justin F.; Gothard, Philip; Polley, Spencer D.; Chiodini, Peter L.

2009-01-01

Treatment of acute malaria caused by Plasmodium falciparum may include long-half-life drugs, such as the antifolate combination sulfadoxine-pyrimethamine (SP), to provide posttreatment chemoprophylaxis against parasite recrudescence or delayed emergence from the liver. An unusual case of P. falciparum recrudescence in a returned British traveler who received such a regimen, as well as a series of 44 parasite isolates from the same hospital, was analyzed by PCR and direct DNA sequencing for the presence of markers of parasite resistance to chloroquine and antifolates. The index patient harbored a mixture of wild-type and resistant pfdhfr and pfdhps alleles upon initial presentation. During his second malaria episode, he harbored only resistant parasites, with the haplotypes IRNI (codons 51, 59, 108, and 164) and SGEAA (codons 436, 437, 540, 581, and 613) at these two loci, respectively. Analysis of isolates from 44 other patients showed that the pfdhfr haplotype IRNI was common (found in 81% of cases). The SGEAA haplotype of pfdhps was uncommon (found only in eight cases of East African origin [17%]). A previously undescribed mutation, I431V, was observed for seven cases of Nigerian origin, occurring as one of two haplotypes, VAGKGS or VAGKAA. The presence of this mutation was also confirmed in isolates of Nigerian origin from the United Kingdom Malaria Reference Laboratory. The presence of the pfdhps haplotype SGEAA in P. falciparum parasites of East African origin appears to compromise the efficacy of treatment regimens that include SP as a means to prevent recrudescence. Parasites with novel pfdhps haplotypes are circulating in West Africa. The response of these parasites to chemotherapy needs to be evaluated. PMID:19433569

Origin and Dissemination of Chloroquine-Resistant Plasmodium falciparum with Mutant pfcrt Alleles in the Philippines

PubMed Central

Chen, Nanhua; Wilson, Danny W.; Pasay, Cielo; Bell, David; Martin, Laura B.; Kyle, Dennis; Cheng, Qin

2005-01-01

The pfcrt allelic type and adjacent microsatellite marker type were determined for 82 Plasmodium falciparum isolates from the Philippines. Mutant pfcrt allelic types P1a and P2a/P2b were dominant in different locations. Microsatellite analysis revealed that P2a/P2b evolved independently in the Philippines, while P1a shared common ancestry with Papua New Guinea chloroquine-resistant parasites. PMID:15855538

Establishment and application of a novel isothermal amplification assay for rapid detection of chloroquine resistance (K76T) in Plasmodium falciparum

PubMed Central

Chahar, Madhvi; Mishra, Neelima; Anvikar, Anup; Dixit, Rajnikant; Valecha, Neena

2017-01-01

Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene. The point mutation at codon 76 (K76T), which has been observed in more than 91% of P. falciparum isolates in India, is the major determinant of CQ resistance. To overcome the limitations and challenges of traditional methods, in this investigation we developed an easy to use loop mediated isothermal amplification (LAMP) protocol for rapid detection of the K76T mutation associated with CQ resistance in P. falciparum with naked eye visualization. In- house designed primers were synthesized and optimized to specifically distinguish the CQ resistant mutants of P. falciparum. The LAMP reaction was optimal at 61 °C for 60 min and calcein dye was added prior to amplification to enable visual detection. We demonstrate the detection limit of <2 ng/μl respectively, supporting the high sensitivity of this calcein based LAMP method. To the best of our knowledge this is the first report on the establishment of an easy, reliable and cost effective LAMP assay for rapid and specific detection of highly CQ resistance in P. falciparum malaria. PMID:28134241

The Hsp40 proteins of Plasmodium falciparum and other apicomplexa: regulating chaperone power in the parasite and the host.

PubMed

Botha, M; Pesce, E-R; Blatch, G L

2007-01-01

Extensive structural and functional remodelling of Plasmodium falciparum (malaria)-infected erythrocytes follows the export of a range of proteins of parasite origin (exportome) across the parasitophorous vacuole into the host erythrocyte. The genome of P. falciparum encodes a diverse chaperone complement including at least 43 members of the heat shock protein 40kDa (Hsp40) family, and six members of the heat shock protein 70kDa (Hsp70) family. Nearly half of the Hsp40 proteins of P. falciparum are predicted to contain a PEXEL/HT (Plasmodium export element/host targeting signal) sequence motif, and hence are likely to be part of the exportome. In this review we critically evaluate the classification, sequence similarity and clustering, and possible interactors of the P. falciparum Hsp40 chaperone machinery. In addition to the types I, II and III Hsp40 proteins all exhibiting the signature J-domain, the P. falciparum genome also encodes a number of specialized Hsp40 proteins with a J-like domain, which we have categorized as type IV Hsp40 proteins. Analysis of the potential P. falciparum Hsp40 protein interaction network revealed connections predominantly with cytoskeletal and membrane proteins, transcriptional machinery, DNA repair and replication machinery, translational machinery, the proteasome and proteolytic enzymes, and enzymes involved in cellular physiology. Comparison of the Hsp40 proteins of P. falciparum to those of other apicomplexa reveals that most of the proteins (especially the PEXEL/HT-containing proteins) are unique to P. falciparum. Furthermore, very few of the P. falciparum Hsp40 proteins have human homologs, except for those proteins implicated in fundamental biological processes. Our analysis suggests that P. falciparum has evolved an expanded and specialized Hsp40 protein machinery to enable it successfully to invade and remodel the human erythrocyte, and we propose a model in which these proteins are involved in chaperone-mediated translocation, folding, assembly and regulation of parasite and host proteins.

Clinical trial of extended-dose chloroquine for treatment of resistant falciparum malaria among Afghan refugees in Pakistan.

PubMed

Howard, Natasha; Durrani, Naeem; Sanda, Sanda; Beshir, Khalid; Hallett, Rachel; Rowland, Mark

2011-06-23

Falciparum malaria is a significant problem for Afghan refugees in Pakistan. Refugee treatment guidelines recommended standard three-day chloroquine treatment (25 mg/kg) for first episodes and extended five-day treatment (40 mg/kg) for recrudescent infections, based on the assumption that a five-day course would more likely achieve a cure. An in-vivo randomized controlled trial was conducted among refugees with uncomplicated falciparum malaria to determine whether five-day treatment (CQ40) was more effective than standard treatment (CQ25). 142 falciparum patients were recruited into CQ25 or CQ40 treatment arms and followed up to 60 days with regular blood smears. The primary outcome was parasitological cure without recrudescence. Treatment failures were retreated with CQ40. PCR genotyping of 270 samples, from the same and nearby sites, was used to support interpretation of outcomes. 84% of CQ25 versus 51% of CQ40 patients experienced parasite recrudescence during follow-up (adjusted odds ratio 0.17, 95%CI 0.08-0.38). Cure rates were significantly improved with CQ40, particularly among adults. Fever clearance time, parasite clearance time, and proportions gametocytaemic post-treatment were similar between treatment groups. Second-line CQ40 treatment resulted in higher failure rates than first-line CQ40 treatment. CQ-resistance marker pfcrt 76T was found in all isolates analysed, while pfmdr1 86Y and 184Y were found in 18% and 37% of isolates respectively. CQ is not suitable for first-line falciparum treatment in Afghan refugee communities. The extended-dose CQ regimen can overcome 39% of resistant infections that would recrudesce under the standard regimen, but the high failure rate after directly observed treatment demonstrates its use is inappropriate.

Conserved mosquito/parasite interactions affect development of Plasmodium falciparum in Africa.

PubMed

Mendes, Antonio M; Schlegelmilch, Timm; Cohuet, Anna; Awono-Ambene, Parfait; De Iorio, Maria; Fontenille, Didier; Morlais, Isabelle; Christophides, George K; Kafatos, Fotis C; Vlachou, Dina

2008-05-16

In much of sub-Saharan Africa, the mosquito Anopheles gambiae is the main vector of the major human malaria parasite, Plasmodium falciparum. Convenient laboratory studies have identified mosquito genes that affect positively or negatively the developmental cycle of the model rodent parasite, P. berghei. Here, we use transcription profiling and reverse genetics to explore whether five disparate mosquito gene regulators of P. berghei development are also pertinent to A. gambiae/P. falciparum interactions in semi-natural conditions, using field isolates of this parasite and geographically related mosquitoes. We detected broadly similar albeit not identical transcriptional responses of these genes to the two parasite species. Gene silencing established that two genes affect similarly both parasites: infections are hindered by the intracellular local activator of actin cytoskeleton dynamics, WASP, but promoted by the hemolymph lipid transporter, ApoII/I. Since P. berghei is not a natural parasite of A. gambiae, these data suggest that the effects of these genes have not been drastically altered by constant interaction and co-evolution of A. gambiae and P. falciparum; this conclusion allowed us to investigate further the mode of action of these two genes in the laboratory model system using a suite of genetic tools and infection assays. We showed that both genes act at the level of midgut invasion during the parasite's developmental transition from ookinete to oocyst. ApoII/I also affects the early stages of oocyst development. These are the first mosquito genes whose significant effects on P. falciparum field isolates have been established by direct experimentation. Importantly, they validate for semi-field human malaria transmission the concept of parasite antagonists and agonists.

Artemisinin resistance at the China-Myanmar border and association with mutations in the K13 propeller gene.

PubMed

Wang, Zenglei; Wang, Yingna; Cabrera, Mynthia; Zhang, Yanmei; Gupta, Bhavna; Wu, Yanrui; Kemirembe, Karen; Hu, Yue; Liang, Xiaoying; Brashear, Awtum; Shrestha, Sony; Li, Xiaolian; Miao, Jun; Sun, Xiaodong; Yang, Zhaoqing; Cui, Liwang

2015-11-01

Artemisinin resistance in Plasmodium falciparum parasites in Southeast Asia is a major concern for malaria control. Its emergence at the China-Myanmar border, where there have been more than 3 decades of artemisinin use, has yet to be investigated. Here, we comprehensively evaluated the potential emergence of artemisinin resistance and antimalarial drug resistance status in P. falciparum using data and parasites from three previous efficacy studies in this region. These efficacy studies of dihydroartemisinin-piperaquine combination and artesunate monotherapy of uncomplicated falciparum malaria in 248 P. falciparum patients showed an overall 28-day adequate clinical and parasitological response of >95% and day 3 parasite-positive rates of 6.3 to 23.1%. Comparison of the 57 K13 sequences (24 and 33 from day 3 parasite-positive and -negative cases, respectively) identified nine point mutations in 38 (66.7%) samples, of which F446I (49.1%) and an N-terminal NN insertion (86.0%) were predominant. K13 propeller mutations collectively, the F446I mutation alone, and the NN insertion all were significantly associated with day 3 parasite positivity. Increased ring-stage survival determined using the ring-stage survival assay (RSA) was highly associated with the K13 mutant genotype. Day 3 parasite-positive isolates had ∼10 times higher ring survival rates than day 3 parasite-negative isolates. Divergent K13 mutations suggested independent evolution of artemisinin resistance. Taken together, this study confirmed multidrug resistance and emergence of artemisinin resistance in P. falciparum at the China-Myanmar border. RSA and K13 mutations are useful phenotypic and molecular markers for monitoring artemisinin resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

K13 mutations and pfmdr1 copy number variation in Plasmodium falciparum malaria in Myanmar.

PubMed

Win, Aye A; Imwong, Mallika; Kyaw, Myat P; Woodrow, Charles J; Chotivanich, Kesinee; Hanboonkunupakarn, Borimas; Pukrittayakamee, Sasithon

2016-02-24

Artemisinin-based combination therapy has been first-line treatment for falciparum malaria in Myanmar since 2005. The wide extent of artemisinin resistance in the Greater Mekong sub-region and the presence of mefloquine resistance at the Myanmar-Thailand border raise concerns over resistance patterns in Myanmar. The availability of molecular markers for resistance to both drugs enables assessment even in remote malaria-endemic areas. A total of 250 dried blood spot samples collected from patients with Plasmodium falciparum malarial infection in five malaria-endemic areas across Myanmar were analysed for kelch 13 sequence (k13) and pfmdr1 copy number variation. K13 mutations in the region corresponding to amino acids 210-726 (including the propeller region of the protein) were detected by nested PCR amplification and sequencing, and pfmdr1 copy number variation by real-time PCR. In two sites, a sub-set of patients were prospectively followed up for assessment of day-3 parasite clearance rates after a standard course of artemether-lumefantrine. K13 mutations and pfmdr1 amplification were successfully analysed in 206 and 218 samples, respectively. Sixty-nine isolates (33.5 %) had mutations within the k13 propeller region with 53 of these (76.8 %) having mutations already known to be associated with artemisinin resistance. F446I (32 isolates) and P574L (15 isolates) were the most common examples. K13 mutation was less common in sites in western border regions (29 of 155 isolates) compared to samples from the east and north (40 of 51 isolates; p < 0.0001). The overall proportion of parasites with multiple pfmdr1 copies (greater than 1.5) was 5.5 %. Seven samples showed both k13 mutation and multiple copies of pfmdr1. Only one of 36 patients followed up after artemether-lumefantrine treatment still had parasites at day 3; molecular analysis indicated wild-type k13 and single copy pfmdr1. The proportion of P. falciparum isolates with mutations in the propeller region of k13 indicates that artemisinin resistance extends across much of Myanmar. There is a low prevalence of parasites with multiple pfmdr1 copies across the country. The efficacy of artemisinin-based combination therapy containing mefloquine and lumefantrine is, therefore, expected to be high, although regular monitoring of efficacy will be important.

Molecular surveillance for drug-resistant Plasmodium falciparum in clinical and subclinical populations from three border regions of Burma/Myanmar: cross-sectional data and a systematic review of resistance studies.

PubMed

Brown, Tyler; Smith, Linda S; Oo, Eh Kalu Shwe; Shawng, Kum; Lee, Thomas J; Sullivan, David; Beyrer, Chris; Richards, Adam K

2012-09-19

Confirmation of artemisinin-delayed parasite clearance in Plasmodium falciparum along the Thai-Myanmar border has inspired a global response to contain and monitor drug resistance to avert the disastrous consequences of a potential spread to Africa. However, resistance data from Myanmar are sparse, particularly from high-risk areas where limited health services and decades of displacement create conditions for resistance to spread. Subclinical infections may represent an important reservoir for resistance genes that confer a fitness disadvantage relative to wild-type alleles. This study estimates the prevalence of resistance genotypes in three previously unstudied remote populations in Myanmar and tests the a priori hypothesis that resistance gene prevalence would be higher among isolates collected from subclinical infections than isolates collected from febrile clinical patients. A systematic review of resistance studies is provided for context. Community health workers in Karen and Kachin States and an area spanning the Indo-Myanmar border collected dried blood spots from 988 febrile clinical patients and 4,591 villagers with subclinical infection participating in routine prevalence surveys. Samples positive for P. falciparum 18 s ribosomal RNA by real-time PCR were genotyped for P. falciparum multidrug resistance protein (pfmdr1) copy number and the pfcrt K76T polymorphism using multiplex real-time PCR. Pfmdr1 copy number increase and the pfcrt K76 polymorphism were determined for 173 and 269 isolates, respectively. Mean pfmdr1 copy number was 1.2 (range: 0.7 to 3.7). Pfmdr1 copy number increase was present in 17.5%, 9.6% and 11.1% of isolates from Karen and Kachin States and the Indo-Myanmar border, respectively. Pfmdr1 amplification was more prevalent in subclinical isolates (20.3%) than clinical isolates (6.4%, odds ratio 3.7, 95% confidence interval 1.1 - 12.5). Pfcrt K76T prevalence ranged from 90-100%. Community health workers can contribute to molecular surveillance of drug resistance in remote areas of Myanmar. Marginal and displaced populations under-represented among previous resistance investigations can and should be included in resistance surveillance efforts, particularly once genetic markers of artemisinin-delayed parasite clearance are identified. Subclinical infections may contribute to the epidemiology of drug resistance, but determination of gene amplification from desiccated filter samples requires further validation when DNA concentration is low.

Pathogenicity Determinants of the Human Malaria Parasite Plasmodium falciparum Have Ancient Origins

PubMed Central

Brazier, Andrew J.; Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell

2017-01-01

ABSTRACT Plasmodium falciparum, the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi. We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum. Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum-infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum, it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P. falciparum. Our findings suggest that parasite adhesion traits associated with both mild and severe malaria have much earlier origins than previously appreciated and have important implications for virulence evolution in a major human pathogen. PMID:28101534

Pathogenicity Determinants of the Human Malaria Parasite Plasmodium falciparum Have Ancient Origins.

PubMed

Brazier, Andrew J; Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell; Smith, Joseph D

2017-01-01

Plasmodium falciparum , the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi . We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum . Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum -infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum , it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P. falciparum . Our findings suggest that parasite adhesion traits associated with both mild and severe malaria have much earlier origins than previously appreciated and have important implications for virulence evolution in a major human pathogen.

Limited geographical origin and global spread of sulfadoxine-resistant dhps alleles in Plasmodium falciparum populations.

PubMed

Mita, Toshihiro; Venkatesan, Meera; Ohashi, Jun; Culleton, Richard; Takahashi, Nobuyuki; Tsukahara, Takahiro; Ndounga, Mathieu; Dysoley, Lek; Endo, Hiroyoshi; Hombhanje, Francis; Ferreira, Marcelo U; Plowe, Christopher V; Tanabe, Kazuyuki

2011-12-15

Plasmodium falciparum malaria resistant to chloroquine and pyrimethamine originated in limited foci and migrated to Africa. It remains unresolved whether P. falciparum resistance to sulfadoxine, which is conferred by mutations in dihydropteroate synthase (DHPS), evolved following a similar pattern. The dhps locus of 893 P. falciparum isolates from 12 countries in Asia, the Pacific Islands, Africa, and South America was sequenced. Haplotypes of 6 microsatellite loci flanking the dhps locus were determined to define the genetic relationships among sulfadoxine-resistant lineages. Six distinct sulfadoxine-resistant lineages were identified. Highly resistant lineages appear to have originated only in Southeast Asia and South America. Two resistant lineages found throughout Southeast Asia have been introduced to East Africa, where they appear to have spread. The infrequent selection of parasites highly resistant to sulfadoxine and the subsequent migration of resistant lineages from Asia to Africa are similar to the patterns observed in chloroquine and pyrimethamine resistance. These findings strongly suggest that the global migration of resistant parasites has played a decisive role in the establishment of drug-resistant P. falciparum parasites, and that similar patterns may be anticipated for the spread of artemisinin resistance.

Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests

PubMed Central

2013-01-01

Background An accurate diagnosis is essential for the rapid and appropriate treatment of malaria. The accuracy of the histidine-rich protein 2 (PfHRP2)-based rapid diagnostic test (RDT) Palutop+4® was assessed here. One possible factor contributing to the failure to detect malaria by this test is the diversity of the parasite PfHRP2 antigens. Methods PfHRP2 detection with the Palutop+4® RDT was carried out. The pfhrp2 and pfhrp3 genes were amplified and sequenced from 136 isolates of Plasmodium falciparum that were collected in Dakar, Senegal from 2009 to 2011. The DNA sequences were determined and statistical analyses of the variation observed between these two genes were conducted. The potential impact of PfHRP2 and PfHRP3 sequence variation on malaria diagnosis was examined. Results Seven P. falciparum isolates (5.9% of the total isolates, regardless of the parasitaemia; 10.7% of the isolates with parasitaemia ≤0.005% or ≤250 parasites/μl) were undetected by the PfHRP2 Palutop+4® RDT. Low parasite density is not sufficient to explain the PfHRP2 detection failure. Three of these seven samples showed pfhrp2 deletion (2.4%). The pfhrp3 gene was deleted in 12.8%. Of the 122 PfHRP2 sequences, 120 unique sequences were identified. Of the 109 PfHRP3 sequences, 64 unique sequences were identified. Using the Baker’s regression model, at least 7.4% of the P. falciparum isolates in Dakar were likely to be undetected by PfHRP2 at a parasite density of ≤250 parasites/μl (slightly lower than the evaluated prevalence of 10.7%). This predictive prevalence increased significantly between 2009 and 2011 (P = 0.0046). Conclusion In the present work, 10.7% of the isolates with a parasitaemia ≤0.005% (≤250 parasites/μl) were undetected by the PfHRP2 Palutop+4® RDT (7.4% by the predictive Baker’model). In addition, all of the parasites with pfhrp2 deletion (2.4% of the total samples) and 2.1% of the parasites with parasitaemia >0.005% and presence of pfhrp2 were not detected by PfHRP2 RDT. PfHRP2 is highly polymorphic in Senegal. Efforts should be made to more accurately determine the prevalence of non-sensitive parasites to pfHRP2. PMID:23347727

Cytoadherence of Plasmodium falciparum to intercellular adhesion molecule 1 and chondroitin-4-sulfate expressed by the syncytiotrophoblast in the human placenta.

PubMed Central

Maubert, B; Guilbert, L J; Deloron, P

1997-01-01

Late stages of Plasmodium falciparum-infected erythrocytes (IRBCs) frequently sequester in the placentas of pregnant women, a phenomenon associated with low birth weight of the offspring. To investigate the physiological mechanism of this sequestration, we developed an in vitro assay for studying the cytoadherence of IRBCs to cultured term human trophoblasts. The capacity for binding to the syncytiotrophoblast varied greatly among P. falciparum isolates and was mediated by intercellular adhesion molecule 1 (ICAM-1), as binding was totally inhibited by 84H10, a monoclonal antibody specific for ICAM-1. Binding of the P. falciparum line RP5 to the syncytiotrophoblast involves chondroitin-4-sulfate (CSA), as this binding was dramatically impaired by addition of free CSA to the binding medium or by preincubation of the syncytiotrophoblast with chondroitinase ABC. ICAM-1 and CSA were visualized on the syncytiotrophoblast by immunofluorescence, while CD36, E-selectin, and vascular cell adhesion molecule 1 were not expressed even on tumor necrosis factor alpha (TNF-alpha)-stimulated syncytiotrophoblast tissue, and monoclonal antibodies against these cell adhesion molecules did not inhibit cytoadherence. ICAM-1 expression and cytoadherence of wild isolates was upregulated by TNF-alpha, a cytokine that can be secreted by the numerous mononuclear phagocytes present in malaria-infected placentas. These results suggest that cytoadherence may be involved in the placental sequestration and broaden the understanding of the physiopathology of the malaria-infected placenta. PMID:9119459

Reduced ex vivo susceptibility of Plasmodium falciparum after oral artemether-lumefantrine treatment in Mali.

PubMed

Dama, Souleymane; Niangaly, Hamidou; Ouattara, Amed; Sagara, Issaka; Sissoko, Sekou; Traore, Oumar Bila; Bamadio, Amadou; Dara, Niawanlou; Djimde, Moussa; Alhousseini, Mohamed Lamine; Goita, Siaka; Maiga, Hamma; Dara, Antoine; Doumbo, Ogobara K; Djimde, Abdoulaye A

2017-02-02

Artemisinin-based combination therapy is the recommended first-line treatment for uncomplicated falciparum malaria worldwide. However, recent studies conducted in Mali showed an increased frequency of recurrent parasitaemia following artemether-lumefantrine (AL) treatment. Study samples were collected during a large WANECAM study. Ex-vivo Plasmodium falciparum sensitivity to artemether and lumefantrine was assessed using the tritiated hypoxanthine-based assay. The prevalence of molecular markers of anti-malarial drug resistance (pfcrt K76T, pfmdr1 N86Y and K13-propeller) were measured by PCR and/or sequencing. Overall 61 samples were successfully analysed in ex vivo studies. Mean IC 50 s increased significantly between baseline and recurrent parasites for both artemether (1.6 nM vs 3.2 nM, p < 0.001) and lumefantrine (1.4 nM vs 3.4 nM, p = 0.004). Wild type Pfmdr1 N86 allele was selected after treatment (71 vs 91%, 112 of 158 vs 95 of 105, p < 0.001) but not the wild type pfcrt K76 variant (23.5 vs 24.8%, 40 of 170 vs 26 of 105, p = 0.9). Three non-synonymous K13-propeller SNPs (A522C, A578S, and G638R) were found with allele frequencies <2%. Malian post-AL P. falciparum isolates were less susceptible to artemether and lumefantrine than baseline isolates.

Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia.

PubMed

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

2017-05-12

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 resistance and development of strategies to prevent or overcome anti-malarial resistance.

Insight into k13-propeller gene polymorphism and ex vivo DHA-response profiles from Cameroonian isolates.

PubMed

Menard, Sandie; Tchoufack, Joëlle Njila; Maffo, Christelle Ngou; Nsango, Sandrine E; Iriart, Xavier; Abate, Luc; Tsapi, Majoline Tchioffo; Awono-Ambéné, Parfait H; Abega Mekongo, Francis A; Morlais, Isabelle; Berry, Antoine

2016-11-26

The spread of Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia is a major source of concern and the emergence of resistance in Africa would have dramatic consequences, by increasing malaria mortality and morbidity. It is therefore urgent to implement regular monitoring in sentinel sites in sub-Saharan Africa using robust and easy-to-implement tools. The prevalence of k13-propeller mutations and the phenotypic profiles are poorly known in sub-Saharan Africa. Here, the k13-propeller polymorphism was compared to both ex vivo susceptibility to DHA and early parasitological and clinical responses to artemisinin combination therapy (ACT). Plasmodium falciparum isolates were collected in 2015 in Yaoundé (Cameroon) from patients treated with dihydroartemisinin-piperaquine combination. Samples were analysed for their susceptibility to artemisinin using the k13-propeller sequencing, the ex vivo ring-stage survival assay, the in vivo parasite positive rate and the clinical statute at day 2. None of the collected isolates revealed the presence of resistance mutations in the k13-propeller sequence. The median ring-stage survival rate for all the 64 interpretable isolates after a 6-hour pulse of 700 nM dihydroartemisinin was low, 0.49% (IQR: 0-1.3). Total parasite clearance was observed for 87.5% of patients and the remaining parasitaemic isolates (12.5%) showed a high reduction of parasite load, ranging from 97.5 to 99.9%. Clinical symptoms disappeared in 92.8% of cases. This study demonstrated the absence of k13-resistant genotypes in P. falciparum isolates from Cameroon. Only synonymous mutations were found with a low prevalence (4.3%). A good association between k13 genotypes and the ex vivo ring-stage survival assay or parasitological and clinical data was obtained. These results give a baseline for the long-term monitoring of artemisinin derivative efficacy in Africa.

On the Diversity of Malaria Parasites in African Apes and the Origin of Plasmodium falciparum from Bonobos

PubMed Central

Pacheco, M. Andreina; Mugisha, Lawrence; André, Claudine; Halbwax, Michel; Fischer, Anne; Krief, Jean-Michel; Kasenene, John M.; Crandfield, Mike; Cornejo, Omar E.; Chavatte, Jean-Marc; Lin, Clara; Letourneur, Franck; Grüner, Anne Charlotte; McCutchan, Thomas F.; Rénia, Laurent; Snounou, Georges

2010-01-01

The origin of Plasmodium falciparum, the etiological agent of the most dangerous forms of human malaria, remains controversial. Although investigations of homologous parasites in African Apes are crucial to resolve this issue, studies have been restricted to a chimpanzee parasite related to P. falciparum, P. reichenowi, for which a single isolate was available until very recently. Using PCR amplification, we detected Plasmodium parasites in blood samples from 18 of 91 individuals of the genus Pan, including six chimpanzees (three Pan troglodytes troglodytes, three Pan t. schweinfurthii) and twelve bonobos (Pan paniscus). We obtained sequences of the parasites' mitochondrial genomes and/or from two nuclear genes from 14 samples. In addition to P. reichenowi, three other hitherto unknown lineages were found in the chimpanzees. One is related to P. vivax and two to P. falciparum that are likely to belong to distinct species. In the bonobos we found P. falciparum parasites whose mitochondrial genomes indicated that they were distinct from those present in humans, and another parasite lineage related to P. malariae. Phylogenetic analyses based on this diverse set of Plasmodium parasites in African Apes shed new light on the evolutionary history of P. falciparum. The data suggested that P. falciparum did not originate from P. reichenowi of chimpanzees (Pan troglodytes), but rather evolved in bonobos (Pan paniscus), from which it subsequently colonized humans by a host-switch. Finally, our data and that of others indicated that chimpanzees and bonobos maintain malaria parasites, to which humans are susceptible, a factor of some relevance to the renewed efforts to eradicate malaria. PMID:20169187

Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants

PubMed Central

Safeukui, Innocent; Millet, Pascal; Boucher, Sébastien; Melinard, Laurence; Fregeville, Frédéric; Receveur, Marie-Catherine; Pistone, Thierry; Fialon, Pierre; Vincendeau, Philippe; Fleury, Hervé; Malvy, Denis

2008-01-01

Background A simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) in febrile returning travellers and migrants was developed and evaluated. Methods Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth Plasmodium species, while the acceptor probe sequence was designed for P. falciparum over a region containing one mismatched, which allowed differentiation of the three other Plasmodium species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria. Results Blood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5°C (0.6°C) for P. falciparum infection and 64.6°C (1.8°C) for non-P. falciparum species. These Tms values of the P. falciparum or non-P. falciparum species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 – 1) or P. falciparum-specific (0.91, 0.8 – 1) primers, or with the microscopy results (0.70, 0.6 – 0.8). The real-time assay was 100% sensitive and specific for differentiation of P. falciparum to non-P. falciparum species, compared with conventional PCR or microscopy. The real-time PCR assay can also detect individuals with mixed infections (P. falciparum and non-P. falciparum sp.) in the same sample. Conclusion This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of P. falciparum to other Plasmodium species. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed. PMID:18442362

Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates.

PubMed

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

2015-01-23

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.

Investigation of a Sudden Malaria Outbreak in the Isolated Amazonian Village of Saül, French Guiana, January–April 2009

PubMed Central

Berger, Franck; Flamand, Claude; Musset, Lise; Djossou, Félix; Rosine, Jacques; Sanquer, Marie-Anne; Dusfour, Isabelle; Legrand, Eric; Ardillon, Vanessa; Rabarison, Patrick; Grenier, Claire; Girod, Romain

2012-01-01

Malaria is endemic in French Guiana. Plasmodium falciparum and Plasmodium vivax are the predominant species responsible and Anopheles darlingi is described as the major vector. In mid-August 2008, an increase in malaria incidence was observed in Saül. A retrospective cohort survey was performed. In vitro susceptibility profiles to antimalarials were determined on P. falciparum isolates. Collections of mosquitoes were organized. The malaria attack rate reached 70.6/100. The risk of malaria increased for people between 40 and 49 years of age, living in a house not subjected to a recent indoor residual insecticide spraying or staying overnight in the surrounding forest. All isolates were susceptible. Anopheles darlingi females and larvae were collected in the village suggesting a local transmission. Our results strongly support a role of illegal mining activities in the emergence of new foci of malaria. Therefore, public health authorities should define policies to fight malaria at a transborder level. PMID:22492141

A SYBR Green 1-based in vitro test of susceptibility of Ghanaian Plasmodium falciparum clinical isolates to a panel of anti-malarial drugs.

PubMed

Quashie, Neils B; Duah, Nancy O; Abuaku, Benjamin; Quaye, Lydia; Ayanful-Torgby, Ruth; Akwoviah, George A; Kweku, Margaret; Johnson, Jacob D; Lucchi, Naomi W; Udhayakumar, Venkatachalam; Duplessis, Christopher; Kronmann, Karl C; Koram, Kwadwo A

2013-12-17

Based on report of declining efficacy of chloroquine, Ghana shifted to the use of artemisinin-based combination therapy (ACT) in 2005 as the first-line anti-malarial drug. Since then, there has not been any major evaluation of the efficacy of anti-malarial drugs in Ghana in vitro. The sensitivity of Ghanaian Plasmodium falciparum isolates to anti-malarial drugs was, therefore, assessed and the data compared with that obtained prior to the change in the malaria treatment policy. A SYBR Green 1 fluorescent-based in vitro drug sensitivity assay was used to assess the susceptibility of clinical isolates of P. falciparum to a panel of 12 anti-malarial drugs in three distinct eco-epidemiological zones in Ghana. The isolates were obtained from children visiting health facilities in sentinel sites located in Hohoe, Navrongo and Cape Coast municipalities. The concentration of anti-malarial drug inhibiting parasite growth by 50% (IC50) for each drug was estimated using the online program, ICEstimator. Pooled results from all the sentinel sites indicated geometric mean IC50 values of 1.60, 3.80, 4.00, 4.56, 5.20, 6.11, 10.12, 28.32, 31.56, 93.60, 107.20, and 8952.50 nM for atovaquone, artesunate, dihydroartemisin, artemether, lumefantrine, amodiaquine, mefloquine, piperaquine, chloroquine, tafenoquine, quinine, and doxycycline, respectively. With reference to the literature threshold value indicative of resistance, the parasites showed resistance to all the test drugs except the artemisinin derivatives, atovaquone and to a lesser extent, lumefantrine. There was nearly a two-fold decrease in the IC50 value determined for chloroquine in this study compared to that determined in 2004 (57.56 nM). This observation is important, since it suggests a significant improvement in the efficacy of chloroquine, probably as a direct consequence of reduced drug pressure after cessation of its use. Compared to that measured prior to the change in treatment policy, significant elevation of artesunate IC50 value was observed. The results also suggest the existence of possible cross-resistance among some of the test drugs. Ghanaian P. falciparum isolates, to some extent, have become susceptible to chloroquine in vitro, however the increasing trend in artesunate IC50 value observed should be of concern. Continuous monitoring of ACT in Ghana is recommended.

Plasmodium falciparum msp1 and msp2 genetic diversity and allele frequencies in parasites isolated from symptomatic malaria patients in Bobo-Dioulasso, Burkina Faso.

PubMed

Somé, Anyirékun Fabrice; Bazié, Thomas; Zongo, Issaka; Yerbanga, R Serge; Nikiéma, Frédéric; Neya, Cathérine; Taho, Liz Karen; Ouédraogo, Jean-Bosco

2018-05-30

In Burkina Faso, malaria remains the overall leading cause of morbidity and mortality accounting for 35.12% of consultations, 40.83% of hospitalizations and 37.5% of deaths. Genotyping of malaria parasite populations remains an important tool to determine the types and number of parasite clones in an infection. The present study aimed to evaluate the merozoite surface protein 1 (msp1) and merozoite surface protein 2 (msp2) genetic diversity and allele frequencies in Bobo-Dioulasso, Burkina Faso. Dried blood spots (DBS) were collected at baseline from patients with uncomplicated malaria in urban health centers in Bobo-Dioulasso. Parasite DNA was extracted using chelex-100 and species were identified using nested PCR. Plamodium falciparum msp1 and msp2 genes were amplified by nested polymerase chain reaction (PCR) and PCR products were analyzed by electrophoresis on a 2.5% agarose gel. Alleles were categorized according to their molecular weight. A total of 228 blood samples were analyzed out of which 227 (99.9%) were confirmed as P. falciparum-positive and one sample classified as mixed infection for P. malaria and P. falciparum. In msp1, the K1 allelic family was predominant with 77.4% (162/209) followed respectively by the MAD20 allelic family with 41.3% and R033 allelic family with 36%. In msp2, the 3D7 allelic family was the most frequently detected with 93.1 % compared to FC27 with 41.3%. Twenty-one different alleles were observed in msp1 with 9 alleles for K1, 8 alleles for MAD20 and 4 alleles for R033. In msp2, 25 individual alleles were detected with 10 alleles for FC27 and 15 alleles for 3D7. The mean multiplicity of falciparum infection was 1.95 with respectively 1.8 (1.76-1.83) and 2.1 (2.03-2.16) for msp1 and msp2 (P = 0.01). Our study showed high genetic diversity and allelic frequencies of msp1 and msp2 in Plasmodium falciparum isolates from symptomatic malaria patients in Bobo-Dioulasso.

The MSPDBL2 Codon 591 Polymorphism Is Associated with Lumefantrine In Vitro Drug Responses in Plasmodium falciparum Isolates from Kilifi, Kenya

PubMed Central

Okombo, John; Mwai, Leah; Kiara, Steven M.; Pole, Lewa; Tetteh, Kevin K. A.; Nzila, Alexis; Marsh, Kevin

2014-01-01

The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.04). The high frequency of Pfmspdbl2 codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem). PMID:25534732

Molecular surveillance for drug-resistant Plasmodium falciparum in clinical and subclinical populations from three border regions of Burma/Myanmar: cross-sectional data and a systematic review of resistance studies

PubMed Central

2012-01-01

Background Confirmation of artemisinin-delayed parasite clearance in Plasmodium falciparum along the Thai-Myanmar border has inspired a global response to contain and monitor drug resistance to avert the disastrous consequences of a potential spread to Africa. However, resistance data from Myanmar are sparse, particularly from high-risk areas where limited health services and decades of displacement create conditions for resistance to spread. Subclinical infections may represent an important reservoir for resistance genes that confer a fitness disadvantage relative to wild-type alleles. This study estimates the prevalence of resistance genotypes in three previously unstudied remote populations in Myanmar and tests the a priori hypothesis that resistance gene prevalence would be higher among isolates collected from subclinical infections than isolates collected from febrile clinical patients. A systematic review of resistance studies is provided for context. Methods Community health workers in Karen and Kachin States and an area spanning the Indo-Myanmar border collected dried blood spots from 988 febrile clinical patients and 4,591 villagers with subclinical infection participating in routine prevalence surveys. Samples positive for P. falciparum 18 s ribosomal RNA by real-time PCR were genotyped for P. falciparum multidrug resistance protein (pfmdr1) copy number and the pfcrt K76T polymorphism using multiplex real-time PCR. Results Pfmdr1 copy number increase and the pfcrt K76 polymorphism were determined for 173 and 269 isolates, respectively. Mean pfmdr1 copy number was 1.2 (range: 0.7 to 3.7). Pfmdr1 copy number increase was present in 17.5%, 9.6% and 11.1% of isolates from Karen and Kachin States and the Indo-Myanmar border, respectively. Pfmdr1 amplification was more prevalent in subclinical isolates (20.3%) than clinical isolates (6.4%, odds ratio 3.7, 95% confidence interval 1.1 - 12.5). Pfcrt K76T prevalence ranged from 90-100%. Conclusions Community health workers can contribute to molecular surveillance of drug resistance in remote areas of Myanmar. Marginal and displaced populations under-represented among previous resistance investigations can and should be included in resistance surveillance efforts, particularly once genetic markers of artemisinin-delayed parasite clearance are identified. Subclinical infections may contribute to the epidemiology of drug resistance, but determination of gene amplification from desiccated filter samples requires further validation when DNA concentration is low. PMID:22992214

Genetic structure of Plasmodium falciparum populations across the Honduras-Nicaragua border.

PubMed

Larrañaga, Nerea; Mejía, Rosa E; Hormaza, José I; Montoya, Alberto; Soto, Aida; Fontecha, Gustavo A

2013-10-04

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.

Genetic polymorphism of merozoite surface protein-1 and merozoite surface protein-2 in Plasmodium falciparum isolates from Brazzaville, Republic of Congo

PubMed Central

2011-01-01

Background The characterization of malaria parasite populations circulating in an area is part of site characterization, as a basis for evaluating the impact of malaria interventions on genetic diversity, parasite species, and multiplicity of infection. The present study was aimed at analysing genetic diversity of Plasmodium falciparum merozoite surface proteins 1 and 2 (MSP-1 and MSP-2) and to determine the multiplicity of infection in clinical isolates collected from children living in the Southern district of Brazzaville in the Republic of Congo. Methods A total of 125 isolates from patients with uncomplicated malaria attending Terinkyo and Madibou health centres were collected between January and June 2005 while evaluating the therapeutic efficacy of amodiaquine-artesunate combination. DNA was extracted and msp-1 and msp-2 genes were genotyped using allele-specific nested-PCR. Results Out of 468 distinct fragments detected, 15 msp-1 and 20 msp-2 genotypes were identified. For the msp-1 gene, K1 family was the predominant allelic type carried alone or in association with RO33 and Mad20 types, whereas the 3D7 family was the most prevalent in the msp-2 gene. Overall, the mean multiplicity of infection was 2.2. Out of 125 samples, 104 (83%) harboured more than one parasite genotype. There was no statistical significant difference in the multiplicity of infection by either sex or age of patients. However, a statistically significant correlation was found between parasite densities and the number of genotypes. Conclusion Polymorphism in P. falciparum clinical isolates from Brazzaville was high and mainly of multiple clones. The basis for the positive association between parasite densities and multiplicity of infection is discussed. PMID:21936949

Structure-activity relationships of C6-uridine derivatives targeting plasmodia orotidine monophosphate decarboxylase.

PubMed

Bello, Angelica M; Poduch, Ewa; Liu, Yan; Wei, Lianhu; Crandall, Ian; Wang, Xiaoyang; Dyanand, Christopher; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2008-02-14

Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on human health, especially due to multidrug resistance. In Plasmodia, orotidine 5'-monophosphate decarboxylase (ODCase) is an essential enzyme for the de novo synthesis of uridine 5'-monophosphate. Impairing ODCase in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure-activity relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6- N-methylamino, and 6- N, N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial ODCase. 6-Azidouridine 5'-monophosphate is a potent covalent inhibitor of P. falciparum ODCase. 6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6- N-Methylamino and 6- N, N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.

Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA▿†‡

PubMed Central

Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W.; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M.; Villegas, Leopoldo; Escalante, Ananias A.; Kachur, S. Patrick; Barnwell, John W.; Peterson, David S.; Udhayakumar, Venkatachalam; Kissinger, Jessica C.

2011-01-01

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms. PMID:21525225

Antiplasmodial activities of dyes against Plasmodium falciparum asexual and sexual stages: Contrasted uptakes of triarylmethanes Brilliant green, Green S (E142), and Patent Blue V (E131) by erythrocytes.

PubMed

Leba, Louis-Jérôme; Popovici, Jean; Estevez, Yannick; Pelleau, Stéphane; Legrand, Eric; Musset, Lise; Duplais, Christophe

2017-12-01

The search for safe antimalarial compounds acting against asexual symptom-responsible stages and sexual transmission-responsible forms of Plasmodium species is one of the major challenges in malaria elimination programs. So far, among current drugs approved for human use, only primaquine has transmission-blocking activity. The discovery of small molecules targeting different Plasmodium falciparum life stages remains a priority in antimalarial drug research. In this context, several independent studies have recently reported antiplasmodial and transmission-blocking activities of commonly used stains, dyes and fluorescent probes against P. falciparum including chloroquine-resistant isolates. Herein we have studied the antimalarial activities of dyes with different scaffold and we report that the triarylmethane dye (TRAM) Brilliant green inhibits the growth of asexual stages (IC 50  ≤ 2 μM) and has exflagellation-blocking activity (IC 50  ≤ 800 nM) against P. falciparum reference strains (3D7, 7G8) and chloroquine-resistant clinical isolate (Q206). In a second step we have investigated the antiplasmodial activities of two polysulfonated triarylmethane food dyes. Green S (E142) is weakly active against P. falciparum asexual stage (IC 50 ≃ 17 μM) whereas Patent Blue V (E131) is inactive in both antimalarial assays. By applying liquid chromatography techniques for the culture supernatant analysis after cell washings and lysis, we report the detection of Brilliant green in erythrocytes, the selective uptake of Green S (E142) by infected erythrocytes, whereas Patent Blue V (E131) could not be detected within non-infected and 3D7-infected erythrocytes. Overall, our results suggest that two polysulfonated food dyes might display different affinity with transporters or channels on infected RBC membrane. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Comparative sequence analysis of domain I of Plasmodium falciparum apical membrane antigen 1 from Saudi Arabia and worldwide isolates.

PubMed

Al-Qahtani, Ahmed A; Abdel-Muhsin, Abdel-Muhsin A; Dajem, Saad M Bin; AlSheikh, Adel Ali H; Bohol, Marie Fe F; Al-Ahdal, Mohammed N; Putaporntip, Chaturong; Jongwutiwes, Somchai

2016-04-01

The apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) plays a crucial role in erythrocyte invasion and is a target of protective antibodies. Although domain I of PfAMA1 has been considered a promising vaccine component, extensive sequence diversity in this domain could compromise an effective vaccine design. To explore the extent of sequence diversity in domain I of PfAMA1, P. falciparum-infected blood samples from Saudi Arabia collected between 2007 and 2009 were analyzed and compared with those from worldwide parasite populations. Forty-six haplotypes and a novel codon change (M190V) were found among Saudi Arabian isolates. The haplotype diversity (0.948±0.004) and nucleotide diversity (0.0191±0.0008) were comparable to those from African hyperendemic countries. Positive selection in domain I of PfAMA1 among Saudi Arabian parasite population was observed because nonsynonymous nucleotide substitutions per nonsynonymous site (dN) significantly exceeded synonymous nucleotide substitutions per synonymous site (dS) and Tajima's D and its related statistics significantly deviated from neutrality in the positive direction. Despite a relatively low prevalence of malaria in Saudi Arabia, a minimum of 17 recombination events occurred in domain I. Genetic differentiation was significant between P. falciparum in Saudi Arabia and parasites from other geographic origins. Several shared or closely related haplotypes were found among parasites from different geographic areas, suggesting that vaccine derived from multiple shared epitopes could be effective across endemic countries. Copyright © 2016 Elsevier B.V. All rights reserved.

Prevalence of antifolate resistance mutations in Plasmodium falciparum isolates in Afghanistan

PubMed Central

2013-01-01

Background Artesunate plus sulphadoxine-pyrimethamine (AS+SP) is now first-line treatment for Plasmodium falciparum infection in several south Asian countries, including Afghanistan. Molecular studies provide a sensitive means to investigate the current state of drug susceptibility to the SP component, and can also provide information on the likely efficacy of other potential forms of artemisinin-combination therapy. Methods During the years 2007 to 2010, 120 blood spots from patients with P. falciparum malaria were obtained in four provinces of Afghanistan. PCR-based methods were used to detect drug-resistance mutations in dhfr, dhps, pfcrt and pfmdr1, as well as to determine copy number of pfmdr1. Results The majority (95.5%) of infections had a double mutation in the dhfr gene (C59R, S108N); no mutations at dhfr positions 16, 51 or 164 were seen. Most isolates were wild type across the dhps gene, but five isolates from the provinces of Kunar and Nangarhar in eastern Afghanistan had the triple mutation A437G / K540E / A581G; all five cases were successfully treated with three receiving AS+SP and two receiving dihydroartemisinin-piperaquine. All isolates showed the pfcrt SVNMT chloroquine resistance haplotype. Five of 79 isolates had the pfmdr1 N86Y mutation, while 52 had pfmdr1 Y184F; positions 1034, 1042 and 1246 were wild type in all isolates. The pfmdr1 gene was not amplified in any sample. Conclusions This study indicates that shortly after the adoption of AS+SP as first-line treatment in Afghanistan, most parasites had a double mutation haplotype in dhfr, and a small number of isolates from eastern Afghanistan harboured a triple mutation haplotype in dhps. The impact of these mutations on the efficacy of AS+SP remains to be assessed in significant numbers of patients, but these results are clearly concerning since they suggest a higher degree of SP resistance than previously detected. Further focused molecular and clinical studies in this region are urgently required. PMID:23497229

Antioxidant and Antiplasmodial Activities of Bergenin and 11-O-Galloylbergenin Isolated from Mallotus philippensis

PubMed Central

Khan, Hamayun; Amin, Hazrat; Ullah, Asad; Saba, Sumbal; Rafique, Jamal; Khan, Khalid; Ahmad, Nasir; Badshah, Syed Lal

2016-01-01

Two important biologically active compounds were isolated from Mallotus philippensis. The isolated compounds were characterized using spectroanalytical techniques and found to be bergenin (1) and 11-O-galloylbergenin (2). The in vitro antioxidant and antiplasmodial activities of the isolated compounds were determined. For the antioxidant potential, three standard analytical protocols, namely, DPPH radical scavenging activity (RSA), reducing power assay (RPA), and total antioxidant capacity (TAC) assay, were adopted. The results showed that compound 2 was found to be more potent antioxidant as compared to 1. Fascinatingly, compound 2 displayed better EC50 results as compared to α-tocopherol while being comparable with ascorbic acid. The antiplasmodial assay data showed that both the compound exhibited good activity against chloroquine sensitive strain of Plasmodium falciparum (D10) and IC50 values were found to be less than 8 μM. The in silico molecular docking analyses were also performed for the determination of binding affinity of the isolated compounds using P. falciparum proteins PfLDH and Pfg27. The results showed that compound 2 has high docking score and binding affinity to both protein receptors as compared to compound 1. The demonstrated biological potentials declared that compound 2 could be the better natural antioxidant and antiplasmodial candidate. PMID:26998192

Molecular Epidemiology of Malaria in Cameroon and Côte d'Ivoire. XXXI. Kelch 13 Propeller Sequences in Plasmodium falciparum Isolates before and after Implementation of Artemisinin-Based Combination Therapy.

PubMed

Djaman, Joseph Allico; Olefongo, Dagnogo; Ako, Aristide Berenger; Roman, Jocelyne; Ngane, Vincent Foumane; Basco, Leonardo K; Tahar, Rachida

2017-07-01

Artemisinin-resistant malaria has not been reported from Africa, but resistance can possibly spread from Asia or arise independently in Africa. The emergence of artemisinin resistance in Africa can be monitored by molecular assay of Kelch 13 (K13) propeller sequences. A total of 251 archived DNA samples of Plasmodium falciparum isolates collected in 2002, 2003, and 2006 in Yaounde, Cameroon, and 47 samples collected in 2006 and 2013 in Abidjan, Côte d'Ivoire, were analyzed for K13-propeller sequence polymorphism. Only one isolate carried a mutant K13-propeller allele (E602D). None of the isolates carried the key mutant alleles (Y493H, R539T, I543T, and C580Y) associated with artemisinin resistance in Cambodia. The presence of the mutant allele was not correlated with in vitro response to dihydroartemisinin determined by the classical hypoxanthine incorporation assay. There was no evidence of K13 mutations associated with artemisinin resistance before and soon after the introduction of artemisinin-based combination therapies in Cameroon and Côte d'Ivoire.

In vitro antiplasmodial activity of bacterium RJAUTHB 14 associated with marine sponge Haliclona Grant against Plasmodium falciparum.

PubMed

Jacob Inbaneson, Samuel; Ravikumar, Sundaram

2012-06-01

Malaria is the most important parasitic disease, leading to annual death of about one million people, and the Plasmodium falciparum develops resistance to well-established antimalarial drugs. The newest antiplasmodial drug from a marine microorganism helps in addressing this problem. In the present study, Haliclona Grant were collected and subjected for enumeration and isolation of associated bacteria. The count of bacterial isolates was maximum in November 2007 (18 × 10(4) colony-forming units (CFU) g(-1), and the average count was maximum during the monsoon season (117 × 10(3) CFU g(-1)). Thirty-three morphologically different bacterial isolates were isolated from Haliclona Grant, and the extracellular ethyl acetate extracts were screened for antiplasmodial activity against P. falciparum. The antiplasmodial activity of bacterium RJAUTHB 14 (11.98 μg[Symbol: see text]ml(-1)) is highly comparable with the positive control chloroquine (IC(50) 19.59 μg[Symbol: see text]ml(-1)), but the other 21 bacterial extracts showed an IC(50) value of more than 100 μg[Symbol: see text]ml(-1). Statistical analysis reveals that significant in vitro antiplasmodial activity (P < 0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes showed no morphological changes in erythrocytes by the ethyl acetate extract of bacterial isolates after 48 h of incubation. The in vitro antiplasmodial activity might be due to the presence of reducing sugars and alkaloids in the ethyl acetate extracts of bacterium RJAUTHB 14. The 16S rRNA gene partial sequence of bacterium RJAUTHB 14 is deposited in NCBI (GenBank accession no. GU269569). It is concluded from the present study that the ethyl acetate extracts of bacterium RJAUTHB 14 possess lead compounds for the development of antiplasmodial drugs.

More than just immune evasion: Hijacking complement by Plasmodium falciparum.

PubMed

Schmidt, Christoph Q; Kennedy, Alexander T; Tham, Wai-Hong

2015-09-01

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.

The MSPDBL2 codon 591 polymorphism is associated with lumefantrine in vitro drug responses in Plasmodium falciparum isolates from Kilifi, Kenya.

PubMed

Ochola-Oyier, Lynette Isabella; Okombo, John; Mwai, Leah; Kiara, Steven M; Pole, Lewa; Tetteh, Kevin K A; Nzila, Alexis; Marsh, Kevin

2015-03-01

The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.04). The high frequency of Pfmspdbl2 codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem). Copyright © 2015, Ochola-Oyier et al.

Genetic Structure of Plasmodium falciparum and Elimination of Malaria, Comoros Archipelago

PubMed Central

Rebaudet, Stanislas; Bogreau, Hervé; Silaï, Rahamatou; Lepère, Jean-François; Bertaux, Lionel; Pradines, Bruno; Delmont, Jean; Gautret, Philippe; Parola, Philippe

2010-01-01

The efficacy of malaria control and elimination on islands may depend on the intensity of new parasite inflow. On the Comoros archipelago, where falciparum malaria remains a major public health problem because of spread of drug resistance and insufficient malaria control, recent interventions for malaria elimination were planned on Moheli, 1 of 4 islands in the Comoros archipelago. To assess the relevance of such a local strategy, we performed a population genetics analysis by using multilocus microsatellite and resistance genotyping of Plasmodium falciparum sampled from each island of the archipelago. We found a contrasted population genetic structure explained by geographic isolation, human migration, malaria transmission, and drug selective pressure. Our findings suggest that malaria elimination interventions should be implemented simultaneously on the entire archipelago rather than restricted to 1 island and demonstrate the necessity for specific chemoresistance surveillance on each of the 4 Comorian islands. PMID:21029525

B-Cell Responses to Pregnancy-Restricted and -Unrestricted Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigens in Ghanaian Women Naturally Exposed to Malaria Parasites

PubMed Central

Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F.; Barfod, Lea

2014-01-01

Protective immunity to Plasmodium falciparum malaria acquired after natural exposure is largely antibody mediated. IgG-specific P. falciparum EMP1 (PfEMP1) proteins on the infected erythrocyte surface are particularly important. The transient antibody responses and the slowly acquired protective immunity probably reflect the clonal antigenic variation and allelic polymorphism of PfEMP1. However, it is likely that other immune-evasive mechanisms are also involved, such as interference with formation and maintenance of immunological memory. We measured PfEMP1-specific antibody levels by enzyme-linked immunosorbent assay (ELISA) and memory B-cell frequencies by enzyme-linked immunosorbent spot (ELISPOT) assay in a cohort of P. falciparum-exposed nonpregnant Ghanaian women. The antigens used were a VAR2CSA-type PfEMP1 (IT4VAR04) with expression restricted to parasites infecting the placenta, as well as two commonly recognized PfEMP1 proteins (HB3VAR06 and IT4VAR60) implicated in rosetting and not pregnancy restricted. This enabled, for the first time, a direct comparison in the same individuals of immune responses specific for a clinically important parasite antigen expressed only during well-defined periods (pregnancy) to responses specific for comparable antigens expressed independent of pregnancy. Our data indicate that PfEMP1-specific B-cell memory is adequately acquired even when antigen exposure is infrequent (e.g., VAR2CSA-type PfEMP1). Furthermore, immunological memory specific for VAR2CSA-type PfEMP1 can be maintained for many years without antigen reexposure and after circulating antigen-specific IgG has disappeared. The study provides evidence that natural exposure to P. falciparum leads to formation of durable B-cell immunity to clinically important PfEMP1 antigens. This has encouraging implications for current efforts to develop PfEMP1-based vaccines. PMID:24566620

Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax

PubMed Central

Pakalapati, Deepak; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Subudhi, Amit K; Boopathi, Arunachalam P; Saxena, Vishal; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

2013-01-01

The 28S rRNA gene was amplified and sequenced from P. falciparum and P. vivax isolates collected from northwest India. Based upon the sequence diversity of the Plasmodium 28SrRNA gene in comparison with its human counterpart, various nested polymerase chain reaction (PCR) primers were designed from the 3R region of the 28SrRNA gene and evaluated on field isolates. This is the first report demonstrating the utility of this gene for species-specific diagnosis of malaria for these two species, prevalent in India. The initial evaluation on 363 clinical isolates indicated that, in comparison with microscopy, which showed sensitivity and specificity of 85.39% and 100% respectively, the sensitivity and specificity of the nested PCR assay was found to be 99.08% and 100% respectively. This assay was also successful in detecting mixed infections that are undetected by microscopy. Our results demonstrate the utility of the 28S rRNA gene as a diagnostic target for the detection of the major plasmodial species infecting humans. PMID:23816509

Parasites bearing a single copy of the multi-drug resistance gene (pfmdr-1) with wild-type SNPs predominate amongst Plasmodium falciparum isolates from Malawi.

PubMed

Nkhoma, Standwell; Nair, Shalini; Mukaka, Mavuto; Molyneux, Malcolm E; Ward, Stephen A; Anderson, Timothy J C

2009-07-01

We genotyped 160 P. falciparum infections from Malawi for pfmdr-1 copy number changes and SNPs associated with in vivo tolerance and poor in vitro sensitivity to the component drugs of Coartem. We also measured in vitro susceptibility of 49 of these isolates to a variety of drugs in clinical use or with a potential for use in Africa. All 160 infections carried a single copy of pfmdr-1 but 34% exhibited sequence variation at 4 of the 5 polymorphic sites in pfmdr-1. Isolates carrying 86-Asn and 184-Tyr pfmdr-1 alleles were significantly less sensitive (p<0.001) to mefloquine, lumefantrine, artemether and dihydroartemisinin compared with those bearing 86-Tyr and 184-Phe polymorphisms. This study provides baseline measures prior to policy change: continued surveillance for changes in baseline drug susceptibility, pfmdr-1 copy number and SNPs, and other putative Coartem resistance loci will be necessary to provide an early warning of emerging Coartem resistance in this setting.

Genetic diversity in the merozoite surface protein 1 and 2 genes of Plasmodium falciparum from the Artibonite Valley of Haiti.

PubMed

Londono-Renteria, Berlin; Eisele, Thomas P; Keating, Joseph; Bennett, Adam; Krogstad, Donald J

2012-01-01

Describing genetic diversity of the Plasmodium falciparum parasite provides important information about the local epidemiology of malaria. In this study, we examined the genetic diversity of P. falciparum isolates from the Artibonite Valley in Haiti using the allelic families of merozoite surface protein 1 and 2 genes (msp-1 and msp-2). The majority of study subjects infected with P. falciparum had a single parasite genotype (56% for msp-1 and 69% for msp-2: n=79); 9 distinct msp-1 genotypes were identified by size differences on agarose gels. K1 was the most polymorphic allelic family with 5 genotypes (amplicons from 100 to 300 base pairs [bp]); RO33 was the least polymorphic, with a single genotype (120-bp). Although both msp-2 alleles (3D7/IC1, FC27) had similar number of genotypes (n=4), 3D7/IC1 was more frequent (85% vs. 26%). All samples were screened for the presence of the K76T mutation on the P. falciparum chloroquine resistance transporter (pfcrt) gene with 10 of 79 samples positive. Of the 2 (out of 10) samples from individuals follow-up for 21 days, P. falciparum parasites were present through day 7 after treatment with chloroquine. No parasites were found on day 21. Our results suggest that the level of genetic diversity is low in this area of Haiti, which is consistent with an area of low transmission. Copyright © 2011 Elsevier B.V. All rights reserved.

Unraveling Haplotype Diversity of the Apical Membrane Antigen-1 Gene in Plasmodium falciparum Populations in Thailand

PubMed Central

Lumkul, Lalita; Sawaswong, Vorthon; Simpalipan, Phumin; Kaewthamasorn, Morakot; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn

2018-01-01

Development of an effective vaccine is critically needed for the prevention of malaria. One of the key antigens for malaria vaccines is the apical membrane antigen 1 (AMA-1) of the human malaria parasite Plasmodium falciparum, the surface protein for erythrocyte invasion of the parasite. The gene encoding AMA-1 has been sequenced from populations of P. falciparum worldwide, but the haplotype diversity of the gene in P. falciparum populations in the Greater Mekong Subregion (GMS), including Thailand, remains to be characterized. In the present study, the AMA-1 gene was PCR amplified and sequenced from the genomic DNA of 65 P. falciparum isolates from 5 endemic areas in Thailand. The nearly full-length 1,848 nucleotide sequence of AMA-1 was subjected to molecular analyses, including nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity and neutrality tests. Phylogenetic analysis and pairwise population differentiation (Fst indices) were performed to infer the population structure. The analyses identified 60 single nucleotide polymorphic loci, predominately located in domain I of AMA-1. A total of 31 unique AMA-1 haplotypes were identified, which included 11 novel ones. The phylogenetic tree of the AMA-1 haplotypes revealed multiple clades of AMA-1, each of which contained parasites of multiple geographical origins, consistent with the Fst indices indicating genetic homogeneity or gene flow among geographically distinct populations of P. falciparum in Thailand’s borders with Myanmar, Laos and Cambodia. In summary, the study revealed novel haplotypes and population structure needed for the further advancement of AMA-1-based malaria vaccines in the GMS. PMID:29742870

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia

PubMed Central

Parobek, Christian M.; Lin, Jessica T.; Saunders, David L.; Barnett, Eric J.; Lon, Chanthap; Lanteri, Charlotte A.; Balasubramanian, Sujata; Brazeau, Nicholas; DeConti, Derrick K.; Garba, Deen L.; Meshnick, Steven R.; Spring, Michele D.; Chuor, Char Meng; Bailey, Jeffrey A.; Juliano, Jonathan J.

2016-01-01

Cambodia, in which both Plasmodium vivax and Plasmodium falciparum are endemic, has been the focus of numerous malaria-control interventions, resulting in a marked decline in overall malaria incidence. Despite this decline, the number of P. vivax cases has actually increased. To understand better the factors underlying this resilience, we compared the genetic responses of the two species to recent selective pressures. We sequenced and studied the genomes of 70 P. vivax and 80 P. falciparum isolates collected between 2009 and 2013. We found that although P. falciparum has undergone population fracturing, the coendemic P. vivax population has grown undisrupted, resulting in a larger effective population size, no discernable population structure, and frequent multiclonal infections. Signatures of selection suggest recent, species-specific evolutionary differences. Particularly, in contrast to P. falciparum, P. vivax transcription factors, chromatin modifiers, and histone deacetylases have undergone strong directional selection, including a particularly strong selective sweep at an AP2 transcription factor. Together, our findings point to different population-level adaptive mechanisms used by P. vivax and P. falciparum parasites. Although population substructuring in P. falciparum has resulted in clonal outgrowths of resistant parasites, P. vivax may use a nuanced transcriptional regulatory approach to population maintenance, enabling it to preserve a larger, more diverse population better suited to facing selective threats. We conclude that transcriptional control may underlie P. vivax’s resilience to malaria control measures. Novel strategies to target such processes are likely required to eradicate P. vivax and achieve malaria elimination. PMID:27911780

Genetic structure of Plasmodium falciparum populations across the Honduras-Nicaragua border

PubMed Central

2013-01-01

Background 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. Methods 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. Results There was a low level of genetic diversity in P. falciparum populations from Honduras and Nicaragua. Expected heterozigosity (He) results were similarly low for both populations. A moderate differentiation was revealed by the FST 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. PMID:24093629

C-terminal polymorphism of Plasmodium falciparium merozoite surface protein-1 (MSP-1) from Tak Province, Thailand.

PubMed

Viputtigul, Kwanjai; Tungpukdee, Noppadon; Ruangareerate, Toon; Luplertlop, Natthanej; Wilairatana, Polrat; Gaywee, Jariyanart; Krudsood, Srivicha

2013-01-01

This study was undertaken to ascertain the extent of polymorphism in the C-terminal region of Plasmodium falciparum merozoite surface protein (MSP-1) from 119 malaria patients in Tak Province on the western border of Thailand, who were admitted to the Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. P. falciparum infection was confirmed by microscopic examination of peripheral blood smears. Clinical manifestations were categorized into 2 groups: uncomplicated (94 cases) and complicated/severe (25 cases). A 1,040 basepair fragment of P. falciparum MSP-1 gene was compared with MSP-1 of reference strains retrieved from GenBank. The consensus sequences of MSP-1 block 16 showed it belonged to MAD20 genotype, which is the major allele of falciparum malaria from the western border of Thailand. MSP-1 block 16 amino acid fragment could be separated into 2 groups: similar and dissimilar to reference sequence. Four variations in MSP-1 block 16 were -1494K, D1510G, D1556N, and K1696I. MSP-1 block 16 diversity is not significantly associated with clinical manifestation although MAD 20 genotype is the predominant genotype in this area. The genetic data of MSP1 gene of faciparum malaria isolated from western Thai border contribute to the existing genetic database of Thai P. falciparum strain.

Development of cultured Plasmodium falciparum blood-stage malaria cell banks for early phase in vivo clinical trial assessment of anti-malaria drugs and vaccines.

PubMed

Stanisic, Danielle I; Liu, Xue Q; De, Sai Lata; Batzloff, Michael R; Forbes, Tanya; Davis, Christopher B; Sekuloski, Silvana; Chavchich, Marina; Chung, Wendy; Trenholme, Katharine; McCarthy, James S; Li, Tao; Sim, B Kim Lee; Hoffman, Stephen L; Good, Michael F

2015-04-07

The ability to undertake controlled human malaria infection (CHMI) studies for preliminary evaluation of malaria vaccine candidates and anti-malaria drug efficacy has been limited by the need for access to sporozoite infected mosquitoes, aseptic, purified, cryopreserved sporozoites or blood-stage malaria parasites derived ex vivo from malaria infected individuals. Three different strategies are described for the manufacture of clinical grade cultured malaria cell banks suitable for use in CHMI studies. Good Manufacturing Practices (GMP)-grade Plasmodium falciparum NF54, clinically isolated 3D7, and research-grade P. falciparum 7G8 blood-stage malaria parasites were cultured separately in GMP-compliant facilities using screened blood components and then cryopreserved to produce three P. falciparum blood-stage malaria cell banks. These cell banks were evaluated according to specific criteria (parasitaemia, identity, viability, sterility, presence of endotoxin, presence of mycoplasma or other viral agents and in vitro anti-malarial drug sensitivity of the cell bank malaria parasites) to ensure they met the criteria to permit product release according to GMP requirements. The P. falciparum NF54, 3D7 and 7G8 cell banks consisted of >78% ring stage parasites with a ring stage parasitaemia of >1.4%. Parasites were viable in vitro following thawing. The cell banks were free from contamination with bacteria, mycoplasma and a broad panel of viruses. The P. falciparum NF54, 3D7 and 7G8 parasites exhibited differential anti-malarial drug susceptibilities. The P. falciparum NF54 and 3D7 parasites were susceptible to all anti-malaria compounds tested, whereas the P. falciparum 7G8 parasites were resistant/had decreased susceptibility to four compounds. Following testing, all defined release criteria were met and the P. falciparum cell banks were deemed suitable for release. Ethical approval has been obtained for administration to human volunteers. The production of cultured P. falciparum blood-stage malaria cell banks represents a suitable approach for the generation of material suitable for CHMI studies. A key feature of this culture-based approach is the ability to take research-grade material through to a product suitable for administration in clinical trials.

Chloroquine-resistant Plasmodium vivax in transmigration settlements of West Kalimantan, Indonesia.

PubMed

Fryauff, D J; Tuti, S; Mardi, A; Masbar, S; Patipelohi, R; Leksana, B; Kain, K C; Bangs, M J; Richie, T L; Baird, J K

1998-10-01

Malariometric surveys were conducted during July 1996 in native Dayak villages and predominantly Javanese transmigration settlements in Ketapang district of West Kalimantan, Indonesia. Malaria prevalence ranged from 0.9% to 2.7% in Dayak villages and from 1% to 20% in the transmigration settlements. Plasmodium falciparum accounted for 67% of the cases among Dayaks but P. vivax was dominant among transmigrants, accounting for more than 72% of the infections. Chloroquine sensitivity/resistance was assessed by 28-day in vivo testing of uncomplicated malaria infections and measurement of chloroquine blood levels in cases where parasitemias reappeared within the 28-day test period. Resistance was based on the appearance of asexual parasites against chloroquine plus desethylchloroquine levels exceeding the minimally effective whole blood concentrations proposed for sensitive parasite strains (P. vivax, 100 ng/ml; P. falciparum, 200 ng/ml). All parasitemias cleared initially within four days of beginning supervised chloroquine therapy (25 mg base/kg over a 48-hr period), but asexual parasites reappeared within 28 days in 27 of 52 P. vivax and three of 12 P. falciparum cases. Chloroquine blood levels at the time of recurrent parasitemias revealed resistance in 12 of the 27 P. vivax cases and in one of the three P. falciparum cases. Genotypes of nine of the 12 recurrent P. vivax isolates matched with their primary isolates and ruled out reinfection. These findings establish the presence of chloroquine-resistant P. vivax on the island of Borneo. The pattern of malaria and the high frequency of chloroquine resistance by P. vivax at the West Kalimantan location may relate to demographic, ecologic, agricultural, and socioeconomic changes associated with transmigration.

Evidence of non-Plasmodium falciparum malaria infection in Kédougou, Sénégal.

PubMed

Daniels, Rachel F; Deme, Awa Bineta; Gomis, Jules F; Dieye, Baba; Durfee, Katelyn; Thwing, Julie I; Fall, Fatou B; Ba, Mady; Ndiop, Medoune; Badiane, Aida S; Ndiaye, Yaye Die; Wirth, Dyann F; Volkman, Sarah K; Ndiaye, Daouda

2017-01-03

Expanded malaria control efforts in Sénégal have resulted in increased use of rapid diagnostic tests (RDT) to identify the primary disease-causing Plasmodium species, Plasmodium falciparum. However, the type of RDT utilized in Sénégal does not detect other malaria-causing species such as Plasmodium ovale spp., Plasmodium malariae, or Plasmodium vivax. Consequently, there is a lack of information about the frequency and types of malaria infections occurring in Sénégal. This study set out to better determine whether species other than P. falciparum were evident among patients evaluated for possible malaria infection in Kédougou, Sénégal. Real-time polymerase chain reaction speciation assays for P. vivax, P. ovale spp., and P. malariae were developed and validated by sequencing and DNA extracted from 475 Plasmodium falciparum-specific HRP2-based RDT collected between 2013 and 2014 from a facility-based sample of symptomatic patients from two health clinics in Kédougou, a hyper-endemic region in southeastern Sénégal, were analysed. Plasmodium malariae (n = 3) and P. ovale wallikeri (n = 2) were observed as co-infections with P. falciparum among patients with positive RDT results (n = 187), including one patient positive for all three species. Among 288 negative RDT samples, samples positive for P. falciparum (n = 24), P. ovale curtisi (n = 3), P. ovale wallikeri (n = 1), and P. malariae (n = 3) were identified, corresponding to a non-falciparum positivity rate of 2.5%. These findings emphasize the limitations of the RDT used for malaria diagnosis and demonstrate that non-P. falciparum malaria infections occur in Sénégal. Current RDT used for routine clinical diagnosis do not necessarily provide an accurate reflection of malaria transmission in Kédougou, Sénégal, and more sensitive and specific methods are required for diagnosis and patient care, as well as surveillance and elimination activities. These findings have implications for other malaria endemic settings where species besides P. falciparum may be transmitted and overlooked by control or elimination activities.

The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity.

PubMed

Delhaes, L; Lazaro, J E; Gay, F; Thellier, M; Danis, M

1999-01-01

Malarial lactate dehydrogenase (LDH), which uses 3-acetyl pyridine adenine dinucleotide as coenzyme in a reaction leading to the formation of pyruvate from L-lactate, may be used to study the susceptibility of Plasmodium falciparum to a drug in vitro. Several methods to determine the activity of this enzyme are available. One, the colorimetric method of Makler and colleagues, was modified slightly, by using sodium-2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5 - carboxanilide (XTT) and following the reaction by measuring the optical density at 450 nm. Using two, culture-adapted strains of P. falciparum, this LDH assay was compared with the unmodified Makler's assay and with the isotopic microtest based on the incorporation of tritium-labelled hypoxanthine. Fresh, clinical P. falciparum isolates were also tested in the presence of several drugs, including chloroquine, mefloquine, quinine, halofantrine, atovaquone and qinghaosu derivatives. The results of the three assays were correlated for all the drugs tested except atovaquone. The two enzymatic assays are non-radioactive, rapid, reliable, inexpensive to perform and semi-automatic. However, they do require an initial parasitaemia of 2% with a haematocrit of 1.8%.

Inhibition of pyrimidine biosynthesis de novo in Plasmodium falciparum by 2-(4-t-butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone in vitro.

PubMed

Hammond, D J; Burchell, J R; Pudney, M

1985-01-01

The effects of the hydroxynaphthoquinone BW58C on some metabolite levels and the flux of H14CO3 through the de novo pyrimidine biosynthetic pathway of intact Plasmodium falciparum have been studied in vitro using HPLC techniques. 800 nM BW58C appeared to have no significant effect on the energy status of isolated P. falciparum, but at 0.1 nM it caused a dramatic decrease in the concentrations of pyrimidine nucleotides, specifically UTP, during 256 min of incubation. Although about one hour was required to achieve a significant decrease in pyrimidine nucleotide concentrations, a much more rapid inhibition of the flux of H14CO3 through the de novo pathway was found upon addition of 0.1 nM BW58C. This inhibition caused about a 10 fold increase in the radioactivity of carbamoyl-aspartate over a 64 min period, and an overall increase in the concentration of this metabolite of about 3 fold during 256 min of incubation. These effects of BW58C against P. falciparum in vitro are discussed in terms of inhibition of de novo pyrimidine biosynthesis at the site of dihydroorotate dehydrogenase.

Malaria epidemic and drug resistance, Djibouti.

PubMed

Rogier, Christophe; Pradines, Bruno; Bogreau, H; Koeck, Jean-Louis; Kamil, Mohamed-Ali; Mercereau-Puijalon, Odile

2005-02-01

Analysis of Plasmodium falciparum isolates collected before, during, and after a 1999 malaria epidemic in Djibouti shows that, despite a high prevalence of resistance to chloroquine, the epidemic cannot be attributed to a sudden increase in drug resistance of local parasite populations.

A four-year surveillance program for detection of Plasmodium falciparum chloroquine resistance in Honduras

PubMed Central

Fontecha, Gustavo A; Sanchez, Ana L; Mendoza, Meisy; Banegas, Engels; Mejía-Torres, Rosa E

2014-01-01

Countries could use the monitoring of drug resistance in malaria parasites as an effective early warning system to develop the timely response mechanisms that are required to avert the further spread of malaria. Drug resistance surveillance is essential in areas where no drug resistance has been reported, especially if neighbouring countries have previously reported resistance. Here, we present the results of a four-year surveillance program based on the sequencing of the pfcrt gene of Plasmodium falciparum populations from endemic areas of Honduras. All isolates were susceptible to chloroquine, as revealed by the pfcrt “CVMNK” genotype in codons 72-76. PMID:25075788

A four-year surveillance program for detection of Plasmodium falciparum chloroquine resistance in Honduras.

PubMed

Fontecha, Gustavo A; Sanchez, Ana L; Mendoza, Meisy; Banegas, Engels; Mejía-Torres, Rosa E

2014-07-01

Countries could use the monitoring of drug resistance in malaria parasites as an effective early warning system to develop the timely response mechanisms that are required to avert the further spread of malaria. Drug resistance surveillance is essential in areas where no drug resistance has been reported, especially if neighbouring countries have previously reported resistance. Here, we present the results of a four-year surveillance program based on the sequencing of the pfcrt gene of Plasmodium falciparum populations from endemic areas of Honduras. All isolates were susceptible to chloroquine, as revealed by the pfcrt "CVMNK" genotype in codons 72-76.

Anti-plasmodial activity of Norcaesalpin D and extracts of four medicinal plants used traditionally for treatment of malaria.

PubMed

Nondo, Ramadhani Selemani Omari; Moshi, Mainen Julius; Erasto, Paul; Masimba, Pax Jessey; Machumi, Francis; Kidukuli, Abdul Waziri; Heydenreich, Matthias; Zofou, Denis

2017-03-24

Malaria is an old life-threatening parasitic disease that is still affecting many people, mainly children living in sub-Saharan Africa. Availability of effective antimalarial drugs played a significant role in the treatment and control of malaria. However, recent information on the emergence of P. falciparum parasites resistant to one of the artemisinin-based combination therapies suggests the need for discovery of new drug molecules. Therefore, this study aimed to evaluate the antiplasmodial activity of extracts, fractions and isolated compound from medicinal plants traditionally used in the treatment of malaria in Tanzania. Dry powdered plant materials were extracted by cold macerations using different solvents. Norcaesalpin D was isolated by column chromatography from dichloromethane root extract of Caesalpinia bonducella and its structure was assigned based on the spectral data. Crude extracts, fractions and isolated compound were evaluated for antiplasmodial activity against chloroquine-sensitive P. falciparum (3D7), chloroquine-resistant P. falciparum (Dd2, K1) and artemisinin-resistant P. falciparum (IPC 5202 Battambang, IPC 4912 Mondolkiri) strains using the parasite lactate dehydrogenase assay. The results indicated that extracts of Erythrina schliebenii, Holarrhena pubescens, Dissotis melleri and C. bonducella exhibited antiplasmodial activity against Dd2 parasites. Ethanolic root extract of E. schliebenii had an IC 50 of 1.87 μg/mL while methanolic and ethanolic root extracts of H. pubescens exhibited an IC 50  = 2.05 μg/mL and IC 50  = 2.43 μg/mL, respectively. Fractions from H. pubescens and C. bonducella roots were found to be highly active against K1, Dd2 and artemisinin-resistant parasites. Norcaesalpin D from C. bonducella root extract was active with IC 50 of 0.98, 1.85 and 2.13 μg/mL against 3D7, Dd2 and IPC 4912-Mondolkiri parasites, respectively. Antiplasmodial activity of norcaesalpin D and extracts of E. schliebenii, H. pubescens, D. melleri and C. bonducella reported in this study requires further attention for the discovery of antimalarial lead compounds for future drug development.

'2TM proteins': an antigenically diverse superfamily with variable functions and export pathways.

PubMed

Kaur, Jasweer; Hora, Rachna

2018-01-01

Malaria is a disease that affects millions of people annually. An intracellular habitat and lack of protein synthesizing machinery in erythrocytes pose numerous difficulties for survival of the human pathogen Plasmodium falciparum . The parasite refurbishes the infected red blood cell (iRBC) by synthesis and export of several proteins in an attempt to suffice its metabolic needs and evade the host immune response. Immune evasion is largely mediated by surface display of highly polymorphic protein families known as variable surface antigens. These include the two trans-membrane (2TM) superfamily constituted by multicopy repetitive interspersed family (RIFINs), subtelomeric variable open reading frame (STEVORs) and Plasmodium falciparum Maurer's cleft two trans-membrane proteins present only in P. falciparum and some simian infecting Plasmodium species. Their hypervariable region flanked by 2TM domains exposed on the iRBC surface is believed to generate antigenic diversity. Though historically named "2TM superfamily," several A-type RIFINs and some STEVORs assume one trans-membrane topology. RIFINs and STEVORs share varied functions in different parasite life cycle stages like rosetting, alteration of iRBC rigidity and immune evasion. Additionally, a member of the STEVOR family has been implicated in merozoite invasion. Differential expression of these families in laboratory strains and clinical isolates propose them to be important for host cell survival and defense. The role of RIFINs in modulation of host immune response and presence of protective antibodies against these surface exposed molecules in patient sera highlights them as attractive targets of antimalarial therapies and vaccines. 2TM proteins are Plasmodium export elements positive, and several of these are exported to the infected erythrocyte surface after exiting through the classical secretory pathway within parasites. Cleaved and modified proteins are trafficked after packaging in vesicles to reach Maurer's clefts, while information regarding delivery to the iRBC surface is sparse. Expression and export timing of the RIFIN and Plasmodium falciparum erythrocyte membrane protein1 families correspond to each other. Here, we have compiled and comprehended detailed information regarding orthologues, domain architecture, surface topology, functions and trafficking of members of the "2TM superfamily." Considering the large repertoire of proteins included in the 2TM superfamily and recent advances defining their function in malaria biology, a surge in research carried out on this important protein superfamily is likely.

Real-time polymerase chain reaction assay for the rapid detection and characterization of chloroquine-resistant Plasmodium falciparum malaria in returned travelers.

PubMed

Farcas, Gabriella A; Soeller, Rainer; Zhong, Kathleen; Zahirieh, Alireza; Kain, Kevin C

2006-03-01

Imported drug-resistant malaria is a growing problem in industrialized countries. Rapid and accurate diagnosis is essential to prevent malaria-associated mortality in returned travelers. However, outside of a limited number of specialized centers, the microscopic diagnosis of malaria is slow, unreliable, and provides little information about drug resistance. Molecular diagnostics have the potential to overcome these limitations. We developed and evaluated a rapid, real-time polymerase chain reaction (PCR) assay to detect Plasmodium falciparum malaria and chloroquine (CQ)-resistance determinants in returned travelers who are febrile. A real-time PCR assay based on detection of the K76T mutation in PfCRT (K76T) of P. falciparum was developed on a LightCycler platform (Roche). The performance characteristics of the real-time assay were compared with those of the nested PCR-restriction fragment-length polymorphism (RFLP) and the sequence analyses of samples obtained from 200 febrile returned travelers, who included 125 infected with P. falciparum (48 of whom were infected CQ-susceptible [K76] and 77 of whom were CQ-resistant [T76] P. falciparum), 22 infected with Plasmodium vivax, 10 infected with Plasmodium ovale, 3 infected with Plasmodium malariae malaria, and 40 infected with other febrile syndromes. All patient samples were coded, and all analyses were performed blindly. The real-time PCR assay detected multiple pfcrt haplotypes associated with CQ resistance in geographically diverse malaria isolates acquired by travelers. Compared with nested-PCR RFLP (the reference standard), the real-time assay was 100% sensitive and 96.2% specific for detection of the P. falciparum K76T mutation. This assay is rapid, sensitive, and specific for the detection and characterization of CQ-resistant P. falciparum malaria in returned travelers. This assay is automated, standardized, and suitable for routine use in clinical diagnostic laboratories.

Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR

PubMed Central

2012-01-01

Background Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. Methods A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. Results The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. Conclusions The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa. PMID:22682065

Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR.

PubMed

Kuamsab, Napaporn; Putaporntip, Chaturong; Pattanawong, Urassaya; Jongwutiwes, Somchai

2012-06-10

Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa.

An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer.

PubMed

Imai, Kazuo; Tarumoto, Norihito; Runtuwene, Lucky Ronald; Sakai, Jun; Hayashida, Kyoko; Eshita, Yuki; Maeda, Ryuichiro; Tuda, Josef; Ohno, Hideaki; Murakami, Takashi; Maesaki, Shigefumi; Suzuki, Yutaka; Yamagishi, Junya; Maeda, Takuya

2018-05-29

The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.

Insights into the Performance of SD Bioline Malaria Ag P.f/Pan Rapid Diagnostic Test and Plasmodium falciparum Histidine-Rich Protein 2 Gene Variation in Madagascar.

PubMed

Willie, Nigani; Mehlotra, Rajeev K; Howes, Rosalind E; Rakotomanga, Tovonahary A; Ramboarina, Stephanie; Ratsimbasoa, Arsène C; Zimmerman, Peter A

2018-06-01

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.

The detection of pfcrt and pfmdr1 point mutations as molecular markers of chloroquine drug resistance, Pahang, Malaysia

PubMed Central

2012-01-01

Background Malaria is still a public health problem in Malaysia with chloroquine (CQ) being the first-line drug in the treatment policy of uncomplicated malaria. There is a scarcity in information about the magnitude of Plasmodium falciparum CQ resistance. This study aims to investigate the presence of single point mutations in the P. falciparum chloroquine-resistance transporter gene (pfcrt) at codons 76, 271, 326, 356 and 371 and in P. falciparum multi-drug resistance-1 gene (pfmdr1) at codons 86 and 1246, as molecular markers of CQ resistance. Methods A total of 75 P. falciparum blood samples were collected from different districts of Pahang state, Malaysia. Single nucleotide polymorphisms in pfcrt gene (codons 76, 271, 326, 356 and 371) and pfmdr1 gene (codons 86 and 1246) were analysed by using mutation-specific nested PCR and restriction fragment length polymorphism (PCR-RFLP) methods. Results Mutations of pfcrt K76T and pfcrt R371I were the most prevalent among pfcrt gene mutations reported by this study; 52% and 77%, respectively. Other codons of the pfcrt gene and the positions 86 and 1246 of the pfmdr1 gene were found mostly of wild type. Significant associations of pfcrt K76T, pfcrt N326S and pfcrt I356T mutations with parasitaemia were also reported. Conclusion The high existence of mutant pfcrt T76 may indicate the low susceptibility of P. falciparum isolates to CQ in Peninsular Malaysia. The findings of this study establish baseline data on the molecular markers of P. falciparum CQ resistance, which may help in the surveillance of drug resistance in Peninsular Malaysia. PMID:22853645

In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger transporter (Pfnhe-1) gene in 393 isolates from Dakar, Senegal.

PubMed

Pascual, Aurélie; Fall, Bécaye; Wurtz, Nathalie; Fall, Mansour; Camara, Cheikhou; Nakoulima, Aminata; Baret, Eric; Diatta, Bakary; Fall, Khadidiatou Ba; Mbaye, Pape Saliou; Diémé, Yaya; Bercion, Raymond; Bogreau, Hervé; Briolant, Sébastien; Rogier, Christophe; Wade, Boubacar; Pradines, Bruno

2013-06-07

Although the World Health Organization recommends replacing quinine (QN) by artesunate due to its increased efficacy and the higher tolerance to the drug in both adults and children, QN remains a first-line treatment for severe malaria, especially in Africa. Investigations of microsatellite Pfnhe-1 ms4760 polymorphisms in culture-adapted isolates from around the world have revealed that an increase in the number of DNNND amino acid motifs was associated with decreased QN susceptibility, whereas an increase in the number of DDNHNDNHNND motifs was associated with increased QN susceptibility. In this context, to further analyse associations between Pfnhe-1 ms4760 polymorphisms and QN susceptibility, 393 isolates freshly collected between October 2009 and January 2010 and July 2010 and February 2011, respectively, at the Hôpital Principal de Dakar, Senegal were assessed ex vivo for QN susceptibility, and their genes were amplified and sequenced. Of the 393 Plasmodium falciparum clinical isolates collected, 145 were successfully cultured. The 145 QN IC50s ranged from 2.1 to 1291 nM, and 17 isolates (11.7%) exceed the QN reduced susceptibility threshold of 611 nM. Among the 393 P. falciparum clinical isolates, 47 different alleles were observed. The three most prevalent profiles were ms4760-1 (no = 72; 18.3%), ms4760-3 (no = 65; 16.5%) and ms4760-7 (no = 40; 10.2%). There were no significant associations observed between QN IC50 values and i) the number of repeats of DNNND in block II (p = 0.0955, Kruskal-Wallis test); ii) the number of repeats of DDNHNDNHNND in block V (p = 0.1455, Kruskal-Wallis test); or iii) ms4760 profiles (p = 0.1809, Kruskal-Wallis test). Pfnhe-1 ms4760 was highly diverse in parasite isolates from Dakar (47 different profiles). Three profiles (ms4760-1, ms4760-3 and ms4760-7) were predominant. The number of repeats for block II (DNNND) or block V (DDNHNDNHNND) was not significantly associated with QN susceptibility. New studies, and especially in vivo studies, are necessary to confirm the role of Pfnhe-1 ms4760 as a marker of QN resistance.

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children.

PubMed

Vignali, Marissa; Armour, Christopher D; Chen, Jingyang; Morrison, Robert; Castle, John C; Biery, Matthew C; Bouzek, Heather; Moon, Wonjong; Babak, Tomas; Fried, Michal; Raymond, Christopher K; Duffy, Patrick E

2011-03-01

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases.

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children

PubMed Central

Vignali, Marissa; Armour, Christopher D.; Chen, Jingyang; Morrison, Robert; Castle, John C.; Biery, Matthew C.; Bouzek, Heather; Moon, Wonjong; Babak, Tomas; Fried, Michal; Raymond, Christopher K.; Duffy, Patrick E.

2011-01-01

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases. PMID:21317536

Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum.

PubMed

Oyebola, Kolapo M; Idowu, Emmanuel T; Olukosi, Yetunde A; Awolola, Taiwo S; Amambua-Ngwa, Alfred

2017-06-29

The burden of falciparum malaria is especially high in sub-Saharan Africa. Differences in pressure from host immunity and antimalarial drugs lead to adaptive changes responsible for high level of genetic variations within and between the parasite populations. Population-specific genetic studies to survey for genes under positive or balancing selection resulting from drug pressure or host immunity will allow for refinement of interventions. We performed a pooled sequencing (pool-seq) of the genomes of 100 Plasmodium falciparum isolates from Nigeria. We explored allele-frequency based neutrality test (Tajima's D) and integrated haplotype score (iHS) to identify genes under selection. Fourteen shared iHS regions that had at least 2 SNPs with a score > 2.5 were identified. These regions code for genes that were likely to have been under strong directional selection. Two of these genes were the chloroquine resistance transporter (CRT) on chromosome 7 and the multidrug resistance 1 (MDR1) on chromosome 5. There was a weak signature of selection in the dihydrofolate reductase (DHFR) gene on chromosome 4 and MDR5 genes on chromosome 13, with only 2 and 3 SNPs respectively identified within the iHS window. We observed strong selection pressure attributable to continued chloroquine and sulfadoxine-pyrimethamine use despite their official proscription for the treatment of uncomplicated malaria. There was also a major selective sweep on chromosome 6 which had 32 SNPs within the shared iHS region. Tajima's D of circumsporozoite protein (CSP), erythrocyte-binding antigen (EBA-175), merozoite surface proteins - MSP3 and MSP7, merozoite surface protein duffy binding-like (MSPDBL2) and serine repeat antigen (SERA-5) were 1.38, 1.29, 0.73, 0.84 and 0.21, respectively. We have demonstrated the use of pool-seq to understand genomic patterns of selection and variability in P. falciparum from Nigeria, which bears the highest burden of infections. This investigation identified known genomic signatures of selection from drug pressure and host immunity. This is evidence that P. falciparum populations explore common adaptive strategies that can be targeted for the development of new interventions.

Identification of Plasmodium falciparum reticulocyte binding protein homologue 5-interacting protein, PfRipr, as a highly conserved blood-stage malaria vaccine candidate.

PubMed

Ntege, Edward H; Arisue, Nobuko; Ito, Daisuke; Hasegawa, Tomoyuki; Palacpac, Nirianne M Q; Egwang, Thomas G; Horii, Toshihiro; Takashima, Eizo; Tsuboi, Takafumi

2016-11-04

Genetic variability in Plasmodium falciparum malaria parasites hampers current malaria vaccine development efforts. Here, we hypothesize that to address the impact of genetic variability on vaccine efficacy in clinical trials, conserved antigen targets should be selected to achieve robust host immunity across multiple falciparum strains. Therefore, suitable vaccine antigens should be assessed for levels of polymorphism and genetic diversity. Using a total of one hundred and two clinical isolates from a region of high malaria transmission in Uganda, we analyzed extent of polymorphism and genetic diversity in four recently reported novel blood-stage malaria vaccine candidate proteins: Rh5 interacting protein (PfRipr), GPI anchored micronemal antigen (PfGAMA), rhoptry-associated leucine zipper-like protein 1 (PfRALP1) and Duffy binding-like merozoite surface protein 1 (PfMSPDBL1). In addition, utilizing the wheat germ cell-free system, we expressed recombinant proteins for the four candidates based on P. falciparum laboratory strain 3D7 sequences, immunized rabbits to obtain specific antibodies (Abs) and performed functional growth inhibition assay (GIA). The GIA activity of the raised Abs was demonstrated using both homologous 3D7 and heterologous FVO strains in vitro. Both pfripr and pfralp1 are less polymorphic but the latter is comparatively more diverse, with varied number of regions having insertions and deletions, asparagine and 6-mer repeats in the coding sequences. Pfgama and pfmspdbl1 are polymorphic and genetically diverse among the isolates with antibodies against the 3D7-based recombinant PfGAMA and PfMSPDBL1 inhibiting merozoite invasion only in the 3D7 but not FVO strain. Moreover, although Abs against the 3D7-based recombinant PfRipr and PfRALP1 proteins potently inhibited merozoite invasion of both 3D7 and FVO, the GIA activity of anti-PfRipr was much higher than that of anti-PfRALP1. Thus, PfRipr is regarded as a promising blood-stage vaccine candidate for next-generation vaccines against P. falciparum. Copyright © 2016 Elsevier Ltd. All rights reserved.

Plasmodium falciparum-Derived Uric Acid Precipitates Induce Maturation of Dendritic Cells

PubMed Central

van de Hoef, Diana L.; Coppens, Isabelle; Holowka, Thomas; Ben Mamoun, Choukri; Branch, OraLee; Rodriguez, Ana

2013-01-01

Malaria is characterized by cyclical fevers and high levels of inflammation, and while an early inflammatory response contributes to parasite clearance, excessive and persistent inflammation can lead to severe forms of the disease. Here, we show that Plasmodium falciparum-infected erythrocytes contain uric acid precipitates in the cytoplasm of the parasitophorous vacuole, which are released when erythrocytes rupture. Uric acid precipitates are highly inflammatory molecules that are considered a danger signal for innate immunity and are the causative agent in gout. We determined that P. falciparum-derived uric acid precipitates induce maturation of human dendritic cells, increasing the expression of cell surface co-stimulatory molecules such as CD80 and CD86, while decreasing human leukocyte antigen-DR expression. In accordance with this, uric acid accounts for a significant proportion of the total stimulatory activity induced by parasite-infected erythrocytes. Moreover, the identification of uric acid precipitates in P. falciparum- and P. vivax-infected erythrocytes obtained directly from malaria patients underscores the in vivo and clinical relevance of our findings. Altogether, our data implicate uric acid precipitates as a potentially important contributor to the innate immune response to Plasmodium infection and may provide a novel target for adjunct therapies. PMID:23405174

Malaria Epidemic and Drug Resistance, Djibouti

PubMed Central

Pradines, Bruno; Bogreau, H.; Koeck, Jean-Louis; Kamil, Mohamed-Ali; Mercereau-Puijalon, Odile

2005-01-01

Analysis of Plasmodium falciparum isolates collected before, during, and after a 1999 malaria epidemic in Djibouti shows that, despite a high prevalence of resistance to chloroquine, the epidemic cannot be attributed to a sudden increase in drug resistance of local parasite populations. PMID:15752455

Cytoadherence and Genotype of Plasmodium falciparum Strains from Symptomatic Children in Franceville, Southeastern Gabon

PubMed Central

Touré, Fousseyni S.; Ouwe-Missi-Oukem-Boyer, Odile; Mezui-Me-Ndong, Jérôme; Ndong-Atome, Guy Roger; Bisvigou, Ulrick; Mazier, Dominique; Bisser, Sylvie

2007-01-01

Background: Plasmodium falciparum causes severe clinical manifestations by sequestering parasitized red blood cells (PRBC) in the microvasculature of major organs such as the brain. This sequestration results from PRBC adherence to vascular endothelial cells via erythrocyte membrane protein 1, a variant parasite surface antigen. Objective: To determine whether P. falciparum multiple genotype infection (MGI) is associated with stronger PRBC cytoadherence and greater clinical severity. Methods: Nested polymerase chain reaction was used to genotype P. falciparum isolates from symptomatic children and to distinguish between single genotype infection (SGI) and MGI. PRBC cytoadhesion was studied with cultured human lung endothelial cells. Results: Analysis of two highly polymorphic regions of the merozoite surface antigen (MSP)-1 and MSP-2 genes and a dimorphic region of the erythrocyte binding antigen-175 gene showed that 21.4% and 78.6% of the 42 children had SGI and MGI, respectively. It also showed that 37 (89%) of the 42 PRBC samples expressed MSP-1 allelic family K1. Cytoadherence values ranged from 58 to 1811 PRBC/mm2 of human lung endothelial cells monolayer in SGI and from 5 to 5744 PRBC/mm2 in MGI. MGI was not associated with higher cytoadherence values or with more severe malaria. Conclusions: These results suggested that infection of the same individual by multiple clones of P. falciparum does not significantly influence PRBC cytoadherence or disease severity and confirmed the predominance of the MSP-1 K1 genotype in southeastern Gabon. PMID:17607045

Antibody Responses to a Novel Plasmodium falciparum Merozoite Surface Protein Vaccine Correlate with Protection against Experimental Malaria Infection in Aotus Monkeys

PubMed Central

Cavanagh, David R.; Kocken, Clemens H. M.; White, John H.; Cowan, Graeme J. M.; Samuel, Kay; Dubbeld, Martin A.; der Wel, Annemarie Voorberg-van; Thomas, Alan W.; McBride, Jana S.; Arnot, David E.

2014-01-01

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. PMID:24421900

Chaperone expression profiles correlate with distinct physiological states of Plasmodium falciparum in malaria patients

PubMed Central

2010-01-01

Background Molecular chaperones have been shown to be important in the growth of the malaria parasite Plasmodium falciparum and inhibition of chaperone function by pharmacological agents has been shown to abrogate parasite growth. A recent study has demonstrated that clinical isolates of the parasite have distinct physiological states, one of which resembles environmental stress response showing up-regulation of specific molecular chaperones. Methods Chaperone networks operational in the distinct physiological clusters in clinical malaria parasites were constructed using cytoscape by utilizing their clinical expression profiles. Results Molecular chaperones show distinct profiles in the previously defined physiologically distinct states. Further, expression profiles of the chaperones from different cellular compartments correlate with specific patient clusters. While cluster 1 parasites, representing a starvation response, show up-regulation of organellar chaperones, cluster 2 parasites, which resemble active growth based on glycolysis, show up-regulation of cytoplasmic chaperones. Interestingly, cytoplasmic Hsp90 and its co-chaperones, previously implicated as drug targets in malaria, cluster in the same group. Detailed analysis of chaperone expression in the patient cluster 2 reveals up-regulation of the entire Hsp90-dependent pro-survival circuitries. In addition, cluster 2 also shows up-regulation of Plasmodium export element (PEXEL)-containing Hsp40s thought to have regulatory and host remodeling roles in the infected erythrocyte. Conclusion In all, this study demonstrates an intimate involvement of parasite-encoded chaperones, PfHsp90 in particular, in defining pathogenesis of malaria. PMID:20719001

Multiple Phenotypic and Genotypic Artemisinin Sensitivity Evaluation of Malian Plasmodium falciparum Isolates.

PubMed

Niaré, Karamoko; Paloque, Lucie; Ménard, Sandie; Tor, Pety; Ramadani, Arba P; Augereau, Jean-Michel; Dara, Antoine; Berry, Antoine; Benoit-Vical, Françoise; Doumbo, Ogobara K

2018-04-01

We assessed the ex vivo/in vitro sensitivity of 54 Malian Plasmodium falciparum isolates to artemisinin for the monitoring of drug resistance in this area. The artemisinin sensitivity of parasites was evaluated using 1) the ex vivo and in vitro parasite recrudescence detection after treatment of the ring stage with 1-200 nM artemisinin for 48 hours and 2) the in vitro parasite recrudescence kinetics assay over 7 days after 6-hour treatment of the ring stage with 700 nM dihydroartemisinin (DHA). In addition, as recommended by the World Health Organization for artemisinin resistance characterization, the ring-stage survival assay (RSA 0-3 h ) was performed and the parasite isolates were sequenced at the kelch 13 propeller locus. No clinical and molecular evidence of artemisinin resistance was observed. However, these isolates present different phenotypic profiles in response to artemisinin treatments. Despite all RSA 0-3 h values less than 1.5%, six out of 46 (13.0%) isolates tested ex vivo and four out of six (66.7%) isolates tested in vitro were able to multiply after 48-hour treatments with 100 nM artemisinin. Moreover, five out of eight isolates tested showed faster parasite recovery after DHA treatment in kinetic assays. The presence of such phenotypes needs to be taken into account in the assessment of the efficacy of artemisinins in Mali. The assays presented here appear as valuable tools for the monitoring of artemisinin sensitivity in the field and thus could help to evaluate the risk of emergence of artemisinin resistance in Africa.

Antiplasmodial activity of two medicinal plants against clinical isolates of Plasmodium falciparum and Plasmodium berghei infected mice.

PubMed

Attemene, Serge David Dago; Beourou, Sylvain; Tuo, Karim; Gnondjui, Albert Alloh; Konate, Abibatou; Toure, Andre Offianan; Kati-Coulibaly, Seraphin; Djaman, Joseph Alico

2018-03-01

Malaria is an infectious and deadly parasitic disease, associated with fever, anaemia and other ailments. Unfortunately the upsurge of plasmodium multidrug resistant constrained researchers to look for new effective drugs. Medicinal plants seem to be an unquenchable source of bioactive principles in the treatment of various diseases. The aim of this study was to assess the antiplasmodial activity of two Ivorian medicinal plants. The in vitro activity was evaluated against clinical isolates and Plasmodium falciparum K1 multidrug resistant strain using the fluorescence based SYBR green I assay. The in vivo bioassay was carried out using the classical 4 day suppressive and curative tests on Plasmodium berghei infected mice. Results showed that the in vitro bioassay of both plant extracts were found to exhibit a promising and moderate antiparasitic effects on clinical isolates (5 µg/mL < IC 50  < 15 µg/mL) and Plasmodium falciparum multidrug resistant K1 strain (15 µg/mL < IC 50  < 50 µg/mL). Furthermore, the in vivo antiplasmodial screening of both extracts showed a significant decrease in parasitemia, which was dose-dependent. Body temperature in mice treated with both extracts at experimental doses increased, compared to the negative control group and was dose-dependent. As for mice body weight a significant decrease ( p  < 0.001) was noticed in the negative control group compared to tested groups of animals. The hydroethanolic stem bark extract of Anthocleista djalonensis A Chev and leaves extract of Ziziphus mauritiana Lam exhibited anti-malarial activities. Therefore, the bioactive compounds of both plant extracts need to be investigated.

Genetic modification of Anopheles stephensi for resistance to multiple Plasmodium falciparum strains does not influence susceptibility to o'nyong'nyong virus or insecticides, or Wolbachia-mediated resistance to the malaria parasite.

PubMed

Pike, Andrew; Dimopoulos, George

2018-01-01

Mosquitoes that have been genetically engineered for resistance to human pathogens are a potential new tool for controlling vector-borne disease. However, genetic modification may have unintended off-target effects that could affect the mosquitoes' utility for disease control. We measured the resistance of five genetically modified Plasmodium-suppressing Anopheles stephensi lines to o'nyong'nyong virus, four classes of insecticides, and diverse Plasmodium falciparum field isolates and characterized the interactions between our genetic modifications and infection with the bacterium Wolbachia. The genetic modifications did not alter the mosquitoes' resistance to either o'nyong'nyong virus or insecticides, and the mosquitoes were equally resistant to all tested P. falciparum strains, regardless of Wolbachia infection status. These results indicate that mosquitoes can be genetically modified for resistance to malaria parasite infection and remain compatible with other vector-control measures without becoming better vectors for other pathogens.

Return of chloroquine sensitivity to Africa? Surveillance of African Plasmodium falciparum chloroquine resistance through malaria imported to China.

PubMed

Lu, Feng; Zhang, Meihua; Culleton, Richard L; Xu, Sui; Tang, Jianxia; Zhou, Huayun; Zhu, Guoding; Gu, Yaping; Zhang, Chao; Liu, Yaobao; Wang, Weiming; Cao, Yuanyuan; Li, Julin; He, Xinlong; Cao, Jun; Gao, Qi

2017-07-26

Chloroquine (CQ) was the cornerstone of anti-malarial treatment in Africa for almost 50 years, but has been widely withdrawn due to the emergence and spread of resistance. Recent reports have suggested that CQ-susceptibility may return following the cessation of CQ usage. Here, we monitor CQ sensitivity and determine the prevalence of genetic polymorphisms in the CQ resistance transporter gene (pfcrt) of Plasmodium falciparum isolates recently imported from Africa to China. Blood samples were collected from falciparum malaria patients returning to China from various countries in Africa. Isolates were tested for their sensitivity to CQ using the SYBR Green I test ex vivo, and for a subset of samples, in vitro following culture adaptation. Mutations at positions 72-76 and codon 220 of the pfcrt gene were analyzed by sequencing and confirmed by PCR-RFLP. Correlations between drug sensitivity and pfcrt polymorphisms were investigated. Of 32 culture adapted isolates assayed, 17 (53.1%), 6 (18.8%) and 9 (28.1%) were classified as sensitive, moderately resistant, and highly resistant, respectively. In vitro CQ susceptibility was related to point mutations in the pfcrt gene, the results indicating a strong association between pfcrt genotype and drug sensitivity. A total of 292 isolates were typed at the pfcrt locus, and the prevalence of the wild type (CQ sensitive) haplotype CVMNK in isolates from East, South, North, West and Central Africa were 91.4%, 80.0%, 73.3%, 53.3% and 51.7%, respectively. The only mutant haplotype observed was CVIET, and this was almost always linked to an additional mutation at A220S. Our results suggest that a reduction in drug pressure following withdrawal of CQ as a first-line drug may lead to a resurgence in CQ sensitive parasites. The prevalence of wild-type pfcrt CQ sensitive parasites from East, South and North Africa was higher than from the West and Central areas, but this varied greatly between countries. Further surveillance is required to assess whether the prevalence of CQ resistant parasites will continue to decrease in the absence of widespread CQ usage.

A histidine-rich protein 2-based malaria drug sensitivity assay for field use.

PubMed

Noedl, Harald; Attlmayr, Bernhard; Wernsdorfer, Walther H; Kollaritsch, Herwig; Miller, Robert S

2004-12-01

With the spread of antimalarial drug resistance, simple and reliable tools for the assessment of antimalarial drug resistance, particularly in endemic regions and under field conditions, have become more important than ever before. We therefore developed a histidine-rich protein 2 (HRP2)-based drug sensitivity assay for testing of fresh isolates of Plasmodium falciparum in the field. In contrast to the HRP2 laboratory assay, the field assay uses a procedure that further simplifies the handling and culturing of malaria parasites by omitting centrifugation, washing, the use of serum, and dilution with uninfected red blood cells. A total of 40 fresh Plasmodium falciparum isolates were successfully tested for their susceptibility to dihydroartemisinin, mefloquine, quinine, and chloroquine (50% inhibitory concentration [IC50] = 3.43, 61.89, 326.75, and 185.31 nM, respectively). Results very closely matched those obtained with a modified World Health Organization schizont maturation assay (R2 = 0.96, P < 0.001; mean log difference at IC50 = 0.054).

Implications of Parasites Lacking Plasmodium falciparum Histidine-Rich Protein 2 on Malaria Morbidity and Control When Rapid Diagnostic Tests Are Used for Diagnosis.

PubMed

Gatton, Michelle L; Dunn, Jessica; Chaudhry, Alisha; Ciketic, Sadmir; Cunningham, Jane; Cheng, Qin

2017-04-01

Rapid diagnostic tests (RDTs) are an important tool for malaria diagnosis, with most using antibodies against Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Reports of P. falciparum lacking this protein are increasing, creating a problem for diagnosis of falciparum malaria in locations without quality-assured microscopy. An agent-based stochastic simulation model of P. falciparum transmission was used to investigate the selective pressure exerted on parasite populations by use of RDTs for diagnosis of symptomatic cases. The model considered parasites with normal, reduced, or no PfHRP2, and diagnosis using PfHRP2-only or combination RDTs. Use of PfHRP2-only RDTs in communities where a PfHRP2-negative parasite was introduced during the simulation resulted in transmission of the parasite in >80% of cases, compared with <30% for normal or PfHRP2-reduced parasites. Using PfHRP2-only RDTs in the presence of PfHRP2-negative parasites caused an increase in prevalence, reduced RDT positivity within symptomatic patients but no change in the number of antimalarial treatments due to false-negative RDT results. Diagnosis with PfHRP2/Pf-Plasmodium lactate dehydrogenase combination RDTs did not select for PfHRP2-negative parasites. The use of PfHRP2-only RDTs is sufficient to select P. falciparum parasites lacking this protein, thus posing a significant public health problem, which could be moderated by using PfHRP2/Pf-Plasmodium lactate dehydrogenase combination RDTs. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Genetic Diversity of Plasmodium falciparum in Haiti: Insights from Microsatellite Markers

PubMed Central

Carter, Tamar E.; Malloy, Halley; Existe, Alexandre; Memnon, Gladys; St. Victor, Yves; Okech, Bernard A.; Mulligan, Connie J.

2015-01-01

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. PMID:26462203

Comparison of a PfHRP2-based rapid diagnostic test and PCR for malaria in a low prevalence setting in rural southern Zambia: implications for elimination.

PubMed

Laban, Natasha M; Kobayashi, Tamaki; Hamapumbu, Harry; Sullivan, David; Mharakurwa, Sungano; Thuma, Philip E; Shiff, Clive J; Moss, William J

2015-01-28

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (PfHRP2) antigen are used to identify individuals with Plasmodium falciparum infection even in low transmission settings seeking to achieve elimination. However, these RDTs lack sensitivity to detect low-density infections, produce false negatives for P. falciparum strains lacking pfhrp2 gene and do not detect species other than P. falciparum. Results of a PfHRP2-based RDT and Plasmodium nested PCR were compared in a region of declining malaria transmission in southern Zambia using samples from community-based, cross-sectional surveys from 2008 to 2012. Participants were tested with a PfHRP2-based RDT and a finger prick blood sample was spotted onto filter paper for PCR analysis and used to prepare blood smears for microscopy. Species-specific, real-time, quantitative PCR (q-PCR) was performed on samples that tested positive either by microscopy, RDT or nested PCR. Of 3,292 total participants enrolled, 12 (0.4%) tested positive by microscopy and 42 (1.3%) by RDT. Of 3,213 (98%) samples tested by nested PCR, 57 (1.8%) were positive, resulting in 87 participants positive by at least one of the three tests. Of these, 61 tested positive for P. falciparum by q-PCR with copy numbers ≤ 2 x 10(3) copies/μL, 5 were positive for both P. falciparum and Plasmodium malariae and 2 were positive for P. malariae alone. RDT detected 32 (53%) of P. falciparum positives, failing to detect three of the dual infections with P. malariae. Among 2,975 participants enrolled during a low transmission period between 2009 and 2012, sensitivity of the PfHRP2-based RDT compared to nested PCR was only 17%, with specificity of >99%. The pfhrp gene was detected in 80% of P. falciparum positives; however, comparison of copy number between RDT negative and RDT positive samples suggested that RDT negatives resulted from low parasitaemia and not pfhrp2 gene deletion. Low-density P. falciparum infections not identified by currently used PfHRP2-based RDTs and the inability to detect non-falciparum malaria will hinder progress to further reduce malaria in low transmission settings of Zambia. More sensitive and specific diagnostic tests will likely be necessary to identify parasite reservoirs and achieve malaria elimination.

In vitro activities of plant extracts from Saudi Arabia against malaria, leishmaniasis, sleeping sickness and Chagas disease.

PubMed

Abdel-Sattar, Essam; Maes, Louis; Salama, Maha Mahmoud

2010-09-01

The in vitro activity of the methanol extracts of 51 plants randomly collected from the Kingdom of Saudi Arabia and some of their fractions (petroleum ether, chloroform, ethyl acetate and aqueous) were evaluated against Plasmodium falciparum, Trypanosoma brucei brucei, T. cruzi and Leishmania infantum, as well as toxicity against MRC-5 fibroblast cells. Ten crude methanolic extracts that demonstrated potent and adequately selective antiprotozoal activity were subjected to solvent fractionation using petroleum ether, ethyl acetate and chloroform. Only three samples showed promising antiprotozoal activity. Argemone ochroleuca (CHCl(3) fraction) showed pronounced activity against P. falciparum(GHA) (IC(50) 0.32 microg/mL) and T. cruzi (IC(50) 0.30 microg/mL) with low cytotoxicity against MRC-5 cells (CC(50) 11.6 microg/mL). Capparis spinosa (EtOAc fraction) showed pronounced activity against P. falciparum(GHA) with an IC(50) 0.50 microg/mL in the absence of toxicity against MRC-5 cell line (CC(50) > 30 microg/mL). Heliotropium curassavicum (CHCl(3) fraction) showed similar activity against P. falciparum (IC(50) 0.65 microg/mL; MRC-5 CC(50) > 30 microg /mL). These three extracts will be subjected for further extensive studies to isolate and identify their active constituents. Copyright 2010 John Wiley & Sons, Ltd.

The Plasmodium PHIST and RESA-Like Protein Families of Human and Rodent Malaria Parasites

PubMed Central

Moreira, Cristina K.; Naissant, Bernina; Coppi, Alida; Bennett, Brandy L.; Aime, Elena; Franke-Fayard, Blandine; Janse, Chris J.; Coppens, Isabelle; Sinnis, Photini; Templeton, Thomas J.

2016-01-01

The phist gene family has members identified across the Plasmodium genus, defined by the presence of a domain of roughly 150 amino acids having conserved aromatic residues and an all alpha-helical structure. The family is highly amplified in P. falciparum, with 65 predicted genes in the genome of the 3D7 isolate. In contrast, in the rodent malaria parasite P. berghei 3 genes are identified, one of which is an apparent pseudogene. Transcripts of the P. berghei phist genes are predominant in schizonts, whereas in P. falciparum transcript profiles span different asexual blood stages and gametocytes. We pursued targeted disruption of P. berghei phist genes in order to characterize a simplistic model for the expanded phist gene repertoire in P. falciparum. Unsuccessful attempts to disrupt P. berghei PBANKA_114540 suggest that this phist gene is essential, while knockout of phist PBANKA_122900 shows an apparent normal progression and non-essential function throughout the life cycle. Epitope-tagging of P. falciparum and P. berghei phist genes confirmed protein export to the erythrocyte cytoplasm and localization with a punctate pattern. Three P. berghei PEXEL/HT-positive exported proteins exhibit at least partial co-localization, in support of a common vesicular compartment in the cytoplasm of erythrocytes infected with rodent malaria parasites. PMID:27022937

In Vitro Activities of Benflumetol against 158 Senegalese Isolates of Plasmodium falciparum in Comparison with Those of Standard Antimalarial Drugs

PubMed Central

Pradines, Bruno; Tall, Adama; Fusai, Thierry; Spiegel, Andre; Hienne, Remi; Rogier, Christophe; Trape, Jean Francois; Le Bras, Jacques; Parzy, Daniel

1999-01-01

The 50% inhibitory concentration (IC50s) of benflumetol (range, 12.5 to 240 nM; mean, 55.1 nM) for 158 Senegalese isolates were evaluated. Ten isolates (6%) showed decreased susceptibility to benflumetol. Benflumetol was slightly more potent against chloroquine-resistant isolates (P < 0.025). No correlation or weak correlations in the responses to benflumetol and pyrimethamine, chloroquine, amodiaquine, artemether, quinine, and pyronaridine were observed, and these correlations are insufficient to suggest cross-resistance. Benflumetol may be an important alternative drug for the treatment of chloroquine-resistant malaria. PMID:9925550

A simple and efficient method for cryopreservation and recovery of viable Plasmodium vivax and P. falciparum sporozoites.

PubMed

Singh, Naresh; Barnes, Samantha J; Jenwithisuk, Rachaneeporn; Sattabongkot, Jetsumon; Adams, John H

2016-10-01

Plasmodium falciparum and Plasmodium vivax sporozoites are the crucial stages of malaria parasites that initiate infection in humans. However, studies to develop new vaccines and drugs targeting these infective stages remain insufficient due to limited availability of sporozoites for research. This is a consequence of relatively few facilities that are established to produce sporozoites of human malaria parasites, sporozoites remaining viable for only a few days, and infected mosquitoes being a biohazard, making them difficult to transport. Cryopreservation of sporozoites offers the potential to alleviate these limitations and enhance sporozoite availability. These experiments were performed to evaluate methods for cryopreservation of P. vivax and P. falciparum sporozoites. Sporozoites, isolated in sterile buffer from infected mosquitoes by manual dissection of salivary glands, were cryopreserved using several types of commercially available serum-free cryoprotective solutions. The efficiency of cryopreservation was validated by a standard in vitro gliding motility assay as a measure of sporozoite activity. Viability of infective sporozoites was defined as percent gliding of sporozoites attached to the coverslip. Significant differences were observed among the cryopreservation media and protocols evaluated, with CryoStor CS2 giving the best results for both P. falciparum and P. vivax, whereas Hestar 200 worked efficiently only for P. vivax sporozoites. Further improvement in recovery of viable sporozoites would be anticipated using automated controlled-rate freezing equipment. Our results demonstrate that cryopreservation provides an alternative for experimental studies that currently rely on fresh P. falciparum and P. vivax sporozoites. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Hyperreactive malarial splenomegaly is associated with low levels of antibodies against red blood cell and Plasmodium falciparum derived glycolipids in Yanomami Amerindians from Venezuela.

PubMed

Vivas, Livia; O'Dea, Kieran P; Noya, Oscar; Pabon, Rosalba; Magris, Magda; Botto, Carlos; Holder, Anthony A; Brown, K Neil

2008-03-01

The immunological basis of the aberrant immune response in hyperreactive malarial splenomegaly (HMS) is poorly understood, but believed to be associated with polyclonal B cell activation by an unidentified malaria mitogen, leading to unregulated immunoglobulin and autoantibody production. HMS has been previously reported in Yanomami communities in the Upper Orinoco region of the Venezuelan Amazon. To investigate a possible association between antibody responses against Plasmodium falciparum and uninfected red blood cell (URBC) glycolipids and splenomegaly, a direct comparison of the parasite versus host anti-glycolipid antibody responses was made in an isolated community of this area. The anti-P. falciparum glycolipid (Pfglp) response was IgG3 dominated, whereas the uninfected red blood cell glycolipid (URBCglp) response showed a predominance of IgG1. The levels of IgG1 against Pfglp, and of IgG4 and IgM against URBCglp were significantly higher in women, while the anti-Pfglp or URBCglp IgM levels were inversely correlated with the degree of splenomegaly. Overall, these results suggest differential regulation of anti-parasite and autoreactive responses and that these responses may be linked to the development and evolution of HMS in this population exposed to endemic malaria. The high mortality rates associated with HMS point out that its early diagnosis together with the implementation of malaria control measures in these isolated Amerindian communities are a priority.

In vitro piperaquine susceptibility is not associated with the Plasmodium falciparum chloroquine resistance transporter gene

PubMed Central

2013-01-01

Background Dihydroartemisinin-piperaquine is a new ACT that is administered as single daily dose for three days and has been demonstrated to be tolerated and highly effective for the treatment of uncomplicated Plasmodium falciparum malaria. Piperaquine was used alone to replace chloroquine as the first-line treatment for uncomplicated malaria in China in response to increasing chloroquine resistance in the 1970s. However, the rapid emergence of piperaquine-resistant strains that resulted in the cessation of its use in China in the 1980s, suggests that there is cross-resistance between piperaquine and chloroquine. Very few data are available on cross-resistance between piperaquine and chloroquine, and the data that do exist are often contradictory. Methods In total, 280 P. falciparum isolates, collected between April 2008 and June 2012 from patients hospitalized in France with imported malaria from a malaria-endemic country, were assessed ex vivo for piperaquine and chloroquine susceptibilities by using the standard 42-hour 3H-hypoxanthine uptake inhibition method. The chloroquine resistance-associated mutation K76T in pfcrt was also investigated for the 280 isolates. Results The IC50 for piperaquine ranged from 9.8 nM to 217.3 nM (mean = 81.3 nM. The IC50 for chloroquine ranged from 5.0 nM to 1,918 nM (mean = 83.6 nM. A significant but low correlation was observed between the Log IC50 values for piperaquine and chloroquine (r = 0.145, p < 0.001). However, the coefficient of determination of 0.021 indicates that only 2.1% of the variation in the response to piperaquine is explained by the variation in the response to chloroquine. The mean value for piperaquine was 74.0 nM in the Pfcrt K76 wild-type group (no = 125) and 87.7 nM in the 76 T mutant group (no = 155). This difference was not significant (p = 0.875, Mann Whitney U test). Conclusions The present work demonstrates that there was no cross-resistance between piperaquine and chloroquine among 280 P. falciparum isolates and that piperaquine susceptibility is not associated with pfcrt, the gene involved in chloroquine resistance. These results confirm the efficacy of piperaquine in association with dihydroartemisinin and support its use in areas in which parasites are resistant to chloroquine. PMID:24274185

Chemical constituents and biological activities from branches of Colubrina asiatica.

PubMed

Sangsopha, Watchara; Kanokmedhakul, Kwanjai; Lekphrom, Ratsami; Kanokmedhakul, Somdej

2018-05-01

Sixteen compounds were isolated from a Thai medicinal plant, Colubrina asiatica. The isolated compounds were elucidated on the basis of spectroscopic methods (IR, 1D and 2D NMR) as six triterpene acids (1-6), five steroids (7-11), one benzoic acid derivative (12), two peptides (13 and 14), one sesquiterpenoid (15) and one jujubogenin (16). Compounds 3 and 10 showed antimalarial activity against Plasmodium falciparum. Compound 5 showed antimycobacterial activity. Moreover, compounds 3, 5, 6, 10 and 14 exhibited weak cytotoxicity against cancer cell lines. Compounds 1-15 have been isolated for the first time from this plant.

Diversity and population structure of Plasmodium falciparum in Thailand based on the spatial and temporal haplotype patterns of the C-terminal 19-kDa domain of merozoite surface protein-1.

PubMed

Simpalipan, Phumin; Pattaradilokrat, Sittiporn; Siripoon, Napaporn; Seugorn, Aree; Kaewthamasorn, Morakot; Butcher, Robert D J; Harnyuttanakorn, Pongchai

2014-02-12

The 19-kDa C-terminal region of the merozoite surface protein-1 of the human malaria parasite Plasmodium falciparum (PfMSP-119) constitutes the major component on the surface of merozoites and is considered as one of the leading candidates for asexual blood stage vaccines. Because the protein exhibits a level of sequence variation that may compromise the effectiveness of a vaccine, the global sequence diversity of PfMSP-119 has been subjected to extensive research, especially in malaria endemic areas. In Thailand, PfMSP-119 sequences have been derived from a single parasite population in Tak province, located along the Thailand-Myanmar border, since 1995. However, the extent of sequence variation and the spatiotemporal patterns of the MSP-119 haplotypes along the Thai borders with Laos and Cambodia are unknown. Sixty-three isolates of P. falciparum from five geographically isolated populations along the Thai borders with Myanmar, Laos and Cambodia in three transmission seasons between 2002 and 2008 were collected and culture-adapted. The msp-1 gene block 17 was sequenced and analysed for the allelic diversity, frequency and distribution patterns of PfMSP-119 haplotypes in individual populations. The PfMSP-119 haplotype patterns were then compared between parasite populations to infer the population structure and genetic differentiation of the malaria parasite. Five conserved polymorphic positions, which accounted for five distinct haplotypes, of PfMSP-119 were identified. Differences in the prevalence of PfMSP-119 haplotypes were detected in different geographical regions, with the highest levels of genetic diversity being found in the Kanchanaburi and Ranong provinces along the Thailand-Myanmar border and Trat province located at the Thailand-Cambodia border. Despite this variability, the distribution patterns of individual PfMSP-119 haplotypes seemed to be very similar across the country and over the three malarial transmission seasons, suggesting that gene flow may operate between parasite populations circulating in Thailand and the three neighboring countries. The major MSP-119 haplotypes of P. falciparum populations in all endemic populations during three transmission seasons in Thailand were identified, providing basic information on the common haplotypes of MSP-119 that is of use for malaria vaccine development and inferring the population structure of P. falciparum populations in Thailand.

Post-translational generation of constitutively active cores from larger phosphatases in the malaria parasite, Plasmodium falciparum: implications for proteomics

PubMed Central

Kumar, Rajinder; Musiyenko, Alla; Oldenburg, Anja; Adams, Brian; Barik, Sailen

2004-01-01

Background Although the complete genome sequences of a large number of organisms have been determined, the exact proteomes need to be characterized. More specifically, the extent to which post-translational processes such as proteolysis affect the synthesized proteins has remained unappreciated. We examined this issue in selected protein phosphatases of the protease-rich malaria parasite, Plasmodium falciparum. Results P. falciparum encodes a number of Ser/Thr protein phosphatases (PP) whose catalytic subunits are composed of a catalytic core and accessory domains essential for regulation of the catalytic activity. Two examples of such regulatory domains are found in the Ca+2-regulated phosphatases, PP7 and PP2B (calcineurin). The EF-hand domains of PP7 and the calmodulin-binding domain of PP2B are essential for stimulation of the phosphatase activity by Ca+2. We present biochemical evidence that P. falciparum generates these full-length phosphatases as well as their catalytic cores, most likely as intermediates of a proteolytic degradation pathway. While the full-length phosphatases are activated by Ca+2, the processed cores are constitutively active and either less responsive or unresponsive to Ca+2. The processing is extremely rapid, specific, and occurs in vivo. Conclusions Post-translational cleavage efficiently degrades complex full-length phosphatases in P. falciparum. In the course of such degradation, enzymatically active catalytic cores are produced as relatively stable intermediates. The universality of such proteolysis in other phosphatases or other multi-domain proteins and its potential impact on the overall proteome of a cell merits further investigation. PMID:15230980

Life-span of in vitro differentiated Plasmodium falciparum gametocytes.

PubMed

Gebru, Tamirat; Lalremruata, Albert; Kremsner, Peter G; Mordmüller, Benjamin; Held, Jana

2017-08-11

The sexual stages (gametocytes) of Plasmodium falciparum do not directly contribute to the pathology of malaria but are essential for transmission of the parasite from the human host to the mosquito. Mature gametocytes circulate in infected human blood for several days and their circulation time has been modelled mathematically from data of previous in vivo studies. This is the first time that longevity of gametocytes is studied experimentally in vitro. The in vitro longevity of P. falciparum gametocytes of 1 clinical isolate and 2 laboratory strains was assessed by three different methods: microscopy, flow cytometry and reverse transcription quantitative real-time PCR (RT-qPCR). Additionally, the rate of gametocytogenesis of the used P. falciparum strains was compared. The maximum in vitro lifespan of P. falciparum gametocytes reached almost 2 months (49 days by flow cytometry, 46 days by microscopy, and at least 52 days by RT-qPCR) from the starting day of gametocyte culture to death of last parasite in the tested strains with an average 50% survival rate of 6.5, 2.6 and 3.5 days, respectively. Peak gametocytaemia was observed on average 19 days after initiation of gametocyte culture followed by a steady decline due to natural decay of the parasites. The rate of gametocytogenesis was highest in the NF54 strain. Plasmodium falciparum mature gametocytes can survive up to 16-32 days (at least 14 days for mature male gametocytes) in vitro in absence of the influence of host factors. This confirms experimentally a previous modelling estimate that used molecular tools for gametocyte detection in treated patients. The survival time might reflect the time the parasite can be transmitted to the mosquito after clearance of asexual parasites. These results underline the importance of efficient transmission blocking agents in the fight against malaria.

Optimizing the HRP-2 In Vitro Malaria Drug Susceptibility Assay Using a Reference Clone to Improve Comparisons of Plasmodium falciparum Field Isolates

DTIC Science & Technology

2012-09-13

6. Desjardins RE, Canfield CJ, Haynes JD, Chulay JD: Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution...vitro antimalarial drug efficacy testing and application to clinical isolates. Antimicrob Agents Chemother 2007, 51:1172–1178. 12. Akala HM, Eyase FL...Cheruiyot AC, Omondi AA, Ogutu BR, Waters NC, Johnson JD, Polhemus ME, Schnabel DC, Walsh DS: Antimalarial drug sensitivity profile of western Kenya

A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein.

PubMed

Tan, Joshua; Sack, Brandon K; Oyen, David; Zenklusen, Isabelle; Piccoli, Luca; Barbieri, Sonia; Foglierini, Mathilde; Fregni, Chiara Silacci; Marcandalli, Jessica; Jongo, Said; Abdulla, Salim; Perez, Laurent; Corradin, Giampietro; Varani, Luca; Sallusto, Federica; Sim, Betty Kim Lee; Hoffman, Stephen L; Kappe, Stefan H I; Daubenberger, Claudia; Wilson, Ian A; Lanzavecchia, Antonio

2018-05-01

Immunization with attenuated Plasmodium falciparum sporozoites (PfSPZs) has been shown to be protective against malaria, but the features of the antibody response induced by this treatment remain unclear. To investigate this response in detail, we isolated IgM and IgG monoclonal antibodies from Tanzanian volunteers who were immunized with repeated injection of Sanaria PfSPZ Vaccine and who were found to be protected from controlled human malaria infection with infectious homologous PfSPZs. All isolated IgG monoclonal antibodies bound to P. falciparum circumsporozoite protein (PfCSP) and recognized distinct epitopes in its N terminus, NANP-repeat region, and C terminus. Strikingly, the most effective antibodies, as determined in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the PfCSP N-terminal junction that is not in the RTS,S vaccine. These dual-specific antibodies were isolated from different donors and were encoded by VH3-30 or VH3-33 alleles that encode tryptophan or arginine at position 52. Using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. Our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies.

UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes.

PubMed

Lim, Michelle Yi-Xiu; LaMonte, Gregory; Lee, Marcus C S; Reimer, Christin; Tan, Bee Huat; Corey, Victoria; Tjahjadi, Bianca F; Chua, Adeline; Nachon, Marie; Wintjens, René; Gedeck, Peter; Malleret, Benoit; Renia, Laurent; Bonamy, Ghislain M C; Ho, Paul Chi-Lui; Yeung, Bryan K S; Chow, Eric D; Lim, Liting; Fidock, David A; Diagana, Thierry T; Winzeler, Elizabeth A; Bifani, Pablo

2016-09-19

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru

PubMed Central

Akinyi, Sheila; Hayden, Tonya; Gamboa, Dionicia; Torres, Katherine; Bendezu, Jorge; Abdallah, Joseph F.; Griffing, Sean M.; Quezada, Wilmer Marquiño; Arrospide, Nancy; De Oliveira, Alexandre Macedo; Lucas, Carmen; Magill, Alan J.; Bacon, David J.; Barnwell, John W.; Udhayakumar, Venkatachalam

2013-01-01

The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times. PMID:24077522

A triazolopyrimidine-based dihydroorotate dehydrogenase inhibitor (DSM421) with improved drug-like properties for treatment and prevention of malaria

PubMed Central

Phillips, Margaret A.; White, Karen L.; Kokkonda, Sreekanth; Deng, Xiaoyi; White, John; Mazouni, Farah El; Marsh, Kennan; Tomchick, Diana R.; Manjalanagara, Krishne; Rudra, Kakali Rani; Wirjanata, Grennady; Noviyanti, Rintis; Price, Ric N; Marfurt, Jutta; Shackleford, David M.; Chiu, Francis C.K.; Campbell, Michael; Jimenez-Diaz, Maria Belen; Bazaga, Santiago Ferrer; Angulo-Barturen, Iñigo; Martinez, Maria Santos; Lafuente-Monasterio, Maria; Kaminsky, Werner; Silue, Kigbafori; Zeeman, Anne-Marie; Kocken, Clemens; Leroy, Didier; Blasco, Benjamin; Rossignol, Emilie; Rueckle, Thomas; Matthews, Dave; Burrows, Jeremy N.; Waterson, David; Palmer, Michael J.; Rathod, Pradipsinh K.; Charman, Susan A.

2016-01-01

The emergence of drug resistant malaria parasites continues to hamper efforts to control this lethal disease. Dihydroorotate dehydrogenase has recently been validated as a new target for the treatment of malaria and a selective inhibitor (DSM265) of the Plasmodium enzyme is currently in clinical development. With the goal of identifying a backup compound to DSM265, we explored replacement of the SF5-aniline moiety of DSM265 with a series of CF3-pyridinyls, while maintaining the core triazolopyrimidine scaffold. This effort led to the identification of DSM421, which has improved solubility, lower intrinsic clearance and increased plasma exposure after oral dosing compared to DSM265, while maintaining a long predicted human half-life. Its improved physical and chemical properties will allow it to be formulated more readily than DSM265. DSM421 showed excellent efficacy in the SCID mouse model of P. falciparum malaria that supports the prediction of a low human dose (<200 mg). Importantly DSM421 showed equal activity against both P. falciparum and P. vivax field isolates, while DSM265 was more active on P. falciparum. DSM421 has the potential to be developed as a single dose cure or once-weekly chemopreventative for both P. falciparum and P. vivax malaria leading to its advancement as a preclinical development candidate. PMID:27641613

Novel Polymorphisms in Plasmodium falciparum ABC Transporter Genes Are Associated with Major ACT Antimalarial Drug Resistance

PubMed Central

Veiga, Maria Isabel; Ferreira, Pedro Eduardo; Jörnhagen, Louise; Malmberg, Maja; Kone, Aminatou; Schmidt, Berit Aydin; Petzold, Max; Björkman, Anders; Nosten, Francois; Gil, Jose Pedro

2011-01-01

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P.falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions. PMID:21633513

Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance.

PubMed

Veiga, Maria Isabel; Ferreira, Pedro Eduardo; Jörnhagen, Louise; Malmberg, Maja; Kone, Aminatou; Schmidt, Berit Aydin; Petzold, Max; Björkman, Anders; Nosten, Francois; Gil, Jose Pedro

2011-01-01

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P.falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions.

Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti

PubMed Central

2012-01-01

Background Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. Methods DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. Results Thirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban vs rural sites (Welch’s T-test p-value = 0.53 and permutations p-value = 0.59). Conclusion This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on alternative malaria treatment options in Haiti. PMID:22889367

Clinical implications of asymptomatic Plasmodium falciparum infections in Malawi.

PubMed

Buchwald, Andrea G; Sixpence, Alick; Chimenya, Mabvuto; Damson, Milius; Sorkin, John D; Wilson, Mark L; Seydel, Karl; Hochman, Sarah; Mathanga, Don P; Taylor, Terrie E; Laufer, Miriam K

2018-05-16

Asymptomatic Plasmodium falciparum infections are common in Malawi, however, the implications of these infections for the burden of malaria illness are unknown. Whether asymptomatic infections eventually progress to malaria illness, persist without causing symptoms, or clear spontaneously remains undetermined. We identified asymptomatic infections and evaluated the associations between persistent asymptomatic infections and malaria illness. Children and adults (N = 120) who presented at a health facility with uncomplicated malaria were followed monthly for two years. During follow-up visits, participants with malaria symptoms were tested and treated if positive. Samples from all visits were tested for parasites using both microscopy and PCR, and all malaria infections underwent genotyping. Cox frailty models were used to estimate the temporal association between asymptomatic infections and malaria illness episodes. Mixed models were used to estimate the odds of clinical symptoms associated with new versus persistent infections. Participants had a median follow-up time of 720 days. Asymptomatic infections were detected during 23% of visits. Persistent asymptomatic infections were associated with decreased risk of malaria illness in all ages (HR 0.50, p < 0.001). When asymptomatic infections preceded malaria illness, newly acquired infections were detected at 92% of subsequent clinical episodes, independent of presence of persistent infections. Malaria illness among children was more likely due to newly acquired infections (OR 1.4, 95%CI 1.3-1.5) than to persistent infections. Asymptomatic P. falciparum infections are associated with decreased incidence of malaria illness but do not protect against disease when new infection occurs.

Epidemiological characterization of Plasmodium falciparum in the Republic of Cabo Verde: implications for potential large-scale re-emergence of malaria

PubMed Central

Alves, Joana; Roque, Ana Luísa; Cravo, Pedro; Valdez, Tomás; Jelinek, Tomas; do Rosário, Virgílio E; Arez, Ana Paula

2006-01-01

Background Malaria has come near eradication at archipelago of Cabo Verde in 1970. Infections are now only observed in Santiago, where outbreaks occur. In these islands, malaria is considered by the international community as being of limited risk and, therefore, no prophylaxis is recommended. Since the understanding of factors that determine malaria outbreaks are crucial for controlling the disease, the present study aimed to investigate if the malaria infections observed in Santiago Island are maintained in isolated foci and in asymptomatic individuals. Methods The occurrence of asymptomatic carriers in villages with history of malaria as well as the level of exposure of these populations were investigated using PCR and serological analyses. Results Results indicate that malaria is maintained as asymptomatic and sub-patent infections and that the majority of the circulating parasite populations harbour chloroquine-resistant mutations. Conclusion These observations highlight the alarming prospect of malaria to become a serious public health problem and underscore the need for a tighter surveillance. PMID:16630349

In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger (Pfnhe-1) gene: the absence of association in clinical isolates from the Republic of Congo

PubMed Central

2011-01-01

Background Quinine is still recommended as an effective therapy for severe cases of Plasmodium falciparum malaria, but the parasite has developed resistance to the drug in some cases. Investigations into the genetic basis for quinine resistance (QNR) suggest that QNR is complex and involves several genes, with either an additive or a pairwise effect. The results obtained when assessing one of these genes, the plasmodial Na+/H+ exchanger, Pfnhe-1, were found to depend upon the geographic origin of the parasite strain. Most of the associations identified have been made in Asian strains; in contrast, in African strains, the influence of Pfnhe on QNR is not apparent. However, a recent study carried out in Kenya did show a significant association between a Pfnhe polymorphism and QNR. As genetic differences may exist across the African continent, more field data are needed to determine if this association exists in other African regions. In the present study, association between Pfnhe and QNR is investigated in a series of isolates from central Africa. Methods The sequence analysis of the polymorphisms at the Pfnhe-1 ms4760 microsatellite and the evaluation of in vitro quinine susceptibility (by isotopic assay) were conducted in 74 P. falciparum isolates from the Republic of Congo. Results Polymorphisms in the number of DNNND or NHNDNHNNDDD repeats in the Pfnhe-1 ms4760 microsatellite were not associated with quinine susceptibility. Conclusions The polymorphism in the microsatellite ms4760 in Pfnhe-1 that cannot be used to monitor quinine response in the regions of the Republic of Congo, where the isolates came from. This finding suggests that there exists a genetic background associated with geographic area for the association that will prevent the use of Pfnhe as a molecular marker for QNR. The contribution of Pfnhe to the in vitro response to quinine remains to be assessed in other regions, including in countries with different levels of drug pressure. PMID:21314947

Differential perpetuation of malaria species among Amazonian Yanomami Amerindians.

PubMed

Laserson, K F; Wypij, D; Petralanda, I; Spielman, A; Maguire, J H

1999-05-01

To determine whether malaria perpetuates within isolated Amerindian villages in the Venezuelan Amazon, we surveyed malaria infection and disease among 1,311 Yanomami in three communities during a 16-month period. Plasmodium vivax was generally present in each of these small, isolated villages; asymptomatic infection was frequent, and clinical disease was most evident among children less than five years of age (odds ratio [OR] = 6.3, 95% confidence interval [CI] = 1.4-29.2) and among persons experiencing parasitemias > or = 1,000 parasites/mm3 of blood (OR = 45.0, 95% CI = 5.5-370.7). Plasmodium falciparum, in contrast, was less prevalent, except during an abrupt outbreak in which 72 infections resulted in symptoms in all age groups and at all levels of parasitemia, and occasionally were life-threatening. The observed endemic pattern of P. vivax infection may derive from the capacity of this pathogen to relapse, while the epidemic pattern of P. falciparum infection may reflect occasional introductions of strains carried by immigrants or residents of distant villages and the subsequent disappearance of this non-relapsing pathogen.

In vitro studies on the sensitivity pattern of Plasmodium falciparum to anti-malarial drugs and local herbal extracts.

PubMed

Olasehinde, Grace I; Ojurongbe, Olusola; Adeyeba, Adegboyega O; Fagade, Obasola E; Valecha, Neena; Ayanda, Isaac O; Ajayi, Adesola A; Egwari, Louis O

2014-02-20

The resistance of human malaria parasites to anti-malarial compounds has become considerable concern, particularly in view of the shortage of novel classes of anti-malarial drugs. One way to prevent resistance is by using new compounds that are not based on existing synthetic antimicrobial agents. Sensitivity of 100 Plasmodium falciparum isolates to chloroquine, quinine, amodiaquine, mefloquine, sulphadoxine/pyrimethamine, artemisinin, Momordica charantia ('Ejirin') Diospyros monbuttensis ('Egun eja') and Morinda lucida ('Oruwo') was determined using the in vitro microtest (Mark III) technique to determine the IC50 of the drugs. All the isolates tested were sensitive to quinine, mefloquine and artesunate. Fifty-one percent of the isolates were resistant to chloroquine, 13% to amodiaquine and 5% to sulphadoxine/pyrimethamine. Highest resistance to chloroquine (68.9%) was recorded among isolates from Yewa zone while highest resistance to amodiaquine (30%) was observed in Ijebu zone. Highest resistance to sulphadoxine/pyrimethamine was recorded in Yewa and Egba zones, respectively. A positive correlation was observed between the responses to artemisinin and mefloquine (P<0.05), artemisinin and quinine (P<0.05) and quinine and mefloquine (P<0.05). A negative correlation was observed between the responses to chloroquine and mefloquine (P>0.05). Highest anti-plasmodial activity was obtained with the ethanolic extract of D. monbuttensis (IC50 = 3.2 nM) while the lowest was obtained from M. lucida (IC50 = 25 nM). Natural products isolated from plants used in traditional medicine, which have potent anti-plasmodial action in vitro, represent potential sources of new anti-malarial drugs.

Enhancing longevity of Plasmodium vivax and P. falciparum sporozoites after dissection from mosquito salivary glands.

PubMed

Lupton, Emily J; Roth, Alison; Patrapuvich, Rapatbhorn; Maher, Steve P; Singh, Naresh; Sattabongkot, Jetsumon; Adams, John H

2015-04-01

The pre-erythrocytic stages of Plasmodium vivax and Plasmodium falciparum remain challenging for experimental research in part due to limited access to sporozoites. An important factor limiting availability is the laboratory support required for producing infected mosquitoes and the ephemeral nature of isolated extracellular sporozoites. This study was undertaken to investigate methods to improve the availability of this limited resource by extending the longevity of the extracellular sporozoites after mosquito dissection. Our goal in this study was to determine whether buffer conditions more closely mimicking the insect microenvironment could prolong longevity of ex vivo P. vivax and P. falciparum sporozoites. The study compared the current standard dissection buffer RPMI1640 to Hank's Balanced Salt Solution with 1g/L glucose (HBSS-1) or 2g/L glucose (HBSS-2) and Grace's Insect Medium for ability to extend longevity of ex vivo P. vivax and P. falciparum sporozoites. The effect of each buffer on sporozoite viability was evaluated by measuring sporozoite gliding motility at 0, 4, 8, and 24h post-dissection from mosquito salivary glands. Comparisons of mean gliding percentages of ex vivo sporozoites in the different buffers and time points found that RPMI and Grace's both showed strong gliding at 0h. In contrast, by 4h post-dissection sporozoites in RPMI consistently had the lowest gliding activity, whereas sporozoites in Grace's had significantly more gliding compared to all other buffers at almost all time points. Our results indicate that P. vivax and P. falciparum sporozoites maintained in insect media rather than the standard dissection buffer RPMI and HBSS retain viability better over time. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Increased pfmdr1 gene copy number and the decline in pfcrt and pfmdr1 resistance alleles in Ghanaian Plasmodium falciparum isolates after the change of anti-malarial drug treatment policy.

PubMed

Duah, Nancy O; Matrevi, Sena A; de Souza, Dziedzom K; Binnah, Daniel D; Tamakloe, Mary M; Opoku, Vera S; Onwona, Christiana O; Narh, Charles A; Quashie, Neils B; Abuaku, Benjamin; Duplessis, Christopher; Kronmann, Karl C; Koram, Kwadwo A

2013-10-30

With the introduction of artemisinin-based combination therapy (ACT) in 2005, monitoring of anti-malarial drug efficacy, which includes the use of molecular tools to detect known genetic markers of parasite resistance, is important for first-hand information on the changes in parasite susceptibility to drugs in Ghana. This study investigated the Plasmodium falciparum multidrug resistance gene (pfmdr1) copy number, mutations and the chloroquine resistance transporter gene (pfcrt) mutations in Ghanaian isolates collected in seven years to detect the trends in prevalence of mutations. Archived filter paper blood blots collected from children aged below five years with uncomplicated malaria in 2003-2010 at sentinel sites were used. Using quantitative real-time polymerase chain reaction (qRT-PCR), 756 samples were assessed for pfmdr1 gene copy number. PCR and restriction fragment length polymorphism (RFLP) were used to detect alleles of pfmdr1 86 in 1,102 samples, pfmdr1 184, 1034, 1042 and 1246 in 832 samples and pfcrt 76 in 1,063 samples. Merozoite surface protein 2 (msp2) genotyping was done to select monoclonal infections for copy number analysis. The percentage of isolates with increased pfmdr1 copy number were 4, 27, 9, and 18% for 2003-04, 2005-06, 2007-08 and 2010, respectively. Significant increasing trends for prevalence of pfmdr1 N86 (×(2) = 96.31, p <0.001) and pfcrt K76 (×(2) = 64.50, p <0.001) and decreasing trends in pfmdr1 Y86 (x(2) = 38.52, p <0.001) and pfcrt T76 (x(2) = 43.49, p <0.001) were observed from 2003-2010. The pfmdr1 F184 and Y184 prevalence showed an increasing and decreasing trends respectively but were not significant (×(2) = 7.39,p=0.060; ×(2) = 7.49, p = 0.057 respectively). The pfmdr1 N86-F184-D1246 haplotype, which is alleged to be selected by artemether-lumefantrine showed a significant increasing trend (×(2) = 20.75, p < 0.001). Increased pfmdr1 gene copy number was observed in the isolates analysed and this finding has implications for the use of ACT in the country although no resistance has been reported. The decreasing trend in the prevalence of chloroquine resistance markers after change of treatment policy presents the possibility for future introduction of chloroquine as prophylaxis for malaria risk groups such as children and pregnant women in Ghana.

Differential T-cell responses to a chimeric Plasmodium falciparum antigen; UB05-09, correlates with acquired immunity to malaria.

PubMed

Dinga, J N; Njimoh, D L; Kiawa, B; Djikeng, A; Nyasa, R B; Nkuo-Akenji, T; Pellé, R; Titanji, V P K

2016-05-01

The development of a sterilizing and cost-effective vaccine against malaria remains a major problem despite recent advances. In this study, it is demonstrated that two antigens of P. falciparum UB05, UB09 and their chimera UB05-09 can serve as protective immunity markers by eliciting higher T-cell responses in malaria semi-immune subjects (SIS) than in frequently sick subjects (FSS) and could be used to distinguish these two groups. UB05, UB09 and UB05-09 were cloned, expressed in E. coli, purified and used to stimulate PBMCs isolated from 63 subjects in a malaria endemic area, for IFN-γ production, which was measured by the ELISpot assay. The polymorphism of UB09 gene in the malaria infected population was also studied by PCR/sequencing of the gene in P. falciparum field isolates. All three antigens were preferentially recognized by PBMCs from SIS. IFN-γ production induced by these antigens correlated with the absence of fever and parasitaemia. UB09 was shown to be relatively well-conserved in nature. It is concluded that UB05, UB09 and the chimera UB05-09 posses T-cell epitopes that are associated with protection against malaria and could thus be used to distinguish SIS from FSS eventhough acute infection with malaria has been shown to reduce cytokine production in some studies. Further investigations of these antigens as potential diagnostic and/or vaccine candidates for malaria are indicated. © 2016 John Wiley & Sons Ltd.

Country-wide assessment of the genetic polymorphism in Plasmodium falciparum and Plasmodium vivax antigens detected with rapid diagnostic tests for malaria

PubMed Central

Mariette, Natacha; Barnadas, Céline; Bouchier, Christiane; Tichit, Magali; Ménard, Didier

2008-01-01

Background Rapid diagnostic tests (RDTs) are becoming increasingly indispensable in malaria management, as a means of increasing the accuracy of diagnosis. The WHO has issued recommendations, but the selection of the most suitable RDT remains difficult for users in endemic countries. The genetic variability of the antigens detected with RDTs has been little studied, but may affect the sensitivity of RDTs. This factor has been studied by comparisons between countries at continental level, but little information is available concerning antigen variability within a given country. Methods A country-wide assessment of polymorphism of the PfHRP2, PfHRP3, pLDH and aldolase antigens was carried out in 260 Plasmodium falciparum and 127 Plasmodium vivax isolates, by sequencing the genes encoding these antigens in parasites originating from the various epidemiological strata for malaria in Madagascar. Results Higher levels of polymorphism were observed for the pfhrp2 and pfhrp3 genes than for the P. falciparum and P. vivax aldolase and pldh genes. Pfhrp2 sequence analysis predicted that 9% of Malagasy isolates would not be detected at parasite densities ≤ 250 parasites/μl (ranging from 6% in the north to 14% in the south), although RDTs based on PfHRP2 detection are now recommended in Madagascar. Conclusion These findings highlight the importance of training of health workers and the end users of RDTs in the provision of information about the possibility of false-negative results for patients with clinical symptoms of malaria, particularly in the south of Madagascar. PMID:18957099

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

PubMed Central

Raabe, Carsten A.; Sanchez, Cecilia P.; Randau, Gerrit; Robeck, Thomas; Skryabin, Boris V.; Chinni, Suresh V.; Kube, Michael; Reinhardt, Richard; Ng, Guey Hooi; Manickam, Ravichandran; Kuryshev, Vladimir Y.; Lanzer, Michael; Brosius, Juergen; Tang, Thean Hock; Rozhdestvensky, Timofey S.

2010-01-01

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense–antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors. PMID:19864253

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum.

PubMed

Raabe, Carsten A; Sanchez, Cecilia P; Randau, Gerrit; Robeck, Thomas; Skryabin, Boris V; Chinni, Suresh V; Kube, Michael; Reinhardt, Richard; Ng, Guey Hooi; Manickam, Ravichandran; Kuryshev, Vladimir Y; Lanzer, Michael; Brosius, Juergen; Tang, Thean Hock; Rozhdestvensky, Timofey S

2010-01-01

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense-antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors.

Wayanin and guaijaverin, two active metabolites found in a Psidium acutangulum Mart. ex DC (syn. P. persoonii McVaugh) (Myrtaceae) antimalarial decoction from the Wayana Amerindians.

PubMed

Houël, Emeline; Nardella, Flore; Jullian, Valérie; Valentin, Alexis; Vonthron-Sénécheau, Catherine; Villa, Pascal; Obrecht, Adeline; Kaiser, Marcel; Bourreau, Eliane; Odonne, Guillaume; Fleury, Marie; Bourdy, Geneviève; Eparvier, Véronique; Deharo, Eric; Stien, Didier

2016-07-01

Psidium acutangulum Mart. ex DC is a small tree used by the Wayana Amerindians from the Upper-Maroni in French Guiana for the treatment of malaria. In a previous study, we highlighted the in vitro antiplasmodial, antioxidant and anti-inflammatory potential of the traditional decoction of P. acutangulum aerial parts. Our goal was then to investigate on the origin of the biological activity of the traditional remedy, and eventually characterize active constituents. Liquid-liquid extractions were performed on the decoction, and the antiplasmodial activity evaluated against chloroquine-resistant FcB1 ([(3)H]-hypoxanthine bioassay) and 7G8 (pLDH bioassay) P. falciparum strains, and on a chloroquine sensitive NF54 ([(3)H]-hypoxanthine bioassay) P. falciparum strain. The ethyl acetate fraction (D) was active and underwent bioguided fractionation. All the isolated compounds were tested on P. falciparum FcB1 strain. In vitro anti-inflammatory activity (IL-1β, IL-6, IL-8, TNFα) of the ethyl acetate fraction and of an anti-Plasmodium active compound, was concurrently assessed on LPS-stimulated human PBMC and NO secretion inhibition was measured on LPS stimulated RAW murine macrophages. Cytotoxicity of the fractions and pure compounds was measured on VERO cells, L6 mammalian cells, PBMCs, and RAW cells. Fractionation of the ethyl acetate soluble fraction (IC50 ranging from 3.4 to <1µg/mL depending on the parasite strain) led to the isolation of six pure compounds: catechin and five glycosylated quercetin derivatives. These compounds have never been isolated from this plant species. Two of these compounds (wayanin and guaijaverin) were found to be moderately active against P. falciparum FcB1 in vitro (IC50 5.5 and 6.9µM respectively). We proposed the name wayanin during public meetings organized in June 2015 in the Upper-Maroni villages, in homage to the medicinal knowledge of the Wayana population. At 50µg/mL, the ethyl acetate fraction (D) significantly inhibited IL-1β secretion (-46%) and NO production (-21%), as previously observed for the decoction. The effects of D and guiajaverin (4) on the secretion of other cytokines or NO production were not significant. The confirmed antiplasmodial activity of the ethyl acetate soluble fraction of the decoction and of the isolated compounds support the previous results obtained on the P. acutangulum decoction. The antiplasmodial activity might be due to a mixture of moderately active non-toxic flavonoids. The anti-inflammatory activities were less marked for ethyl acetate fraction (D) than for the decoction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Implications of Plasmodium vivax Biology for Control, Elimination, and Research

PubMed Central

Olliaro, Piero L.; Barnwell, John W.; Barry, Alyssa; Mendis, Kamini; Mueller, Ivo; Reeder, John C.; Shanks, G. Dennis; Snounou, Georges; Wongsrichanalai, Chansuda

2016-01-01

This paper summarizes our current understanding of the biology of Plasmodium vivax, how it differs from Plasmodium falciparum, and how these differences explain the need for P. vivax-tailored interventions. The article further pinpoints knowledge gaps where investments in research are needed to help identify and develop such specific interventions. The principal obstacles to reduce and eventually eliminate P. vivax reside in 1) its higher vectorial capacity compared with P. falciparum due to its ability to develop at lower temperature and over a shorter sporogonic cycle in the vector, allowing transmission in temperate zones and making it less sensitive to vector control measures that are otherwise effective on P. falciparum; 2) the presence of dormant liver forms (hypnozoites), sustaining multiple relapsing episodes from a single infectious bite that cannot be diagnosed and are not susceptible to any available antimalarial except primaquine, with routine deployment restricted by toxicity; 3) low parasite densities, which are difficult to detect with current diagnostics leading to missed diagnoses and delayed treatments (and protracted transmission), coupled with 4) transmission stages (gametocytes) occurring early in acute infections, before infection is diagnosed. PMID:27799636

Annual Plasmodium falciparum entomological inoculation rates (EIR) across Africa: literature survey, internet access and review

PubMed Central

Hay, Simon I.; Rogers, David J.; Toomer, Jonathan F.; Snow, Robert W.

2011-01-01

This paper presents the results of an extensive search of the formal and informal literature on annual Plasmodium falciparum entomological inoculation rates (EIR) across Africa from 1980 onwards. It first describes how the annual EIR data were collated, summarized, neo-referenced and staged for public access on the internet. Problems of data standardization, reporting accuracy and the subsequent publishing of information on the internet follow. The review was conducted primarily to investigate the spatial heterogeneity of malaria exposure in Africa and supports the idea of highly heterogeneous risk at the continental, regional and country levels. The implications for malaria control of the significant spatial (and seasonal) variation in exposure to infected mosquito bites are discussed. PMID:10897348

Prevalence and patterns of antifolate and chloroquine drug resistance markers in Plasmodium vivax across Pakistan

PubMed Central

2013-01-01

Background Plasmodium vivax is the most prevalent malaria species in Pakistan, with a distribution that coincides with Plasmodium falciparum in many parts of the country. Both species are likely exposed to drug pressure from a number of anti-malarials including chloroquine, sulphadoxine-pyrimethamine (SP), and artemisinin combination therapy, yet little is known regarding the effects of drug pressure on parasite genes associated with drug resistance. The aims of this study were to determine the prevalence of polymorphisms in the SP resistance-associated genes pvdhfr, pvdhps and chloroquine resistance-associated gene pvmdr1 in P. vivax isolates collected from across the country. Methods In 2011, 801 microscopically confirmed malaria-parasite positive filter paper blood samples were collected at 14 sites representing four provinces and the capital city of Islamabad. Species-specific polymerase chain reaction (PCR) was used to identify human Plasmodium species infection. PCR-positive P. vivax isolates were subjected to sequencing of pvdhfr, pvdhps and pvmdr1 and to real-time PCR analysis to assess pvmdr1 copy number variation. Results Of the 801 samples, 536 were determined to be P. vivax, 128 were P. falciparum, 43 were mixed vivax/falciparum infections and 94 were PCR-negative for Plasmodium infection. Of PCR-positive P. vivax samples, 372 were selected for sequence analysis. Seventy-six of the isolates (23%) were double mutant at positions S58R and S117N in pvdhfr. Additionally, two mutations at positions N50I and S93H were observed in 55 (15%) and 24 (7%) of samples, respectively. Three 18 base pair insertion-deletions (indels) were observed in pvdhfr, with two insertions at different nucleotide positions in 36 isolates and deletions in 10. Ninety-two percent of samples contained the pvdhps (S382/A383G/K512/A553/V585) SAKAV wild type haplotype. For pvmdr1, all isolates were wild type at position Y976F and 335 (98%) carried the mutation at codon F1076L. All isolates harboured single copies of the pvmdr1 gene. Conclusions The prevalence of mutations associated with SP resistance in P. vivax is low in Pakistan. The high prevalence of P. vivax mutant pvmdr1 codon F1076L indicates that efficacy of chloroquine plus primaquine could be in danger of being compromised, but further studies are required to assess the clinical relevance of this observation. These findings will serve as a baseline for further monitoring of drug-resistant P. vivax malaria in Pakistan. PMID:24007534

Spatio-temporal analysis of the genetic diversity and complexity of Plasmodium falciparum infections in Kedougou, southeastern Senegal.

PubMed

Niang, Makhtar; Thiam, Laty G; Loucoubar, Cheikh; Sow, Abdourahmane; Sadio, Bacary D; Diallo, Mawlouth; Sall, Amadou A; Toure-Balde, Aissatou

2017-01-19

Genetic analyses of the malaria parasite population and its temporal and spatial dynamics could provide an assessment of the effectiveness of disease control strategies. The genetic diversity of Plasmodium falciparum has been poorly documented in Senegal, and limited data are available from the Kedougou Region. This study examines the spatial and temporal variation of the genetic diversity and complexity of P. falciparum infections in acute febrile patients in Kedougou, southeastern Senegal. A total of 263 sera from patients presenting with acute febrile illness and attending Kedougou health facilities between July 2009 and July 2013 were obtained from a collection established as part of arbovirus surveillance in Kedougou. Samples identified as P. falciparum by nested PCR were characterized for their genetic diversity and complexity using msp-1 and msp-2 polymorphic markers. Samples containing only P. falciparum accounted for 60.83% (160/263) of the examined samples. All three msp-1 allelic families (K1, MAD20 and RO33) and two msp-2 allelic families (FC27 and 3D7) were detected in all villages investigated over the 5-year collection period. The average genotype per allelic family was comparable between villages. Frequencies of msp-1 and msp-2 allelic types showed no correlation with age (Fisher's exact test, P = 0.59) or gender (Fisher's exact test, P = 0.973), and were similarly distributed throughout the 5-year sampling period (Fisher's exact test, P = 0.412) and across villages (Fisher's exact test, P = 0.866). Mean multiplicity of infection (MOI) for both msp-1 and msp-2 was highest in Kedougou village (2.25 and 2.21, respectively) and among younger patients aged ≤ 15 years (2.12 and 2.00, respectively). The mean MOI was highest in 2009 and decreased progressively onward. Characterization of the genetic diversity and complexity of P. falciparum infections in Kedougou revealed no spatio-temporal variation in the genetic diversity of P. falciparum isolates. However, mean MOI varied with time of sera collection and decreased over the course of the study (July 2009 to July 2013). This suggests a slow progressive decrease of malaria transmission intensity in Kedougou Region despite the limited impact of preventive and control measures implemented by the National Malaria Control Programme on malaria morbidity and mortality.

Effect of artesunate-mefloquine fixed-dose combination in malaria transmission in amazon basin communities

PubMed Central

2012-01-01

Background Studies in South-East Asia have suggested that early diagnosis and treatment with artesunate (AS) and mefloquine (MQ) combination therapy may reduce the transmission of Plasmodium falciparum malaria and the progression of MQ resistance. Methods The effectiveness of a fixed-dose combination of AS and MQ (ASMQ) in reducing malaria transmission was tested in isolated communities of the Juruá valley in the Amazon region. Priority municipalities within the Brazilian Legal Amazon area were selected according to pre-specified criteria. Routine national malaria control programmatic procedures were followed. Existing health structures were reinforced and health care workers were trained to treat with ASMQ all confirmed falciparum malaria cases that match inclusion criteria. A local pharmacovigilance structure was implemented. Incidence of malaria and hospitalizations were recorded two years before, during, and after the fixed-dose ASMQ intervention. In total, between July 2006 and December 2008, 23,845 patients received ASMQ. Two statistical modelling approaches were applied to monthly time series of P. falciparum malaria incidence rates, P. falciparum/Plasmodium vivax infection ratio, and malaria hospital admissions rates. All the time series ranged from January 2004 to December 2008, whilst the intervention period span from July 2006 to December 2008. Results The ASMQ intervention had a highly significant impact on the mean level of each time series, adjusted for trend and season, of 0.34 (95%CI 0.20 – 0.58) for the P. falciparum malaria incidence rates, 0.67 (95%CI 0.50 – 0.89) for the P. falciparum/P. vivax infection ratio, and 0.53 (95%CI 0.41 – 0.69) for the hospital admission rates. There was also a significant change in the seasonal (or monthly) pattern of the time series before and after intervention, with the elimination of the malaria seasonal peak in the rainy months of the years following the introduction of ASMQ. No serious adverse events relating to the use of fixed-dose ASMQ were reported. Conclusions In the remote region of the Juruá valley, the early detection of malaria by health care workers and treatment with fixed-dose ASMQ was feasible and efficacious, and significantly reduced the incidence and morbidity of P. falciparum malaria. PMID:22905900

Partner-Drug Resistance and Population Substructuring of Artemisinin-Resistant Plasmodium falciparum in Cambodia

PubMed Central

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.

2017-01-01

Abstract 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. PMID:28854635

Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion.

PubMed

Sim, B K; Orlandi, P A; Haynes, J D; Klotz, F W; Carter, J M; Camus, D; Zegans, M E; Chulay, J D

1990-11-01

The Plasmodium falciparum gene encoding erythrocyte binding antigen-175 (EBA-175), a putative receptor for red cell invasion (Camus, D., and T. J. Hadley. 1985. Science (Wash. DC). 230:553-556.), has been isolated and characterized. DNA sequencing demonstrated a single open reading frame encoding a translation product of 1,435 amino acid residues. Peptides corresponding to regions on the deduced amino acid sequence predicted to be B cell epitopes were assessed for immunogenicity. Immunization of mice and rabbits with EBA-peptide 4, a synthetic peptide encompassing amino acid residues 1,062-1,103, produced antibodies that recognized P. falciparum merozoites in an indirect fluorescent antibody assay. When compared to sera from rabbits immunized with the same adjuvant and carrier protein, sera from rabbits immunized with EBA-peptide 4 inhibited merozoite invasion of erythrocytes in vitro by 80% at a 1:5 dilution. Furthermore, these sera inhibited the binding of purified, authentic EBA-175 to erythrocytes, suggesting that their activity in inhibiting merozoite invasion of erythrocytes is mediated by blocking the binding of EBA-175 to erythrocytes. Since the nucleotide sequence of EBA-peptide 4 is conserved among seven strains of P. falciparum from throughout the world (Sim, B. K. L. 1990. Mol. Biochem. Parasitol. 41:293-296.), these data identify a region of the protein that should be a focus of vaccine development efforts.

Genetic diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and its effect on the performance of PfHRP2-based rapid diagnostic tests.

PubMed

Baker, Joanne; McCarthy, James; Gatton, Michelle; Kyle, Dennis E; Belizario, Vicente; Luchavez, Jennifer; Bell, David; Cheng, Qin

2005-09-01

Rising costs of antimalarial agents are increasing the demand for accurate diagnosis of malaria. Rapid diagnostic tests (RDTs) offer great potential to improve the diagnosis of malaria, particularly in remote areas. Many RDTs are based on the detection of Plasmodium falciparum histidine-rich protein (PfHRP) 2, but reports from field tests have questioned their sensitivity and reliability. We hypothesize that the variability in the results of PfHRP2-based RDTs is related to the variability in the target antigen. We tested this hypothesis by examining the genetic diversity of PfHRP2, which includes numerous amino acid repeats, in 75 P. falciparum lines and isolates originating from 19 countries and testing a subset of parasites by use of 2 PfHRP2-based RDTs. We observed extensive diversity in PfHRP2 sequences, both within and between countries. Logistic regression analysis indicated that 2 types of repeats were predictive of RDT detection sensitivity (87.5% accuracy), with predictions suggesting that only 84% of P. falciparum parasites in the Asia-Pacific region are likely to be detected at densities < or = 250 parasites/microL. Our data also indicated that PfHRP3 may play a role in the performance of PfHRP2-based RDTs. These findings provide an alternative explanation for the variable sensitivity in field tests of malaria RDTs that is not due to the quality of the RDTs.

Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum

PubMed Central

2012-01-01

Background The post-genomic era of malaria research provided unprecedented insights into the biology of Plasmodium parasites. Due to the large evolutionary distance to model eukaryotes, however, we lack a profound understanding of many processes in Plasmodium biology. One example is the cell nucleus, which controls the parasite genome in a development- and cell cycle-specific manner through mostly unknown mechanisms. To study this important organelle in detail, we conducted an integrative analysis of the P. falciparum nuclear proteome. Results We combined high accuracy mass spectrometry and bioinformatic approaches to present for the first time an experimentally determined core nuclear proteome for P. falciparum. Besides a large number of factors implicated in known nuclear processes, one-third of all detected proteins carry no functional annotation, including many phylum- or genus-specific factors. Importantly, extensive experimental validation using 30 transgenic cell lines confirmed the high specificity of this inventory, and revealed distinct nuclear localization patterns of hitherto uncharacterized proteins. Further, our detailed analysis identified novel protein domains potentially implicated in gene transcription pathways, and sheds important new light on nuclear compartments and processes including regulatory complexes, the nucleolus, nuclear pores, and nuclear import pathways. Conclusion Our study provides comprehensive new insight into the biology of the Plasmodium nucleus and will serve as an important platform for dissecting general and parasite-specific nuclear processes in malaria parasites. Moreover, as the first nuclear proteome characterized in any protist organism, it will provide an important resource for studying evolutionary aspects of nuclear biology. PMID:23181666

Antiplasmodial activity of flavonol quercetin and its analogues in Plasmodium falciparum: evidence from clinical isolates in Bangladesh and standardized parasite clones.

PubMed

Ganesh, Deepa; Fuehrer, Hans-Peter; Starzengrüber, Peter; Swoboda, Paul; Khan, Wasif Ali; Reismann, Johannes A B; Mueller, Milena S K; Chiba, Peter; Noedl, Harald

2012-06-01

Malaria is still a major threat in many parts of the world with resistance spreading to almost all classes of antimalarials. The limited arsenal of available antimalarial drugs emphasizes the urgent need for novel antimalarial compounds. Owing to the fact that novel leads from nature have traditionally played a pivotal role in the development of various classes of antimalarials, we investigated a set of eight naturally occurring dietary flavonoids and their analogues for their antiplasmodial activity on clinical field isolates in southeastern Bangladesh and culture-adapted chloroquine-sensitive and chloroquine-resistant parasite clones. Except for taxifolin, all the other flavonoids had 50% inhibitory concentrations below 14 μM, both in the field and laboratory-adapted parasites. Neither of the flavonoids showed any activity correlation with chloroquine. The quercetin analogue rutin (7.10 ± 10.32 μM) was the most active substance in field isolates as well as laboratory-adapted cultures (3.53 ± 13.34 μM in 3D7 and 10.38 ± 15.08 μM in K1), providing the first evidence of its activity against Plasmodium falciparum parasites. Thus, our results provide important evidence of the antimalarial activity of flavonoids in traditional use and thus warrant further investigation of these compounds as potential antiplasmodial agents.

An Analysis of the Binding Characteristics of a Panel of Recently Selected ICAM-1 Binding Plasmodium falciparum Patient Isolates

PubMed Central

Madkhali, Aymen M.; Alkurbi, Mohammed O.; Szestak, Tadge; Bengtsson, Anja; Patil, Pradeep R.; Wu, Yang; Alharthi, Saeed; Jensen, Anja T. R.; Pleass, Richard; Craig, Alister G.

2014-01-01

The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE) from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1) expressed on the surface of the infected erythrocyte membrane. One of the commonly used host receptors is ICAM-1, and it has been suggested that ICAM-1 has a role in cerebral malaria pathology, although the evidence to support this is not conclusive. The current study examined the cytoadherence patterns of lab-adapted patient isolates after selecting on ICAM-1. We investigated the binding phenotypes using variant ICAM-1 proteins including ICAM-1Ref, ICAM-1Kilifi, ICAM-1S22/A, ICAM-1L42/A and ICAM-1L44/A using static assays. The study also examined ICAM-1 blocking by four anti-ICAM-1 monoclonal antibodies (mAb) under static conditions. We also characterised the binding phenotypes using Human Dermal Microvascular Endothelial Cells (HDMEC) under flow conditions. The results show that different isolates have variant-specific binding phenotypes under both static and flow conditions, extending our previous observations that this variation might be due to variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants. PMID:25360558

In vitro studies on the sensitivity pattern of Plasmodium falciparum to anti-malarial drugs and local herbal extracts

PubMed Central

2014-01-01

Background The resistance of human malaria parasites to anti-malarial compounds has become considerable concern, particularly in view of the shortage of novel classes of anti-malarial drugs. One way to prevent resistance is by using new compounds that are not based on existing synthetic antimicrobial agents. Results Sensitivity of 100 Plasmodium falciparum isolates to chloroquine, quinine, amodiaquine, mefloquine, sulphadoxine/pyrimethamine, artemisinin, Momordica charantia (‘Ejirin’) Diospyros monbuttensis (‘Egun eja’) and Morinda lucida (‘Oruwo’) was determined using the in vitro microtest (Mark III) technique to determine the IC50 of the drugs. All the isolates tested were sensitive to quinine, mefloquine and artesunate. Fifty-one percent of the isolates were resistant to chloroquine, 13% to amodiaquine and 5% to sulphadoxine/pyrimethamine. Highest resistance to chloroquine (68.9%) was recorded among isolates from Yewa zone while highest resistance to amodiaquine (30%) was observed in Ijebu zone. Highest resistance to sulphadoxine/pyrimethamine was recorded in Yewa and Egba zones, respectively. A positive correlation was observed between the responses to artemisinin and mefloquine (P<0.05), artemisinin and quinine (P<0.05) and quinine and mefloquine (P<0.05). A negative correlation was observed between the responses to chloroquine and mefloquine (P>0.05). Highest anti-plasmodial activity was obtained with the ethanolic extract of D. monbuttensis (IC50 = 3.2nM) while the lowest was obtained from M. lucida (IC50 =25nM). Conclusions Natural products isolated from plants used in traditional medicine, which have potent anti-plasmodial action in vitro, represent potential sources of new anti-malarial drugs. PMID:24555525

Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase

PubMed Central

Hawkins, Vivian N; Auliff, Alyson; Prajapati, Surendra Kumar; Rungsihirunrat, Kanchana; Hapuarachchi, Hapuarachchige C; Maestre, Amanda; O'Neil, Michael T; Cheng, Qin; Joshi, Hema; Na-Bangchang, Kesara; Sibley, Carol Hopkins

2008-01-01

Background In order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and spread in natural populations. Recent work has demonstrated that pyrimethamine-resistance conferring mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) have arisen rarely de novo, but spread widely in Asia and Africa. The origin and spread of mutations in Plasmodium vivax dhfr were assessed by constructing haplotypes based on sequencing dhfr and its flanking regions. Methods The P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network. Results Synonymous and non-synonymous single nucleotide polymorphisms (SNPs) within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel). SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N) dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T) and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T) mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu. Conclusion It was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts sharply with the selective sweep of rare antifolate resistant alleles observed in the P. falciparum populations in Asia and Africa. The finding of multiple origins of resistance-conferring mutations has important implications for drug policy. PMID:18442404

Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase.

PubMed

Hawkins, Vivian N; Auliff, Alyson; Prajapati, Surendra Kumar; Rungsihirunrat, Kanchana; Hapuarachchi, Hapuarachchige C; Maestre, Amanda; O'Neil, Michael T; Cheng, Qin; Joshi, Hema; Na-Bangchang, Kesara; Sibley, Carol Hopkins

2008-04-28

In order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and spread in natural populations. Recent work has demonstrated that pyrimethamine-resistance conferring mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) have arisen rarely de novo, but spread widely in Asia and Africa. The origin and spread of mutations in Plasmodium vivax dhfr were assessed by constructing haplotypes based on sequencing dhfr and its flanking regions. The P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network. Synonymous and non-synonymous single nucleotide polymorphisms (SNPs) within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel). SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N) dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T) and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T) mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu. It was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts sharply with the selective sweep of rare antifolate resistant alleles observed in the P. falciparum populations in Asia and Africa. The finding of multiple origins of resistance-conferring mutations has important implications for drug policy.

Genome-wide analysis of selection on the malaria parasite Plasmodium falciparum in West African populations of differing infection endemicity.

PubMed

Mobegi, Victor A; Duffy, Craig W; Amambua-Ngwa, Alfred; Loua, Kovana M; Laman, Eugene; Nwakanma, Davis C; MacInnis, Bronwyn; Aspeling-Jones, Harvey; Murray, Lee; Clark, Taane G; Kwiatkowski, Dominic P; Conway, David J

2014-06-01

Locally varying selection on pathogens may be due to differences in drug pressure, host immunity, transmission opportunities between hosts, or the intensity of between-genotype competition within hosts. Highly recombining populations of the human malaria parasite Plasmodium falciparum throughout West Africa are closely related, as gene flow is relatively unrestricted in this endemic region, but markedly varying ecology and transmission intensity should cause distinct local selective pressures. Genome-wide analysis of sequence variation was undertaken on a sample of 100 P. falciparum clinical isolates from a highly endemic region of the Republic of Guinea where transmission occurs for most of each year and compared with data from 52 clinical isolates from a previously sampled population from The Gambia, where there is relatively limited seasonal malaria transmission. Paired-end short-read sequences were mapped against the 3D7 P. falciparum reference genome sequence, and data on 136,144 single nucleotide polymorphisms (SNPs) were obtained. Within-population analyses identifying loci showing evidence of recent positive directional selection and balancing selection confirm that antimalarial drugs and host immunity have been major selective agents. Many of the signatures of recent directional selection reflected by standardized integrated haplotype scores were population specific, including differences at drug resistance loci due to historically different antimalarial use between the countries. In contrast, both populations showed a similar set of loci likely to be under balancing selection as indicated by very high Tajima's D values, including a significant overrepresentation of genes expressed at the merozoite stage that invades erythrocytes and several previously validated targets of acquired immunity. Between-population FST analysis identified exceptional differentiation of allele frequencies at a small number of loci, most markedly for five SNPs covering a 15-kb region within and flanking the gdv1 gene that regulates the early stages of gametocyte development, which is likely related to the extreme differences in mosquito vector abundance and seasonality that determine the transmission opportunities for the sexual stage of the parasite. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genetic mutations in pfcrt and pfmdr1 at the time of artemisinin combination therapy introduction in South Pacific islands of Vanuatu and Solomon Islands.

PubMed

Gresty, Karryn J; Gray, Karen-Ann; Bobogare, Albino; Taleo, George; Hii, Jeffrey; Wini, Lyndes; Cheng, Qin; Waters, Norman C

2014-10-15

Chloroquine (CQ), alone or in combination with sulphadoxine-pyrimethamine, was widely used for the treatment of Plasmodium falciparum and Plasmodium vivax for several decades in both Vanuatu and Solomon Islands prior to the introduction of artemether-lumefantrine (AL) in 2008. However, the effect of chloroquine selection on parasite population, which may affect the efficacy of lumefantrine or other partner drugs of artemisinin, has not been well assessed. This study aims to provide baseline data on molecular markers (pfcrt and pfmdr1), along with the origins of pfcrt, prior to the introduction of AL. Blood spots were obtained from epidemiological surveys conducted on Tanna Island, Tafea Province, Vanuatu and Temotu Province, Solomon Islands in 2008. Additional samples from Malaita Province, Solomon Islands were collected as part of an artemether-lumefantrine efficacy study in 2008. Plasmodium falciparum pfcrt and pfmdr1 genes were examined for polymorphisms. Microsatellite markers flanking pfcrt were also examined to ascertain origins of CQ resistance. Pfcrt analysis revealed 100% of parasites from Tafea Province, Vanuatu and Malaita Province, Solomon Islands and 98% of parasites from Temotu Province, Solomon Islands carried the K76T polymorphism that confers CQ resistance. Comparison of pfcrt allelic patterns and microsatellite markers flanking pfcrt revealed six haplotypes with more than 70% of isolates possessing haplotypes very similar to those observed in Papua New Guinea. The dominant (98.5%) pfmdr1 allele across all island groups was YYCND. Prior to the introduction of AL in the Solomon Islands and Vanuatu, P. falciparum isolates possessed point mutations known to confer CQ resistance and possibly associated with a decreased susceptibility to quinine and halofantrine, but an increased susceptibility to artemisinin and lumefantrine. Overall, pfcrt allelic types and the flanking microsatellite markers exhibited similarities to those of Papua New Guinea, suggesting these parasites share a common ancestry. The current use of AL for both P. falciparum and P. vivax infections will enable changes in these markers, in the absence of CQ pressure, to be monitored.

Assessing the Role of Climate Change in Malaria Transmission in Africa.

PubMed

Ngarakana-Gwasira, E T; Bhunu, C P; Masocha, M; Mashonjowa, E

2016-01-01

The sensitivity of vector borne diseases like malaria to climate continues to raise considerable concern over the implications of climate change on future disease dynamics. The problem of malaria vectors shifting from their traditional locations to invade new zones is of important concern. A mathematical model incorporating rainfall and temperature is constructed to study the transmission dynamics of malaria. The reproduction number obtained is applied to gridded temperature and rainfall datasets for baseline climate and future climate with aid of GIS. As a result of climate change, malaria burden is likely to increase in the tropics, the highland regions, and East Africa and along the northern limit of falciparum malaria. Falciparum malaria will spread into the African highlands; however it is likely to die out at the southern limit of the disease.

Limited artemisinin resistance-associated polymorphisms in Plasmodium falciparum K13-propeller and PfATPase6 gene isolated from Bioko Island, Equatorial Guinea

PubMed Central

Li, Jian; Chen, Jiangtao; Xie, Dongde; Eyi, Urbano Monsuy; Matesa, Rocio Apicante; Ondo Obono, Maximo Miko; Ehapo, Carlos Sala; Yang, Liye; Yang, Huitian; Lin, Min

2016-01-01

Objective With emergence and geographically expanding of antimalarial resistance worldwide, molecular markers are essential tool for surveillance of resistant Plasmodium parasites. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain are shown to be associated with artemisinin (ART) resistance in vivo and in vitro. This study aims to investigate the ART resistance-associated polymorphisms of K13-propeller and PfATPase6 genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea (EG). Methods A total of 172 samples were collected from falciparum malaria patients on Bioko Island between 2013 and 2014. The polymorphisms of K13-propeller and PfATPase6 genes were analyzed by Nest-PCR and sequencing. Results Sequences of K13-propeller and PfATPase6 were obtained from 90.74% (98/108) and 91.45% (139/152) samples, respectively. The 2.04% (2/98) cases had non-synonymous K13-propeller A578S mutation but no found the mutations associated with ART resistance in Southeast Asia. For PfATPase6, the mutations were found at positions N569K and A630S with the mutation prevalence of 7.91% (11/139) and 1.44% (2/139), respectively. In addition, a sample with the mixed type at position I723V was discovered (0.72%, 1/139). Conclusions This study initially offers an insight of K13-propeller and PfATPase6 polymorphisms on Bioko Island, EG. It suggests no widespread ART resistance or tolerance in the region, and might be helpful for developing and updating guidance for the use of ART-based combination therapies (ACTs). PMID:27054064

Limited artemisinin resistance-associated polymorphisms in Plasmodium falciparum K13-propeller and PfATPase6 gene isolated from Bioko Island, Equatorial Guinea.

PubMed

Li, Jian; Chen, Jiangtao; Xie, Dongde; Eyi, Urbano Monsuy; Matesa, Rocio Apicante; Ondo Obono, Maximo Miko; Ehapo, Carlos Sala; Yang, Liye; Yang, Huitian; Lin, Min

2016-04-01

With emergence and geographically expanding of antimalarial resistance worldwide, molecular markers are essential tool for surveillance of resistant Plasmodium parasites. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain are shown to be associated with artemisinin (ART) resistance in vivo and in vitro. This study aims to investigate the ART resistance-associated polymorphisms of K13-propeller and PfATPase6 genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea (EG). A total of 172 samples were collected from falciparum malaria patients on Bioko Island between 2013 and 2014. The polymorphisms of K13-propeller and PfATPase6 genes were analyzed by Nest-PCR and sequencing. Sequences of K13-propeller and PfATPase6 were obtained from 90.74% (98/108) and 91.45% (139/152) samples, respectively. The 2.04% (2/98) cases had non-synonymous K13-propeller A578S mutation but no found the mutations associated with ART resistance in Southeast Asia. For PfATPase6, the mutations were found at positions N569K and A630S with the mutation prevalence of 7.91% (11/139) and 1.44% (2/139), respectively. In addition, a sample with the mixed type at position I723V was discovered (0.72%, 1/139). This study initially offers an insight of K13-propeller and PfATPase6 polymorphisms on Bioko Island, EG. It suggests no widespread ART resistance or tolerance in the region, and might be helpful for developing and updating guidance for the use of ART-based combination therapies (ACTs).

Isolation and Functional Characterization of Two Distinct Sexual-Stage-Specific Promoters of the Human Malaria Parasite Plasmodium falciparum†

PubMed Central

Dechering, Koen J.; Kaan, Anita M.; Mbacham, Wilfred; Wirth, Dyann F.; Eling, Wijnand; Konings, Ruud N. H.; Stunnenberg, Hendrik G.

1999-01-01

Transmission of malaria depends on the successful development of the sexual stages of the parasite within the midgut of the mosquito vector. The differentiation process leading to the production of the sexual stages is delineated by several developmental switches. Arresting the progression through this sexual differentiation pathway would effectively block the spread of the disease. The successful development of such transmission-blocking agents is hampered by the lack of a detailed understanding of the program of gene expression that governs sexual differentiation of the parasite. Here we describe the isolation and functional characterization of the Plasmodium falciparum pfs16 and pfs25 promoters, whose activation marks the developmental switches executed during the sexual differentiation process. We have studied the differential activation of the pfs16 and pfs25 promoters during intraerythrocytic development by transfection of P. falciparum and during gametogenesis and early sporogonic development by transfection of the related malarial parasite P. gallinaceum. Our data indicate that the promoter of the pfs16 gene is activated at the onset of gametocytogenesis, while the activity of the pfs25 promoter is induced following the transition to the mosquito vector. Both promoters have unusual DNA compositions and are extremely A/T rich. We have identified the regions in the pfs16 and pfs25 promoters that are essential for high transcriptional activity. Furthermore, we have identified a DNA-binding protein, termed PAF-1, which activates pfs25 transcription in the mosquito midgut. The data presented here shed the first light on the details of processes of gene regulation in the important human pathogen P. falciparum. PMID:9891033

Molecular epidemiology of malaria in Cameroon. XV. Experimental studies on serum substitutes and supplements and alternative culture media for in vitro drug sensitivity assays using fresh isolates of Plasmodium falciparum.

PubMed

Basco, Leonardo K

2003-08-01

In vitro drug sensitivity assay is an important tool for various on-going studies aiming to establish the correlation between candidate molecular markers for drug resistance and drug response in laboratory-adapted strains and field isolates of Plasmodium falciparum. A widespread use of this technique in the field would require a suitable substitute that can replace human serum. In this study, several alternative sources of serum substitutes and supplements were evaluated for their capacity to sustain parasite growth for a single life cycle and their compatibility with in vitro assays for clinical isolates that have not been adapted to in vitro culture. Albumax, a commercial preparation of lipid-enriched bovine albumin, did not support parasite growth as much as human serum and fetal calf serum in several isolates. Other serum supplements (AmnioMax and Ultroser) supported parasite growth relatively well. The 50% inhibitory concentrations (IC50s) of chloroquine and antifolates determined with 0.05% Albumax were generally two or three times higher than with human serum. With 10% fetal calf serum, IC50s for chloroquine and antifolates were approximately two times higher and three times lower than with human serum, respectively. The use of AmnioMax and OptiMAb resulted in a greater than two-fold increase in IC50s and several uninterpretable assays. Despite possible batch-to-batch differences, fetal calf serum may be a suitable substitute for in vitro drug assays while awaiting the results of further studies on other serum substitutes and supplements.

Micro-epidemiological structuring of Plasmodium falciparum parasite populations in regions with varying transmission intensities in Africa.

PubMed Central

Omedo, Irene; Mogeni, Polycarp; Bousema, Teun; Rockett, Kirk; Amambua-Ngwa, Alfred; Oyier, Isabella; C. Stevenson, Jennifer; Y. Baidjoe, Amrish; de Villiers, Etienne P.; Fegan, Greg; Ross, Amanda; Hubbart, Christina; Jeffreys, Anne; N. Williams, Thomas; Kwiatkowski, Dominic; Bejon, Philip

2017-01-01

Background: The first models of malaria transmission assumed a completely mixed and homogeneous population of parasites.  Recent models include spatial heterogeneity and variably mixed populations. However, there are few empiric estimates of parasite mixing with which to parametize such models. Methods: Here we genotype 276 single nucleotide polymorphisms (SNPs) in 5199 P. falciparum isolates from two Kenyan sites (Kilifi county and Rachuonyo South district) and one Gambian site (Kombo coastal districts) to determine the spatio-temporal extent of parasite mixing, and use Principal Component Analysis (PCA) and linear regression to examine the relationship between genetic relatedness and distance in space and time for parasite pairs. Results: Using 107, 177 and 82 SNPs that were successfully genotyped in 133, 1602, and 1034 parasite isolates from The Gambia, Kilifi and Rachuonyo South district, respectively, we show that there are no discrete geographically restricted parasite sub-populations, but instead we see a diffuse spatio-temporal structure to parasite genotypes.  Genetic relatedness of sample pairs is predicted by relatedness in space and time. Conclusions: Our findings suggest that targeted malaria control will benefit the surrounding community, but unfortunately also that emerging drug resistance will spread rapidly through the population. PMID:28612053

Antimalarial activity of Bidens pilosa L. (Asteraceae) ethanol extracts from wild plants collected in various localities or plants cultivated in humus soil.

PubMed

Andrade-Neto, Valter F; Brandão, Maria G L; Oliveira, Francielda Q; Casali, Vicente W D; Njaine, Brian; Zalis, Mariano G; Oliveira, Luciana A; Krettli, Antoniana U

2004-08-01

Bidens pilosa (Asteraceae), a medicinal plant used worldwide, has antimalarial activity as shown in previous work. This study tested ethanol extracts from wild plants collected in three different regions of Brazil and from plants cultivated in various soil conditions. The extracts were active in mice infected with P. berghei: doses of < or =500 mg/kg administered by oral route reduced malaria parasitaemia and mouse mortality; higher doses were found to be less effective. Tested in vitro against three P. falciparum isolates, two chloroquine resistant and one mefloquine resistant, the plants cultivated under standard conditions, and in humus enriched soil, were active; but the wild plants were the most active. Analysis using thin layer chromatography demonstrated the presence of flavonoids (compounds considered responsible for the antimalarial activity) in all plants tested, even though at different profiles. Because B. pilosa is proven to be active against P. falciparum drug-resistant parasites in vitro, and in rodent malaria in vivo, it is a good candidate for pre-clinical tests as a phytotherapeutic agent or for chemical isolation of the active compounds with the aim of finding new antimalarial drugs. Copyright (c) 2004 John Wiley & Sons, Ltd.

In silico epitope mapping and experimental evaluation of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) as a malaria vaccine candidate.

PubMed

Cravo, Pedro; Machado, Renato B; Leite, Juliana A; Leda, Taizy; Suwanarusk, Rossarin; Bittencourt, Najara; Albrecht, Letusa; Judice, Carla; Lopes, Stefanie C P; Lacerda, Marcus V G; Ferreira, Marcelo U; Soares, Irene S; Goh, Yun Shan; Bargieri, Daniel Y; Nosten, François; Russell, Bruce; Rénia, Laurent; Costa, Fabio T M

2018-01-10

Technical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated. Detailed epitope mapping of MAEBL through immunoinformatics identified several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specific immunity. The MAEBL antigen is promising candidate towards the development of a malaria vaccine.

Implications of Plasmodium vivax Biology for Control, Elimination, and Research.

PubMed

Olliaro, Piero L; Barnwell, John W; Barry, Alyssa; Mendis, Kamini; Mueller, Ivo; Reeder, John C; Shanks, G Dennis; Snounou, Georges; Wongsrichanalai, Chansuda

2016-12-28

This paper summarizes our current understanding of the biology of Plasmodium vivax, how it differs from Plasmodium falciparum, and how these differences explain the need for P. vivax-tailored interventions. The article further pinpoints knowledge gaps where investments in research are needed to help identify and develop such specific interventions. The principal obstacles to reduce and eventually eliminate P. vivax reside in 1) its higher vectorial capacity compared with P. falciparum due to its ability to develop at lower temperature and over a shorter sporogonic cycle in the vector, allowing transmission in temperate zones and making it less sensitive to vector control measures that are otherwise effective on P. falciparum; 2) the presence of dormant liver forms (hypnozoites), sustaining multiple relapsing episodes from a single infectious bite that cannot be diagnosed and are not susceptible to any available antimalarial except primaquine, with routine deployment restricted by toxicity; 3) low parasite densities, which are difficult to detect with current diagnostics leading to missed diagnoses and delayed treatments (and protracted transmission), coupled with 4) transmission stages (gametocytes) occurring early in acute infections, before infection is diagnosed. © The American Society of Tropical Medicine and Hygiene.

Prevalence of K13-propeller gene polymorphisms among Plasmodium falciparum parasites isolated from adult symptomatic patients in northern Uganda.

PubMed

Ocan, Moses; Bwanga, Freddie; Okeng, Alfred; Katabazi, Fred; Kigozi, Edgar; Kyobe, Samuel; Ogwal-Okeng, Jasper; Obua, Celestino

2016-08-19

In the absence of an effective vaccine, malaria treatment and eradication is still a challenge in most endemic areas globally. This is especially the case with the current reported emergence of resistance to artemisinin agents in Southeast Asia. This study therefore explored the prevalence of K13-propeller gene polymorphisms among Plasmodium falciparum parasites in northern Uganda. Adult patients (≥18 years) presenting to out-patients department of Lira and Gulu regional referral hospitals in northern Uganda were randomly recruited. Laboratory investigation for presence of plasmodium infection among patients was done using Plasmodium falciparum exclusive rapid diagnostic test, histidine rich protein-2 (HRP2) (Pf). Finger prick capillary blood from patients with a positive malaria test was spotted on a filter paper Whatman no. 903. The parasite DNA was extracted using chelex resin method and sequenced for mutations in K13-propeller gene using Sanger sequencing. PCR DNA sequence products were analyzed using in DNAsp 5.10.01software, data was further processed in Excel spreadsheet 2007. A total of 60 parasite DNA samples were sequenced. Polymorphisms in the K13-propeller gene were detected in four (4) of the 60 parasite DNA samples sequenced. A non-synonymous polymorphism at codon 533 previously detected in Cambodia was found in the parasite DNA samples analyzed. Polymorphisms at codon 522 (non-synonymous) and codon 509 (synonymous) were also found in the samples analyzed. The study found evidence of positive selection in the Plasmodium falciparum population in northern Uganda (Tajima's D = -1.83205; Fu and Li's D = -1.82458). Polymorphism in the K13-propeller gene previously reported in Cambodia has been found in the Ugandan Plasmodium falciparum parasites. There is need for continuous surveillance for artemisinin resistance gene markers in the country.

Two novel mutations of pfdhps K540T and I588F, affecting sulphadoxine-pyrimethamine-resistant response in uncomplicated falciparum malaria at Banjar district, South Kalimantan Province, Indonesia

PubMed Central

2014-01-01

Background Mutations in pfdhfr and pfdhps genes have been shown to associate with sulphadoxine-pyrimethamine (SP) resistance of Plasmodium falciparum parasites. However, pfdhfr, pfdhps genotypes and the correlations to SP treatment outcome in Indonesia has not yet been well analysed. Methods After obtaining informed consent, 61 uncomplicated falciparum malaria patients were recruited in Banjar district, South Kalimantan Province, Indonesia, from October 2009 to August 2010. They were treated by a single oral dose of SP and its effects on clinical and parasitological status were followed until day 28 after treatment. Occasionally, a thick smear blood film for microscopy observation and blood spot on a filter paper for pfdhfr and pfdhps genotype analysis were collected. Results Pfdhfr and pfdhps genotypes from 24 P. falciparum-infected patients consisting of adequate clinical parasitological response (ACPR) (n = 6; 25.0%) and early treatment failure (ETF) (n = 10; 41.7%) or late parasitological failure (LPF) (n = 8; 33.3%) were obtained by sequencing. Two novel mutations of pfdhps gene, K540T and I588F, were determined in ten and five isolates, respectively. These mutations were present in the pfdhfr/pfdhps combined haplotypes of ANRNI/SGTGA (n = 6), ANRNL/SGTGA (n = 4), and ANRNI/SGEAA(588F) (n = 5), (mutation codons are bold typed); these haplotypes were mostly belonging to parasitological failure (ETF or LPF). The parasites acquiring five mutations in pfdhfr/pfdhps haplotypes and four mutations with additional I588F did not respond adequately to SP treatment. Conclusion Many of Plasmodium falciparum infected patients in Banjar district, South Kalimantan, Indonesia did not respond adequately to SP treatment and these low ineffectiveness of SP in this area was associated with two novel mutations of pfdhps, K540T and I588F. PMID:25187019

Two novel mutations of pfdhps K540T and I588F, affecting sulphadoxine-pyrimethamine-resistant response in uncomplicated falciparum malaria at Banjar district, South Kalimantan Province, Indonesia.

PubMed

Basuki, Sukmawati; Fitriah; Riyanto, Sugeng; Budiono; Dachlan, Yoes P; Uemura, Haruki

2014-04-04

Mutations in pfdhfr and pfdhps genes have been shown to associate with sulphadoxine-pyrimethamine (SP) resistance of Plasmodium falciparum parasites. However, pfdhfr, pfdhps genotypes and the correlations to SP treatment outcome in Indonesia has not yet been well analysed. After obtaining informed consent, 61 uncomplicated falciparum malaria patients were recruited in Banjar district, South Kalimantan Province, Indonesia, from October 2009 to August 2010. They were treated by a single oral dose of SP and its effects on clinical and parasitological status were followed until day 28 after treatment. Occasionally, a thick smear blood film for microscopy observation and blood spot on a filter paper for pfdhfr and pfdhps genotype analysis were collected. Pfdhfr and pfdhps genotypes from 24 P. falciparum-infected patients consisting of adequate clinical parasitological response (ACPR) (n = 6; 25.0%) and early treatment failure (ETF) (n = 10; 41.7%) or late parasitological failure (LPF) (n = 8; 33.3%) were obtained by sequencing. Two novel mutations of pfdhps gene, K540T and I588F, were determined in ten and five isolates, respectively. These mutations were present in the pfdhfr/pfdhps combined haplotypes of ANRNI/SGTGA (n = 6), ANRNL/SGTGA (n = 4), and ANRNI/SGEAA(588F) (n = 5), (mutation codons are bold typed); these haplotypes were mostly belonging to parasitological failure (ETF or LPF). The parasites acquiring five mutations in pfdhfr/pfdhps haplotypes and four mutations with additional I588F did not respond adequately to SP treatment. Many of Plasmodium falciparum infected patients in Banjar district, South Kalimantan, Indonesia did not respond adequately to SP treatment and these low ineffectiveness of SP in this area was associated with two novel mutations of pfdhps, K540T and I588F.

Importation of chloroquine-resistant Plasmodium falciparum by Guatemalan peacekeepers returning from the Democratic Republic of the Congo

PubMed Central

2013-01-01

Background Malaria elimination is being pursued in five of seven Central American countries. Military personnel returning from peacekeeping missions in sub-Saharan Africa could import chloroquine-resistant Plasmodium falciparum, posing a threat to elimination and to the continued efficacy of first-line chloroquine (CQ) treatment in these countries. This report describes the importation of P. falciparum from among 150 Guatemalan army special forces and support staff who spent ten months on a United Nations’ peacekeeping mission in the Democratic Republic of the Congo (DRC) in 2010. Methods Investigators reviewed patients’ medical charts and interviewed members of the contingent to identify malaria cases and risk factors for malaria acquisition. Clinical specimens were tested for malaria; isolated parasites were characterized molecularly for CQ resistance. Results Investigators identified 12 cases (8%) of laboratory-confirmed P. falciparum infection within the contingent; one case was from a soldier infected with a CQ-resistant pfcrt genotype resulting in his death. None of the contingent used an insecticide-treated bed net (ITN) or completely adhered to malaria chemoprophylaxis while in the DRC. Conclusion This report highlights the need to promote use of malaria prevention measures, in particular ITNs and chemoprophylaxis, among peacekeepers stationed in malaria-endemic areas. Countries attempting to eliminate malaria should consider appropriate methods to screen peacekeepers returning from endemic areas for malaria infections. Cases of malaria in travellers, immigrants and soldiers returning to Central America from countries with CQ-resistant malaria should be assumed to be carry resistant parasites and receive appropriate anti-malarial therapy to prevent severe disease and death. PMID:24060234

Microsatellite genotyping and genome-wide single nucleotide polymorphism-based indices of Plasmodium falciparum diversity within clinical infections.

PubMed

Murray, Lee; Mobegi, Victor A; Duffy, Craig W; Assefa, Samuel A; Kwiatkowski, Dominic P; Laman, Eugene; Loua, Kovana M; Conway, David J

2016-05-12

In regions where malaria is endemic, individuals are often infected with multiple distinct parasite genotypes, a situation that may impact on evolution of parasite virulence and drug resistance. Most approaches to studying genotypic diversity have involved analysis of a modest number of polymorphic loci, although whole genome sequencing enables a broader characterisation of samples. PCR-based microsatellite typing of a panel of ten loci was performed on Plasmodium falciparum in 95 clinical isolates from a highly endemic area in the Republic of Guinea, to characterize within-isolate genetic diversity. Separately, single nucleotide polymorphism (SNP) data from genome-wide short-read sequences of the same samples were used to derive within-isolate fixation indices (F ws), an inverse measure of diversity within each isolate compared to overall local genetic diversity. The latter indices were compared with the microsatellite results, and also with indices derived by randomly sampling modest numbers of SNPs. As expected, the number of microsatellite loci with more than one allele in each isolate was highly significantly inversely correlated with the genome-wide F ws fixation index (r = -0.88, P < 0.001). However, the microsatellite analysis revealed that most isolates contained mixed genotypes, even those that had no detectable genome sequence heterogeneity. Random sampling of different numbers of SNPs showed that an F ws index derived from ten or more SNPs with minor allele frequencies of >10 % had high correlation (r > 0.90) with the index derived using all SNPs. Different types of data give highly correlated indices of within-infection diversity, although PCR-based analysis detects low-level minority genotypes not apparent in bulk sequence analysis. When whole-genome data are not obtainable, quantitative assay of ten or more SNPs can yield a reasonably accurate estimate of the within-infection fixation index (F ws).

Increased pfmdr1 gene copy number and the decline in pfcrt and pfmdr1 resistance alleles in Ghanaian Plasmodium falciparum isolates after the change of anti-malarial drug treatment policy

PubMed Central

2013-01-01

Background With the introduction of artemisinin-based combination therapy (ACT) in 2005, monitoring of anti-malarial drug efficacy, which includes the use of molecular tools to detect known genetic markers of parasite resistance, is important for first-hand information on the changes in parasite susceptibility to drugs in Ghana. This study investigated the Plasmodium falciparum multidrug resistance gene (pfmdr1) copy number, mutations and the chloroquine resistance transporter gene (pfcrt) mutations in Ghanaian isolates collected in seven years to detect the trends in prevalence of mutations. Methods Archived filter paper blood blots collected from children aged below five years with uncomplicated malaria in 2003–2010 at sentinel sites were used. Using quantitative real-time polymerase chain reaction (qRT-PCR), 756 samples were assessed for pfmdr1 gene copy number. PCR and restriction fragment length polymorphism (RFLP) were used to detect alleles of pfmdr1 86 in 1,102 samples, pfmdr1 184, 1034, 1042 and 1246 in 832 samples and pfcrt 76 in 1,063 samples. Merozoite surface protein 2 (msp2) genotyping was done to select monoclonal infections for copy number analysis. Results The percentage of isolates with increased pfmdr1 copy number were 4, 27, 9, and 18% for 2003–04, 2005–06, 2007–08 and 2010, respectively. Significant increasing trends for prevalence of pfmdr1 N86 (×2 = 96.31, p <0.001) and pfcrt K76 (×2 = 64.50, p <0.001) and decreasing trends in pfmdr1 Y86 (×2 = 38.52, p <0.001) and pfcrt T76 (×2 = 43.49, p <0.001) were observed from 2003–2010. The pfmdr1 F184 and Y184 prevalence showed an increasing and decreasing trends respectively but were not significant (×2 = 7.39,p=0.060; ×2 = 7.49, p = 0.057 respectively). The pfmdr1 N86-F184-D1246 haplotype, which is alleged to be selected by artemether-lumefantrine showed a significant increasing trend (×2 = 20.75, p < 0.001). Conclusion Increased pfmdr1 gene copy number was observed in the isolates analysed and this finding has implications for the use of ACT in the country although no resistance has been reported. The decreasing trend in the prevalence of chloroquine resistance markers after change of treatment policy presents the possibility for future introduction of chloroquine as prophylaxis for malaria risk groups such as children and pregnant women in Ghana. PMID:24172030

Proteogenomic analysis of the total and surface-exposed proteomes of Plasmodium vivax salivary gland sporozoites

PubMed Central

Swearingen, Kristian E.; Lindner, Scott E.; Flannery, Erika L.; Vaughan, Ashley M.; Morrison, Robert D.; Patrapuvich, Rapatbhorn; Koepfli, Cristian; Muller, Ivo; Jex, Aaron; Moritz, Robert L.; Kappe, Stefan H. I.; Sattabongkot, Jetsumon

2017-01-01

Plasmodium falciparum and Plasmodium vivax cause the majority of human malaria cases. Research efforts predominantly focus on P. falciparum because of the clinical severity of infection and associated mortality rates. However, P. vivax malaria affects more people in a wider global range. Furthermore, unlike P. falciparum, P. vivax can persist in the liver as dormant hypnozoites that can be activated weeks to years after primary infection, causing relapse of symptomatic blood stages. This feature makes P. vivax unique and difficult to eliminate with the standard tools of vector control and treatment of symptomatic blood stage infection with antimalarial drugs. Infection by Plasmodium is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver. The most advanced malaria vaccine for P. falciparum (RTS,S, a subunit vaccine containing of a portion of the major sporozoite surface protein) conferred limited protection in Phase III trials, falling short of WHO-established vaccine efficacy goals. However, blocking the sporozoite stage of infection in P. vivax, before the establishment of the chronic liver infection, might be an effective malaria vaccine strategy to reduce the occurrence of relapsing blood stages. It is also thought that a multivalent vaccine comprising multiple sporozoite surface antigens will provide better protection, but a comprehensive analysis of proteins in P. vivax sporozoites is not available. To inform sporozoite-based vaccine development, we employed mass spectrometry-based proteomics to identify nearly 2,000 proteins present in P. vivax salivary gland sporozoites. Analysis of protein post-translational modifications revealed extensive phosphorylation of glideosome proteins as well as regulators of transcription and translation. Additionally, the sporozoite surface proteins CSP and TRAP, which were recently discovered to be glycosylated in P. falciparum salivary gland sporozoites, were also observed to be similarly modified in P. vivax sporozoites. Quantitative comparison of the P. vivax and P. falciparum salivary gland sporozoite proteomes revealed a high degree of similarity in protein expression levels, including among invasion-related proteins. Nevertheless, orthologs with significantly different expression levels between the two species could be identified, as well as highly abundant, species-specific proteins with no known orthologs. Finally, we employed chemical labeling of live sporozoites to isolate and identify 36 proteins that are putatively surface-exposed on P. vivax salivary gland sporozoites. In addition to identifying conserved sporozoite surface proteins identified by similar analyses of other Plasmodium species, our analysis identified several as-yet uncharacterized proteins, including a putative 6-Cys protein with no known ortholog in P. falciparum. PMID:28759593

Host genotype by parasite genotype interactions underlying the resistance of anopheline mosquitoes to Plasmodium falciparum.

PubMed

Lambrechts, Louis; Halbert, Jean; Durand, Patrick; Gouagna, Louis C; Koella, Jacob C

2005-01-11

Most studies on the resistance of mosquitoes to their malaria parasites focus on the response of a mosquito line or colony against a single parasite genotype. In natural situations, however, it may be expected that mosquito-malaria relationships are based, as are many other host-parasite systems, on host genotype by parasite genotype interactions. In such systems, certain hosts are resistant to one subset of the parasite's genotypes, while other hosts are resistant to a different subset. To test for genotype by genotype interactions between malaria parasites and their anopheline vectors, different genetic backgrounds (families consisting of the F1 offspring of individual females) of the major African vector Anopheles gambiae were challenged with several isolates of the human malaria parasite Plasmodium falciparum (obtained from naturally infected children in Kenya). Averaged across all parasites, the proportion of infected mosquitoes and the number of oocysts found in their midguts were similar in all mosquito families. Both indices of resistance, however, differed considerably among isolates of the parasite. In particular, no mosquito family was most resistant to all parasites, and no parasite isolate was most infectious to all mosquitoes. These results suggest that the level of mosquito resistance depends on the interaction between its own and the parasite's genotype. This finding thus emphasizes the need to take into account the range of genetic diversity exhibited by mosquito and malaria field populations in ideas and studies concerning the control of malaria.

Recombinant Scorpine Produced Using SUMO Fusion Partner in Escherichia coli Has the Activities against Clinically Isolated Bacteria and Inhibits the Plasmodium falciparum Parasitemia In Vitro

PubMed Central

Gu, Yaping; Zhou, Huayun; Cao, Jun; Gao, Qi

2014-01-01

Scorpine, a small cationic peptide from the venom of Pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. However, the isolation of scorpine from natural sources is inefficient and time-consuming. Here, we first report the expression and purification of recombinant scorpine in Escherichia coli, using small ubiquitin-related modifier (SUMO) fusion partner. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blotting analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni2+–NTA) resin chromatography. After the SUMO-scorpine fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni2+–NTA column. Tricine/SDS-PAGE gel results indicated that Scorpine had been purified successfully to more than 95% purity. The recombinantly expressed Scorpine showed anti-bacterial activity against two standard bacteria including Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606, and clinically isolated bacteria including S. aureus S, S. aureus R, A. baumannii S, and A. baumannii R. It also produced 100% reduction in Plasmodium falciparum parasitemia in vitro. Thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant Scorpine for pharmaceutical applications in the future. PMID:25068263

Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum.

PubMed

Niemand, J; Louw, A I; Birkholtz, L; Kirk, K

2012-09-01

Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular polyamine pool via salvage of exogenous polyamines from the host. However, the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study, the uptake of the polyamines, putrescine and spermidine, into Plasmodium falciparum parasites functionally isolated from their host erythrocyte was investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competition between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na(+) but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depolarization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energised by the parasite's inwardly negative membrane potential. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Antiplasmodial, cytotoxic activities and characterization of a new naturally occurring quinone methide pentacyclic triterpenoid derivative isolated from Salacia leptoclada Tul. (Celastraceae) originated from Madagascar

PubMed Central

Ruphin, Fatiany Pierre; Baholy, Robijaona; Emmanue, Andrianarivo; Amelie, Raharisololalao; Martin, Marie-Therese; Koto-te-Nyiwa, Ngbolua

2013-01-01

Objective To validate scientifically the traditional use of Salacia leptoclada Tul. (Celastraceae) (S. leptoclada) and to isolate and elucidate the structure of the biologically active compound. Methods Bioassay-guided fractionation of the acetonic extract of the stem barks of S. leptoclada was carried out by a combination of chromatography technique and biological experiments in viro using Plasmodium falciparum and P388 leukemia cell lines as models. The structure of the biologically active pure compound was elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. Results Biological screening of S. leptoclada extracts resulted in the isolation of a pentacyclic triterpenic quinone methide. The pure compound exhibited both in vitro a cytotoxic effect on murine P388 leukemia cells with IC50 value of (0.041±0.020) µg/mL and an antiplasmodial activity against the chloroquine-resistant strain FC29 of Plasmodium falciparum with an IC50 value of (0.052±0.030) µg/mL. Despite this interesting anti-malarial property of the lead compound, the therapeutic index was weak (0.788). In the best of our knowledge, the quinone methide pentacyclic triterpenoid derivative compound is reported for the first time in S. leptoclada. Conclusions The results suggest that furthers studies involving antineoplastic activity is needed for the development of this lead compound as anticancer drug. PMID:24075342

Antiplasmodial, cytotoxic activities and characterization of a new naturally occurring quinone methide pentacyclic triterpenoid derivative isolated from Salacia leptoclada Tul. (Celastraceae) originated from Madagascar.

PubMed

Ruphin, Fatiany Pierre; Baholy, Robijaona; Emmanue, Andrianarivo; Amelie, Raharisololalao; Martin, Marie-Therese; Koto-te-Nyiwa, Ngbolua

2013-10-01

To validate scientifically the traditional use of Salacia leptoclada Tul. (Celastraceae) (S. leptoclada) and to isolate and elucidate the structure of the biologically active compound. Bioassay-guided fractionation of the acetonic extract of the stem barks of S. leptoclada was carried out by a combination of chromatography technique and biological experiments in viro using Plasmodium falciparum and P388 leukemia cell lines as models. The structure of the biologically active pure compound was elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. Biological screening of S. leptoclada extracts resulted in the isolation of a pentacyclic triterpenic quinone methide. The pure compound exhibited both in vitro a cytotoxic effect on murine P388 leukemia cells with IC50 value of (0.041±0.020) μg/mL and an antiplasmodial activity against the chloroquine-resistant strain FC29 of Plasmodium falciparum with an IC50 value of (0.052±0.030) μg/mL. Despite this interesting anti-malarial property of the lead compound, the therapeutic index was weak (0.788). In the best of our knowledge, the quinone methide pentacyclic triterpenoid derivative compound is reported for the first time in S. leptoclada. The results suggest that furthers studies involving antineoplastic activity is needed for the development of this lead compound as anticancer drug. Copyright © 2013 Asian Pacific Tropical Biomedical Magazine. Published by Elsevier B.V. All rights reserved.

Antibodies to Polymorphic Invasion-Inhibitory and Non-Inhibitory Epitopes of Plasmodium falciparum Apical Membrane Antigen 1 in Human Malaria

PubMed Central

Mugyenyi, Cleopatra K.; Elliott, Salenna R.; McCallum, Fiona J.; Anders, Robin F.; Marsh, Kevin; Beeson, James G.

2013-01-01

Background Antibodies to P. falciparum apical membrane protein 1 (AMA1) may contribute to protective immunity against clinical malaria by inhibiting blood stage growth of P. falciparum, and AMA1 is a leading malaria vaccine candidate. Currently, there is limited knowledge of the acquisition of strain-specific and cross-reactive antibodies to AMA1 in humans, or the acquisition of invasion-inhibitory antibodies to AMA1. Methodology/Findings We examined the acquisition of human antibodies to specific polymorphic invasion-inhibitory and non-inhibitory AMA1 epitopes, defined by the monoclonal antibodies 1F9 and 2C5, respectively. Naturally acquired antibodies were measured in cohorts of Kenyan children and adults. Antibodies to the invasion-inhibitory 1F9 epitope and non-inhibitory 2C5 epitope were measured indirectly by competition ELISA. Antibodies to the 1F9 and 2C5 epitopes were acquired by children and correlated with exposure, and higher antibody levels and prevalence were observed with increasing age and with active P. falciparum infection. Of note, the prevalence of antibodies to the inhibitory 1F9 epitope was lower than antibodies to AMA1 or the 2C5 epitope. Antibodies to AMA1 ectodomain, the 1F9 or 2C5 epitopes, or a combination of responses, showed some association with protection from P. falciparum malaria in a prospective longitudinal study. Furthermore, antibodies to the invasion-inhibitory 1F9 epitope were positively correlated with parasite growth-inhibitory activity of serum antibodies. Conclusions/Significance Individuals acquire antibodies to functional, polymorphic epitopes of AMA1 that may contribute to protective immunity, and these findings have implications for AMA1 vaccine development. Measuring antibodies to the 1F9 epitope by competition ELISA may be a valuable approach to assessing human antibodies with invasion-inhibitory activity in studies of acquired immunity and vaccine trials of AMA1. PMID:23861883

Analysis of the interactome of the Ser/Thr Protein Phosphatase type 1 in Plasmodium falciparum.

PubMed

Hollin, Thomas; De Witte, Caroline; Lenne, Astrid; Pierrot, Christine; Khalife, Jamal

2016-03-17

Protein Phosphatase 1 (PP1) is an enzyme essential to cell viability in the malaria parasite Plasmodium falciparum (Pf). The activity of PP1 is regulated by the binding of regulatory subunits, of which there are up to 200 in humans, but only 3 have been so far reported for the parasite. To better understand the P. falciparum PP1 (PfPP1) regulatory network, we here report the use of three strategies to characterize the PfPP1 interactome: co-affinity purified proteins identified by mass spectrometry, yeast two-hybrid (Y2H) screening and in silico analysis of the P. falciparum predicted proteome. Co-affinity purification followed by MS analysis identified 6 PfPP1 interacting proteins (Pips) of which 3 contained the RVxF consensus binding, 2 with a Fxx[RK]x[RK] motif, also shown to be a PP1 binding motif and one with both binding motifs. The Y2H screens identified 134 proteins of which 30 present the RVxF binding motif and 20 have the Fxx[RK]x[RK] binding motif. The in silico screen of the Pf predicted proteome using a consensus RVxF motif as template revealed the presence of 55 potential Pips. As further demonstration, 35 candidate proteins were validated as PfPP1 interacting proteins in an ELISA-based assay. To the best of our knowledge, this is the first study on PfPP1 interactome. The data reports several conserved PP1 interacting proteins as well as a high number of specific interactors to PfPP1. Their analysis indicates a high diversity of biological functions for PP1 in Plasmodium. Based on the present data and on an earlier study of the Pf interactome, a potential implication of Pips in protein folding/proteolysis, transcription and pathogenicity networks is proposed. The present work provides a starting point for further studies on the structural basis of these interactions and their functions in P. falciparum.

The effect of dams and seasons on malaria incidence and anopheles abundance in Ethiopia

PubMed Central

2013-01-01

Background Reservoirs created by damming rivers are often believed to increase malaria incidence risk and/or stretch the period of malaria transmission. In this paper, we report the effects of a mega hydropower dam on P. falciparum malaria incidence in Ethiopia. Methods A longitudinal cohort study was conducted over a period of 2 years to determine Plasmodium falciparum malaria incidence among children less than 10 years of age living near a mega hydropower dam in Ethiopia. A total of 2080 children from 16 villages located at different distances from a hydropower dam were followed up from 2008 to 2010 using active detection of cases based on weekly house to house visits. Of this cohort of children, 951 (48.09%) were females and 1059 (51.91%) were males, with a median age of 5 years. Malaria vectors were simultaneously surveyed in all the 16 study villages. Frailty models were used to explore associations between time-to-malaria and potential risk factors, whereas, mixed-effects Poisson regression models were used to assess the effect of different covariates on anopheline abundance. Results Overall, 548 (26.86%) children experienced at least one clinical malaria episode during the follow up period with mean incidence rate of 14.26 cases/1000 child-months at risk (95% CI: 12.16 - 16.36). P. falciparum malaria incidence showed no statistically significant association with distance from the dam reservoir (p = 0.32). However, P. falciparum incidence varied significantly between seasons (p < 0.01). The malaria vector, Anopheles arabiensis, was however more abundant in villages nearer to the dam reservoir. Conclusions P. falciparum malaria incidence dynamics were more influenced by seasonal drivers than by the dam reservoir itself. The findings could have implications in timing optimal malaria control interventions and in developing an early warning system in Ethiopia. PMID:23566411

Re-evaluation of how artemisinins work in light of emerging evidence of in vitro resistance

PubMed Central

Krishna, Sanjeev; Woodrow, Charles J.; Staines, Henry M.; Haynes, Richard K.; Mercereau-Puijalon, Odile

2006-01-01

There are more than half a billion cases of malaria every year. Combinations of an artemisinin with other antimalarial drugs are now recommended treatments for Plasmodium falciparum malaria in most endemic areas. These treatment regimens act rapidly to relieve symptoms and effect cure. There is considerable controversy on how artemisinins work and over emerging indications of resistance to this class of antimalarial drugs. Several individual molecules have been proposed as targets for artemisinins, in addition to the idea that artemisinins might have many targets at the same time. Our suggestion that artemisinins inhibit the parasite-encoded sarco–endoplasmic reticulum Ca2+-ATPase (SERCA) PfATP6 has gained support from recent observations that a polymorphism in the gene encoding PfATP6 is associated with in vitro resistance to artemether in field isolates of P. falciparum. PMID:16616639

Increased frequency of chloroquine resistant P. falciparum on a rubber estate in Peninsular Malaysia during two years of systematic chloroquine treatment.

PubMed

Dondero, T J; Parsons, R E; O'Holohan, D R

1975-12-01

Chloroquine pressure was applied over a 22 month period on a somewhat isolated, malarious rubber estate by examination of residents at 4-week intervals and treatment of parasitaemias with chloroquine. During this time the monthly attack rate for P. falciparum rose four-fold to an average of nearly 18% per month, while that of P. vivax remained relatively constant at about 8%. Eight in vivo chloroquine resistance studies, which allowed both detection of late recrudescing R-I resistance and estimation of the risk of reinfection, showed an apparent rise in the drug resistance rate, from 12% to 20% prior to the study to the range of 40-50%. Virtually all resistance encountered was R-I in nature. There was no convincing evidence of chloroquine resistance among 148 tested P. vivax infections.

Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax

PubMed Central

Hupalo, Daniel N; Luo, Zunping; Melnikov, Alexandre; Sutton, Patrick L; Rogov, Peter; Escalante, Ananias; Vallejo, Andrés F; Herrera, Sócrates; Arévalo-Herrera, Myriam; Fan, Qi; Wang, Ying; Cui, Liwang; Lucas, Carmen M; Durand, Salomon; Sanchez, Juan F; Baldeviano, G Christian; Lescano, Andres G; Laman, Moses; Barnadas, Celine; Barry, Alyssa; Mueller, Ivo; Kazura, James W; Eapen, Alex; Kanagaraj, Deena; Valecha, Neena; Ferreira, Marcelo U; Roobsoong, Wanlapa; Nguitragool, Wang; Sattabonkot, Jetsumon; Gamboa, Dionicia; Kosek, Margaret; Vinetz, Joseph M; González-Cerón, Lilia; Birren, Bruce W; Neafsey, Daniel E; Carlton, Jane M

2017-01-01

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally. PMID:27348298

Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax.

PubMed

Hupalo, Daniel N; Luo, Zunping; Melnikov, Alexandre; Sutton, Patrick L; Rogov, Peter; Escalante, Ananias; Vallejo, Andrés F; Herrera, Sócrates; Arévalo-Herrera, Myriam; Fan, Qi; Wang, Ying; Cui, Liwang; Lucas, Carmen M; Durand, Salomon; Sanchez, Juan F; Baldeviano, G Christian; Lescano, Andres G; Laman, Moses; Barnadas, Celine; Barry, Alyssa; Mueller, Ivo; Kazura, James W; Eapen, Alex; Kanagaraj, Deena; Valecha, Neena; Ferreira, Marcelo U; Roobsoong, Wanlapa; Nguitragool, Wang; Sattabonkot, Jetsumon; Gamboa, Dionicia; Kosek, Margaret; Vinetz, Joseph M; González-Cerón, Lilia; Birren, Bruce W; Neafsey, Daniel E; Carlton, Jane M

2016-08-01

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.

Synthesis of 3-(1,2,3-triazol-1-yl)- and 3-(1,2,3-triazol-4-yl)-substituted pyrazolo[3,4-d]pyrimidin-4-amines via click chemistry: potential inhibitors of the Plasmodium falciparum PfPK7 protein kinase.

PubMed

Klein, Michael; Dinér, Peter; Dorin-Semblat, Dominique; Doerig, Christian; Grøtli, Morten

2009-09-07

Efficient routes to 3-(1,2,3-triazol-1-yl)- and 3-(1,2,3-triazol-4-yl)pyrazolo[3,4-d]pyrimidin-4-amines using a one-pot two-step reaction are presented. The two routes give easy access to two different isomers of 1,4-disubstituted triazoles and the target compounds are obtained from a variety of readily available aromatic and aliphatic halides without isolation of potentially unstable organic azide intermediates. Two compounds show activity towards the PfPK7 kinase (IC(50) 10-20 microM) of P. falciparum, the organism responsible for the most virulent form of malaria, and can be regarded as hits useful for further development into lead compounds.

The traditional medicine Spilanthes acmella, and the alkylamides spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide, demonstrate in vitro and in vivo anti-malarial activity

PubMed Central

Spelman, Kevin; Depoix, Delphine; McCray, Megan; Mouray, Elisabeth; Grellier, Philippe

2010-01-01

Spilanthes spp. are used as traditional herbal medicines in Africa and India to treat malaria. Yet, to date, there is no data on active constituents or most effective extraction methods for this indication. The isolated alkylamides, spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide, found in S. acmella Murr., were shown to have IC50s of 16.5 μg/mL and 41.4 μg/mL on Plasmodium falciparum strain PFB and IC50s of 5.8 μg/mL and 16.3 μg/mL for the chloroquine resistant P. falciparum K1 strain, respectively. Further investigations revealed that at relatively low concentrations, spilanthol and the water extract of S. acmella reduced the parasitemia 59% and 53% in mice infected with P. yoelii yoelii 17XNL at 5 mg/kg and 50 mg/kg, respectively. Unexpectedly, the 95% ethanol extract of S. acmella was less effective (36% reduction in parasitemia) at 50 mg/kg. These results provide the first evidence supporting S. acmella against malaria and demonstrating active constituents in S. acmella against P. falciparum. PMID:22692989

Effect of naturally occurring Wolbachia in Anopheles gambiae s.l. mosquitoes from Mali on Plasmodium falciparum malaria transmission

PubMed Central

Gomes, Fabio M.; Hixson, Bretta L.; Tyner, Miles D. W.; Ramirez, Jose Luis; Canepa, Gaspar E.; Alves e Silva, Thiago Luiz; Molina-Cruz, Alvaro; Keita, Moussa; Kane, Fouseyni; Traoré, Boïssé; Sogoba, Nafomon; Barillas-Mury, Carolina

2017-01-01

A naturally occurring Wolbachia strain (wAnga-Mali) was identified in mosquitoes of the Anopheles gambiae complex collected in the Malian villages of Dangassa and Kenieroba. Phylogenetic analysis of the nucleotide sequence of two 16S rRNA regions showed that wAnga-Mali clusters with Wolbachia strains from supergroup A and has the highest homology to a Wolbachia strain isolated from cat fleas (Ctenocephalides). wAnga-Mali is different from two Wolbachia strains previously reported in A. gambiae from Burkina Faso (wAnga_VK5_STP and wAnga_VK5_3.1a). Quantitative analysis of Wolbachia and Plasmodium sporozoite infection in field-collected mosquitoes indicates that the prevalence and intensity of Plasmodium falciparum sporozoite infection is significantly lower in Wolbachia-infected females. The presence of Wolbachia in females from a laboratory Anopheles coluzzii (A. gambiae, M form) colony experimentally infected with P. falciparum (NF54 strain) gametocyte cultures slightly enhanced oocyst infection. However, Wolbachia infection significantly reduced the prevalence and intensity of sporozoite infection, as observed in the field. This indicates that wAnga-Mali infection does not limit early stages of Plasmodium infection in the mosquito, but it has a strong deleterious effect on sporozoites and reduces malaria transmission. PMID:29114059

Plasmodium glyceraldehyde-3-phosphate dehydrogenase: A potential malaria diagnostic target.

PubMed

Krause, Robert G E; Hurdayal, Ramona; Choveaux, David; Przyborski, Jude M; Coetzer, Theresa H T; Goldring, J P Dean

2017-08-01

Malaria rapid diagnostic tests (RDTs) are immunochromatographic tests detecting Plasmodial histidine-rich protein 2 (HRP2), lactate dehydrogenase (LDH) and aldolase. HRP2 is only expressed by Plasmodium falciparum parasites and the protein is not expressed in several geographic isolates. LDH-based tests lack sensitivity compared to HRP2 tests. This study explored the potential of the Plasmodial glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a new malaria diagnostic biomarker. The P. falciparum and P. yoelii proteins were recombinantly expressed in BL21(DE3) Escherischia coli host cells and affinity purified. Two epitopes (CADGFLLIGEKKVSVFA and CAEKDPSQIPWGKCQV) specific to P. falciparum GAPDH and one common to all mammalian malaria species (CKDDTPIYVMGINH) were identified. Antibodies were raised in chickens against the two recombinant proteins and the three epitopes and affinity purified. The antibodies detected the native protein in parasite lysates as a 38 kDa protein and immunofluorescence verified a parasite cytosolic localization for the native protein. The antibodies suggested a 4-6 fold higher concentration of native PfGAPDH compared to PfLDH in immunoprecipitation and ELISA formats, consistent with published proteomic data. PfGAPDH shows interesting potential as a malaria diagnostic biomarker. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic diversity and population structure of Plasmodium falciparum over space and time in an African archipelago.

PubMed

Salgueiro, Patrícia; Vicente, José Luís; Figueiredo, Rita Carrilho; Pinto, João

2016-09-01

The archipelago of São Tomé and Principe (STP), West Africa, has suffered the heavy burden of malaria since the 16th century. Until the last decade, when after a successful control program STP has become a low transmission country and one of the few nations with decreases of more than 90% in malaria admission and death rates. We carried out a longitudinal study to determine the genetic structure of STP parasite populations over time and space. Twelve microsatellite loci were genotyped in Plasmodium falciparum samples from two islands collected in 1997, 2000 and 2004. Analysis was performed on proportions of mixed genotype infections, allelic diversity, population differentiation, effective population size and bottleneck effects. We have found high levels of genetic diversity and minimal inter-population genetic differentiation typical of African continental regions with intense and stable malaria transmission. We detected significant differences between the years, with special emphasis for 1997 that showed the highest proportion of samples infected with P. falciparum and the highest mean number of haplotypes per isolate. This study establishes a comprehensive genetic data baseline of a pre-intervention scenario for future studies; taking into account the most recent and successful control intervention on the territory. Copyright © 2016 Elsevier B.V. All rights reserved.

Na+ Influx Induced by New Antimalarials Causes Rapid Alterations in the Cholesterol Content and Morphology of Plasmodium falciparum

PubMed Central

Das, Sudipta; Bhatanagar, Suyash; Morrisey, Joanne M.; Daly, Thomas M.; Burns, James M.; Coppens, Isabelle; Vaidya, Akhil B.

2016-01-01

Among the several new antimalarials discovered over the past decade are at least three clinical candidate drugs, each with a distinct chemical structure, that disrupt Na+ homeostasis resulting in a rapid increase in intracellular Na+ concentration ([Na+]i) within the erythrocytic stages of Plasmodium falciparum. At present, events triggered by Na+ influx that result in parasite demise are not well-understood. Here we report effects of two such drugs, a pyrazoleamide and a spiroindolone, on intraerythrocytic P. falciparum. Within minutes following the exposure to these drugs, the trophozoite stage parasite, which normally contains little cholesterol, was made permeant by cholesterol-dependent detergents, suggesting it acquired a substantial amount of the lipid. Consistently, the merozoite surface protein 1 and 2 (MSP1 and MSP2), glycosylphosphotidylinositol (GPI)-anchored proteins normally uniformly distributed in the parasite plasma membrane, coalesced into clusters. These alterations were not observed following drug treatment of P. falciparum parasites adapted to grow in a low [Na+] growth medium. Both cholesterol acquisition and MSP1 coalescence were reversible upon the removal of the drugs, implicating an active process of cholesterol exclusion from trophozoites that we hypothesize is inhibited by high [Na+]i. Electron microscopy of drug-treated trophozoites revealed substantial morphological changes normally seen at the later schizont stage including the appearance of partial inner membrane complexes, dense organelles that resemble “rhoptries” and apparent nuclear division. Together these results suggest that [Na+]i disruptor drugs by altering levels of cholesterol in the parasite, dysregulate trophozoite to schizont development and cause parasite demise. PMID:27227970

South American Plasmodium falciparum after the Malaria Eradication Era: Clonal Population Expansion and Survival of the Fittest Hybrids

PubMed Central

Griffing, Sean M.; Mixson-Hayden, Tonya; Sridaran, Sankar; Alam, Md Tauqeer; McCollum, Andrea M.; Cabezas, César; Marquiño Quezada, Wilmer; Barnwell, John W.; Macedo De Oliveira, Alexandre; Lucas, Carmen; Arrospide, Nancy; Escalante, Ananias A.; Bacon, David J.; Udhayakumar, Venkatachalam

2011-01-01

Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradication's successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999–2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006–2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase. PMID:21949680

Absence of association between polymorphisms in the K13 gene and the presence of Plasmodium falciparum parasites at day 3 after treatment with artemisinin derivatives in Senegal.

PubMed

Madamet, Marylin; Kounta, Mame Bou; Wade, Khalifa Ababacar; Lo, Gora; Diawara, Silman; Fall, Mansour; Bercion, Raymond; Nakoulima, Aminata; Fall, Khadidiatou Ba; Benoit, Nicolas; Gueye, Mamadou Wague; Fall, Bécaye; Diatta, Bakary; Pradines, Bruno

2017-06-01

In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy as first-line treatment for uncomplicated malaria. In addition, intravenous (i.v.) injection of artesunate and artemether has gradually replaced quinine for the treatment of severe malaria. Mutations in the propeller domain of the Kelch 13 gene (K13-propeller, PF3D71343700), such as Y493H, R539T, I543T and C580Y, were recently associated with in vivo and in vitro resistance to artemisinin in Southeast Asia. However, these mutations were not identified in Africa. In total, 181 isolates of Plasmodium falciparum from 161 patients from Dakar, Senegal, were collected between August 2015 and January 2016. The K13-propeller gene of the isolates was sequenced. A search for non-synonymous mutations in the propeller region of K13 was performed in the 181 isolates collected from Dakar from 2015 to 2016. Three synonymous mutations were detected (D464D, C469C and R471R). Of 119 patients treated with i.v. artesunate or intramuscular artemether followed by artemether/lumefantrine, 9 patients were still parasitaemic on Day 3. Parasites from these nine patients were wild-type for K13-propeller. None of the polymorphisms known to be involved in artemisinin resistance in Asia were detected. These results suggest that K13 is not the best predictive marker for artemisinin resistance in Africa. More isolates from clinical failure cases or patients with delayed parasite clearance after treatment with artemisinin derivatives are necessary to identify new molecular markers. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Patterns of mixed Plasmodium species infections among children six years and under in selected malaria hyper-endemic communities of Zambia: population-based survey observations.

PubMed

Sitali, Lungowe; Chipeta, James; Miller, John M; Moonga, Hawela B; Kumar, Nirbhay; Moss, William J; Michelo, Charles

2015-05-02

Although malaria is preventable and treatable, it still claims 660,000 lives every year globally with children under five years of age having the highest burden. In Zambia, malaria rapid diagnostic tests (RDTs) that only detect Plasmodium falciparum are the main confirmatory means for malaria diagnosis in most health facilities without microscopy services. As a consequence of this P. falciparum species diagnostic approach, non-falciparum malaria is not only under-diagnosed but entirely missed, thereby making the exact disease burden unknown. We thus investigated the prevalence of various Plasmodium spp. and associated burden of infection in selected communities in Zambia. Data from two malaria hyper-endemic provinces (Eastern and Luapula) of the 2012 National Malaria Indicator Survey (MIS), conducted between April and May 2012, were used. The MIS is a nationally representative, two-stage cluster survey conducted to coincide with the end of the malaria transmission season. Social, behavioural and background information were collected from households as part of the survey. Thick blood smears, RDTs and dried blood spots (DBS) were collected from children below six years of age. Slides were stained using Giemsa and examined by microscopy while polymerase chain reaction (PCR) was used to analyse the DBS for malaria Plasmodium spp. Multivariate logistic regression was employed to examine the association between background factors and malaria. Overall, 873 children younger than six years of age were surveyed. The overall prevalence of Plasmodium spp. by PCR was 54.3% (95% CI 51-57.6%). Of the total Plasmodium isolates, 88% were P. falciparum, 10.6% were mixed infections and 1.4% were non-falciparum mono infections. Among the mixed infections, the majority were a combination of P. falciparum and P. malariae (6.5% of all mixed infections). Children two years and older (2-5 years) had three-fold higher risk of mixed malaria infections (aOR 2.8 CI 1.31-5.69) than children younger than two years of age. The high prevalence of mixed Plasmodium spp. infections in this population stresses review of the current malaria RDT diagnostic approaches. The observed less incidence of mixed infections in children under two years of age compared to their older two-to-five-year-old counterparts is probably due to the protective maternal passive immunity, among other factors, in that age group.

Genetic Diversity of Plasmodium falciparum Populations in Malaria Declining Areas of Sabah, East Malaysia

PubMed Central

Mohd Abd Razak, Mohd Ridzuan; Sastu, Umi Rubiah; Norahmad, Nor Azrina; Abdul-Karim, Abass; Muhammad, Amirrudin; Muniandy, Prem Kumar; Jelip, Jenarun; Rundi, Christina; Imwong, Mallika; Mudin, Rose Nani; Abdullah, Noor Rain

2016-01-01

Malaysia has a national goal to eliminate malaria by 2020. Understanding the genetic diversity of malaria parasites in residual transmission foci can provide invaluable information which may inform the intervention strategies used to reach elimination targets. This study was conducted to determine the genetic diversity level of P. falciparum isolates in malaria residual foci areas of Sabah. Malaria active case detection was conducted in Kalabakan and Kota Marudu. All individuals in the study sites were screened for malaria infection by rapid diagnostic test. Blood from P. falciparum-infected individuals were collected on filter paper prior to DNA extraction. Genotyping was performed using merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2), glutamate rich protein (GLURP) and 10 neutral microsatellite loci markers. The size of alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (He), linkage disequilibrium (LD) and genetic differentiation (FST) were determined. In Kalabakan, the MSP-1 and MSP-2 alleles were predominantly K1 and FC27 family types, respectively. The GLURP genotype VI (751–800 bp) was predominant. The MOI for MSP-1 and MSP-2 were 1.65 and 1.20, respectively. The Na per microsatellite locus was 1.70. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.17, 0.37, 0.70 and 0.33, respectively. In Kota Marudu, the MSP-1 and MSP-2 alleles were predominantly MAD20 and 3D7 family types, respectively. The GLURP genotype IV (651–700 bp) was predominant. The MOI for both MSP-1 and MSP-2 was 1.05. The Na per microsatellite locus was 3.60. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.24, 0.25, 0.69 and 0.30, respectively. A significant LD was observed in Kalabakan (0.495, p<0.01) and Kota Marudu P. falciparum populations (0.601, p<0.01). High genetic differentiation between Kalabakan and Kota Marudu P. falciparum populations was observed (FST = 0.532). The genetic data from the present study highlighted the limited diversity and contrasting genetic pattern of P. falciparum populations in the malaria declining areas of Sabah. PMID:27023787

Dihydroartemisinin–piperaquine resistance in Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study

PubMed Central

Amaratunga, Chanaki; Lim, Pharath; Suon, Seila; Sreng, Sokunthea; Mao, Sivanna; Sopha, Chantha; Sam, Baramey; Dek, Dalin; Try, Vorleak; Amato, Roberto; Blessborn, Daniel; Song, Lijiang; Tullo, Gregory S; Fay, Michael P; Anderson, Jennifer M; Tarning, Joel; Fairhurst, Rick M

2016-01-01

Background Artemisinin resistance in Plasmodium falciparum threatens to reduce the efficacy of artemisinin combination therapies (ACTs), thus compromising global efforts to eliminate malaria. Recent treatment failures with dihydroartemisinin-piperaquine, the current first-line ACT in Cambodia, suggest that piperaquine resistance may be emerging in this country. We explored the relation between artemisinin resistance and dihydroartemisinin–piperaquine failures, and sought to confirm the presence of piperaquine-resistant P falciparum infections in Cambodia. Methods In this prospective cohort study, we enrolled patients aged 2–65 years with uncomplicated P falciparum malaria in three Cambodian provinces: Pursat, Preah Vihear, and Ratanakiri. Participants were given standard 3-day courses of dihydroartemisinin–piperaquine. Peripheral blood parasite densities were measured until parasites cleared and then weekly to 63 days. The primary outcome was recrudescent P falciparum parasitaemia within 63 days. We measured piperaquine plasma concentrations at baseline, 7 days, and day of recrudescence. We assessed phenotypic and genotypic markers of drug resistance in parasite isolates. The study is registered with ClinicalTrials.gov, number NCT01736319. Findings Between Sept 4, 2012, and Dec 31, 2013, we enrolled 241 participants. In Pursat, where artemisinin resistance is entrenched, 37 (46%) of 81 patients had parasite recrudescence. In Preah Vihear, where artemisinin resistance is emerging, ten (16%) of 63 patients had recrudescence and in Ratanakiri, where artemisinin resistance is rare, one (2%) of 60 patients did. Patients with recrudescent P falciparum infections were more likely to have detectable piperaquine plasma concentrations at baseline compared with non-recrudescent patients, but did not differ significantly in age, initial parasite density, or piperaquine plasma concentrations at 7 days. Recrudescent parasites had a higher prevalence of kelch13 mutations, higher piperaquine 50% inhibitory concentration (IC50) values, and lower mefloquine IC50 values; none had multiple pfmdr1 copies, a genetic marker of mefloquine resistance. Interpretation Dihydroartemisinin–piperaquine failures are caused by both artemisinin and piperaquine resistance, and commonly occur in places where dihydroartemisinin–piperaquine has been used in the private sector. In Cambodia, artesunate plus mefloquine may be a viable option to treat dihydroartemisinin–piperaquine failures, and a more effective first-line ACT in areas where dihydroartemisinin–piperaquine failures are common. The use of single low-dose primaquine to eliminate circulating gametocytes is needed in areas where artemisinin and ACT resistance is prevalent. Funding National Institute of Allergy and Infectious Diseases. PMID:26774243

Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia.

PubMed

Lim, Pharath; Alker, Alisa P; Khim, Nimol; Shah, Naman K; Incardona, Sandra; Doung, Socheat; Yi, Poravuth; Bouth, Denis Mey; Bouchier, Christiane; Puijalon, Odile Mercereau; Meshnick, Steven R; Wongsrichanalai, Chansuda; Fandeur, Thierry; Le Bras, Jacques; Ringwald, Pascal; Ariey, Frédéric

2009-01-12

The combination of artesunate and mefloquine was introduced as the national first-line treatment for Plasmodium falciparum malaria in Cambodia in 2000. However, recent clinical trials performed at the Thai-Cambodian border have pointed to the declining efficacy of both artesunate-mefloquine and artemether-lumefantrine. Since pfmdr1 modulates susceptibility to mefloquine and artemisinin derivatives, the aim of this study was to assess the link between pfmdr1 copy number, in vitro susceptibility to individual drugs and treatment failure to combination therapy. Blood samples were collected from P. falciparum-infected patients enrolled in two in vivo efficacy studies in north-western Cambodia: 135 patients were treated with artemether-lumefantrine (AL group) in Sampovloun in 2002 and 2003, and 140 patients with artesunate-mefloquine (AM group) in Sampovloun and Veal Veng in 2003 and 2004. At enrollment, the in vitro IC50 was tested and the strains were genotyped for pfmdr1 copy number by real-time PCR. The pfmdr1 copy number was analysed for 115 isolates in the AM group, and for 109 isolates in the AL group. Parasites with increased pfmdr1 copy number had significantly reduced in vitro susceptibility to mefloquine, lumefantrine and artesunate. There was no association between pfmdr1 polymorphisms and in vitro susceptibilities. In the patients treated with AM, the mean pfmdr1copy number was lower in subjects with adequate clinical and parasitological response compared to those who experienced late treatment failure (n = 112, p < 0.001). This was not observed in the patients treated with AL (n = 96, p = 0.364). The presence of three or more copies of pfmdr1 were associated with recrudescence in artesunate-mefloquine treated patients (hazard ratio (HR) = 7.80 [95%CI: 2.09-29.10], N = 115), p = 0.002) but not with recrudescence in artemether-lumefantrine treated patients (HR = 1.03 [95%CI: 0.24-4.44], N = 109, p = 0.969). This study shows that pfmdr1 copy number is a molecular marker of AM treatment failure in falciparum malaria on the Thai-Cambodian border. However, while it is associated with increased IC50 for lumefantrine, pfmdr1 copy number is not associated with AL treatment failure in the area, suggesting involvement of other molecular mechanisms in AL treatment failures in Cambodia.

A high-content phenotypic screen reveals the disruptive potency of quinacrine and 3',4'-dichlorobenzamil on the digestive vacuole of Plasmodium falciparum.

PubMed

Lee, Yan Quan; Goh, Amanda S P; Ch'ng, Jun Hong; Nosten, François H; Preiser, Peter Rainer; Pervaiz, Shazib; Yadav, Sanjiv Kumar; Tan, Kevin S W

2014-01-01

Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca(2+). This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3',4'-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.

Synthesis of antimalarial amide analogues based on the plant serrulatane diterpenoid 3,7,8-trihydroxyserrulat-14-en-19-oic acid.

PubMed

Kumar, Rohitesh; Duffy, Sandra; Avery, Vicky M; Davis, Rohan A

2017-09-01

A plant-derived natural product scaffold, 3,7,8-trihydroxyserrulat-14-en-19-oic acid (1) was isolated in high yield from the aerial parts of the endemic Australian desert plant Eremophila microtheca. This scaffold (1) was subsequently used in the generation of a series of new amide analogues via a one-pot mixed anhydride amidation using pivaloyl chloride. The structures of all analogues were characterized using MS, NMR, and UV data. The major serrulatane natural products (1-3), isolated from the plant extract, and all amide analogues (6-15) together with several pivaloylated derivatives of 3,7,8-trihydroxyserrulat-14-en-19-oic acid (16-18) were evaluated for their antimalarial activity against 3D7 (chloroquine sensitive) and Dd2 (chloroquine resistant) Plasmodium falciparum strains, and preliminary cytotoxicity data were also acquired using the human embryonic kidney cell line HEK293. The natural product scaffold (1) did not display any antimalarial activity at 10µM. Replacing the carboxylic acid of 1 with various amides resulted in moderate activity against the P. falciparum 3D7 strain with IC 50 values ranging from 1.25 to 5.65µM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Methylene blue inhibits the asexual development of vivax malaria parasites from a region of increasing chloroquine resistance

PubMed Central

Suwanarusk, Rossarin; Russell, Bruce; Ong, Alice; Sriprawat, Kanlaya; Chu, Cindy S.; PyaePhyo, Aung; Malleret, Benoit; Nosten, François; Renia, Laurent

2015-01-01

Objectives Methylene blue, once discarded due to its unsettling yet mild side effects, has now found a renewed place in the pharmacopoeia of modern medicine. The continued spread of drug-resistant Plasmodium vivax and Plasmodium falciparum has also led to a recent re-examination of methylene blue's potent antimalarial properties. Here we examine the ex vivo susceptibility profile of Plasmodium spp. isolates to methylene blue; the isolates were from a region on the Thai–Myanmar border where there are increasing rates of failure when treating vivax malaria with chloroquine. Methods To do this we used a newly developed ex vivo susceptibility assay utilizing flow cytometry and a portable flow cytometer with a near-UV laser. Results P. vivax (median methylene blue IC50 3.1 nM, IQR 1.7–4.3 nM) and P. falciparum (median methylene blue IC50 1.8 nM, IQR 1.6–2.3 nM) are susceptible to methylene blue treatment at physiologically relevant levels. Unfortunately, the addition of chloroquine to combination treatments with methylene blue significantly reduces the ex vivo effectiveness of this molecule. Conclusions Our data support further efforts to employ methylene blue as a safe, low-cost antimalarial to treat drug-resistant malaria. PMID:25150147

Natural selection of K13 mutants of Plasmodium falciparum in response to artemisinin combination therapies in Thailand.

PubMed

Putaporntip, C; Kuamsab, N; Kosuwin, R; Tantiwattanasub, W; Vejakama, P; Sueblinvong, T; Seethamchai, S; Jongwutiwes, S; Hughes, A L

2016-03-01

Resistance of Plasmodium falciparum to artemisinin combination therapy (ACT) in Southeast Asia can have a devastating impact on chemotherapy and control measures. In this study, the evolution of artemisinin-resistant P. falciparum in Thailand was assessed by exploring mutations in the K13 locus believed to confer drug resistance phenotype. P. falciparum-infected blood samples were obtained from patients in eight provinces of Thailand over two decades (1991-2014; n = 904). Analysis of the K13 gene was performed by either sequencing the complete coding region (n = 259) or mutation-specific PCR-restriction fragment length polymorphism method (n = 645). K13 mutations related to artesunate resistance were detected in isolates from Trat province bordering Cambodia in 1991, about 4 years preceding widespread deployment of ACT in Thailand and increased in frequency over time. Nonsynonymous nucleotide diversity exceeded synonymous nucleotide diversity in the propeller region of the K13 gene, supporting the hypothesis that this diversity was driven by natural selection. No single mutant appeared to be favoured in every population, and propeller-region mutants were rarely observed in linkage with each other in the same haplotype. On the other hand, there was a highly significant association between the occurrence of a propeller mutant and the insertion of two or three asparagines after residue 139 of K13. Whether this insertion plays a compensatory role for deleterious effects of propeller mutants on the function of the K13 protein requires further investigation. However, modification of duration of ACT from 2-day to 3-day regimens in 2008 throughout the country does not halt the increase in frequency of mutants conferring artemisinin resistance phenotype. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.

Effects of larvicidal and larval nutritional stresses on Anopheles gambiae development, survival and competence for Plasmodium falciparum.

PubMed

Vantaux, Amélie; Ouattarra, Issiaka; Lefèvre, Thierry; Dabiré, Kounbobr Roch

2016-04-23

Many studies have shown that the environment in which larvae develop can influence adult characteristics with consequences for the transmission of pathogens. We investigated how two environmental stresses (larviciding and nutritional stress) interact to affect Anopheles gambiae (previously An. gambiae S molecular form) life history traits and its susceptibility for field isolates of its natural malaria agent Plasmodium falciparum. Larvae were reared in the presence or not of a sub-lethal concentration of larvicide and under a high and low food regimen. Development time, individual size, adult survival and competence for P. falciparum were assessed. Individuals under low food regimen took more time to develop, had a lower development success and were smaller while there was no main effect of larvicide exposure on these traits. However, larvicide exposure impacted individual size in interaction with nutritional stress. Female survival was affected by the interaction between gametocytemia, parasite exposure and larval diet, as well as the interaction between gametocytemia, parasite exposure and larvicidal stress, and the interaction between gametocytemia, larvicidal exposure and larval diet. Among the 951 females dissected 7 days post-infection, 559 (58.78%) harboured parasites. Parasite prevalence was significantly affected by the interaction between larvicidal stress and larval diet. Indeed, females under low food regimen had a higher prevalence than females under high food regimen and this difference was greater under larvicidal stress. The two stresses did not impact parasite intensity. We found that larval nutritional and larvicidal stresses affect mosquito life history traits in complex ways, which could greatly affect P. falciparum transmission. Further studies combining field-based trials on larvicide use and mosquito experimental infections would give a more accurate understanding of the effects of this vector control tool on malaria transmission.

Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009-2013).

PubMed

Mohon, Abu Naser; Alam, Mohammad Shafiul; Bayih, Abebe Genetu; Folefoc, Asongna; Shahinas, Dea; Haque, Rashidul; Pillai, Dylan R

2014-11-18

Bangladesh is a malaria hypo-endemic country sharing borders with India and Myanmar. Artemisinin combination therapy (ACT) remains successful in Bangladesh. An increase of artemisinin-resistant malaria parasites on the Thai-Cambodia and Thai-Myanmar borders is worrisome. K13 propeller gene (PF3D7_1343700 or PF13_0238) mutations have been linked to both in vitro artemisinin resistance and in vivo slow parasite clearance rates. This group undertook to evaluate if mutations seen in Cambodia have emerged in Bangladesh where ACT use is now standard for a decade. Samples were obtained from Plasmodium falciparum-infected malaria patients from Upazila health complexes (UHC) between 2009 and 2013 in seven endemic districts of Bangladesh. These districts included Khagrachari (Matiranga UHC), Rangamati (Rajasthali UHC), Cox's Bazar (Ramu and Ukhia UHC), Bandarban (Lama UHC), Mymensingh (Haluaghat UHC), Netrokona (Durgapur and Kalmakanda UHC), and Moulvibazar (Sreemangal and Kamalganj UHC). Out of 296 microscopically positive P. falciparum samples, 271 (91.6%) were confirmed as mono-infections by both real-time PCR and nested PCR. The K13 propeller gene from 253 (93.4%) samples was sequenced bi-directionally. One non-synonymous mutation (A578S) was found in Bangladeshi clinical isolates. The A578S mutation was confirmed and lies adjacent to the C580Y mutation, the major mutation causing delayed parasite clearance in Cambodia. Based on computational modeling A578S should have a significant effect on tertiary structure of the protein. The data suggest that P. falciparum in Bangladesh remains free of the C580Y mutation linked to delayed parasite clearance. However, the mutation A578S is present and based on structural analysis could affect K13 gene function. Further in vivo clinical studies are required to validate the effect of this mutation.

Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain

PubMed Central

Gelabert, Pere; Sandoval-Velasco, Marcela; Olalde, Iñigo; Fregel, Rosa; Rieux, Adrien; Escosa, Raül; Aranda, Carles; Paaijmans, Krijn; Mueller, Ivo; Gilbert, M. Thomas P.; Lalueza-Fox, Carles

2016-01-01

Phylogenetic analysis of Plasmodium parasites has indicated that their modern-day distribution is a result of a series of human-mediated dispersals involving transport between Africa, Europe, America, and Asia. A major outstanding question is the phylogenetic affinity of the malaria causing parasites Plasmodium vivax and falciparum in historic southern Europe—where it was endemic until the mid-20th century, after which it was eradicated across the region. Resolving the identity of these parasites will be critical for answering several hypotheses on the malaria dispersal. Recently, a set of slides with blood stains of malaria-affected people from the Ebro Delta (Spain), dated between 1942 and 1944, have been found in a local medical collection. We extracted DNA from three slides, two of them stained with Giemsa (on which Plasmodium parasites could still be seen under the microscope) and another one consisting of dried blood spots. We generated the data using Illumina sequencing after using several strategies aimed at increasing the Plasmodium DNA yield: depletion of the human genomic (g)DNA content through hybridization with human gDNA baits, and capture-enrichment using gDNA derived from P. falciparum. Plasmodium mitochondrial genome sequences were subsequently reconstructed from the resulting data. Phylogenetic analysis of the eradicated European P. vivax mtDNA genome indicates that the European isolate is closely related to the most common present-day American haplotype and likely entered the American continent post-Columbian contact. Furthermore, the European P. falciparum mtDNA indicates a link with current Indian strains that is in agreement with historical accounts. PMID:27671660

Clinical trials of artesunate plus sulfadoxine-pyrimethamine for Plasmodium falciparum malaria in Afghanistan: maintained efficacy a decade after introduction.

PubMed

Awab, Ghulam Rahim; Imwong, Mallika; Pukrittayakamee, Sasithon; Alim, Fazel; Hanpithakpong, Warunee; Tarning, Joel; Dondorp, Arjen M; Day, Nicholas P J; White, Nicholas J; Woodrow, Charles J

2016-02-25

Combination therapy with artesunate plus sulfadoxine-pyrimethamine (SP) was adopted as recommended treatment for Plasmodium falciparum infection in Afghanistan in 2003. A series of prospective clinical studies examining the efficacy of artesunate plus sulfadoxine-pyrimethamine (AS + SP) against P. falciparum were undertaken in sentinel sites in Afghanistan from 2007 to 2014, accompanied by relevant molecular studies. The first study was a randomized trial of AS + SP versus dihydroartemisinin-piperaquine, while two subsequent studies were standard therapeutic efficacy studies of AS + SP. Three hundred and three patients were enrolled across four provinces in the north and east of the country. Curative efficacy was high in all the trials, with an adequate clinical and parasitological response (ACPR) of more than 95 % in all groups and trial stages. Genotyping for drug-resistance alleles at dhfr indicated fixation of the S108 N mutation and a prevalence of the C59R mutation of approximately 95 % across all sites. Other mutations in dhfr and dhps remained rare or absent entirely, although five isolates from the first trial carried the dhps triple mutant SGEGA haplotype. In the last study undertaken in 2012-2014 the K13 artemisinin resistance marker was examined; only two of 60 successfully sequenced samples carried a K13-propeller mutation. These data confirm maintained efficacy 10 years after introduction of artesunate plus SP as combination treatment of P. falciparum in Afghanistan. The molecular data indicate that despite a substantial fall in incidence, resistance has not developed to artemisinins, or intensified to the ACT partner drug components. Trial Registration http://www.clinicaltrials.gov/ct NCT00682578, NCT01115439 and NCT01707199.

Acquired Antibodies to Merozoite Antigens in Children from Uganda with Uncomplicated or Severe Plasmodium falciparum Malaria

PubMed Central

Ahmed Ismail, Hodan; Ribacke, Ulf; Reiling, Linda; Normark, Johan; Egwang, Tom; Kironde, Fred; Beeson, James G.; Wahlgren, Mats

2013-01-01

Malaria can present itself as an uncomplicated or severe disease. We have here studied the quantity and quality of antibody responses against merozoite antigens, as well as multiplicity of infection (MOI), in children from Uganda. We found higher levels of IgG antibodies toward erythrocyte-binding antigen EBA181, MSP2 of Plasmodium falciparum 3D7 and FC27 (MSP2-3D7/FC27), and apical membrane antigen 1 (AMA1) in patients with uncomplicated malaria by enzyme-linked immunosorbent assay (ELISA) but no differences against EBA140, EBA175, MSP1, and reticulocyte-binding protein homologues Rh2 and Rh4 or for IgM against MSP2-3D7/FC27.Patients with uncomplicated malaria were also shown to have higher antibody affinities for AMA1 by surface plasmon resonance (SPR). Decreased invasion of two clinical P. falciparum isolates in the presence of patient plasma correlated with lower initial parasitemia in the patients, in contrast to comparisons of parasitemia to ELISA values or antibody affinities, which did not show any correlations. Analysis of the heterogeneity of the infections revealed a higher MOI in patients with uncomplicated disease, with the P. falciparum K1 MSP1 (MSP1-K1) and MSP2-3D7 being the most discriminative allelic markers. Higher MOIs also correlated positively with higher antibody levels in several of the ELISAs. In conclusion, certain antibody responses and MOIs were associated with differences between uncomplicated and severe malaria. When different assays were combined, some antibodies, like those against AMA1, seemed particularly discriminative. However, only decreased invasion correlated with initial parasitemia in the patient, signaling the importance of functional assays in understanding development of immunity against malaria and in evaluating vaccine candidates. PMID:23740926

Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain.

PubMed

Gelabert, Pere; Sandoval-Velasco, Marcela; Olalde, Iñigo; Fregel, Rosa; Rieux, Adrien; Escosa, Raül; Aranda, Carles; Paaijmans, Krijn; Mueller, Ivo; Gilbert, M Thomas P; Lalueza-Fox, Carles

2016-10-11

Phylogenetic analysis of Plasmodium parasites has indicated that their modern-day distribution is a result of a series of human-mediated dispersals involving transport between Africa, Europe, America, and Asia. A major outstanding question is the phylogenetic affinity of the malaria causing parasites Plasmodium vivax and falciparum in historic southern Europe-where it was endemic until the mid-20th century, after which it was eradicated across the region. Resolving the identity of these parasites will be critical for answering several hypotheses on the malaria dispersal. Recently, a set of slides with blood stains of malaria-affected people from the Ebro Delta (Spain), dated between 1942 and 1944, have been found in a local medical collection. We extracted DNA from three slides, two of them stained with Giemsa (on which Plasmodium parasites could still be seen under the microscope) and another one consisting of dried blood spots. We generated the data using Illumina sequencing after using several strategies aimed at increasing the Plasmodium DNA yield: depletion of the human genomic (g)DNA content through hybridization with human gDNA baits, and capture-enrichment using gDNA derived from P. falciparum Plasmodium mitochondrial genome sequences were subsequently reconstructed from the resulting data. Phylogenetic analysis of the eradicated European P. vivax mtDNA genome indicates that the European isolate is closely related to the most common present-day American haplotype and likely entered the American continent post-Columbian contact. Furthermore, the European P. falciparum mtDNA indicates a link with current Indian strains that is in agreement with historical accounts.

Genome-wide profiling of chromosome interactions in Plasmodium falciparum characterizes nuclear architecture and reconfigurations associated with antigenic variation

PubMed Central

Lemieux, Jacob E; Kyes, Sue A; Otto, Thomas D; Feller, Avi I; Eastman, Richard T; Pinches, Robert A; Berriman, Matthew; Su, Xin-zhuan; Newbold, Chris I

2013-01-01

Spatial relationships within the eukaryotic nucleus are essential for proper nuclear function. In Plasmodium falciparum, the repositioning of chromosomes has been implicated in the regulation of the expression of genes responsible for antigenic variation, and the formation of a single, peri-nuclear nucleolus results in the clustering of rDNA. Nevertheless, the precise spatial relationships between chromosomes remain poorly understood, because, until recently, techniques with sufficient resolution have been lacking. Here we have used chromosome conformation capture and second-generation sequencing to study changes in chromosome folding and spatial positioning that occur during switches in var gene expression. We have generated maps of chromosomal spatial affinities within the P. falciparum nucleus at 25 Kb resolution, revealing a structured nucleolus, an absence of chromosome territories, and confirming previously identified clustering of heterochromatin foci. We show that switches in var gene expression do not appear to involve interaction with a distant enhancer, but do result in local changes at the active locus. These maps reveal the folding properties of malaria chromosomes, validate known physical associations, and characterize the global landscape of spatial interactions. Collectively, our data provide critical information for a better understanding of gene expression regulation and antigenic variation in malaria parasites. PMID:23980881

Plasmodium falciparum malaria in 1st-2nd century CE southern Italy.

PubMed

Marciniak, Stephanie; Prowse, Tracy L; Herring, D Ann; Klunk, Jennifer; Kuch, Melanie; Duggan, Ana T; Bondioli, Luca; Holmes, Edward C; Poinar, Hendrik N

2016-12-05

The historical record attests to the devastation malaria exacted on ancient civilizations, particularly the Roman Empire [1]. However, evidence for the presence of malaria during the Imperial period in Italy (1st-5th century CE) is based on indirect sources, such as historical, epigraphic, or skeletal evidence. Although these sources are crucial for revealing the context of this disease, they cannot establish the causative species of Plasmodium. Importantly, definitive evidence for the presence of malaria is now possible through the implementation of ancient DNA technology. As malaria is presumed to have been at its zenith during the Imperial period [1], we selected first or second molars from 58 adults from three cemeteries from this time: Isola Sacra (associated with Portus Romae, 1st-3rd century CE), Velia (1st-2nd century CE), and Vagnari (1st-4th century CE). We performed hybridization capture using baits designed from the mitochondrial (mtDNA) genomes of Plasmodium spp. on a prioritized subset of 11 adults (informed by metagenomic sequencing). The mtDNA sequences generated provided compelling phylogenetic evidence for the presence of P. falciparum in two individuals. This is the first genomic data directly implicating P. falciparum in Imperial period southern Italy in adults. Copyright © 2016 Elsevier Ltd. All rights reserved.

Apoptosis and dysfunction of blood dendritic cells in patients with falciparum and vivax malaria

PubMed Central

Woodberry, Tonia; Kienzle, Vivian; McPhun, Virginia; Minigo, Gabriela; Lampah, Daniel A.; Kenangalem, Enny; Engwerda, Christian; López, J. Alejandro; Anstey, Nicholas M.

2013-01-01

Malaria causes significant morbidity worldwide and a vaccine is urgently required. Plasmodium infection causes considerable immune dysregulation, and elicitation of vaccine immunity remains challenging. Given the central role of dendritic cells (DCs) in initiating immunity, understanding their biology during malaria will improve vaccination outcomes. Circulating DCs are particularly important, as they shape immune responses in vivo and reflect the functional status of other subpopulations. We performed cross-sectional and longitudinal assessments of the frequency, phenotype, and function of circulating DC in 67 Papuan adults during acute uncomplicated P. falciparum, P. vivax, and convalescent P. falciparum infections. We demonstrate that malaria patients display a significant reduction in circulating DC numbers and the concurrent accumulation of immature cells. Such alteration is associated with marked levels of spontaneous apoptosis and impairment in the ability of DC to mature, capture, and present antigens to T cells. Interestingly, sustained levels of plasma IL-10 were observed in patients with acute infection and were implicated in the induction of DC apoptosis. DC apoptosis was reversed upon IL-10 blockade, and DC function recovered when IL-10 levels returned to baseline by convalescence. Our data provide key information on the mechanisms behind DC suppression during malaria and will assist in developing strategies to better harness DC’s immunotherapeutic potential. PMID:23835848

Population structuring of multi-copy, antigen-encoding genes in Plasmodium falciparum

PubMed Central

Artzy-Randrup, Yael; Rorick, Mary M; Day, Karen; Chen, Donald; Dobson, Andrew P; Pascual, Mercedes

2012-01-01

The coexistence of multiple independently circulating strains in pathogen populations that undergo sexual recombination is a central question of epidemiology with profound implications for control. An agent-based model is developed that extends earlier ‘strain theory’ by addressing the var gene family of Plasmodium falciparum. The model explicitly considers the extensive diversity of multi-copy genes that undergo antigenic variation via sequential, mutually exclusive expression. It tracks the dynamics of all unique var repertoires in a population of hosts, and shows that even under high levels of sexual recombination, strain competition mediated through cross-immunity structures the parasite population into a subset of coexisting dominant repertoires of var genes whose degree of antigenic overlap depends on transmission intensity. Empirical comparison of patterns of genetic variation at antigenic and neutral sites supports this role for immune selection in structuring parasite diversity. DOI: http://dx.doi.org/10.7554/eLife.00093.001 PMID:23251784

Household level spatio-temporal analysis of Plasmodium falciparum and Plasmodium vivax malaria in Ethiopia.

PubMed

Seyoum, Dinberu; Yewhalaw, Delenasaw; Duchateau, Luc; Brandt, Patrick; Rosas-Aguirre, Angel; Speybroeck, Niko

2017-04-20

The global decline of malaria burden and goals for elimination has led to an increased interest in the fine-scale epidemiology of malaria. Micro-geographic heterogeneity of malaria infection could have implications for designing targeted small-area interventions. Two-year longitudinal cohort study data were used to explore the spatial and spatio-temporal distribution of malaria episodes in 2040 children aged < 10 years in 16 villages near the Gilgel-Gibe hydropower dam in Southwest Ethiopia. All selected households (HHs) were geo-referenced, and children were followed up through weekly house-to-house visits for two consecutive years to identify febrile episodes of P. falciparum and P. vivax infections. After confirming the spatial dependence of malaria episodes with Ripley's K function, SatScan TM was used to identify purely spatial and space-time clusters (hotspots) of annual malaria incidence for 2 years follow-up: year 1 (July 2008-June 2009) and year 2 (July 2009-June 2010). In total, 685 P. falciparum episodes (in 492 HHs) and 385 P. vivax episodes (in 290 HHs) were identified, representing respectively incidence rates of 14.6 (95% CI: 13.4-15.6) and 8.2 (95% CI: 7.3-9.1) per 1000 child-months at risk. In year 1, the most likely (128 HHs with 63 episodes, RR = 2.1) and secondary (15 HHs with 12 episodes, RR = 5.31) clusters of P. vivax incidence were found respectively in southern and north-western villages; while in year 2, the most likely cluster was located only in north-western villages (85 HHs with 16 episodes, RR = 4.4). Instead, most likely spatial clusters of P. falciparum incidence were consistently located in villages south of the dam in both years: year 1 (167 HHs with 81 episodes, RR = 1.8) and year 2 (133 HHs with 67 episodes, RR = 2.2). Space-time clusters in southern villages for P. vivax were found in August-November 2008 in year 1 and between November 2009 and February 2010 in year 2; while for P. falciparum, they were found in September-November 2008 in year 1 and October-November 2009 in year 2. Hotspots of P. falciparum incidence in children were more stable at the geographical level and over time compared to those of P. vivax incidence during the study period.

Non-falciparum malaria infections in pregnant women in West Africa.

PubMed

Williams, John; Njie, Fanta; Cairns, Matthew; Bojang, Kalifa; Coulibaly, Sheick Oumar; Kayentao, Kassoum; Abubakar, Ismaela; Akor, Francis; Mohammed, Khalifa; Bationo, Richard; Dabira, Edgar; Soulama, Alamissa; Djimdé, Moussa; Guirou, Etienne; Awine, Timothy; Quaye, Stephen L; Ordi, Jaume; Doumbo, Ogobara; Hodgson, Abraham; Oduro, Abraham; Magnussen, Pascal; Ter Kuile, Feiko O; Woukeu, Arouna; Milligan, Paul; Tagbor, Harry; Greenwood, Brian; Chandramohan, Daniel

2016-01-29

Non-Plasmodium falciparum malaria infections are found in many parts of sub-Saharan Africa but little is known about their importance in pregnancy. Blood samples were collected at first antenatal clinic attendance from 2526 women enrolled in a trial of intermittent screening and treatment of malaria in pregnancy (ISTp) versus intermittent preventive treatment (IPTp) conducted in Burkina Faso, The Gambia, Ghana and Mali. DNA was extracted from blood spots and tested for P. falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale using a nested PCR test. Risk factors for a non-falciparum malaria infection were investigated and the influence of these infections on the outcome of pregnancy was determined. P. falciparum infection was detected frequently (overall prevalence by PCR: 38.8 %, [95 % CI 37.0, 40.8]), with a prevalence ranging from 10.8 % in The Gambia to 56.1 % in Ghana. Non-falciparum malaria infections were found only rarely (overall prevalence 1.39 % [95 % CI 1.00, 1.92]), ranging from 0.17 % in the Gambia to 3.81 % in Mali. Ten non-falciparum mono-infections and 25 mixed falciparum and non-falciparum infections were found. P. malariae was the most frequent non-falciparum infection identified; P. vivax was detected only in Mali. Only four of the non-falciparum mono-infections were detected by microscopy or rapid diagnostic test. Recruitment during the late rainy season and low socio-economic status were associated with an increased risk 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. Non-falciparum infections were infrequent in the populations studied, rarely detected when present as a mono-infection and unlikely to have had an important impact on the outcome of pregnancy in the communities studied due to the small number of women infected with non-falciparum parasites.

Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2.

PubMed

Leow, Chiuan Herng; Jones, Martina; Cheng, Qin; Mahler, Stephen; McCarthy, James

2014-07-18

Early and accurate diagnosis of Plasmodium falciparum infection is important for providing appropriate treatment to patients with malaria. However, technical limitations of currently available diagnostic tests limit their use in control programs. One possible explanation for the vulnerability of current antibodies used in RDTs is their propensity to degrade at high ambient temperatures. Isolation of new antibodies with better thermal stability represents an appealing approach to improve the performance of RDTs. In this study, phage display technology was deployed to isolate novel binders by screening a human naïve scFv antibody library against recombinant Plasmodium falciparum histidine rich protein 2 (rPfHRP2). The isolated scFv clones were reformatted to whole IgG and the recombinant mAbs were produced in a mammalian CHO cell expression system. To verify the biological activity of these purified recombinant mAbs, range of functional assays were characterized. Two unique clones (D2 and F9) were isolated after five rounds of biopanning. The reformatted and expressed antibodies demonstrated high binding specificity to malaria recombinant PfHRP2 and native proteins. When 5 μg/mL of mAbs applied, mAb C1-13 had the highest sensitivity, with an OD value of 1, the detection achieved 5 ng/mL of rPfHRP2, followed by mAbs D2 and F9 at 10 ng/mL and 100 ng/mL of rPfHRP2, respectively. Although the sensitivity of mAbs D2 and F9 was lower than the control, these recombinant human mAbs have shown better stability compared to mouse mAb C1-13 at various temperatures in DSC and blot assays. In view of epitope mapping, the predominant motif of rPfHRP2 recognized by mAb D2 was AHHAADAHHA, whereas mAb F9 was one amino acid shorter, resulting in AHHAADAHH. mAb F9 had the strongest binding affinity to rPfHRP2 protein, with a KD value of 4.27 × 10(-11) M, followed by control mAb C1-13 at 1.03 × 10(-10) M and mAb D2 at 3.05 × 10(-10) M. Overall, the performance of these mAbs showed comparability to currently available PfHRP2-specific mouse mAb C1-13. The stability of these novel binders indicate that they merit further work to evaluate their utility in the development of new generation point of care diagnosis of malaria.

Screening and evaluation of antiparasitic and in vitro anticancer activities of Panamanian endophytic fungi.

PubMed

Martínez-Luis, Sergio; Cherigo, Lilia; Higginbotham, Sarah; Arnold, Elizabeth; Spadafora, Carmenza; Ibañez, Alicia; Gerwick, William H; Cubilla-Rios, Luis

2011-06-01

Many compounds produced by fungi have relevant pharmaceutical applications. The purpose of this study was to collect and isolate endophytic fungi from different regions of Panama and then to test their potential therapeutic activities against Leishmania donovani, Plasmodium falciparum, and Trypanosoma cruzi as well as their anticancer activities in MCF-7 cells. Of the 25 fungal isolates obtained, ten of them had good anti-parasitic potential, showing selective activity against L. donovani; four had significant anti-malarial activity; and three inhibited the growth of T. cruzi. Anticancer activity was demonstrated in four isolates. Of the active isolates, Edenia sp. strain F0755, Xylaria sp. strain F1220, Aspergillus sp. strain F1544, Mycoleptodiscus sp. strain F0194, Phomopsis sp. strain F1566, Pycnoporus sp. strain F0305, and Diaporthe sp. strain F1647 showed the most promise based on their selective bioactivity and lack of toxicity in the assays.

Plasmodium falciparum diagnostic tools in HIV-positive under-5-year-olds in two ART clinics in Ghana: are there missed infections?

PubMed

Owusu, Ewurama D A; Djonor, Samson K; Brown, Charles A; Grobusch, Martin P; Mens, Petra F

2018-02-23

Plasmodium falciparum, the most dominant species in sub-Saharan Africa, causes the most severe clinical malaria manifestations. In resource-limited Ghana, where malaria and HIV geographically overlap, histidine-rich protein 2 (HRP2)-based rapid diagnostic test (RDT) is a faster, easier and cheaper alternative to clinical gold standard light microscopy. However, mutations in parasite hrp2 gene may result in missed infections, which have severe implications for malaria control. The performance of a common HRP2-based RDT and expert light microscopy in HIV-positive and HIV-negative children under 5 years old was compared with PCR as laboratory gold standard. Finger-prick capillary blood was tested with First Response ® Malaria Ag P. falciparum (HRP2). Giemsa-stained thick and thin blood films were examined with ≥ 200 high power fields and parasites counted per 200 white blood cells. Nested PCR species identification of P. falciparum was performed and resolved on agarose gel. False negatives from RDT were further tested for deleted pfhrp2/3 and flanking genes, using PCR. The study was performed in two anti-retroviral therapy clinics in Accra and Atibie. Out of 401 participants enrolled, 150 were HIV positive and 251 HIV negative. Malaria was more prevalent in children without HIV. Microscopy had a higher sensitivity [100% (99-100)] than RDT [83% (53.5-100)]. Parasites with pfhrp2/3 deletions contributed to missed infections from RDT false negatives. Circulation of malaria parasites with pfrhp2/3 deletions in this population played a role in missed infections with RDT. This ought to be addressed if further strides in malaria control are to be made.

Studies of Toxoplasma gondii and Plasmodium falciparum enoyl acyl carrier protein reductase and implications for the development of antiparasitic agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muench, Stephen P.; Prigge, Sean T.; McLeod, Rima

2007-03-01

The crystal structures of T. gondii and P. falciparum ENR in complex with NAD{sup +} and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively. Recent studies have demonstrated that submicromolar concentrations of the biocide triclosan arrest the growth of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii and inhibit the activity of the apicomplexan enoyl acyl carrier protein reductase (ENR). The crystal structures of T. gondii and P. falciparum ENR in complex with NAD{sup +} and triclosan and of T. gondii ENR in an apo form have beenmore » solved to 2.6, 2.2 and 2.8 Å, respectively. The structures of T. gondii ENR have revealed that, as in its bacterial and plant homologues, a loop region which flanks the active site becomes ordered upon inhibitor binding, resulting in the slow tight binding of triclosan. In addition, the T. gondii ENR–triclosan complex reveals the folding of a hydrophilic insert common to the apicomplexan family that flanks the substrate-binding domain and is disordered in all other reported apicomplexan ENR structures. Structural comparison of the apicomplexan ENR structures with their bacterial and plant counterparts has revealed that although the active sites of the parasite enzymes are broadly similar to those of their bacterial counterparts, there are a number of important differences within the drug-binding pocket that reduce the packing interactions formed with several inhibitors in the apicomplexan ENR enzymes. Together with other significant structural differences, this provides a possible explanation of the lower affinity of the parasite ENR enzyme family for aminopyridine-based inhibitors, suggesting that an effective antiparasitic agent may well be distinct from equivalent antimicrobials.« less

Ex Vivo Activity of Endoperoxide Antimalarials, Including Artemisone and Arterolane, against Multidrug-Resistant Plasmodium falciparum Isolates from Cambodia

DTIC Science & Technology

2014-10-01

OCT 2014 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Ex Vivo Activity of Endoperoxide Antimalarials , Including Artemisone...Prescribed by ANSI Std Z39-18 Ex Vivo Activity of Endoperoxide Antimalarials , Including Artemisone and Arterolane, against Multidrug-Resistant...potent antimalarial activity (2, 3). Despite having a rapid mecha- nism of action, artemisinin resistance eventually emerged and was first detected

Role of Pfmdr1 in In Vitro Plasmodium falciparum Susceptibility to Chloroquine, Quinine, Monodesethylamodiaquine, Mefloquine, Lumefantrine, and Dihydroartemisinin

PubMed Central

Wurtz, Nathalie; Fall, Bécaye; Pascual, Aurélie; Fall, Mansour; Baret, Eric; Camara, Cheikhou; Nakoulima, Aminata; Diatta, Bakary; Fall, Khadidiatou Ba; Mbaye, Pape Saliou; Diémé, Yaya; Bercion, Raymond; Wade, Boubacar

2014-01-01

The involvement of Pfmdr1 (Plasmodium falciparum multidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms in Pfmdr1 (N86Y, Y184F, S1034C, N1042D, and D1246Y) and Pfcrt (K76T) and in vitro responses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174 Plasmodium falciparum isolates from Dakar, Senegal. The Pfmdr1 86Y mutation was identified in 14.9% of the samples, and the 184F mutation was identified in 71.8% of the isolates. No 1034C, 1042N, or 1246Y mutations were detected. The Pfmdr1 86Y mutation was significantly associated with increased susceptibility to MDAQ (P = 0.0023), LMF (P = 0.0001), DHA (P = 0.0387), and MQ (P = 0.00002). The N86Y mutation was not associated with CQ (P = 0.214) or QN (P = 0.287) responses. The Pfmdr1 184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs (P = 0.168 for CQ, 0.778 for MDAQ, 0.324 for LMF, 0.961 for DHA, 0.084 for QN, and 0.298 for MQ). The Pfmdr1 86Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ (P = 0.0136), LMF (P = 0.0019), and MQ (P = 0.0001). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to MDAQ (P < 0.0001), LMF (P < 0.0001), MQ (P < 0.0001), and QN (P = 0.0026) in wild-type Pfcrt K76 parasites. The additional Pfmdr1 86Y mutation significantly increased the in vitro susceptibility to CQ (P = 0.0179) in Pfcrt 76T CQ-resistant parasites. PMID:25199781

Efficacy and safety of methylene blue in the treatment of malaria: a systematic review.

PubMed

Lu, G; Nagbanshi, M; Goldau, N; Mendes Jorge, M; Meissner, P; Jahn, A; Mockenhaupt, F P; Müller, O

2018-04-25

Methylene blue (MB) was the first synthetic antimalarial to be discovered and was used during the late 19th and early 20th centuries against all types of malaria. MB has been shown to be effective in inhibiting Plasmodium falciparum in culture, in the mouse model and in rhesus monkeys. MB was also shown to have a potent ex vivo activity against drug-resistant isolates of P. falciparum and P. vivax. In preclinical studies, MB acted synergistically with artemisinin derivates and demonstrated a strong effect on gametocyte reduction in P. falciparum. MB has, thus, been considered a potentially useful partner drug for artemisinin-based combination therapy (ACT), particularly when elimination is the final goal. The aim of this study was to review the scientific literature published until early 2017 to summarise existing knowledge on the efficacy and safety of MB in the treatment of malaria. This systematic review followed PRISMA guidelines. Studies reporting on the efficacy and safety of MB were systematically searched for in relevant electronic databases according to a pre-designed search strategy. The search (without language restrictions) was limited to studies of humans published until February 2017. Out of 474 studies retrieved, a total of 22 articles reporting on 21 studies were eligible for analysis. The 21 included studies that reported data on 1504 malaria patients (2/3 were children). Older studies were case series and reports on MB monotherapy while recent studies were mainly controlled trials of combination regimens. MB was consistently shown to be highly effective in all endemic areas and demonstrated a strong effect on P. falciparum gametocyte reduction and synergy with ACT. MB treatment was associated with mild urogenital and gastrointestinal symptoms as well as blue coloration of urine. In G6PD-deficient African individuals, MB caused a slight but clinically non-significant haemoglobin reduction. More studies are needed to define the effects of MB in P. falciparum malaria in areas outside Africa and against P. vivax malaria. Adding MB to ACT could be a valuable approach for the prevention of resistance development and for transmission reduction in control and elimination programs. This study is registered at PROSPERO (registration number CRD42017062349 ).

Plasmodium falciparum population structure in Sudan post artemisinin-based combination therapy.

PubMed

Bakhiet, Amani M A; Abdel-Muhsin, Abdel-Muhsin A; Elzaki, Salah-Eldin G; Al-Hashami, Zainab; Albarwani, Hamida S; AlQamashoui, Badar A; Al-Hamidhi, Salama; Idris, Mohamed A; Elagib, Atif A; Beja-Pereira, Albano; Babiker, Hamza A

2015-08-01

Over the past decade, Sudan has stepped up malaria control backed by WHO, and this has resulted in significant reduction in parasite rate, malaria morbidity and mortality. The present study analyzed Plasmodium falciparum parasites in four geographical separated areas, to examine whether the success in malaria control following the use of artemisinin-based combination therapy (ACT) has disrupted the population structure and evolution of the parasite. We examined 319 P. falciparum isolates collected between October 2009 and October 2012 in four different areas in Sudan (Jazira [central Sudan], Southern Darfur [western Sudan], Upper Nile [southern Sudan] and Kasala [eastern Sudan]). Twelve microsatellites were analyzed for allelic diversity, multi-locus haplotype and inter-population differentiation. Level of diversity was compared to that detected for three of the above microsatellites among P. falciparum parasites in central and eastern Sudan in 1999, prior to introduction of ACT. Diversity at each locus (unbiased heterozygosity [H]) was high in all areas (Jazira, H=0.67), (Southern Darfur, H=0.71), (Upper Nile, H=0.71), and (Kasala, H=0.63). Microsatellites were distributed widely and private alleles, detected in a single population, were rare. The extent of diversity in the above sites was similar to that seen, in 1999, in central (Khartoum, H=0.73) and eastern Sudan (Gedaref, H=0.75). Significant Linkage disequilibrium (LD) was observed between the microsatellites in all populations. Pairwise FST analysis revealed that parasites in the four areas could be considered as one population. However, the parasites in Sudan clustered away from parasites in West Africa and the Arabian Peninsula. Despite marked reduction in malaria risk in Sudan, the extent of diversity and parasite genetic structure are indicative of a large population size. Further considerable reduction in transmission would be needed before fragmented sub-population can be seen. In addition, the large divergence of P. falciparum in Sudan from West Africa and Arabian Peninsula populations may result from differential evolutionary pressures acting at the population level, which shall be considered in eradication plans. Copyright © 2015 Elsevier B.V. All rights reserved.

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

PubMed Central

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike; Ockenhouse, Christian F.; Emerling, Daniel; Glanville, Jacob; Volkmuth, Wayne; Flores-Garcia, Yevel; Zavala, Fidel; Ward, Andrew B.; King, C. Richter; Wilson, Ian A.

2017-01-01

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development in vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA)3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Together, these structural insights may aid in the design of a next-generation malaria vaccine. PMID:29138320

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

DOE PAGES

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike; ...

2017-11-14

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here in this paper, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development inmore » vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA) 3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Lastly, together, these structural insights may aid in the design of a next-generation malaria vaccine.« less

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here in this paper, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development inmore » vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA) 3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Lastly, together, these structural insights may aid in the design of a next-generation malaria vaccine.« less

Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology.

PubMed

Callaghan, Paul S; Siriwardana, Amila; Hassett, Matthew R; Roepe, Paul D

2016-03-31

Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). The approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter. During analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions. These data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.

Comparative Assessment of Transmission-Blocking Vaccine Candidates against Plasmodium falciparum

PubMed Central

Kapulu, M. C.; Da, D. F.; Miura, K.; Li, Y; Blagborough, A. M.; Churcher, T. S.; Nikolaeva, D.; Williams, A. R.; Goodman, A. L.; Sangare, I.; Turner, A. V.; Cottingham, M. G.; Nicosia, A.; Straschil, U.; Tsuboi, T.; Gilbert, S. C.; Long, Carole A.; Sinden, R. E.; Draper, S. J.; Hill, A. V. S.; Cohuet, A.; Biswas, S.

2015-01-01

Malaria transmission-blocking vaccines (TBVs) target the development of Plasmodium parasites within the mosquito, with the aim of preventing malaria transmission from one infected individual to another. Different vaccine platforms, mainly protein-in-adjuvant formulations delivering the leading candidate antigens, have been developed independently and have reported varied transmission-blocking activities (TBA). Here, recombinant chimpanzee adenovirus 63, ChAd63, and modified vaccinia virus Ankara, MVA, expressing AgAPN1, Pfs230-C, Pfs25, and Pfs48/45 were generated. Antibody responses primed individually against all antigens by ChAd63 immunization in BALB/c mice were boosted by the administration of MVA expressing the same antigen. These antibodies exhibited a hierarchy of inhibitory activity against the NF54 laboratory strain of P. falciparum in Anopheles stephensi mosquitoes using the standard membrane feeding assay (SMFA), with anti-Pfs230-C and anti-Pfs25 antibodies giving complete blockade. The observed rank order of inhibition was replicated against P. falciparum African field isolates in A. gambiae in direct membrane feeding assays (DMFA). TBA achieved was IgG concentration dependent. This study provides the first head-to-head comparative analysis of leading antigens using two different parasite sources in two different vector species, and can be used to guide selection of TBVs for future clinical development using the viral-vectored delivery platform. PMID:26063320

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters.

PubMed

Kraft, Thomas E; Heitmeier, Monique R; Putanko, Marina; Edwards, Rachel L; Ilagan, Ma Xenia G; Payne, Maria A; Autry, Joseph M; Thomas, David D; Odom, Audrey R; Hruz, Paul W

2016-12-01

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z' factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC 50 ) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Sequence analysis of DBL2β domain of vargene of Indonesian Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Sulistyaningsih, E.; Romadhon, B. D.; Palupi, I.; Hidayah, F.; Dewi, R.; Prasetyo, A.

2018-03-01

Malaria is a major health problem in tropical countries including Indonesia. The most deadly agent is Plasmodium falciparum. In P. falciparum infection, PfEMP1 is supposed to play an important role in the pathogenesis of malaria. PfEMP1 is encoded by var gene family, it is a polymorphic protein where the extra-cellular portion contains of three distinct binding domains: Duffy binding-like (DBL), Cysteine-rich interdomain regions (CIDR) and C2. PfEMP1 varies in domain composition and binding specificity. The study explored the characteristic of Indonesian DBL2β-var genes and investigated its role to the malaria outcome. Twenty blood samples from clinically mild to severe malaria patients in Jember, East Java were collected for DNA extraction. Diagnosis was confirmed by Giemsa-stained thick blood smear. PCR was conducted using specific primer targeting on the full-length of DBL2ß and resulted approximately single band of 1,7 kb in a sample. This band was observed only from severe malaria sample. Sequence analysis directly from PCR product showed 74-99% similarities with previous sequences in Gene Bank. In conclusion, the DBL2β domain of vargene of Indonesian isolates was 1603 nucleotides in length and there was a possible association of the existence of DBL2β domain with the severity of malaria outcome.

UCT943, a next generation Plasmodium falciparum PI4K inhibitor preclinical candidate for the treatment of malaria.

PubMed

Brunschwig, Christel; Lawrence, Nina; Taylor, Dale; Abay, Efrem; Njoroge, Mathew; Basarab, Gregory S; Le Manach, Claire; Paquet, Tanya; Gonzàlez Cabrera, Diego; Nchinda, Aloysius T; de Kock, Carmen; Wiesner, Lubbe; Denti, Paolo; Waterson, David; Blasco, Benjamin; Leroy, Didier; Witty, Michael J; Donini, Cristina; Duffy, James; Wittlin, Sergio; White, Karen L; Charman, Susan A; Jiménez-Díaz, Maria Belén; Angulo-Barturen, Iñigo; Herreros, Esperanza; Gamo, Francisco Javier; Rochford, Rosemary; Mancama, Dalu; Coetzer, Theresa L; van der Watt, Mariëtte E; Reader, Janette; Birkholtz, Lyn-Marie; Marsh, Kennan C; Solapure, Suresh M; Vanaerschot, Manu; Fidock, David A; Fish, Paul V; Siegl, Peter; Smith, Dennis A; Wirjanata, Grennady; Noviyanti, Rintis; Price, Ric N; Marfurt, Jutta; Silue, Kigbafori D; Street, Leslie J; Chibale, Kelly

2018-06-25

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite lifecycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activity than MMV048 and was more potent against resistant P. falciparum and P. vivax clinical isolates. Excellent in vitro antiplasmodial activity translated into high efficacy in P. berghei and humanized P. falciparum NOD- scid IL-2Rγ null mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate in vitro intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next generation Plasmodium PI4K inhibitor, the combined preclinical data suggest that UCT943 has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent and block the transmission of malaria. Copyright © 2018 American Society for Microbiology.

RTS,S/AS01 malaria vaccine mismatch observed among Plasmodium falciparum isolates from southern and central Africa and globally.

PubMed

Pringle, Julia C; Carpi, Giovanna; Almagro-Garcia, Jacob; Zhu, Sha Joe; Kobayashi, Tamaki; Mulenga, Modest; Bobanga, Thierry; Chaponda, Mike; Moss, William J; Norris, Douglas E

2018-04-26

The RTS,S/AS01 malaria vaccine encompasses the central repeats and C-terminal of Plasmodium falciparum circumsporozoite protein (PfCSP). Although no Phase II clinical trial studies observed evidence of strain-specific immunity, recent studies show a decrease in vaccine efficacy against non-vaccine strain parasites. In light of goals to reduce malaria morbidity, anticipating the effectiveness of RTS,S/AS01 is critical to planning widespread vaccine introduction. We deep sequenced C-terminal Pfcsp from 77 individuals living along the international border in Luapula Province, Zambia and Haut-Katanga Province, the Democratic Republic of the Congo (DRC) and compared translated amino acid haplotypes to the 3D7 vaccine strain. Only 5.2% of the 193 PfCSP sequences from the Zambia-DRC border region matched 3D7 at all 84 amino acids. To further contextualize the genetic diversity sampled in this study with global PfCSP diversity, we analyzed an additional 3,809 Pfcsp sequences from the Pf3k database and constructed a haplotype network representing 15 countries from Africa and Asia. The diversity observed in our samples was similar to the diversity observed in the global haplotype network. These observations underscore the need for additional research assessing genetic diversity in P. falciparum and the impact of PfCSP diversity on RTS,S/AS01 efficacy.

Molecular surveillance of Plasmodium falciparum drug resistance in the Republic of Congo: four and nine years after the introduction of artemisinin-based combination therapy.

PubMed

Koukouikila-Koussounda, Felix; Jeyaraj, Sankarganesh; Nguetse, Christian N; Nkonganyi, Charles Nchotebah; Kokou, Kossiwa Clarisse; Etoka-Beka, Mandingha K; Ntoumi, Francine; Velavan, Thirumalaisamy P

2017-04-19

Resistance to anti-malarial drugs hinders efforts on malaria elimination and eradication. Following the global spread of chloroquine-resistant parasites, the Republic of Congo adopted artemisinin-based combination therapy (ACT) in 2006 as a first-line treatment for uncomplicated malaria. To assess the impacts after implementation of ACT, a molecular surveillance for anti-malarial drug resistance was conducted in Congo 4 and 9 years after the introduction of ACT. Blood samples of 431 febrile children aged 1-10 years were utilized from two previous studies conducted in 2010 (N = 311) and 2015 (N = 120). All samples were screened for malaria parasites using nested PCR. Direct sequencing was used to determine the frequency distribution of genetic variants in the anti-malarial drug-resistant Plasmodium falciparum genes (Pfcrt, Pfmdr1, Pfatp6, Pfk13) in malaria-positive isolates. One-hundred and nineteen (N = 70 from 2010 and N = 49 from 2015) samples were positive for P. falciparum. A relative decrease in the proportion of chloroquine-resistant haplotype (CVIET) from 100% in 2005, 1 year before the introduction and implementation of ACT in 2006, to 98% in 2010 to 71% in 2015 was observed. Regarding the multidrug transporter gene, a considerable reduction in the frequency of the mutations N86Y (from 73 to 27%) and D1246Y (from 22 to 0%) was observed. However, the prevalence of the Y184F mutation remained stable (49% in 2010 compared to 54% in 2015). Isolates carrying the Pfatp6 H243Y was 25% in 2010 and this frequency was reduced to null in 2015. None of the parasites harboured the Pfk13 mutations associated with prolonged artemisinin clearance in Southeast Asia. Nevertheless, 13 new Pfk13 variants are reported among the investigated isolates. The implementation of ACT has led to the decline in prevalence of chloroquine-resistant parasites in the Republic of Congo. However, the constant prevalence of the PfMDR1 Y184F mutation, associated with lumefantrine susceptibility, indicate a selective drug pressure still exists. Taken together, this study could serve as the basis for epidemiological studies monitoring the distribution of molecular markers of artemisinin resistance in the Republic of Congo.

Dibenzylideneacetone analogues as novel Plasmodium falciparum inhibitors.

PubMed

Aher, Rahul Balasaheb; Wanare, Gajanan; Kawathekar, Neha; Kumar, Ravi Ranjan; Kaushik, Naveen Kumar; Sahal, Dinkar; Chauhan, Virander Singh

2011-05-15

A series of dibenzylideneacetones (A1-A12) and some of their pyrazolines (B1-B4) were synthesized and evaluated in vitro for blood stage antiplasmodial properties in Plasmodium falciparum culture using SYBR-green-I fluorescence assay. The compound (1E, 4E)-1,5-bis(3,4-dimethoxyphenyl)penta-1,4-dien-3-one (A9) was found to be the most active with IC(50) of 1.97 μM against chloroquine-sensitive strain (3D7) and 1.69 μM against chloroquine-resistant field isolate (RKL9). The MTT based cytotoxicity assay on HeLa cell line has confirmed that A9 is selective in its action against malaria parasite (with a therapeutic index of 166). Our results revealed that these compounds exhibited promising antiplasmodial activities which can be further explored as potential leads for the development of cheaper, safe, effective and potent drugs against chloroquine-resistant malarial parasites. Copyright © 2011. Published by Elsevier Ltd.

A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

NASA Astrophysics Data System (ADS)

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

2014-01-01

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.

Antiplasmodial activity of compounds from Sloanea rhodantha (Baker) Capuron var. rhodantha from the Madagascar rain forest.

PubMed

Cao, Shugeng; Ranarivelo, Lalasoa; Ratsimbason, Michel; Randrianasolo, Sennen; Ratovoson, Fidy; Andrianjafy, Mamisoa; Kingston, David G I

2006-12-01

Bioassay-directed separation of the butanol-soluble portion of an extract of Sloanea rhodantha (Baker) Capuron var. Rhodantha (Elaeocarpaceae) active against the drug-sensitive HB3 strain of Plasmodium falciparum led to the isolation of seven phenolic compounds, gallic acid (1), 3,5-di-O-galloylquinic acid (2), 1,6-di-O-galloyl glucopyranoside (3), 3,4,5-tri-O-galloylquinic acid (4), 1-O-eudesmoylquinic acid (5), 1,2,3,6-tetra-O-galloyl glucopyranoside (6), and 3,4,5-trimethoxyphenyl-(6'-O-galloyl)-O-b-D-glucopyranoside (7). The structure of the new compound 5 was established on the basis of interpretation of its 1D and 2D NMR spectroscopic data. Compounds 2, 3, 4, 6, and 7 showed weak inhibitory activity against the drug-sensitive HB3 and the drug-resistant FCM29 strains of P. falciparum, with IC (50) values ranging from 8.0 - 43.0 and 16.1 - 93.0 microg/mL, respectively.

A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

PubMed Central

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

2016-01-01

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

Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

PubMed Central

Ismail, Hanafy M.; Barton, Victoria; Phanchana, Matthew; Charoensutthivarakul, Sitthivut; Wong, Michael H. L.; Hemingway, Janet; Biagini, Giancarlo A.; O’Neill, Paul M.; Ward, Stephen A.

2016-01-01

The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs. PMID:26858419

Minor oxygenated cannabinoids from high potency Cannabis sativa L.

PubMed

Ahmed, Safwat A; Ross, Samir A; Slade, Desmond; Radwan, Mohamed M; Khan, Ikhlas A; ElSohly, Mahmoud A

2015-09-01

Nine oxygenated cannabinoids were isolated from a high potency Cannabis sativa L. variety. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR, HRMS and GC-MS. These minor compounds include four hexahydrocannabinols, four tetrahydrocannabinols, and one hydroxylated cannabinol, namely 9α-hydroxyhexahydrocannabinol, 7-oxo-9α-hydroxyhexa-hydrocannabinol, 10α-hydroxyhexahydrocannabinol, 10aR-hydroxyhexahydrocannabinol, Δ(9)-THC aldehyde A, 8-oxo-Δ(9)-THC, 10aα-hydroxy-10-oxo-Δ(8)-THC, 9α-hydroxy-10-oxo-Δ(6a,10a)-THC, and 1'S-hydroxycannabinol, respectively. The latter compound showed moderate anti-MRSa (IC50 10.0 μg/mL), moderate antileishmanial (IC50 14.0 μg/mL) and mild antimalarial activity against Plasmodium falciparum (D6 clone) and P. falciparum (W2 clone) with IC50 values of 3.4 and 2.3 μg/mL, respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

In-vitro interaction of tafenoquine and chloroquine in Plasmodium falciparum from northwestern Thailand.

PubMed

Vollnberg, Anke; Prajakwong, Somsak; Sirichaisinthop, Jeeraphat; Wiedermann, Gerhard; Wernsdorfer, Gunther; Wernsdorfer, Walther H

2003-01-01

The blood schizontocidal, pharmacodynamic interaction between tafenoquine (WR 238605--a 5-phenoxyprimaquine derivative--and chloroquine was investigated, using an in-vitro test for the inhibition of schizont maturation, in 15 fresh isolates of Plasmodium falciparum that originated from northwestern Thailand and neighbouring Myanmar. In this area the parasite is highly resistant to chloroquine. The geometric mean cut-off concentrations of schizont maturation for tafenoquine and chloroquine were 5261 nM and 7638 nM, respectively. With a mixture of tafenoquine and chloroquine, the mean cut-off concentration was 5252 nM, corresponding to 389 nM tafenoquine + 4863 nM chloroquine. Further analysis showed that the interaction between tafenoquine and chloroquine was additive within the range of EC20 and EC77. At concentrations higher than the EC77, interaction was moderately synergistic. While tafenoquine did not reverse the resistance to chloroquine to the degree of clinically relevant sensitivity, there was evidence that the blood schizontocidal efficacy of tafenoquine would be enhanced in the presence of chloroquine.

Syncarpamide, a new antiplasmodial (+)-norepinephrine derivative from Zanthoxylum syncarpum.

PubMed

Ross, Samir A; Sultana, Gazi N N; Burandt, Charles L; ElSohly, Mahmoud A; Marais, Jannie P J; Ferreira, Daneel

2004-01-01

A new (+)-norepinephrine derivative, syncarpamide (1), along with a known coumarin, (+)-S-marmesin (2), and one known alkaloid, decarine (3), have been isolated from the stem of Zanthoxylum syncarpum. The structure of compound 1 was elucidated on the basis of 1D and 2D NMR, MS, IR, optical rotation, and CD analyses. Its absolute stereochemistry was elucidated by synthesis of its enantiomer and subsequent comparison of CD data. Characterizations of compounds 2 and 3 were based on spectral analysis and comparison with reported data. Compounds 1 and 3 showed antiplasmodial activity, with IC(50) values of 2.04 and 1.44 microM against Plasmodium falciparum D(6) clone and 3.06 and 0.88 microM against P. falciparum W(2) clone, respectively. Compound 3 showed cytotoxicity at 56.42 microM, whereas compound 1 was not cytotoxic at 10.42 microM. Compound 1 was tested for hypotensive activity, but no activity was observed. Compound 2 showed no antiplasmodial or antimicrobial activities.

Heme-binding activity of methoxyflavones from Pentzia monodiana Maire (Asteraceae).

PubMed

Ortiz, Sergio; Dali-Yahia, Kamel; Vasquez-Ocmin, Pedro; Grougnet, Raphaël; Grellier, Philippe; Michel, Sylvie; Maciuk, Alexandre; Boutefnouchet, Sabrina

2017-04-01

A heme-binding assay based on mass spectrometry was performed on P. monodiana Maire (Asteraceae) extracts to identify metabolites able to form adducts with heminic part of haemoglobin, as potential antimalarial drugs. Main adducts were characterized and their stability was measured. Isolation of main constituents of P. monodiana Maire lead to identification of the two methoxyflavones 3'-O-methyleupatorin (7) and artemetin (8) involved in the adducts formation. Four seco-tanapartholides (1-4), a guaianolide (5), a germacranolide (6) and two other methoxyflavones (9, 10) were also characterized. Evaluation of isolated compounds on P. falciparum and T. brucei brucei showed a moderate antiprotozoal activity of the two methoxyflavones. Copyright © 2017. Published by Elsevier B.V.

Ceanothane-type triterpenoids from Cyphostemma adenocaule.

PubMed

Chouna, Jean Rodolphe; Nardella, Flore; Lenta, Bruno Ndjakou; Vonthron-Sénécheau, Catherine; Nkeng-Efouet-Alango, Pépin; Sewald, Norbert

2016-07-26

Phytochemical investigation of the methanol extract of Cyphostemma adenocaule liana (bark and wood) led to the isolation of two new ceanothane-type triterpenoids, cyphostemmic acid A 1 and cyphostemmic acid B 2, together with the known triterpenoids 3-7, β-sitosterol and its glucoside. The structures of the isolated compounds were established by 1D- and 2D-NMR spectroscopy. Ozonolysis of cyphostemmic acid A 1, epigouanic acid A 3 and betulin 6 yielded semisynthetic derivatives, cyphostemmic acid C 8, cyphostemmic acid D 9, and 3β,28-dihydroxy-30-norlupan-20-one 10 respectively. Compounds 1-4, 6, 8-10 were tested in vitro, for their antiplasmodial activity against Plasmodium falciparum 3D7 strain and showed weak activity.

Plasmodium malariae and Plasmodium ovale infections in the China-Myanmar border area.

PubMed

Li, Peipei; Zhao, Zhenjun; Xing, Hua; Li, Wenli; Zhu, Xiaotong; Cao, Yaming; Yang, Zhaoqing; Sattabongkot, Jetsumon; Yan, Guiyun; Fan, Qi; Cui, Liwang

2016-11-15

The Greater Mekong Subregion is aiming to achieve regional malaria elimination by 2030. Though a shift in malaria parasite species predominance by Plasmodium vivax has been recently documented, the transmission of the two minor Plasmodium species, Plasmodium malariae and Plasmodium ovale spp., is poorly characterized in the region. This study aims to determine the prevalence of these minor species in the China-Myanmar border area and their genetic diversity. Epidemiology study was conducted during passive case detection in hospitals and clinics in Myanmar and four counties in China along the China-Myanmar border. Cross-sectional surveys were conducted in villages and camps for internally displaced persons to determine the prevalence of malaria infections. Malaria infections were diagnosed initially by microscopy and later in the laboratory using nested PCR for the SSU rRNA genes. Plasmodium malariae and P. ovale infections were confirmed by sequencing the PCR products. The P. ovale subtypes were determined by sequencing the Pocytb, Pocox1 and Pog3p genes. Parasite populations were evaluated by PCR amplification and sequencing of the MSP-1 genes. Antifolate sensitivity was assessed by sequencing the dhfr-ts and dhps genes from the P. malariae and P. ovale isolates. Analysis of 2701 blood samples collected from the China-Myanmar border by nested PCR targeting the parasite SSU rRNA genes identified 561 malaria cases, including 161 Plasmodium falciparum, 327 P. vivax, 66 P. falciparum/P. vivax mixed infections, 4 P. malariae and 3 P. ovale spp. P. vivax and P. falciparum accounted for >60 and ~30% of all malaria cases, respectively. In comparison, the prevalence of P. malariae and P. ovale spp. was very low and only made up ~1% of all PCR-positive cases. Nevertheless, these two species were often misidentified as P. vivax infections or completely missed by microscopy even among symptomatic patients. Phylogenetic analysis of the SSU rRNA, Pocytb, Pocox1 and Pog3p genes confirmed that the three P. ovale spp. isolates belonged to the subtype P. ovale curtisi. Low-level genetic diversity was detected in the MSP-1, dhfr and dhps genes of these minor parasite species, potentially stemming from the low prevalence of these parasites preventing their mixing. Whereas most of the dhfr and dhps positions equivalent to those conferring antifolate resistance in P. falciparum and P. vivax were wild type, a new mutation S113C corresponding to the S108 position in pfdhfr was identified in two P. ovale curtisi isolates. The four human malaria parasite species all occurred sympatrically at the China-Myanmar border. While P. vivax has become the predominant species, the two minor parasite species also occurred at very low prevalence but were often misidentified or missed by conventional microscopy. These minor parasite species displayed low levels of polymorphisms in the msp-1, dhfr and dhps genes.

Molecular surveillance of Plasmodium falciparum drug resistance markers reveals partial recovery of chloroquine susceptibility but sustained sulfadoxine-pyrimethamine resistance at two sites of different malaria transmission intensities in Rwanda.

PubMed

Kateera, Fredrick; Nsobya, Sam L; Tukwasibwe, Steven; Hakizimana, Emmanuel; Mutesa, Leon; Mens, Petra F; Grobusch, Martin P; van Vugt, Michèle; Kumar, Nirbhay

2016-12-01

Faced with intense levels of chloroquine (CQ) resistance in Plasmodium falciparum malaria, Rwanda replaced CQ with amodiaquine (AQ)+sulfadoxine-pyrimethamine (SP) in 2001, and subsequently with artemether-lumefantrine (AL) in 2006, as first-line treatments for uncomplicated malaria. Following years of discontinuation of CQ use, re-emergence of CQ-susceptible parasites has been reported in countries including Malawi, Kenya and Tanzania. In contrast, high levels of SP resistant mutant parasites continue to be reported even in countries of presumed reduced SP drug selection pressure. The prevalence and distributions of genetic polymorphisms linked with CQ and SP resistance at two sites of different malaria transmission intensities are described here to better understand drug-related genomic adaptations over time and exposure to varying drug pressures in Rwanda. Using filter paper blood isolates collected from P. falciparum infected patients, DNA was extracted and a nested PCR performed to identify resistance-mediating polymorphisms in the pfcrt, pfmdr1, pfdhps and pfdhfr genes. Amplicons from a total of 399 genotyped samples were analysed by ligase detection reaction fluorescent microsphere assay. CQ susceptible pfcrt 76K and pfmdr1 86N wild-type parasites were found in about 50% and 81% of isolates, respectively. Concurrently, SP susceptible pfdhps double (437G-540E), pfdhfr triple (108N-51I-59R), quintuple pfdhps 437G-540E/pfdhfr 51I-59R-108N and sextuple haplotypes were found in about 84%, 85%, 74% and 18% of isolates, respectively. High-level SP resistance associated pfdhfr 164L and pfdhps 581G mutant prevalences were noted to decline. Mutations pfcrt 76T, pfdhfr 59R and pfdhfr 164L were found differentially distributed between the two study sites with the pfdhfr 164L mutants found only at Ruhuha site, eastern Rwanda. Overall, sustained intense levels of SP resistance mutations and a recovery of CQ susceptible parasites were found in this study following 7 years and 14 years of drug withdrawal from use, respectively. Most likely, the sustained high prevalence of resistant parasites is due to the use of DHFR/DHPS inhibitors like trimethoprim-sulfamethoxazole (TS) for the treatment of and prophylaxis against bacterial infections among HIV infected individuals as well as the continued use of IPTp-SP within the East and Central African regions for malaria prevention among pregnant women. With regard to CQ, the slow recovery of CQ susceptible parasites may have been caused partly by the continued use of CQ and/or CQ mimicking antimalarial drugs like AQ in spite of policies to withdraw it from Rwanda and the neighbouring countries of Uganda and Tanzania. Continued surveillance of P. falciparum CQ and SP associated polymorphisms is recommended for guiding future rational drug policy-making and mitigation of future risk of anti-malaria drug resistance development. Copyright © 2016 Elsevier B.V. All rights reserved.

The antiplasmodial agents of the stem bark of Entandrophragma angolense (Meliaceae).

PubMed

Bickii, Jean; Tchouya, Guy Raymond Feuya; Tchouankeu, Jean Claude; Tsamo, Etienne

2006-11-13

In the search of active principles from the stem bark of Entandrophragma angolense, we submitted the compounds isolated from the dichloromethane-methanol (1:1) extract of the stem bark to antimalarial test against chloroquine resistant strain W2 of Plasmodium falciparum malaria parasite. Only 7alpha-obacunyl acetate and a cycloartane derivative exhibited a good activity, with IC(50)s of 2 and 5.4 microg/ml respectively. Other compounds were moderately active.

The Plasmodium falciparum transcriptome in severe malaria reveals altered expression of genes involved in important processes including surface antigen–encoding var genes

PubMed Central

Tonkin-Hill, Gerry Q.; Trianty, Leily; Noviyanti, Rintis; Nguyen, Hanh H. T.; Sebayang, Boni F.; Lampah, Daniel A.; Marfurt, Jutta; Cobbold, Simon A.; Rambhatla, Janavi S.; McConville, Malcolm J.; Rogerson, Stephen J.; Brown, Graham V.; Day, Karen P.; Price, Ric N.; Anstey, Nicholas M.

2018-01-01

Within the human host, the malaria parasite Plasmodium falciparum is exposed to multiple selection pressures. The host environment changes dramatically in severe malaria, but the extent to which the parasite responds to—or is selected by—this environment remains unclear. From previous studies, the parasites that cause severe malaria appear to increase expression of a restricted but poorly defined subset of the PfEMP1 variant, surface antigens. PfEMP1s are major targets of protective immunity. Here, we used RNA sequencing (RNAseq) to analyse gene expression in 44 parasite isolates that caused severe and uncomplicated malaria in Papuan patients. The transcriptomes of 19 parasite isolates associated with severe malaria indicated that these parasites had decreased glycolysis without activation of compensatory pathways; altered chromatin structure and probably transcriptional regulation through decreased histone methylation; reduced surface expression of PfEMP1; and down-regulated expression of multiple chaperone proteins. Our RNAseq also identified novel associations between disease severity and PfEMP1 transcripts, domains, and smaller sequence segments and also confirmed all previously reported associations between expressed PfEMP1 sequences and severe disease. These findings will inform efforts to identify vaccine targets for severe malaria and also indicate how parasites adapt to—or are selected by—the host environment in severe malaria. PMID:29529020

Methylene blue inhibits the asexual development of vivax malaria parasites from a region of increasing chloroquine resistance.

PubMed

Suwanarusk, Rossarin; Russell, Bruce; Ong, Alice; Sriprawat, Kanlaya; Chu, Cindy S; PyaePhyo, Aung; Malleret, Benoit; Nosten, François; Renia, Laurent

2015-01-01

Methylene blue, once discarded due to its unsettling yet mild side effects, has now found a renewed place in the pharmacopoeia of modern medicine. The continued spread of drug-resistant Plasmodium vivax and Plasmodium falciparum has also led to a recent re-examination of methylene blue's potent antimalarial properties. Here we examine the ex vivo susceptibility profile of Plasmodium spp. isolates to methylene blue; the isolates were from a region on the Thai-Myanmar border where there are increasing rates of failure when treating vivax malaria with chloroquine. To do this we used a newly developed ex vivo susceptibility assay utilizing flow cytometry and a portable flow cytometer with a near-UV laser. P. vivax (median methylene blue IC50 3.1 nM, IQR 1.7-4.3 nM) and P. falciparum (median methylene blue IC50 1.8 nM, IQR 1.6-2.3 nM) are susceptible to methylene blue treatment at physiologically relevant levels. Unfortunately, the addition of chloroquine to combination treatments with methylene blue significantly reduces the ex vivo effectiveness of this molecule. Our data support further efforts to employ methylene blue as a safe, low-cost antimalarial to treat drug-resistant malaria. © 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: journals.permissions@oup.com.

3-Halo Chloroquine Derivatives Overcome Plasmodium falciparum Chloroquine Resistance Transporter-Mediated Drug Resistance in P. falciparum.

PubMed

Edaye, Sonia; Tazoo, Dagobert; Bohle, D Scott; Georges, Elias

2015-12-01

Polymorphism in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) was shown to cause chloroquine resistance. In this report, we examined the antimalarial potential of novel 3-halo chloroquine derivatives (3-chloro, 3-bromo, and 3-iodo) against chloroquine-susceptible and -resistant P. falciparum. All three derivatives inhibited the proliferation of P. falciparum; with 3-iodo chloroquine being most effective. Moreover, 3-iodo chloroquine was highly effective at potentiating and reversing chloroquine toxicity of drug-susceptible and -resistant P. falciparum. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Accuracy of an HRP-2/panLDH rapid diagnostic test to detect peripheral and placental Plasmodium falciparum infection in Papua New Guinean women with anaemia or suspected malaria.

PubMed

Umbers, Alexandra J; Unger, Holger W; Rosanas-Urgell, Anna; Wangnapi, Regina A; Kattenberg, Johanna H; Jally, Shadrach; Silim, Selina; Lufele, Elvin; Karl, Stephan; Ome-Kaius, Maria; Robinson, Leanne J; Rogerson, Stephen J; Mueller, Ivo

2015-10-19

The diagnosis of malaria during pregnancy is complicated by placental sequestration, asymptomatic infection, and low-density peripheral parasitaemia. Where intermittent preventive treatment (IPT) with sulfadoxine-pyrimethamine is threatened by drug resistance, or is inappropriate due to low transmission, intermittent screening and treatment (ISTp) with rapid diagnostic tests for malaria (RDT) could be a valuable alternative. Therefore, the accuracy of RDTs to detect peripheral and placental infection was assessed in a declining transmission setting in Papua New Guinea (PNG). The performance of a combination RDT detecting histidine-rich protein-2 (HRP-2) and Plasmodium lactate dehydrogenase (pLDH), and light microscopy (LM), to diagnose peripheral Plasmodium falciparum and Plasmodium vivax infections during pregnancy, were assessed using quantitative real-time PCR (qPCR) as the reference standard. Participants in a malaria prevention trial in PNG with a haemoglobin ≤90 g/L, or symptoms suggestive of malaria, were tested. Ability of RDT and LM to detect active placental infection on histology was evaluated in some participants. Among 876 women, 1162 RDTs were undertaken (anaemia: 854 [73.5 %], suspected malaria: 308 [26.5 %]). qPCR detected peripheral infection during 190 RDT episodes (165 P. falciparum, 19 P. vivax, 6 mixed infections). Overall, RDT detected peripheral P. falciparum infection with 45.6 % sensitivity (95 % CI 38.0-53.4), a specificity of 96.4 % (95.0-97.4), a positive predictive value of 68.4 % (59.1-76.8), and a negative predictive value of 91.1 % (89.2-92.8). RDT performance to detect P. falciparum was inferior to LM, more so amongst anaemic women (18.6 vs 45.3 % sensitivity, Liddell's exact test, P < 0.001) compared to symptomatic women (72.9 vs 82.4 % sensitivity, P = 0.077). RDT and LM missed 88.0 % (22/25) and 76.0 % (19/25) of P. vivax infections, respectively. In a subset of women tested at delivery and who had placental histology (n = 158) active placental infection was present in 19.6 %: all three peripheral blood infection detection methods (RDT, LM, qPCR) missed >50 % of these infections. In PNG, HRP-2/pLDH RDTs may be useful to diagnose peripheral P. falciparum infections in symptomatic pregnant women. However, they are not sufficiently sensitive for use in intermittent screening amongst asymptomatic (anaemic) women. These findings have implications for the management of malaria in pregnancy. The adverse impact of infections undetected by RDT or LM on pregnancy outcomes needs further evaluation.

Life cycle-dependent cytoskeletal modifications in Plasmodium falciparum infected erythrocytes.

PubMed

Shi, Hui; Liu, Zhuo; Li, Ang; Yin, Jing; Chong, Alvin G L; Tan, Kevin S W; Zhang, Yong; Lim, Chwee Teck

2013-01-01

Plasmodium falciparum infection of human erythrocytes is known to result in the modification of the host cell cytoskeleton by parasite-coded proteins. However, such modifications and corresponding implications in malaria pathogenesis have not been fully explored. Here, we probed the gradual modification of infected erythrocyte cytoskeleton with advancing stages of infection using atomic force microscopy (AFM). We reported a novel strategy to derive accurate and quantitative information on the knob structures and their connections with the spectrin network by performing AFM-based imaging analysis of the cytoplasmic surface of infected erythrocytes. Significant changes on the red cell cytoskeleton were observed from the expansion of spectrin network mesh size, extension of spectrin tetramers and the decrease of spectrin abundance with advancing stages of infection. The spectrin network appeared to aggregate around knobs but also appeared sparser at non-knob areas as the parasite matured. This dramatic modification of the erythrocyte skeleton during the advancing stage of malaria infection could contribute to the loss of deformability of the infected erythrocyte.

Highly potent anti-proliferative effects of a gallium(III) complex with 7-chloroquinoline thiosemicarbazone as a ligand: synthesis, cytotoxic and antimalarial evaluation.

PubMed

Kumar, Kewal; Schniper, Sarah; González-Sarrías, Antonio; Holder, Alvin A; Sanders, Natalie; Sullivan, David; Jarrett, William L; Davis, Krystyn; Bai, Fengwei; Seeram, Navindra P; Kumar, Vipan

2014-10-30

A gallium(III) complex with 7-chloroquinoline thiosemicarbazone was synthesized and characterized. The complex proved to be thirty-one times more potent on colon cancer cell line, HCT-116, with considerably less cytotoxicity on non-cancerous colon fibroblast, CCD-18Co, when compared to etoposide. Its anti-malarial potential on 3D7 isolate of Plasmodium falciparum was better than lumefantrine. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Ex Vivo Drug Sensitivity Profiles of Plasmodium falciparum Field Isolates from Cambodia and Thailand, 2005 to 2010, Determined by a Histidine-Rich Protein-2 Assay

DTIC Science & Technology

2012-06-13

References 1. Desjardins RE, Canfield CJ, Haynes JD, Chulay JD: Quantitative assessment of antimalarial activity in vitro by a semiautomated...potentially concerning. As before, un- regulated availability of antimalarial medications during that period may have been a contributing factor. Not- ably...et al. observed a similar dip in IC50 values for a range of antimalarial drugs in 2006, and attributed the observa- tion to sampling bias since most

Activity of Ocimum basilicum, Ocimum canum, and Cymbopogon citratus essential oils against Plasmodium falciparum and mature-stage larvae of Anopheles funestus s.s.

PubMed

Akono Ntonga, Patrick; Baldovini, Nicolas; Mouray, Elisabeth; Mambu, Lengo; Belong, Philippe; Grellier, Philippe

2014-01-01

The biological activities of essential oils from three plants grown in Cameroon: Ocimum basilicum, Ocimum canum, and Cymbopogon citratus were tested against Plasmodium falciparum and mature-stage larvae of Anopheles funestus. Gas chromatography and gas chromatography - mass spectrometry analyses showed that the main compounds are geranial, 1,8-cineole and linalool in C. citratus, O. canum and O. basilicum, respectively. Larvicidal tests carried out according to the protocol recommended by the World Health Organization showed that the essential oil of leaves of C. citratus is the most active against larvae of An. funestus (LC50 values = 35.5 ppm and 34.6 ppm, respectively, for larval stages III and IV after 6 h of exposure). Besides, the in vitro anti-plasmodial activity evaluated by the radioisotopic method showed that the C. citratus oil is the most active against P. falciparum, with an IC50 value of 4.2 ± 0.5 μg/mL compared with O. canum (20.6 ± 3.4 μg/mL) and O. basilicum (21 ± 4.6 μg/mL). These essential oils can be recommended for the development of natural biocides for fighting the larvae of malaria vectors and for the isolation of natural products with anti-malarial activity. © P. Akono Ntonga et al., published by EDP Sciences, 2014.

Activity of Ocimum basilicum, Ocimum canum, and Cymbopogon citratus essential oils against Plasmodium falciparum and mature-stage larvae of Anopheles funestus s.s.

PubMed Central

Akono Ntonga, Patrick; Baldovini, Nicolas; Mouray, Elisabeth; Mambu, Lengo; Belong, Philippe; Grellier, Philippe

2014-01-01

The biological activities of essential oils from three plants grown in Cameroon: Ocimum basilicum, Ocimum canum, and Cymbopogon citratus were tested against Plasmodium falciparum and mature-stage larvae of Anopheles funestus. Gas chromatography and gas chromatography – mass spectrometry analyses showed that the main compounds are geranial, 1,8-cineole and linalool in C. citratus, O. canum and O. basilicum, respectively. Larvicidal tests carried out according to the protocol recommended by the World Health Organization showed that the essential oil of leaves of C. citratus is the most active against larvae of An. funestus (LC50 values = 35.5 ppm and 34.6 ppm, respectively, for larval stages III and IV after 6 h of exposure). Besides, the in vitro anti-plasmodial activity evaluated by the radioisotopic method showed that the C. citratus oil is the most active against P. falciparum, with an IC50 value of 4.2 ± 0.5 μg/mL compared with O. canum (20.6 ± 3.4 μg/mL) and O. basilicum (21 ± 4.6 μg/mL). These essential oils can be recommended for the development of natural biocides for fighting the larvae of malaria vectors and for the isolation of natural products with anti-malarial activity. PMID:24995776

Detection of specific antibodies to Plasmodium falciparum in blood bank donors from malaria-endemic and non-endemic areas of Venezuela.

PubMed

Contreras, C E; Pance, A; Marcano, N; González, N; Bianco, N

1999-06-01

Malaria antibody detection is valuable in providing retrospective confirmation of an attack of malaria. Blood bank screening is another area were malaria serology is potentially useful. In the present study, we tested the presence of antibodies to Plasmodium falciparum in sera from blood bank donors of non-endemic and malaria-endemic areas of Venezuela. Sera from 1,000 blood donors were tested by an indirect immunofluorescent antibody (IFA) assay and an IgG-ELISA for the presence of malaria antibodies using a synchronized in vitro-cultured Venezuelan isolate of P. falciparum as the antigen source. A selected group of positive and negative sera (n = 100) was also tested by a dot-IgG-ELISA. Positive results (reciprocal titer > or = 40) were found in 0.8% and 3.8% of blood donors when tested by the IFA assay and in 0.8% and 2% (optical density > or = 0.2) when tested by the IgG-ELISA in Caracas (non-endemic area) and Bolivar City (endemic area), respectively. The presence of anti-malarial antibodies in some sera from non-endemic areas such as Caracas reflects the increased potential risk of post-transfusional malaria in those areas due to the mobility of the blood donors. The data obtained indicate the need to implement new blood donor policy in blood banks in developing areas. Our results also indicate that the IFA assay is the most reliable test to use in malaria serodiagnosis.

Declining Efficacy of Artemisinin Combination Therapy Against P. Falciparum Malaria on the Thai-Myanmar Border (2003-2013): The Role of Parasite Genetic Factors.

PubMed

Phyo, Aung Pyae; Ashley, Elizabeth A; Anderson, Tim J C; Bozdech, Zbynek; Carrara, Verena I; Sriprawat, Kanlaya; Nair, Shalini; White, Marina McDew; Dziekan, Jerzy; Ling, Clare; Proux, Stephane; Konghahong, Kamonchanok; Jeeyapant, Atthanee; Woodrow, Charles J; Imwong, Mallika; McGready, Rose; Lwin, Khin Maung; Day, Nicholas P J; White, Nicholas J; Nosten, Francois

2016-09-15

Deployment of mefloquine-artesunate (MAS3) on the Thailand-Myanmar border has led to a sustained reduction in falciparum malaria, although antimalarial efficacy has declined substantially in recent years. The role of Plasmodium falciparum K13 mutations (a marker of artemisinin resistance) in reducing treatment efficacy remains controversial. Between 2003 and 2013, we studied the efficacy of MAS3 in 1005 patients with uncomplicated P. falciparum malaria in relation to molecular markers of resistance. Polymerase chain reaction (PCR)-adjusted cure rates declined from 100% in 2003 to 81.1% in 2013 as the proportions of isolates with multiple Pfmdr1 copies doubled from 32.4% to 64.7% and those with K13 mutations increased from 6.7% to 83.4%. K13 mutations conferring moderate artemisinin resistance (notably E252Q) predominated initially but were later overtaken by propeller mutations associated with slower parasite clearance (notably C580Y). Those infected with both multiple Pfmdr1 copy number and a K13 propeller mutation were 14 times more likely to fail treatment. The PCR-adjusted cure rate was 57.8% (95% confidence interval [CI], 45.4, 68.3) compared with 97.8% (95% CI, 93.3, 99.3) in patients with K13 wild type and Pfmdr1 single copy. K13 propeller mutation alone was a strong risk factor for recrudescence (P = .009). The combined population attributable fraction of recrudescence associated with K13 mutation and Pfmdr1 amplification was 82%. The increasing prevalence of K13 mutations was the decisive factor for the recent and rapid decline in efficacy of artemisinin-based combination (MAS3) on the Thailand-Myanmar border. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

KBE009: An antimalarial bestatin-like inhibitor of the Plasmodium falciparum M1 aminopeptidase discovered in an Ugi multicomponent reaction-derived peptidomimetic library.

PubMed

González-Bacerio, Jorge; Maluf, Sarah El Chamy; Méndez, Yanira; Pascual, Isel; Florent, Isabelle; Melo, Pollyana M S; Budu, Alexandre; Ferreira, Juliana C; Moreno, Ernesto; Carmona, Adriana K; Rivera, Daniel G; Alonso Del Rivero, Maday; Gazarini, Marcos L

2017-09-01

Malaria is a global human parasitic disease mainly caused by the protozoon Plasmodium falciparum. Increased parasite resistance to current drugs determines the relevance of finding new treatments against new targets. A novel target is the M1 alanyl-aminopeptidase from P. falciparum (PfA-M1), which is essential for parasite development in human erythrocytes and is inhibited by the pseudo-peptide bestatin. In this work, we used a combinatorial multicomponent approach to produce a library of peptidomimetics and screened it for the inhibition of recombinant PfA-M1 (rPfA-M1) and the in vitro growth of P. falciparum erythrocytic stages (3D7 and FcB1 strains). Dose-response studies with selected compounds allowed identifying the bestatin-based peptidomimetic KBE009 as a submicromolar rPfA-M1 inhibitor (K i =0.4μM) and an in vitro antimalarial compound as potent as bestatin (IC 50 =18μM; without promoting erythrocyte lysis). At therapeutic-relevant concentrations, KBE009 is selective for rPfA-M1 over porcine APN (a model of these enzymes from mammals), and is not cytotoxic against HUVEC cells. Docking simulations indicate that this compound binds PfA-M1 without Zn 2+ coordination, establishing mainly hydrophobic interactions and showing a remarkable shape complementarity with the active site of the enzyme. Moreover, KBE009 inhibits the M1-type aminopeptidase activity (Ala-7-amido-4-methylcoumarin substrate) in isolated live parasites with a potency similar to that of the antimalarial activity (IC 50 =82μM), strongly suggesting that the antimalarial effect is directly related to the inhibition of the endogenous PfA-M1. These results support the value of this multicomponent strategy to identify PfA-M1 inhibitors, and make KBE009 a promising hit for drug development against malaria. Copyright © 2017 Elsevier Ltd. All rights reserved.

To Live Like a Pig and Die Like a Dog: Environmental Implications for World War I in East Africa

DTIC Science & Technology

2009-12-03

held decisive advantages including greater numbers of troops, more robust logistics, and unchallenged control of the sea lines of communications...The Center for Disease Control defines malaria as “a serious and sometimes fatal disease caused by a parasite that commonly infects a certain type of...malaria parasites that can infect humans, Plasmodium falciparum remains indigenous to east 32 Center for Disease Control, “Malaria Home > Frequently

Sequence homology and structural analysis of plasmepsin 4 isolated from Indian Plasmodium vivax isolates.

PubMed

Rawat, Manmeet; Vijay, Sonam; Gupta, Yash; Dixit, Rajnikant; Tiwari, P K; Sharma, Arun

2011-07-01

Plasmodium vivax malaria is a globally widespread disease responsible for 50% of human malaria cases in Central and South America, South East Asia and Indian subcontinent. The rising severity of the disease and emerging resistance of the parasite has emphasized the need for the search of novel therapeutic targets to combat P. vivax malaria. Plasmepsin 4 (PM4) a food vacuole aspartic protease is essential in parasite functions and viability such as initiating hemoglobin digestion and processing of proteins and is being looked upon as potential drug target. Although the plasmepsins of Plasmodium falciparum have been extensively studied, the plasmepsins of P. vivax are not well characterized. This is the first report detailing complete PM4 gene analysis from Indian P. vivax isolates. Blast results of sequences of P. vivax plasmepsin 4 (PvPM4) shows 100% homology among isolates of P. vivax collected from different geographical regions of India. All of the seven Indian isolates did not contain intron within the coding region. Interestingly, PvPM4 sequence analysis showed a very high degree of homology with all other sequences of Plasmodium species available in the genebank. Our results strongly suggest that PvPM4 are highly conserved except a small number of amino acid substitutions that did not modify key motifs at active site formation for the function or the structure of the enzymes. Furthermore, our study shows that PvPM4 occupies unique phylogenetic status within Plasmodium group and sufficiently differ from the most closely related human aspartic protease, cathepsin D. The analysis of 3D model of PM4 showed a typical aspartic protease structure with bi-lobed, compact and distinct peptide binding cleft in both P. vivax and P. falciparum. In order to validate appropriate use of PM4 as potential anti-malarial drug target, studies on genetic and structural variations among P. vivax plasmepsins (PvPMs) from different geographical regions are of utmost importance for drugs and vaccine designs for anti-malarial strategies. Copyright © 2011 Elsevier B.V. All rights reserved.

Mosquito Vectors and the Globalization of Plasmodium falciparum Malaria.

PubMed

Molina-Cruz, Alvaro; Zilversmit, Martine M; Neafsey, Daniel E; Hartl, Daniel L; Barillas-Mury, Carolina

2016-11-23

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.

Prolonged Plasmodium falciparum Infection in Immigrants, Paris

PubMed Central

Godineau, Nadine; Fontanet, Arnaud; Houze, Sandrine; Bouchaud, Olivier; Matheron, Sophie; Le Bras, Jacques

2008-01-01

Few immigrant travelers have Plasmodium falciparum infections >2 months after leaving malaria-endemic areas. We conducted a case–control study to identify factors associated with prolonged P. falciparum infection in immigrant travelers. Results suggest that P. falciparum infection should be systematically suspected, even months after travel, especially in pregnant women and first-arrival immigrants. PMID:18258132

Treatment of Chronic Asymptomatic Plasmodium falciparum Infection Does Not Increase the Risk of Clinical Malaria Upon Reinfection.

PubMed

Portugal, Silvia; Tran, Tuan M; Ongoiba, Aissata; Bathily, Aboudramane; Li, Shanping; Doumbo, Safiatou; Skinner, Jeff; Doumtabe, Didier; Kone, Younoussou; Sangala, Jules; Jain, Aarti; Davies, D Huw; Hung, Christopher; Liang, Li; Ricklefs, Stacy; Homann, Manijeh Vafa; Felgner, Philip L; Porcella, Stephen F; Färnert, Anna; Doumbo, Ogobara K; Kayentao, Kassoum; Greenwood, Brian M; Traore, Boubacar; Crompton, Peter D

2017-03-01

Chronic asymptomatic Plasmodium falciparum infections are common in endemic areas and are thought to contribute to the maintenance of malaria immunity. Whether treatment of these infections increases the subsequent risk of clinical episodes of malaria is unclear. In a 3-year study in Mali, asymptomatic individuals with or without P. falciparum infection at the end of the 6-month dry season were identified by polymerase chain reaction (PCR), and clinical malaria risk was compared during the ensuing 6-month malaria transmission season. At the end of the second dry season, 3 groups of asymptomatic children were identified: (1) children infected with P. falciparum as detected by rapid diagnostic testing (RDT) who were treated with antimalarials (n = 104), (2) RDT-negative children whose untreated P. falciparum infections were detected retrospectively by PCR (n = 55), and (3) uninfected children (RDT/PCR negative) (n = 434). Clinical malaria risk during 2 subsequent malaria seasons was compared. Plasmodium falciparum-specific antibody kinetics during the dry season were compared in children who did or did not harbor asymptomatic P. falciparum infections. Chronic asymptomatic P. falciparum infection predicted decreased clinical malaria risk during the subsequent malaria season(s); treatment of these infections did not alter this reduced risk. Plasmodium falciparum-specific antibodies declined similarly in children who did or did not harbor chronic asymptomatic P. falciparum infection during the dry season. These findings challenge the notion that chronic asymptomatic P. falciparum infection maintains malaria immunity and suggest that mass drug administration during the dry season should not increase the subsequent risk of clinical malaria. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Sarcocystis neurona: molecular characterization of enolase domain I region and a comparison to other protozoa.

PubMed

Bolten, K E; Marsh, A E; Reed, S M; Dubey, J P; Toribio, R E; Saville, W J A

2008-09-01

Sarcocystis neurona causes protozoal myeloencephalitis and has the ability to infect a wide host range in contrast to other Sarcocystis species. In the current study, five S. neurona isolates from a variety of sources, three Sarcocystis falcatula, one Sarcocystis dasypi/S. neurona-like isolate, and one Besnoitia darlingi isolate were used to compare the enolase 2 gene segment containing the domain I region to previously sequenced enolase genes from Neospora caninum, Neospora hughesi, Toxoplasma gondii, Plasmodium falciparum, and Trypanosoma cruzi; enolase 2 segment containing domain I region is highly conserved amongst these parasites of veterinary and medical importance. Immunohistochemistry results indicates reactivity of T. gondii enolase 1 and 2 antibodies to S. neurona merozoites and metrocytes, but no reactivity of anti-enolase 1 to the S. neurona bradyzoite stage despite reactivity to T. gondii bradyzoites, suggesting expression differences between organisms.

Falciparum malaria infection with invasive pulmonary aspergillosis in immunocompetent host – case report

NASA Astrophysics Data System (ADS)

Andriyani, Y.

2018-03-01

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.

Fatty Acid Synthesis and Pyruvate Metabolism Pathways Remain Active in Dihydroartemisinin-Induced Dormant Ring Stages of Plasmodium falciparum

PubMed Central

Chen, Nanhua; LaCrue, Alexis N.; Teuscher, Franka; Waters, Norman C.; Gatton, Michelle L.; Kyle, Dennis E.

2014-01-01

Artemisinin (ART)-based combination therapy (ACT) is used as the first-line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action, there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART-induced ring-stage dormancy and recovery have been implicated as possible causes of recrudescence; however, little is known about the characteristics of dormant parasites, including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA)-induced dormancy and recovery. Transcription analysis showed an immediate downregulation for 10 genes following exposure to DHA but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly of genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, was also maintained. Additions of inhibitors for biotin acetyl-coenzyme A (CoA) carboxylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively, following DHA treatment. Our results demonstrate that most metabolic pathways are downregulated in DHA-induced dormant parasites. In contrast, fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment. PMID:24913167

Plasmodium falciparum signal peptide peptidase cleaves malaria heat shock protein 101 (HSP101). Implications for gametocytogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baldwin, Michael; Russo, Crystal; Li, Xuerong

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 reticulummore » 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.« less

Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL) Confer Multidrug Resistance.

PubMed

LaMonte, Gregory; Lim, Michelle Yi-Xiu; Wree, Melanie; Reimer, Christin; Nachon, Marie; Corey, Victoria; Gedeck, Peter; Plouffe, David; Du, Alan; Figueroa, Nelissa; Yeung, Bryan; Bifani, Pablo; Winzeler, Elizabeth A

2016-07-05

Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites. Several previous in vitro evolution studies have implicated the Plasmodium falciparum cyclic amine resistance locus (PfCARL) as a potential target of imidazolopiperazines, potent antimalarial compounds with broad activity against different parasite life cycle stages. Given that the imidazolopiperazines are currently being tested in clinical trials, understanding their mechanism of resistance and the cellular processes involved will allow more effective clinical usage. Copyright © 2016 LaMonte et al.

Plasmodium falciparum malaria skews globin gene expression balance in in-vitro haematopoietic stem cell culture system: Its implications in malaria associated anemia.

PubMed

Pathak, Vrushali; Colah, Roshan; Ghosh, Kanjaksha

2018-02-01

Understanding the pathophysiology and associated host parasite interactions of the malaria infection is the prerequisite for developing effective prevention and treatment strategies. The exact mechanism underlying malaria associated ineffective and dyserythropoiesis is not yet fully understood. Being an important protein, haemoglobin serves as the main amino acid reservoir available to the intra-erythrocytic plasmodium. It is important to check the expression profiling of globin genes which may help us to understand host parasite interactions and its potential contribution to both infection and disease. Here, an in-vitro culture system was used to study the effect of different doses of Plasmodium falciparum on haematopoietic stem cell expansion, differentiation and expression of globin genes. Upon exposure to the different doses of P. falciparum parasites of strains 3D7, Dd2 and RKL9 (intact and lysed form) at different stages of erythroid development, cells demonstrated suppression in growth and differentiation. At almost all stages of erythroid development upon parasite exposure, the γ globin gene was found to be downregulated and the α/β as well as α/non- α globin mRNA ratios in late stage erythroid cells were found to be reduced (p < .01) compared to the untreated controls. The imbalance in globin chain expression might be considered as one of the factors involved in malaria associated inappropriate erythropoietic responses. Copyright © 2018 Elsevier Inc. All rights reserved.

Effect of chemotherapy on malaria transmission among Yanomami Amerindians: simulated consequences of placebo treatment.

PubMed

Freeman, J; Laserson, K F; Petralanda, I; Spielman, A

1999-05-01

To determine whether chemotherapy effectively reduces Plasmodium falciparum malaria transmission in isolated human populations, we followed two abrupt sequential outbreaks of malaria infection among Yanomami Amerindians and modeled the effect of chemotherapy and the consequences if no drug was available. A Macdonald-type mathematical model demonstrated that both outbreaks comprised a single epidemic event linked by an invisible outbreak in vector mosquitoes. The basic reproductive number, R0, from fitted values based on the treated epidemic was 2 during the initial phase of the epidemic, and waned as vector density decreased with the onset of the dry season. In the observed epidemic, 60 (45%) of 132 village residents were affected, and the treated outbreak ended after two months. Although the initial chemotherapy regimen was only marginally effective, the duration of human infectivity was reduced from an expected nine months to two weeks. In the absence of this intervention, the initial R0 value would have been 40, more than 60% of the population would have been infected, and more than 30% would have remained parasitemic until the next rainy season (about six months later). Another outbreak would then have ensued, and malaria probably would have remained endemic in this village. Our simulated placebo treatment permits us to conclude that even partially effective chemotherapeutic interventions, such as those in our study, interrupt serial transmission of P. falciparum among isolated human populations that are exposed to infection seasonally.

Reemergence of chloroquine-sensitive pfcrt K76 Plasmodium falciparum genotype in southeastern Cameroon.

PubMed

Ndam, Nicaise Tuikue; Basco, Leonardo K; Ngane, Vincent Foumane; Ayouba, Ahidjo; Ngolle, Eitel Mpoudi; Deloron, Philippe; Peeters, Martine; Tahar, Rachida

2017-03-27

Chloroquine had been used extensively during the last five decades in Cameroon. Its decreasing clinical effectiveness, supported by high proportions of clinical isolates carrying the mutant pfcrt haplotype (CVIET), led the health authorities to resort to amodiaquine monotherapy in 2002 and artemisinin-based combination therapy (ACT) in 2004 (artesunate-amodiaquine, with artemether-lumefantrine as an alternative since 2006) as the first-line treatment of uncomplicated malaria. The aim of the present study was to investigate whether the withdrawal of chloroquine was associated with a reduction in pfcrt mutant parasite population and reemergence of chloroquine-sensitive parasites in southeastern Cameroon between 2003 and 2012. The frequency of pfcrt haplotypes at positions 72-76 in Plasmodium falciparum isolates collected from individuals in 2003 and 2012 in southeastern Cameroon was determined by sequence specific oligonucleotide probes-enzyme linked immunosorbent assay (SSOP-ELISA). The proportions of parasites carrying the mutant haplotype CVIET and the wild-type CVMNK were 53.0 and 28.0% in 2003, respectively. The proportion of the mutant haplotype in samples collected 9 years later decreased to 25.3% whereas the proportion of parasites carrying the wild-type CVMNK haplotype was 53.7%. Even though the proportion of chloroquine-sensitive parasites seems to be increasing in southeastern Cameroon, a reintroduction of chloroquine cannot be recommended at present in Cameroon. The current national anti-malarial drug policy should be implemented and reinforced to combat drug-resistant malaria.

Sustained Ex Vivo Susceptibility of Plasmodium falciparum to Artemisinin Derivatives but Increasing Tolerance to Artemisinin Combination Therapy Partner Quinolines in The Gambia

PubMed Central

Okebe, Joseph; Mbye, Haddijatou; Ceesay, Sukai; El-Fatouri, Fatima; Joof, Fatou; Nyang, Haddy; Janha, Ramatoulie; Affara, Muna; Ahmad, Abdullahi; Kolly, Olimatou; Nwakanma, Davis; D'Alessandro, Umberto

2017-01-01

ABSTRACT Antimalarial interventions have yielded a significant decline in malaria prevalence in The Gambia, where artemether-lumefantrine (AL) has been used as a first-line antimalarial for a decade. Clinical Plasmodium falciparum isolates collected from 2012 to 2015 were analyzed ex vivo for antimalarial susceptibility and genotyped for drug resistance markers (pfcrt K76T, pfmdr1 codons 86, 184, and 1246, and pfk13) and microsatellite variation. Additionally, allele frequencies of single nucleotide polymorphisms (SNPs) from other drug resistance-associated genes were compared from genomic sequence data sets from 2008 (n = 79) and 2014 (n = 168). No artemisinin resistance-associated pfk13 mutation was found, and only 4% of the isolates tested in 2015 showed significant growth after exposure to dihydroartemisinin. Conversely, the 50% inhibitory concentrations (IC50s) of amodiaquine and lumefantrine increased within this period. pfcrt 76T and pfmdr1 184F mutants remained at a prevalence above 80%. pfcrt 76T was positively associated with higher IC50s to chloroquine. pfmdr1 NYD increased in frequency between 2012 and 2015 due to lumefantrine selection. The TNYD (pfcrt 76T and pfmdr1 NYD wild-type haplotype) also increased in frequency following AL implementation in 2008. These results suggest selection for pfcrt and pfmdr1 genotypes that enable tolerance to lumefantrine. Increased tolerance to lumefantrine calls for sustained chemotherapeutic monitoring in The Gambia to minimize complete artemisinin combination therapy (ACT) failure in the future. PMID:28971859

Prevalence of pfhrp2 and pfhrp3 gene deletions in Puerto Lempira, Honduras.

PubMed

Abdallah, Joseph F; Okoth, Sheila Akinyi; Fontecha, Gustavo A; Torres, Rosa Elena Mejia; Banegas, Engels I; Matute, María Luisa; Bucheli, Sandra Tamara Mancero; Goldman, Ira F; de Oliveira, Alexandre Macedo; Barnwell, John W; Udhayakumar, Venkatachalam

2015-01-21

Recent studies have demonstrated the deletion of the histidine-rich protein 2 (PfHRP2) gene (pfhrp2) in field isolates of Plasmodium falciparum, which could result in false negative test results when PfHRP2-based rapid diagnostic tests (RDTs) are used for malaria diagnosis. Although primary diagnosis of malaria in Honduras is determined based on microscopy, RDTs may be useful in remote areas. In this study, it was investigated whether there are deletions of the pfhrp2, pfhrp3 and their respective flanking genes in 68 P. falciparum parasite isolates collected from the city of Puerto Lempira, Honduras. In addition, further investigation considered the possible correlation between parasite population structure and the distribution of these gene deletions by genotyping seven neutral microsatellites. Sixty-eight samples used in this study, which were obtained from a previous chloroquine efficacy study, were utilized in the analysis. All samples were genotyped for pfhrp2, pfhrp3 and flanking genes by PCR. The samples were then genotyped for seven neutral microsatellites in order to determine the parasite population structure in Puerto Lempira at the time of sample collection. It was found that all samples were positive for pfhrp2 and its flanking genes on chromosome 8. However, only 50% of the samples were positive for pfhrp3 and its neighboring genes while the rest were either pfhrp3-negative only or had deleted a combination of pfhrp3 and its neighbouring genes on chromosome 13. Population structure analysis predicted that there are at least two distinct parasite population clusters in this sample population. It was also determined that a greater proportion of parasites with pfhrp3-(and flanking gene) deletions belonged to one cluster compared to the other. The findings indicate that the P. falciparum parasite population in the municipality of Puerto Lempira maintains the pfhrp2 gene and that PfHRP2-based RDTs could be considered for use in this region; however continued monitoring of parasite population will be useful to detect any parasites with deletions of pfhrp2.

Antiplasmodial activity of Mezoneuron benthamianum leaves and identification of its active constituents.

PubMed

Jansen, Olivia; Tchinda, Alembert T; Loua, Jean; Esters, Virginie; Cieckiewicz, Ewa; Ledoux, Allison; Toukam, Paul D; Angenot, Luc; Tits, Monique; Balde, Aliou M; Frédérich, Michel

2017-05-05

Decoctions of the leaves of M. benthamianum Baill. are used by traditional healers in Guinea to treat malaria and this use was validated by a preliminary clinical assay. To evaluate the in vitro antiplasmodial activity and to identify active compounds from extracts of M. benthamianum leaves. Antiplasmodial activity of extracts, fractions and pure compounds was evaluated in vitro against a chloroquine-sensitive strain of Plasmodium falciparum (3D7) using the measurement of the plasmodial lactate dehydrogenase activity. Selectivity of extracts and purified compounds for Plasmodium parasites was evaluated by using WST-1 test on HeLa human cells. Compounds were isolated using normal phase silica gel column chromatography and prepHPLC and their structures elucidated using extensive spectroscopic analysis. Hydroethanolic extracts (70% v/v) of M. benthamianum leaves showed a moderate in vitro activity against P. falciparum 3D7, with IC 50 in the range 22.5 - 32.6µg/mL, depending on the batch; while a dark precipitate formed during ethanol evaporation showed higher activity (IC 50 =6.5µg/mL). The fractionation was performed on this most active fraction and was followed by in vitro antiplasmodial assay. Active compounds (5, 7, 8) belong to several phytochemical classes, contributing together to the global antiplasmodial activity of the hydroethanolic extract against P. falciparum parasite. This study finally allowed the isolation of three diterpenes including two new compounds named Mezobenthamic acids A (1) and B (2) and neocaesalpin H (3), as well as quercetin (4), kaempferol (7), resveratrol (6), gallic acid (9) and its ethylester (5), β-sitosterol glucoside (10) and 13b-hydroxy-pheophorbide a (8). This study gives some concrete evidence to support the ethnopharmacological use of Mezoneuron benthamianum leaves extract in the management of malaria. The active compounds can be further studied for their antiplasmodial potential, as well as their suitability to be used as quality markers for the standardization of this herbal drug from the Guinean traditional pharmacopeia. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Drug Evaluation in the Plasmodium Falciparum-Aotus Model.

DTIC Science & Technology

1986-09-30

strains of Plasmodium falciparum, Uganda Palo Alto ( chloroquine sensi- tive) or Vietnam Smith (chioroquine resistant), in Aotus trivirgatus, were used...Plasmodium falciparum in the Panamanian owl monkey Aotus Two strains of falciparum malaria, Uganda Palo Alto (sensitive to chloroquine anfd quinine...resistant to pyrimetha- mine) and Vietnam Smith (resistant to chloroquine , quinine and pyrimethamine) were used. Previous evaluation of two stereoisomers of

Artemisinin-Naphthoquine versus Artemether-Lumefantrine for Uncomplicated Malaria in Papua New Guinean Children: An Open-Label Randomized Trial

PubMed Central

Laman, Moses; Moore, Brioni R.; Benjamin, John M.; Yadi, Gumul; Bona, Cathy; Warrel, Jonathan; Kattenberg, Johanna H.; Koleala, Tamarah; Manning, Laurens; Kasian, Bernadine; Robinson, Leanne J.; Sambale, Naomi; Lorry, Lina; Karl, Stephan; Davis, Wendy A.; Rosanas-Urgell, Anna; Mueller, Ivo; Siba, Peter M.; Betuela, Inoni; Davis, Timothy M. E.

2014-01-01

Background Artemisinin combination therapies (ACTs) with broad efficacy are needed where multiple Plasmodium species are transmitted, especially in children, who bear the brunt of infection in endemic areas. In Papua New Guinea (PNG), artemether-lumefantrine is the first-line treatment for uncomplicated malaria, but it has limited efficacy against P. vivax. Artemisinin-naphthoquine should have greater activity in vivax malaria because the elimination of naphthoquine is slower than that of lumefantrine. In this study, the efficacy, tolerability, and safety of these ACTs were assessed in PNG children aged 0.5–5 y. Methods and Findings An open-label, randomized, parallel-group trial of artemether-lumefantrine (six doses over 3 d) and artemisinin-naphthoquine (three daily doses) was conducted between 28 March 2011 and 22 April 2013. Parasitologic outcomes were assessed without knowledge of treatment allocation. Primary endpoints were the 42-d P. falciparum PCR-corrected adequate clinical and parasitologic response (ACPR) and the P. vivax PCR-uncorrected 42-d ACPR. Non-inferiority and superiority designs were used for falciparum and vivax malaria, respectively. Because the artemisinin-naphthoquine regimen involved three doses rather than the manufacturer-specified single dose, the first 188 children underwent detailed safety monitoring. Of 2,542 febrile children screened, 267 were randomized, and 186 with falciparum and 47 with vivax malaria completed the 42-d follow-up. Both ACTs were safe and well tolerated. P. falciparum ACPRs were 97.8% and 100.0% in artemether-lumefantrine and artemisinin-naphthoquine-treated patients, respectively (difference 2.2% [95% CI −3.0% to 8.4%] versus −5.0% non-inferiority margin, p = 0.24), and P. vivax ACPRs were 30.0% and 100.0%, respectively (difference 70.0% [95% CI 40.9%–87.2%], p<0.001). Limitations included the exclusion of 11% of randomized patients with sub-threshold parasitemias on confirmatory microscopy and direct observation of only morning artemether-lumefantrine dosing. Conclusions Artemisinin-naphthoquine is non-inferior to artemether-lumefantrine in PNG children with falciparum malaria but has greater efficacy against vivax malaria, findings with implications in similar geo-epidemiologic settings within and beyond Oceania. Trial registration Australian New Zealand Clinical Trials Registry ACTRN12610000913077 Please see later in the article for the Editors' Summary PMID:25549086

In vitro anti-plasmodial activity of some traditionally used medicinal plants against Plasmodium falciparum.

PubMed

Venkatesalu, V; Gopalan, N; Pillai, C R; Singh, Vineeta; Chandrasekaran, M; Senthilkumar, A; Chandramouli, N

2012-07-01

The anti-plasmodial activity of different solvent extracts of Adhatoda vasica (root), Caesalpinia pulcherrima (leaf), Carica papaya (pulp), Erythroxylum monogynum (leaf), Lantana camara (whole plant), Ocimum sanctum (root) and Phyllanthus niruri (whole plant) were studied against Plasmodium falciparum. Of the 35 extracts tested, seven extracts showed good anti-plasmodial activity. Methanol extract of C. pulcherrima showed the lowest IC50 value (10.96 μg/mL) followed by methanol extract of A. vasica (IC(50)=11.1 μg/mL), chloroform extract of O. sanctum (IC(50)=11.47 μg/mL), methanol extract of E. monogynum (IC(50)=12.23 μg/mL), acetone extract of C. pulcherrima (IC(50)=12.49 μg/mL), methanol extract of O. sanctum and acetone extract of A. vasica (IC(50)=14.04 μg/mL). The results of the present study justify the use of these medicinal plants in traditional practice, and also, a further study on the isolation of anti-plasmodial molecules from their active crude extracts is in progress.

Genomic Analysis Reveals a Common Breakpoint in Amplifications of the Plasmodium vivax Multidrug Resistance 1 Locus in Thailand

PubMed Central

Auburn, Sarah; Serre, David; Pearson, Richard D.; Amato, Roberto; Sriprawat, Kanlaya; To, Sheren; Handayuni, Irene; Suwanarusk, Rossarin; Russell, Bruce; Drury, Eleanor; Stalker, Jim; Miotto, Olivo; Kwiatkowski, Dominic P.; Nosten, Francois; Price, Ric N.

2016-01-01

In regions of coendemicity for Plasmodium falciparum and Plasmodium vivax where mefloquine is used to treat P. falciparum infection, drug pressure mediated by increased copy numbers of the multidrug resistance 1 gene (pvmdr1) may select for mefloquine-resistant P. vivax. Surveillance is not undertaken routinely owing in part to methodological challenges in detection of gene amplification. Using genomic data on 88 P. vivax samples from western Thailand, we identified pvmdr1 amplification in 17 isolates, all exhibiting tandem copies of a 37.6–kilobase pair region with identical breakpoints. A novel breakpoint-specific polymerase chain reaction assay was designed to detect the amplification. The assay demonstrated high sensitivity, identifying amplifications in 13 additional, polyclonal infections. Application to 132 further samples identified the common breakpoint in all years tested (2003–2015), with a decline in prevalence after 2012 corresponding to local discontinuation of mefloquine regimens. Assessment of the structure of pvmdr1 amplification in other geographic regions will yield information about the population-specificity of the breakpoints and underlying amplification mechanisms. PMID:27456706

Discordant Temporal Evolution of Pfcrt and Pfmdr1 Genotypes and Plasmodium falciparum In Vitro Drug Susceptibility to 4-Aminoquinolines after Drug Policy Change in French Guiana

PubMed Central

Yrinesi, Joséphine; Ekala, Marie-Thérèse; Péneau, Julie; Volney, Béatrice; Berger, Franck; Bouchier, Christiane; Bertani, Stéphane; Musset, Lise; Meynard, Jean-Baptiste; Mercereau-Puijalon, Odile

2012-01-01

Analysis of the evolution of drug target genes under changing drug policy is needed to assist monitoring of Plasmodium falciparum drug resistance in the field. Here we genotype Pfcrt and Pfdmr1 of 700 isolates collected in French Guiana from 2000 (5 years after withdrawal of chloroquine) to 2008, i.e., the period when the artemether-lumefantrine combination was progressively introduced and mefloquine was abandoned. Gene sequencing showed fixation of the 7G8-type Pfcrt SMVNT resistance haplotype and near fixation of the NYCDY Pfdmr1 haplotype. Pfdmr1 gene copy number correlated with 50% inhibitory concentrations of mefloquine and halofantrine (r = 0.64 and 0.47, respectively, n = 547); its temporal changes paralleled changes in in vitro mefloquine susceptibility. However, the molecular parameters studied did not account for the regained in vitro susceptibility to chloroquine and showed a poor correlation with susceptibility to artemether, lumefantrine, or quinine. Identification of novel markers of resistance to these antimalarials is needed in this South American area. PMID:22232280

Dynamics of the Major Histocompatibility Complex Class I Processing and Presentation Pathway in the Course of Malaria Parasite Development in Human Hepatocytes: Implications for Vaccine Development

PubMed Central

Ma, Jinxia; Trop, Stefanie; Baer, Samantha; Rakhmanaliev, Elian; Arany, Zita; Dumoulin, Peter; Zhang, Hao; Romano, Julia; Coppens, Isabelle; Levitsky, Victor; Levitskaya, Jelena

2013-01-01

Control of parasite replication exerted by MHC class I restricted CD8+ T-cells in the liver is critical for vaccination-induced protection against malaria. While many intracellular pathogens subvert the MHC class I presentation machinery, its functionality in the course of malaria replication in hepatocytes has not been characterized. Using experimental systems based on specific identification, isolation and analysis of human hepatocytes infected with P. berghei ANKA GFP or P. falciparum 3D7 GFP sporozoites we demonstrated that molecular components of the MHC class I pathway exhibit largely unaltered expression in malaria-infected hepatocytes until very late stages of parasite development. Furthermore, infected cells showed no obvious defects in their capacity to upregulate expression of different molecular components of the MHC class I machinery in response to pro-inflammatory lymphokines or trigger direct activation of allo-specific or peptide-specific human CD8+ T-cells. We further demonstrate that ectopic expression of circumsporozoite protein does not alter expression of critical genes of the MHC class I pathway and its response to pro-inflammatory cytokines. In addition, we identified supra-cellular structures, which arose at late stages of parasite replication, possessed the characteristic morphology of merosomes and exhibited nearly complete loss of surface MHC class I expression. These data have multiple implications for our understanding of natural T-cell immunity against malaria and may promote development of novel, efficient anti-malaria vaccines overcoming immune escape of the parasite in the liver. PMID:24086507

Evaluation of antiplasmodial and antileishmanial activities of herbal medicine Pseudelephantopus spiralis (Less.) Cronquist and isolated hirsutinolide-type sesquiterpenoids.

PubMed

Girardi, Cynthia; Fabre, Nicolas; Paloque, Lucie; Ramadani, Arba Pramundita; Benoit-Vical, Françoise; González-Aspajo, German; Haddad, Mohamed; Rengifo, Elsa; Jullian, Valérie

2015-07-21

Pseudelephantopus spiralis (Less.) Cronquist is distributed in the Caribbean, Mesoamerica and Latin America. Preparations of the plant are traditionally used in Latin America for the treatment of various diseases including fever, malaria, and spleen or liver inflammations. Aerial parts of P. spiralis were extracted with either ethanol or distilled water. Seven hirsutinolide-type sesquiterpenoids were isolated: 8-acetyl-13-ethoxypiptocarphol (1), diacetylpiptocarphol (2), piptocarphins A (3), F (4) and D (5), (1S(*),4R(*),8S(*),10R(*))-1,4-epoxy-13-ethoxy-1,8,10-trihydroxygermacra-5E,7(11)-dien-6,12-olide (6), and piptocarphol (7). Extracts and isolated compounds (2, 3, 5-7) were screened for their in vitro antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum strain FcM29-Cameroon and antileishmanial activity against three stages of Leishmania infantum. Their cytotoxicities were also evaluated against healthy VERO cell lines and J774A.1 macrophages, the host cells of the Leishmania parasites in humans. Aqueous extracts showed a greater inhibitory effect than alcoholic extracts, with IC50 on P. falciparum of 3.0µg/mL versus 21.1µg/mL, and on L. infantum of 13.4µg/mL versus >50µg/mL. Both extracts were found to be cytotoxic to VERO cells (CC50<3µg/mL). Sesquiterpene lactones 2 and 3 showed the best activity against both parasites but failed in selectivity. Carbon 8 hydroxylated hirsutinolides 5-7 presented the particularity of exhibiting two conformers observed in solution during extensive NMR analyses in CD3OD and UHPLC-MS. The presence of a hydroxyl function at C-8 decreased the activity of 5-7 on the two parasites and also on VERO cells. The antiplasmodial activity displayed by the aqueous extract explains the traditional use of P. spiralis in the treatment of malaria. This activity seems to be attributable to the presence of sesquiterpene lactones 2 and 3, the most active against P. falciparum. Aqueous extract and compounds 2, 3 and 6 were also active against L. infantum but lacked in selectivity due to their cytotoxicity towards macrophages. Exploring the safety and antiplasmodial efficacy of this traditional remedy will require further toxicological and in vivo studies in the light of the cytotoxicity towards healthy cell lines displayed by the aqueous extract and compounds 2 and 3. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The Plasmodium gaboni genome illuminates allelic dimorphism of immunologically important surface antigens in P. falciparum.

PubMed

Roy, Scott William

2015-12-01

In the deadly human malaria parasite Plasmodium falciparum, several major merozoite surface proteins (MSPs) show a striking pattern of allelic diversity called allelic dimorphism (AD). In AD, the vast majority of observed alleles fall into two highly divergent allelic classes, with recombinant alleles being rare or not observed, presumably due to repression by natural selection (recombination suppression, or RS). The three AD loci, merozoite surface proteins (MSPs) 1, 2, and 6, along with MSP3, which also exhibits RS among four allelic classes, can be collectively called AD/RS. The causes of AD/RS and the evolutionary history of allelic diversity at these loci remain mysterious. The few available sequences from a single closely related chimpanzee parasite, P. reichenowi, have suggested that for 3/4 loci, AD/RS is an ancient state that has been retained in P. falciparum since well before the P. falciparum-P. reichenowi ancestor. On the other hand, based on comparative sequence analysis, we recently suggested that (i) AD/RS P. falciparum loci have undergone interallelic recombination over longer evolutionary times (on the timescale of recent speciation events), and thus (ii) AD/RS may be a recent phenomenon. The recent publication of genomic sequencing efforts for P. gaboni, an outgroup to P. falciparum and P. reichenowi, allows for improved reconstruction of the evolutionary history of these loci. In this work, I report genic sequence for P. gaboni for all four AD/RS P. falciparum loci (MSP1, 2, 3, and 6). Comparison of these sequences with available P. falciparum and P. reichenowi data strengthens the evidence for interallelic recombination over the evolutionary history of these species and also strengthens the case that AD/RS at these loci is ancient. Combined with previous results, these data provide evidence that AD/RS at different loci has evolved at several different times in the evolutionary history of P. falciparum: (i) before the P. gaboni-P. falciparum divergence, for much of MSP1 and MSP3; (ii) between the P. gaboni-P. falciparum and P. reichenowi-P. falciparum divergences, for the 5' end of the AD region of MSP6 and block 3 of MSP1; (iii) near the P. reichenowi-P. falciparum divergence, for the 3' end of the AD region of MSP6; and (iv) after the P. reichenowi-P. falciparum divergence, for MSP2. Based on these results, I suggest a new hypothesis for long-term evolutionary maintenance of AD/RS by recombination within allelic groups. Copyright © 2015 Elsevier B.V. All rights reserved.

Plasmodium falciparum in the southeastern Atlantic forest: a challenge to the bromeliad-malaria paradigm?

PubMed

Laporta, Gabriel Zorello; Burattini, Marcelo Nascimento; Levy, Debora; Fukuya, Linah Akemi; de Oliveira, Tatiane Marques Porangaba; Maselli, Luciana Morganti Ferreira; Conn, Jan Evelyn; Massad, Eduardo; Bydlowski, Sergio Paulo; Sallum, Maria Anice Mureb

2015-04-25

Recently an unexpectedly high prevalence of Plasmodium falciparum was found in asymptomatic blood donors living in the southeastern Brazilian Atlantic forest. The bromeliad-malaria paradigm assumes that transmission of Plasmodium vivax and Plasmodium malariae involves species of the subgenus Kerteszia of Anopheles and only a few cases of P. vivax malaria are reported annually in this region. The expectations of this paradigm are a low prevalence of P. vivax and a null prevalence of P. falciparum. Therefore, the aim of this study was to verify if P. falciparum is actively circulating in the southeastern Brazilian Atlantic forest remains. In this study, anophelines were collected with Shannon and CDC-light traps in seven distinct Atlantic forest landscapes over a 4-month period. Field-collected Anopheles mosquitoes were tested by real-time PCR assay in pools of ten, and then each mosquito from every positive pool, separately for P. falciparum and P. vivax. Genomic DNA of P. falciparum or P. vivax from positive anophelines was then amplified by traditional PCR for sequencing of the 18S ribosomal DNA to confirm Plasmodium species. Binomial probabilities were calculated to identify non-random results of the P. falciparum-infected anopheline findings. The overall proportion of anophelines naturally infected with P. falciparum was 4.4% (21/480) and only 0.8% (4/480) with P. vivax. All of the infected mosquitoes were found in intermixed natural and human-modified environments and most were Anopheles cruzii (22/25 = 88%, 18 P. falciparum plus 4 P. vivax). Plasmodium falciparum was confirmed by sequencing in 76% (16/21) of positive mosquitoes, whereas P. vivax was confirmed in only 25% (1/4). Binomial probabilities suggest that P. falciparum actively circulates throughout the region and that there may be a threshold of the forested over human-modified environment ratio upon which the proportion of P. falciparum-infected anophelines increases significantly. These results show that P. falciparum actively circulates, in higher proportion than P. vivax, among Anopheles mosquitoes of fragments of the southeastern Brazilian Atlantic forest. This finding challenges the classical bromeliad-malaria paradigm, which considers P. vivax circulation as the driver for the dynamics of residual malaria transmission in this region.

Spatial and temporal distribution of falciparum malaria in China

PubMed Central

Lin, Hualiang; Lu, Liang; Tian, Linwei; Zhou, Shuisen; Wu, Haixia; Bi, Yan; Ho, Suzanne C; Liu, Qiyong

2009-01-01

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 characterized by the predominance of adult men cases. Imported falciparum malaria in the non-endemic area of China, affected mainly by the malaria transmission in Yunnan, has increased both spatially and temporally. Specific intervention measures targeted at the mobile population groups are warranted. PMID:19523209

Plasmodium falciparum, anaemia and cognitive and educational performance among school children in an area of moderate malaria transmission: baseline results of a cluster randomized trial on the coast of Kenya

PubMed Central

Halliday, Katherine E; Karanja, Peris; Turner, Elizabeth L; Okello, George; Njagi, Kiambo; Dubeck, Margaret M; Allen, Elizabeth; Jukes, Matthew CH; Brooker, Simon J

2012-01-01

Objectives Studies have typically investigated health and educational consequences of malaria among school-aged children in areas of high malaria transmission, but few have investigated these issues in moderate transmission settings. This study investigates the patterns of and risks for Plasmodium falciparum and anaemia and their association with cognitive and education outcomes on the Kenyan coast, an area of moderate malaria transmission. Methods As part of a cluster randomised trial, a baseline cross-sectional survey assessed the prevalence of and risk factors for P. falciparum infection and anaemia and the associations between health status and measures of cognition and educational achievement. Results are presented for 2400 randomly selected children who were enrolled in the 51 intervention schools. Results The overall prevalence of P. falciparum infection and anaemia was 13.0% and 45.5%, respectively. There was marked heterogeneity in the prevalence of P. falciparum infection by school. In multivariable analysis, being male, younger age, not sleeping under a mosquito net and household crowding were adjusted risk factors for P. falciparum infection, whilst P. falciparum infection, being male and indicators of poor nutritional intake were risk factors for anaemia. No association was observed between either P. falciparum or anaemia and performance on tests of sustained attention, cognition, literacy or numeracy. Conclusion The results indicate that in this moderate malaria transmission setting, P. falciparum is strongly associated with anaemia, but there is no clear association between health status and education. Intervention studies are underway to investigate whether removing the burden of chronic asymptomatic P. falciparum and related anaemia can improve education outcomes. PMID:22950512

Plasmodium falciparum, anaemia and cognitive and educational performance among school children in an area of moderate malaria transmission: baseline results of a cluster randomized trial on the coast of Kenya.

PubMed

Halliday, Katherine E; Karanja, Peris; Turner, Elizabeth L; Okello, George; Njagi, Kiambo; Dubeck, Margaret M; Allen, Elizabeth; Jukes, Matthew C H; Brooker, Simon J

2012-05-01

Studies have typically investigated health and educational consequences of malaria among school-aged children in areas of high malaria transmission, but few have investigated these issues in moderate transmission settings. This study investigates the patterns of and risks for Plasmodium falciparum and anaemia and their association with cognitive and education outcomes on the Kenyan coast, an area of moderate malaria transmission. As part of a cluster randomised trial, a baseline cross-sectional survey assessed the prevalence of and risk factors for P. falciparum infection and anaemia and the associations between health status and measures of cognition and educational achievement. Results are presented for 2400 randomly selected children who were enrolled in the 51 intervention schools. The overall prevalence of P. falciparum infection and anaemia was 13.0% and 45.5%, respectively. There was marked heterogeneity in the prevalence of P. falciparum infection by school. In multivariable analysis, being male, younger age, not sleeping under a mosquito net and household crowding were adjusted risk factors for P. falciparum infection, whilst P. falciparum infection, being male and indicators of poor nutritional intake were risk factors for anaemia. No association was observed between either P. falciparum or anaemia and performance on tests of sustained attention, cognition, literacy or numeracy. The results indicate that in this moderate malaria transmission setting, P. falciparum is strongly associated with anaemia, but there is no clear association between health status and education. Intervention studies are underway to investigate whether removing the burden of chronic asymptomatic P. falciparum and related anaemia can improve education outcomes. © 2012 Blackwell Publishing Ltd.

Low sensitivity of malaria rapid diagnostic tests stored at room temperature in the Brazilian Amazon Region.

PubMed

Gomes, Luciano T; Tada, Mauro S; Katsuragawa, Tony H; Povoa, Marinete M; Viana, Giselle Mr; Alecrim, Maria das Gracas C; De Santana-Filho, Frankllin S; Arcanjo, Ana Ruth L; Couto, Alvaro A R A; Calvosa, Vanja S P; Nery, Andreia F; Fontes, Cor J F

2013-03-14

In remote areas of the Amazon Region, diagnosis of malaria by microscopy is practically impossible. This study aimed to evaluate the performance of two rapid diagnostic tests (RDTs) targeting different malaria antigens stored at room temperature in the Brazilian Amazon Region. Performance of the OptiMal Pf/Pan test and ICT-Now Pf/Pan test was analyzed retrospectively in 1,627 and 1,602 blood samples, respectively. Tests were performed over a 15-month period. Kits were stored at room temperature in five community health centres located in the Brazilian Amazon Region. RDT results were compared with thick blood smear (TBS) results to determine sensitivity, specificity, and accuracy of the RDT. The sensitivities of the OptiMal Pf/Pan test were 79.7% for Plasmodium falciparum malaria diagnosis and 85.7% for non-P. falciparum infections. The results showed a crude agreement of 88.5% for P. falciparum, and 88.3% for non-P. falciparum infections (Kappa index = 0.74 and 0.75, respectively). For the ICT-Now Pf/Pan test (CI 95%), the sensitivities were 87.9% for P. falciparum malaria diagnosis and 72.5% for non-P. falciparum infection. Crude agreement between the ICT-Now Pf/Pan test and TBS was 91.4% for P. falciparum and 79.7% for non-P. falciparum infection. The Kappa index was 0.81 and 0.59 for the final diagnosis of P. falciparum and non-P. falciparum, respectively. Higher levels of parasitaemia were associated with higher crude agreement between RDT and TBS. The sensitivities of RDTs stored at room temperature over a 15-month period and performed in field conditions were lower than those previously reported.

[Construction of Plasmodium falciparum signal peptide peptidase-GFP mutant and its expression analysis in the malaria parasite].

PubMed

Li, Xue-rong; Wu, Yin-juan; Shang, Mei; Li, Ye; Xu, Jin; Yu, Xin-bing; Athar, Chishti

2014-08-01

To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum (PJSPP) and GFP, and transfect into P. falciparum (3D7 strain) to obtain mutant parasites which can express PJSPP-GFP. Plasmodium falciparum(3D7 strain) genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PJSPP, an 883 bp gene fragment was amplified by PCR, and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR, double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol, stained with DAPI, and observed under immunofluorescence microscope. The PJSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting. The C-terminal region of PJSPP was amplified from P.falciparum (3D7 strain) genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening, double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy, and mainly located in the cytoplasm. The PJSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64,000. Recombinant vector PJSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.

CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum.

PubMed

Bryant, Jessica M; Regnault, Clément; Scheidig-Benatar, Christine; Baumgarten, Sebastian; Guizetti, Julien; Scherf, Artur

2017-07-11

Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes-the intron-in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression. Copyright © 2017 Bryant et al.

Erythrocytic adenosine monophosphate as an alternative purine source in Plasmodium falciparum.

PubMed

Cassera, María B; Hazleton, Keith Z; Riegelhaupt, Paul M; Merino, Emilio F; Luo, Minkui; Akabas, Myles H; Schramm, Vern L

2008-11-21

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Comparison of Clinical Profile between P. vivax and P. falciparum Malaria in Children: A Tertiary Care Centre Perspective from India.

PubMed

Goyal, Jagdish Prasad; Makwana, Aarti M

2014-01-01

Background. Malaria is a one of the leading causes of morbidity and mortality in tropical countries. Plasmodium vivax (P. vivax) is usually thought to be causing benign malaria with low incidence of complications as compared to Plasmodium falciparum (P. falciparum). Methods. This retrospective observational study included malaria patients who were admitted to K.T. Children Hospital and P.D.U. Government Medical College, Rajkot, a tertiary care teaching hospital, Gujarat, western India, during the period January 2012 to December 2012. Inclusion criteria were patients in whom either P. falciparum or P. vivax was positive on rapid malaria antigen test and peripheral blood smear. Patients showing mixed infections were excluded from study. Results. A total of 79 subjects (mean age 5.4 ± 3.6 years) were included in the study. It consisted of 47 P. vivax and 32 P. falciparum cases. The P. vivax cases consisted of 33 (70.2%) males and 11 (19.8%) females while P. falciparum cases consisted of 14 (43.8%) males and 18 (56.2%) females. One patient of each P. vivax and P. falciparum expired. There was no statistical significant difference found between complications such as anemia, thrombocytopenia, liver and renal dysfunction, ARDS, and cerebral malaria between P. vivax and P. falciparum. Conclusion. We conclude that P. vivax monoinfection tends to have as similar course and complications as compared to malaria due to P. falciparum monoinfection.

Performance of "VIKIA Malaria Ag Pf/Pan" (IMACCESS®), a new malaria rapid diagnostic test for detection of symptomatic malaria infections.

PubMed

Chou, Monidarin; Kim, Saorin; Khim, Nimol; Chy, Sophy; Sum, Sarorn; Dourng, Dany; Canier, Lydie; Nguon, Chea; Ménard, Didier

2012-08-24

Recently, IMACCESS® developed a new malaria test (VIKIA Malaria Ag Pf/Pan™), based on the detection of falciparum malaria (HRP-2) and non-falciparum malaria (aldolase). The performance of this new malaria rapid diagnostic test (RDT) was assessed using 1,000 febrile patients seeking malaria treatment in four health centres in Cambodia from August to December 2011. The results of the VIKIA Malaria Ag Pf/Pan were compared with those obtained by microscopy, the CareStart Malaria™ RDT (AccessBio®) which is currently used in Cambodia, and real-time PCR (as "gold standard"). The best performances of the VIKIA Malaria Ag Pf/Pan™ test for detection of both Plasmodium falciparum and non-P. falciparum were with 20-30 min reading times (sensitivity of 93.4% for P. falciparum and 82.8% for non-P. falciparum and specificity of 98.6% for P. falciparum and 98.9% for non-P. falciparum) and were similar to those for the CareStart Malaria™ test. This new RDT performs similarly well as other commercially available tests (especially the CareStart Malaria™ test, used as comparator), and conforms to the World Health Organization's recommendations for RDT performance. It is a good alternative tool for the diagnosis of malaria in endemic areas.

In Vivo and In Vitro Antimalarial Properties of Azithromycin-Chloroquine Combinations That Include the Resistance Reversal Agent Amlodipine ▿ †

PubMed Central

Pereira, Marcus R.; Henrich, Philipp P.; Sidhu, Amar bir Singh; Johnson, David; Hardink, Joel; Van Deusen, Jeffrey; Lin, Jian; Gore, Katrina; O'Brien, Connor; Wele, Mamadou; Djimde, Abdoulaye; Chandra, Richa; Fidock, David A.

2011-01-01

Evidence of emerging Plasmodium falciparum resistance to artemisinin-based combination therapies, documented in western Cambodia, underscores the continuing need to identify new antimalarial combinations. Given recent reports of the resurgence of chloroquine-sensitive P. falciparum parasites in Malawi, after the enforced and prolonged withdrawal of this drug, and indications of a possible synergistic interaction with the macrolide azithromycin, we sought to further characterize chloroquine-azithromycin combinations for their in vitro and in vivo antimalarial properties. In vitro 96-h susceptibility testing of chloroquine-azithromycin combinations showed mostly additive interactions against freshly cultured P. falciparum field isolates obtained from Mali. Some evidence of synergy, however, was apparent at the fractional 90% inhibitory concentration level. Additional in vitro testing highlighted the resistance reversal properties of amlodipine for both chloroquine and quinine. In vivo experiments, using the Peters 4-day suppressive test in a P. yoelii mouse model, revealed up to 99.9% suppression of parasitemia following treatment with chloroquine-azithromycin plus the R enantiomer of amlodipine. This enantiomer was chosen because it does not manifest the cardiac toxicities observed with the racemic mixture. Pharmacokinetic/pharmacodynamic analyses in this rodent model and subsequent extrapolation to a 65-kg adult led to the estimation that 1.8 g daily of R-amlodipine would be required to achieve similar efficacy in humans, for whom this is likely an unsafe dose. While these data discount amlodipine as an additional partner for chloroquine-based combination therapy, our studies continue to support azithromycin as a safe and effective addition to antimalarial combination therapies. PMID:21464242

Methionine transport in the malaria parasite Plasmodium falciparum.

PubMed

Cobbold, Simon A; Martin, Rowena E; Kirk, Kiaran

2011-01-01

The intraerythrocytic malaria parasite, Plasmodium falciparum, derives amino acids from the digestion of host cell haemoglobin. However, it also takes up amino acids from the extracellular medium. Isoleucine is absent from adult human haemoglobin and an exogenous source of isoleucine is essential for parasite growth. An extracellular source of methionine is also important for the normal growth of at least some parasite strains. In this study we have characterised the uptake of methionine by P. falciparum-infected human erythrocytes, and by parasites functionally isolated from their host cells by saponin-permeabilization of the erythrocyte membrane. Infected erythrocytes take up methionine much faster than uninfected erythrocytes, with the increase attributable to the flux of this amino acid via the New Permeability Pathways induced by the parasite in the erythrocyte membrane. Having entered the infected cell, methionine is taken up by the intracellular parasite via a saturable, temperature-dependent process that is independent of ATP, Na(+) and H(+). Substrate competition studies, and comparison of the transport of methionine with that of isoleucine and leucine, yielded results consistent with the hypothesis that the parasite has at its surface one or more transporters which mediate the flux into and out of the parasite of a broad range of neutral amino acids. These transporters function most efficiently when exchanging one neutral amino acid for another, thus providing a mechanism whereby the parasite is able to import important exogenous amino acids in exchange for surplus neutral amino acids liberated from the digestion of host cell haemoglobin. Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Baseline in vivo, ex vivo and molecular responses of Plasmodium falciparum to artemether and lumefantrine in three endemic zones for malaria in Colombia.

PubMed

Aponte, Samanda; Guerra, Ángela Patricia; Álvarez-Larrotta, Catalina; Bernal, Sindy Durley; Restrepo, César; González, Camila; Yasnot, María Fernanda; Knudson-Ospina, Angélica

2017-02-01

Colombia began using artemisinin-based combination therapies for the treatment of uncomplicated Plasmodium falciparum malaria in 2006. It is necessary to implement resistance surveillance to antimalarial drugs in order to promptly detect changes in parasite susceptibility. The aim of this study was to establish a susceptibility baseline of P. falciparum to artemether-lumefantrine using three monitoring tools. Patients with uncomplicated malaria treated with artemether-lumefantrine underwent clinical and parasitological follow-up over 28 days. Ex vivo test was performed using the microtest technique for chloroquine, arthemeter, dihydroartemisinin and lumefantrine. Pfmdr1 copy number and polymorphisms in Pfk13, Pfatp6, Pfcrt and Pfmdr1 genes were analyzed. From a total of 150 screened patients, 49 completed follow-up for 28 days. All treated patients had adequate clinical and parasitological responses. Parasitic clearance showed a drastic reduction of parasite biomass at 24 hours and complete elimination at 48 hours. One hundred eleven isolates were processed, all exhibited high susceptibility to artemisinins and a slight decrease in susceptibility to lumefantrine. No genetic polymorphisms associated with resistance to artemisinin were found. This study generated a susceptibility baseline in response to therapy with Coartem (artemether-lumefantrine) with numerical reference values, which will allow data comparison with future studies to systematically monitor changes in the parasite and to provide an early alert to the health authorities. © The Author 2017. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Antimalarial activity of sesquiterpene lactones from Vernonia cinerea.

PubMed

Chea, Aun; Hout, Sotheara; Long, Christophe; Marcourt, Laurence; Faure, Robert; Azas, Nadine; Elias, Riad

2006-10-01

Two new sesquiterpene lactones, vernolides C and D as well as six known ones were isolated from the dichloromethane fraction of an aqueous extract from Vernonia cinerea. Their structures were elucidated by spectroscopic methods. Among the known sesquiterpene lactones, three of them were described in this plant for the first time. In vitro antiplasmodial evaluation showed that the three major compounds 1, 7 and 8 were active against chloroquine resistant Plasmodium falciparum strain (W2) with IC(50) 3.9, 3.7 and 3.5 microM, respectively.

Trafficking of the signature protein of intra-erythrocytic Plasmodium berghei-induced structures, IBIS1, to P. falciparum Maurer's clefts.

PubMed

Petersen, Wiebke; Matuschewski, Kai; Ingmundson, Alyssa

2015-01-01

Remodeling of the host red blood cell by Plasmodium falciparum is well established and crucial for infection and parasite virulence. Host cell modifications are not exclusive to human Plasmodium parasites and also occur in hepatocytes and erythrocytes infected with murine Plasmodium parasites. The recently described intra-erythrocytic P. berghei-induced structures (IBIS) share similarities to P. falciparum Maurer's clefts. It is shown here that a potential candidate IBIS1 homologue in P. falciparum, PfHYP12 (PF3D7_1301400), is partially exported into the erythrocyte cytoplasm. To analyze a potential similarity between IBIS and Maurer's clefts we expressed the signature protein of IBIS in P. falciparum parasites. Visualization of the tagged protein revealed that PbIBIS1 can be exported by P. falciparum and localizes to Maurer's clefts in P. falciparum-infected erythrocytes, which indicates that IBIS and Maurer's clefts may be evolutionarily conserved parasite-induced structures in infected erythrocytes. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Changes in Malaria Parasite Drug Resistance in an Endemic Population Over a 25-Year Period With Resulting Genomic Evidence of Selection

PubMed Central

Nwakanma, Davis C.; Duffy, Craig W.; Amambua-Ngwa, Alfred; Oriero, Eniyou C.; Bojang, Kalifa A.; Pinder, Margaret; Drakeley, Chris J.; Sutherland, Colin J.; Milligan, Paul J.; MacInnis, Bronwyn; Kwiatkowski, Dominic P.; Clark, Taane G.; Greenwood, Brian M.; Conway, David J.

2014-01-01

Background. Analysis of genome-wide polymorphism in many organisms has potential to identify genes under recent selection. However, data on historical allele frequency changes are rarely available for direct confirmation. Methods. We genotyped single nucleotide polymorphisms (SNPs) in 4 Plasmodium falciparum drug resistance genes in 668 archived parasite-positive blood samples of a Gambian population between 1984 and 2008. This covered a period before antimalarial resistance was detected locally, through subsequent failure of multiple drugs until introduction of artemisinin combination therapy. We separately performed genome-wide sequence analysis of 52 clinical isolates from 2008 to prospect for loci under recent directional selection. Results. Resistance alleles increased from very low frequencies, peaking in 2000 for chloroquine resistance-associated crt and mdr1 genes and at the end of the survey period for dhfr and dhps genes respectively associated with pyrimethamine and sulfadoxine resistance. Temporal changes fit a model incorporating likely selection coefficients over the period. Three of the drug resistance loci were in the top 4 regions under strong selection implicated by the genome-wide analysis. Conclusions. Genome-wide polymorphism analysis of an endemic population sample robustly identifies loci with detailed documentation of recent selection, demonstrating power to prospectively detect emerging drug resistance genes. PMID:24265439

Efficacy of chloroquine for the treatment of uncomplicated Plasmodium falciparum malaria in Honduras.

PubMed

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

2013-05-01

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.

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

PubMed Central

Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

2008-01-01

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

Cytokine profiles among patients co-infected with Plasmodium falciparum malaria and soil borne helminths attending Kampala International University Teaching Hospital, in Uganda.

PubMed

Bwanika, Richard; Kato, Charles D; Welishe, Johnson; Mwandah, Daniel C

2018-01-01

Malaria and helminths share the same geographical distribution in tropical Africa. Studies of the interaction of helminth and malaria co-infection in humans have been few and are mainly epidemiological, with little information on cellular immune responses. This study aimed to determine Cytokine profiles among patients co-infected with Plasmodium falciparum malaria and soil borne helminth attending Kampala International University Teaching Hospital (KIU). A case control study of 240 patients were recruited at KIU teaching hospital. Patients with Plasmodium falciparum malaria were 55 (22.9%) and those with soil-borne helminths were 63 (26.3%). The controls were 89 (37.1%), while those co-infected with Plasmodium falciparum malaria and soil-borne helminths were 33 (13.8%). Cases were defined as having a positive blood smear for P. falciparum malaria, those with helminths or co-infections of the two. Negative controls were those with a negative blood smear for P. falciparum malaria and those with no stool parasitic infections. Patients presenting with signs and symptoms of malaria or those suspected of having helminths were recruited for the study. A panel of five cytokines (IFN-γ, TNF-α, IL-6, TGF-β and IL-10) were assayed from plasma samples in patients with and without Plasmodium falciparum malaria, patients with and without helminth, and then those co-infected with the two diseases diagnosis was done using thick blood smears stained with 10% Giemsa and stool examination was done following the Kato Katz technique following standard procedures. The prevalence of Plasmodium falciparum malaria by sex was 28 (11.7%) and 27 (11.3%) in male and female respectively. The overall prevalence of soil borne helminth was 26.3%, and among those harbouring helminths, 13.8% were co-infected with Plasmodium falciparum. Cytokine levels significantly differed across Plasmodium falciparum malaria, soil borne helminth infected patients and health controls for IFN-γ (P = 0.023), IL-10 (P = 0.008) and TGF-β (P = 0.0001). Cytokine levels significantly differed across Plasmodium falciparum malaria, soil borne helminth infected patients and patients co-infected with Plasmodium falciparum malaria and soil borne helminth for IL-10 (P = 0.004), IL-6 (P = 0.011) and TGF-β (P = 0.003). An up-regulation of IFN-γ during Plasmodium falciparum malaria and an up-regulation of IL-10 and TGF-β in soil borne helminth infections was demonstrated. We demonstrate that co-infections of Plasmodium falciparum and soil borne helminth lead to an up-regulation of IL-10 and IL-6 and a down-regulation of TGF-β. Trial registration No17/10-16.

Effects of novel triple-stage antimalarial ionic liquids on lipid membrane models.

PubMed

Ferraz, Ricardo; Pinheiro, Marina; Gomes, Ana; Teixeira, Cátia; Prudêncio, Cristina; Reis, Salette; Gomes, Paula

2017-09-01

Primaquine-based ionic liquids, obtained by acid-base reaction between parent primaquine and cinnamic acids, were recently found as triple-stage antimalarial hits. These ionic compounds displayed significant activity against both liver- and blood-stage Plasmodium parasites, as well as against stage V P. falciparum parasites. Remarkably, blood-stage activity of the ionic liquids against both chloroquine-sensitive (3D7) and resistant (Dd2) P. falciparum strains was clearly superior to those of the respective covalent (amide) analogues and of parent primaquine. Having hypothesized that such behaviour might be ascribed to an enhanced ability of the ionic compounds to permeate into Plasmodium-infected erythrocytes, we have carried out a differential scanning calorimetry-based study of the interactions between the ionic liquids and membrane models. Results provide evidence, at the molecular level, that the primaquine-derived ionic liquids may contribute to an increased permeation of the parent drug into malaria-infected erythrocytes, which has relevant implications towards novel antimalarial approaches based on ionic liquids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of a novel trafficking pathway exporting a replication protein, Orc2 to nucleus via classical secretory pathway in Plasmodium falciparum.

PubMed

Sharma, Rahul; Sharma, Bhumika; Gupta, Ashish; Dhar, Suman Kumar

2018-05-01

Malaria parasites use an extensive secretory pathway to traffic a number of proteins within itself and beyond. In higher eukaryotes, Endoplasmic Reticulum (ER) membrane bound transcription factors such as SREBP are reported to get processed en route and migrate to nucleus under the influence of specific cues. However, a protein constitutively trafficked to the nucleus via classical secretory pathway has not been reported. Herein, we report the presence of a novel trafficking pathway in an apicomplexan, Plasmodium falciparum where a homologue of an Origin Recognition Complex 2 (Orc2) goes to the nucleus following its association with the ER. Our work highlights the unconventional role of ER in protein trafficking and reports for the first time an ORC homologue getting trafficked through such a pathway to the nucleus where it may be involved in DNA replication and other ancillary functions. Such trafficking pathways may have a profound impact on the cell biology of a malaria parasite and have significant implications in strategizing new antimalarials. Copyright © 2018 Elsevier B.V. All rights reserved.

Procalcitonin as a biomarker for severe Plasmodium falciparum disease: a critical appraisal of a semi-quantitative point-of-care test in a cohort of travellers with imported malaria.

PubMed

Hesselink, Dennis A; Burgerhart, Jan-Steven; Bosmans-Timmerarends, Hanna; Petit, Pieter; van Genderen, Perry J J

2009-09-01

Imported malaria occurs as a relatively rare event in developed countries. As a consequence, most clinicians have little experience in making clinical assessments of disease severity and decisions regarding the need for parenteral therapy or high-level monitoring. In this study, the diagnostic accuracy of procalcitonin (PCT) for severe Plasmodium falciparum disease was assessed in a cohort of 100 consecutive travellers with various species of imported malaria. In all patients, PCT was measured on admission with a semi-quantitative 'point-of-care' test. Patients with severe P. falciparum malaria had significantly higher median PCT levels on admission as compared with patients with uncomplicated P. falciparum disease. In addition, PCT levels in patients with non-falciparum malaria were also higher compared with patients with non-severe falciparum malaria but lower compared with severe P. falciparum malaria. At a cut-off point of 10 ng/mL, PCT had a sensitivity of 0,67 and a specificity of 0,94 for severe falciparum disease. However, at lower cut-off points the specificity and positive predictive value were rather poor although the sensitivity and negative predictive value remained high. Potential drawbacks in the interpretation of elevated PCT levels on admission may be caused by infections with non-falciparum species and by concomitant bacterial infections. Semi-quantitative determination of PCT on admission is of limited use in the initial clinical assessment of disease severity in travellers with imported malaria, especially in settings with limited experience with the treatment of malaria.

The weekly associations between climatic factors and Plasmodium vivax and Plasmodium falciparum malaria in China, 2005-2014.

PubMed

Hundessa, Samuel; Williams, Gail; Li, Shanshan; Guo, Jinpeng; Zhang, Wenyi; Guo, Yuming

2017-05-01

Meteorological factors play a crucial role in malaria transmission, but limited evidence is available from China. This study aimed to estimate the weekly associations between meteorological factors and Plasmodium vivax and Plasmodium falciparum malaria in China. The Distributed Lag Non-Linear Model was used to examine non-linearity and delayed effects of average temperature, rainfall, relative humidity, sunshine hours, wind speed and atmospheric pressure on malaria. Average temperature was associated with P. vivax and P. falciparum cases over long ranges of lags. The effect was more immediate on P. vivax (0-6 weeks) than on P. falciparum (1-9 weeks). Relative humidity was associated with P. vivax and P. falciparum over 8-10 weeks and 5-8 weeks lag, respectively. A significant effect of wind speed on P. vivax was observed at 0-2 weeks lag, but no association was found with P. falciparum. Rainfall had a decreasing effect on P. vivax, but no association was found with P. falciparum. Sunshine hours were negatively associated with P. falciparum, but the association was unclear for P. vixax. However, the effects of atmospheric pressure on both malaria types were not significant at any lag. Our study highlights a substantial effect of weekly climatic factors on P. vivax and P. falciparum malaria transmission in China, with different lags. This provides an evidence base for health authorities in developing a malaria early-warning system. © The Author 2017. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Elucidation of sulfadoxine resistance with structural models of the bifunctional Plasmodium falciparum dihydropterin pyrophosphokinase-dihydropteroate synthase.

PubMed

de Beer, Tjaart A P; Louw, Abraham I; Joubert, Fourie

2006-07-01

Resistance of the most virulent human malaria parasite, Plasmodium falciparum, to antifolates is spreading with increasing speed, especially in Africa. Antifolate resistance is mainly caused by point mutations in the P. falciparum dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) target proteins. Homology models of the bifunctional P. falciparum dihydropterin pyrophosphokinase-dihydropteroate synthase (PPPK-DHPS) enzyme as well as the separate domains complete with bound substrates were constructed using the crystal structures of Saccharomyces cerevisiae (PPPK-DHPS), Mycobacterium tuberculosis (DHPS), Bacillus anthracis (DHPS), and Escherichia coli (PPPK) as templates. The resulting structures were subsequently solvated and refined using molecular dynamics. The active site residues of DHPS are highly conserved in S. cerevisiae, M. tuberculosis, E. coli, S. aureus, and B. anthracis, an attribute also shared by P. falciparum DHPS. Sulfadoxine was superimposed into the equivalent position of the p-aminobenzoic acid substrate and its binding parameters were refined using minimization and molecular dynamics. Sulfadoxine appears to interact mainly with P. falciparum DHPS mainly through hydrophobic interactions. Rational explanations are provided by the model for the sulfadoxine resistance-causing effects of four of the five known mutations in P. falciparum DHPS. A possible structure for the bifunctional PPPK-DHPS was derived from the structure from the S. cerevisiae bifunctional enzyme. The active site residues of P. falciparum PPPK are also conserved when compared to S. cerevisiae, Haemophilus influenzae, and E. coli. The informative nature of these models opens up avenues for structure-based drug design approaches toward the development of alternative and more effective inhibitors of P. falciparum PPPK-DHPS.

Prevalence of molecular markers of anti-malarial drug resistance in Plasmodium vivax and Plasmodium falciparum in two districts of Nepal

PubMed Central

2011-01-01

Background Sulphadoxine-pyrimethamine (SP) and chloroquine (CQ) have been used in treatment of falciparum and vivax malaria in Nepal. Recently, resistance to both drugs have necessitated a change towards artemisinin combination therapy (ACT) against Plasmodium falciparum in highly 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 CQ and SP resistance in P. falciparum and Plasmodium vivax 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 and P. vivax for CQ (Pfcrt, Pfmdr1, Pvmdr1) and SP (Pfdhfr, Pfdhps, Pvdhfr), using various PCR-based methods. Results and discussion Positive P. vivax and P. falciparum infections were identified by PCR in 92 and 41 samples respectively. However, some of these were negative in subsequent PCRs. Based 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 the Pfmdr1 wild type haplotype was relatively low (35%). Molecular level of SP resistance in P. falciparum was found to be high. The most prevalent Pfdhfr haplotype was double mutant CNRNI (91%), while frequency of Pfdhps double mutant SGEAA and AGEAA were 38% and 33% respectively. Combined, the frequency of quadruple mutations (CNRNI-SGEAA/AGEAA) was 63%. Based on P. vivax samples, low CQ and SP resistance were most likely due to low prevalence of Pvmdr1 Y976F mutation (5%) and absence of triple/quadruple mutations in Pvdhfr. Conclusions Based on the limited number of samples, prevalence of CQ and SP resistance at molecular levels in the population in the study area were determined as high in P. falciparum and low in P. vivax. Therefore, CQ could still be used in the treatment of P. vivax infections, but this remains to be tested in vivo while the change to ACT for P. falciparum seems justified. PMID:21457533

New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach.

PubMed

Mata-Cantero, Lydia; Azkargorta, Mikel; Aillet, Fabienne; Xolalpa, Wendy; LaFuente, Maria J; Elortza, Felix; Carvalho, Ana Sofia; Martin-Plaza, Julio; Matthiesen, Rune; Rodriguez, Manuel S

2016-04-29

Malaria, caused by Plasmodium falciparum (P. falciparum), ranks as one of the most baleful infectious diseases worldwide. New antimalarial treatments are needed to face existing or emerging drug resistant strains. Protein degradation appears to play a significant role during the asexual intraerythrocytic developmental cycle (IDC) of P. falciparum. Inhibition of the ubiquitin proteasome system (UPS), a major intracellular proteolytic pathway, effectively reduces infection and parasite replication. P. falciparum and erythrocyte UPS coexist during IDC but the nature of their relationship is largely unknown. We used an approach based on Tandem Ubiquitin-Binding Entities (TUBEs) and 1D gel electrophoresis followed by mass spectrometry to identify major components of the TUBEs-associated ubiquitin proteome of both host and parasite during ring, trophozoite and schizont stages. Ring-exported protein (REX1), a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, was found to reach a maximum level of ubiquitylation in trophozoites stage. The Homo sapiens (H. sapiens) TUBEs associated ubiquitin proteome decreased during the infection, whereas the equivalent P. falciparum TUBEs-associated ubiquitin proteome counterpart increased. Major cellular processes such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. In this work we analyze for the first time the interconnection between Plasmodium and human red blood cells ubiquitin-regulated proteins in the context of infection. We identified a number of human and Plasmodium proteins whose ubiquitylation pattern changes during the asexual infective stage. We demonstrate that ubiquitylation of REX1, a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, peaks in trophozoites stage. The ubiquitin-proteome from P. falciparum infected red blood cells (iRBCs) revealed a significant host-parasite crosstalk, underlining the importance of ubiquitin-regulated proteolytic activities during the intraerythrocytic developmental cycle (IDC) of P. falciparum. Major cellular processes defined from gene ontology such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. Given the importance of ubiquitylation in the development of infectious diseases, this work provides a number of potential drug-target candidates that should be further explored. Copyright © 2016 Elsevier B.V. All rights reserved.

Performance of “VIKIA Malaria Ag Pf/Pan” (IMACCESS®), a new malaria rapid diagnostic test for detection of symptomatic malaria infections

PubMed Central

2012-01-01

Background Recently, IMACCESS® developed a new malaria test (VIKIA Malaria Ag Pf/Pan™), based on the detection of falciparum malaria (HRP-2) and non-falciparum malaria (aldolase). Methods The performance of this new malaria rapid diagnostic test (RDT) was assessed using 1,000 febrile patients seeking malaria treatment in four health centres in Cambodia from August to December 2011. The results of the VIKIA Malaria Ag Pf/Pan were compared with those obtained by microscopy, the CareStart Malaria™ RDT (AccessBio®) which is currently used in Cambodia, and real-time PCR (as “gold standard”). Results The best performances of the VIKIA Malaria Ag Pf/Pan™ test for detection of both Plasmodium falciparum and non-P. falciparum were with 20–30 min reading times (sensitivity of 93.4% for P. falciparum and 82.8% for non-P. falciparum and specificity of 98.6% for P. falciparum and 98.9% for non-P. falciparum) and were similar to those for the CareStart Malaria™ test. Conclusions This new RDT performs similarly well as other commercially available tests (especially the CareStart Malaria™ test, used as comparator), and conforms to the World Health Organization’s recommendations for RDT performance. It is a good alternative tool for the diagnosis of malaria in endemic areas. PMID:22920654

Associations Between Helminth Infections, Plasmodium falciparum Parasite Carriage and Antibody Responses to Sexual and Asexual Stage Malarial Antigens.

PubMed

Ateba-Ngoa, Ulysse; Jones, Sophie; Zinsou, Jeannot Fréjus; Honkpehedji, Josiane; Adegnika, Ayola Akim; Agobe, Jean-Claude Dejon; Massinga-Loembe, Marguerite; Mordmüller, Benjamin; Bousema, Teun; Yazdanbakhsh, Maria

2016-08-03

Infections with helminths and Plasmodium spp. overlap in their geographical distribution. It has been postulated that helminth infections may influence malarial transmission by altering Plasmodium falciparum gametocytogenesis. This cross-sectional study assessed the effect of helminth infections on P. falciparum gametocyte carriage and on humoral immune responses to sexual stage antigens in Gabon. Schistosoma haematobium and filarial infections as well as P. falciparum asexual forms and gametocyte carriage were determined. The antibody responses measured were to sexual (Pfs230, Pfs48/45) and asexual P. falciparum antigens (AMA1, MSP1, and GLURP). A total of 287 subjects were included. The prevalence of microscopically detectable P. falciparum asexual parasites was higher in S. haematobium-infected subjects in comparison to their uninfected counterparts (47% versus 26%, P = 0.003), but this was not different when filarial infections were considered. Plasmodium falciparum gametocyte carriage was similar between Schistosoma- or filaria-infected and uninfected subjects. We observed a significant decrease of Pfs48/45 immunoglobulin G titer in S. haematobium-infected subjects (P = 0.037), whereas no difference was seen for Pfs230 antibody titer, nor for antibodies to AMA1, MSP1, or GLURP. Our findings suggest an effect of S. haematobium on antibody responses to some P. falciparum gametocyte antigens that may have consequences for transmission-blocking immunity. © The American Society of Tropical Medicine and Hygiene.

Evaluation of wondfo rapid diagnostic kit (Pf-HRP2/PAN-pLDH) for diagnosis of malaria by using nano-gold immunochromatographic assay.

PubMed

Wu, Junlin; Peng, Yunping; Liu, Xiaoyun; Li, Wenmei; Tang, Shixing

2014-06-01

Prompt and accurate diagnosis is necessary to start adequate treatment for different affecting species including P. falciparum and P. vivax. Here we described the Wondfo Rapid diagnostic Kit (Pf-HRP2/PAN-pLDH) for the detection of P. falciparum and pan-plasmodium in patient specimen by using a nano-gold immunochromatographic assay. Our rapid assay adapted nano-gold labeling techniques and the monoclonal antibodies (mAbs) against both histidine rich protein-2 (Pf HRP-2) of P. falciparum and pan plasmodium-specific pLDH (pan pLDH). The established two-antibody sandwich immunochromatographic assay could detect P. falciparum and pan-plasmodium. The sensitivity and specificity of Wondfo rapid diagnostic kit were determined by comparing with the "gold standard" of microscopic examination of blood smears. In this study1023 blood samples were collected from outpatient clinics in China and Burma, and detected by both Wondfo kit and microscopic examination. The detection sensitivity and specificity of Wondfo rapid diagnostic kit were 96.46% and 99.67% for P. falciparum (HRP2), 95.03% and 99.24% for pLDH, 96.83% and 99.74% for non-falciparum species, 96.70% and 99.74% for P. vivax, respectively. These results indicate that Wondfo rapid diagnostic assay may be useful for detecting P. falciparum and non-P. falciparum (especially P.v.) in patient specimen.

Host age and Plasmodium falciparum multiclonality are associated with gametocyte prevalence: a 1-year prospective cohort study.

PubMed

Adomako-Ankomah, Yaw; Chenoweth, Matthew S; Tocker, Aaron M; Doumbia, Saibou; Konate, Drissa; Doumbouya, Mory; Keita, Abdoul S; Anderson, Jennifer M; Fairhurst, Rick M; Diakite, Mahamadou; Miura, Kazutoyo; Long, Carole A

2017-11-21

Since Plasmodium falciparum transmission relies exclusively on sexual-stage parasites, several malaria control strategies aim to disrupt this step of the life cycle. Thus, a better understanding of which individuals constitute the primary gametocyte reservoir within an endemic population, and the temporal dynamics of gametocyte carriage, especially in seasonal transmission settings, will not only support the effective implementation of current transmission control programmes, but also inform the design of more targeted strategies. A 1-year prospective cohort study was initiated in June 2013 with the goal of assessing the longitudinal dynamics of P. falciparum gametocyte carriage in a village in Mali with intense seasonal malaria transmission. A cohort of 500 individuals aged 1-65 years was recruited for this study. Gametocyte prevalence was measured monthly using Pfs25-specific RT-PCR, and analysed for the effects of host age and gender, seasonality, and multiclonality of P. falciparum infection over 1 year. Most P. falciparum infections (51-89%) in this population were accompanied by gametocytaemia throughout the 1-year period. Gametocyte prevalence among P. falciparum-positive individuals (proportion of gametocyte positive infections) was associated with age (p = 0.003) but not with seasonality (wet vs. dry) or gender. The proportion of gametocyte positive infections were similarly high in children aged 1-17 years (74-82% on median among 5 age groups), while older individuals had relatively lower proportion, and those aged > 35 years (median of 43%) had significantly lower than those aged 1-17 years (p < 0.05). Plasmodium falciparum-positive individuals with gametocytaemia were found to have significantly higher P. falciparum multiclonality than those without gametocytaemia (p < 0.033 in two different analyses). Taken together, these results suggest that a substantial proportion of Pf-positive individuals carries gametocytes throughout the year, and that age is a significant determinant of gametocyte prevalence among these P. falciparum-positive individuals. Furthermore, the presence of multiple P. falciparum genotypes in an infection, a common feature of P. falciparum infections in high transmission areas, is associated with gametocyte prevalence.

Fatty acid synthesis and pyruvate metabolism pathways remain active in dihydroartemisinin-induced dormant ring stages of Plasmodium falciparum.

PubMed

Chen, Nanhua; LaCrue, Alexis N; Teuscher, Franka; Waters, Norman C; Gatton, Michelle L; Kyle, Dennis E; Cheng, Qin

2014-08-01

Artemisinin (ART)-based combination therapy (ACT) is used as the first-line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action, there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART-induced ring-stage dormancy and recovery have been implicated as possible causes of recrudescence; however, little is known about the characteristics of dormant parasites, including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA)-induced dormancy and recovery. Transcription analysis showed an immediate downregulation for 10 genes following exposure to DHA but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly of genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, was also maintained. Additions of inhibitors for biotin acetyl-coenzyme A (CoA) carboxylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively, following DHA treatment. Our results demonstrate that most metabolic pathways are downregulated in DHA-induced dormant parasites. In contrast, fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum

PubMed Central

Martin, Rowena E; Henry, Roselani I; Abbey, Janice L; Clements, John D; Kirk, Kiaran

2005-01-01

Background The uptake of nutrients, expulsion of metabolic wastes and maintenance of ion homeostasis by the intraerythrocytic malaria parasite is mediated by membrane transport proteins. Proteins of this type are also implicated in the phenomenon of antimalarial drug resistance. However, the initial annotation of the genome of the human malaria parasite Plasmodium falciparum identified only a limited number of transporters, and no channels. In this study we have used a combination of bioinformatic approaches to identify and attribute putative functions to transporters and channels encoded by the malaria parasite, as well as comparing expression patterns for a subset of these. Results A computer program that searches a genome database on the basis of the hydropathy plots of the corresponding proteins was used to identify more than 100 transport proteins encoded by P. falciparum. These include all the transporters previously annotated as such, as well as a similar number of candidate transport proteins that had escaped detection. Detailed sequence analysis enabled the assignment of putative substrate specificities and/or transport mechanisms to all those putative transport proteins previously without. The newly-identified transport proteins include candidate transporters for a range of organic and inorganic nutrients (including sugars, amino acids, nucleosides and vitamins), and several putative ion channels. The stage-dependent expression of RNAs for 34 candidate transport proteins of particular interest are compared. Conclusion The malaria parasite possesses substantially more membrane transport proteins than was originally thought, and the analyses presented here provide a range of novel insights into the physiology of this important human pathogen. PMID:15774027

Drug resistance associated genetic polymorphisms in Plasmodium falciparum and Plasmodium vivax collected in Honduras, Central America.

PubMed

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

2011-12-19

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. falciparum and P. vivax is therefore essential also in Honduras.

Drug resistance associated genetic polymorphisms in Plasmodium falciparum and Plasmodium vivax collected in Honduras, Central America

PubMed Central

2011-01-01

Background 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. Methods 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. Results 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. Conclusion 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. falciparum and P. vivax is therefore essential also in Honduras. PMID:22183028

P. falciparum Modulates Erythroblast Cell Gene Expression in Signaling and Erythrocyte Production Pathways

PubMed Central

Tamez, Pamela A.; Liu, Hui; Wickrema, Amittha; Haldar, Kasturi

2011-01-01

Global, genomic responses of erythrocytes to infectious agents have been difficult to measure because these cells are e-nucleated. We have previously demonstrated that in vitro matured, nucleated erythroblast cells at the orthochromatic stage can be efficiently infected by the human malaria parasite Plasmodium falciparum. We now show that infection of orthochromatic cells induces change in 609 host genes. 592 of these transcripts are up-regulated and associated with metabolic and chaperone pathways unique to P. falciparum infection, as well as a wide range of signaling pathways that are also induced in related apicomplexan infections of mouse hepatocytes or human fibroblast cells. Our data additionally show that polychromatophilic cells, which precede the orthochromatic stage and are not infected when co-cultured with P. falciparum, up-regulate a small set of genes, at least two of which are associated with pathways of hematopoiesis and/or erythroid cell development. These data support the idea that P. falciparum affects erythropoiesis at multiple stages during erythroblast differentiation. Further P. falciparum may modulate gene expression in bystander erythroblasts and thus influence pathways of erythrocyte development. This study provides a benchmark of the host erythroblast cell response to infection by P. falciparum. PMID:21573240

Structural insights into a key carotenogenesis related enzyme phytoene synthase of P. falciparum: a novel drug target for malaria.

PubMed

Agarwal, Shalini; Sharma, Vijeta; Phulera, Swastik; Abdin, M Z; Ayana, R; Singh, Shailja

2015-12-01

Carotenoids represent a diverse group of pigments derived from the common isoprenoid precursors and fulfill a variety of critical functions in plants and animals. Phytoene synthase (PSY), a transferase enzyme that catalyzes the first specific step in carotenoid biosynthesis plays a central role in the regulation of a number of essential functions mediated via carotenoids. PSYs have been deeply investigated in plants, bacteria and algae however in apicomplexans it is poorly studied. In an effort to characterize PSY in apicomplexans especially the malaria parasite Plasmodium falciparum (P. falciparum), a detailed bioinformatics analysis is undertaken. We have analysed the Phylogenetic relationship of PSY also referred to as octaprenyl pyrophosphate synthase (OPPS) in P. falciparum with other taxonomic groups. Further, we in silico characterized the secondary and tertiary structures of P. falciparum PSY/OPPS and compared the tertiary structures with crystal structure of Thermotoga maritima (T. maritima) OPPS. Our results evidenced the resemblance of P. falciparum PSY with the active site of T. maritima OPPS. Interestingly, the comparative structural analysis revealed an unconserved unique loop in P. falciparum OPPS/PSY. Such structural insights might contribute novel accessory functions to the protein thus, offering potential drug targets.

Geographic Structuring of the Plasmodium falciparum Sarco(endo)plasmic Reticulum Ca2+ ATPase (PfSERCA) Gene Diversity

PubMed Central

Pinto, João; Gribaldo, Simonetta; Legrand, Eric; Niang, Makhtar; Kim, Nimol; Pharath, Lim; Volnay, Béatrice; Ekala, Marie Therese; Bouchier, Christiane; Fandeur, Thierry; Berzosa, Pedro; Benito, Agustin; Ferreira, Isabel Dinis; Ferreira, Cynthia; Vieira, Pedro Paulo; Alecrim, Maria das Graças; Mercereau-Puijalon, Odile; Cravo, Pedro

2010-01-01

Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp), of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates). This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte. PMID:20195531

Genetic variability and population structure of Plasmodium falciparum parasite populations from different malaria ecological regions of Kenya.

PubMed

Ingasia, Luicer A; Cheruiyot, Jelagat; Okoth, Sheila Akinyi; Andagalu, Ben; Kamau, Edwin

2016-04-01

Transmission intensity, movement of human and vector hosts, biogeographical features, and malaria control measures are some of the important factors that determine Plasmodium falciparum parasite genetic variability and population structure. Kenya has different malaria ecologies which might require different disease intervention methods. Refined parasite population genetic studies are critical for informing malaria control and elimination strategies. This study describes the genetic diversity and population structure of P. falciparum parasites from the different malaria ecological zones in Kenya. Twelve multi-locus microsatellite (MS) loci previously described were genotyped in 225 P. falciparum isolates collected between 2012 and 2013 from five sites; three in lowland endemic regions (Kisumu, Kombewa, and Malindi) and two in highland, epidemic regions (Kisii and Kericho). Parasites from the lowland endemic and highland epidemic regions of western Kenya had high genetic diversity compared to coastal lowland endemic region of Kenya [Malindi]. The Kenyan parasites had a mean genetic differentiation index (FST) of 0.072 (p=0.011). The multi-locus genetic analysis of the 12 MS revealed all the parasites had unique haplotypes. Significant linkage disequilibrium (LD) was observed in all the five parasite populations. Kisumu had the most significant index of association values (0.16; p<0.0001) whereas Kisii had the least significant index of association values (0.03; p<0.0001). Our data suggest high genetic diversity in Kenyan parasite population with the exception of parasite from Malindi where malaria has been on the decline. The presence of significant LD suggests that there is occurrence of inbreeding in the parasite population. Parasite populations from Kisii showed the strongest evidence for epidemic population structure whereas the rest of the regions showed panmixia. Defining the genetic diversity of the parasites in different ecological regions of Kenya after introduction of the artemether-lumefantrine is important in refining the spread of drug resistant strains and malaria transmission for more effective control and eventual elimination of malaria in Kenya. Copyright © 2015. Published by Elsevier B.V.

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand

PubMed Central

Sawaswong, Vorthon; Simpalipan, Phumin; Siripoon, Napaporn; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn

2015-01-01

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. PMID:25925176

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand.

PubMed

Sawaswong, Vorthon; Simpalipan, Phumin; Siripoon, Napaporn; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn

2015-04-01

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.

Higher Complexity of Infection and Genetic Diversity of Plasmodium vivax Than Plasmodium falciparum Across All Malaria Transmission Zones of Papua New Guinea.

PubMed

Fola, Abebe A; Harrison, G L Abby; Hazairin, Mita Hapsari; Barnadas, Céline; Hetzel, Manuel W; Iga, Jonah; Siba, Peter M; Mueller, Ivo; Barry, Alyssa E

2017-03-01

Abstract Plasmodium falciparum and Plasmodium vivax have varying transmission dynamics that are informed by molecular epidemiology. This study aimed to determine the complexity of infection and genetic diversity of P. vivax and P. falciparum throughout Papua New Guinea (PNG) to evaluate transmission dynamics across the country. In 2008-2009, a nationwide malaria indicator survey collected 8,936 samples from all 16 endemic provinces of PNG. Of these, 892 positive P. vivax samples were genotyped at PvMS16 and PvmspF3 , and 758 positive P. falciparum samples were genotyped at Pfmsp2 . The data were analyzed for multiplicity of infection (MOI) and genetic diversity. Overall, P. vivax had higher polyclonality (71%) and mean MOI (2.32) than P. falciparum (20%, 1.39). These measures were significantly associated with prevalence for P. falciparum but not for P. vivax . The genetic diversity of P. vivax ( PvMS16 : expected heterozygosity = 0.95, 0.85-0.98; PvMsp1F3 : 0.78, 0.66-0.89) was higher and less variable than that of P. falciparum ( Pfmsp2 : 0.89, 0.65-0.97). Significant associations of MOI with allelic richness (rho = 0.69, P = 0.009) and expected heterozygosity (rho = 0.87, P < 0.001) were observed for P. falciparum . Conversely, genetic diversity was not correlated with polyclonality nor mean MOI for P. vivax . The results demonstrate higher complexity of infection and genetic diversity of P. vivax across the country. Although P. falciparum shows a strong association of these parameters with prevalence, a lack of association was observed for P. vivax and is consistent with higher potential for outcrossing of this species.

Impact of Malaria Preexposure on Antiparasite Cellular and Humoral Immune Responses after Controlled Human Malaria Infection

PubMed Central

Obiero, Joshua M.; Shekalaghe, Seif; Hermsen, Cornelus C.; Mpina, Maxmillian; Bijker, Else M.; Roestenberg, Meta; Teelen, Karina; Billingsley, Peter F.; Sim, B. Kim Lee; James, Eric R.; Daubenberger, Claudia A.; Hoffman, Stephen L.; Abdulla, Salim

2015-01-01

To understand the effect of previous malaria exposure on antiparasite immune responses is important for developing successful immunization strategies. Controlled human malaria infections (CHMIs) using cryopreserved Plasmodium falciparum sporozoites provide a unique opportunity to study differences in acquisition or recall of antimalaria immune responses in individuals from different transmission settings and genetic backgrounds. In this study, we compared antiparasite humoral and cellular immune responses in two cohorts of malaria-naive Dutch volunteers and Tanzanians from an area of low malarial endemicity, who were subjected to the identical CHMI protocol by intradermal injection of P. falciparum sporozoites. Samples from both trials were analyzed in parallel in a single center to ensure direct comparability of immunological outcomes. Within the Tanzanian cohort, we distinguished one group with moderate levels of preexisting antibodies to asexual P. falciparum lysate and another that, based on P. falciparum serology, resembled the malaria-naive Dutch cohort. Positive P. falciparum serology at baseline was associated with a lower parasite density at first detection by quantitative PCR (qPCR) after CHMI than that for Tanzanian volunteers with negative serology. Post-CHMI, both Tanzanian groups showed a stronger increase in anti-P. falciparum antibody titers than Dutch volunteers, indicating similar levels of B-cell memory independent of serology. In contrast to the Dutch, Tanzanians failed to increase P. falciparum-specific in vitro recall gamma interferon (IFN-γ) production after CHMI, and innate IFN-γ responses were lower in P. falciparum lysate-seropositive individuals than in seronegative individuals. In conclusion, positive P. falciparum lysate serology can be used to identify individuals with better parasite control but weaker IFN-γ responses in circulating lymphocytes, which may help to stratify volunteers in future CHMI trials in areas where malaria is endemic. PMID:25776749

Population genetics of Plasmodium falciparum and Plasmodium vivax and asymptomatic malaria in Temotu Province, Solomon Islands

PubMed Central

2013-01-01

Background Temotu Province, Solomon Islands is progressing toward malaria elimination. A baseline survey conducted in 2008 showed that most Plasmodium infections in the province were of low parasite density and asymptomatic infections. To better understand mechanisms underlying these malaria transmission characteristics genetic diversity and relationships among Plasmodium falciparum and Plasmodium vivax populations in the province were examined. Methods Forty-five P. falciparum and 67 P. vivax samples collected in the 2008 baseline survey were successfully genotyped using eight P. falciparum and seven P. vivax microsatellite markers. Genetic diversity, relationships and distribution of both P. falciparum and P. vivax populations were analysed. Results Plasmodium falciparum population exhibited low diversity with 19 haplotypes identified and had closely related clusters indicating clonal expansion. Interestingly, a dominant haplotype was significantly associated with fever and high parasite density. In contrast, the P. vivax population was highly diverse with 58 haplotypes identified that were not closely related. Parasite populations between different islands in the province showed low genetic differentiation. Conclusion The low diversity and clonal population of P. falciparum population may partially account for clinical immunity developed against illness. However, it is possible that importation of a new P. falciparum strain was the major cause of illness. High diversity in P. vivax population and low relatedness between strains suggested clinical immunity to P. vivax may be maintained by different mechanisms. The genetic diversity, population structure and distribution of strains indicate that transmission of P. falciparum was low, but that of P. vivax was still high in 2008. These data will be useful for assessing changes in malaria transmission resulting from interventions. PMID:24261646

An interplay between 2 signaling pathways: Melatonin-cAMP and IP{sub 3}–Ca{sup 2+} signaling pathways control intraerythrocytic development of the malaria parasite Plasmodium falciparum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furuyama, Wakako; Enomoto, Masahiro; Mossaad, Ehab

Highlights: • A melatonin receptor antagonist blocked Ca{sup 2+} oscillation in P. falciparum and inhibited parasite growth. • P. falciparum development is controlled by Ca{sup 2+}- and cAMP-signaling pathways. • The cAMP-signaling pathway at ring form and late trophozoite stages governs parasite growth of P. falciparum. - Abstract: Plasmodium falciparum spends most of its asexual life cycle within human erythrocytes, where proliferation and maturation occur. Development into the mature forms of P. falciparum causes severe symptoms due to its distinctive sequestration capability. However, the physiological roles and the molecular mechanisms of signaling pathways that govern development are poorly understood. Ourmore » previous study showed that P. falciparum exhibits stage-specific spontaneous Calcium (Ca{sup 2+}) oscillations in ring and early trophozoites, and the latter was essential for parasite development. In this study, we show that luzindole (LZ), a selective melatonin receptor antagonist, inhibits parasite growth. Analyses of development and morphology of LZ-treated P. falciparum revealed that LZ severely disrupted intraerythrocytic maturation, resulting in parasite death. When LZ was added at ring stage, the parasite could not undergo further development, whereas LZ added at the trophozoite stage inhibited development from early into late schizonts. Live-cell Ca{sup 2+} imaging showed that LZ treatment completely abolished Ca{sup 2+} oscillation in the ring forms while having little effect on early trophozoites. Further, the melatonin-induced cAMP increase observed at ring and late trophozoite stage was attenuated by LZ treatment. These suggest that a complex interplay between IP{sub 3}–Ca{sup 2+} and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum.« less

Evaluation of cysteine proteases of Plasmodium vivax as antimalarial drug targets: sequence analysis and sensitivity to cysteine protease inhibitors.

PubMed

Na, Byoung-Kuk; Kim, Tong-Soo; Rosenthal, Philip J; Lee, Jong-Koo; Kong, Yoon

2004-10-01

Cysteine proteases perform critical roles in the life cycles of malaria parasites. In Plasmodium falciparum, treatment of cysteine protease inhibitors inhibits hemoglobin hydrolysis and blocks the parasite development in vitro and in vivo, suggesting that plasmodial cysteine proteases may be interesting targets for new chemotherapeutics. To determine whether sequence diversity may limit chemotherapy against Plasmodium vivax, we analyzed sequence variations in the genes encoding three cysteine proteases, vivapain-1, -2 and -3, in 22 wild isolates of P. vivax. The sequences were highly conserved among wild isolates. A small number of substitutions leading to amino acid changes were found, while they did not modify essential residues for the function or structure of the enzymes. The substrate specificities and sensitivities to synthetic cysteine protease inhibitors of vivapain-2 and -3 from wild isolates were also very similar. These results support the suggestion that cysteine proteases of P. vivax are promising antimalarial chemotherapeutic targets.

The efficiency of sporozoite transmission in the human malarias, Plasmodium falciparum and P. vivax*

PubMed Central

Burkot, T. R.; Graves, P. M.; Cattan, J. A.; Wirtz, R. A.; Gibson, F. D.

1987-01-01

Reported are malaria sporozoite and inoculation rates over a 1-year period in eight epidemiologically defined villages of different endemicity in Madang Province, Papua New Guinea. In the study, more than 41 000 wild-caught mosquitos were analysed for Plasmodium falciparum and P. vivax sporozoites by ELISA. In a given village the entomological inoculation rates correlated strongly with the prevalences of both these malarial parasites in children. However, the prevalence of P. falciparum infections in children was much higher than that of P. vivax, despite similar inoculation rates for the two species. These data suggest that in Papua New Guinea P. falciparum is more efficiently transmitted than P. vivax from mosquito to man. The increased efficiency of transmission of P. falciparum may be due to the heavier sporozoite densities in wild-caught mosquitos naturally infected with P. falciparum sporozoites that were tenfold greater than the sporozoite densities in mosquitos infected with P. vivax. PMID:3311441

Efficacy of Chloroquine for the Treatment of Uncomplicated Plasmodium falciparum Malaria in Honduras

PubMed Central

Torres, Rosa Elena Mejia; 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

2013-01-01

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. PMID:23458957

Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum

PubMed Central

Chiang, Annette N.; Valderramos, Juan-Carlos; Balachandran, Raghavan; Chovatiya, Raj J.; Mead, Brian P.; Schneider, Corinne; Bell, Samantha L.; Klein, Michael G.; Huryn, Donna M.; Chen, Xiaojiang S.; Day, Billy W.; Fidock, David A.; Wipf, Peter; Brodsky, Jeffrey L.

2009-01-01

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC50 values from 30 nM to 1.6 μM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents. PMID:19195901

HIV infection deregulates innate immunity to malaria despite combination antiretroviral therapy.

PubMed

Finney, Constance A M; Ayi, Kodjo; Wasmuth, James D; Sheth, Prameet M; Kaul, Rupert; Loutfy, Mona R; Kain, Kevin C; Serghides, Lena

2013-01-28

Malaria and HIV-1 adversely interact, with HIV-positive individuals suffering higher parasite burdens and worse clinical outcomes. However, the mechanisms underlying these disease interactions are unclear. We hypothesized that HIV coinfection impairs the innate immune response to malaria, and that combination antiretroviral therapy (cART) may restore this response. Our aim was to examine the innate inflammatory response of natural killer (NK), natural killer T (NKT), and γδ T-cells isolated from the peripheral blood of HIV-infected therapy-naive donors to malaria parasites, and determine the effect of cART on these responses. Freshly isolated peripheral blood mononuclear cells from 25 HIV-infected individuals pre-cART (month 0) and post-cART (months 3 and 6), and HIV-negative individuals at matched time-points, were cultured in the presence of Plasmodium falciparum parasitized erythrocytes. Supernatants and cells were collected to assess cytokine production and phenotypic changes. Compared to HIV-negative participants, NKT, NK, and γδ T-cell subsets from participants with chronic HIV infection showed marked differences, including decreased production of interferon γ (IFNγ) and tumor necrosis factor (TNF) in response to malaria parasites. IFNγ production was linked to interleukin-18 receptor (IL-18R) expression in all three cell types studied. Six months of cART provided partial cellular reconstitution but had no effect on IL-18R expression, or IFNγ and TNF production. These data suggest that HIV infection impairs the inflammatory response of innate effector cells to malaria, and that the response is not fully restored within 6 months of cART. This may contribute to higher parasite burdens and ineffective immune responses, and have implications for vaccination initiatives in coinfected individuals.

Phenylpropanoids and furanocoumarins as antibacterial and antimalarial constituents of the Bhutanese medicinal plant Pleurospermum amabile.

PubMed

Wangchuk, Phurpa; Pyne, Stephen G; Keller, Paul A; Taweechotipatr, Malai; Kamchonwongpaisane, Sumalee

2014-07-01

With the objective of determining safety and verifying the traditional uses of the Bhutanese medicinal plant, Pleurospermum amabile Craib & W. W. Smith, we investigated its crude extracts and the isolated phytochemicals for their biological activities. Four phenylpropanoids [(E)-isomyristicin (1), (E)-isoapiol (2), methyl eugenol (3) and (E)-isoelemicin (4)] and six furanocoumarins [psoralen (5), bergapten (6), isoimperatorin (7), isopimpinellin (8), oxypeucedanin hydrate (9) and oxypeucedanin methanolate (10)] were isolated from this plant. Among the test samples, compound 10 showed weak antibacterial activity against Bacillus subtilis and best antimalarial activity against the Plasmodium falciparum strains, TM4/8.2 (chloroquine and antifolate sensitive) and K1CB1 (multidrug resistant). None of the test samples showed cytotoxicity. This study generated scientific data that support the traditional medical uses of the plant.

Plasmodium falciparum malaria: Convergent evolutionary trajectories towards delayed clearance following artemisinin treatment.

PubMed

Wilairat, Prapon; Kümpornsin, Krittikorn; Chookajorn, Thanat

2016-05-01

Malaria is a major global health challenge with 300million new cases every year. The most effective regimen for treating Plasmodium falciparum malaria is based on artemisinin and its derivatives. The drugs are highly effective, resulting in rapid clearance of parasites even in severe P. falciparum malaria patients. During the last five years, artemisinin-resistant parasites have begun to emerge first in Cambodia and now in Thailand and Myanmar. At present, the level of artemisinin resistance is relatively low with clinical presentation of delayed artemisinin clearance (a longer time to reduce parasite load) and a small decrease in artemisinin sensitivity in cultured isolates. Nevertheless, multiple genetic loci associated with delayed parasite clearance have been reported, but they cannot account for a large portion of cases. Even the most well-studied kelch 13 propeller mutations cannot always predict the outcome of artemisinin treatment in vitro and in vivo. Here we propose that delayed clearance by artemisinin could be the result of convergent evolution, driven by multiple trajectories to overcome artemisinin-induced stress, but precluded to become full blown resistance by high fitness cost. Genetic association studies by several genome-wide approaches reveal linkage disequilibrium between multiple loci and delayed parasite clearance. Genetic manipulations at some of these loci already have resulted in loss in artemisinin sensitivity. The notion presented here is by itself consistent with existing evidence on artemisinin resistance and has the potential to be explored using available genomic data. Most important of all, molecular surveillance of artemisinin resistance based on multi-genic markers could be more informative than relying on any one particular molecular marker. Copyright © 2016 Elsevier Ltd. All rights reserved.

Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription

PubMed Central

2011-01-01

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 of pathogens with drug resistance phenotypes in vivo. PMID:21810278

Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

PubMed Central

Dogovski, Con; Xie, Stanley C.; Burgio, Gaetan; Bridgford, Jess; Mok, Sachel; McCaw, James M.; Chotivanich, Kesinee; Kenny, Shannon; Gnädig, Nina; Straimer, Judith; Bozdech, Zbynek; Fidock, David A.; Simpson, Julie A.; Dondorp, Arjen M.; Foote, Simon; Klonis, Nectarios; Tilley, Leann

2015-01-01

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance. PMID:25901609

A phenolic glycoside from Flacourtia indica induces heme mediated oxidative stress in Plasmodium falciparum and attenuates malaria pathogenesis in mice.

PubMed

Singh, Shiv Vardan; Manhas, Ashan; Singh, Suriya P; Mishra, Sonali; Tiwari, Nimisha; Kumar, Parmanand; Shanker, Karuna; Srivastava, Kumkum; Sashidhara, Koneni V; Pal, Anirban

2017-07-01

Flacourtia indica is especially popular among the various communities of many African countries where it is being used traditionally for the treatment of malaria. In our previous report, we have identified some phenolic glycosides from the aerial parts of F. indica as promising antiplasmodial agents under in vitro conditions. Antimalarial bioprospection of F. indica derived phenolic glycoside in Swiss mice (in vivo) with special emphasis on its mode of action. Chloroquine sensitive strain of Plasmodium falciparum was routinely cultured and used for the in vitro studies. The in vivo antimalarial potential of phenolic glycoside was evaluated against P. berghei in Swiss mice through an array of parameters viz., hematological, biochemical, chemo-suppression and mean survival time. 2-(6-benzoyl-β-d-glucopyranosyloxy)-7-(1α, 2α, 6α-trihydroxy-3-oxocyclohex-4-enoyl)-5-hydroxybenzyl alcohol (CPG), a phenolic glycoside isolated from the aerial parts of F. indica was found to exhibit promising antiplasmodial activity by arresting the P. falciparum growth at the trophozoite stage. Spectroscopic investigations reveal that CPG possesses a strong binding affinity with free heme moieties. In addition, these interactions lead to the inhibition of heme polymerization in malaria parasite, augmenting oxidative stress, and delaying the rapid growth of parasite. Under in-vivo condition, CPG exhibited significant antimalarial activity against P. berghei at 50 and 75mg/kg body weight through chemo-suppression of parasitemia and ameliorating the parasite induced inflammatory and oxidative (hepatic) imbalance in the experimental mice. CPG was found to be a potential antimalarial constituent of F. indica with an explored mechanism of action, which also offers the editing choices for developing CPG based antimalarial chemotypes. Copyright © 2017 Elsevier GmbH. All rights reserved.

ent-Pimarane and ent-Kaurane Diterpenes from Aldama discolor (Asteraceae) and Their Antiprotozoal Activity.

PubMed

Nogueira, Mauro S; Da Costa, Fernando B; Brun, Reto; Kaiser, Marcel; Schmidt, Thomas J

2016-09-15

Aldama discolor (syn.Viguiera discolor) is an endemic Asteraceae from the Brazilian "Cerrado", which has not previously been investigated for its chemical constituents and biological activity. Diterpenes are common secondary metabolites found in Aldama species, some of which have been reported to present potential antiprotozoal and antimicrobial activities. In this study, the known ent-3-α-hydroxy-kaur-16-en-18-ol (1), as well as three new diterpenes, namely, ent-7-oxo-pimara-8,15-diene-18-ol (2), ent-2S,4S-2-19-epoxy-pimara-8(3),15-diene-7β-ol (3) and ent-7-oxo-pimara-8,15-diene-3β-ol (4), were isolated from the dichloromethane extract of A. discolor leaves and identified by means of MS and NMR. The compounds were assayed in vitro against Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani, Plasmodium falciparum and also tested for cytotoxicity against mammalian cells (L6 cell line). The ent-kaurane 1 showed significant in vitro activity against both P. falciparum (IC 50 = 3.5 μ M) and L. donovani (IC 50 = 2.5 μ M) and ent-pimarane 2 against P. falciparum (IC 50 = 3.8 μ M). Both compounds returned high selectivity indices (SI >10) in comparison with L6 cells, which makes them interesting candidates for in vivo tests. In addition to the diterpenes, the sesquiterpene lactone budlein A (5), which has been reported to possess a strong anti-T. b. rhodesiense activity, was identified as major compound in the A. discolor extract and explains its high activity against this parasite (100% growth inhibition at 2 μ g/mL).

A quantitative liquid chromatography tandem mass spectrometry method for metabolomic analysis of Plasmodium falciparum lipid related metabolites.

PubMed

Vo Duy, S; Besteiro, S; Berry, L; Perigaud, C; Bressolle, F; Vial, H J; Lefebvre-Tournier, I

2012-08-20

Plasmodium falciparum is the causative agent of malaria, a deadly infectious disease for which treatments are scarce and drug-resistant parasites are now increasingly found. A comprehensive method of identifying and quantifying metabolites of this intracellular parasite could expand the arsenal of tools to understand its biology, and be used to develop new treatments against the disease. Here, we present two methods based on liquid chromatography tandem mass spectrometry for reliable measurement of water-soluble metabolites involved in phospholipid biosynthesis, as well as several other metabolites that reflect the metabolic status of the parasite including amino acids, carboxylic acids, energy-related carbohydrates, and nucleotides. A total of 35 compounds was quantified. In the first method, polar compounds were retained by hydrophilic interaction chromatography (amino column) and detected in negative mode using succinic acid-(13)C(4) and fluorovaline as internal standards. In the second method, separations were carried out using reverse phase (C18) ion-pair liquid chromatography, with heptafluorobutyric acid as a volatile ion pairing reagent in positive detection mode, using d(9)-choline and 4-aminobutanol as internal standards. Standard curves were performed in P. falciparum-infected and uninfected red blood cells using standard addition method (r(2)>0.99). The intra- and inter-day accuracy and precision as well as the extraction recovery of each compound were determined. The lower limit of quantitation varied from 50pmol to 100fmol/3×10(7)cells. These methods were validated and successfully applied to determine intracellular concentrations of metabolites from uninfected host RBCs and isolated Plasmodium parasites. Copyright © 2012 Elsevier B.V. All rights reserved.

COMPARISON OF A GENUS-SPECIFIC CONVENTIONAL PCR AND A SPECIES-SPECIFIC NESTED-PCR FOR MALARIA DIAGNOSIS USING FTA COLLECTED SAMPLES FROM KINGDOM OF SAUDI ARABIA.

PubMed

Al-Harthi, Saeed A

2015-12-01

Molecular tools are increasingly accepted as the most sensitive and reliable techniques for malaria diagnosis and epidemiological surveys. Also, collection of finger prick blood spots onto filter papers is the most simple and affordable method for samples preservation and posterior molecular analysis, especially in rural endemic regions where malaria remains a major health problem. Two malaria molecular diagnostic tests, a Plasmodium genus-specific conventional PCR and a Plasmodium species-specific Nested PCR, were evaluated using DNA templates prepared from Whatman-FTA cards' dry blood spots using both, Methanol-fixation/Heat-extraction and FTA commercial purification kit. A total of 121 blood samples were collected from six Saudi south-western endemic districts both, as thick and thin films for routine microscopic screening and onto FTA cards for molecular studies. Out of the 121 samples, 75 were P. falciparum positive by at least one technique. No other species of Plasmodium were detected. P. falciparum parasites were identified in 69/75 (92%) samples by microscopic screening in health care centers. P. genus-specific PCR was able to amplify P. falciparum DNA in 41/75 (55%) and 59/75 (79%) samples using Methanol-fixation/Heat-extraction and FTA purification kit, respectively. P. species-specific Nested PCR revealed 68/75 (91%) and 75/75 (100%) positive samples using DNA templates were isolated by Methanol-fixation/Heat- extraction and FTA purification methods, respectively. The species-specific Nested PCR applied to Whatman-FTA preserved and processed blood samples represents the best alternative to classical microscopy for malaria diagnosis, particularly in epidemiological screening.

Antimalarial activity of Garcinia mangostana L rind and its synergistic effect with artemisinin in vitro.

PubMed

Tjahjani, Susy

2017-02-28

Malaria especially falciparum malaria still causes high morbidity and mortality in tropical countries. Several factors have been linked to this situation and the most important one is the rapid spread of parasite resistance to the currently available antimalarials, including artemisinin. Artemisinin is the main component of the currently recommended antimalarial, artemisinin based combination therapy (ACT), and it is a free radical generating antimalarial. Garcinia mangostana L (mangosteen) rind contain a lot of xanthone compounds acting as an antioxidant and exhibited antimalarial activity. The aim of this study was to evaluate the antimalarial activity of mangosteen rind extract and its fractions and their interaction with artemisinin against the 3D7 clone of Plasmodium falciparum in vitro. Dry ripe mangosteen rind was extracted with ethanol followed by fractionation with hexane, ethylacetate, buthanol, and water consecutively to get ethanol extract, hexane, athylacetate, buthanol, and water fractions. Each of these substances was diluted in DMSO and examined for antimalarial activity either singly or in combination with artemisinin in vitro against Plasmodium falciparum 3D7 clone. Synergism between these substances with artemisinin was evaluated according to certain formula to get the sum of fractional inhibitory concentration 50 (∑FIC 50 ). Analysis of the parasite growth in vitro indicated that IC 50 of these mangosteen rind extract, hexane, ethylacetate, buthanol, and water fraction ranged from 0.41 to > 100 μg/mL. All of the ∑FIC50 were <1. This study demonstrated a promising antimalarial activity of the extract and fractions of G.mangostana L rind and its synergistic effect with artemisinin. Further study using lead compound(s) isolated from extract and fractions should be performed to identify more accurately their mechanism of antimalarial activities.

Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria.

PubMed

Lucchi, Naomi W; Demas, Allison; Narayanan, Jothikumar; Sumari, Deborah; Kabanywanyi, Abdunoor; Kachur, S Patrick; Barnwell, John W; Udhayakumar, Venkatachalam

2010-10-29

Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.

Imported Malaria in Turkey: The Importance of Diagnosis and Treatment of Plasmodium falciparum/Plasmodium vivax Mixed Infection.

PubMed

Tünger, Özlem; Çakmak, Akide; Özbilgin, Ahmet; Tunalı, Varol; Çetin, Çiğdem Banu

2018-05-21

The most common types of malaria in the world are Plasmodium vivax and P. falciparum. In countries where both species are endemic, P. vivax and P. falciparum coinfection also occurs. Thus, the possibility of mixed malaria in Turkey should always be considered in cases with a traveling history to these countries. Here, we report a case of P. vivax/P. falciparum mixed infection that was diagnosed as P. falciparum malaria in Ethiopia. However, the administered treatment was inadequate, and infection recurred because of the miss in the diagnosis of P. vivax malaria, for which an effective drug for hypnozoites was not administered. This case report emphasizes the importance of diagnosis, correct and adequate treatment of infections, and a close follow-up of diseases.

Prevalence and Clinical Manifestations of Malaria in Aligarh, India

PubMed Central

Asma, Umm-e; Taufiq, Farha

2014-01-01

Malaria is one of the most widespread infectious diseases of tropical countries with an estimated 207 million cases globally. In India, there are endemic pockets of this disease, including Aligarh. Hundreds of Plasmodium falciparum and P. vivax cases with severe pathological conditions are recorded every year in this district. The aim of this study is to find out changes in liver enzymes and kidney markers. Specific diagnosis for P. falciparum and P. vivax was made by microscopic examination of Giemsa stained slides. Clinical symptoms were observed in both of these infections. Liver enzymes, such as AST, ALT, and ALP, and kidney function markers, such as creatinine and urea, were estimated by standard biochemical techniques. In Aligarh district, P. vivax, P. falciparum, and mixed infections were 64%, 34%, and 2%, respectively. In case of P. falciparum infection, the incidences of anemia, splenomegaly, renal failure, jaundice, and neurological sequelae were higher compared to those in P. vivax infection. Recrudescence and relapse rates were 18% and 20% in P. falciparum and P. vivax infections, respectively. Liver dysfunctions and renal failures were more common in P. falciparum patients, particularly in elderly patients. Artesunate derivatives must, therefore, be introduced for the treatment of P. falciparum as they resist to chloroquine as well as sulfadoxine-pyrimethamine combinations. PMID:25548413

Prevalence and clinical manifestations of malaria in Aligarh, India.

PubMed

Asma, Umm-e; Taufiq, Farha; Khan, Wajihullah

2014-12-01

Malaria is one of the most widespread infectious diseases of tropical countries with an estimated 207 million cases globally. In India, there are endemic pockets of this disease, including Aligarh. Hundreds of Plasmodium falciparum and P. vivax cases with severe pathological conditions are recorded every year in this district. The aim of this study is to find out changes in liver enzymes and kidney markers. Specific diagnosis for P. falciparum and P. vivax was made by microscopic examination of Giemsa stained slides. Clinical symptoms were observed in both of these infections. Liver enzymes, such as AST, ALT, and ALP, and kidney function markers, such as creatinine and urea, were estimated by standard biochemical techniques. In Aligarh district, P. vivax, P. falciparum, and mixed infections were 64%, 34%, and 2%, respectively. In case of P. falciparum infection, the incidences of anemia, splenomegaly, renal failure, jaundice, and neurological sequelae were higher compared to those in P. vivax infection. Recrudescence and relapse rates were 18% and 20% in P. falciparum and P. vivax infections, respectively. Liver dysfunctions and renal failures were more common in P. falciparum patients, particularly in elderly patients. Artesunate derivatives must, therefore, be introduced for the treatment of P. falciparum as they resist to chloroquine as well as sulfadoxine-pyrimethamine combinations.

Genome-Level Determination of Plasmodium falciparum Blood-Stage Targets of Malarial Clinical Immunity in the Peruvian Amazon

PubMed Central

Torres, Katherine J.; Castrillon, Carlos E.; Moss, Eli L.; Saito, Mayuko; Tenorio, Roy; Molina, Douglas M.; Davies, Huw; Neafsey, Daniel E.; Felgner, Philip; Vinetz, Joseph M.; Gamboa, Dionicia

2015-01-01

Background. Persons with blood-stage Plasmodium falciparum parasitemia in the absence of symptoms are considered to be clinically immune. We hypothesized that asymptomatic subjects with P. falciparum parasitemia would differentially recognize a subset of P. falciparum proteins on a genomic scale. Methods and Findings. Compared with symptomatic subjects, sera from clinically immune, asymptomatically infected individuals differentially recognized 51 P. falciparum proteins, including the established vaccine candidate PfMSP1. Novel, hitherto unstudied hypothetical proteins and other proteins not previously recognized as potential vaccine candidates were also differentially recognized. Genes encoding the proteins differentially recognized by the Peruvian clinically immune individuals exhibited a significant enrichment of nonsynonymous nucleotide variation, an observation consistent with these genes undergoing immune selection. Conclusions. A limited set of P. falciparum protein antigens was associated with the development of naturally acquired clinical immunity in the low-transmission setting of the Peruvian Amazon. These results imply that, even in a low-transmission setting, an asexual blood-stage vaccine designed to reduce clinical malaria symptoms will likely need to contain large numbers of often-polymorphic proteins, a finding at odds with many current efforts in the design of vaccines against asexual blood-stage P. falciparum. PMID:25381370

Towards development of aptamers that specifically bind to lactate dehydrogenase of Plasmodium falciparum through epitopic targeting.

PubMed

Frith, Kelly-Anne; Fogel, Ronen; Goldring, J P Dean; Krause, Robert G E; Khati, Makobetsa; Hoppe, Heinrich; Cromhout, Mary E; Jiwaji, Meesbah; Limson, Janice L

2018-05-03

Early detection is crucial for the effective treatment of malaria, particularly in those cases infected with Plasmodium falciparum. There is a need for diagnostic devices with the capacity to distinguish P. falciparum from other strains of malaria. Here, aptamers generated against targeted species-specific epitopes of P. falciparum lactate dehydrogenase (rPfLDH) are described. Two classes of aptamers bearing high binding affinity and specificity for recombinant P. falciparum lactate dehydrogenase (rPfLDH) and P. falciparum-specific lactate dehydrogenase epitopic oligopeptide (LDHp) were separately generated. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers reported here and previously published, confirming their importance in recognition of the target, while novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Aptamers with diagnostically-supportive functions were synthesized, prime examples of which are the aptamers designated as LDHp 1, LDHp 11 and rLDH 4 and rLDH 15 in work presented herein. Of the sampled aptamers raised against the recombinant protein, rLDH 4 showed the highest binding to the target rPfLDH in the ELONA assay, with both rLDH 4 and rLDH 15 indicating an ability to discriminate between rPfLDH and rPvLDH. LDHp 11 was generated against a peptide selected as a unique P. falciparum LDH peptide. The aptamer, LDHp 11, like antibodies against the same peptide, only detected rPfLDH and discriminated between rPfLDH and rPvLDH. This was supported by affinity binding experiments where only aptamers generated against a unique species-specific epitope showed an ability to preferentially bind to rPfLDH relative to rPvLDH rather than those generated against the whole recombinant protein. In addition, rLDH 4 and LDHp 11 demonstrated in situ binding to P. falciparum cells during confocal microscopy. The utilization and application of LDHp 11, an aptamer generated against a unique species-specific epitope of P. falciparum LDH indicated the ability to discriminate between recombinant P. falciparum and Plasmodium vivax LDH. This aptamer holds promise as a biorecognition element in malaria diagnostic devices for the detection, and differentiation, of P. falciparum and P. vivax malaria infections. This study paves the way to explore aptamer generation against targeted species-specific epitopes of other Plasmodium species.

Effect of combining mosquito repellent and insecticide treated net on malaria prevalence in Southern Ethiopia: a cluster-randomised trial.

PubMed

Deressa, Wakgari; Yihdego, Yemane Y; Kebede, Zelalem; Batisso, Esey; Tekalegne, Agonafer; Dagne, Getachew A

2014-03-28

A mosquito repellent has the potential to prevent malaria infection, but there has been few studies demonstrating the effectiveness of combining this strategy with the highly effective long-lasting insecticidal nets (LLINs). This study aimed to determine the effect of combining community-based mosquito repellent with LLINs in the reduction of malaria. A community-based clustered-randomised trial was conducted in 16 rural villages with 1,235 households in southern Ethiopia between September and December of 2008. The villages were randomly assigned to intervention (mosquito repellent and LLINs, eight villages) and control (LLINs alone, eight villages) groups. Households in the intervention villages received mosquito repellent (i.e., Buzz-Off petroleum jelly, essential oil blend) applied every evening. The baseline survey was followed by two follow-up surveys, at one month interval. The primary outcome was detection of Plasmodium falciparum, Plasmodium vivax, or both parasites, through microscopic examination of blood slides. Analysis was by intention to treat. Baseline imbalances and clustering at individual, household and village levels were adjusted using a generalized linear mixed model. 3,078 individuals in intervention and 3,004 in control group were enrolled into the study. Compared with the control arm, the combined use of mosquito repellent and LLINs significantly reduced malaria infection of all types over time [adjusted Odds Ratio (aOR) = 0.66; 95% CI = 0.45-0.97]. Similarly, a substantial reduction in P. falciparum malaria infection during the follow-up surveys was observed in the intervention group (aOR = 0.53, 95% CI = 0.31-0.89). The protective efficacy of using mosquito repellent and LLINs against malaria infection of both P. falciparum/P. vivax and P. falciparum was 34% and 47%, respectively. Daily application of mosquito repellent during the evening followed by the use of LLINs during bedtime at community level has significantly reduced malaria infection. The finding has strong implication particularly in areas where malaria vectors feed mainly in the evening before bedtime. ClinicalTrials.gov identifier: NCT01160809.

Molecular evidence of high rates of asymptomatic P. vivax infection and very low P. falciparum malaria in Botswana.

PubMed

Motshoge, Thato; Ababio, Grace K; Aleksenko, Larysa; Read, John; Peloewetse, Elias; Loeto, Mazhani; Mosweunyane, Tjantilili; Moakofhi, Kentse; Ntebele, Davies S; Chihanga, Simon; Motlaleng, Mpho; Chinorumba, Anderson; Vurayai, Moses; Pernica, Jeffrey M; Paganotti, Giacomo M; Quaye, Isaac K

2016-09-29

Botswana is one of eight SADC countries targeting malaria elimination by 2018. Through spirited upscaling of control activities and passive surveillance, significant reductions in case incidence of Plasmodium falciparum (0.96 - 0.01) was achieved between 2008 and 2012. As part of the elimination campaign, active detection of asymptomatic Plasmodium species by a highly sensitive method was deemed necessary. This study was carried out to determine asymptomatic Plasmodium species carriage by nested PCR in the country, in 2012. A cross-sectional study involving 3924 apparently healthy participants were screened for Plasmodium species in 14 districts (5 endemic: Okavango, Ngami, Tutume, Boteti and Bobirwa; and 9 epidemic: North East, Francistown, Serowe-Palapye, Ghanzi, Kweneng West, Kweneng East, Kgatleng, South East, and Good Hope). Venous blood was taken from each participant for a nested PCR detection of Plasmodium species. The parasite rates of asymptomatic Plasmodium species detected were as follows: Plasmodium falciparum, 0.16 %; Plasmodium vivax, 4.66 %; Plasmodium malariae, (Pm) 0.16 %; Plasmodium ovale, 0 %, mixed infections (P. falciparum and P. vivax), 0.055 %; and (P. vivax and P. malariae), 0.027 %, (total: 5.062 %). The high proportion of asymptomatic reservoir of P. vivax was clustered in the East, South Eastern and Central districts of the country. There appeared to be a correlation between the occurrence of P. malariae infection with P. vivax infection, with the former only occurring in districts that had substantial P. vivax circulation. The median age among 2-12 year olds for P. vivax infection was 5 years (Mean 5.13 years, interquartile range 3-7 years). The odds of being infected with P. vivax decreased by 7 % for each year increase in age (OR 0.93, 95 % CI 0.87-1.00, p = 0.056). We have confirmed low parasite rate of asymptomatic Plasmodium species in Botswana, with the exception of P.vivax which was unexpectedly high. This has implication for the elimination campaign so a follow up study is warranted to inform decisions on new strategies that take this evidence into account in the elimination campaign.

Molecular determinants of sulfadoxine-pyrimethamine resistance in Plasmodium falciparum in Nigeria and the regional emergence of dhps 431V.

PubMed

Oguike, Mary C; Falade, Catherine O; Shu, Elvis; Enato, Izehiuwa G; Watila, Ismaila; Baba, Ebenezer S; Bruce, Jane; Webster, Jayne; Hamade, Prudence; Meek, Sylvia; Chandramohan, Daniel; Sutherland, Colin J; Warhurst, David; Roper, Cally

2016-12-01

There are few published reports of mutations in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes in P. falciparum populations in Nigeria, but one previous study has recorded a novel dhps mutation at codon 431 among infections imported to the United Kingdom from Nigeria. To assess how widespread this mutation is among parasites in different parts of the country and consequently fill the gap in sulfadoxine-pyrimethamine (SP) resistance data in Nigeria, we retrospectively analysed 1000 filter paper blood spots collected in surveys of pregnant women and children with uncomplicated falciparum malaria between 2003 and 2015 from four sites in the south and north. Genomic DNA was extracted from filter paper blood spots and placental impressions. Point mutations at codons 16, 50, 51, 59, 108, 140 and 164 of the dhfr gene and codons 431, 436, 437, 540, 581 and 613 of the dhps gene were evaluated by nested PCR amplification followed by direct sequencing. The distribution of the dhps-431V mutation was widespread throughout Nigeria with the highest prevalence in Enugu (46%). In Ibadan where we had sequential sampling, its prevalence increased from 0% to 6.5% between 2003 and 2008. Although there were various combinations of dhps mutations with 431V, the combination 431V + 436A + 437G+581G+613S was the most common. All these observations support the view that dhps-431V is on the increase. In addition, P. falciparum DHPS crystal structure modelling shows that the change from Isoleucine to Valine (dhps-431V) could alter the effects of both S436A/F and A437G, which closely follow the 2nd β-strand. Consequently, it is now a research priority to assess the implications of dhps-VAGKGS mutant haplotype on continuing use of SP in seasonal malaria chemoprevention (SMC) and intermittent preventive treatment in pregnancy (IPTp). Our data also provides surveillance data for SP resistance markers in Nigeria between 2003 and 2015. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity.

PubMed

Bello, Angelica M; Poduch, Ewa; Fujihashi, Masahiro; Amani, Merhnaz; Li, Yan; Crandall, Ian; Hui, Raymond; Lee, Ping I; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2007-03-08

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.

A HECT Ubiquitin-Protein Ligase as a Novel Candidate Gene for Altered Quinine and Quinidine Responses in Plasmodium falciparum

PubMed Central

Sanchez, Cecilia P.; Cyrklaff, Marek; Mu, Jianbing; Ferdig, Michael T.; Stein, Wilfred D.; Lanzer, Michael

2014-01-01

The emerging resistance to quinine jeopardizes the efficacy of a drug that has been used in the treatment of malaria for several centuries. To identify factors contributing to differential quinine responses in the human malaria parasite Plasmodium falciparum, we have conducted comparative quantitative trait locus analyses on the susceptibility to quinine and also its stereoisomer quinidine, and on the initial and steady-state intracellular drug accumulation levels in the F1 progeny of a genetic cross. These data, together with genetic screens of field isolates and laboratory strains associated differential quinine and quinidine responses with mutated pfcrt, a segment on chromosome 13, and a novel candidate gene, termed MAL7P1.19 (encoding a HECT ubiquitin ligase). Despite a strong likelihood of association, episomal transfections demonstrated a role for the HECT ubiquitin-protein ligase in quinine and quinidine sensitivity in only a subset of genetic backgrounds, and here the changes in IC50 values were moderate (approximately 2-fold). These data show that quinine responsiveness is a complex genetic trait with multiple alleles playing a role and that more experiments are needed to unravel the role of the contributing factors. PMID:24830312

Bifurcatriol, a New Antiprotozoal Acyclic Diterpene from the Brown Alga Bifurcaria bifurcata

PubMed Central

Smyrniotopoulos, Vangelis; Merten, Christian; Kaiser, Marcel; Tasdemir, Deniz

2017-01-01

Linear diterpenes that are commonly found in brown algae are of high chemotaxonomic and ecological importance. This study reports bifurcatriol (1), a new linear diterpene featuring two stereogenic centers isolated from the Irish brown alga Bifurcaria bifurcata. The gross structure of this new natural product was elucidated based on its spectroscopic data (IR, 1D and 2D-NMR, HRMS). Its absolute configuration was identified by experimental and computational vibrational circular dichroism (VCD) spectroscopy, combined with the calculation of 13C-NMR chemical shielding constants. Bifurcatriol (1) was tested for in vitro antiprotozoal activity towards a small panel of parasites (Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi, and Leishmania donovani) and cytotoxicity against mammalian primary cells. The highest activity was exerted against the malaria parasite P. falciparum (IC50 value 0.65 μg/mL) with low cytotoxicity (IC50 value 56.6 μg/mL). To our knowledge, this is the first successful application of VCD and DP4 probability analysis of the calculated 13C-NMR chemical shifts for the simultaneous assignment of the absolute configuration of multiple stereogenic centers in a long-chain acyclic natural product. PMID:28767061

Structural and Immunological Characterization of Recombinant 6-Cysteine Domains of the Plasmodium falciparum Sexual Stage Protein Pfs230*

PubMed Central

MacDonald, Nicholas J.; Nguyen, Vu; Shimp, Richard; Reiter, Karine; Herrera, Raul; Burkhardt, Martin; Muratova, Olga; Kumar, Krishan; Aebig, Joan; Rausch, Kelly; Lambert, Lynn; Dawson, Nikiah; Sattabongkot, Jetsumon; Ambroggio, Xavier; Duffy, Patrick E.; Wu, Yimin; Narum, David L.

2016-01-01

Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel® appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine. Pfs230, a 230-kDa sexual stage protein expressed in gametocytes is an alternative vaccine candidate. A unique 6-cysteine-rich domain structure within Pfs230 have thwarted its recombinant expression and characterization for clinical evaluation for nearly a quarter of a century. Here, we report on the identification, biochemical, biophysical, and immunological characterization of recombinant Pfs230 domains. Rabbit antibodies generated against recombinant Pfs230 domains blocked mosquito transmission of a laboratory strain and two field isolates using an ex vivo assay. A planned clinical trial of the Pfs230 vaccine is a significant step toward the potential development of a transmission blocking vaccine to eliminate malaria. PMID:27432885

Genomic Analysis Reveals a Common Breakpoint in Amplifications of the Plasmodium vivax Multidrug Resistance 1 Locus in Thailand.

PubMed

Auburn, Sarah; Serre, David; Pearson, Richard D; Amato, Roberto; Sriprawat, Kanlaya; To, Sheren; Handayuni, Irene; Suwanarusk, Rossarin; Russell, Bruce; Drury, Eleanor; Stalker, Jim; Miotto, Olivo; Kwiatkowski, Dominic P; Nosten, Francois; Price, Ric N

2016-10-15

In regions of coendemicity for Plasmodium falciparum and Plasmodium vivax where mefloquine is used to treat P. falciparum infection, drug pressure mediated by increased copy numbers of the multidrug resistance 1 gene (pvmdr1) may select for mefloquine-resistant P. vivax Surveillance is not undertaken routinely owing in part to methodological challenges in detection of gene amplification. Using genomic data on 88 P. vivax samples from western Thailand, we identified pvmdr1 amplification in 17 isolates, all exhibiting tandem copies of a 37.6-kilobase pair region with identical breakpoints. A novel breakpoint-specific polymerase chain reaction assay was designed to detect the amplification. The assay demonstrated high sensitivity, identifying amplifications in 13 additional, polyclonal infections. Application to 132 further samples identified the common breakpoint in all years tested (2003-2015), with a decline in prevalence after 2012 corresponding to local discontinuation of mefloquine regimens. Assessment of the structure of pvmdr1 amplification in other geographic regions will yield information about the population-specificity of the breakpoints and underlying amplification mechanisms. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

High Levels of Plasmodium falciparum Rosetting in All Clinical Forms of Severe Malaria in African Children

PubMed Central

Doumbo, Ogobara K.; Thera, Mahamadou A.; Koné, Abdoulaye K.; Raza, Ahmed; Tempest, Louisa J.; Lyke, Kirsten E.; Plowe, Christopher V.; Rowe, J. Alexandra

2010-01-01

Plasmodium falciparum rosetting (the spontaneous binding of infected erythrocytes to uninfected erythrocytes) is a well-recognized parasite virulence factor. However, it is currently unclear whether rosetting is associated with all clinical forms of severe malaria, or only with specific syndromes such as cerebral malaria. We investigated the relationship between rosetting and clinical malaria in 209 Malian children enrolled in a case-control study of severe malaria. Rosetting was significantly higher in parasite isolates from severe malaria cases compared with non-severe hyperparasitemia and uncomplicated malaria controls (F2,117 = 8.15, P < 0.001). Analysis of sub-categories of severe malaria (unrousable coma, severe anemia, non-comatose neurological impairment, repeated seizures or a small heterogeneous group with signs of renal failure or jaundice) showed high levels of rosetting in all sub-categories, and no statistically significant differences in rosetting between sub-categories (F4,67 = 1.28, P = 0.28). Thus rosetting may contribute to the pathogenesis of all severe malaria syndromes in African children, and interventions to disrupt rosetting could be potential adjunctive therapies for all forms of severe malaria in Africa. PMID:19996426

Longitudinal study assessing the return of chloroquine susceptibility of Plasmodium falciparum in isolates from travellers returning from West and Central Africa, 2000–2011

PubMed Central

2013-01-01

Background Chloroquine (CQ) was the main malaria therapy worldwide from the 1940s until the 1990s. Following the emergence of CQ-resistant Plasmodium falciparum, most African countries discontinued the use of CQ, and now promote artemisinin-based combination therapy as the first-line treatment. This change was generally initiated during the last decade in West and Central Africa. The aim of this study is to describe the changes in CQ susceptibility in this African region, using travellers returning from this region as a sentinel system. Methods The study was conducted by the Malaria National Reference Centre, France. The database collated the pfcrtK76T molecular marker for CQ susceptibility and the in vitro response to CQ of parasites from travellers’ isolates returning from Senegal, Mali, Ivory Coast or Cameroon. As a proxy of drug pressure, data regarding CQ intake in febrile children were collated for the study period. Logistic regression models were used to detect trends in the proportions of CQ resistant isolates. Results A total of 2874 parasite isolates were genotyped between 2000–2011. The prevalence of the pfcrt76T mutant genotype significantly decreased for Senegal (from 78% to 47%), Ivory Coast (from 63% to 37%), Cameroon (from 90% to 59%) and remained stable for Mali. The geometric mean of the 50% inhibitory concentration (IC50) of CQ in vitro susceptibility and the proportion of resistant isolates (defining resistance as an IC50 value > 100 nM) significantly decreased for Senegal (from 86 nM (59%) to 39 nM (25%)), Mali (from 84 nM (50%) to 51 nM (31%)), Ivory Coast (from 75 nM (59%) to 29 nM (16%)) and Cameroon (from 181 nM (75%) to 51 nM (37%)). Both analyses (molecular and in vitro susceptibility) were performed for the 2004–2011 period, after the four countries had officially discontinued CQ and showed an accelerated decline of the resistant isolates for the four countries. Meanwhile, CQ use among children significantly deceased in this region (fixed effects slope = −0.3, p < 10-3). Conclusions An increase in CQ susceptibility following official withdrawal of the drug was observed in travellers returning from West and Central African countries. The same trends were observed for molecular and in vitro analysis between 2004-2011and they correlated to the decrease of the drug pressure. PMID:23351608

Longitudinal study assessing the return of chloroquine susceptibility of Plasmodium falciparum in isolates from travellers returning from West and Central Africa, 2000-2011.

PubMed

Gharbi, Myriam; Flegg, Jennifer A; Hubert, Véronique; Kendjo, Eric; Metcalf, Jessica E; Bertaux, Lionel; Guérin, Philippe J; Le Bras, Jacques; Aboubaca, Ahmed; Agnamey, Patrice; Angoulvant, Adela; Barbut, Patricia; Basset, Didier; Belkadi, Ghania; Bellanger, Anne Pauline; Bemba, Dieudonné; Benoit-Vica, Françoise; Berry, Antoine; Bigel, Marie-Laure; Bonhomme, Julie; Botterel, Françoise; Bouchaud, Olivier; Bougnoux, Marie-Elisabeth; Bourée, Patrice; Bourgeois, Nathalie; Branger, Catherine; Bret, Laurent; Buret, Bernadette; Casalino, Enrique; Chevrier, Sylviane; Conquere de Monbrison, Frédérique; Cuisenier, Bernadette; Danis, Martin; Darde, Marie-Laure; De Gentile, Ludovic; Delarbre, Jean-Marie; Delaunay, Pascal; Delaval, Anne; Desoubeaux, Guillaume; Develoux, Michel; Dunand, Jean; Durand, Rémy; Eloy, Odile; Fauchet, Nathalie; Faugere, Bernard; Faye, Alber; Fenneteau, Odile; Flori, Pierre; Fontrouge, Madeleine; Garabedian, Chantal; Gayandrieu, Françoise; Godineau, Nadine; Houzé, Pascal; Houzé, Sandrine; Hurst, Jean-Pierre; Ichou, Houria; Lachaud, Laurence; Lebuisson, Agathe; Lefevre, Magalie; LeGuern, Anne-Sophie; Le Moal, Gwenaë; Lusina, Daniel; Machouart, Marie-Claude; Malvy, Denis; Matheron, Sophie; Maubon, Danièle; Mechali, Denis; Megarbane, Bruno; Menard, Guillaume; Millon, Laurence; Aiach, Muriel Mimoun; Minodier, Philippe; Morelle, Christelle; Nevez, Gilles; Parola, Philippe; Parzy, Daniel; Patey, Olivier; Patoz, Pierre; Penn, Pascale; Perignon, Alice; Picot, Stéphane; Pilo, Jean-Etienne; Poilane, Isabelle; Pons, Denis; Poupart, Marie; Pradines, Bruno; Raffenot, Didier; Rapp, Christophe; Receveur, Marie-Catherine; Sarfati, Claudine; Senghor, Yaye; Simon, Fabrice; Siriez, Jean-Yves; Taudon, Nicolas; Thellier, Marc; Thouvenin, Maxime; Toubas, Dominique

2013-01-25

Chloroquine (CQ) was the main malaria therapy worldwide from the 1940s until the 1990s. Following the emergence of CQ-resistant Plasmodium falciparum, most African countries discontinued the use of CQ, and now promote artemisinin-based combination therapy as the first-line treatment. This change was generally initiated during the last decade in West and Central Africa. The aim of this study is to describe the changes in CQ susceptibility in this African region, using travellers returning from this region as a sentinel system. The study was conducted by the Malaria National Reference Centre, France. The database collated the pfcrtK76T molecular marker for CQ susceptibility and the in vitro response to CQ of parasites from travellers' isolates returning from Senegal, Mali, Ivory Coast or Cameroon. As a proxy of drug pressure, data regarding CQ intake in febrile children were collated for the study period. Logistic regression models were used to detect trends in the proportions of CQ resistant isolates. A total of 2874 parasite isolates were genotyped between 2000-2011. The prevalence of the pfcrt76T mutant genotype significantly decreased for Senegal (from 78% to 47%), Ivory Coast (from 63% to 37%), Cameroon (from 90% to 59%) and remained stable for Mali. The geometric mean of the 50% inhibitory concentration (IC50) of CQ in vitro susceptibility and the proportion of resistant isolates (defining resistance as an IC50 value > 100 nM) significantly decreased for Senegal (from 86 nM (59%) to 39 nM (25%)), Mali (from 84 nM (50%) to 51 nM (31%)), Ivory Coast (from 75 nM (59%) to 29 nM (16%)) and Cameroon (from 181 nM (75%) to 51 nM (37%)). Both analyses (molecular and in vitro susceptibility) were performed for the 2004-2011 period, after the four countries had officially discontinued CQ and showed an accelerated decline of the resistant isolates for the four countries. Meanwhile, CQ use among children significantly deceased in this region (fixed effects slope = -0.3, p < 10-3). An increase in CQ susceptibility following official withdrawal of the drug was observed in travellers returning from West and Central African countries. The same trends were observed for molecular and in vitro analysis between 2004-2011 and they correlated to the decrease of the drug pressure.

Hyperreactive malarial splenomegaly in Venezuela.

PubMed

Torres, J; Noya, O; Mondolfi, A; Peceño, C; Botto, C

1988-07-01

A cross-sectional seroepidemiological survey seeking hyperreactive malarial splenomegaly was carried out in isolated Yanomami hamlets in Amazonas Territory in Venezuela. All 110 inhabitants greater than 1 year of age were evaluated clinically and 98 were studied immunologically. The spleen index for individuals greater than 10 years of age was 44%. Only 3 patients had Plasmodium spp. on thick blood smears. All had serological evidence of infection with Plasmodium falciparum and P. vivax. Twenty-three patients were considered to show hyperreactive malarial splenomegaly. Clinical manifestations of the syndrome did not differ from those described in other parts of the world.

In vitro anti-plasmodial activity of Dicoma anomala subsp. gerrardii (Asteraceae): identification of its main active constituent, structure-activity relationship studies and gene expression profiling.

PubMed

Becker, John V W; van der Merwe, Marina M; van Brummelen, Anna C; Pillay, Pamisha; Crampton, Bridget G; Mmutlane, Edwin M; Parkinson, Chris; van Heerden, Fanie R; Crouch, Neil R; Smith, Peter J; Mancama, Dalu T; Maharaj, Vinesh J

2011-10-11

Anti-malarial drug resistance threatens to undermine efforts to eliminate this deadly disease. The resulting omnipresent requirement for drugs with novel modes of action prompted a national consortium initiative to discover new anti-plasmodial agents from South African medicinal plants. One of the plants selected for investigation was Dicoma anomala subsp. gerrardii, based on its ethnomedicinal profile. Standard phytochemical analysis techniques, including solvent-solvent extraction, thin-layer- and column chromatography, were used to isolate the main active constituent of Dicoma anomala subsp. gerrardii. The crystallized pure compound was identified using nuclear magnetic resonance spectroscopy, mass spectrometry and X-ray crystallography. The compound was tested in vitro on Plasmodium falciparum cultures using the parasite lactate dehydrogenase (pLDH) assay and was found to have anti-malarial activity. To determine the functional groups responsible for the activity, a small collection of synthetic analogues was generated - the aim being to vary features proposed as likely to be related to the anti-malarial activity and to quantify the effect of the modifications in vitro using the pLDH assay. The effects of the pure compound on the P. falciparum transcriptome were subsequently investigated by treating ring-stage parasites (alongside untreated controls), followed by oligonucleotide microarray- and data analysis. The main active constituent was identified as dehydrobrachylaenolide, a eudesmanolide-type sesquiterpene lactone. The compound demonstrated an in vitro IC50 of 1.865 μM against a chloroquine-sensitive strain (D10) of P. falciparum. Synthetic analogues of the compound confirmed an absolute requirement that the α-methylene lactone be present in the eudesmanolide before significant anti-malarial activity was observed. This feature is absent in the artemisinins and suggests a different mode of action. Microarray data analysis identified 572 unique genes that were differentially expressed as a result of the treatment and gene ontology analysis identified various biological processes and molecular functions that were significantly affected. Comparison of the dehydrobrachylaenolide treatment transcriptional dataset with a published artesunate (also a sesquiterpene lactone) dataset revealed little overlap. These results strengthen the notion that the isolated compound and the artemisinins have differentiated modes of action. The novel mode of action of dehydrobrachylaenolide, detected during these studies, will play an ongoing role in advancing anti-plasmodial drug discovery efforts.

Analysis of polymorphisms in Plasmodium falciparum genes related to drug resistance: a survey over four decades under different treatment policies in Brazil.

PubMed

Inoue, Juliana; Lopes, Dinora; do Rosário, Virgílio; Machado, Marta; Hristov, Angélica D; Lima, Giselle Fmc; Costa-Nascimento, Maria J; Segurado, Aluísio C; Di Santi, Silvia M

2014-09-19

Anti-malarial resistance in Plasmodium falciparum remains an obstacle for malaria control. Resistance-associated genes were analysed in Brazilian samples over four decades to evaluate the impact of different treatment regimens on the parasite genetic profile. Samples were collected on filter paper from patients infected in the Amazon region from 1984 to 2011. DNA was extracted with Chelex® 100 and monoinfection confirmed by PCR. SNPs in the pfcrt, pfmdr1, pfdhfr and pfdhps genes were assessed by PCR-RFLP. The pfmdr1 copy number was estimated using real time quantitative PCR with SYBR® Green. Parasite response was assessed ex vivo with seven concentrations of each anti-malarial. Patients were treated according to Brazilian guidelines: quinine plus tetracycline or mefloquine in period 1 and ACT in period 2. All 96 samples presented the pfcrt 76T mutant throughout the assessed periods. In addition, all isolates showed ex vivo chloroquine resistance. The pfmdr1 86Y was detected in 1.5% of samples in period 1, and in 25% in period 2. All samples presented the pfmdr1 1246Y. The analysis of pfmdr1 copy number showed amplification in 37.3% in period 1 and in 42% in period 2. Mutations in pfdhfr were shown as follows: 51I in all samples in period 1 and in 81.2% in period 2; 59R in 6.4% in period 2. The pfdhfr 108N and the pfdhps 437G were seen in all samples along time; the pfdhps 540E in 93.7% in period 1 and in 75% in period 2. The 76T mutation associated to chloroquine resistance is still present in the parasite population, although this anti-malarial was withdrawn from the chemotherapy of P. falciparum in Brazil in the mid-1980s. All isolates assayed ex vivo for chloroquine showed resistant phenotype and 76T. No association was observed between pfmdr1 mutations and resistance to quinine, mefloquine and artemisinin derivatives. Additionally, the pfdhfr 108N mutation was detected in all samples throughout the evaluated periods, demonstrating fixation of the mutant allele in the parasite population. Changes in Brazilian national guidelines for the malaria chemotherapy in the last 27 years yielded a discreet genetic impact in the parasite population.

A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs

PubMed Central

2011-01-01

Background Mounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation. Results We used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere. Conclusions We have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation. PMID:21689454

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

PubMed Central

Ronander, Elena; Bengtsson, Dominique C.; Joergensen, Louise; Jensen, Anja T. R.; Arnot, David E.

2012-01-01

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1. Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types. PMID:23070076

Plasmodium falciparum Infection Status among Children with Schistosoma in Sub-Saharan Africa: A Systematic Review and Meta-analysis

PubMed Central

Degarege, Abraham; Degarege, Dawit; Veledar, Emir; Erko, Berhanu; Nacher, Mathieu; Beck-Sague, Consuelo M.; Madhivanan, Purnima

2016-01-01

Background It has been suggested that Schistosoma infection may be associated with Plasmodium falciparum infection or related reduction in haemoglobin level, but the nature of this interaction remains unclear. This systematic review synthesized evidence on the relationship of S. haematobium or S. mansoni infection with the occurrence of P. falciparum malaria, Plasmodium density and related reduction in haemoglobin level among children in sub-Saharan Africa (SSA). Methodology/Principal findings A systematic review in according with PRISMA guidelines was conducted. All published articles available in PubMed, Embase, Cochrane library and CINAHL databases before May 20, 2015 were searched without any limits. Two reviewers independently screened, reviewed and assessed all the studies. Cochrane Q and Moran’s I2 were used to assess heterogeneity and the Egger test was used to examine publication bias. The summary odds ratio (OR), summary regression co-efficient (β) and 95% confidence intervals (CI) were estimated using a random-effects model. Out of 2,920 citations screened, 12 articles (five cross-sectional, seven prospective cohort) were eligible to be included in the systematic review and 11 in the meta-analysis. The 12 studies involved 9,337 children in eight SSA countries. Eight studies compared the odds of asymptomatic/uncomplicated P. falciparum infection, two studies compared the incidence of uncomplicated P. falciparum infection, six studies compared P. falciparum density and four studies compared mean haemoglobin level between children infected and uninfected with S. haematobium or S. mansoni. Summary estimates of the eight studies based on 6,018 children showed a higher odds of asymptomatic/uncomplicated P. falciparum infection in children infected with S. mansoni or S. haematobium compared to those uninfected with Schistosoma (summary OR: 1.82; 95%CI: 1.41, 2.35; I2: 52.3%). The increase in odds of asymptomatic/uncomplicated P. falciparum infection among children infected with Schistosoma remained significant when subgroup analysis was conducted for S. haematobium (summary OR: 1.68; 95%CI: 1.18, 2.41; I2: 53.2%) and S. mansoni (summary OR: 2.15; 95%CI: 1.89, 2.46: I2: 0.0%) infection. However, the density of P. falciparum infection was lower in children co-infected with S. haematobium compared to those uninfected with Schistosoma (summary-β: -0.14; 95% CI: -0.24, -0.01; I2: 39.7%). The mean haemoglobin level was higher among children co-infected with S. haematobium and P. falciparum than those infected with only P. falciparum (summary-mean haemoglobin difference: 0.49; 95% CI: 0.04, 0.95; I2: 66.4%) Conclusions/Significance The current review suggests S. mansoni or S. haematobium co-infection may be associated with increased prevalence of asymptomatic/uncomplicated P. falciparum infection in children, but may protect against high density P. falciparum infection and related reduction in haemoglobin level. PMID:27926919

Drug Evaluation in the Plasmodium falciparum - Aotus Model

DTIC Science & Technology

1984-09-01

consecutive days to Colombian Aotus. Six amodiaquin analogues were evaluated for their capacity to cure in- fections of chloroquine -sensitive and...AMODIAQUIN ANALOGUES AND AMODIAQUIN AGAINST INFECTIONS OF CHLOROQUINE -SENSITIVE AND CHLOROQUINE -RESISTANT STRAINS OF PLASMODIUM FALCIPARUM 14...AMODIAQUIN ANALOGUES AND AMOOIAQUIN AGAINST INFECTIONS OF CHLOROQUINE -SENSITIVE AND CHLOROQUINE - RESISTANT STRAINS OF PLASMODIUM FALCIPARUM Following

Spatial mapping and prediction of Plasmodium falciparum infection risk among school-aged children in Côte d'Ivoire.

PubMed

Houngbedji, Clarisse A; Chammartin, Frédérique; Yapi, Richard B; Hürlimann, Eveline; N'Dri, Prisca B; Silué, Kigbafori D; Soro, Gotianwa; Koudou, Benjamin G; Assi, Serge-Brice; N'Goran, Eliézer K; Fantodji, Agathe; Utzinger, Jürg; Vounatsou, Penelope; Raso, Giovanna

2016-09-07

In Côte d'Ivoire, malaria remains a major public health issue, and thus a priority to be tackled. The aim of this study was to identify spatially explicit indicators of Plasmodium falciparum infection among school-aged children and to undertake a model-based spatial prediction of P. falciparum infection risk using environmental predictors. A cross-sectional survey was conducted, including parasitological examinations and interviews with more than 5,000 children from 93 schools across Côte d'Ivoire. A finger-prick blood sample was obtained from each child to determine Plasmodium species-specific infection and parasitaemia using Giemsa-stained thick and thin blood films. Household socioeconomic status was assessed through asset ownership and household characteristics. Children were interviewed for preventive measures against malaria. Environmental data were gathered from satellite images and digitized maps. A Bayesian geostatistical stochastic search variable selection procedure was employed to identify factors related to P. falciparum infection risk. Bayesian geostatistical logistic regression models were used to map the spatial distribution of P. falciparum infection and to predict the infection prevalence at non-sampled locations via Bayesian kriging. Complete data sets were available from 5,322 children aged 5-16 years across Côte d'Ivoire. P. falciparum was the predominant species (94.5 %). The Bayesian geostatistical variable selection procedure identified land cover and socioeconomic status as important predictors for infection risk with P. falciparum. Model-based prediction identified high P. falciparum infection risk in the north, central-east, south-east, west and south-west of Côte d'Ivoire. Low-risk areas were found in the south-eastern area close to Abidjan and the south-central and west-central part of the country. The P. falciparum infection risk and related uncertainty estimates for school-aged children in Côte d'Ivoire represent the most up-to-date malaria risk maps. These tools can be used for spatial targeting of malaria control interventions.

P. falciparum infection and maternofetal antibody transfer in malaria-endemic settings of varying transmission

PubMed Central

Stanisic, Danielle; McGready, Rose; Chotivanich, Kesinee; Clapham, Caroline; Baiwog, Francesca; Pimanpanarak, Mupawjay; Siba, Peter; Mueller, Ivo; King, Christopher L.; Nosten, François; Beeson, James G.; Rogerson, Stephen; Simpson, Julie A.; Fowkes, Freya J. I.

2017-01-01

Introduction 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. Methods 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. Results 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. Discussion 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 the majority of the reduction in maternofetal antibody transfer efficiency with placental infection. PMID:29028827

P. falciparum infection and maternofetal antibody transfer in malaria-endemic settings of varying transmission.

PubMed

McLean, Alistair R D; Stanisic, Danielle; McGready, Rose; Chotivanich, Kesinee; Clapham, Caroline; Baiwog, Francesca; Pimanpanarak, Mupawjay; Siba, Peter; Mueller, Ivo; King, Christopher L; Nosten, François; Beeson, James G; Rogerson, Stephen; Simpson, Julie A; Fowkes, Freya J I

2017-01-01

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 the majority of the reduction in maternofetal antibody transfer efficiency with placental infection.

Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

PubMed

Wijayalath, Wathsala; Majji, Sai; Villasante, Eileen F; Brumeanu, Teodor D; Richie, Thomas L; Casares, Sofia

2014-09-30

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 of protection and to identify protective malaria antigens.

Computational synchronization of microarray data with application to Plasmodium falciparum.

PubMed

Zhao, Wei; Dauwels, Justin; Niles, Jacquin C; Cao, Jianshu

2012-06-21

Microarrays are widely used to investigate the blood stage of Plasmodium falciparum infection. Starting with synchronized cells, gene expression levels are continually measured over the 48-hour intra-erythrocytic cycle (IDC). However, the cell population gradually loses synchrony during the experiment. As a result, the microarray measurements are blurred. In this paper, we propose a generalized deconvolution approach to reconstruct the intrinsic expression pattern, and apply it to P. falciparum IDC microarray data. We develop a statistical model for the decay of synchrony among cells, and reconstruct the expression pattern through statistical inference. The proposed method can handle microarray measurements with noise and missing data. The original gene expression patterns become more apparent in the reconstructed profiles, making it easier to analyze and interpret the data. We hypothesize that reconstructed gene expression patterns represent better temporally resolved expression profiles that can be probabilistically modeled to match changes in expression level to IDC transitions. In particular, we identify transcriptionally regulated protein kinases putatively involved in regulating the P. falciparum IDC. By analyzing publicly available microarray data sets for the P. falciparum IDC, protein kinases are ranked in terms of their likelihood to be involved in regulating transitions between the ring, trophozoite and schizont developmental stages of the P. falciparum IDC. In our theoretical framework, a few protein kinases have high probability rankings, and could potentially be involved in regulating these developmental transitions. This study proposes a new methodology for extracting intrinsic expression patterns from microarray data. By applying this method to P. falciparum microarray data, several protein kinases are predicted to play a significant role in the P. falciparum IDC. Earlier experiments have indeed confirmed that several of these kinases are involved in this process. Overall, these results indicate that further functional analysis of these additional putative protein kinases may reveal new insights into how the P. falciparum IDC is regulated.

Spatial Distribution of Falciparum Malaria Infections in Zanzibar: Implications for Focal Drug Administration Strategies Targeting Asymptomatic Parasite Carriers

PubMed Central

Cook, Jackie; Sturrock, Hugh; Msellem, Mwinyi; Ali, Abdullah; Xu, Weiping; Molteni, Fabrizio; Gosling, Roly; Drakeley, Chris; Mårtensson, Andreas

2017-01-01

Abstract Background. 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. Methods. 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. Results. 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 <300 m, compared with >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. Conclusions. 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. PMID:28431115

Origins of the current outbreak of multidrug-resistant malaria in southeast Asia: a retrospective genetic study.

PubMed

Amato, Roberto; Pearson, Richard D; Almagro-Garcia, Jacob; Amaratunga, Chanaki; Lim, Pharath; Suon, Seila; Sreng, Sokunthea; Drury, Eleanor; Stalker, Jim; Miotto, Olivo; Fairhurst, Rick M; Kwiatkowski, Dominic P

2018-03-01

Antimalarial resistance is rapidly spreading across parts of southeast Asia where dihydroartemisinin-piperaquine is used as first-line treatment for Plasmodium falciparum malaria. The first published reports about resistance to antimalarial drugs came from western Cambodia in 2013. Here, we analyse genetic changes in the P falciparum population of western Cambodia in the 6 years before those reports. We analysed genome sequence data on 1492 P falciparum samples from 11 locations across southeast Asia, including 464 samples collected in western Cambodia between 2007 and 2013. Different epidemiological origins of resistance were identified by haplotypic analysis of the kelch13 artemisinin resistance locus and the plasmepsin 2-3 piperaquine resistance locus. We identified more than 30 independent origins of artemisinin resistance, of which the KEL1 lineage accounted for 140 (91%) of 154 parasites resistant to dihydroartemisinin-piperaquine. In 2008, KEL1 combined with PLA1, the major lineage associated with piperaquine resistance. By 2013, the KEL1/PLA1 co-lineage had reached a frequency of 63% (24/38) in western Cambodia and had spread to northern Cambodia. The KEL1/PLA1 co-lineage emerged in the same year that dihydroartemisinin-piperaquine became the first-line antimalarial drug in western Cambodia and spread rapidly thereafter, displacing other artemisinin-resistant parasite lineages. These findings have important implications for management of the global health risk associated with the current outbreak of multidrug-resistant malaria in southeast Asia. Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, UK Department for International Development, and the Intramural Research Program of the National Institute of Allergy and Infectious Diseases. Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.

Evaluation of a rapid and inexpensive dipstick assay for the diagnosis of Plasmodium falciparum malaria.

PubMed Central

Mills, C. D.; Burgess, D. C.; Taylor, H. J.; Kain, K. C.

1999-01-01

Rapid, accurate and affordable methods are needed for the diagnosis of malaria. Reported here is an evaluation of a new immunochromatographic strip, the PATH Falciparum Malaria IC Strip, which is impregnated with an immobilized IgM monoclonal antibody that binds to the HRP-II antigen of Plasmodium falciparum. In contrast to other commercially available kits marketed for the rapid diagnosis of falciparum malaria, this kit should be affordable in the malaria-endemic world. Using microscopy and polymerase chain reaction (PCR)-based methods as reference standards, we compared two versions of the PATH test for the detection of P. falciparum infection in 200 febrile travellers. As determined by PCR and microscopy, 148 travellers had malaria, 50 of whom (33.8%) were infected with P. falciparum. Compared with PCR, the two versions of the PATH test had initial sensitivities of 90% and 88% and specificities of 97% and 96%, respectively, for the detection of falciparum malaria. When discrepant samples were retested blindly with a modified procedure (increased sample volume and longer washing step) the sensitivity and specificity of both kits improved to 96% and 99%, respectively. The two remaining false negatives occurred in samples with < 100 parasites per microliter of blood. The accuracy, simplicity and predicted low cost may make this test a useful diagnostic tool in malaria-endemic areas. PMID:10444878

Genome-level determination of Plasmodium falciparum blood-stage targets of malarial clinical immunity in the Peruvian Amazon.

PubMed

Torres, Katherine J; Castrillon, Carlos E; Moss, Eli L; Saito, Mayuko; Tenorio, Roy; Molina, Douglas M; Davies, Huw; Neafsey, Daniel E; Felgner, Philip; Vinetz, Joseph M; Gamboa, Dionicia

2015-04-15

Persons with blood-stage Plasmodium falciparum parasitemia in the absence of symptoms are considered to be clinically immune. We hypothesized that asymptomatic subjects with P. falciparum parasitemia would differentially recognize a subset of P. falciparum proteins on a genomic scale. Compared with symptomatic subjects, sera from clinically immune, asymptomatically infected individuals differentially recognized 51 P. falciparum proteins, including the established vaccine candidate PfMSP1. Novel, hitherto unstudied hypothetical proteins and other proteins not previously recognized as potential vaccine candidates were also differentially recognized. Genes encoding the proteins differentially recognized by the Peruvian clinically immune individuals exhibited a significant enrichment of nonsynonymous nucleotide variation, an observation consistent with these genes undergoing immune selection. A limited set of P. falciparum protein antigens was associated with the development of naturally acquired clinical immunity in the low-transmission setting of the Peruvian Amazon. These results imply that, even in a low-transmission setting, an asexual blood-stage vaccine designed to reduce clinical malaria symptoms will likely need to contain large numbers of often-polymorphic proteins, a finding at odds with many current efforts in the design of vaccines against asexual blood-stage P. falciparum. © 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: journals.permissions@oup.com.

[Evaluation of effect of prevention and control system for imported falciparum malaria in Hanjiang District].

PubMed

She, Guo-lin; Ma, Yu-Cai; Wang, Fu-biao

2013-08-01

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.

Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification.

PubMed

Poon, Leo L M; Wong, Bonnie W Y; Ma, Edmund H T; Chan, Kwok H; Chow, Larry M C; Abeyewickreme, Wimal; Tangpukdee, Noppadon; Yuen, Kwok Y; Guan, Yi; Looareesuwan, Sornchai; Peiris, J S Malik

2006-02-01

Malaria is one of the most important parasitic infections in humans. A sensitive diagnostic test for malaria that could be applied at the community level could be useful in programs to control the disease. The aim of the present work was to develop a simple, inexpensive molecular test for Plasmodium falciparum. Blood was collected from controls (n = 100) and from patients diagnosed with falciparum malaria infection (n = 102), who were recruited to the study. Heat-treated blood samples were tested by a loop-mediated isothermal amplification (LAMP) assay for P. falciparum. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. To evaluate the assay, DNA from these samples was purified and tested by PCR. Results from the LAMP and PCR assays were compared. The LAMP assay detected P. falciparum directly from heat-treated blood. The quantitative data from the assay correlated to the parasite counts obtained by blood-film microscopic analyses. When we used the PCR assay as the comparison method, the sensitivity and specificity of the LAMP assay were 95% and 99%, respectively. Unlike PCR, the LAMP assay does not require purified DNA for efficient DNA amplification, thereby reducing the cost and turnaround time for P. falciparum diagnosis. The assay requires only basic instruments, and assay positivity can be verified by visual inspection.

Routine parallel diagnosis of malaria using microscopy and the malaria rapid diagnostic test SD 05FK60: the experience of Médecins Sans Frontières in Myanmar.

PubMed

Kosack, Cara S; Naing, Wint Thu; Piriou, Erwan; Shanks, Leslie

2013-05-21

Malaria rapid diagnostic tests (RDTs) are commonly used in Médecins Sans Frontières (MSF) programmes to detect acute malaria infection. Programmes in regions with both Plasmodium falciparum and non-falciparum malaria (i.e. Plasmodium ovale, Plasmodium malariae and Plasmodium vivax) use a three-band P. falciparum/Pan test such as the SD Bioline Malaria Ag P.f/Pan 05FK60 (Standard Diagnostics, Kyonggi, Republic of Korea), hereafter referred to as SD 05FK60, as used by the MSF-Holland clinics in Rakhine state, Myanmar. In spite of published reports of generally good test performance, medical and paramedical staff on the ground often doubt the diagnostic accuracy of these RDTs. Parallel testing with malaria microscopy and RDT was conducted at two clinics in Rakhine state, Myanmar, for a period of 14 months as a programmatic response due to doubts and concerns of medical and paramedical staff into malaria RDTs. A total of 2,585 blood samples from non-pregnant suspected malaria patients were examined by the SD 05FK60 RDT and microscopy at two clinics in Myanmar from October 2010 to December 2011. The reference standard microscopy diagnosed 531 P. falciparum and 587 P. vivax or P. malariae mono-infections. The overall sensitivity for P. falciparum detection by the SD 05FK60 was 90.2% (95% CI: 87.4-92.6) and for P. vivax/P. malariae 79.4% (95% CI: 75.9-82.6). The overall specificity for P. falciparum detection by the SD 05FK60 was 98.5% (95% CI: 97.7-99.1) and for P. vivax/P. malariae 98.7% (95% CI: 97.9-99.2). The sensitivity for P. falciparum was >91% for parasitaemia levels of >100-1,000 parasites/μl and increased for P. vivax/P. malariae with the parasitaemia level but was overall lower than for P. falciparum 25/408 and 13/420 cases, respectively, of P. falciparum and non-falciparum malaria were missed by the RDT. In field conditions in Myanmar, the SD 05FK60 malaria RDT performed consistent with other reports. The test detected malaria caused by P. vivax/P. malariae to a lesser extent than P. falciparum infection. Sensitivity improved with increasing parasitaemia level, however even at higher levels some infections were missed. The SD 05FK60 is adequate for use in settings where high quality microscopy is not available.

A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

PubMed Central

2012-01-01

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. PMID:23190769

Very high carriage of gametocytes in asymptomatic low-density Plasmodium falciparum and P. vivax infections in western Thailand.

PubMed

Nguitragool, Wang; Mueller, Ivo; Kumpitak, Chalermpon; Saeseu, Teerawat; Bantuchai, Sirasate; Yorsaeng, Ritthideach; Yimsamran, Surapon; Maneeboonyang, Wanchai; Sa-Angchai, Patiwat; Chaimungkun, Wutthichai; Rukmanee, Prasert; Puangsa-Art, Supalarp; Thanyavanich, Nipon; Koepfli, Cristian; Felger, Ingrid; Sattabongkot, Jetsumon; Singhasivanon, Pratap

2017-10-24

Low-density asymptomatic infections of Plasmodium spp. are common in low endemicity areas worldwide, but outside Africa, their contribution to malaria transmission is poorly understood. Community-based studies with highly sensitive molecular diagnostics are needed to quantify the asymptomatic reservoir of Plasmodium falciparum and P. vivax infections in Thai communities. A cross-sectional survey of 4309 participants was conducted in three endemic areas in Kanchanaburi and Ratchaburi provinces of Thailand in 2012. The presence of P. falciparum and P. vivax parasites was determined using 18S rRNA qPCR. Gametocytes were also detected by pfs25 / pvs25 qRT-PCRs. A total of 133 individuals were found infected with P. vivax (3.09%), 37 with P. falciparum (0.86%), and 11 with mixed P. vivax/ P. falciparum (0.26%). The clear majority of both P. vivax (91.7%) and P. falciparum (89.8%) infections were not accompanied by any febrile symptoms. Infections with either species were most common in adolescent and adult males. Recent travel to Myanmar was highly associated with P. falciparum (OR = 9.0, P = 0.001) but not P. vivax infections (P = 0.13). A large number of P. vivax (71.5%) and P. falciparum (72.0%) infections were gametocyte positive by pvs25/pfs25 qRT-PCR. Detection of gametocyte-specific pvs25 and pfs25 transcripts was strongly dependent on parasite density. pvs25 transcript numbers, a measure of gametocyte density, were also highly correlated with parasite density (r 2  = 0.82, P < 0.001). Asymptomatic infections with Plasmodium spp. were common in western Thai communities in 2012. The high prevalence of gametocytes indicates that these infections may contribute substantially to the maintenance of local malaria transmission.

Submicroscopic placental infection by non-falciparum Plasmodium spp.

PubMed

Doritchamou, Justin Y A; Akuffo, Richard A; Moussiliou, Azizath; Luty, Adrian J F; Massougbodji, Achille; Deloron, Philippe; Tuikue Ndam, Nicaise G

2018-02-01

Among the Plasmodium species that infect humans, adverse effects of P. falciparum and P. vivax have been extensively studied and reported with respect to poor outcomes particularly in first time mothers and in pregnant women living in areas with unstable malaria transmission. Although, other non-falciparum malaria infections during pregnancy have sometimes been reported, little is known about the dynamics of these infections during pregnancy. Using a quantitative PCR approach, blood samples collected from Beninese pregnant women during the first antenatal visit (ANV) and at delivery including placental blood were screened for Plasmodium spp. Risk factors associated with Plasmodium spp. infection during pregnancy were assessed as well as the relationships with pregnancy outcomes. P. falciparum was the most prevalent Plasmodium species detected during pregnancy, irrespective either of parity, of age or of season during which the infection occurred. Although no P. vivax infections were detected in this cohort, P. malariae (9.2%) and P. ovale (5.8%) infections were observed in samples collected during the first ANV. These non-falciparum infections were also detected in maternal peripheral blood (1.3% for P. malariae and 1.2% for P. ovale) at delivery. Importantly, higher prevalence of P. malariae (5.5%) was observed in placental than peripheral blood while that of P. ovale was similar (1.8% in placental blood). Among the non-falciparum infected pregnant women with paired peripheral and placental samples, P. malariae infections in the placental blood was significantly higher than in the peripheral blood, suggesting a possible affinity of P. malariae for the placenta. However, no assoctiation of non-falciparum infections and the pregnancy outcomes was observed. Overall this study provided insights into the molecular epidemiology of Plasmodium spp. infection during pregnancy, indicating placental infection by non-falciparum Plasmodium and the lack of association of these infections with adverse pregnancy outcomes.

Spatial and space-time distribution of Plasmodium vivax and Plasmodium falciparum malaria in China, 2005-2014.

PubMed

Hundessa, Samuel H; Williams, Gail; Li, Shanshan; Guo, Jinpeng; Chen, Linping; Zhang, Wenyi; Guo, Yuming

2016-12-19

Despite the declining burden of malaria in China, the disease remains a significant public health problem with periodic outbreaks and spatial variation across the country. A better understanding of the spatial and temporal characteristics of malaria is essential for consolidating the disease control and elimination programme. This study aims to understand the spatial and spatiotemporal distribution of Plasmodium vivax and Plasmodium falciparum malaria in China during 2005-2009. Global Moran's I statistics was used to detect a spatial distribution of local P. falciparum and P. vivax malaria at the county level. Spatial and space-time scan statistics were applied to detect spatial and spatiotemporal clusters, respectively. Both P. vivax and P. falciparum malaria showed spatial autocorrelation. The most likely spatial cluster of P. vivax was detected in northern Anhui province between 2005 and 2009, and western Yunnan province between 2010 and 2014. For P. falciparum, the clusters included several counties of western Yunnan province from 2005 to 2011, Guangxi from 2012 to 2013, and Anhui in 2014. The most likely space-time clusters of P. vivax malaria and P. falciparum malaria were detected in northern Anhui province and western Yunnan province, respectively, during 2005-2009. The spatial and space-time cluster analysis identified high-risk areas and periods for both P. vivax and P. falciparum malaria. Both malaria types showed significant spatial and spatiotemporal variations. Contrary to P. vivax, the high-risk areas for P. falciparum malaria shifted from the west to the east of China. Further studies are required to examine the spatial changes in risk of malaria transmission and identify the underlying causes of elevated risk in the high-risk areas.

Field performance of malaria rapid diagnostic test for the detection of Plasmodium falciparum infection in Odisha State, India.

PubMed

Sahu, S S; Gunasekaran, K; Jambulingam, P

2015-12-01

Rapid diagnostic tests (RDTs) have become an essential surveillance tool in the malaria control programme in India. The current study aimed to assess the performance of ParaHIT-f, a rapid test in diagnosis of Plasmodium falciparum infection through detecting its specific antigen, histidine rich protein 2 (PfHRP-2), in Odisha State, India. The study was undertaken in eight falciparum malaria endemic southern districts of Odisha State. Febrile patients included through active case detection, were diagnosed by Accredited Social Health Activists (ASHAs) for P. falciparum infection using the RDT, ParaHIT-f. The performance of ParaHIT-f was evaluated using microscopy as the gold standard. A total of 1030 febrile patients were screened by both microscopy and the RDT for P. falciparum infection. The sensitivity of ParaHIT-f was 63.6% (95% CI: 56.0-70.6) and specificity was 98.9% (95% CI: 97.9-99.5), with positive and negative predictive values (PPV and NPV) of 92.6% (95% CI: 86.0-96.3) and 93.0% (95% CI: 91.0-94.5), respectively. When related to parasitaemia, the RDT sensitivity was 47.8% at the low parasitaemia of 4 to 40 parasites/µl of blood. The results showed that the performance of the RDT, ParaHIT-f, was not as sensitive as microscopy in detecting true falciparum infections; a high specificity presented a low frequency of false-positive RDT results. t0 he sensitivity of ParaHIT-f was around 60 per cent. It is, therefore, essential to improve the efficiency (sensitivity) of the kit so that the true falciparum infections will not be missed especially in areas where P. falciparum has been the predominant species causing cerebral malaria.

Characterization of asymptomatic Plasmodium falciparum infection and its risk factors in pregnant women from the Republic of Congo.

PubMed

Francine, Ntoumi; Damien, Bakoua; Anna, Fesser; Michael, Kombo; Christevy, Vouvoungui J; Felix, Koukouikila-Koussounda

2016-01-01

Malaria in pregnancy remains a serious public health problem in the Republic of Congo despite the implementation of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) in 2006. The aim of this cross-sectional study was to characterize Plasmodium falciparum infections and determine possible risk factors in pregnant Congolese women attending an antenatal clinic in a periurban area of southern Brazzaville. This study was conducted from March 2012 to December 2013 in a site where several years ago, high malaria resistance to SP was reported. Pregnant women were enrolled during antenatal visits and the number of received IPTp-SP doses was recorded as well as individual sociodemographic data. Peripheral blood was collected and P. falciparum infection was checked by microscopy and by PCR targeting P. falciparum merozoite surface protein gene (msp2). Haemoglobin concentration was measured and P. falciparum positive samples were typed for msp2 allelic diversity. A total of 363 pregnant women were recruited. The prevalence of asymptomatic P. falciparum infection was 7% and 19% by microscopy and by PCR, respectively. More than one half (51.5%) of the pregnant women were anaemic. Multivariate analysis indicated that P. falciparum infection was associated with anaemia. It was also observed that women who have received IPTp-SP have significantly lower prevalence of infection. The administration of IPTp-SP did not influence the multiplicity of infection (MOI). This first study investigating asymptomatic malaria infection on pregnant women of the Republic of Congo shows that P. falciparum infections were clearly associated with maternal anaemia, and use of IPTp-SP reduced the risk of carrying asymptomatic infections. Copyright © 2015 Elsevier B.V. All rights reserved.

High prevalence of asymptomatic Plasmodium falciparum infection in Gabonese adults.

PubMed

Dal-Bianco, Matthias P; Köster, Kai B; Kombila, Ulrich D; Kun, Jürgen F J; Grobusch, Martin P; Ngoma, Ghyslain Mombo; Matsiegui, Pierre B; Supan, Christian; Salazar, Carmen L Ospina; Missinou, Michel A; Issifou, Saadou; Lell, Bertrand; Kremsner, Peter

2007-11-01

Plasmodium falciparum, the most common malarial parasite in sub-Saharan Africa, accounts for a high number of deaths in children less than five years of age. In malaria-endemic countries with stable transmission, semi-immunity is usually acquired after childhood. For adults, severe malaria is rare. Infected adults have either uncomplicated malaria or asymptomatic parasitemia. During a period of one year, we screened 497 afebrile males to investigate the prevalence of asymptomatic P. falciparum parasitemia in villages near Lambaréné, Gabon by use of three different methods. A total of 52% of the individuals had parasites detected by a subtelomeric variable open reading frame polymerase chain reaction (stevor-PCR), 27% of the rapid diagnostic test results were positive, and 12% of the thick blood smears with low parasitemias had P. falciparum. Most positive cases were only detected by the stevor-PCR. Asymptomatic P. falciparum parasitemia in adults living in a malaria-endemic country is frequent.

On Programmed Cell Death in Plasmodium falciparum: Status Quo

PubMed Central

Engelbrecht, Dewaldt; Durand, Pierre Marcel; Coetzer, Thérèsa Louise

2012-01-01

Conflicting arguments and results exist regarding the occurrence and phenotype of programmed cell death (PCD) in the malaria parasite Plasmodium falciparum. Inconsistencies relate mainly to the number and type of PCD markers assessed and the different methodologies used in the studies. In this paper, we provide a comprehensive overview of the current state of knowledge and empirical evidence for PCD in the intraerythrocytic stages of P. falciparum. We consider possible reasons for discrepancies in the data and offer suggestions towards more standardised investigation methods in this field. Furthermore, we present genomic evidence for PCD machinery in P. falciparum. We discuss the potential adaptive or nonadaptive role of PCD in the parasite life cycle and its possible exploitation in the development of novel drug targets. Lastly, we pose pertinent unanswered questions concerning the PCD phenomenon in P. falciparum to provide future direction. PMID:22287973

Chloroquine-resistant falciparum malaria in East Kalimantan, Indonesia.

PubMed

Verdrager, J; Arwati; Simanjuntak, C H; Saroso, J S

1976-03-01

Following the discovery of four imported chloroquine-resistant P. falciparum infections in the Province of Yogyakarta (Island of Java) sensitivity tests were carried out in the Province of East Kalimantan Island of Borneo). Twenty subjects were given 25 mg. of chloroquine base per kilogram of body weight over three days. Two infections were found resistant at the RII level and a third at the RI level with early recrudescence on day 7. In the other 17 cases followed up to day 21, six were found again with asexual parasites between day 9 and day 14 and a seventh on day 21. These results confirm the presence of chloroquine resistance in P. falciparum in East Kalimantan and, together with previous findings, suggest a widespread distribution of chloroquine-resistant falciparum malaria in this Province of Indonesia. It is particularly interesting to note that chloroquine-resistant falciparum malaria has now been detected in almost all the area of dispersion of A. balabacensis.

Hemozoin (Malarial Pigment) Directly Promotes Apoptosis of Erythroid Precursors

PubMed Central

Lamikanra, Abigail A.; Theron, Michel; Kooij, Taco W. A.; Roberts, David J.

2009-01-01

Severe malarial anemia is the most common syndrome of severe malaria in endemic areas. The pathophysiology of chronic malaria is characterised by a striking degree of abnormal development of erythroid precursors (dyserythropoiesis) and an inadequate erythropoietic response in spite of elevated levels of erythropoietin. The cause of dyserythropoiesis is unclear although it has been suggested that bone-marrow macrophages release cytokines, chemokines or lipo-peroxides after exposure to hemozoin, a crystalloid form of undigested heme moieties from malarial infected erythrocytes, and so inhibit erythropoiesis. However, we have previously shown that hemozoin may directly inhibit erythroid development in vitro and the levels of hemozoin in plasma from patients with malarial anemia and hemozoin within the bone marrow was associated with reduced reticulocyte response. We hypothesized that macrophages may reduce, not enhance, the inhibitory effect of hemozoin on erythropoiesis. In an in vitro model of erythropoiesis, we now show that inhibition of erythroid cell development by hemozoin isolated from P. falciparum is characterised by delayed expression of the erythroid markers and increased apoptosis of progenitor cells. Crucially, macrophages appear to protect erythroid cells from hemozoin, consistent with a direct contribution of hemozoin to the depression of reticulocyte output from the bone marrow in children with malarial anemia. Moreover, hemozoin isolated from P. falciparum in vitro inhibits erythroid development independently of inflammatory mediators by inducing apoptotic pathways that not only involve activation of caspase 8 and cleavage of caspase 3 but also loss of mitochondrial potential. Taken together these data are consistent with a direct effect of hemozoin in inducing apoptosis in developing erythroid cells in malarial anemia. Accumulation of hemozoin in the bone marrow could therefore result in inadequate reticulocytosis in children that have adequate levels of circulating erythropoietin. PMID:20041181

Development, standardization and validation of molecular techniques for malaria vector species identification, trophic preferences, and detection of Plasmodium falciparum.

PubMed

Rath, Animesha; Prusty, Manas R; Barik, Sushanta K; Das, Mumani; Tripathy, Hare K; Mahapatra, Namita; Hazra, Rupenangshu K

2017-01-01

Knowledge on prevalence of malaria vector species of a certain area provides important information for implementation of appropriate control strategies. The present study describes a rapid method for screening of major Anopheline vector species and at the same time detection of Plasmodium falciparum sporozoite infection and blood meal preferences/trophic preferences. The study was carried from February 2012 to March 2013 in three seasons, i.e. rainy, winter and summer in Jhumpura PHC of Keonjhar district, Odisha, India. Processing of mosquitoes was carried out in two different methods, viz. mosquito pool (P1) and mosquito DNA pool (P2). Pool size for both the methods was standardized for DNA isolation and multiplex PCR assay. This PCR based assay was employed to screen the major vector com- position in three different seasons of four different ecotypes of Keonjhar district. Pearson's correlation coefficient was determined for a comparative analysis of the morphological identification with the pool prevalence assay for each ecotype. A pool size of 10 was standardized for DNA isolation as well as PCR. PCR assay revealed that the average pool prevalence for all ecotypes was highest for An. annularis in winter and summer whereas for An. culicifacies it was rainy season. Foothill and plain ecotypes contributed to highest and lowest vectorial abundance respectively. The results of the prevalence of vector species in pool from PCR based assay were found to be highly correlated with that of the results of morphological identification. Screening by pool based PCR assay is relatively rapid as compared to conventional identification and can be employed as an important tool in malaria control programmes.

Antiprotozoal Diterpenes from Perovskia abrotanoides.

PubMed

Tabefam, Marzieh; Farimani, Mahdi Moridi; Danton, Ombeline; Ramseyer, Justine; Kaiser, Marcel; Ebrahimi, Samad N; Salehi, Peyman; Batooli, Hossien; Potterat, Olivier; Hamburger, Matthias

2018-04-26

As part of a screening for new antiparasitic natural products from Iranian plants, n -hexane and ethyl acetate extracts from the aerial parts of Perovskia abrotanoides were found to exhibit strong inhibitory activity against Trypanosoma brucei rhodesiense and Leishmania donovani . The activity was tracked by high-performance liquid chromatography (HPLC)-based activity profiling. Preparative isolation by a combination of silica gel column chromatography and HPLC afforded 17 diterpenoids (1: -17: ), including 14 abietane-, two icetexane-, and one isopimarane-type derivatives. Among these, (5 R ,10 S )-11-hydroxy-12-methoxy-20-norabieta-8,11,13-triene (2: ), 12-hydroxy-norabieta-1(10),8,11,13-tetraene-1,11-furan (6: ), and 12-methoxybarbatusol (9: ) were new compounds, the structure of which was established by comprehensive spectroscopic data analysis (one- and two-dimensional nuclear magnetic resonance, high-resolution electrospray ionization mass spectrometry, electronic circular dichroism). The antiprotozoal activity of the isolated compounds was evaluated against T. b. rhodesiense, Trypanosoma cruzi, L. donovani , and Plasmodium falciparum . Selectivity indexes (SI) were calculated in comparison to cytotoxicity on rat myoblast (L6) cells. Particularly active were 7 α -ethoxyrosmanol (4: ) with an IC 50 of 0.8 µM against T. b. rhodesiense (SI 14.9) and an IC 50 of 1.8 µM (SI 6.9) against L. donovani , ferruginol (8: ) with an IC 50 of 2.9 µM (SI 19.2) against P. falciparum , and miltiodiol (10: ) with an IC 50 of 0.5 µM (SI 10.5) against T. b. rhodesiense . None of the compounds exhibited selective toxicity against T. cruzi (SI ≤ 1.6). Georg Thieme Verlag KG Stuttgart · New York.