A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle.

PubMed

Vaughan, Ashley M; Mikolajczak, Sebastian A; Camargo, Nelly; Lakshmanan, Viswanathan; Kennedy, Mark; Lindner, Scott E; Miller, Jessica L; Hume, Jen C C; Kappe, Stefan H I

2012-12-01

Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP-luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages. Copyright © 2012 Elsevier B.V. All rights reserved.

Short report: entomologic inoculation rates and Plasmodium falciparum malaria prevalence in Africa.

PubMed

Beier, J C; Killeen, G F; Githure, J I

1999-07-01

Epidemiologic patterns of malaria infection are governed by environmental parameters that regulate vector populations of Anopheles mosquitoes. The intensity of malaria parasite transmission is normally expressed as the entomologic inoculation rate (EIR), the product of the vector biting rate times the proportion of mosquitoes infected with sporozoite-stage malaria parasites. Malaria transmission intensity in Africa is highly variable with annual EIRs ranging from < 1 to > 1,000 infective bites per person per year. Malaria control programs often seek to reduce morbidity and mortality due to malaria by reducing or eliminating malaria parasite transmission by mosquitoes. This report evaluates data from 31 sites throughout Africa to establish fundamental relationships between annual EIRs and the prevalence of Plasmodium falciparum malaria infection. The majority of sites fitted a linear relationship (r2 = 0.71) between malaria prevalence and the logarithm of the annual EIR. Some sites with EIRs < 5 infective bites per year had levels of P. falciparum prevalence exceeding 40%. When transmission exceeded 15 infective bites per year, there were no sites with prevalence rates < 50%. Annual EIRs of 200 or greater were consistently associated with prevalence rates > 80%. The basic relationship between EIR and P. falciparum prevalence, which likely holds in east and west Africa, and across different ecologic zones, shows convincingly that substantial reductions in malaria prevalence are likely to be achieved only when EIRs are reduced to levels less than 1 infective bite per person per year. The analysis also highlights that the EIR is a more direct measure of transmission intensity than traditional measures of malaria prevalence or hospital-based measures of infection or disease incidence. As such, malaria field programs need to consider both entomologic and clinical assessments of the efficacy of transmission control measures.

The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum.

PubMed

Robbins, Jonathan A; Absalon, Sabrina; Streva, Vincent A; Dvorin, Jeffrey D

2017-06-13

All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G 1 -, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G 1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways. IMPORTANCE Human infection by the eukaryotic parasite Plasmodium falciparum causes malaria. Most well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs) to promote essential cell division processes. Remarkably, there are no identifiable cyclins that are predicted to control the cell cycle in the malaria parasite genome. Thus, our knowledge regarding the basic mechanisms of the malaria parasite cell cycle remains unsatisfactory. We

Artemisinin-Resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes

PubMed Central

Hott, Amanda; Casandra, Debora; Sparks, Kansas N.; Morton, Lindsay C.; Castanares, Geocel-Grace; Rutter, Amanda

2015-01-01

Artemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance. PMID:25779582

DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

PubMed Central

Lee, Andrew H.; Symington, Lorraine S.

2014-01-01

SUMMARY Research into the complex genetic underpinnings of the malaria parasite Plasmodium falciparum is entering a new era with the arrival of site-specific genome engineering. Previously restricted only to model systems but now expanded to most laboratory organisms, and even to humans for experimental gene therapy studies, this technology allows researchers to rapidly generate previously unattainable genetic modifications. This technological advance is dependent on DNA double-strand break repair (DSBR), specifically homologous recombination in the case of Plasmodium. Our understanding of DSBR in malaria parasites, however, is based largely on assumptions and knowledge taken from other model systems, which do not always hold true in Plasmodium. Here we describe the causes of double-strand breaks, the mechanisms of DSBR, and the differences between model systems and P. falciparum. These mechanisms drive basic parasite functions, such as meiosis, antigen diversification, and copy number variation, and allow the parasite to continually evolve in the contexts of host immune pressure and drug selection. Finally, we discuss the new technologies that leverage DSBR mechanisms to accelerate genetic investigations into this global infectious pathogen. PMID:25184562

The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bakkouri, Majida El; Pow, Andre; Mulichak, Anne

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, weremore » also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.« less

The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum.

PubMed

El Bissati, Kamal; Zufferey, Rachel; Witola, William H; Carter, Nicola S; Ullman, Buddy; Ben Mamoun, Choukri

2006-06-13

The human malaria parasite Plasmodium falciparum relies on the acquisition of host purines for its survival within human erythrocytes. Purine salvage by the parasite requires specialized transporters at the parasite plasma membrane (PPM), but the exact mechanism of purine entry into the infected erythrocyte, and the primary purine source used by the parasite, remain unknown. Here, we report that transgenic parasites lacking the PPM transporter PfNT1 (P. falciparum nucleoside transporter 1) are auxotrophic for hypoxanthine, inosine, and adenosine under physiological conditions and are viable only if these normally essential nutrients are provided at excess concentrations. Transport measurements across the PPM revealed a severe reduction in hypoxanthine uptake in the knockout, whereas adenosine and inosine transport were only partially affected. These data provide compelling evidence for a sequential pathway for exogenous purine conversion into hypoxanthine using host enzymes followed by PfNT1-mediated transport into the parasite. The phenotype of the conditionally lethal mutant establishes PfNT1 as a critical component of purine salvage in P. falciparum and validates PfNT1 as a potential therapeutic target.

Influence of host factors and parasite biomass on the severity of imported Plasmodium falciparum malaria

PubMed Central

Kendjo, Eric; Augé-Courtoi, Claire; Cojean, Sandrine; Clain, Jérôme; Houzé, Pascal; Thellier, Marc; Hubert, Veronique; Deloron, Philippe; Houzé, Sandrine

2017-01-01

Objectives Imported malaria in France is characterized by various clinical manifestations observed in a heterogeneous population of patients such as travelers/expatriates and African migrants. In this population, host factors and parasite biomass associated with severe imported malaria are poorly known. Methods From data collected by the Centre National de Référence du Paludisme, we identified epidemiological, demographic and biological features including parasite biomass and anti-plasmodial antibody levels (negative, positive and strongly positive serology) associated with different disease severity groups (very severe, moderately severe, and uncomplicated malaria) in 3 epidemiological groups (travelers/expatriates, first- and second-generation migrants). Results Age, ethnicity, absence of prior infection with P. falciparum, antibody levels, plasma PfHRP2 levels, total and circulating parasite biomass were related to severe malaria onset. Sequestered parasite biomass tended to be increased in very severe malaria, and was strongly correlated to the antibody level of the host. Conclusions Prior exposure to P. falciparum is associated with high anti-plasmodial antibody levels which influence clinical presentation of imported malaria and its correlated circulating and sequestered parasite burden. PMID:28410415

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

The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites

PubMed Central

Charnaud, Sarah C.; Dixon, Matthew W. A.; Nie, Catherine Q.; Chappell, Lia; Sanders, Paul R.; Nebl, Thomas; Hanssen, Eric; Berriman, Matthew; Chan, Jo-Anne; Blanch, Adam J.; Beeson, James G.; Rayner, Julian C.; Przyborski, Jude M.; Tilley, Leann; Crabb, Brendan S.

2017-01-01

Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins. PMID:28732045

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.

Molecular Genetic Analysis of Parasite Survival in P. falciparum Malaria.

DTIC Science & Technology

1991-03-15

conducting research utilizing recombinant DNA technology , the investigator(s) adhered to current guidelines promulgated by the National Institute of Health...oligonucleotides unrelated to the conserved elements of Plasmodium falciparum were used (lane marked random oligo). Furthermore, extracts prepared...once with Ix Trager’s buffer. The following steps were carried out on ice. Erythrocytes were lysed in 0.05% saponin (19). The released parasites were

In-depth comparative analysis of malaria parasite genomes reveals protein-coding genes linked to human disease in Plasmodium falciparum genome.

PubMed

Liu, Xuewu; Wang, Yuanyuan; Liang, Jiao; Wang, Luojun; Qin, Na; Zhao, Ya; Zhao, Gang

2018-05-02

Plasmodium falciparum is the most virulent malaria parasite capable of parasitizing human erythrocytes. The identification of genes related to this capability can enhance our understanding of the molecular mechanisms underlying human malaria and lead to the development of new therapeutic strategies for malaria control. With the availability of several malaria parasite genome sequences, performing computational analysis is now a practical strategy to identify genes contributing to this disease. Here, we developed and used a virtual genome method to assign 33,314 genes from three human malaria parasites, namely, P. falciparum, P. knowlesi and P. vivax, and three rodent malaria parasites, namely, P. berghei, P. chabaudi and P. yoelii, to 4605 clusters. Each cluster consisted of genes whose protein sequences were significantly similar and was considered as a virtual gene. Comparing the enriched values of all clusters in human malaria parasites with those in rodent malaria parasites revealed 115 P. falciparum genes putatively responsible for parasitizing human erythrocytes. These genes are mainly located in the chromosome internal regions and participate in many biological processes, including membrane protein trafficking and thiamine biosynthesis. Meanwhile, 289 P. berghei genes were included in the rodent parasite-enriched clusters. Most are located in subtelomeric regions and encode erythrocyte surface proteins. Comparing cluster values in P. falciparum with those in P. vivax and P. knowlesi revealed 493 candidate genes linked to virulence. Some of them encode proteins present on the erythrocyte surface and participate in cytoadhesion, virulence factor trafficking, or erythrocyte invasion, but many genes with unknown function were also identified. Cerebral malaria is characterized by accumulation of infected erythrocytes at trophozoite stage in brain microvascular. To discover cerebral malaria-related genes, fast Fourier transformation (FFT) was introduced to extract

Parasite Sequestration in Plasmodium falciparum Malaria: Spleen and Antibody Modulation of Cytoadherence of Infected Erythrocytes

NASA Astrophysics Data System (ADS)

David, Peter H.; Hommel, Marcel; Miller, Louis H.; Udeinya, Iroka J.; Oligino, Lynette D.

1983-08-01

Sequestration, the adherence of infected erythrocytes containing late developmental stages of the parasite (trophozoites and schizonts) to the endothelium of capillaries and venules, is characteristic of Plasmodium falciparum infections. We have studied two host factors, the spleen and antibody, that influence sequestration of P. falciparum in the squirrel monkey. Sequestration of trophozoite/schizont-infected erythrocytes that occurs in intact animals is reduced in splenectomized animals; in vitro, when infected blood is incubated with monolayers of human melanoma cells, trophozoite/schizont-infected erythrocytes from intact animals but not from splenectomized animals bind to the melanoma cells. The switch in cytoadherence characteristics of the infected erythrocytes from nonbinding to binding occurs with a cloned parasite. Immune serum can inhibit and reverse in vitro binding to melanoma cells of infected erythrocytes from intact animals. Similarly, antibody can reverse in vivo sequestration as shown by the appearance of trophozoite/schizont-infected erythrocytes in the peripheral blood of an intact animal after inoculation with immune serum. These results indicate that the spleen modulates the expression of parasite alterations of the infected erythrocyte membrane responsible for sequestration and suggest that the prevention and reversal of sequestration could be one of the effector mechanisms involved in antibody-mediated protection against P. falciparum malaria.

Infectivity of Plasmodium falciparum in Malaria-Naive Individuals Is Related to Knob Expression and Cytoadherence of the Parasite

PubMed Central

Stanisic, Danielle I.; Gerrard, John; Fink, James; Griffin, Paul M.; Liu, Xue Q.; Sundac, Lana; Sekuloski, Silvana; Rodriguez, Ingrid B.; Pingnet, Jolien; Yang, Yuedong; Zhou, Yaoqi; Trenholme, Katharine R.; Wang, Claire Y. T.; Hackett, Hazel; Chan, Jo-Anne A.; Langer, Christine; Hanssen, Eric; Hoffman, Stephen L.; Beeson, James G.; McCarthy, James S.

2016-01-01

Plasmodium falciparum is the most virulent human malaria parasite because of its ability to cytoadhere in the microvasculature. Nonhuman primate studies demonstrated relationships among knob expression, cytoadherence, and infectivity. This has not been examined in humans. Cultured clinical-grade P. falciparum parasites (NF54, 7G8, and 3D7B) and ex vivo-derived cell banks were characterized. Knob and knob-associated histidine-rich protein expression, CD36 adhesion, and antibody recognition of parasitized erythrocytes (PEs) were evaluated. Parasites from the cell banks were administered to malaria-naive human volunteers to explore infectivity. For the NF54 and 3D7B cell banks, blood was collected from the study participants for in vitro characterization. All parasites were infective in vivo. However, infectivity of NF54 was dramatically reduced. In vitro characterization revealed that unlike other cell bank parasites, NF54 PEs lacked knobs and did not cytoadhere. Recognition of NF54 PEs by immune sera was observed, suggesting P. falciparum erythrocyte membrane protein 1 expression. Subsequent recovery of knob expression and CD36-mediated adhesion were observed in PEs derived from participants infected with NF54. Knobless cell bank parasites have a dramatic reduction in infectivity and the ability to adhere to CD36. Subsequent infection of malaria-naive volunteers restored knob expression and CD36-mediated cytoadherence, thereby showing that the human environment can modulate virulence. PMID:27382019

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

Focused Screening and Treatment (FSAT): A PCR-Based Strategy to Detect Malaria Parasite Carriers and Contain Drug Resistant P. falciparum, Pailin, Cambodia

PubMed Central

Hoyer, Stefan; Nguon, Sokomar; Kim, Saorin; Habib, Najibullah; Khim, Nimol; Sum, Sarorn; Christophel, Eva-Maria; Bjorge, Steven; Thomson, Andrew; Kheng, Sim; Chea, Nguon; Yok, Sovann; Top, Samphornarann; Ros, Seyha; Sophal, Uth; Thompson, Michelle M.; Mellor, Steve; Ariey, Frédéric; Witkowski, Benoit; Yeang, Chhiang; Yeung, Shunmay; Duong, Socheat; Newman, Robert D.; Menard, Didier

2012-01-01

Recent studies have shown that Plasmodium falciparum malaria parasites in Pailin province, along the border between Thailand and Cambodia, have become resistant to artemisinin derivatives. To better define the epidemiology of P. falciparum populations and to assess the risk of the possible spread of these parasites outside Pailin, a new epidemiological tool named “Focused Screening and Treatment” (FSAT), based on active molecular detection of asymptomatic parasite carriers was introduced in 2010. Cross-sectional malariometric surveys using PCR were carried out in 20 out of 109 villages in Pailin province. Individuals detected as P. falciparum carriers were treated with atovaquone-proguanil combination plus a single dose of primaquine if the patient was non-G6PD deficient. Interviews were conducted to elicit history of cross-border travel that might contribute to the spread of artemisinin-resistant parasites. After directly observed treatment, patients were followed up and re-examined on day 7 and day 28. Among 6931 individuals screened, prevalence of P. falciparum carriers was less than 1%, of whom 96% were asymptomatic. Only 1.6% of the individuals had a travel history or plans to go outside Cambodia, with none of those tested being positive for P. falciparum. Retrospective analysis, using 2010 routine surveillance data, showed significant differences in the prevalence of asymptomatic carriers discovered by FSAT between villages classified as “high risk” and “low risk” based on malaria incidence data. All positive individuals treated and followed-up until day 28 were cured. No mutant-type allele related to atovaquone resistance was found. FSAT is a potentially useful tool to detect, treat and track clusters of asymptomatic carriers of P. falciparum along with providing valuable epidemiological information regarding cross-border movements of potential malaria parasite carriers and parasite gene flow. PMID:23049687

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.

Decreased growth rate of P. falciparum blood stage parasitemia with age in a holoendemic population.

PubMed

Pinkevych, Mykola; Petravic, Janka; Chelimo, Kiprotich; Vulule, John; Kazura, James W; Moormann, Ann M; Davenport, Miles P

2014-04-01

In malaria holoendemic settings, decreased parasitemia and clinical disease is associated with age and cumulative exposure. The relative contribution of acquired immunity against various stages of the parasite life cycle is not well understood. In particular, it is not known whether changes in infection dynamics can be best explained by decreasing rates of infection, or by decreased growth rates of parasites in blood. Here, we analyze the dynamics of Plasmodium falciparum infection after treatment in a cohort of 197 healthy study participants of different ages. We use both polymerase chain reaction (PCR) and microscopy detection of parasitemia in order to understand parasite growth rates and infection rates over time. The more sensitive PCR assay detects parasites earlier than microscopy, and demonstrates a higher overall prevalence of infection than microscopy alone. The delay between PCR and microscopy detection is significantly longer in adults compared with children, consistent with slower parasite growth with age. We estimated the parasite multiplication rate from delay to PCR and microscopy detections of parasitemia. We find that both the delay between PCR and microscopy infection as well as the differing reinfection dynamics in different age groups are best explained by a slowing of parasite growth with age.

Persistence of Plasmodium falciparum parasites in infected pregnant Mozambican women after delivery.

PubMed

Serra-Casas, Elisa; Menéndez, Clara; Dobaño, Carlota; Bardají, Azucena; Quintó, Llorenç; Quintó, Llorençc; Ordi, Jaume; Sigauque, Betuel; Cisteró, Pau; Mandomando, Inacio; Alonso, Pedro L; Mayor, Alfredo

2011-01-01

Pregnant women are susceptible to Plasmodium falciparum parasites that sequester in the placenta. The massive accumulation of infected erythrocytes in the placenta has been suggested to trigger the deleterious effects of malaria in pregnant women and their offspring. The risk of malaria is also high during the postpartum period, although mechanisms underlying this susceptibility are not known. Here, we aimed to identify host factors contributing to the risk of postpartum infections and to determine the origin of postpartum parasites by comparing their genotypes with those present at the time of delivery. To address this, blood samples were collected at delivery (n = 402) and postpartum (n = 354) from Mozambican women enrolled in a trial of intermittent preventive treatment in pregnancy (IPTp). P. falciparum was detected by real-time quantitative PCR (qPCR), and the parasite merozoite surface protein 1 (msp-1) and msp-2 genes were genotyped. Fifty-seven out of 354 (16%) women were infected postpartum as assessed by qPCR, whereas prevalence by optical microscopy was only 4%. Risk of postpartum infection was lower in older women (odds ratio [OR] = 0.34, 95% confidence interval [CI] = 0.15 to 0.81) and higher in women with a placental infection at delivery (OR = 4.20, 95% CI = 2.19 to 8.08). Among 24 women with matched infections, 12 (50%) were infected postpartum with at least one parasite strain that was also present in their placentas. These results suggest that parasites infecting pregnant women persist after delivery and increase the risk of malaria during the postpartum period. Interventions that reduce malaria during pregnancy may translate into a lower risk of postpartum infection.

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.

Inability to detect transferrin receptors on P. falciparum parasitized red cells.

PubMed

Pollack, S; Schnelle, V

1988-01-01

The mechanism by which P. falciparum takes up iron from transferrin has been explored. Binding of 125I labelled transferrin to parasitized red cells at 37 degrees C is two-fold greater than to control cells; at 0 degrees C there is no significant difference. The binding is non-specific as judged from the following: it is not saturable; it is not limited to transferrin as lactoferrin (which has iron binding domains) and bovine serum albumin (which does not) also bind in excess to parasitized red cells. A transferrin receptor complex could not be demonstrated when parasitized red cells, to which 125I transferrin was bound, were solubilized in Triton X100. Previous observation showed that uptake of transferrin iron by parasitized red cells is not accompanied by equimolar uptake of transferrin protein. We therefore suggest that nonspecifically bound transferrin is endocytosed, that the protein is degraded and the iron selectively retained.

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.

Comparison of detection methods to estimate asexual Plasmodium falciparum parasite prevalence and gametocyte carriage in a community survey in Tanzania.

PubMed

Mwingira, Felista; Genton, Blaise; Kabanywanyi, Abdu-Noor M; Felger, Ingrid

2014-11-18

The use of molecular techniques to detect malaria parasites has been advocated to improve the accuracy of parasite prevalence estimates, especially in moderate to low endemic settings. Molecular work is time-consuming and costly, thus the effective gains of this technique need to be carefully evaluated. Light microscopy (LM) and rapid diagnostic tests (RDT) are commonly used to detect malaria infection in resource constrained areas, but their limited sensitivity results in underestimation of the proportion of people infected with Plasmodium falciparum. This study aimed to evaluate the extent of missed infections via a community survey in Tanzania, using polymerase chain reaction (PCR) to detect P. falciparum parasites and gametocytes. Three hundred and thirty individuals of all ages from the Kilombero and Ulanga districts (Tanzania) were enrolled in a cross-sectional survey. Finger prick blood samples were collected for parasite detection by RDT, LM and molecular diagnosis using quantitative 18S rRNA PCR and msp2 nPCR. Gametocytes were detected by LM and by amplifying transcripts of the gametocyte-specific marker pfs25. Results from all three diagnostic methods were available for a subset of 226 individuals. Prevalence of P. falciparum was 38% (86/226; 95% CI 31.9-44.4%) by qPCR, 15.9% (36/226; 95% CI 11.1-20.7%) by RDT and 5.8% (13/226; 95% CI 2.69- 8.81%) by LM. qPCR was positive for 72% (26/36) of the RDT-positive samples. Gametocyte prevalence was 10.6% (24/226) by pfs25-qRT-PCR and 1.2% by LM. LM showed the poorest performance, detecting only 15% of P. falciparum parasite carriers identified by PCR. Thus, LM is not a sufficiently accurate technique from which to inform policies and malaria control or elimination efforts. The diagnostic performance of RDT was superior to that of LM. However, it is also insufficient when precise prevalence data are needed for monitoring intervention success or for determining point prevalence rates in countrywide surveillance

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

K13-Propeller Polymorphisms in Plasmodium falciparum Parasites From Sub-Saharan Africa

PubMed Central

Kamau, Edwin; Campino, Susana; Amenga-Etego, Lucas; Drury, Eleanor; Ishengoma, Deus; Johnson, Kimberly; Mumba, Dieudonne; Kekre, Mihir; Yavo, William; Mead, Daniel; Bouyou-Akotet, Marielle; Apinjoh, Tobias; Golassa, Lemu; Randrianarivelojosia, Milijaona; Andagalu, Ben; Maiga-Ascofare, Oumou; Amambua-Ngwa, Alfred; Tindana, Paulina; Ghansah, Anita; MacInnis, Bronwyn; Kwiatkowski, Dominic; Djimde, Abdoulaye A.

2015-01-01

Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa. PMID:25367300

Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites

PubMed Central

2015-01-01

New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z′ factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z′ factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing d-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level. PMID:25102353

Plasmodium falciparum-like parasites infecting wild apes in southern Cameroon do not represent a recurrent source of human malaria

PubMed Central

Sundararaman, Sesh A.; Liu, Weimin; Keele, Brandon F.; Learn, Gerald H.; Bittinger, Kyle; Mouacha, Fatima; Ahuka-Mundeke, Steve; Manske, Magnus; Sherrill-Mix, Scott; Li, Yingying; Malenke, Jordan A.; Delaporte, Eric; Laurent, Christian; Mpoudi Ngole, Eitel; Kwiatkowski, Dominic P.; Shaw, George M.; Rayner, Julian C.; Peeters, Martine; Sharp, Paul M.; Bushman, Frederic D.; Hahn, Beatrice H.

2013-01-01

Wild-living chimpanzees and gorillas harbor a multitude of Plasmodium species, including six of the subgenus Laverania, one of which served as the progenitor of Plasmodium falciparum. Despite the magnitude of this reservoir, it is unknown whether apes represent a source of human infections. Here, we used Plasmodium species-specific PCR, single-genome amplification, and 454 sequencing to screen humans from remote areas of southern Cameroon for ape Laverania infections. Among 1,402 blood samples, we found 1,000 to be Plasmodium mitochondrial DNA (mtDNA) positive, all of which contained human parasites as determined by sequencing and/or restriction enzyme digestion. To exclude low-abundance infections, we subjected 514 of these samples to 454 sequencing, targeting a region of the mtDNA genome that distinguishes ape from human Laverania species. Using algorithms specifically developed to differentiate rare Plasmodium variants from 454-sequencing error, we identified single and mixed-species infections with P. falciparum, Plasmodium malariae, and/or Plasmodium ovale. However, none of the human samples contained ape Laverania parasites, including the gorilla precursor of P. falciparum. To characterize further the diversity of P. falciparum in Cameroon, we used single-genome amplification to amplify 3.4-kb mtDNA fragments from 229 infected humans. Phylogenetic analysis identified 62 new variants, all of which clustered with extant P. falciparum, providing further evidence that P. falciparum emerged following a single gorilla-to-human transmission. Thus, unlike Plasmodium knowlesi-infected macaques in southeast Asia, African apes harboring Laverania parasites do not seem to serve as a recurrent source of human malaria, a finding of import to ongoing control and eradication measures. PMID:23569255

G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets.

PubMed

Harris, Lynne M; Monsell, Katelyn R; Noulin, Florian; Famodimu, M Toyin; Smargiasso, Nicolas; Damblon, Christian; Horrocks, Paul; Merrick, Catherine J

2018-03-01

G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic acids. These four-stranded structures, composed of stacked quartets of guanine bases, can be highly stable and have been demonstrated to occur in vivo in the DNA of human cells and other systems, where they play important biological roles, influencing processes such as telomere maintenance, DNA replication and transcription, or, in the case of RNA G-quadruplexes, RNA translation and processing. We report for the first time that DNA G-quadruplexes can be detected in the nuclei of the malaria parasite Plasmodium falciparum , which has one of the most A/T-biased genomes sequenced and therefore possesses few guanine-rich sequences with the potential to form G-quadruplexes. We show that despite this paucity of putative G-quadruplex-forming sequences, P. falciparum parasites are sensitive to several G-quadruplex-stabilizing drugs, including quarfloxin, which previously reached phase 2 clinical trials as an anticancer drug. Quarfloxin has a rapid initial rate of kill and is active against ring stages as well as replicative stages of intraerythrocytic development. We show that several G-quadruplex-stabilizing drugs, including quarfloxin, can suppress the transcription of a G-quadruplex-containing reporter gene in P. falciparum but that quarfloxin does not appear to disrupt the transcription of rRNAs, which was proposed as its mode of action in both human cells and trypanosomes. These data suggest that quarfloxin has potential for repositioning as an antimalarial with a novel mode of action. Furthermore, G-quadruplex biology in P. falciparum may present a target for development of other new antimalarial drugs. Copyright © 2018 American Society for Microbiology.

Glucose-6-phosphate metabolism in Plasmodium falciparum.

PubMed

Preuss, Janina; Jortzik, Esther; Becker, Katja

2012-07-01

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based. Copyright © 2012 Wiley Periodicals, Inc.

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

Return of chloroquine-sensitive Plasmodium falciparum parasites and emergence of chloroquine-resistant Plasmodium vivax in Ethiopia

PubMed Central

2014-01-01

Background Increased resistance by Plasmodium falciparum parasites led to the withdrawal of the antimalarial drugs chloroquine and sulphadoxine-pyrimethamine in Ethiopia. Since 2004 artemether-lumefantrine has served to treat uncomplicated P. falciparum malaria. However, increasing reports on delayed parasite clearance to artemisinin opens up a new challenge in anti-malarial therapy. With the complete withdrawal of CQ for the treatment of Plasmodium falciparum malaria, this study assessed the evolution of CQ resistance by investigating the prevalence of mutant alleles in the pfmdr1 and pfcrt genes in P. falciparum and pvmdr1 gene in Plasmodium vivax in Southern and Eastern Ethiopia. Methods Of the 1,416 febrile patients attending primary health facilities in Southern Ethiopia, 329 febrile patients positive for P. falciparum or P. vivax were recruited. Similarly of the 1,304 febrile patients from Eastern Ethiopia, 81 febrile patients positive for P. falciparum or P. vivax were included in the study. Of the 410 finger prick blood samples collected from malaria patients, we used direct sequencing to investigate the prevalence of mutations in pfcrt and pfmdr1. This included determining the gene copy number in pfmdr1 in 195 P. falciparum clinical isolates, and mutations in the pvmdr1 locus in 215 P. vivax clinical isolates. Results The pfcrt K76 CQ-sensitive allele was observed in 84.1% of the investigated P.falciparum clinical isolates. The pfcrt double mutations (K76T and C72S) were observed less than 3%. The pfcrt SVMNT haplotype was also found to be present in clinical isolates from Ethiopia. The pfcrt CVMNK-sensitive haplotypes were frequently observed (95.9%). The pfmdr1 mutation N86Y was observed only in 14.9% compared to 85.1% of the clinical isolates that carried sensitive alleles. Also, the sensitive pfmdr1 Y184 allele was more common, in 94.9% of clinical isolates. None of the investigated P. falciparum clinical isolates carried S1034C, N1042D and D1246Y

Ned-19 inhibition of parasite growth and multiplication suggests a role for NAADP mediated signalling in the asexual development of Plasmodium falciparum.

PubMed

Suárez-Cortés, Pablo; Gambara, Guido; Favia, Annarita; Palombi, Fioretta; Alano, Pietro; Filippini, Antonio

2017-09-12

Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca 2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca 2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca 2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca 2+ levels. This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca 2+ oscillations suggests a potential role of NAADP in regulating Ca 2+ signalling of P. falciparum.

Plasmodium falciparum infection and age influence parasite growth inhibition mediated by IgG in Beninese infants.

PubMed

Adamou, Rafiou; Chénou, Francine; Sadissou, Ibrahim; Sonon, Paulin; Dechavanne, Célia; Djilali-Saïah, Abdelkader; Cottrell, Gilles; Le Port, Agnès; Massougbodji, Achille; Remarque, Edmond J; Luty, Adrian J F; Sanni, Ambaliou; Garcia, André; Migot-Nabias, Florence; Milet, Jacqueline; Courtin, David

2016-07-01

Antibodies that impede the invasion of Plasmodium falciparum (P. falciparum) merozoites into erythrocytes play a critical role in anti-malarial immunity. The Growth Inhibition Assay (GIA) is an in vitro measure of the functional capacity of such antibodies to limit erythrocyte invasion and/or parasite growth. Up to now, it is unclear whether growth-inhibitory activity correlates with protection from clinical disease and there are inconsistent results from studies performed with GIA. Studies that have focused on the relationship between IgGs and their in vitro parasite Growth Inhibition Activity (GIAc) in infants aged less than two years old are rare. Here, we used clinical and parasitological data to precisely define symptomatic or asymptomatic infection with P. falciparum in groups of infants followed-up actively for 18 months post-natally. We quantified the levels of IgG1 and IgG3 directed to a panel of candidate P. falciparum vaccine antigens (AMA-1, MSP1, 2, 3 and GLURP) using ELISA and the functional activity of IgG was quantified using GIA. Data were then correlated with individuals' infection status. At 18 months of age, infants harbouring infections at the time of blood sampling had an average 19% less GIAc than those not infected (p=0.004, multivariate linear regression). GIAc decreased from 12 to 18 months of age (p=0.003, Wilcoxon matched pairs test). Antibody levels quantified at 18 months in infants were strongly correlated with their exposure to malarial infection, however GIAc was not correlated with malaria infectious status (asymptomatic and symptomatic groups). In conclusion, both infection status at blood draw and age influence parasite growth inhibition mediated by IgG in the GIA. Both factors must be taken into account when correlations between GIAc and anti-malarial protection or vaccine efficacy have to be made. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Sugar nucleotide quantification by liquid chromatography tandem mass spectrometry reveals a distinct profile in Plasmodium falciparum sexual stage parasites

PubMed Central

López-Gutiérrez, Borja; Dinglasan, Rhoel R.

2017-01-01

The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties in obtaining sufficient amounts of biological material have hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the presence of N-, O-, and other glycans in key parasite proteins. In this work, we adapted a liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on the use of porous graphitic carbon (PGC) columns and MS-friendly solvents to quantify sugar nucleotides in the malaria parasite. We report the thorough quantification of the pools of these metabolites throughout the intraerythrocytic cycle of P. falciparum. The sensitivity of the method enabled, for the first time, the targeted analysis of these glycosylation precursors in gametocytes, the parasite sexual stages that are transmissible to the mosquito vector. PMID:28104756

Gametocytes of the Malaria Parasite Plasmodium falciparum Interact With and Stimulate Bone Marrow Mesenchymal Cells to Secrete Angiogenetic Factors

PubMed Central

Messina, Valeria; Valtieri, Mauro; Rubio, Mercedes; Falchi, Mario; Mancini, Francesca; Mayor, Alfredo; Alano, Pietro; Silvestrini, Francesco

2018-01-01

The gametocytes of Plasmodium falciparum, responsible for the transmission of this malaria parasite from humans to mosquitoes, accumulate and mature preferentially in the human bone marrow. In the 10 day long sexual development of P. falciparum, the immature gametocytes reach and localize in the extravascular compartment of this organ, in contact with several bone marrow stroma cell types, prior to traversing the endothelial lining and re-entering in circulation at maturity. To investigate the host parasite interplay underlying this still obscure process, we developed an in vitro tridimensional co-culture system in a Matrigel scaffold with P. falciparum gametocytes and self-assembling spheroids of human bone marrow mesenchymal cells (hBM-MSCs). Here we show that this co-culture system sustains the full maturation of the gametocytes and that the immature, but not the mature, gametocytes adhere to hBM-MSCs via trypsin-sensitive parasite ligands exposed on the erythrocyte surface. Analysis of a time course of gametocytogenesis in the co-culture system revealed that gametocyte maturation is accompanied by the parasite induced stimulation of hBM-MSCs to secrete a panel of 14 cytokines and growth factors, 13 of which have been described to play a role in angiogenesis. Functional in vitro assays on human bone marrow endothelial cells showed that supernatants from the gametocyte mesenchymal cell co-culture system enhance ability of endothelial cells to form vascular tubes. These results altogether suggest that the interplay between immature gametocytes and hBM-MSCs may induce functional and structural alterations in the endothelial lining of the human bone marrow hosting the P. falciparum transmission stages. PMID:29546035

Pfhrp2-Deleted Plasmodium falciparum Parasites in the Democratic Republic of the Congo: A National Cross-sectional Survey.

PubMed

Parr, Jonathan B; Verity, Robert; Doctor, Stephanie M; Janko, Mark; Carey-Ewend, Kelly; Turman, Breanna J; Keeler, Corinna; Slater, Hannah C; Whitesell, Amy N; Mwandagalirwa, Kashamuka; Ghani, Azra C; Likwela, Joris L; Tshefu, Antoinette K; Emch, Michael; Juliano, Jonathan J; Meshnick, Steven R

2017-07-01

Rapid diagnostic tests (RDTs) account for more than two-thirds of malaria diagnoses in Africa. Deletions of the Plasmodium falciparum hrp2 (pfhrp2) gene cause false-negative RDT results and have never been investigated on a national level. Spread of pfhrp2-deleted P. falciparum mutants, resistant to detection by HRP2-based RDTs, would represent a serious threat to malaria elimination efforts. Using a nationally representative cross-sectional study of 7,137 children under five years of age from the Democratic Republic of Congo (DRC), we tested 783 subjects with RDT-/PCR+ results using PCR assays to detect and confirm deletions of the pfhrp2 gene. Spatial and population genetic analyses were employed to examine the distribution and evolution of these parasites. We identified 149 pfhrp2-deleted parasites, representing 6.4% of all P. falciparum infections country-wide (95% confidence interval 5.1-8.0%). Bayesian spatial analyses identified statistically significant clustering of pfhrp2 deletions near Kinshasa and Kivu. Population genetic analysis revealed significant genetic differentiation between wild-type and pfhrp2-deleted parasite populations (GST = .046, p ≤ .00001). Pfhrp2-deleted P. falciparum is a common cause of RDT-/PCR+ malaria among asymptomatic children in the DRC and appears to be clustered within select communities. Surveillance for these deletions is needed, and alternatives to HRP2-specific RDTs may be necessary. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

An enhancer-like region regulates hrp3 promoter stage-specific gene expression in the human malaria parasite Plasmodium falciparum

PubMed Central

López-Estraño, Carlos; Gopalakrishnan, Anusha M.; Semblat, Jean-Philippe; Fergus, M. Ross; Mazier, Dominique; Haldar, Kasturi

2008-01-01

The asexual blood stage of Plasmodium falciparum is comprised of morphologically distinct ring, trophozoite and schizont stages. Each of these developmental stages possesses a distinct pattern of gene expression. Regulation of P. falciparum gene expression is thought to occur, at least in part, at the promoter level. Previously, we have found that although the RNA of the P. falciparum hrp3 gene is only seen in ring-stage parasites, deletion of a specific sequensce in the 5’ end of the promoter region decreased ring-stage expression of hrp3 and enabled detection of its transcripts in trophozoite-stage parasites. In order to investigate this stage specific regulation of gene expression, we employed a series of nested deletions of the 1.7-kb hrp3 promoter. Firefly luciferase gene was used as a reporter to evaluate the role of promoter sequences in gene regulation. Using this approach, we identified a ring-stage specific regulatory region on the hrp3 promoter located between -1.7-kb and -1.1-kb from the ATG initiation codon. Small 100–150 bp truncations on this enhancer-like region failed to uncover discrete regulatory sequences, suggesting the multipartite nature of this element. The data presented in this study demonstrates that stage specific promoter activity of the hrp3 gene in P. falciparum blood stage parasites is supported, at least in-part, by a small promoter region that can function in the absence of a larger chromosomal context. PMID:17570541

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

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

Size and sequence polymorphisms in the glutamate-rich protein gene of the human malaria parasite Plasmodium falciparum in Thailand.

PubMed

Pattaradilokrat, Sittiporn; Trakoolsoontorn, Chawinya; Simpalipan, Phumin; Warrit, Natapot; Kaewthamasorn, Morakot; Harnyuttanakorn, Pongchai

2018-01-22

The glutamate-rich protein (GLURP) of the malaria parasite Plasmodium falciparum is a key surface antigen that serves as a component of a clinical vaccine. Moreover, the GLURP gene is also employed routinely as a genetic marker for malarial genotyping in epidemiological studies. While extensive size polymorphisms in GLURP are well recorded, the extent of the sequence diversity of this gene is rarely investigated. The present study aimed to explore the genetic diversity of GLURP in natural populations of P. falciparum. The polymorphic C-terminal repetitive R2 region of GLURP sequences from 65 P. falciparum isolates in Thailand were generated and combined with the data from 103 worldwide isolates to generate a GLURP database. The collection was comprised of 168 alleles, encoding 105 unique GLURP subtypes, characterized by 18 types of amino acid repeat units (AAU). Of these, 28 GLURP subtypes, formed by 10 AAU types, were detected in P. falciparum in Thailand. Among them, 19 GLURP subtypes and 2 AAU types are described for the first time in the Thai parasite population. The AAU sequences were highly conserved, which is likely due to negative selection. Standard Fst analysis revealed the shared distributions of GLURP types among the P. falciparum populations, providing evidence of gene flow among the different demographic populations. Sequence diversity causing size variations in GLURP in Thai P. falciparum populations were detected, and caused by non-synonymous substitutions in repeat units and some insertion/deletion of aspartic acid or glutamic acid codons between repeat units. The P. falciparum population structure based on GLURP showed promising implications for the development of GLURP-based vaccines and for monitoring vaccine efficacy.

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

Characterization of the Plasmodium falciparum and P. berghei glycerol 3-phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast.

PubMed

Shears, Melanie J; MacRae, James I; Mollard, Vanessa; Goodman, Christopher D; Sturm, Angelika; Orchard, Lindsey M; Llinás, Manuel; McConville, Malcolm J; Botté, Cyrille Y; McFadden, Geoffrey I

2017-01-01

Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti-malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3-phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N-terminal targeting sequence to GFP and 3' tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site-directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3-phosphate acyltransferase in malaria parasites. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

Malaria case clinical profiles and Plasmodium falciparum parasite genetic diversity: a cross sectional survey at two sites of different malaria transmission intensities in Rwanda.

PubMed

Kateera, Fredrick; Nsobya, Sam L; Tukwasibwe, Stephen; Mens, Petra F; Hakizimana, Emmanuel; Grobusch, Martin P; Mutesa, Leon; Kumar, Nirbhay; van Vugt, Michele

2016-04-26

Malaria remains a public health challenge in sub-Saharan Africa with Plasmodium falciparum being the principal cause of malaria disease morbidity and mortality. Plasmodium falciparum virulence is attributed, in part, to its population-level genetic diversity-a characteristic that has yet to be studied in Rwanda. Characterizing P. falciparum molecular epidemiology in an area is needed for a better understand of malaria transmission and to inform choice of malaria control strategies. In this health-facility based survey, malaria case clinical profiles and parasite densities as well as parasite genetic diversity were compared among P. falciparum-infected patients identified at two sites of different malaria transmission intensities in Rwanda. Data on demographics and clinical features and finger-prick blood samples for microscopy and parasite genotyping were collected(.) Nested PCR was used to genotype msp-2 alleles of FC27 and 3D7. Patients' variables of age group, sex, fever (both by patient report and by measured tympanic temperatures), parasite density, and bed net use were found differentially distributed between the higher endemic (Ruhuha) and lower endemic (Mubuga) sites. Overall multiplicity of P. falciparum infection (MOI) was 1.73 but with mean MOI found to vary significantly between 2.13 at Ruhuha and 1.29 at Mubuga (p < 0.0001). At Ruhuha, expected heterozygosity (EH) for FC27 and 3D7 alleles were 0.62 and 0.49, respectively, whilst at Mubuga, EH for FC27 and 3D7 were 0.26 and 0.28, respectively. In this study, a higher geometrical mean parasite counts, more polyclonal infections, higher MOI, and higher allelic frequency were observed at the higher malaria-endemic (Ruhuha) compared to the lower malaria-endemic (Mubuga) area. These differences in malaria risk and MOI should be considered when choosing setting-specific malaria control strategies, assessing p. falciparum associated parameters such as drug resistance, immunity and impact of used

H2O2 dynamics in the malaria parasite Plasmodium falciparum

PubMed Central

Rahbari, Mahsa; Bogeski, Ivan

2017-01-01

Hydrogen peroxide is an important antimicrobial agent but is also crucially involved in redox signaling and pathogen-host cell interactions. As a basis for systematically investigating intracellular H2O2 dynamics and regulation in living malaria parasites, we established the genetically encoded fluorescent H2O2 sensors roGFP2-Orp1 and HyPer-3 in Plasmodium falciparum. Both ratiometric redox probes as well as the pH control SypHer were expressed in the cytosol of blood-stage parasites. Both redox sensors showed reproducible sensitivity towards H2O2 in the lower micromolar range in vitro and in the parasites. Due to the pH sensitivity of HyPer-3, we used parasites expressing roGFP2-Orp1 for evaluation of short-, medium-, and long-term effects of antimalarial drugs on H2O2 levels and detoxification in Plasmodium. None of the quinolines or artemisinins tested had detectable direct effects on the H2O2 homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H2O2. The systematic evaluation and comparison of the two genetically encoded cytosolic H2O2 probes in malaria parasites provides a basis for studying parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity. PMID:28369083

K13-propeller polymorphisms in Plasmodium falciparum parasites from sub-Saharan Africa.

PubMed

Kamau, Edwin; Campino, Susana; Amenga-Etego, Lucas; Drury, Eleanor; Ishengoma, Deus; Johnson, Kimberly; Mumba, Dieudonne; Kekre, Mihir; Yavo, William; Mead, Daniel; Bouyou-Akotet, Marielle; Apinjoh, Tobias; Golassa, Lemu; Randrianarivelojosia, Milijaona; Andagalu, Ben; Maiga-Ascofare, Oumou; Amambua-Ngwa, Alfred; Tindana, Paulina; Ghansah, Anita; MacInnis, Bronwyn; Kwiatkowski, Dominic; Djimde, Abdoulaye A

2015-04-15

Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa. © 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.

Identification and characterization of ARS-like sequences as putative origin(s) of replication in human malaria parasite Plasmodium falciparum.

PubMed

Agarwal, Meetu; Bhowmick, Krishanu; Shah, Kushal; Krishnamachari, Annangarachari; Dhar, Suman Kumar

2017-08-01

DNA replication is a fundamental process in genome maintenance, and initiates from several genomic sites (origins) in eukaryotes. In Saccharomyces cerevisiae, conserved sequences known as autonomously replicating sequences (ARSs) provide a landing pad for the origin recognition complex (ORC), leading to replication initiation. Although origins from higher eukaryotes share some common sequence features, the definitive genomic organization of these sites remains elusive. The human malaria parasite Plasmodium falciparum undergoes multiple rounds of DNA replication; therefore, control of initiation events is crucial to ensure proper replication. However, the sites of DNA replication initiation and the mechanism by which replication is initiated are poorly understood. Here, we have identified and characterized putative origins in P. falciparum by bioinformatics analyses and experimental approaches. An autocorrelation measure method was initially used to search for regions with marked fluctuation (dips) in the chromosome, which we hypothesized might contain potential origins. Indeed, S. cerevisiae ARS consensus sequences were found in dip regions. Several of these P. falciparum sequences were validated with chromatin immunoprecipitation-quantitative PCR, nascent strand abundance and a plasmid stability assay. Subsequently, the same sequences were used in yeast to confirm their potential as origins in vivo. Our results identify the presence of functional ARSs in P. falciparum and provide meaningful insights into replication origins in these deadly parasites. These data could be useful in designing transgenic vectors with improved stability for transfection in P. falciparum. © 2017 Federation of European Biochemical Societies.

Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle.

PubMed

Collins, Christine R; Das, Sujaan; Wong, Eleanor H; Andenmatten, Nicole; Stallmach, Robert; Hackett, Fiona; Herman, Jean-Paul; Müller, Sylke; Meissner, Markus; Blackman, Michael J

2013-05-01

Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes. © 2013 John Wiley & Sons Ltd.

Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krungkrai, Sudaratana R.; Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871; Tokuoka, Keiji

Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation. Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 asmore » a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V{sub M} = 2.3 Å{sup 3} Da{sup −1})« less

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

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.

The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

DOE Office of Scientific and Technical Information (OSTI.GOV)

M El Bakkouri; A Pow; A Mulichak

The Clpchaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clpchaperones and proteases in the humanmalariaparasitePlasmodiumfalciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clpchaperonesmore » and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.« less

Plasmodium falciparum in vitro continuous culture conditions: A comparison of parasite susceptibility and tolerance to anti-malarial drugs throughout the asexual intra-erythrocytic life cycle.

PubMed

Duffy, Sandra; Avery, Vicky M

2017-12-01

The continuous culture of Plasmodium falciparum is often seen as a means to an end, that end being to probe the biology of the parasite in question, and ultimately for many in the malaria drug discovery arena, to identify means of killing the parasite in order to treat malaria. In vitro continuous culture of Plasmodium falciparum is a fundamental requirement when undertaking malaria research where the primary objectives utilise viable parasites of a desired lifecycle stage. This investigation, and resulting data, compared the impact culturing Plasmodium falciparum long term (4 months) in different environmental conditions had on experimental outcomes and thus conclusions. The example presented here focused specifically on the effect culture conditions had on the in vitro tolerance of Plasmodium falciparum to standard anti-malarial drugs, including artemisinin and lumefantrine. Historical data from an independent experiment for 3D7-ALB (5% O 2 ) was also compared with that obtained from this study. We concluded that parasites cultured for several months in media supplemented with a serum substitute such as Albumax II ® or within hyperoxic conditions (21% O 2 ), demonstrate highly variable responses to artemisinin and lumefantrine but not all anti-malarial drugs, when compared to those cultured in human serum in combination with Albumax II ® under normoxic conditions (5% O 2 ) for the parasite. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

In vitro growth of Plasmodium falciparum in neonatal blood.

PubMed

Sauerzopf, Ulrich; Honkpehedji, Yabo J; Adgenika, Ayôla A; Feugap, Elianne N; Ngoma, Ghyslain Mombo; Mackanga, Jean-Rodolphe; Lötsch, Felix; Loembe, Marguerite M; Kremsner, Peter G; Mordmüller, Benjamin; Ramharter, Michael

2014-11-18

Children below the age of six months suffer less often from malaria than older children in sub-Saharan Africa. This observation is commonly attributed to the persistence of foetal haemoglobin (HbF), which is considered not to permit growth of Plasmodium falciparum and therefore providing protection against malaria. Since this concept has recently been challenged, this study evaluated the effect of HbF erythrocytes and maternal plasma on in vitro parasite growth of P. falciparum in Central African Gabon. Umbilical cord blood and peripheral maternal blood were collected at delivery at the Albert Schweitzer Hospital in Gabon. Respective erythrocyte suspension and plasma were used in parallel for in vitro culture. In vitro growth rates were compared between cultures supplemented with either maternal or cord erythrocytes. Plasma of maternal blood and cord blood was evaluated. Parasite growth rates were assessed by the standard HRP2-assay evaluating the increase of HRP2 concentration in Plasmodium culture. Culture of P. falciparum using foetal erythrocytes led to comparable growth rates (mean growth rate = 4.2, 95% CI: 3.5 - 5.0) as cultures with maternal red blood cells (mean growth rate =4.2, 95% CI: 3.4 - 5.0) and those from non-malaria exposed individuals (mean growth rate = 4.6, 95% CI: 3.8 - 5.5). Standard in vitro culture of P. falciparum supplemented with either maternal or foetal plasma showed both significantly lower growth rates than a positive control using non-malaria exposed donor plasma. These data challenge the concept of HbF serving as intrinsic inhibitor of P. falciparum growth in the first months of life. Erythrocytes containing HbF are equally permissive to P. falciparum growth in vitro. However, addition of maternal and cord plasma led to reduced in vitro growth which may translate to protection against clinical disease or show synergistic effects with HbF in vivo. Further studies are needed to elucidate the pathophysiology of innate and acquired

Antimalarial Activity of KAF156 in Falciparum and Vivax Malaria.

PubMed

White, Nicholas J; Duong, Tran T; Uthaisin, Chirapong; Nosten, François; Phyo, Aung P; Hanboonkunupakarn, Borimas; Pukrittayakamee, Sasithon; Jittamala, Podjanee; Chuthasmit, Kittiphum; Cheung, Ming S; Feng, Yiyan; Li, Ruobing; Magnusson, Baldur; Sultan, Marc; Wieser, Daniela; Xun, Xiaolei; Zhao, Rong; Diagana, Thierry T; Pertel, Peter; Leong, F Joel

2016-09-22

KAF156 belongs to a new class of antimalarial agents (imidazolopiperazines), with activity against asexual and sexual blood stages and the preerythrocytic liver stages of malarial parasites. We conducted a phase 2, open-label, two-part study at five centers in Thailand and Vietnam to assess the antimalarial efficacy, safety, and pharmacokinetic profile of KAF156 in adults with acute Plasmodium vivax or P. falciparum malaria. Assessment of parasite clearance rates in cohorts of patients with vivax or falciparum malaria who were treated with multiple doses (400 mg once daily for 3 days) was followed by assessment of the cure rate at 28 days in a separate cohort of patients with falciparum malaria who received a single dose (800 mg). Median parasite clearance times were 45 hours (interquartile range, 42 to 48) in 10 patients with falciparum malaria and 24 hours (interquartile range, 20 to 30) in 10 patients with vivax malaria after treatment with the multiple-dose regimen and 49 hours (interquartile range, 42 to 54) in 21 patients with falciparum malaria after treatment with the single dose. Among the 21 patients who received the single dose and were followed for 28 days, 1 had reinfection and 7 had recrudescent infections (cure rate, 67%; 95% credible interval, 46 to 84). The mean (±SD) KAF156 terminal elimination half-life was 44.1±8.9 hours. There were no serious adverse events in this small study. The most common adverse events included sinus bradycardia, thrombocytopenia, hypokalemia, anemia, and hyperbilirubinemia. Vomiting of grade 2 or higher occurred in 2 patients, 1 of whom discontinued treatment because of repeated vomiting after receiving the single 800-mg dose. More adverse events were reported in the single-dose cohort, which had longer follow-up, than in the multiple-dose cohorts. KAF156 showed antimalarial activity without evident safety concerns in a small number of adults with uncomplicated P. vivax or P. falciparum malaria. (Funded by Novartis and

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

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.

Chloroquine diphosphate bearing dextran nanoparticles augmented drug delivery and overwhelmed drug resistance in Plasmodium falciparum parasites.

PubMed

Kashyap, Aman; Kaur, Rupinder; Baldi, Ashish; Jain, Upendra Kumar; Chandra, Ramesh; Madan, Jitender

2018-07-15

Chloroquine diphosphate (CHQ) is primarily used for the treatment of Plasmodium falciparum malaria at the dose of 500mg orally or 10mg/kg parenterally. However, point mutations in Plasmodiumfalciparum chloroquine resistance transporter (PfCRT) protein and Plasmodium falciparum multidrug resistance protein 1 (Pfmdr1) localized in digestive vacuole membrane, are responsible for CHQ resistance. Therefore, in present investigation, dextran nanoparticles bearing chloroquine diphosphate (CHQ-DEX-NPs) were formulated by solvent diffusion method of size below 70nm with zeta-potential of -20.1±3.2mV. FT-IR, DSC and PXRD techniques confirmed the successful loading of drug in nanomatrix system with amorphous attributes. In vitro drug release analysis indicated the Higuchi pattern with diffusion controlled drug release. The IC 50 of CHQ-DEX-NPs in sensitive (3D7) and resistant (RKL9) Plasmodium falciparum strains was estimated to be 0.031-μg/ml and 0.13-μg/ml significantly lower than 0.059-μg/ml and 0.36-μg/ml of CHQ. The augmented therapeutic efficacy of CHQ-DEX-NPs may be credited to deposition of tailored nanoparticles in food vacuoles of malaria parasites owing to the affinity of parasite towards DEX that consequently lower the drug resistance and improved the therapeutic index. In conclusion, CHQ-DEX-NPs must be evaluated under a set of stringent in vivo parameters to establish its therapeutic efficacy in preclinical model. Copyright © 2018 Elsevier B.V. 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

Co-expression network with protein-protein interaction and transcription regulation in malaria parasite Plasmodium falciparum.

PubMed

Yu, Fu-Dong; Yang, Shao-You; Li, Yuan-Yuan; Hu, Wei

2013-04-10

Malaria continues to be one of the most severe global infectious diseases, as a major threat to human health and economic development. Network-based biological analysis is a promising approach to uncover key genes and biological processes from a network viewpoint, which could not be recognized from individual gene-based signatures. We integrated gene co-expression profile with protein-protein interaction and transcriptional regulation information to construct a comprehensive gene co-expression network of Plasmodium falciparum. Based on this network, we identified 10 core modules by using ICE (Iterative Clique Enumeration) algorithm, which were essential for malaria parasite development in intraerythrocytic developmental cycle (IDC) stages. In each module, all genes were highly correlated probably due to co-regulation or formation of a protein complex. Some of these genes were recognized to be differentially coexpressed among three close-by IDC stages. The gene of prpf8 (PFD0265w) encoding pre-mRNA processing splicing factor 8 product was identified as DCGs (differentially co-expressed genes) among IDC stages, although this gene function was seldom reported in previous researches. Integrating the species-specific gene prediction and differential co-expression gene detection, we found some modules could perform species-specific functions according to some of genes in these modules were species-specific genes, like the module 10. Furthermore, in order to reveal the underlying mechanisms of the erythrocyte invasion by P. falciparum, Steiner Tree algorithm was employed to identify the invasion subnetwork from our gene co-expression network. The subnetwork-based analysis indicated that some important Plasmodium parasite specific genes could corporate with each other and be co-regulated during the parasite invasion process, which including a head-to-head gene pair of PfRH2a (PF13_0198) and PfRH2b (MAL13P1.176). This study based on gene co-expression network could shed new

Plasmodium falciparum: attenuation by irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waki, S.; Yonome, I.; Suzuki, M.

The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed tomore » doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10(6) parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.« less

The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny, duplication rate and parasite virulence

PubMed Central

Morlon-Guyot, Juliette; Bordat, Yann; Lebrun, Maryse; Gubbels, Marc-Jan; Doerig, Christian; Daher, Wassim

2016-01-01

Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function, and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora-related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In T. gondii, we show that TgArk2 and TgArk3 concentrate at specific sub-cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein (GFP) in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock-down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3-depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites, and highlights Aurora kinase 3 as potential drug target. PMID:26833682

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.

High rate of adaptation of mammalian proteins that interact with Plasmodium and related parasites

PubMed Central

Telis, Natalie; Petrov, Dmitri A.

2017-01-01

Plasmodium parasites, along with their Piroplasm relatives, have caused malaria-like illnesses in terrestrial mammals for millions of years. Several Plasmodium-protective alleles have recently evolved in human populations, but little is known about host adaptation to blood parasites over deeper evolutionary timescales. In this work, we analyze mammalian adaptation in ~500 Plasmodium- or Piroplasm- interacting proteins (PPIPs) manually curated from the scientific literature. We show that (i) PPIPs are enriched for both immune functions and pleiotropy with other pathogens, and (ii) the rate of adaptation across mammals is significantly elevated in PPIPs, compared to carefully matched control proteins. PPIPs with high pathogen pleiotropy show the strongest signatures of adaptation, but this pattern is fully explained by their immune enrichment. Several pieces of evidence suggest that blood parasites specifically have imposed selection on PPIPs. First, even non-immune PPIPs that lack interactions with other pathogens have adapted at twice the rate of matched controls. Second, PPIP adaptation is linked to high expression in the liver, a critical organ in the parasite life cycle. Finally, our detailed investigation of alpha-spectrin, a major red blood cell membrane protein, shows that domains with particularly high rates of adaptation are those known to interact specifically with P. falciparum. Overall, we show that host proteins that interact with Plasmodium and Piroplasm parasites have experienced elevated rates of adaptation across mammals, and provide evidence that some of this adaptation has likely been driven by blood parasites. PMID:28957326

Chloroquine transport in Plasmodium falciparum. 1. Influx and efflux kinetics for live trophozoite parasites using a novel fluorescent chloroquine probe.

PubMed

Cabrera, Mynthia; Natarajan, Jayakumar; Paguio, Michelle F; Wolf, Christian; Urbach, Jeffrey S; Roepe, Paul D

2009-10-13

Several models for how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Distinguishing between these models requires detailed analysis of high-resolution CQ transport data that is unfortunately impossible to obtain with traditional radio-tracer methods. Thus, we have designed and synthesized fluorescent CQ analogues for drug transport studies. One probe places a NBD (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) group at the tertiary aliphatic N of CQ, via a flexible 6 C amide linker. This probe localizes to the malarial parasite digestive vacuole (DV) during initial perfusion under physiologic conditions and exhibits similar pharmacology relative to CQ, vs both CQ-sensitive (CQS) and CQ-resistant (CQR) parasites. Using live, synchronized intraerythrocytic parasites under continuous perfusion, we define NBD-CQ influx and efflux kinetics for CQS vs CQR parasites. Since this fluorescence approach provides data at much higher kinetic resolution relative to fast-filtration methods using (3)H-CQ, rate constants vs linear initial rates for CQ probe flux can be analyzed in detail. Importantly, we find that CQR parasites have a decreased rate constant for CQ influx into the DV and that this is due to mutation of PfCRT. Analysis of zero trans efflux for CQS and CQR parasites suggests that distinguishing between bound vs free pools of intra-DV drug probe is essential for proper kinetic analysis of efflux. The accompanying paper (DOI 10.1021/bi901035j ) further probes efflux kinetics for proteoliposomes containing purified, reconstituted PfCRT.

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

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

Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of /sup 125/I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of /sup 125/I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes.more » By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen.« less

Plasmodium falciparum parasite population structure and gene flow associated to anti-malarial drugs resistance in Cambodia.

PubMed

Dwivedi, Ankit; Khim, Nimol; Reynes, Christelle; Ravel, Patrice; Ma, Laurence; Tichit, Magali; Bourchier, Christiane; Kim, Saorin; Dourng, Dany; Khean, Chanra; Chim, Pheaktra; Siv, Sovannaroth; Frutos, Roger; Lek, Dysoley; Mercereau-Puijalon, Odile; Ariey, Frédéric; Menard, Didier; Cornillot, Emmanuel

2016-06-14

Western Cambodia is recognized as the epicentre of emergence of Plasmodium falciparum multi-drug resistance. The emergence of artemisinin resistance has been observed in this area since 2008-2009 and molecular signatures associated to artemisinin resistance have been characterized in k13 gene. At present, one of the major threats faced, is the possible spread of Asian artemisinin resistant parasites over the world threatening millions of people and jeopardizing malaria elimination programme efforts. To anticipate the diffusion of artemisinin resistance, the identification of the P. falciparum population structure and the gene flow among the parasite population in Cambodia are essential. To this end, a mid-throughput PCR-LDR-FMA approach based on LUMINEX technology was developed to screen for genetic barcode in 533 blood samples collected in 2010-2011 from 16 health centres in malaria endemics areas in Cambodia. Based on successful typing of 282 samples, subpopulations were characterized along the borders of the country. Each 11-loci barcode provides evidence supporting allele distribution gradient related to subpopulations and gene flow. The 11-loci barcode successfully identifies recently emerging parasite subpopulations in western Cambodia that are associated with the C580Y dominant allele for artemisinin resistance in k13 gene. A subpopulation was identified in northern Cambodia that was associated to artemisinin (R539T resistant allele of k13 gene) and mefloquine resistance. The gene flow between these subpopulations might have driven the spread of artemisinin resistance over Cambodia.

Plasmodium falciparum incidence relative to entomologic inoculation rates at a site proposed for testing malaria vaccines in western Kenya.

PubMed

Beier, J C; Oster, C N; Onyango, F K; Bales, J D; Sherwood, J A; Perkins, P V; Chumo, D K; Koech, D V; Whitmire, R E; Roberts, C R

1994-05-01

Relationships between Plasmodium falciparum incidence and entomologic inoculation rates (EIRs) were determined for a 21-month period in Saradidi, western Kenya, in preparation for malaria vaccine field trials. Children, ranging in age from six months to six years and treated to clear malaria parasites, were monitored daily for up to 12 weeks to detect new malaria infections. Overall, new P. falciparum infections were detected in 77% of 809 children. The percentage of children that developed infections per two-week period averaged 34.7%, ranging from 7.3% to 90.9%. Transmission by vector populations was detected in 86.4% (38 of 44) of the two-week periods, with daily EIRs averaging 0.75 infective bites per person. Periods of intense transmission during April to August, and from November to January, coincided with seasonal rains. Relationships between daily malaria attack rates and EIRs indicated that an average of only 7.5% (1 in 13) of the sporozoite inoculations produced new infections in children. Regression analysis demonstrated that EIRs accounted for 74% of the variation in attack rates. One of the components of the EIR, the human-biting rate, alone accounted for 68% of the variation in attack rates. Thus, measurements of either the EIR or the human-biting rate can be used to predict corresponding attack rates in children. These baseline epidemiologic studies indicate that the intense transmission patterns of P. falciparum in Saradidi will provide excellent conditions for evaluating malaria vaccine efficacy.

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.

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

Interrogating alkyl and arylalkylpolyamino (bis)urea and (bis)thiourea isosteres as potent antimalarial chemotypes against multiple lifecycle forms of Plasmodium falciparum parasites.

PubMed

Verlinden, Bianca K; de Beer, Marna; Pachaiyappan, Boobalan; Besaans, Ethan; Andayi, Warren A; Reader, Janette; Niemand, Jandeli; van Biljon, Riette; Guy, Kiplin; Egan, Timothy; Woster, Patrick M; Birkholtz, Lyn-Marie

2015-08-15

A new series of potent potent aryl/alkylated (bis)urea- and (bis)thiourea polyamine analogues were synthesized and evaluated in vitro for their antiplasmodial activity. Altering the carbon backbone and terminal substituents increased the potency of analogues in the compound library 3-fold, with the most active compounds, 15 and 16, showing half-maximal inhibitory concentrations (IC50 values) of 28 and 30 nM, respectively, against various Plasmodium falciparum parasite strains without any cross-resistance. In vitro evaluation of the cytotoxicity of these analogues revealed marked selectivity towards targeting malaria parasites compared to mammalian HepG2 cells (>5000-fold lower IC50 against the parasite). Preliminary biological evaluation of the polyamine analogue antiplasmodial phenotype revealed that (bis)urea compounds target parasite asexual proliferation, whereas (bis)thiourea compounds of the same series have the unique ability to block transmissible gametocyte forms of the parasite, indicating pluripharmacology against proliferative and non-proliferative forms of the parasite. In this manuscript, we describe these results and postulate a refined structure-activity relationship (SAR) model for antiplasmodial polyamine analogues. The terminally aryl/alkylated (bis)urea- and (bis)thiourea-polyamine analogues featuring a 3-5-3 or 3-6-3 carbon backbone represent a structurally novel and distinct class of potential antiplasmodials with activities in the low nanomolar range, and high selectivity against various lifecycle forms of P. falciparum parasites. Copyright © 2015 Elsevier Ltd. All rights reserved.

Spread of artemisinin resistance in Plasmodium falciparum malaria.

PubMed

Ashley, Elizabeth A; Dhorda, Mehul; Fairhurst, Rick M; Amaratunga, Chanaki; Lim, Parath; Suon, Seila; Sreng, Sokunthea; Anderson, Jennifer M; Mao, Sivanna; Sam, Baramey; Sopha, Chantha; Chuor, Char Meng; Nguon, Chea; Sovannaroth, Siv; Pukrittayakamee, Sasithon; Jittamala, Podjanee; Chotivanich, Kesinee; Chutasmit, Kitipumi; Suchatsoonthorn, Chaiyaporn; Runcharoen, Ratchadaporn; Hien, Tran Tinh; Thuy-Nhien, Nguyen Thanh; Thanh, Ngo Viet; Phu, Nguyen Hoan; Htut, Ye; Han, Kay-Thwe; Aye, Kyin Hla; Mokuolu, Olugbenga A; Olaosebikan, Rasaq R; Folaranmi, Olaleke O; Mayxay, Mayfong; Khanthavong, Maniphone; Hongvanthong, Bouasy; Newton, Paul N; Onyamboko, Marie A; Fanello, Caterina I; Tshefu, Antoinette K; Mishra, Neelima; Valecha, Neena; Phyo, Aung Pyae; Nosten, Francois; Yi, Poravuth; Tripura, Rupam; Borrmann, Steffen; Bashraheil, Mahfudh; Peshu, Judy; Faiz, M Abul; Ghose, Aniruddha; Hossain, M Amir; Samad, Rasheda; Rahman, M Ridwanur; Hasan, M Mahtabuddin; Islam, Akhterul; Miotto, Olivo; Amato, Roberto; MacInnis, Bronwyn; Stalker, Jim; Kwiatkowski, Dominic P; Bozdech, Zbynek; Jeeyapant, Atthanee; Cheah, Phaik Yeong; Sakulthaew, Tharisara; Chalk, Jeremy; Intharabut, Benjamas; Silamut, Kamolrat; Lee, Sue J; Vihokhern, Benchawan; Kunasol, Chanon; Imwong, Mallika; Tarning, Joel; Taylor, Walter J; Yeung, Shunmay; Woodrow, Charles J; Flegg, Jennifer A; Das, Debashish; Smith, Jeffery; Venkatesan, Meera; Plowe, Christopher V; Stepniewska, Kasia; Guerin, Philippe J; Dondorp, Arjen M; Day, Nicholas P; White, Nicholas J

2014-07-31

Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; Clinical

Genome content analysis yields new insights into the relationship between the human malaria parasite Plasmodium falciparum and its anopheline vectors.

PubMed

Oppenheim, Sara J; Rosenfeld, Jeffrey A; DeSalle, Rob

2017-02-27

The persistent and growing gap between the availability of sequenced genomes and the ability to assign functions to sequenced genes led us to explore ways to maximize the information content of automated annotation for studies of anopheline mosquitos. Specifically, we use genome content analysis of a large number of previously sequenced anopheline mosquitos to follow the loss and gain of protein families over the evolutionary history of this group. The importance of this endeavor lies in the potential for comparative genomic studies between Anopheles and closely related non-vector species to reveal ancestral genome content dynamics involved in vector competence. In addition, comparisons within Anopheles could identify genome content changes responsible for variation in the vectorial capacity of this family of important parasite vectors. The competence and capacity of P. falciparum vectors do not appear to be phylogenetically constrained within the Anophelinae. Instead, using ancestral reconstruction methods, we suggest that a previously unexamined component of vector biology, anopheline nucleotide metabolism, may contribute to the unique status of anophelines as P. falciparum vectors. While the fitness effects of nucleotide co-option by P. falciparum parasites on their anopheline hosts are not yet known, our results suggest that anopheline genome content may be responding to selection pressure from P. falciparum. Whether this response is defensive, in an attempt to redress improper nucleotide balance resulting from P. falciparum infection, or perhaps symbiotic, resulting from an as-yet-unknown mutualism between anophelines and P. falciparum, is an open question that deserves further study. Clearly, there is a wealth of functional information to be gained from detailed manual genome annotation, yet the rapid increase in the number of available sequences means that most researchers will not have the time or resources to manually annotate all the sequence data they

Periodic and chaotic host-parasite interactions in human malaria.

PubMed Central

Kwiatkowski, D; Nowak, M

1991-01-01

It has been recognized since ancient times that malaria fever is highly periodic but the mechanism has been poorly understood. Malaria fever is related to the parasite growth cycle in erythrocytes. After a fixed period of replication, a mature parasite (schizont) causes the infected erythrocyte to rupture, releasing progeny that quickly invade other erythrocytes. Simultaneous rupture of a large number of schizonts stimulates a host fever response. Febrile temperatures are damaging to Plasmodium falciparum, particularly in the second half of its 48-hr replicative cycle. Using a mathematical model, we show that these interactions naturally tend to generate periodic fever. The model predicts chaotic parasite population dynamics at high multiplication rates, consistent with the classical observation that P. falciparum causes less regular fever than other species of parasite. PMID:2052590

A high parasite density environment induces transcriptional changes and cell death in Plasmodium falciparum blood stages.

PubMed

Chou, Evelyn S; Abidi, Sabia Z; Teye, Marian; Leliwa-Sytek, Aleksandra; Rask, Thomas S; Cobbold, Simon A; Tonkin-Hill, Gerry Q; Subramaniam, Krishanthi S; Sexton, Anna E; Creek, Darren J; Daily, Johanna P; Duffy, Michael F; Day, Karen P

2018-03-01

Transient regulation of Plasmodium numbers below the density that induces fever has been observed in chronic malaria infections in humans. This species transcending control cannot be explained by immunity alone. Using an in vitro system we have observed density dependent regulation of malaria population size as a mechanism to possibly explain these in vivo observations. Specifically, Plasmodium falciparum blood stages from a high but not low-density environment exhibited unique phenotypic changes during the late trophozoite (LT) and schizont stages of the intraerythrocytic cycle. These included in order of appearance: failure of schizonts to mature and merozoites to replicate, apoptotic-like morphological changes including shrinking, loss of mitochondrial membrane potential, and blebbing with eventual release of aberrant parasites from infected erythrocytes. This unique death phenotype was triggered in a stage-specific manner by sensing of a high-density culture environment. Conditions of glucose starvation, nutrient depletion, and high lactate could not induce the phenotype. A high-density culture environment induced rapid global changes in the parasite transcriptome including differential expression of genes involved in cell remodeling, clonal antigenic variation, metabolism, and cell death pathways including an apoptosis-associated metacaspase gene. This transcriptional profile was also characterized by concomitant expression of asexual and sexual stage-specific genes. The data show strong evidence to support our hypothesis that density sensing exists in P. falciparum. They indicate that an apoptotic-like mechanism may play a role in P. falciparum density regulation, which, as in yeast, has features quite distinguishable from mammalian apoptosis. Gene expression data are available in the GEO databases under the accession number GSE91188. © 2017 Federation of European Biochemical Societies.

A secretory multifunctional serine protease, DegP of Plasmodium falciparum, plays an important role in thermo-oxidative stress, parasite growth and development.

PubMed

Sharma, Shweta; Jadli, Mohit; Singh, Anu; Arora, Kavita; Malhotra, Pawan

2014-03-01

Plasmodium falciparum heat shock proteins and proteases are known for their indispensable roles in parasite virulence and survival in the host cell. They neutralize various host-derived stress responses that are deleterious for parasite growth and invasion. We report identification and functional characterization of the first DegP from an apicomplexan (P. falciparum). To determine the molecular identity and functions of the parasite-encoded DegP, we complemented the Escherichia coli degP null mutant with a putative PfdegP gene, and the results showed that PfDegP complements the growth defect of the temperature sensitive DegP-deficient mutant and imparts resistance to non-permissive temperatures and oxidative stress. Molecular interaction studies showed that PfDegP exists as a complex with parasite-encoded heat shock protein 70, iron superoxide dismutase and enolase. DegP expression is significantly induced in parasite culture upon heat shock/oxidative stress. Our data suggest that the PfDegP protein may play a role in the growth and development of P. falciparum through its ability to confer protection against thermal/oxidative stress. Antibody against DegP showed anti-plasmodial activity against blood-stage parasites in vitro, suggesting that PfDegP and its associated complex may be a potential focus for new anti-malarial therapies. ●PfDegP physically interacts with PfHsp70 and PfEno by anti-bait co-immunoprecipitation (View interaction) ●PfDegP physically interacts with PfEno, PfSod, PfOat, PfHsp70, PfLDH and PfGpi by anti-bait co-immunoprecipitation (View interaction) ●PfHsp-70 and PfDegP co-localize by fluorescence microscopy (View interaction) ●PfDegP physically interacts with PfOat, PfHsp70, PfEno, PfSod, PfGpi and PfLDH by surface plasmon resonance (View interaction) ●PfEno and PfDegP co-localize by fluorescence microscopy (View interaction) ●PfDegP and PfHsp70 co-localize by co-sedimentation through density gradient (View interaction). © 2014

Positive Selection of Plasmodium falciparum Parasites With Multiple var2csa-Type PfEMP1 Genes During the Course of Infection in Pregnant Women

PubMed Central

Salanti, Ali; Lavstsen, Thomas; Nielsen, Morten A.; Theander, Thor G.; Leke, Rose G. F.; Lo, Yeung Y.; Bobbili, Naveen; Arnot, David E.; Taylor, Diane W.

2011-01-01

Placental malaria infections are caused by Plasmodium falciparum–infected red blood cells sequestering in the placenta by binding to chondroitin sulfate A, mediated by VAR2CSA, a variant of the PfEMP1 family of adhesion antigens. Recent studies have shown that many P. falciparum genomes have multiple genes coding for different VAR2CSA proteins, and parasites with >1 var2csa gene appear to be more common in pregnant women with placental malaria than in nonpregnant individuals. We present evidence that, in pregnant women, parasites containing multiple var2csa-type genes possess a selective advantage over parasites with a single var2csa gene. Accumulation of parasites with multiple copies of the var2csa gene during the course of pregnancy was also correlated with the development of antibodies involved in blocking VAR2CSA adhesion. The data suggest that multiplicity of var2csa-type genes enables P. falciparum parasites to persist for a longer period of time during placental infections, probably because of their greater capacity for antigenic variation and evasion of variant-specific immune responses. PMID:21592998

Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum

PubMed Central

Painter, Heather J.; Carrasquilla, Manuela; Llinás, Manuel

2017-01-01

To capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from preexisting mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. Using the pfs16 gametocyte-specific promoter to express FCU-GFP in 3D7 parasites, we found that sexual stage commitment is governed by transcriptional reprogramming and stabilization of a subset of essential gametocyte transcripts. We also measured mRNA dynamics in F12 gametocyte-deficient parasites and demonstrate that the transcriptional program required for sexual commitment and maturation is initiated but likely aborted due to the absence of the PfAP2-G transcriptional regulator and a lack of gametocyte-specific mRNA stabilization. Biosynthetic labeling of Plasmodium mRNAs is incredibly versatile, can be used to measure transcriptional dynamics at any stage of parasite development, and will allow for future applications to comprehensively measure RNA-protein interactions in the malaria parasite. PMID:28416533

Polymorphisms in the K13-propeller gene in artemisinin-susceptible Plasmodium falciparum parasites from Bougoula-Hameau and Bandiagara, Mali.

PubMed

Ouattara, Amed; Kone, Aminatou; Adams, Matthew; Fofana, Bakary; Maiga, Amelia Walling; Hampton, Shay; Coulibaly, Drissa; Thera, Mahamadou A; Diallo, Nouhoum; Dara, Antoine; Sagara, Issaka; Gil, Jose Pedro; Bjorkman, Anders; Takala-Harrison, Shannon; Doumbo, Ogobara K; Plowe, Christopher V; Djimde, Abdoulaye A

2015-06-01

Artemisinin-resistant Plasmodium falciparum malaria has been documented in southeast Asia and may already be spreading in that region. Molecular markers are important tools for monitoring the spread of antimalarial drug resistance. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain were shown to be associated with artemisinin resistance in vivo and in vitro. The prevalence and role of K13-propeller mutations are poorly known in sub-Saharan Africa. K13-propeller mutations were genotyped by direct sequencing of nested polymerase chain reaction (PCR) amplicons from dried blood spots of pre-treatment falciparum malaria infections collected before and after the use of artemisinin-based combination therapy (ACT) as first-line therapy in Mali. Although K13-propeller mutations previously associated with delayed parasite clearance in Cambodia were not identified, 26 K13-propeller mutations were identified in both recent samples and pre-ACT infections. Parasite clearance time was comparable between infections with non-synonymous K13-propeller mutations and infections with the reference allele. These findings suggest that K13-propeller mutations are present in artemisinin-sensitive parasites and that they preceded the wide use of ACTs in Mali. © The American Society of Tropical Medicine and Hygiene.

Initial Characterization of the Pf-Int Recombinase from the Malaria Parasite Plasmodium falciparum

PubMed Central

Ghorbal, Mehdi; Scheidig-Benatar, Christine; Bouizem, Salma; Thomas, Christophe; Paisley, Genevieve; Faltermeier, Claire; Liu, Melanie; Scherf, Artur; Lopez-Rubio, Jose-Juan; Gopaul, Deshmukh N.

2012-01-01

Background Genetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs) that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes. A gene homologous to a tyrosine site-specific recombinase has been identified in the genome of Plasmodium falciparum. The sequence is highly conserved among five other members of Plasmodia. Methodology/Principal Findings The predicted open reading frame encodes for a ∼57 kDa protein containing a C-terminal domain including the putative tyrosine recombinase conserved active site residues R-H-R-(H/W)-Y. The N-terminus has the typical alpha-helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. Pf-Int mRNA is expressed differentially during the P. falciparum erythrocytic life stages, peaking in the schizont stage. Recombinant Pf-Int and affinity chromatography of DNA from genomic or synthetic origin were used to identify potential DNA targets after sequencing or micro-array hybridization. Interestingly, the sequences captured also included highly variable subtelomeric genes such as var, rif, and stevor sequences. Electrophoretic mobility shift assays with DNA were carried out to verify Pf-Int/DNA binding. Finally, Pf-Int knock-out parasites were created in order to investigate the biological role of Pf-Int. Conclusions/Significance Our data identify for the first time a malaria parasite gene with structural and functional features of recombinases. Pf-Int may bind to and alter DNA, either in a sequence specific or in a non-specific fashion, and may contribute to programmed or random DNA rearrangements. Pf-Int is the first molecular player identified with a potential role in genome plasticity in this pathogen. Finally, Pf-Int knock-out parasite is viable showing no detectable impact on blood stage development, which is

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.

Plasmodium falciparum uses vitamin E to avoid oxidative stress.

PubMed

Sussmann, Rodrigo A C; Fotoran, Wesley L; Kimura, Emilia A; Katzin, Alejandro M

2017-10-10

Plasmodium falciparum is sensitive to oxidative stress in vitro and in vivo, and many drugs such as artemisinin, chloroquine and cercosporin interfere in the parasite's redox system. To minimize the damage caused by reactive radicals, antioxidant enzymes and their substrates found in parasites and in erythrocytes must be functionally active. It was shown that P. falciparum synthesizes vitamin E and that usnic acid acts as an inhibitor of its biosynthesis. Vitamin E is a potent antioxidant that protects polyunsaturated fatty acids from lipid peroxidation, and this activity can be measured by detecting its oxidized product and by evaluating reactive oxygen species (ROS) levels. Here, we demonstrated that ROS levels increased in P. falciparum when vitamin E biosynthesis was inhibited by usnic acid treatment and decreased to basal levels if exogenous vitamin E was added. Furthermore, we used metabolic labelling to demonstrate that vitamin E biosynthesized by the parasite acts as an antioxidant since we could detect its radiolabeled oxidized product. The treatment with chloroquine or cercosporin of the parasites increased the ratio between α-tocopherolquinone and α-tocopherol. Our findings demonstrate that vitamin E produced endogenously by P. falciparum is active as an antioxidant, probably protecting the parasite from the radicals generated by drugs.

Quantifying the biophysical characteristics of Plasmodium-falciparum-parasitized red blood cells in microcirculation

PubMed Central

Fedosov, D. A.; Caswell, B.; Suresh, S.; Karniadakis, G. E.

2011-01-01

The pathogenicity of Plasmodium falciparum (Pf) malaria results from the stiffening of red blood cells (RBCs) and its ability to adhere to endothelial cells (cytoadherence). The dynamics of Pf-parasitized RBCs is studied by three-dimensional mesoscopic simulations of flow in cylindrical capillaries in order to predict the flow resistance enhancement at different parasitemia levels. In addition, the adhesive dynamics of Pf-RBCs is explored for various parameters revealing several types of cell dynamics such as firm adhesion, very slow slipping along the wall, and intermittent flipping. The parasite inside the RBC is modeled explicitly in order to capture phenomena such as “hindered tumbling” motion of the RBC and the sudden transition from firm RBC cytoadherence to flipping on the endothelial surface. These predictions are in quantitative agreement with recent experimental observations, and thus the three-dimensional modeling method presented here provides new capabilities for guiding and interpreting future in vitro and in vivo studies of malaria. PMID:21173269

Nanopore sequencing of drug-resistance-associated genes in malaria parasites, Plasmodium falciparum.

PubMed

Runtuwene, Lucky R; Tuda, Josef S B; Mongan, Arthur E; Makalowski, Wojciech; Frith, Martin C; Imwong, Mallika; Srisutham, Suttipat; Nguyen Thi, Lan Anh; Tuan, Nghia Nguyen; Eshita, Yuki; Maeda, Ryuichiro; Yamagishi, Junya; Suzuki, Yutaka

2018-05-29

Here, we report the application of a portable sequencer, MinION, for genotyping the malaria parasite Plasmodium falciparum. In the present study, an amplicon mixture of nine representative genes causing resistance to anti-malaria drugs is diagnosed. First, we developed the procedure for four laboratory strains (3D7, Dd2, 7G8, and K1), and then applied the developed procedure to ten clinical samples. We sequenced and re-sequenced the samples using the obsolete flow cell R7.3 and the most recent flow cell R9.4. Although the average base-call accuracy of the MinION sequencer was 74.3%, performing >50 reads at a given position improves the accuracy of the SNP call, yielding a precision and recall rate of 0.92 and 0.8, respectively, with flow cell R7.3. These numbers increased significantly with flow cell R9.4, in which the precision and recall are 1 and 0.97, respectively. Based on the SNP information, the drug resistance status in ten clinical samples was inferred. We also analyzed K13 gene mutations from 54 additional clinical samples as a proof of concept. We found that a novel amino-acid changing variation is dominant in this area. In addition, we performed a small population-based analysis using 3 and 5 cases (K13) and 10 and 5 cases (PfCRT) from Thailand and Vietnam, respectively. We identified distinct genotypes from the respective regions. This approach will change the standard methodology for the sequencing diagnosis of malaria parasites, especially in developing countries.

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

Relationship between the entomologic inoculation rate and the force of infection for Plasmodium falciparum malaria.

PubMed

Smith, Thomas; Maire, Nicolas; Dietz, Klaus; Killeen, Gerry F; Vounatsou, Penelope; Molineaux, Louis; Tanner, Marcel

2006-08-01

We propose a stochastic model for the relationship between the entomologic inoculation rate (EIR) for Plasmodium falciparum malaria and the force of infection in endemic areas. The model incorporates effects of increased exposure to mosquito bites as a result of the growth in body surface area with the age of the host, naturally acquired pre-erythrocytic immunity, and the reduction in the proportion of entomologically assessed inoculations leading to infection, as the EIR increases. It is fitted to multiple datasets from field studies of the relationship between malaria infection and the EIR. We propose that this model can account for non-monotonic relationships between the age of the host and the parasite prevalence and incidence of disease. It provides a parsimonious explanation for the faster acquisition of natural immunity in adults than in children exposed to high EIRs. This forms one component of a new stochastic model for the entire transmission cycle of P. falciparum that we have derived to estimate the potential epidemiologic impact of malaria vaccines and other malaria control interventions.

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

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.

Mitotic Evolution of Plasmodium falciparum Shows a Stable Core Genome but Recombination in Antigen Families

PubMed Central

Bopp, Selina E. R.; Manary, Micah J.; Bright, A. Taylor; Johnston, Geoffrey L.; Dharia, Neekesh V.; Luna, Fabio L.; McCormack, Susan; Plouffe, David; McNamara, Case W.; Walker, John R.; Fidock, David A.; Denchi, Eros Lazzerini; Winzeler, Elizabeth A.

2013-01-01

Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases) revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone). In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1) on chromosome 1. We observed 18 large-scale (>1 kb on average) deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10−6 structural variants per base pair per generation). Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0–9.7×10−9 mutations per base pair per generation), we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum evolves to

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology

PubMed Central

Gabryszewski, Stanislaw J.; Dhingra, Satish K.; Lewis, Ian A.; Callaghan, Paul S.; Hassett, Matthew R.; Siriwardana, Amila; Henrich, Philipp P.; Lee, Andrew H.; Gnädig, Nina F.; Musset, Lise; Llinás, Manuel; Egan, Timothy J.; Roepe, Paul D.

2016-01-01

Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize

Hidden reservoir of resistant parasites: the missing link in the elimination of falciparum malaria.

PubMed

Abdul-Ghani, Rashad; Mahdy, Mohammed A K; Beier, John C; Basco, Leonardo K

2017-02-06

To successfully eliminate malaria, an integrated system that includes a number of approaches and interventions-aimed at overcoming the threat of antimalarial drug resistance-is required. Significant progress has been made in reducing malaria incidence through large-scale use of artemisinin-based combination therapies and insecticide-treated nets. To consolidate these gains, attention should be paid to the missing links in the elimination of malaria. One of these gaps is the residual reservoir of submicroscopic resistant parasites, which remains after case management or other control measures have been carried out. Therefore, the present opinion piece highlights the importance of exploring the role that submicroscopic resistant parasites could play in hindering malaria elimination by allowing the persistence of transmission, particularly in areas of low transmission or in the pre-elimination and/or elimination phase. If malaria elimination interventions are to be effective, the relative role of the hidden reservoir of resistant parasites needs to be assessed, particularly in regions that are low-transmission settings and/or in pre-elimination and/or elimination phases. Various ongoing studies are focusing on the role of submicroscopic malaria infections in malaria transmission but overlook the possible build-up of resistance to antimalarial drugs among submicroscopic parasite populations. This is an important factor as it may eventually limit the effectiveness of malaria elimination strategies. An evidence-based estimation of the "true" reservoir of resistant parasites can help target the existing and emerging foci of resistant parasites before they spread. Emergence and spread of artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia underline the need to contain drug resistance.

Identification of New Human Malaria Parasite Plasmodium Falciparum Dihydroorotate Dehydrogenase Inhibitors by Pharmacophore and Structure-Based Virtual Screening

PubMed Central

Pavadai, Elumalai; El Mazouni, Farah; Wittlin, Sergio; de Kock, Carmen; Phillips, Margaret A.; Chibale, Kelly

2016-01-01

Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH), a key enzyme in the de novo pyrimidine biosynthesis pathway, which the Plasmodium falciparum relies on exclusively for survival, has emerged as a promising target for antimalarial drugs. In an effort to discover new and potent PfDHODH inhibitors, 3D-QSAR pharmacophore models were developed based on the structures of known PfDHODH inhibitors and the validated Hypo1 model was used as a 3D search query for virtual screening of the National Cancer Institute database. The virtual hit compounds were further filtered based on molecular docking and Molecular Mechanics/Generalized Born Surface Area binding energy calculations. The combination of the pharmacophore and structure-based virtual screening resulted in the identification of nine new compounds that showed >25% inhibition of PfDHODH at a concentration of 10 μM, three of which exhibited IC50 values in the range of 0.38–20 μM. The most active compound, NSC336047, displayed species-selectivity for PfDHODH over human DHODH and inhibited parasite growth with an IC50 of 26 μM. In addition to this, thirteen compounds inhibited parasite growth with IC50 values of ≤ 50 μM, four of which showed IC50 values in the range of 5–12 μM. These compounds could be further explored in the identification and development of more potent PfDHODH and parasite growth inhibitors. PMID:26915022

Syk inhibitors interfere with erythrocyte membrane modification during P falciparum growth and suppress parasite egress.

PubMed

Pantaleo, Antonella; Kesely, Kristina R; Pau, Maria Carmina; Tsamesidis, Ioannis; Schwarzer, Evelin; Skorokhod, Oleksii A; Chien, Huynh D; Ponzi, Marta; Bertuccini, Lucia; Low, Philip S; Turrini, Francesco M

2017-08-24

Band 3 (also known as the anion exchanger, SLCA1, AE1) constitutes the major attachment site of the spectrin-based cytoskeleton to the erythrocyte's lipid bilayer and thereby contributes critically to the stability of the red cell membrane. During the intraerythrocytic stage of Plasmodium falciparum 's lifecycle, band 3 becomes tyrosine phosphorylated in response to oxidative stress, leading to a decrease in its affinity for the spectrin/actin cytoskeleton and causing global membrane destabilization. Because this membrane weakening is hypothesized to facilitate parasite egress and the consequent dissemination of released merozoites throughout the bloodstream, we decided to explore which tyrosine kinase inhibitors might block the kinase-induced membrane destabilization. We demonstrate here that multiple Syk kinase inhibitors both prevent parasite-induced band 3 tyrosine phosphorylation and inhibit parasite-promoted membrane destabilization. We also show that the same Syk kinase inhibitors suppress merozoite egress near the end of the parasite's intraerythrocytic lifecycle. Because the entrapped merozoites die when prevented from escaping their host erythrocytes and because some Syk inhibitors have displayed long-term safety in human clinical trials, we suggest Syk kinase inhibitors constitute a promising class of antimalarial drugs that can suppress parasitemia by inhibiting a host target that cannot be mutated by the parasite to evolve drug resistance. © 2017 by The American Society of Hematology.

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.

Plasmodium falciparum: a simplified technique for obtaining singly infected erythrocytes.

PubMed

Puthia, Manoj K; Tan, Kevin S W

2005-02-01

We report the development of a simple technique involving 15 ml polypropylene tubes and a rotatory incubator for obtaining erythrocytes singly infected with Plasmodium falciparum. This technique will be useful for cloning of the parasite. Our finding that P. falciparum merozoite invasion is inhibited during rotation suggests that this method may also be useful for the study of parasite-erythrocyte interactions under dynamic circulatory conditions.

Plasmodium falciparum Malaria Endemicity in Indonesia in 2010

PubMed Central

Elyazar, Iqbal R. F.; Gething, Peter W.; Patil, Anand P.; Rogayah, Hanifah; Kusriastuti, Rita; Wismarini, Desak M.; Tarmizi, Siti N.; Baird, J. Kevin; Hay, Simon I.

2011-01-01

Background Malaria control programs require a detailed understanding of the contemporary spatial distribution of infection risk to efficiently allocate resources. We used model based geostatistics (MBG) techniques to generate a contemporary map of Plasmodium falciparum malaria risk in Indonesia in 2010. Methods Plasmodium falciparum Annual Parasite Incidence (PfAPI) data (2006–2008) were used to map limits of P. falciparum transmission. A total of 2,581 community blood surveys of P. falciparum parasite rate (PfPR) were identified (1985–2009). After quality control, 2,516 were included into a national database of age-standardized 2–10 year old PfPR data (PfPR2–10) for endemicity mapping. A Bayesian MBG procedure was used to create a predicted surface of PfPR2–10 endemicity with uncertainty estimates. Population at risk estimates were derived with reference to a 2010 human population count surface. Results We estimate 132.8 million people in Indonesia, lived at risk of P. falciparum transmission in 2010. Of these, 70.3% inhabited areas of unstable transmission and 29.7% in stable transmission. Among those exposed to stable risk, the vast majority were at low risk (93.39%) with the reminder at intermediate (6.6%) and high risk (0.01%). More people in western Indonesia lived in unstable rather than stable transmission zones. In contrast, fewer people in eastern Indonesia lived in unstable versus stable transmission areas. Conclusion While further feasibility assessments will be required, the immediate prospects for sustained control are good across much of the archipelago and medium term plans to transition to the pre-elimination phase are not unrealistic for P. falciparum. Endemicity in areas of Papua will clearly present the greatest challenge. This P. falciparum endemicity map allows malaria control agencies and their partners to comprehensively assess the region-specific prospects for reaching pre-elimination, monitor and evaluate the effectiveness of

Kinetics of B cell responses to Plasmodium falciparum erythrocyte membrane protein 1 in Ghanaian women naturally exposed to malaria parasites.

PubMed

Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F; Barfod, Lea; Hviid, Lars

2014-06-01

Naturally acquired protective immunity to Plasmodium falciparum malaria takes years to develop. It relies mainly on Abs, particularly IgG specific for Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins on the infected erythrocyte surface. It is only partially understood why acquisition of clinical protection takes years to develop, but it probably involves a range of immune-evasive parasite features, not least of which are PfEMP1 polymorphism and clonal variation. Parasite-induced subversion of immunological memory and expansion of "atypical" memory B cells may also contribute. In this first, to our knowledge, longitudinal study of its kind, we measured B cell subset composition, as well as PfEMP1-specific Ab levels and memory B cell frequencies, in Ghanaian women followed from early pregnancy up to 1 y after delivery. Cell phenotypes and Ag-specific B cell function were assessed three times during and after pregnancy. Levels of IgG specific for pregnancy-restricted, VAR2CSA-type PfEMP1 increased markedly during pregnancy and declined after delivery, whereas IgG levels specific for two PfEMP1 proteins not restricted to pregnancy did not. Changes in VAR2CSA-specific memory B cell frequencies showed typical primary memory induction among primigravidae and recall expansion among multigravidae, followed by contraction postpartum in all. No systematic changes in the frequencies of memory B cells specific for the two other PfEMP1 proteins were identified. The B cell subset analysis confirmed earlier reports of high atypical memory B cell frequencies among residents of P. falciparum-endemic areas, and indicated an additional effect of pregnancy. Our study provides new knowledge regarding immunity to P. falciparum malaria and underpins efforts to develop PfEMP1-based vaccines against this disease. Copyright © 2014 by The American Association of Immunologists, Inc.

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

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.

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

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.

Structure-activity analysis of CJ-15,801 analogues that interact with Plasmodium falciparum pantothenate kinase and inhibit parasite proliferation.

PubMed

Spry, Christina; Sewell, Alan L; Hering, Yuliya; Villa, Mathew V J; Weber, Jonas; Hobson, Stephen J; Harnor, Suzannah J; Gul, Sheraz; Marquez, Rodolfo; Saliba, Kevin J

2018-01-01

Survival of the human malaria parasite Plasmodium falciparum is dependent on pantothenate (vitamin B 5 ), a precursor of the fundamental enzyme cofactor coenzyme A. CJ-15,801, an enamide analogue of pantothenate isolated from the fungus Seimatosporium sp. CL28611, was previously shown to inhibit P. falciparum proliferation in vitro by targeting pantothenate utilization. To inform the design of next generation analogues, we set out to synthesize and test a series of synthetic enamide-bearing pantothenate analogues. We demonstrate that conservation of the R-pantoyl moiety and the trans-substituted double bond of CJ-15,801 is important for the selective, on-target antiplasmodial effect, while replacement of the carboxyl group is permitted, and, in one case, favored. Additionally, we show that the antiplasmodial potency of CJ-15,801 analogues that retain the R-pantoyl and trans-substituted enamide moieties correlates with inhibition of P. falciparum pantothenate kinase (PfPanK)-catalyzed pantothenate phosphorylation, implicating the interaction with PfPanK as a key determinant of antiplasmodial activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

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.

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

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.

Molecular inference of sources and spreading patterns of Plasmodium falciparum malaria parasites in internally displaced persons settlements in Myanmar-China border area.

PubMed

Lo, Eugenia; Zhou, Guofa; Oo, Winny; Lee, Ming-Chieh; Baum, Elisabeth; Felgner, Philip L; Yang, Zhaoqing; Cui, Liwang; Yan, Guiyun

2015-07-01

In Myanmar, civil unrest and establishment of internally displaced persons (IDP) settlement along the Myanmar-China border have impacted malaria transmission. The growing IDP populations raise deep concerns about health impact on local communities. Microsatellite markers were used to examine the source and spreading patterns of Plasmodium falciparum between IDP settlement and surrounding villages in Myanmar along the China border. Genotypic structure of P. falciparum was compared over the past three years from the same area and the demographic history was inferred to determine the source of recent infections. In addition, we examined if border migration is a factor of P. falciparum infections in China by determining gene flow patterns across borders. Compared to local community, the IDP samples showed a reduced and consistently lower genetic diversity over the past three years. A strong signature of genetic bottleneck was detected in the IDP samples. P. falciparum infections from the border regions in China were genetically similar to Myanmar and parasite gene flow was not constrained by geographical distance. Reduced genetic diversity of P. falciparum suggested intense malaria control within the IDP settlement. Human movement was a key factor to the spread of malaria both locally in Myanmar and across the international border. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Survival of Plasmodium falciparum in human blood during refrigeration.

PubMed

Chattopadhyay, Rana; Majam, Victoria F; Kumar, Sanjai

2011-03-01

Transfusion-transmitted malaria remains a serious concern for blood safety. Viable Plasmodium parasites must be present in human blood to transmit malaria, but their survival in blood over time stored under refrigeration has never been carefully investigated. We spiked leukoreduced normal human blood with Plasmodium falciparum (3D7 strain) asexual ring-stage parasites and stored it at 4 °C for 28 days, taking samples at different days intervals. We evaluated the samples for parasitemia by blood film microscopy and by culturing red blood cells (RBCs) to allow further development of parasites. We observed a significant reduction in parasitemia (0.5% vs. 0.12%) after only 1 day in storage at 4 °C. Thereafter, reduction in parasitemia was relatively gradual. Microscopically detectable parasites were present even after 28 days of storage. However, after storing for more than 14 days at 4 °C, parasites no longer replicated when cultured in vitro. Although the storage of asexual blood-stage P. falciparum parasites at 4 °C is detrimental to their survival (a 7.1-fold reduction in parasitemia after 14 days in storage), parasites remained microscopically detectable for 28 days, the end time point of our study. Further in vitro and in vivo studies will be needed to confirm loss of viability of P. falciparum after 14 days in storage, but our initial efforts repeatedly failed to show maturation and development of the parasites in cultured RBCs after that time. © 2010 American Association of Blood Banks.

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

Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle.

PubMed

Hallée, Stéphanie; Richard, Dave

2015-01-01

Invasion of a red blood cell by Plasmodium falciparum merozoites is an essential step in the malaria lifecycle. Several of the proteins involved in this process are stored in the apical complex of the merozoite, a structure containing secretory organelles that are released at specific times during invasion. The molecular players involved in erythrocyte invasion thus represent potential key targets for both therapeutic and vaccine-based strategies to block parasite development. In our quest to identify and characterize new effectors of invasion, we investigated the P. falciparum homologue of a P. berghei protein putatively localized to the rhoptries, the Putative rhoptry protein 2 (PbPRP2). We show that in P. falciparum, the protein colocalizes extensively with the Golgi apparatus across the asexual erythrocytic cycle. Furthermore, imaging of merozoites caught at different times during invasion show that PfPRP2 is not secreted during the process instead staying associated with the Golgi apparatus. Our evidence therefore suggests that PfPRP2 is a Golgi protein and that it is likely not a direct effector in the process of merozoite invasion.

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.

The Rheopathobiology of Plasmodium vivax and Other Important Primate Malaria Parasites.

PubMed

Russell, Bruce M; Cooke, Brian M

2017-04-01

Our current understanding of how malaria parasites remodel their host red blood cells (RBCs) and ultimately cause disease is largely based on studies of Plasmodium falciparum. In this review, we expand our knowledge to include what is currently known about pathophysiological changes to RBCs that are infected by non-falciparum malaria parasites. We highlight the potential folly of making generalizations about the rheology of malaria infection, and emphasize the need for more systematic studies into the erythrocytic biology of non-falciparum malaria parasites. We propose that a better understanding of the mechanisms that underlie the changes to RBCs induced by malaria parasites other than P. falciparum may be highly informative for the development of therapeutics that specifically disrupt the altered rheological profile of RBCs infected with either sexual- or asexual-stage parasites, resulting in drugs that block transmission, reduce disease severity, and help delay the onset of resistance to current and future anti-malaria drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate

PubMed Central

Counihan, Natalie A; Chisholm, Scott A; Bullen, Hayley E; Srivastava, Anubhav; Sanders, Paul R; Jonsdottir, Thorey K; Weiss, Greta E; Ghosh, Sreejoyee; Crabb, Brendan S; Creek, Darren J; Gilson, Paul R; de Koning-Ward, Tania F

2017-01-01

Plasmodium falciparum parasites, the causative agents of malaria, modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste. Here we investigate the contribution of the rhoptry protein RhopH2, in the formation of new permeability pathways (NPPs) in Plasmodium-infected erythrocytes. We show RhopH2 interacts with RhopH1, RhopH3, the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling. Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two. There is also a significant impact on parasite growth, replication and transition into cycle three. The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown. These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity and highlight the importance of NPPs to parasite survival. DOI: http://dx.doi.org/10.7554/eLife.23217.001 PMID:28252383

Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro.

PubMed

Dumoulin, Peter C; Trop, Stefanie A; Ma, Jinxia; Zhang, Hao; Sherman, Matthew A; Levitskaya, Jelena

2015-01-01

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden. The liver stage of Plasmodium falciparum infection is a leading target for immunological and pharmacological interventions. Therefore, novel approaches providing specific detection and isolation of live P. falciparum exoerythrocytic forms (EEFs) are warranted. Utilizing a recently generated parasite strain expressing green fluorescent protein (GFP) we established a method which, allows for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. Here, we show that EEFs can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Finally, we validated the practical applications of our approach by re-examining the importance of host ligand CD81 for hepatocyte infection by P. falciparum sporozoites in vitro and assessment of the inhibitory activity of anti-sporozoite antibodies. This methodology provides us with the tools to study both, the basic biology of the P. falciparum liver stage and the effects of host-derived factors on the development of P. falciparum EEFs.

Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro

PubMed Central

Dumoulin, Peter C.; Trop, Stefanie A.; Ma, Jinxia; Zhang, Hao; Sherman, Matthew A.; Levitskaya, Jelena

2015-01-01

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden. The liver stage of Plasmodium falciparum infection is a leading target for immunological and pharmacological interventions. Therefore, novel approaches providing specific detection and isolation of live P. falciparum exoerythrocytic forms (EEFs) are warranted. Utilizing a recently generated parasite strain expressing green fluorescent protein (GFP) we established a method which, allows for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. Here, we show that EEFs can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Finally, we validated the practical applications of our approach by re-examining the importance of host ligand CD81 for hepatocyte infection by P. falciparum sporozoites in vitro and assessment of the inhibitory activity of anti-sporozoite antibodies. This methodology provides us with the tools to study both, the basic biology of the P. falciparum liver stage and the effects of host-derived factors on the development of P. falciparum EEFs. PMID:26070149

The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences.

PubMed

Vinetz, J M; Dave, S K; Specht, C A; Brameld, K A; Xu, B; Hayward, R; Fidock, D A

1999-11-23

Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC(50) (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.

The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences

PubMed Central

Vinetz, Joseph M.; Dave, Sanat K.; Specht, Charles A.; Brameld, Kenneth A.; Xu, Bo; Hayward, Rhian; Fidock, David A.

1999-01-01

Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC50 (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase. PMID:10570198

Impact on Malaria Parasite Multiplication Rates in Infected Volunteers of the Protein-in-Adjuvant Vaccine AMA1-C1/Alhydrogel+CPG 7909

PubMed Central

Duncan, Christopher J. A.; Sheehy, Susanne H.; Ewer, Katie J.; Douglas, Alexander D.; Collins, Katharine A.; Halstead, Fenella D.; Elias, Sean C.; Lillie, Patrick J.; Rausch, Kelly; Aebig, Joan; Miura, Kazutoyo; Edwards, Nick J.; Poulton, Ian D.; Hunt-Cooke, Angela; Porter, David W.; Thompson, Fiona M.; Rowland, Ros; Draper, Simon J.; Gilbert, Sarah C.; Fay, Michael P.; Long, Carole A.; Zhu, Daming; Wu, Yimin; Martin, Laura B.; Anderson, Charles F.; Lawrie, Alison M.; Hill, Adrian V. S.; Ellis, Ruth D.

2011-01-01

Background Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria. Methods In this phase I/IIa open-label clinical trial five healthy malaria-naive volunteers were immunised with AMA1/C1-Alhydrogel+CPG 7909, and together with three unvaccinated controls were challenged by intravenous inoculation of P. falciparum infected erythrocytes. Results A significant correlation was observed between parasite multiplication rate in 48 hours (PMR) and both vaccine-induced growth-inhibitory activity (Pearson r = −0.93 [95% CI: −1.0, −0.27] P = 0.02) and AMA1 antibody titres in the vaccine group (Pearson r = −0.93 [95% CI: −0.99, −0.25] P = 0.02). However immunisation failed to reduce overall mean PMR in the vaccine group in comparison to the controls (vaccinee 16 fold [95% CI: 12, 22], control 17 fold [CI: 0, 65] P = 0.70). Therefore no impact on pre-patent period was observed (vaccine group median 8.5 days [range 7.5–9], control group median 9 days [range 7–9]). Conclusions Despite the first observation in human experimental malaria infection of a significant association between vaccine-induced in vitro growth inhibitory activity and in vivo parasite multiplication rate, this did not translate into any observable clinically relevant vaccine effect in this small group of volunteers. Trial Registration ClinicalTrials.gov [NCT00984763] PMID:21799809

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

Expressed var gene repertoire and variant surface antigen diversity in a shrinking Plasmodium falciparum population.

PubMed

Carlos, Bianca C; Fotoran, Wesley L; Menezes, Maria J; Cabral, Fernanda J; Bastos, Marcele F; Costa, Fabio T M; Sousa-Neto, Jayme A; Ribolla, Paulo E M; Wunderlich, Gerhard; Ferreira, Marcelo U

2016-11-01

The var gene-encoded erythrocyte membrane protein-1 of Plasmodium falciparum (PfEMP-1) is the main variant surface antigen (VSA) expressed on infected erythrocytes. The rate at which antibody responses to VSA expressed by circulating parasites are acquired depends on the size of the local VSA repertoire and the frequency of exposure to new VSA. Because parasites from areas with declining malaria endemicity, such as the Amazon, typically express a restricted PfEMP-1 repertoire, we hypothesized that Amazonians would rapidly acquire antibodies to most locally circulating VSA. Consistent with our expectations, the analysis of 5878 sequence tags expressed by 10 local P. falciparum samples revealed little PfEMP-1 DBL1α domain diversity. Among the most commonly expressed DBL1α types, 45% were shared by two or more independent parasite lines. Nevertheless, Amazonians displayed major gaps in their repertoire of anti-VSA antibodies, although the breadth of anti-VSA antibody responses correlated positively with their cumulative exposure to malaria. We found little antibody cross-reactivity even when testing VSA from related parasites expressing the same dominant DBL1α types. We conclude that variant-specific immunity to P. falciparum VSAs develops slowly despite the relatively restricted PfEMP-1 repertoire found in low-endemicity settings. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Plasmodium falciparum Calcium-Dependent Protein Kinase 2 Is Critical for Male Gametocyte Exflagellation but Not Essential for Asexual Proliferation

PubMed Central

Molina-Cruz, Alvaro; Brzostowski, Joseph; Mu, Jianbing

2017-01-01

ABSTRACT Drug development efforts have focused mostly on the asexual blood stages of the malaria parasite Plasmodium falciparum. Except for primaquine, which has its own limitations, there are no available drugs that target the transmission of the parasite to mosquitoes. Therefore, there is a need to validate new parasite proteins that can be targeted for blocking transmission. P. falciparum calcium-dependent protein kinases (PfCDPKs) play critical roles at various stages of the parasite life cycle and, importantly, are absent in the human host. These features mark them as attractive drug targets. In this study, using CRISPR/Cas9 we successfully knocked out PfCDPK2 from blood-stage parasites, which was previously thought to be an indispensable protein. The growth rate of the PfCDPK2 knockout (KO) parasites was similar to that of wild-type parasites, confirming that PfCDPK2 function is not essential for the asexual proliferation of the parasite in vitro. The mature male and female gametocytes of PfCDPK2 KO parasites become round after induction. However, they fail to infect female Anopheles stephensi mosquitoes due to a defect(s) in male gametocyte exflagellation and possibly in female gametes. PMID:29042501

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

Transgenic Parasites Stably Expressing Full-Length Plasmodium falciparum Circumsporozoite Protein as a Model for Vaccine Down-Selection in Mice Using Sterile Protection as an Endpoint

PubMed Central

Porter, Michael D.; Nicki, Jennifer; Pool, Christopher D.; DeBot, Margot; Illam, Ratish M.; Brando, Clara; Bozick, Brooke; De La Vega, Patricia; Angra, Divya; Spaccapelo, Roberta; Crisanti, Andrea; Murphy, Jittawadee R.; Bennett, Jason W.; Schwenk, Robert J.; Ockenhouse, Christian F.

2013-01-01

Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations. PMID:23536694

High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography

NASA Astrophysics Data System (ADS)

Kim, Kyoohyun; Yoon, HyeOk; Diez-Silva, Monica; Dao, Ming; Dasari, Ramachandra R.; Park, YongKeun

2014-01-01

We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated.

The Exported Chaperone PfHsp70x Is Dispensable for the Plasmodium falciparum Intraerythrocytic Life Cycle.

PubMed

Cobb, David W; Florentin, Anat; Fierro, Manuel A; Krakowiak, Michelle; Moore, Julie M; Muralidharan, Vasant

2017-01-01

Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum . IMPORTANCE Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is

Plasmodium falciparum erythrocyte membrane protein-1 specifically suppresses early production of host interferon-gamma.

PubMed

D'Ombrain, Marthe C; Voss, Till S; Maier, Alexander G; Pearce, J Andrew; Hansen, Diana S; Cowman, Alan F; Schofield, Louis

2007-08-16

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a variable antigen expressed by P. falciparum, the malarial parasite. PfEMP-1, present on the surface of infected host erythrocytes, mediates erythrocyte binding to vascular endothelium, enabling the parasite to avoid splenic clearance. In addition, PfEMP-1 is proposed to regulate host immune responses via interactions with the CD36 receptor on antigen-presenting cells. We investigated the immunoregulatory function of PfEMP-1 by comparing host cell responses to erythrocytes infected with either wild-type parasites or transgenic parasites lacking PfEMP-1. We showed that PfEMP-1 suppresses the production of the cytokine interferon-gamma by human peripheral blood mononuclear cells early after exposure to P. falciparum. Suppression of this rapid proinflammatory response was CD36 independent and specific to interferon-gamma production by gammadelta-T, NK, and alphabeta-T cells. These data demonstrate a parasite strategy for downregulating the proinflammatory interferon-gamma response and further establish transgenic parasites lacking PfEMP-1 as powerful tools for elucidating PfEMP-1 functions.

Human Monoclonal Antibodies to Pf 155, a Major Antigen of Malaria Parasite Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Udomsangpetch, Rachanee; Lundgren, Katarina; Berzins, Klavs; Wahlin, Birgitta; Perlmann, Hedvig; Troye-Blomberg, Marita; Carlsson, Jan; Wahlgren, Mats; Perlmann, Peter; Bjorkman, Anders

1986-01-01

Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

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

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.

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

PubMed

Akide-Ndunge, Oscar Bate; Tambini, Elisa; Giribaldi, Giuliana; McMillan, Paul J; Müller, Sylke; Arese, Paolo; Turrini, Francesco

2009-05-29

Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of

Malaria control by chlorproguanil. I. Clinical effects and susceptibility of Plasmodium falciparum in vivo after seven years of monthly chlorproguanil administration to children in a Liberian village.

PubMed

Björkman, A; Brohult, J; Willcox, M; Pehrson, P O; Rombo, L; Hedman, P; Hetland, G; Kollie, E; Hanson, A P; Bengtsson, E

1985-12-01

For seven years, chlorproguanil (1.0 to 2.0 mg kg-1) was administered monthly to the children below 15 years of age in a village with holoendemic malaria. Malariometric indices were recorded every six months. Susceptibility in vivo was monitored by the clearance of Plasmodium falciparum parasitaemia after drug intake. Three parasite species were found initially: P. falciparum (52%), P. malariae (8%) and P. ovale (4%). The parasites found during the study were mainly P. falciparum, and parasite rates ranged from 37 to 87% at the different surveys one month after respective drug intake. A fifty-fold decrease of mean parasite density was generally observed seven days after drug intake. Splenomegaly was initially recorded in all two to nine year old children, with a mean size of 2.64 according to Hackett's index. From 18 months onwards as the mean spleen index was 1.15 in the same age group. Chlorproguanil may represent an important alternative drug to groups at risk in malaria control schemes.

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

Structure of Plasmodium falciparum orotate phosphoribosyltransferase with autologous inhibitory protein–protein interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Shiva; Krishnamoorthy, Kalyanaraman; Mudeppa, Devaraja G.

P. falciparum orotate phosphoribosyltransferase, a potential target for antimalarial drugs and a conduit for prodrugs, crystallized as a structure with eight molecules per asymmetric unit that included some unique parasite-specific auto-inhibitory interactions between catalytic dimers. The most severe form of malaria is caused by the obligate parasite Plasmodium falciparum. Orotate phosphoribosyltransferase (OPRTase) is the fifth enzyme in the de novo pyrimidine-synthesis pathway in the parasite, which lacks salvage pathways. Among all of the malaria de novo pyrimidine-biosynthesis enzymes, the structure of P. falciparum OPRTase (PfOPRTase) was the only one unavailable until now. PfOPRTase that could be crystallized was obtained aftermore » some low-complexity sequences were removed. Four catalytic dimers were seen in the asymmetic unit (a total of eight polypeptides). In addition to revealing unique amino acids in the PfOPRTase active sites, asymmetric dimers in the larger structure pointed to novel parasite-specific protein–protein interactions that occlude the catalytic active sites. The latter could potentially modulate PfOPRTase activity in parasites and possibly provide new insights for blocking PfOPRTase functions.« less

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

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

Prevalence of G6PD deficiency and Plasmodium falciparum parasites in asymptomatic school children living in southern Ghana.

PubMed

Amoah, Linda Eva; Opong, Akua; Ayanful-Torgby, Ruth; Abankwa, Joana; Acquah, Festus K

2016-07-26

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder that results in impaired enzyme activity. Although G6PD deficiency is globally distributed it is more prevalent in malaria-endemic countries. Several mutations have been identified in the G6PD gene, which alter enzyme activity. The G6PD genotype predominantly found in sub-Saharan Africa is the G6PDB (G6PD376A) with (G6PD376G) and G6PDA- (G6PD376G/202A, G6PD376G/542T, G6PD376G/680T and G6PD376G/968C) occurring at lower frequencies. The aim of this study was to identify the prevalence of G6PD deficiency and asymptomatic Plasmodium falciparum carriage in children living in southern Ghana and determine whether G6PD deficiency influences asymptomatic carriage of P. falciparum parasites. Blood samples were obtained once a month from 170 healthy Ghanaian school children aged between 5 and 12 years from Basic schools in two communities Obom and Abura with similar rainfall patterns and malaria peak seasons. G6PD enzyme activity was assessed using the qualitative G6PD RDT kit (AccessBIO). G6PD genotyping and asymptomatic parasite carriage was determined by PCR followed by restriction fragment length polymorphism (RFLP) of DNA extracted from dried blood spots. The only sub-Saharan G6PD A- allele detected was the A376G/G202A found in 12.4 % (21/170), of the children and distributed as 4.1 % (7/170) A-, 1.8 % (3/170) A-/A- homozygous deficient males and females and 6.5 % (11/170) A/A- and B/A- heterozygous deficient females. Phenotypically, 10.6 % (15/142) of the children were G6PD deficient. The asymptomatic carriage of P. falciparum by PCR was 50, 29.4, 38.2 and 38.8 % over the months of February through May 2015, respectively, and 28.8, 22.4, 25.9 and 5.9 % by microscopy during the same periods. G6PD deficiency was significantly associated with a lowered risk of PCR-estimated asymptomatic P. falciparum carriage in children during the off peak malaria season in Southern Ghana.

RTS,S vaccination is associated with serologic evidence of decreased exposure to Plasmodium falciparum liver- and blood-stage parasites.

PubMed

Campo, Joe J; Aponte, John J; Skinner, Jeff; Nakajima, Rie; Molina, Douglas M; Liang, Li; Sacarlal, Jahit; Alonso, Pedro L; Crompton, Peter D; Felgner, Philip L; Dobaño, Carlota

2015-03-01

The leading malaria vaccine candidate, RTS,S, targets the sporozoite and liver stages of the Plasmodium falciparum life cycle, yet it provides partial protection against disease associated with the subsequent blood stage of infection. Antibodies against the vaccine target, the circumsporozoite protein, have not shown sufficient correlation with risk of clinical malaria to serve as a surrogate for protection. The mechanism by which a vaccine that targets the asymptomatic sporozoite and liver stages protects against disease caused by blood-stage parasites remains unclear. We hypothesized that vaccination with RTS,S protects from blood-stage disease by reducing the number of parasites emerging from the liver, leading to prolonged exposure to subclinical levels of blood-stage parasites that go undetected and untreated, which in turn boosts pre-existing antibody-mediated blood-stage immunity. To test this hypothesis, we compared antibody responses to 824 P. falciparum antigens by protein array in Mozambican children 6 months after receiving a full course of RTS,S (n = 291) versus comparator vaccine (n = 297) in a Phase IIb trial. Moreover, we used a nested case-control design to compare antibody responses of children who did or did not experience febrile malaria. Unexpectedly, we found that the breadth and magnitude of the antibody response to both liver and asexual blood-stage antigens was significantly lower in RTS,S vaccinees, with the exception of only four antigens, including the RTS,S circumsporozoite antigen. Contrary to our initial hypothesis, these findings suggest that RTS,S confers protection against clinical malaria by blocking sporozoite invasion of hepatocytes, thereby reducing exposure to the blood-stage parasites that cause disease. We also found that antibody profiles 6 months after vaccination did not distinguish protected and susceptible children during the subsequent 12-month follow-up period but were strongly associated with exposure. Together

Plasmodium falciparum, but not P. vivax, can induce erythrocytic apoptosis.

PubMed

Totino, Paulo Renato Rivas; Magalhães, Aline das Dores; Alves, Eliana Brasil; Costa, Monica Regina Farias; de Lacerda, Marcus Vinícius Guimarães; Daniel-Ribeiro, Cláudio Tadeu; Ferreira-da-Cruz, Maria de Fátima

2014-10-18

Apoptosis can occur in red blood cells (RBC) and seems to be involved in hematologic disorders related to many diseases. In malaria it is known that parasitized RBC (pRBC) is involved in the development of anemia and thrombosis; however, non-parasitized RBC (nRBC) apoptosis could amplify these malaria-associated hematologic events. In fact, in experimental malaria, increased levels of apoptosis were observed in nRBC during lethal Plasmodium yoelii 17XL infection, but in human malaria erythrocytic apoptosis has never been studied. The present study was performed to investigate if nRBC apoptosis also occurs in P. vivax and P. falciparum infections. Apoptosis of nRBC was evaluated in blood samples of P. vivax malaria patients and clinically healthly individuals living in Manaus, Brazil, both ex vivo and after incubation of RBC for 24 h. Additionally, the capacity of plasma from P. vivax or P. falciparum patients was tested for induction of in vitro apoptosis of normal RBC from a clinically healthy individual living in a non-endemic malaria region. Apoptosis was detected by flow cytometry using annexin V staining. In contrast to experimental malaria that significantly increased the levels of apoptotic nRBC both ex-vivo and after 24 h of incubation, no significant alteration on apoptotic nRBC rates was detected in P. vivax infected patients when compared with non-infected control individuals. Similar results were observed when plasma of these P. vivax patients was incubated with normal RBC. Conversely, plasma from P. falciparum-infected subjects induced significant apoptosis of these cells. Apoptosis of normal RBC can be induced by plasma from individuals with P. falciparum (but not with P. vivax) malaria. This finding could reflect the existence of erythrocytic apoptosis during infection that could contribute to the pathogenesis of hematological and vascular complications associated with falciparum malaria.

High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography

PubMed Central

Kim, Kyoohyun; Yoon, HyeOk; Diez-Silva, Monica; Dao, Ming; Dasari, Ramachandra R.

2013-01-01

Abstract. We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated. PMID:23797986

The structurally related auxin and melatonin tryptophan-derivatives and their roles in Arabidopsis thaliana and in the human malaria parasite Plasmodium falciparum.

PubMed

Koyama, Fernanda C; Carvalho, Thais L G; Alves, Eduardo; da Silva, Henrique B; de Azevedo, Mauro F; Hemerly, Adriana S; Garcia, Célia R S

2013-01-01

Indole compounds are involved in a range of functions in many organisms. In the human malaria parasite Plasmodium falciparum, melatonin and other tryptophan derivatives are able to modulate its intraerythrocytic cycle, increasing the schizont population as well as parasitemia, likely through ubiquitin-proteasome system (UPS) gene regulation. In plants, melatonin regulates root development, in a similar way to that described for indoleacetic acid, suggesting that melatonin and indoleacetic acid could co-participate in some physiological processes due to structural similarities. In the present work, we evaluate whether the chemical structure similarity found in indoleacetic acid and melatonin can lead to similar effects in Arabidopsis thaliana lateral root formation and P. falciparum cell cycle modulation, as well as in the UPS of gene regulation, by qRT-PCR. Our data show that P. falciparum is not able to respond to indoleacetic acid either in the modulation of the intraerythrocytic cycle or in the gene regulation mediated by the UPS as observed for melatonin. The similarities of these indole compounds are not sufficient to confer synergistic functions in P. falciparum cell cycle modulation, but could interplay in A. thaliana lateral root formation. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

Plasmodium falciparum exhibits markers of regulated cell death at high population density in vitro.

PubMed

Engelbrecht, Dewaldt; Coetzer, Thérèsa Louise

2016-12-01

The asexual erythrocytic cycle of the protozoan parasite Plasmodium falciparum is responsible for the pathogenesis of malaria and causes the overwhelming majority of malaria deaths. Rapidly increasing parasitaemia during this 48hour cycle threatens the survival of the human host and the parasite prior to transmission of the slow-maturing sexual stages to the mosquito host. The parasite may utilise regulated cell death (RCD) to control the burden of infection on the host and thus aid its own survival and transmission. The occurrence of RCD in P. falciparum remains a controversial topic. We provide strong evidence for the occurrence of an apoptosis-like phenotype of RCD in P. falciparum under conditions of high parasite density. P. falciparum was maintained in vitro and stressed by allowing growth to an unrestricted peak parasitaemia. Cell death markers, including morphological changes, DNA fragmentation, mitochondrial polarisation and phosphatidylserine externalisation were used to characterise parasite death at the time of peak parasitaemia and 24h later. At peak parasitaemia, mitochondrial depolarisation was observed, together with phosphatidylserine externalisation in both parasitised- and neighbouring non-infected erythrocytes. DNA fragmentation coincided with a decline in parasitaemia. Fewer merozoites were observed in mature schizonts at peak parasitaemia. Growth recovery to near-peak parasitaemia was noted within two intraerythrocytic cycles. The combination and chronological order of the biochemical markers of cell death suggest the occurrence of an apoptosis-like phenotype. The identification of a RCD pathway in P. falciparum may provide novel drug targets, particularly if the pathway differs from the host machinery. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Acyclic Immucillin Phosphonates. Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hazelton, Keith Z.; Ho, Meng-Chaio; Cassera, Maria B.

We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPsmore » are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.« less

Efficacy of oral single dose therapy with artemisinin-naphthoquine phosphate in uncomplicated falciparum malaria.

PubMed

Tun, Thein; Tint, Hla Soe; Lin, Khin; Kyaw, Thar Tun; Myint, Moe Kyaw; Khaing, Win; Tun, Zaw Win

2009-09-01

All artemisinin-based combination therapies (ACTs), recommended by the World Health Organization, are 3-day regimens. A considerable level of non-compliance on ACTs has been reported from some countries. The study aimed to assess the therapeutic efficacy of single dose treatment with new generation ACT containing artemisinin plus naphthoquine. An oral single dose of eight tablets (400 mg of naphthoquine+1000 mg artemisinin) of the combination drug was administered to adult uncomplicated falciparum malaria patients. Observations of fever, parasite clearance and reappearance, and other clinical manifestations were made on Days 0, 1, 2, 3, 7, 14, 21 and 28. Fifty-three adult falciparum positive cases, with fever or history of fever within the previous 24 h, were included in the final evaluation of the study. Mean fever clearance time, parasite clearance time were 18.2+/-8.6 h and 34.6+/-14.3 h, respectively. Adequate clinical and parasitological response was achieved in 52 cases, the rate being 98.1% (95% CI, 91.1-99.9). One patient was classified as late parasitological failure because of the reappearance of falciparum parasite on Day 14. The drug was well tolerated and no adverse reactions were detected in the patients. Since it is a single dose therapy, health workers can administer the drug as directly observed treatment.

Investigation and control of a Plasmodium falciparum malaria outbreak in Shan Special Region II of Myanmar along the China-Myanmar Border from June to December 2014.

PubMed

Liu, Hui; Xu, Jian-Wei; Yang, Heng-Lin; Li, Mei; Sun, Cheng-De; Yin, Yi-Jie; Zheng, Zhi-Liang; Zhang, Guang-Yun; Yu, Ai-Shui; Yang, Yong-Hui; Li, Chun-Hui; Ai, Shui

2016-04-25

From 2007 to 2013, intensive control measures reduced malaria burden by 90 % along the China-Myanmar border. However, despite these measures a P. falciparum malaria outbreak was reported in the Shan Special Region II of Myanmar in June of 2014. Epidemiological, parasitological and entomological investigations were performed. Dihydroartemisinin piperaquine (DAPQ) was immediately administered to treat parasite positive individuals. Long lasting insecticidal nets (LLIN), indoor residual spraying (IRS) with insecticides and behavior change communication (BCC) were also provided for outbreak control. An embedded efficacy study was conducted evaluating DP. Molecular genotyping via polymerase chain reaction (PCR) was performed on the Kelch gene on chromosome 13. All infections were identified as Plasmodium falciparum by RDT and microscopy. Two fatalities resulted from the outbreak. The attack rate was 72.8 % (67/92) and the incidence density rate was 14.2 per 100 person-weeks. The positive rate of rapid diagnostic test (RDT) was 72.2 % (65/90) and microscopically-determine parasite rate 42.2 % (38/90). Adjusted odds ratio (OR) of multivariate logistic regression analysis for aged <15 years, 15-45 years, inappropriate treatment from a private healer and lack of bed nets were 13.51 (95 % confidence interval, 2.21-105.89), 7.75 (1.48-44.97), 3.78 (1.30-46.18) and 3.21(1.21-15.19) respectively. In the six surrounding communities of the outbreak site, positive RDT rate was 1.2 % (4/328) and microscopically-determine parasite rate 0.6 % (2/328). Two light traps collected a total of 110 anopheline mosquitoes including local vectors, An. minimus, An. sinensis and An. maculates. After intensive control, the detection of malaria attacks, parasites and antigen were reduced to zero between July 1 and December 1, 2014. The cure rate of P. falciparum patients at day 42 was 94.3 % (95 % CI, 80.8-99.3 %). The PCR did not detect K13-propeller mutations. Imported P. falciparum caused the

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

Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum

PubMed Central

Tiffert, Teresa; Ginsburg, Hagai; Krugliak, Miriam; Elford, Barry C.; Lew, Virgilio L.

2000-01-01

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC50), assessed by parasite incorporation of [3H]hypoxanthine, were between 0.2 and 1.1 μM. CLT concentrations of 2 μM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (≈48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 μM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria. PMID:10618418

Validation of isoleucine utilization targets in Plasmodium falciparum

PubMed Central

Istvan, Eva S.; Dharia, Neekesh V.; Bopp, Selina E.; Gluzman, Ilya; Winzeler, Elizabeth A.; Goldberg, Daniel E.

2011-01-01

Intraerythrocytic malaria parasites can obtain nearly their entire amino acid requirement by degrading host cell hemoglobin. The sole exception is isoleucine, which is not present in adult human hemoglobin and must be obtained exogenously. We evaluated two compounds for their potential to interfere with isoleucine utilization. Mupirocin, a clinically used antibacterial, kills Plasmodium falciparum parasites at nanomolar concentrations. Thiaisoleucine, an isoleucine analog, also has antimalarial activity. To identify targets of the two compounds, we selected parasites resistant to either mupirocin or thiaisoleucine. Mutants were analyzed by genome-wide high-density tiling microarrays, DNA sequencing, and copy number variation analysis. The genomes of three independent mupirocin-resistant parasite clones had all acquired either amplifications encompassing or SNPs within the chromosomally encoded organellar (apicoplast) isoleucyl-tRNA synthetase. Thiaisoleucine-resistant parasites had a mutation in the cytoplasmic isoleucyl-tRNA synthetase. The role of this mutation in thiaisoleucine resistance was confirmed by allelic replacement. This approach is generally useful for elucidation of new targets in P. falciparum. Our study shows that isoleucine utilization is an essential pathway that can be targeted for antimalarial drug development. PMID:21205898

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.

Metabolic QTL Analysis Links Chloroquine Resistance in Plasmodium falciparum to Impaired Hemoglobin Catabolism

PubMed Central

Olszewski, Kellen L.; Cobbold, Simon A.; Baska, Katelynn S.; Tan, Asako; Ferdig, Michael T.; Llinás, Manuel

2014-01-01

Drug resistant strains of the malaria parasite, Plasmodium falciparum, have rendered chloroquine ineffective throughout much of the world. In parts of Africa and Asia, the coordinated shift from chloroquine to other drugs has resulted in the near disappearance of chloroquine-resistant (CQR) parasites from the population. Currently, there is no molecular explanation for this phenomenon. Herein, we employ metabolic quantitative trait locus mapping (mQTL) to analyze progeny from a genetic cross between chloroquine-susceptible (CQS) and CQR parasites. We identify a family of hemoglobin-derived peptides that are elevated in CQR parasites and show that peptide accumulation, drug resistance, and reduced parasite fitness are all linked in vitro to CQR alleles of the P. falciparum chloroquine resistance transporter (pfcrt). These findings suggest that CQR parasites are less fit because mutations in pfcrt interfere with hemoglobin digestion by the parasite. Moreover, our findings may provide a molecular explanation for the reemergence of CQS parasites in wild populations. PMID:24391526

A Chimeric Plasmodium falciparum Merozoite Surface Protein Vaccine Induces High Titers of Parasite Growth Inhibitory Antibodies

PubMed Central

Alaro, James R.; Partridge, Andrea; Miura, Kazutoyo; Diouf, Ababacar; Lopez, Ana M.; Angov, Evelina; Long, Carole A.

2013-01-01

The C-terminal 19-kDa domain of Plasmodium falciparum merozoite surface protein 1 (PfMSP119) is an established target of protective antibodies. However, clinical trials of PfMSP142, a leading blood-stage vaccine candidate which contains the protective epitopes of PfMSP119, revealed suboptimal immunogenicity and efficacy. Based on proof-of-concept studies in the Plasmodium yoelii murine model, we produced a chimeric vaccine antigen containing recombinant PfMSP119 (rPfMSP119) fused to the N terminus of P. falciparum merozoite surface protein 8 that lacked its low-complexity Asn/Asp-rich domain, rPfMSP8 (ΔAsn/Asp). Immunization of mice with the chimeric rPfMSP1/8 vaccine elicited strong T cell responses to conserved epitopes associated with the rPfMSP8 (ΔAsn/Asp) fusion partner. While specific for PfMSP8, this T cell response was adequate to provide help for the production of high titers of antibodies to both PfMSP119 and rPfMSP8 (ΔAsn/Asp) components. This occurred with formulations adjuvanted with either Quil A or with Montanide ISA 720 plus CpG oligodeoxynucleotide (ODN) and was observed in both inbred and outbred strains of mice. PfMSP1/8-induced antibodies were highly reactive with two major alleles of PfMSP119 (FVO and 3D7). Of particular interest, immunization with PfMSP1/8 elicited higher titers of PfMSP119-specific antibodies than a combined formulation of rPfMSP142 and rPfMSP8 (ΔAsn/Asp). As a measure of functionality, PfMSP1/8-specific rabbit IgG was shown to potently inhibit the in vitro growth of blood-stage parasites of the FVO and 3D7 strains of P. falciparum. These data support the further testing and evaluation of this chimeric PfMSP1/8 antigen as a component of a multivalent vaccine for P. falciparum malaria. PMID:23897613

Complete Plasmodium falciparum liver-stage development in liver-chimeric mice.

PubMed

Vaughan, Ashley M; Mikolajczak, Sebastian A; Wilson, Elizabeth M; Grompe, Markus; Kaushansky, Alexis; Camargo, Nelly; Bial, John; Ploss, Alexander; Kappe, Stefan H I

2012-10-01

Plasmodium falciparum, which causes the most lethal form of human malaria, replicates in the host liver during the initial stage of infection. However, in vivo malaria liver-stage (LS) studies in humans are virtually impossible, and in vitro models of LS development do not reconstitute relevant parasite growth conditions. To overcome these obstacles, we have adopted a robust mouse model for the study of P. falciparum LS in vivo: the immunocompromised and fumarylacetoacetate hydrolase-deficient mouse (Fah-/-, Rag2-/-, Il2rg-/-, termed the FRG mouse) engrafted with human hepatocytes (FRG huHep). FRG huHep mice supported vigorous, quantifiable P. falciparum LS development that culminated in complete maturation of LS at approximately 7 days after infection, providing a relevant model for LS development in humans. The infections allowed observations of previously unknown expression of proteins in LS, including P. falciparum translocon of exported proteins 150 (PTEX150) and exported protein-2 (EXP-2), components of a known parasite protein export machinery. LS schizonts exhibited exoerythrocytic merozoite formation and merosome release. Furthermore, FRG mice backcrossed to the NOD background and repopulated with huHeps and human red blood cells supported reproducible transition from LS infection to blood-stage infection. Thus, these mice constitute reliable models to study human LS directly in vivo and demonstrate utility for studies of LS-to-blood-stage transition of a human malaria parasite.

Dynamic epigenetic regulation of gene expression during the life cycle of malaria parasite Plasmodium falciparum.

PubMed

Gupta, Archna P; Chin, Wai Hoe; Zhu, Lei; Mok, Sachel; Luah, Yen-Hoon; Lim, Eng-How; Bozdech, Zbynek

2013-02-01

Epigenetic mechanisms are emerging as one of the major factors of the dynamics of gene expression in the human malaria parasite, Plasmodium falciparum. To elucidate the role of chromatin remodeling in transcriptional regulation associated with the progression of the P. falciparum intraerythrocytic development cycle (IDC), we mapped the temporal pattern of chromosomal association with histone H3 and H4 modifications using ChIP-on-chip. Here, we have generated a broad integrative epigenomic map of twelve histone modifications during the P. falciparum IDC including H4K5ac, H4K8ac, H4K12ac, H4K16ac, H3K9ac, H3K14ac, H3K56ac, H4K20me1, H4K20me3, H3K4me3, H3K79me3 and H4R3me2. While some modifications were found to be associated with the vast majority of the genome and their occupancy was constant, others showed more specific and highly dynamic distribution. Importantly, eight modifications displaying tight correlations with transcript levels showed differential affinity to distinct genomic regions with H4K8ac occupying predominantly promoter regions while others occurred at the 5' ends of coding sequences. The promoter occupancy of H4K8ac remained unchanged when ectopically inserted at a different locus, indicating the presence of specific DNA elements that recruit histone modifying enzymes regardless of their broad chromatin environment. In addition, we showed the presence of multivalent domains on the genome carrying more than one histone mark, highlighting the importance of combinatorial effects on transcription. Overall, our work portrays a substantial association between chromosomal locations of various epigenetic markers, transcriptional activity and global stage-specific transitions in the epigenome.

The pathogenesis of Plasmodium falciparum malaria in humans: insights from splenic physiology

PubMed Central

Safeukui, Innocent; Deplaine, Guillaume; Brousse, Valentine; Prendki, Virginie; Thellier, Marc; Turner, Gareth D.; Mercereau-Puijalon, Odile

2011-01-01

Clinical manifestations of Plasmodium falciparum infection are induced by the asexual stages of the parasite that develop inside red blood cells (RBCs). Because splenic microcirculatory beds filter out altered RBCs, the spleen can innately clear subpopulations of infected or uninfected RBC modified during falciparum malaria. The spleen appears more protective against severe manifestations of malaria in naïve than in immune subjects. The spleen-specific pitting function accounts for a large fraction of parasite clearance in artemisinin-treated patients. RBC loss contributes to malarial anemia, a clinical form associated with subacute progression, frequent splenomegaly, and relatively low parasitemia. Stringent splenic clearance of ring-infected RBCs and uninfected, but parasite-altered, RBCs, may altogether exacerbate anemia and reduce the risks of severe complications associated with high parasite loads, such as cerebral malaria. The age of the patient directly influences the risk of severe manifestations. We hypothesize that coevolution resulting in increased splenic clearance of P. falciparum–altered RBCs in children favors the survival of the host and, ultimately, sustained parasite transmission. This analysis of the RBC–spleen dynamic interactions during P falciparum infection reflects both data and hypotheses, and provides a framework on which a more complete immunologic understanding of malaria pathogenesis may be elaborated. PMID:20852127

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.

A combined strategy for screening a clustered mobile population returning from highly endemic areas for Plasmodium falciparum.

PubMed

Li, Mei; Li, Jun; Xia, Zhigui; Xiao, Ning; Jiang, Weikang; Wen, Yongkang

2017-04-30

Early and accurate diagnosis of imported malaria cases in clusters is crucial for protecting the health of patients and local populations, especially confirmed parasitic persons who are asymptomatic. A total of 226 gold miners who had stayed in highly endemic areas of Ghana for more than six months and returned in clusters were selected randomly. Blood samples from them were tested with microscopy, nest polymerase chain reaction, and rapid diagnostic test (RDT). The sensitivity, specificity, predictive values, agreement rate, and Youden's index of each of three diagnostic methods were calculated and compared with the defined gold standard. A quick and efficient way to respond to screening such a clustered mobile population was predicted and analyzed by evaluating two assumed results of combining microscopy and RDT with or without symptoms of illness. The rate of the carriers of malaria parasites in the populations of gold miners was 19.47%, including 39 P. falciparum. Among the three diagnostic methods, the microscopy method showed the highest specificity, while the RDT method showed the highest sensitivity but the lowest specificity in detecting P. falciparum. The assumed results of combining RDT and microscopy with symptoms showed the best results among all the test results in screening P. falciparum. It was too complex and difficult to catch all parasite carriers in a short period of time among populations with such a complicated situation as that in Shanglin County. A strategy of combing microscopy and RDT for diagnosis is highly recommended.

Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X.

PubMed

Li, Fengwu; Bounkeua, Viengngeun; Pettersen, Kenneth; Vinetz, Joseph M

2016-02-24

Plasmodium invasion of the mosquito midgut is a population bottleneck in the parasite lifecycle. Interference with molecular mechanisms by which the ookinete invades the mosquito midgut is one potential approach to developing malaria transmission-blocking strategies. Plasmodium aspartic proteases are one such class of potential targets: plasmepsin IV (known to be present in the asexual stage food vacuole) was previously shown to be involved in Plasmodium gallinaceum infection of the mosquito midgut, and plasmepsins VII and plasmepsin X (not known to be present in the asexual stage food vacuole) are upregulated in Plasmodium falciparum mosquito stages. These (and other) parasite-derived enzymes that play essential roles during ookinete midgut invasion are prime candidates for transmission-blocking vaccines. Reverse transcriptase PCR (RT-PCR) was used to determine timing of P. falciparum plasmepsin VII (PfPM VII) and plasmepsin X (PfPM X) mRNA transcripts in parasite mosquito midgut stages. Protein expression was confirmed by western immunoblot and immunofluorescence assays (IFA) using anti-peptide monoclonal antibodies (mAbs) against immunogenic regions of PfPM VII and PfPM X. These antibodies were also used in standard membrane feeding assays (SMFA) to determine whether inhibition of these proteases would affect parasite transmission to mosquitoes. The Mann-Whitney U test was used to analyse mosquito transmission assay results. RT-PCR, western immunoblot and immunofluorescence assay confirmed expression of PfPM VII and PfPM X in mosquito stages. Whereas PfPM VII was expressed in zygotes and ookinetes, PfPM X was expressed in gametes, zygotes, and ookinetes. Antibodies against PfPM VII and PfPM X decreased P. falciparum invasion of the mosquito midgut when used at high concentrations, indicating that these proteases play a role in Plasmodium mosquito midgut invasion. Failure to generate genetic knockouts of these genes limited determination of the precise role of

Plasmodium falciparum malaria importation from Africa to China and its mortality: an analysis of driving factors

NASA Astrophysics Data System (ADS)

Lai, Shengjie; Wardrop, Nicola A.; Huang, Zhuojie; Bosco, Claudio; Sun, Junling; Bird, Tomas; Wesolowski, Amy; Zhou, Sheng; Zhang, Qian; Zheng, Canjun; Li, Zhongjie; Tatem, Andrew J.; Yu, Hongjie

2016-12-01

Plasmodium falciparum malaria importation from Africa to China is rising with increasing Chinese overseas investment and international travel. Identifying networks and drivers of this phenomenon as well as the contributors to high case-fatality rate is a growing public health concern to enable efficient response. From 2011-2015, 8653 P. falciparum cases leading to 98 deaths (11.3 per 1000 cases) were imported from 41 sub-Saharan countries into China, with most cases (91.3%) occurring in labour-related Chinese travellers. Four strongly connected groupings of origin African countries with destination Chinese provinces were identified, and the number of imported cases was significantly associated with the volume of air passengers to China (P = 0.006), parasite prevalence in Africa (P < 0.001), and the amount of official development assistance from China (P < 0.001) with investment in resource extraction having the strongest relationship with parasite importation. Risk factors for deaths from imported cases were related to the capacity of malaria diagnosis and diverse socioeconomic factors. The spatial heterogeneity uncovered, principal drivers explored, and risk factors for mortality found in the rising rates of P. falciparum malaria importation to China can serve to refine malaria elimination strategies and the management of cases, and high risk groups and regions should be targeted.

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

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

Calcium homeostasis in intraerythrocytic malaria parasites.

PubMed

Garcia, C R; Dluzewski, A R; Catalani, L H; Burting, R; Hoyland, J; Mason, W T

1996-12-01

The fluorescent indicator, fura-2, AM, was used to measure free calcium concentrations in the intraerythrocytic malaria parasites of Plasmodium chabaudi and Plasmodium falciparum. In both species the free cytosolic calcium concentration was maintained at low levels (between 40 and 100 nM throughout the maturation process. Digital image analysis of the indicator fluorescence was performed on parasites and evaluated with the aid of a calibration of the calcium response, based on permeabilized parasites, exposed to calcium buffers. This again revealed that free calcium concentrations in the intact parasite are maintained at a predetermined level, regardless of the free calcium in the surrounding milieu. Both species of parasites are thus capable of regulating their internal free calcium levels with high precision, presumably by means of calcium pump ATPases. A small but significant elevation of the cytosolic free calcium concentration by the tumor promoter, thapsigargin, may be taken to reflect the presence of calcium stores in the endoplasmic reticulum in P. falciparum.

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.

Prevalence of glucose-6-phosphate dehydrogenase deficiency and its association with Plasmodium falciparum infection among children in Iganga distric in Uganda.

PubMed

Bwayo, Denis; Kaddumukasa, Mark; Ddungu, Henry; Kironde, Fred

2014-06-18

Glucose-6-phosphate dehydrogenase (G6PD) is a metabolic enzyme involved in the pentose phosphate pathway, its especially important in red blood cell metabolism. Glucose-6-phosphate dehydrogenase deficiency is an X-linked recessive hereditary disease characterised by abnormally low levels of G6PD. About 400 million people worldwide have a deficiency of this enzyme. The remarkable geographic correlation of G6PD deficiency distribution with historical endemicity patterns of malaria has led to suggestions that the two could be linked. Some studies have concluded that G6PD deficiency confers resistance to malaria. To determine the prevalence of G6PD deficiency, and determine its relationship with prevalence and incidence of P. falciparum infection among children in Uganda. This was longitudinal study involving 245 children, 135 were actively followed up for 12 months. G6PD status was assessed for using PCR-RFLP method. A thick smear was done to determine presence of plasmodium trophozoites and parasite densities. A total of 245 children between 6 months and 9 years were recruited. Of these 46.5% were males. Overall prevalence for the X-linked G6PD A- mutation was; 79.59% wild type, 12.65% heterozygous and 7.76% homozygous or hemizygous. Among the males 14% were hemizygous. At baseline, 40.8% had asymptomatic P falciparum infection. There was no statistically significant difference in prevalence and incidence rates of malaria infection among the different G6PD genotypes with prevalence among heterozygous, homozygous, and wild type being 29%, 42.6% and 43% respectively (p = 0.11) and incidence among heterozygous and wild type being 0.56 and 0.52 episodes/year (p = 0.5). The heterozygous G6PD A- females had a lower parasite density compared to the wild type (2505 vs 941 parasites/μL; P = 0.024). This study showed that 20.41% of the population in this part of Uganda carry the G6PD A-mutation, within the range of 15-32% seen in other parts of Africa. P

A new world malaria map: Plasmodium falciparum endemicity in 2010.

PubMed

Gething, Peter W; Patil, Anand P; Smith, David L; Guerra, Carlos A; Elyazar, Iqbal R F; Johnston, Geoffrey L; Tatem, Andrew J; Hay, Simon I

2011-12-20

Transmission intensity affects almost all aspects of malaria epidemiology and the impact of malaria on human populations. Maps of transmission intensity are necessary to identify populations at different levels of risk and to evaluate objectively options for disease control. To remain relevant operationally, such maps must be updated frequently. Following the first global effort to map Plasmodium falciparum malaria endemicity in 2007, this paper describes the generation of a new world map for the year 2010. This analysis is extended to provide the first global estimates of two other metrics of transmission intensity for P. falciparum that underpin contemporary questions in malaria control: the entomological inoculation rate (PfEIR) and the basic reproductive number (PfR). Annual parasite incidence data for 13,449 administrative units in 43 endemic countries were sourced to define the spatial limits of P. falciparum transmission in 2010 and 22,212 P. falciparum parasite rate (PfPR) surveys were used in a model-based geostatistical (MBG) prediction to create a continuous contemporary surface of malaria endemicity within these limits. A suite of transmission models were developed that link PfPR to PfEIR and PfR and these were fitted to field data. These models were combined with the PfPR map to create new global predictions of PfEIR and PfR. All output maps included measured uncertainty. An estimated 1.13 and 1.44 billion people worldwide were at risk of unstable and stable P. falciparum malaria, respectively. The majority of the endemic world was predicted with a median PfEIR of less than one and a median PfRc of less than two. Values of either metric exceeding 10 were almost exclusive to Africa. The uncertainty described in both PfEIR and PfR was substantial in regions of intense transmission. The year 2010 has a particular significance as an evaluation milestone for malaria global health policy. The maps presented here contribute to a rational basis for control and

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.

Identification of O-GlcNAcylated proteins in Plasmodium falciparum.

PubMed

Kupferschmid, Mattis; Aquino-Gil, Moyira Osny; Shams-Eldin, Hosam; Schmidt, Jörg; Yamakawa, Nao; Krzewinski, Frédéric; Schwarz, Ralph T; Lefebvre, Tony

2017-11-29

Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans. The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS). While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins. This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.

Plasmodium falciparum mitochondria import tRNAs along with an active phenylalanyl-tRNA synthetase.

PubMed

Sharma, Arvind; Sharma, Amit

2015-02-01

The Plasmodium falciparum protein translation enzymes aminoacyl-tRNA synthetases (aaRSs) are an emergent family of drug targets. The aaRS ensemble catalyses transfer of amino acids to cognate tRNAs, thus providing charged tRNAs for ribosomal consumption. P. falciparum proteome expression relies on a total of 36 aaRSs for the three translationally independent compartments of cytoplasm, apicoplast and mitochondria. In the present study, we show that, of this set of 36, a single genomic copy of mitochondrial phenylalanyl-tRNA synthetase (mFRS) is targeted to the parasite mitochondria, and that the mFRS gene is exclusive to malaria parasites within the apicomplexan phyla. Our protein cellular localization studies based on immunofluorescence data show that, along with mFRS, P. falciparum harbours two more phenylalanyl-tRNA synthetase (FRS) assemblies that are localized to its apicoplast and cytoplasm. The 'extra' mFRS is found in mitochondria of all asexual blood stage parasites and is competent in aminoacylation. We show further that the parasite mitochondria import tRNAs from the cytoplasmic tRNA pool. Hence drug targeting of FRSs presents a unique opportunity to potentially stall protein production in all three parasite translational compartments.

Development of loop-mediated isothermal amplification with Plasmodium falciparum unique genes for molecular diagnosis of human malaria.

PubMed

Zhang, Yijing; Yao, Yi; Du, Weixing; Wu, Kai; Xu, Wenyue; Lin, Min; Tan, Huabing; Li, Jian

2017-07-01

In order to achieve better outcomes for treatment and in the prophylaxis of malaria, it is imperative to develop a sensitive, specific, and accurate assay for early diagnosis of Plasmodium falciparum infection, which is the major cause of malaria. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay with P. falciparum unique genes for sensitive, specific, and accurate detection of P. falciparum infection. The unique genes of P. falciparum were randomly selected from PlasmoDB. The LAMP primers of the unique genes were designed using PrimerExplorer V4. LAMP assays with primers from unique genes of P. falciparum and conserved 18S rRNA gene were developed and their sensitivity was assessed. The specificity of the most sensitive LAMP assay was further examined using genomic DNA from Plasmodium vivax, Plasmodium yoelii and Toxoplasma gondii. Finally, the unique gene-based LAMP assay was validated using clinical samples of P. falciparum infection cases. A total of 31 sets of top-scored LAMP primers from nine unique genes were selected from the pools of designed primers. The LAMP assay with PF3D7_1253300-5 was the most sensitive with the detection limit 5 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. vivax, P. yoelii, and T. gondii. The LAMP assay with PF3D7_0112300 (18S rRNA) was less sensitive with the detection limit 50 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. yoelii and T. gondii, but displayed positive LAMP detection with P. vivax. The positive detection rate of the LAMP assay with PF3D7_1253300-5 was 90% (27/30), higher than that (80%, 24/30) of the positive rate of PF3D7_0112300 (18S rRNA) in examining clinical samples of P. falciparum infection cases. The LAMP assay with the primer set PF3D7_1253300-5 was more sensitive, specific, and accurate than those with PF3D7_0112300 (18S rRNA) in examining P. falciparum infection, and therefore it is a

Rapid, low dose X-ray diffractive imaging of the malaria parasite Plasmodium falciparum.

PubMed

Jones, Michael W M; Dearnley, Megan K; van Riessen, Grant A; Abbey, Brian; Putkunz, Corey T; Junker, Mark D; Vine, David J; McNulty, Ian; Nugent, Keith A; Peele, Andrew G; Tilley, Leann

2014-08-01

Phase-diverse X-ray coherent diffractive imaging (CDI) provides a route to high sensitivity and spatial resolution with moderate radiation dose. It also provides a robust solution to the well-known phase-problem, making on-line image reconstruction feasible. Here we apply phase-diverse CDI to a cellular sample, obtaining images of an erythrocyte infected by the sexual stage of the malaria parasite, Plasmodium falciparum, with a radiation dose significantly lower than the lowest dose previously reported for cellular imaging using CDI. The high sensitivity and resolution allow key biological features to be identified within intact cells, providing complementary information to optical and electron microscopy. This high throughput method could be used for fast tomographic imaging, or to generate multiple replicates in two-dimensions of hydrated biological systems without freezing or fixing. This work demonstrates that phase-diverse CDI is a valuable complementary imaging method for the biological sciences and ready for immediate application. © 2013 Elsevier B.V. All rights reserved.

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum.

PubMed

Garg, Aprajita; Wesolowski, Donna; Alonso, Dulce; Deitsch, Kirk W; Ben Mamoun, Choukri; Altman, Sidney

2015-09-22

Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome.

Phenotypic and genotypic characterization of Thai isolates of Plasmodium falciparum after an artemisinin resistance containment project.

PubMed

Thita, Thunyapit; Jadsri, Pimrat; Thamkhantho, Jarupatr; Ruang-Areerate, Toon; Suwandittakul, Nantana; Sitthichot, Naruemon; Mahotorn, Kittiya; Tan-Ariya, Peerapan; Mungthin, Mathirut

2018-05-15

In Thailand, artemisinin-based combination therapy (ACT) has been used to treat uncomplicated falciparum malaria since 1995. Unfortunately, artemisinin resistance has been reported from Thailand and other Southeast Asian countries since 2003. Malarone ® , a combination of atovaquone-proguanil (ATQ-PG), has been used to cease artemisinin pressure in some areas along Thai-Cambodia border, as part of an artemisinin resistance containment project since 2009. This study aimed to determine genotypes and phenotypes of Plasmodium falciparum isolates collected from the Thai-Cambodia border after the artemisinin resistance containment project compared with those collected before. One hundred and nine of P. falciparum isolates collected from Thai-Cambodia border from Chanthaburi and Trat provinces during 1988-2016 were used in this study. Of these, 58 isolates were collected after the containment. These parasite isolates were characterized for in vitro antimalarial sensitivities including chloroquine (CQ), quinine (QN), mefloquine (MQ), piperaquine (PPQ), artesunate (AS), dihydroartemisinin (DHA), ATQ and PG and genetic markers for drug resistance including the Kelch13 (k13), Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1) and cytochrome b (cytb) genes. Mean CQ, QN, MQ, PPQ and AS IC 50 s of the parasite isolates collected from 2009 to 2016 exhibited significantly higher than those of parasites collected before 2009. Approximately 57% exhibited in vitro MQ resistance. Approximately 94% of the isolates collected from 2009 to 2016 contained the pfmdr1 184F allele. Mutations of the k13 gene were detected in approximately 90% of the parasites collected from 2009 to 2016 which were significantly higher than the parasite isolates collected before. No ATQ-resistant genotype and phenotype of P. falciparum were found among the isolates collected after the containment project. Although the containment project had been

Amodiaquine failure associated with erythrocytic glutathione in Plasmodium falciparum malaria

PubMed Central

Zuluaga, Lina; Pabón, Adriana; López, Carlos; Ochoa, Aleida; Blair, Silvia

2007-01-01

Objective To establish the relationship between production of glutathione and the therapeutic response to amodiaquine (AQ) monotherapy in Plasmodium falciparum non-complicated malaria patients. Methodology Therapeutic response to AQ was evaluated in 32 patients with falciparum malaria in two townships of Antioquia, Colombia, and followed-up for 28 days. For every patient, total glutathione and enzymatic activity (glutathione reductase, GR, and γ-glutamylcysteine synthetase, γ-GCS) were determined in parasitized erythrocytes, non-infected erythrocytes and free parasites, on the starting day (day zero, before ingestion of AQ) and on the day of failure (in case of occurrence). Results There was found an AQ failure of 31.25%. Independent of the therapeutic response, on the starting day and on the day of failure, lower total glutathione concentration and higher GR activities in parasitized erythrocytes were found, compared with non-infected erythrocytes (p < 0.003). In addition, only on the day of failure, γ-GCS activity of parasitized erythrocytes was higher, compared with that of healthy erythrocytes (p = 0.01). Parasitized and non-parasitized erythrocytes in therapeutic failure patients (TF) had higher total glutathione on the starting day compared with those of adequate clinical response (ACR) (p < 0.02). Parasitized erythrocytes of TF patients showed lower total glutathione on the failure day, compared with starting day (p = 0.017). No differences was seen in the GR and γ-GCS activities by compartment, neither between the two therapeutic response groups nor between the two treatment days. Conclusion This study is a first approach to explaining P. falciparum therapeutic failure in humans through differences in glutathione metabolism in TF and ACR patients. These results suggest a role for glutathione in the therapeutic failure to antimalarials. PMID:17451604

Biosynthesis of GDP-fucose and Other Sugar Nucleotides in the Blood Stages of Plasmodium falciparum*

PubMed Central

Sanz, Sílvia; Bandini, Giulia; Ospina, Diego; Bernabeu, Maria; Mariño, Karina; Fernández-Becerra, Carmen; Izquierdo, Luis

2013-01-01

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions. PMID:23615908

Biosynthesis of GDP-fucose and other sugar nucleotides in the blood stages of Plasmodium falciparum.

PubMed

Sanz, Sílvia; Bandini, Giulia; Ospina, Diego; Bernabeu, Maria; Mariño, Karina; Fernández-Becerra, Carmen; Izquierdo, Luis

2013-06-07

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions.

The Plasmodium falciparum pseudoprotease SERA5 regulates the kinetics and efficiency of malaria parasite egress from host erythrocytes

PubMed Central

Hackett, Fiona; Atid, Jonathan; Tan, Michele Ser Ying

2017-01-01

Egress of the malaria parasite Plasmodium falciparum from its host red blood cell is a rapid, highly regulated event that is essential for maintenance and completion of the parasite life cycle. Egress is protease-dependent and is temporally associated with extensive proteolytic modification of parasite proteins, including a family of papain-like proteins called SERA that are expressed in the parasite parasitophorous vacuole. Previous work has shown that the most abundant SERA, SERA5, plays an important but non-enzymatic role in asexual blood stages. SERA5 is extensively proteolytically processed by a parasite serine protease called SUB1 as well as an unidentified cysteine protease just prior to egress. However, neither the function of SERA5 nor the role of its processing is known. Here we show that conditional disruption of the SERA5 gene, or of both the SERA5 and related SERA4 genes simultaneously, results in a dramatic egress and replication defect characterised by premature host cell rupture and the failure of daughter merozoites to efficiently disseminate, instead being transiently retained within residual bounding membranes. SERA5 is not required for poration (permeabilization) or vesiculation of the host cell membrane at egress, but the premature rupture phenotype requires the activity of a parasite or host cell cysteine protease. Complementation of SERA5 null parasites by ectopic expression of wild-type SERA5 reversed the egress defect, whereas expression of a SERA5 mutant refractory to processing failed to rescue the phenotype. Our findings implicate SERA5 as an important regulator of the kinetics and efficiency of egress and suggest that proteolytic modification is required for SERA5 function. In addition, our study reveals that efficient egress requires tight control of the timing of membrane rupture. PMID:28683142

Optimal sampling designs for estimation of Plasmodium falciparum clearance rates in patients treated with artemisinin derivatives

PubMed Central

2013-01-01

Background The emergence of Plasmodium falciparum resistance to artemisinins in Southeast Asia threatens the control of malaria worldwide. The pharmacodynamic hallmark of artemisinin derivatives is rapid parasite clearance (a short parasite half-life), therefore, the in vivo phenotype of slow clearance defines the reduced susceptibility to the drug. Measurement of parasite counts every six hours during the first three days after treatment have been recommended to measure the parasite clearance half-life, but it remains unclear whether simpler sampling intervals and frequencies might also be sufficient to reliably estimate this parameter. Methods A total of 2,746 parasite density-time profiles were selected from 13 clinical trials in Thailand, Cambodia, Mali, Vietnam, and Kenya. In these studies, parasite densities were measured every six hours until negative after treatment with an artemisinin derivative (alone or in combination with a partner drug). The WWARN Parasite Clearance Estimator (PCE) tool was used to estimate “reference” half-lives from these six-hourly measurements. The effect of four alternative sampling schedules on half-life estimation was investigated, and compared to the reference half-life (time zero, 6, 12, 24 (A1); zero, 6, 18, 24 (A2); zero, 12, 18, 24 (A3) or zero, 12, 24 (A4) hours and then every 12 hours). Statistical bootstrap methods were used to estimate the sampling distribution of half-lives for parasite populations with different geometric mean half-lives. A simulation study was performed to investigate a suite of 16 potential alternative schedules and half-life estimates generated by each of the schedules were compared to the “true” half-life. The candidate schedules in the simulation study included (among others) six-hourly sampling, schedule A1, schedule A4, and a convenience sampling schedule at six, seven, 24, 25, 48 and 49 hours. Results The median (range) parasite half-life for all clinical studies combined was 3.1 (0

Optimal sampling designs for estimation of Plasmodium falciparum clearance rates in patients treated with artemisinin derivatives.

PubMed

Flegg, Jennifer A; Guérin, Philippe J; Nosten, Francois; Ashley, Elizabeth A; Phyo, Aung Pyae; Dondorp, Arjen M; Fairhurst, Rick M; Socheat, Duong; Borrmann, Steffen; Björkman, Anders; Mårtensson, Andreas; Mayxay, Mayfong; Newton, Paul N; Bethell, Delia; Se, Youry; Noedl, Harald; Diakite, Mahamadou; Djimde, Abdoulaye A; Hien, Tran T; White, Nicholas J; Stepniewska, Kasia

2013-11-13

The emergence of Plasmodium falciparum resistance to artemisinins in Southeast Asia threatens the control of malaria worldwide. The pharmacodynamic hallmark of artemisinin derivatives is rapid parasite clearance (a short parasite half-life), therefore, the in vivo phenotype of slow clearance defines the reduced susceptibility to the drug. Measurement of parasite counts every six hours during the first three days after treatment have been recommended to measure the parasite clearance half-life, but it remains unclear whether simpler sampling intervals and frequencies might also be sufficient to reliably estimate this parameter. A total of 2,746 parasite density-time profiles were selected from 13 clinical trials in Thailand, Cambodia, Mali, Vietnam, and Kenya. In these studies, parasite densities were measured every six hours until negative after treatment with an artemisinin derivative (alone or in combination with a partner drug). The WWARN Parasite Clearance Estimator (PCE) tool was used to estimate "reference" half-lives from these six-hourly measurements. The effect of four alternative sampling schedules on half-life estimation was investigated, and compared to the reference half-life (time zero, 6, 12, 24 (A1); zero, 6, 18, 24 (A2); zero, 12, 18, 24 (A3) or zero, 12, 24 (A4) hours and then every 12 hours). Statistical bootstrap methods were used to estimate the sampling distribution of half-lives for parasite populations with different geometric mean half-lives. A simulation study was performed to investigate a suite of 16 potential alternative schedules and half-life estimates generated by each of the schedules were compared to the "true" half-life. The candidate schedules in the simulation study included (among others) six-hourly sampling, schedule A1, schedule A4, and a convenience sampling schedule at six, seven, 24, 25, 48 and 49 hours. The median (range) parasite half-life for all clinical studies combined was 3.1 (0.7-12.9) hours. Schedule A1

Complete Plasmodium falciparum liver-stage development in liver-chimeric mice

PubMed Central

Vaughan, Ashley M.; Mikolajczak, Sebastian A.; Wilson, Elizabeth M.; Grompe, Markus; Kaushansky, Alexis; Camargo, Nelly; Bial, John; Ploss, Alexander; Kappe, Stefan H.I.

2012-01-01

Plasmodium falciparum, which causes the most lethal form of human malaria, replicates in the host liver during the initial stage of infection. However, in vivo malaria liver-stage (LS) studies in humans are virtually impossible, and in vitro models of LS development do not reconstitute relevant parasite growth conditions. To overcome these obstacles, we have adopted a robust mouse model for the study of P. falciparum LS in vivo: the immunocompromised and fumarylacetoacetate hydrolase–deficient mouse (Fah–/–, Rag2–/–, Il2rg–/–, termed the FRG mouse) engrafted with human hepatocytes (FRG huHep). FRG huHep mice supported vigorous, quantifiable P. falciparum LS development that culminated in complete maturation of LS at approximately 7 days after infection, providing a relevant model for LS development in humans. The infections allowed observations of previously unknown expression of proteins in LS, including P. falciparum translocon of exported proteins 150 (PTEX150) and exported protein-2 (EXP-2), components of a known parasite protein export machinery. LS schizonts exhibited exoerythrocytic merozoite formation and merosome release. Furthermore, FRG mice backcrossed to the NOD background and repopulated with huHeps and human red blood cells supported reproducible transition from LS infection to blood-stage infection. Thus, these mice constitute reliable models to study human LS directly in vivo and demonstrate utility for studies of LS–to–blood-stage transition of a human malaria parasite. PMID:22996664

Genetic investigation of tricarboxylic acid metabolism during the Plasmodium falciparum life cycle.

PubMed

Ke, Hangjun; Lewis, Ian A; Morrisey, Joanne M; McLean, Kyle J; Ganesan, Suresh M; Painter, Heather J; Mather, Michael W; Jacobs-Lorena, Marcelo; Llinás, Manuel; Vaidya, Akhil B

2015-04-07

New antimalarial drugs are urgently needed to control drug-resistant forms of the malaria parasite Plasmodium falciparum. Mitochondrial electron transport is the target of both existing and new antimalarials. Herein, we describe 11 genetic knockout (KO) lines that delete six of the eight mitochondrial tricarboxylic acid (TCA) cycle enzymes. Although all TCA KOs grew normally in asexual blood stages, these metabolic deficiencies halted life-cycle progression in later stages. Specifically, aconitase KO parasites arrested as late gametocytes, whereas α-ketoglutarate-dehydrogenase-deficient parasites failed to develop oocysts in the mosquitoes. Mass spectrometry analysis of (13)C-isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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

Transcriptional Profiling Defines Histone Acetylation as a Regulator of Gene Expression during Human-to-Mosquito Transmission of the Malaria Parasite Plasmodium falciparum

PubMed Central

Ngwa, Che J.; Kiesow, Meike J.; Papst, Olga; Orchard, Lindsey M.; Filarsky, Michael; Rosinski, Alina N.; Voss, Till S.; Llinás, Manuel; Pradel, Gabriele

2017-01-01

Transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by the intraerythrocytic gametocytes, which, once taken up during a blood meal, become activated to initiate sexual reproduction. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they are crucial for spreading the tropical disease. During gametocyte maturation, different repertoires of genes are switched on and off in a well-coordinated sequence, pointing to regulatory mechanisms of gene expression. While epigenetic gene control has been studied during erythrocytic schizogony of P. falciparum, little is known about this process during human-to-mosquito transmission of the parasite. To unveil the potential role of histone acetylation during gene expression in gametocytes, we carried out a microarray-based transcriptome analysis on gametocytes treated with the histone deacetylase inhibitor trichostatin A (TSA). TSA-treatment impaired gametocyte maturation and lead to histone hyper-acetylation in these stages. Comparative transcriptomics identified 294 transcripts, which were more than 2-fold up-regulated during gametocytogenesis following TSA-treatment. In activated gametocytes, which were less sensitive to TSA, the transcript levels of 48 genes were increased. TSA-treatment further led to repression of ~145 genes in immature and mature gametocytes and 7 genes in activated gametocytes. Up-regulated genes are mainly associated with functions in invasion, cytoadherence, and protein export, while down-regulated genes could particularly be assigned to transcription and translation. Chromatin immunoprecipitation demonstrated a link between gene activation and histone acetylation for selected genes. Among the genes up-regulated in TSA-treated mature gametocytes was a gene encoding the ring finger (RING)-domain protein PfRNF1, a putative E3 ligase of the ubiquitin-mediated signaling pathway. Immunochemistry demonstrated PfRNF1

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

PubMed

Price, Ric N; Uhlemann, Anne-Catrin; Brockman, Alan; McGready, Rose; Ashley, Elizabeth; Phaipun, Lucy; Patel, Rina; Laing, Kenneth; Looareesuwan, Sornchai; White, Nicholas J; Nosten, François; Krishna, Sanjeev

The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with

Correlation between 'H' blood group antigen and Plasmodium falciparum invasion.

PubMed

Pathak, Vrushali; Colah, Roshan; Ghosh, Kanjaksha

2016-06-01

The ABO blood group system is the most important blood group system in clinical practice. The relationship between Plasmodium falciparum and ABO blood groups has been studied for many years. This study was undertaken to investigate the abilities of different blood group erythrocytes to support in vitro growth of P. falciparum parasites. P. falciparum parasites of four different strains (3D7, 7G8, Dd2 and RKL9) were co-cultured with erythrocytes of blood group 'A', 'B', 'O' (n = 10 for each) and 'O(h)' (Bombay group) (n = 7) for 5 days. Statistically significant differences were observed on the fourth day among the mean percent parasitemias of 'O', non-'O' ('A' and 'B') and 'O(h)' group cultures. The parasitemias of four strains ranged from 12.23 to 14.66, 11.68 to 13.24, 16.89 to 22.3, and 7.37 to 11.27 % in 'A', 'B', 'O' and Bombay group cultures, respectively. As the expression of H antigen decreased from 'O' blood group to 'A' and 'B' and then to Bombay blood group, parasite invasion (percent parasitemia) also decreased significantly (p < 0.01) and concomitantly, indicating the association of parasite invasion with the amount of H antigen present on the surface of erythrocyte. Thus, the question arises, could H antigen be involved in P. falciparum invasion? To evaluate erythrocyte invasion inhibition, 'O' group erythrocytes were virtually converted to Bombay group-like erythrocytes by the treatment of anti-H lectins extracted from Ulex europaeus seeds. Mean percent parasitemia of lectin-treated cultures on the fourth day was significantly lower (p < 0.05) than that of non-treated cultures and was found to be similar with the mean percent parasitemia demonstrated by the Bombay group erythrocyte cultures, thus further strengthening the hypothesis.

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.

Absence of Putative Artemisinin Resistance Mutations Among Plasmodium falciparum in Sub-Saharan Africa: A Molecular Epidemiologic Study

PubMed Central

Taylor, Steve M.; Parobek, Christian M.; DeConti, Derrick K.; Kayentao, Kassoum; Coulibaly, Sheick Oumar; Greenwood, Brian M.; Tagbor, Harry; Williams, John; Bojang, Kalifa; Njie, Fanta; Desai, Meghna; Kariuki, Simon; Gutman, Julie; Mathanga, Don P.; Mårtensson, Andreas; Ngasala, Billy; Conrad, Melissa D.; Rosenthal, Philip J.; Tshefu, Antoinette K.; Moormann, Ann M.; Vulule, John M.; Doumbo, Ogobara K.; ter Kuile, Feiko O.; Meshnick, Steven R.; Bailey, Jeffrey A.; Juliano, Jonathan J.

2015-01-01

Plasmodium falciparum parasites that are resistant to artemisinins have been detected in Southeast Asia. Resistance is associated with several polymorphisms in the parasite's K13-propeller gene. The molecular epidemiology of these artemisinin resistance genotypes in African parasite populations is unknown. We developed an assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies, validated it by evaluating mixtures of laboratory parasite strains, and then used it to screen P. falciparum parasites from >1100 African infections collected since 2002 from 14 sites across sub-Saharan Africa. We found no mutations in African parasite populations that are associated with artemisinin resistance in Southeast Asian parasites. However, we observed 15 coding mutations, including 12 novel mutations, and limited allele sharing between parasite populations, consistent with a large reservoir of naturally occurring K13-propeller variation. Although polymorphisms associated with artemisinin resistance in P. falciparum in Southeast Asia are not prevalent in sub-Saharan Africa, numerous K13-propeller coding polymorphisms circulate in Africa. Although their distributions do not support a widespread selective sweep for an artemisinin-resistant phenotype, the impact of these mutations on artemisinin susceptibility is unknown and will require further characterization. Rapid, scalable molecular surveillance offers a useful adjunct in tracking and containing artemisinin resistance. PMID:25180240

Plasmodium falciparum Serine/Threonine Phosphoprotein Phosphatases (PPP): From Housekeeper to 'Holy Grail'

USDA-ARS?s Scientific Manuscript database

Availability of complete genome sequence for Plasmodium falciparum has been useful in drawing a comprehensive metabolic map of the parasite. Distinct and unique metabolic characteristics of the parasite may be exploited as potential targets for new antimalarial drug discovery research. Reversible ph...

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

Genetic architecture of artemisinin-resistant Plasmodium falciparum

PubMed Central

Miotto, Olivo; Amato, Roberto; Ashley, Elizabeth A; MacInnis, Bronwyn; Almagro-Garcia, Jacob; Amaratunga, Chanaki; Lim, Pharath; Mead, Daniel; Oyola, Samuel O; Dhorda, Mehul; Imwong, Mallika; Woodrow, Charles; Manske, Magnus; Stalker, Jim; Drury, Eleanor; Campino, Susana; Amenga-Etego, Lucas; Thanh, Thuy-Nhien Nguyen; Tran, Hien Tinh; Ringwald, Pascal; Bethell, Delia; Nosten, Francois; Phyo, Aung Pyae; Pukrittayakamee, Sasithon; Chotivanich, Kesinee; Chuor, Char Meng; Nguon, Chea; Suon, Seila; Sreng, Sokunthea; Newton, Paul N; Mayxay, Mayfong; Khanthavong, Maniphone; Hongvanthong, Bouasy; Htut, Ye; Han, Kay Thwe; Kyaw, Myat Phone; Faiz, Md Abul; Fanello, Caterina I; Onyamboko, Marie; Mokuolu, Olugbenga A; Jacob, Christopher G; Takala-Harrison, Shannon; Plowe, Christopher V; Day, Nicholas P; Dondorp, Arjen M; Spencer, Chris C A; McVean, Gilean; Fairhurst, Rick M; White, Nicholas J; Kwiatkowski, Dominic P

2015-01-01

We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population. PMID:25599401

The shape of the iceberg: quantification of submicroscopic Plasmodium falciparum and Plasmodium vivax parasitaemia and gametocytaemia in five low endemic settings in Ethiopia.

PubMed

Tadesse, Fitsum G; van den Hoogen, Lotus; Lanke, Kjerstin; Schildkraut, Jodie; Tetteh, Kevin; Aseffa, Abraham; Mamo, Hassen; Sauerwein, Robert; Felger, Ingrid; Drakeley, Chris; Gadissa, Endalamaw; Bousema, Teun

2017-03-03

The widespread presence of low-density asymptomatic infections with concurrent gametocytes may be a stumbling block for malaria elimination. This study investigated the asymptomatic reservoir of Plasmodium falciparum and Plasmodium vivax infections in schoolchildren from five settings in northwest Ethiopia. Two cross-sectional surveys were conducted in June and November 2015, enrolling 551 students from five schools and 294 students from three schools, respectively. Finger prick whole blood and plasma samples were collected. The prevalence and density of P. falciparum and P. vivax parasitaemia and gametocytaemia were determined by 18S rRNA quantitative PCR (qPCR) and pfs25 and pvs25 reverse transcriptase qPCR. Antibodies against blood stage antigens apical membrane antigen-1 (AMA-1) and merozoite surface protein-1 (MSP-1 19 ) were measured for both species. Whilst only 6 infections were detected by microscopy in 881 slides (0.7%), 107 of 845 blood samples (12.7%) were parasite positive by (DNA-based) qPCR. qPCR parasite prevalence between sites and surveys ranged from 3.8 to 19.0% for P. falciparum and 0.0 to 9.0% for P. vivax. The median density of P. falciparum infections (n = 85) was 24.4 parasites/µL (IQR 18.0-34.0) and the median density of P. vivax infections (n = 28) was 16.4 parasites/µL (IQR 8.8-55.1). Gametocyte densities by (mRNA-based) qRT-PCR were strongly associated with total parasite densities for both P. falciparum (correlation coefficient = 0.83, p = 0.010) and P. vivax (correlation coefficient = 0.58, p = 0.010). Antibody titers against P. falciparum AMA-1 and MSP-1 19 were higher in individuals who were P. falciparum parasite positive in both surveys (p < 0.001 for both comparisons). This study adds to the available evidence on the wide-scale presence of submicroscopic parasitaemia by quantifying submicroscopic parasite densities and concurrent gametocyte densities. There was considerable heterogeneity in the occurrence of P

Declining responsiveness of Plasmodium falciparum infections to artemisinin-based combination treatments on the Kenyan coast.

PubMed

Borrmann, Steffen; Sasi, Philip; Mwai, Leah; Bashraheil, Mahfudh; Abdallah, Ahmed; Muriithi, Steven; Frühauf, Henrike; Schaub, Barbara; Pfeil, Johannes; Peshu, Judy; Hanpithakpong, Warunee; Rippert, Anja; Juma, Elizabeth; Tsofa, Benjamin; Mosobo, Moses; Lowe, Brett; Osier, Faith; Fegan, Greg; Lindegårdh, Niklas; Nzila, Alexis; Peshu, Norbert; Mackinnon, Margaret; Marsh, Kevin

2011-01-01

The emergence of artemisinin-resistant P. falciparum malaria in South-East Asia highlights the need for continued global surveillance of the efficacy of artemisinin-based combination therapies. On the Kenyan coast we studied the treatment responses in 474 children 6-59 months old with uncomplicated P. falciparum malaria in a randomized controlled trial of dihydroartemisinin-piperaquine vs. artemether-lumefantrine from 2005 to 2008. (ISRCTN88705995). The proportion of patients with residual parasitemia on day 1 rose from 55% in 2005-2006 to 87% in 2007-2008 (odds ratio, 5.4, 95%CI, 2.7-11.1; P<0.001) and from 81% to 95% (OR, 4.1, 95%CI, 1.7-9.9; P = 0.002) in the DHA-PPQ and AM-LM groups, respectively. In parallel, Kaplan-Meier estimated risks of apparent recrudescent infection by day 84 increased from 7% to 14% (P = 0.1) and from 6% to 15% (P = 0.05) with DHA-PPQ and AM-LM, respectively. Coinciding with decreasing transmission in the study area, clinical tolerance to parasitemia (defined as absence of fever) declined between 2005-2006 and 2007-2008 (OR body temperature >37.5°C, 2.8, 1.9-4.1; P<0.001). Neither in vitro sensitivity of parasites to DHA nor levels of antibodies against parasite extract accounted for parasite clearance rates or changes thereof. The significant, albeit small, decline through time of parasitological response rates to treatment with ACTs may be due to the emergence of parasites with reduced drug sensitivity, to the coincident reduction in population-level clinical immunity, or both. Maintaining the efficacy of artemisinin-based therapy in Africa would benefit from a better understanding of the mechanisms underlying reduced parasite clearance rates. Controlled-Trials.com ISRCTN88705995.

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

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.

Reduced Parasite Burden in Children with Falciparum Malaria and Bacteremia Coinfections: Role of Mediators of Inflammation

DOE PAGES

Davenport, Gregory C.; Hittner, James B.; Otieno, Vincent; ...

2016-01-01

Bmore » acteremia and malaria coinfection is a common and life-threatening condition in children residing in sub-Saharan Africa. We previously showed that coinfection with Gram negative (G[−]) enteric acilli and Plasmodium falciparum ( Pf [+]) was associated with reduced high-density parasitemia (HDP, >10,000 parasites/ μ L), enhanced respiratory distress, and severe anemia. Since inflammatory mediators are largely unexplored in such coinfections, circulating cytokines were determined in four groups of children ( n = 206 , aged <3 yrs): healthy; Pf [+] alone; G[−] coinfected; and G[+] coinfected. Staphylococcus aureus and non-Typhi Salmonella were the most frequently isolated G[+] and G[−] organisms, respectively. Coinfected children, particularly those with G[−] pathogens, had lower parasite burden (peripheral and geometric mean parasitemia and HDP). In addition, both coinfected groups had increased IL-4, IL-5, IL-7, IL-12, IL-15, IL-17, IFN- γ , and IFN- α and decreased TNF- α relative to malaria alone. Children with G[−] coinfection had higher IL-1 β and IL-1Ra and lower IL-10 than the Pf [+] group and higher IFN- γ than the G[+] group. To determine how the immune response to malaria regulates parasitemia, cytokine production was investigated with a multiple mediation model. Cytokines with the greatest mediational impact on parasitemia were IL-4, IL-10, IL-12, and IFN- γ . Results here suggest that enhanced immune activation, especially in G[−] coinfected children, acts to reduce malaria parasite burden.« less

Reduced Parasite Burden in Children with Falciparum Malaria and Bacteremia Coinfections: Role of Mediators of Inflammation.

PubMed

Davenport, Gregory C; Hittner, James B; Otieno, Vincent; Karim, Zachary; Mukundan, Harshini; Fenimore, Paul W; Hengartner, Nicolas W; McMahon, Benjamin H; Kempaiah, Prakasha; Ong'echa, John M; Perkins, Douglas J

2016-01-01

Bacteremia and malaria coinfection is a common and life-threatening condition in children residing in sub-Saharan Africa. We previously showed that coinfection with Gram negative (G[-]) enteric Bacilli and Plasmodium falciparum (Pf[+]) was associated with reduced high-density parasitemia (HDP, >10,000 parasites/μL), enhanced respiratory distress, and severe anemia. Since inflammatory mediators are largely unexplored in such coinfections, circulating cytokines were determined in four groups of children (n = 206, aged <3 yrs): healthy; Pf[+] alone; G[-] coinfected; and G[+] coinfected. Staphylococcus aureus and non-Typhi Salmonella were the most frequently isolated G[+] and G[-] organisms, respectively. Coinfected children, particularly those with G[-] pathogens, had lower parasite burden (peripheral and geometric mean parasitemia and HDP). In addition, both coinfected groups had increased IL-4, IL-5, IL-7, IL-12, IL-15, IL-17, IFN-γ, and IFN-α and decreased TNF-α relative to malaria alone. Children with G[-] coinfection had higher IL-1β and IL-1Ra and lower IL-10 than the Pf[+] group and higher IFN-γ than the G[+] group. To determine how the immune response to malaria regulates parasitemia, cytokine production was investigated with a multiple mediation model. Cytokines with the greatest mediational impact on parasitemia were IL-4, IL-10, IL-12, and IFN-γ. Results here suggest that enhanced immune activation, especially in G[-] coinfected children, acts to reduce malaria parasite burden.

Reduced Parasite Burden in Children with Falciparum Malaria and Bacteremia Coinfections: Role of Mediators of Inflammation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davenport, Gregory C.; Hittner, James B.; Otieno, Vincent

Bmore » acteremia and malaria coinfection is a common and life-threatening condition in children residing in sub-Saharan Africa. We previously showed that coinfection with Gram negative (G[−]) enteric acilli and Plasmodium falciparum ( Pf [+]) was associated with reduced high-density parasitemia (HDP, >10,000 parasites/ μ L), enhanced respiratory distress, and severe anemia. Since inflammatory mediators are largely unexplored in such coinfections, circulating cytokines were determined in four groups of children ( n = 206 , aged <3 yrs): healthy; Pf [+] alone; G[−] coinfected; and G[+] coinfected. Staphylococcus aureus and non-Typhi Salmonella were the most frequently isolated G[+] and G[−] organisms, respectively. Coinfected children, particularly those with G[−] pathogens, had lower parasite burden (peripheral and geometric mean parasitemia and HDP). In addition, both coinfected groups had increased IL-4, IL-5, IL-7, IL-12, IL-15, IL-17, IFN- γ , and IFN- α and decreased TNF- α relative to malaria alone. Children with G[−] coinfection had higher IL-1 β and IL-1Ra and lower IL-10 than the Pf [+] group and higher IFN- γ than the G[+] group. To determine how the immune response to malaria regulates parasitemia, cytokine production was investigated with a multiple mediation model. Cytokines with the greatest mediational impact on parasitemia were IL-4, IL-10, IL-12, and IFN- γ . Results here suggest that enhanced immune activation, especially in G[−] coinfected children, acts to reduce malaria parasite burden.« less

DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

PubMed Central

Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

2014-01-01

Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells.

PubMed

Waller, Karena L; Nunomura, Wataru; An, Xiuli; Cooke, Brian M; Mohandas, Narla; Coppel, Ross L

2003-09-01

The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a 19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a 51-residue region encoded by exon 10 of the 4.1R gene. The 3D structure of this region reveals that the MESA binding site overlaps the region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex. Further binding studies using p55, 4.1R, and MESA showed competition between p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal binding domains involved in critical protein interactions in IRBCs may aid the development of novel therapies for falciparum malaria.

Persistent Plasmodium falciparum infection in women with an intent to become pregnant is a risk factor for pregnancy-associated malaria.

PubMed

Tuikue Ndam, Nicaise; Tornyigah, Bernard; Dossou, Akpéyédjé Yannelle; Escriou, Guillaume; Nielsen, Morten A; Salanti, Ali; Issifou, Saadou; Massougbodji, Achille; Chippaux, Jean-Philippe; Deloron, Philippe

2018-05-04

Pregnant women are more susceptible to P. falciparum than before pregnancy, and infection has consequences for both mother and offspring. WHO recommends that pregnant woman in areas of transmission receive intermittent preventive treatment starting in the second trimester. Consequently, women are not protected during the first trimester, although P. falciparum infections are both frequent and harmful. A cohort of nulligravidae women was followed during subsequent pregnancy. Malaria was diagnosed by microscopy and PCR. Parasites were genotyped at polymorphic loci. Among 275 nulligravidae enrolled, 68 women became pregnant and were followed during pregnancy. Before pregnancy, P. falciparum prevalence rates were 15% by microscopy and 66% by PCR. Microscopic infection rates increased to 29% until IPT administration, while their density increased by 20-fold. Conversely, submicroscopic infections decreased. Following IPT administration, all types of infections decreased, but increased again late in pregnancy. The risk of infection during pregnancy was higher in women with a microscopic (OR = 6.5, p = 0.047) or submicroscopic (OR = 3.06, p = 0.05) infection before pregnancy and was not related to the season of occurrence. Most infections during pregnancy were persistent infections acquired before pregnancy. Microscopic and sub-microscopic malaria infections were frequent in nulligravidae women from south Benin. During the first trimester of pregnancy, microscopic infections were more frequent, with a higher parasite density, and mainly derived from parasites infecting the woman before conception. Prevention strategies targeting non-pregnant women with a desire of conception need to be designed.

Effect of sodium citrate based anticoagulants on the growth activity of malaria parasites.

PubMed

Liu, Zhong-xiang; Wang, Xian-feng; Li, Shu-mei; Li, Xun; Xue, Cai-fang; Miao, Jun

2004-12-01

To study the effect of anticoagulants based on sodium citrate on the growth activity of malaria parasites. The parasites were treated with 3 anticoagulants (ACD, CD and SC), respectively, and the parasitemia was determined to measure the effect of the anticoagulants on the growth of the parasites. Unsynchronized Plasmodium falciparum was treated with the anticoagulants at different concentrations for 3 h at 37 degrees C. Treated erythrocytes were mixed with normal parasites or treated parasites with normal erythrocytes, which was followed by parasitemia determination of the two cultures to determine the cell target of the anticoagulants. Stage-synchronized parasites (ring, trophozoite and schizont) were treated as above to investigate the stage target. P. berghei was also treated with anticoagulants and inoculated in mice to detect the effect of anticoagulants on the animal malaria parasite by counting the parasitemia. All 3 anticoagulants inhibited falciparum parasite growth and ACD had the strongest potency. The treatments of the erythrocyte and the parasite with anticoagulants respectively showed that the anticoagulants targeted the parasites rather than normal erythrocytes. Stage-synchronized parasite treatment suggested anticoagulants primarily inhibited schizonts. The effect of anticoagulants on P. berghei was similar to that on P. falciparum. ACD showed the most significant inhibitive effect on the growth of malaria parasites and SC was the best anticoagulant based on sodium citrate for malarial experiments.

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.

Plasmodium falciparum Malaria, Southern Algeria, 2007

PubMed Central

Gassen, Ibrahim; Khechache, Yacine; Lamali, Karima; Tchicha, Boualem; Brengues, Cécile; Menegon, Michela; Severini, Carlo; Fontenille, Didier; Harrat, Zoubir

2010-01-01

An outbreak of Plasmodium falciparum malaria occurred in Tinzaouatine in southern Algeria in 2007. The likely vector, Anopheles gambiae mosquitoes, had not been detected in Algeria. Genes for resistance to chloroquine were detected in the parasite. The outbreak shows the potential for an increase in malaria vectors in Algeria. PMID:20113565

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.

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

Plasmodium falciparum coronin organizes arrays of parallel actin filaments potentially guiding directional motility in invasive malaria parasites.

PubMed

Olshina, Maya A; Angrisano, Fiona; Marapana, Danushka S; Riglar, David T; Bane, Kartik; Wong, Wilson; Catimel, Bruno; Yin, Meng-Xin; Holmes, Andrew B; Frischknecht, Friedrich; Kovar, David R; Baum, Jake

2015-07-18

Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin. Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion. A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions

Blood Stage Plasmodium falciparum Exhibits Biological Responses to Direct Current Electric Fields

PubMed Central

Coronado, Lorena M.; Montealegre, Stephania; Chaverra, Zumara; Mojica, Luis; Espinosa, Carlos; Almanza, Alejandro; Correa, Ricardo; Stoute, José A.; Gittens, Rolando A.

2016-01-01

The development of resistance to insecticides by the vector of malaria and the increasingly faster appearance of resistance to antimalarial drugs by the parasite can dangerously hamper efforts to control and eradicate the disease. Alternative ways to treat this disease are urgently needed. Here we evaluate the in vitro effect of direct current (DC) capacitive coupling electrical stimulation on the biology and viability of Plasmodium falciparum. We designed a system that exposes infected erythrocytes to different capacitively coupled electric fields in order to evaluate their effect on P. falciparum. The effect on growth of the parasite, replication of DNA, mitochondrial membrane potential and level of reactive oxygen species after exposure to electric fields demonstrate that the parasite is biologically able to respond to stimuli from DC electric fields involving calcium signaling pathways. PMID:27537497

Blood Stage Plasmodium falciparum Exhibits Biological Responses to Direct Current Electric Fields.

PubMed

Coronado, Lorena M; Montealegre, Stephania; Chaverra, Zumara; Mojica, Luis; Espinosa, Carlos; Almanza, Alejandro; Correa, Ricardo; Stoute, José A; Gittens, Rolando A; Spadafora, Carmenza

2016-01-01

The development of resistance to insecticides by the vector of malaria and the increasingly faster appearance of resistance to antimalarial drugs by the parasite can dangerously hamper efforts to control and eradicate the disease. Alternative ways to treat this disease are urgently needed. Here we evaluate the in vitro effect of direct current (DC) capacitive coupling electrical stimulation on the biology and viability of Plasmodium falciparum. We designed a system that exposes infected erythrocytes to different capacitively coupled electric fields in order to evaluate their effect on P. falciparum. The effect on growth of the parasite, replication of DNA, mitochondrial membrane potential and level of reactive oxygen species after exposure to electric fields demonstrate that the parasite is biologically able to respond to stimuli from DC electric fields involving calcium signaling pathways.

Induction of Multidrug Tolerance in Plasmodium falciparum by Extended Artemisinin Pressure

PubMed Central

Ménard, Sandie; Ben Haddou, Tanila; Ramadani, Arba Pramundita; Ariey, Frédéric; Iriart, Xavier; Beghain, Johann; Bouchier, Christiane; Witkowski, Benoit; Berry, Antoine; Mercereau-Puijalon, Odile

2015-01-01

Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia threatens global malaria control strategies. Whether delayed parasite clearance, which exposes larger parasite numbers to artemisinins for longer times, selects higher-grade resistance remains unexplored. We investigated whether long-lasting artemisinin pressure selects a novel multidrug-tolerance profile. Although 50% inhibitory concentrations for 10 antimalarial drugs tested were unchanged, drug-tolerant parasites showed higher recrudescence rates for endoperoxides, quinolones, and an antifolate, including partner drugs of recommended combination therapies, but remained susceptible to atovaquone. Moreover, the age range of intraerythrocytic stages able to resist artemisinin was extended to older ring forms and trophozoites. Multidrug tolerance results from drug-induced quiescence, which enables parasites to survive exposure to unrelated antimalarial drugs that inhibit a variety of metabolic pathways. This novel resistance pattern should be urgently monitored in the field because this pattern is not detected by current assays and represents a major threat to antimalarial drug policy. PMID:26401601

African great apes are natural hosts of multiple related malaria species, including Plasmodium falciparum

PubMed Central

Prugnolle, Franck; Durand, Patrick; Neel, Cécile; Ollomo, Benjamin; Ayala, Francisco J.; Arnathau, Céline; Etienne, Lucie; Mpoudi-Ngole, Eitel; Nkoghe, Dieudonné; Leroy, Eric; Delaporte, Eric; Peeters, Martine; Renaud, François

2010-01-01

Plasmodium reichenowi, a chimpanzee parasite, was until very recently the only known close relative of Plasmodium falciparum, the most virulent agent of human malaria. Recently, Plasmodium gaboni, another closely related chimpanzee parasite, was discovered, suggesting that the diversity of Plasmodium circulating in great apes in Africa might have been underestimated. It was also recently shown that P. reichenowi is a geographically widespread and genetically diverse chimpanzee parasite and that the world diversity of P. falciparum is fully included within the much broader genetic diversity of P. reichenowi. The evidence indicates that all extant populations of P. falciparum originated from P. reichenowi, likely by a single transfer from chimpanzees. In this work, we have studied the diversity of Plasmodium species infecting chimpanzees and gorillas in Central Africa (Cameroon and Gabon) from both wild-living and captive animals. The studies in wild apes used noninvasive sampling methods. We confirm the presence of P. reichenowi and P. gaboni in wild chimpanzees. Moreover, our results reveal the existence of an unexpected genetic diversity of Plasmodium lineages circulating in gorillas. We show that gorillas are naturally infected by two related lineages of parasites that have not been described previously, herein referred to as Plasmodium GorA and P. GorB, but also by P. falciparum, a species previously considered as strictly human specific. The continuously increasing contacts between humans and primate populations raise concerns about further reciprocal host transfers of these pathogens. PMID:20133889

Parasite burden and severity of malaria in Tanzanian children.

PubMed

Gonçalves, Bronner P; Huang, Chiung-Yu; Morrison, Robert; Holte, Sarah; Kabyemela, Edward; Prevots, D Rebecca; Fried, Michal; Duffy, Patrick E

2014-05-08

Severe Plasmodium falciparum malaria is a major cause of death in children. The contribution of the parasite burden to the pathogenesis of severe malaria has been controversial. We documented P. falciparum infection and disease in Tanzanian children followed from birth for an average of 2 years and for as long as 4 years. Of the 882 children in our study, 102 had severe malaria, but only 3 had more than two episodes. More than half of first episodes of severe malaria occurred after a second infection. Although parasite levels were higher on average when children had severe rather than mild disease, most children (67 of 102) had high-density infection (>2500 parasites per 200 white cells) with only mild symptoms before severe malaria, after severe malaria, or both. The incidence of severe malaria decreased considerably after infancy, whereas the incidence of high-density infection was similar among all age groups. Infections before and after episodes of severe malaria were associated with similar parasite densities. Nonuse of bed nets, placental malaria at the time of a woman's second or subsequent delivery, high-transmission season, and absence of the sickle cell trait increased severe-malaria risk and parasite density during infections. Resistance to severe malaria was not acquired after one or two mild infections. Although the parasite burden was higher on average during episodes of severe malaria, a high parasite burden was often insufficient to cause severe malaria even in children who later were susceptible. The diverging rates of severe disease and high-density infection after infancy, as well as the similar parasite burdens before and after severe malaria, indicate that naturally acquired resistance to severe malaria is not explained by improved control of parasite density. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests

PubMed Central

2010-01-01

Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation. PMID:20470441

Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol.

PubMed

Mogollon, Catherin Marin; van Pul, Fiona J A; Imai, Takashi; Ramesar, Jai; Chevalley-Maurel, Séverine; de Roo, Guido M; Veld, Sabrina A J; Kroeze, Hans; Franke-Fayard, Blandine M D; Janse, Chris J; Khan, Shahid M

2016-01-01

The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters.

Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol

PubMed Central

Mogollon, Catherin Marin; van Pul, Fiona J. A.; Imai, Takashi; Ramesar, Jai; Chevalley-Maurel, Séverine; de Roo, Guido M.; Veld, Sabrina A. J.; Kroeze, Hans; Franke-Fayard, Blandine M. D.; Janse, Chris J.

2016-01-01

The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters. PMID:27997583

Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

PubMed Central

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

2002-01-01

Background Reversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level. Results We have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA) led to inhibition of parasite DNA synthesis. Conclusions The high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes. PMID:12057017

Purification and partial characterization of PfHRP-II protein of Plasmodium falciparum.

PubMed

Ghimire, Prakash; Samantaray, J C; Mirdha, B R; Patra, A K; Panda, A K

2003-12-01

The human malarial parasite Plasmodium falciparum secretes various intra-and extra-cellular proteins during its asexual life cycle in human RBC. Histidine rich protein-II (HRP-II) is one of the most prominent proteins, found to be secreted by P. falciparum throughout the asexual cycle with the peak during mature schizont stage of the parasite development in human IRBC. The high histidine content (35% of the total amino acids in protein) of this protein suggested the potential to bind divalent metal ions. We have demonstrated by metal chelate chromatography, an extraordinary capacity of HRP-II to bind nickel ions (Ni++) and employed this characteristic to purify the extra-cellular HRP-II protein secreted by P. falciparum from culture supernatant. The identity of the purified protein was verified by the relative molecular weight on SDS-PAGE, by reacting with polyclonal antibodies directed against it using Western blot technique.

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

Absence of putative artemisinin resistance mutations among Plasmodium falciparum in Sub-Saharan Africa: a molecular epidemiologic study.

PubMed

Taylor, Steve M; Parobek, Christian M; DeConti, Derrick K; Kayentao, Kassoum; Coulibaly, Sheick Oumar; Greenwood, Brian M; Tagbor, Harry; Williams, John; Bojang, Kalifa; Njie, Fanta; Desai, Meghna; Kariuki, Simon; Gutman, Julie; Mathanga, Don P; Mårtensson, Andreas; Ngasala, Billy; Conrad, Melissa D; Rosenthal, Philip J; Tshefu, Antoinette K; Moormann, Ann M; Vulule, John M; Doumbo, Ogobara K; Ter Kuile, Feiko O; Meshnick, Steven R; Bailey, Jeffrey A; Juliano, Jonathan J

2015-03-01

Plasmodium falciparum parasites that are resistant to artemisinins have been detected in Southeast Asia. Resistance is associated with several polymorphisms in the parasite's K13-propeller gene. The molecular epidemiology of these artemisinin resistance genotypes in African parasite populations is unknown. We developed an assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies, validated it by evaluating mixtures of laboratory parasite strains, and then used it to screen P. falciparum parasites from >1100 African infections collected since 2002 from 14 sites across sub-Saharan Africa. We found no mutations in African parasite populations that are associated with artemisinin resistance in Southeast Asian parasites. However, we observed 15 coding mutations, including 12 novel mutations, and limited allele sharing between parasite populations, consistent with a large reservoir of naturally occurring K13-propeller variation. Although polymorphisms associated with artemisinin resistance in P. falciparum in Southeast Asia are not prevalent in sub-Saharan Africa, numerous K13-propeller coding polymorphisms circulate in Africa. Although their distributions do not support a widespread selective sweep for an artemisinin-resistant phenotype, the impact of these mutations on artemisinin susceptibility is unknown and will require further characterization. Rapid, scalable molecular surveillance offers a useful adjunct in tracking and containing artemisinin resistance. © 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.

In vitro adaptation of Plasmodium falciparum reveal variations in cultivability.

PubMed

White, John; Mascarenhas, Anjali; Pereira, Ligia; Dash, Rashmi; Walke, Jayashri T; Gawas, Pooja; Sharma, Ambika; Manoharan, Suresh Kumar; Guler, Jennifer L; Maki, Jennifer N; Kumar, Ashwani; Mahanta, Jagadish; Valecha, Neena; Dubhashi, Nagesh; Vaz, Marina; Gomes, Edwin; Chery, Laura; Rathod, Pradipsinh K

2016-01-22

Culture-adapted Plasmodium falciparum parasites can offer deeper understanding of geographic variations in drug resistance, pathogenesis and immune evasion. To help ground population-based calculations and inferences from culture-adapted parasites, the complete range of parasites from a study area must be well represented in any collection. To this end, standardized adaptation methods and determinants of successful in vitro adaption were sought. Venous blood was collected from 33 P. falciparum-infected individuals at Goa Medical College and Hospital (Bambolim, Goa, India). Culture variables such as whole blood versus washed blood, heat-inactivated plasma versus Albumax, and different starting haematocrit levels were tested on fresh blood samples from patients. In vitro adaptation was considered successful when two four-fold or greater increases in parasitaemia were observed within, at most, 33 days of attempted culture. Subsequently, parasites from the same patients, which were originally cryopreserved following blood draw, were retested for adaptability for 45 days using identical host red blood cells (RBCs) and culture media. At a new endemic area research site, ~65% of tested patient samples, with varied patient history and clinical presentation, were successfully culture-adapted immediately after blood collection. Cultures set up at 1% haematocrit and 0.5% Albumax adapted most rapidly, but no single test condition was uniformly fatal to culture adaptation. Success was not limited by low patient parasitaemia nor by patient age. Some parasites emerged even after significant delays in sample processing and even after initiation of treatment with anti-malarials. When 'day 0' cryopreserved samples were retested in parallel many months later using identical host RBCs and media, speed to adaptation appeared to be an intrinsic property of the parasites collected from individual patients. Culture adaptation of P. falciparum in a field setting is formally shown to be

Platelet factor 4 activity against P. falciparum and its translation to nonpeptidic mimics as antimalarials.

PubMed

Love, Melissa S; Millholland, Melanie G; Mishra, Satish; Kulkarni, Swapnil; Freeman, Katie B; Pan, Wenxi; Kavash, Robert W; Costanzo, Michael J; Jo, Hyunil; Daly, Thomas M; Williams, Dewight R; Kowalska, M Anna; Bergman, Lawrence W; Poncz, Mortimer; DeGrado, William F; Sinnis, Photini; Scott, Richard W; Greenbaum, Doron C

2012-12-13

Plasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV). PF4 rapidly accumulates only within infected erythrocytes and is required for parasite killing in infected erythrocyte-platelet cocultures. To exploit this antimalarial mechanism, we tested a library of small, nonpeptidic mimics of HDPs (smHDPs) and identified compounds that kill P. falciparum by rapidly lysing the parasite DV while sparing the erythrocyte plasma membrane. Lead smHDPs also reduced parasitemia in a murine malaria model. Thus, identifying host molecules that control parasite growth can further the development of related molecules with therapeutic potential. Copyright © 2012 Elsevier Inc. All rights reserved.

A Lectin-Like Receptor is Involved in Invasion of Erythrocytes by Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Jungery, M.; Pasvol, G.; Newbold, C. I.; Weatherall, D. J.

1983-02-01

Glycophorin both in solution and inserted into liposomes blocks invasion of erythrocytes by the malaria parasite Plasmodium falciparum. Furthermore, one sugar, N-acetyl-D-glucosamine (GlcNAc), completely blocks invasion of the erythrocyte by this parasite. GlcNAc coupled to bovine serum albumin to prevent the sugar entering infected erythrocytes was at least 100,000 times more effective than GlcNAc alone. Bovine serum albumin coupled to lactose or bovine serum albumin alone had no effect on invasion. These results suggest that the binding of P. falciparum to erythrocytes is lectin-like and is determined by carbohydrates on glycophorin.

Multiplicity and molecular epidemiology of Plasmodium vivax and Plasmodium falciparum infections in East Africa.

PubMed

Zhong, Daibin; Lo, Eugenia; Wang, Xiaoming; Yewhalaw, Delenasaw; Zhou, Guofa; Atieli, Harrysone E; Githeko, Andrew; Hemming-Schroeder, Elizabeth; Lee, Ming-Chieh; Afrane, Yaw; Yan, Guiyun

2018-05-02

Parasite genetic diversity and multiplicity of infection (MOI) affect clinical outcomes, response to drug treatment and naturally-acquired or vaccine-induced immunity. Traditional methods often underestimate the frequency and diversity of multiclonal infections due to technical sensitivity and specificity. Next-generation sequencing techniques provide a novel opportunity to study complexity of parasite populations and molecular epidemiology. Symptomatic and asymptomatic Plasmodium vivax samples were collected from health centres/hospitals and schools, respectively, from 2011 to 2015 in Ethiopia. Similarly, both symptomatic and asymptomatic Plasmodium falciparum samples were collected, respectively, from hospitals and schools in 2005 and 2015 in Kenya. Finger-pricked blood samples were collected and dried on filter paper. Long amplicon (> 400 bp) deep sequencing of merozoite surface protein 1 (msp1) gene was conducted to determine multiplicity and molecular epidemiology of P. vivax and P. falciparum infections. The results were compared with those based on short amplicon (117 bp) deep sequencing. A total of 139 P. vivax and 222 P. falciparum samples were pyro-sequenced for pvmsp1 and pfmsp1, yielding a total of 21 P. vivax and 99 P. falciparum predominant haplotypes. The average MOI for P. vivax and P. falciparum were 2.16 and 2.68, respectively, which were significantly higher than that of microsatellite markers and short amplicon (117 bp) deep sequencing. Multiclonal infections were detected in 62.2% of the samples for P. vivax and 74.8% of the samples for P. falciparum. Four out of the five subjects with recurrent P. vivax malaria were found to be a relapse 44-65 days after clearance of parasites. No difference was observed in MOI among P. vivax patients of different symptoms, ages and genders. Similar patterns were also observed in P. falciparum except for one study site in Kenyan lowland areas with significantly higher MOI. The study used a novel method

Hyperparasitaemia during clinical malaria episodes in infants aged 0-24 months and its association with in utero exposure to Plasmodium falciparum.

PubMed

Sylvester, Boniphace; Gasarasi, Dinah B; Aboud, Said; Tarimo, Donath; Massawe, Siriel; Mpembeni, Rose; Swedberg, Gote

2018-04-04

Existing information has shown that infants who are prenatally exposed to P. falciparum are susceptible to subsequent malaria infections. However, the effect of prenatal exposure to P. falciparum on parasite density during clinical malaria episodes has not been fully elucidated. This study is a component of a prospective cohort study for which initial results have been published. This component was designed to determine the effect of prenatal exposure to P. falciparum on parasite density during clinical malaria episodes in the first 24 months of life. A total of 215 infants were involved and monitored for clinical malaria episodes defined by fever (≥ 37 °C) and parasitaemia. The geometric mean parasite counts between exposed and unexposed infants were compared using independent samples t test. The effect of in utero exposure to P. falciparum on parasite density was assessed using binary logistic regression. The geometric mean parasite count per µl of blood during clinical malaria episodes in exposed infants was 24,889 (95% CI 18,286-31,490) while in unexposed infants it was 14,035 (95% CI 12,111-15,960), P < 0.05. Prenatal exposure to P. falciparum was associated with hyperparasitaemia during clinical malaria episodes (OR 7.04, 95% CI 2.31-21.74), while other factors were not significantly associated (P > 0.05).

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

Site-Specific Editing of the Plasmodium falciparum Genome Using Engineered Zinc-Finger Nucleases

PubMed Central

Straimer, Judith; Lee, Marcus CS; Lee, Andrew H; Zeitler, Bryan; Williams, April E; Pearl, Jocelynn R; Zhang, Lei; Rebar, Edward J; Gregory, Philip D; Llinás, Manuel; Urnov, Fyodor D; Fidock, David A

2013-01-01

Malaria afflicts over 200 million people worldwide and its most lethal etiologic agent, Plasmodium falciparum, is evolving to resist even the latest-generation therapeutics. Efficient tools for genome-directed investigations of P. falciparum pathogenesis, including drug resistance mechanisms, are clearly required. Here we report rapid and targeted genetic engineering of this parasite, using zinc-finger nucleases (ZFNs) that produce a double-strand break in a user-defined locus and trigger homology-directed repair. Targeting an integrated egfp locus, we obtained gene deletion parasites with unprecedented speed (two weeks), both with and without direct selection. ZFNs engineered against the endogenous parasite gene pfcrt, responsible for chloroquine treatment escape, rapidly produced parasites that carried either an allelic replacement or a panel of specified point mutations. The efficiency, versatility and precision of this method will enable a diverse array of genome editing approaches to interrogate this human pathogen. PMID:22922501

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

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.

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.

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number

PubMed Central

Brockman, Alan; McGready, Rose; Ashley, Elizabeth; Phaipun, Lucy; Patel, Rina; Laing, Kenneth; Looareesuwan, Sornchai; White, Nicholas J; Nosten, François; Krishna, Sanjeev

2015-01-01

Summary Background The borders of Thailand harbour the world’s most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. Methods The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Findings Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6·3 (95% CI 2·9–13·8, p<0·001) after mefloquine monotherapy and 5·4 (2·0-14·6, p=0·001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Interpretation Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Relevance to practice Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a

Computational identification of signalling pathways in Plasmodium falciparum.

PubMed

Oyelade, Jelili; Ewejobi, Itunu; Brors, Benedikt; Eils, Roland; Adebiyi, Ezekiel

2011-06-01

Malaria is one of the world's most common and serious diseases causing death of about 3 million people each year. Its most severe occurrence is caused by the protozoan Plasmodium falciparum. Reports have shown that the resistance of the parasite to existing drugs is increasing. Therefore, there is a huge and urgent need to discover and validate new drug or vaccine targets to enable the development of new treatments for malaria. The ability to discover these drug or vaccine targets can only be enhanced from our deep understanding of the detailed biology of the parasite, for example how cells function and how proteins organize into modules such as metabolic, regulatory and signal transduction pathways. It has been noted that the knowledge of signalling transduction pathways in Plasmodium is fundamental to aid the design of new strategies against malaria. This work uses a linear-time algorithm for finding paths in a network under modified biologically motivated constraints. We predicted several important signalling transduction pathways in Plasmodium falciparum. We have predicted a viable signalling pathway characterized in terms of the genes responsible that may be the PfPKB pathway recently elucidated in Plasmodium falciparum. We obtained from the FIKK family, a signal transduction pathway that ends up on a chloroquine resistance marker protein, which indicates that interference with FIKK proteins might reverse Plasmodium falciparum from resistant to sensitive phenotype. We also proposed a hypothesis that showed the FIKK proteins in this pathway as enabling the resistance parasite to have a mechanism for releasing chloroquine (via an efflux process). Furthermore, we also predicted a signalling pathway that may have been responsible for signalling the start of the invasion process of Red Blood Cell (RBC) by the merozoites. It has been noted that the understanding of this pathway will give insight into the parasite virulence and will facilitate rational vaccine design

Guide RNA selection for CRISPR-Cas9 transfections in Plasmodium falciparum.

PubMed

Ribeiro, Jose M; Garriga, Meera; Potchen, Nicole; Crater, Anna K; Gupta, Ankit; Ito, Daisuke; Desai, Sanjay A

2018-06-12

CRISPR-Cas9 mediated genome editing is addressing key limitations in the transfection of malaria parasites. While this method has already simplified the needed molecular cloning and reduced the time required to generate mutants in the human pathogen Plasmodium falciparum, optimal selection of required guide RNAs and guidelines for successful transfections have not been well characterized, leading workers to use time-consuming trial and error approaches. We used a genome-wide computational approach to create a comprehensive and publicly accessible database of possible guide RNA sequences in the P. falciparum genome. For each guide, we report on-target efficiency and specificity scores as well as information about the genomic site relevant to optimal design of CRISPR-Cas9 transfections to modify, disrupt, or conditionally knockdown any gene. As many antimalarial drug and vaccine targets are encoded by multigene families, we also developed a new paralog specificity score that should facilitate modification of either a single family member of interest or multiple paralogs that serve overlapping roles. Finally, we tabulated features of successful transfections in our laboratory, providing broadly useful guidelines for parasite transfections. Molecular studies aimed at understanding parasite biology or characterizing drug and vaccine targets in P. falciparum should be facilitated by this comprehensive database. Published by Elsevier Ltd.

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

Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum

PubMed Central

Murphy, Sean C.; Fernandez-Pol, Sebastian; Chung, Paul H.; Prasanna Murthy, S. N.; Milne, Stephen B.; Salomao, Marcela; Brown, H. Alex; Lomasney, Jon W.; Mohandas, Narla

2007-01-01

Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non–receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP2) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP2, although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection. PMID:17526861

Genetic diversity of Plasmodium falciparum populations in southeast and western Myanmar.

PubMed

Soe, Than Naing; Wu, Yanrui; Tun, Myo Win; Xu, Xin; Hu, Yue; Ruan, Yonghua; Win, Aung Ye Naung; Nyunt, Myat Htut; Mon, Nan Cho Nwe; Han, Kay Thwe; Aye, Khin Myo; Morris, James; Su, Pincan; Yang, Zhaoqing; Kyaw, Myat Phone; Cui, Liwang

2017-07-04

The genetic diversity of malaria parasites reflects the complexity and size of the parasite populations. This study was designed to explore the genetic diversity of Plasmodium falciparum populations collected from two southeastern areas (Shwekyin and Myawaddy bordering Thailand) and one western area (Kyauktaw bordering Bangladesh) of Myanmar. A total of 267 blood samples collected from patients with acute P. falciparum infections during 2009 and 2010 were used for genotyping at the merozoite surface protein 1 (Msp1), Msp2 and glutamate-rich protein (Glurp) loci. One hundred and eighty four samples were successfully genotyped at three genes. The allelic distributions of the three genes were all significantly different among three areas. MAD20 and 3D7 were the most prevalent alleles in three areas for Msp1 and Msp2, respectively. The Glurp allele with a bin size of 700-750 bp was the most prevalent both in Shwekyin and Myawaddy, whereas two alleles with bin sizes of 800-850 bp and 900-1000 bp were the most prevalent in the western site Kyauktaw. Overall, 73.91% of samples contained multiclonal infections, resulting in a mean multiplicity of infection (MOI) of 1.94. Interestingly, the MOI level presented a rising trend with the order of Myawaddy, Kyauktaw and Shwekyin, which also paralleled with the increasing frequencies of Msp1 RO33 and Msp2 FC27 200-250 bp alleles. Msp1 and Msp2 genes displayed higher levels of diversity and higher MOI rates than Glurp. PCR revealed four samples (two from Shwekyin and two from Myawaddy) with mixed infections of P. falciparum and P. vivax. This study genotyped parasite clinical samples from two southeast regions and one western state of Myanmar at the Msp1, Msp2 and Glurp loci, which revealed high levels of genetic diversity and mixed-strain infections of P. falciparum populations at these sites. The results indicated that malaria transmission intensity in these regions remained high and more strengthened control efforts are

PfPK7, an atypical MEK-related protein kinase, reflects the absence of classical three-component MAPK pathways in the human malaria parasite Plasmodium falciparum.

PubMed

Dorin, Dominique; Semblat, Jean-Philippe; Poullet, Patrick; Alano, Pietro; Goldring, J P Dean; Whittle, Christina; Patterson, Shelley; Chakrabarti, Debopam; Doerig, Christian

2005-01-01

Two members of the mitogen-activated protein kinase (MAPK) family have been previously characterized in Plasmodium falciparum, but in vitro attempts at identifying MAP kinase kinase (MAPKK) homologues have failed. Here we report the characterization of a novel plasmodial protein kinase, PfPK7, whose top scores in blastp analysis belong to the MAPKK3/6 subgroup of MAPKKs. However, homology to MAPKKs is restricted to regions of the C-terminal lobe of the kinase domain, whereas the N-terminal region is closer to fungal protein kinase A enzymes (PKA, members of the AGC group of protein kinases). Hence, PfPK7 is a 'composite' enzyme displaying regions of similarity to more than one protein kinase family, similar to a few other plasmodial protein kinases. PfPK7 is expressed in several developmental stages of the parasite, both in the mosquito vector and in the human host. Recombinant PfPK7 displayed kinase activity towards a variety of substrates, but was unable to phosphorylate the two P. falciparum MAPK homologues in vitro, and was insensitive to PKA and MEK inhibitors. Together with the absence of a typical MAPKK activation site in its T-loop, this suggests that PfPK7 is not a MAPKK orthologue, despite the fact that this enzyme is the most 'MAPKK-like' enzyme encoded in the P. falciparum genome. This is consistent with recent observations that the plasmodial MAPKs are not true orthologues of the ERK1/2, p38 or JNK MAPKs, and strengthens the evidence that classical three-component module-dependent MAPK signalling pathways do not operate in malaria parasites, a feature that has not been described in any other eukaryote.

A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections

PubMed Central

Kemleu, Sylvie; Guelig, Dylan; Eboumbou Moukoko, Carole; Essangui, Estelle; Diesburg, Steven; Mouliom, Abas; Melingui, Bernard; Manga, Jeanne; Donkeu, Christiane; Epote, Annie; Texier, Gaëtan; LaBarre, Paul; Burton, Robert

2016-01-01

Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar’s test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar’s test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in

Differences in trans-stimulated chloroquine efflux kinetics are linked to PfCRT in Plasmodium falciparum

PubMed Central

Sanchez, Cecilia P.; Rohrbach, Petra; McLean, Jeremy E.; Fidock, David A.; Stein, Wilfred D.; Lanzer, Michael

2010-01-01

Summary The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently discussed models include a carrier or a channel for chloroquine, the former actively expelling the drug, the latter facilitating its passive diffusion, out of the parasite’s food vacuole, where chloroquine accumulates and inhibits haem detoxification. Here we have challenged both models using an established trans-stimulation efflux protocol. While carriers may demonstrate trans-stimulation, channels do not. Our data reveal that extracellular chloroquine stimulates chloroquine efflux in the presence and absence of metabolic energy in both chloroquine-sensitive and -resistant parasites, resulting in a hyperbolic increase in the apparent initial efflux rates as the concentration of external chloroquine increases. In the absence of metabolic energy, the apparent initial efflux rates were comparable in both parasites. Significant differences were only observed in the presence of metabolic energy, where consistently higher apparent initial efflux rates were found in chloroquine-resistant parasites. As trans-stimulation is characteristic of a carrier, and not a channel, we interpret our data in favour of a carrier for chloroquine being present in both chloroquine-sensitive and -resistant parasites, however, with different transport modalities. PMID:17493125

Ape parasite origins of human malaria virulence genes

PubMed Central

Larremore, Daniel B.; Sundararaman, Sesh A.; Liu, Weimin; Proto, William R.; Clauset, Aaron; Loy, Dorothy E.; Speede, Sheri; Plenderleith, Lindsey J.; Sharp, Paul M.; Hahn, Beatrice H.; Rayner, Julian C.; Buckee, Caroline O.

2015-01-01

Antigens encoded by the var gene family are major virulence factors of the human malaria parasite Plasmodium falciparum, exhibiting enormous intra- and interstrain diversity. Here we use network analysis to show that var architecture and mosaicism are conserved at multiple levels across the Laverania subgenus, based on var-like sequences from eight single-species and three multi-species Plasmodium infections of wild-living or sanctuary African apes. Using select whole-genome amplification, we also find evidence of multi-domain var structure and synteny in Plasmodium gaboni, one of the ape Laverania species most distantly related to P. falciparum, as well as a new class of Duffy-binding-like domains. These findings indicate that the modular genetic architecture and sequence diversity underlying var-mediated host-parasite interactions evolved before the radiation of the Laverania subgenus, long before the emergence of P. falciparum. PMID:26456841

Manual exchange transfusion for severe imported falciparum malaria: a retrospective study.

PubMed

Lin, Jinfeng; Huang, Xiaoying; Qin, Gang; Zhang, Suyan; Sun, Weiwei; Wang, Yadong; Ren, Ke; Xu, Junxian; Han, Xudong

2018-01-16

This study was designed to evaluate the efficacy of exchange transfusion in patients with severe imported falciparum malaria. Twelve patients who met the diagnostic criteria for severe malaria were treated with exchange transfusion 14 times according to a conventional anti-malarial treatment. This study evaluated the efficacy of exchange transfusion for severe imported falciparum malaria. Clinical data of severe imported falciparum malaria patients admitted to the intensive care unit (ICU) of Nantong Third People's Hospital from January 2007 to December 2016 were investigated in this retrospective study. Patients were divided into the intervention group, which received exchange transfusion, and the control group. This study assessed parasite clearance and outcomes of the two groups, and levels of erythrocytes, haemoglobin, platelets, coagulation, liver function, lactate, C-reactive protein, and procalcitonin, before and after exchange transfusion in the intervention group. There was no significant difference in the severity of admitted patients. Exchange transfusion was successfully applied 14 times in the intervention group. Differences in the levels of erythrocytes, haemoglobin and platelets did not reach statistical significance. Exchange transfusion improved coagulation, liver function, lactic acid, C-reactive protein, and procalcitonin. No differences were observed in parasite clearance, ICU and hospital length of stay, in-hospital mortality, and costs of hospitalization between the two groups. Exchange transfusion as adjunctive therapy for severe malaria was observed to be safe in this setting. Exchange transfusion can improve liver function and coagulation and reduce inflammation, but it failed to improve parasite clearance and the outcomes of severe imported falciparum malaria in this case series.

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.

A nested real-time PCR assay for the quantification of Plasmodium falciparum DNA extracted from dried blood spots.

PubMed

Tran, Tuan M; Aghili, Amirali; Li, Shanping; Ongoiba, Aissata; Kayentao, Kassoum; Doumbo, Safiatou; Traore, Boubacar; Crompton, Peter D

2014-10-04

As public health efforts seek to eradicate malaria, there has been an emphasis on eliminating low-density parasite reservoirs in asymptomatic carriers. As such, diagnosing submicroscopic Plasmodium infections using PCR-based techniques has become important not only in clinical trials of malaria vaccines and therapeutics, but also in active malaria surveillance campaigns. However, PCR-based quantitative assays that rely on nucleic acid extracted from dried blood spots (DBS) have demonstrated lower sensitivity than assays that use cryopreserved whole blood as source material. The density of Plasmodium falciparum asexual parasites was quantified using genomic DNA extracted from dried blood spots (DBS) and the sensitivity of two approaches was compared: quantitative real-time PCR (qPCR) targeting the P. falciparum 18S ribosomal RNA gene, either with an initial conventional PCR amplification prior to qPCR (nested qPCR), or without an initial amplification (qPCR only). Parasite densities determined by nested qPCR, qPCR only, and light microscopy were compared. Nested qPCR results in 10-fold higher sensitivity (0.5 parasites/μl) when compared to qPCR only (five parasites/ul). Among microscopy-positive samples, parasite densities calculated by nested qPCR correlated strongly with microscopy for both asymptomatic (Pearson's r=0.58, P<0.001) and symptomatic (Pearson's r=0.70, P<0.0001) P. falciparum infections. Nested qPCR improves the sensitivity for the detection of P. falciparum blood-stage infection from clinical DBS samples. This approach may be useful for active malaria surveillance in areas where submicroscopic asymptomatic infections are prevalent.

Chromosome End Repair and Genome Stability in Plasmodium falciparum.

PubMed

Calhoun, Susannah F; Reed, Jake; Alexander, Noah; Mason, Christopher E; Deitsch, Kirk W; Kirkman, Laura A

2017-08-08

The human malaria parasite Plasmodium falciparum replicates within circulating red blood cells, where it is subjected to conditions that frequently cause DNA damage. The repair of DNA double-stranded breaks (DSBs) is thought to rely almost exclusively on homologous recombination (HR), due to a lack of efficient nonhomologous end joining. However, given that the parasite is haploid during this stage of its life cycle, the mechanisms involved in maintaining genome stability are poorly understood. Of particular interest are the subtelomeric regions of the chromosomes, which contain the majority of the multicopy variant antigen-encoding genes responsible for virulence and disease severity. Here, we show that parasites utilize a competitive balance between de novo telomere addition, also called "telomere healing," and HR to stabilize chromosome ends. Products of both repair pathways were observed in response to DSBs that occurred spontaneously during routine in vitro culture or resulted from experimentally induced DSBs, demonstrating that both pathways are active in repairing DSBs within subtelomeric regions and that the pathway utilized was determined by the DNA sequences immediately surrounding the break. In combination, these two repair pathways enable parasites to efficiently maintain chromosome stability while also contributing to the generation of genetic diversity. IMPORTANCE Malaria is a major global health threat, causing approximately 430,000 deaths annually. This mosquito-transmitted disease is caused by Plasmodium parasites, with infection with the species Plasmodium falciparum being the most lethal. Mechanisms underlying DNA repair and maintenance of genome integrity in P. falciparum are not well understood and represent a gap in our understanding of how parasites survive the hostile environment of their vertebrate and insect hosts. Our work examines DNA repair in real time by using single-molecule real-time (SMRT) sequencing focused on the subtelomeric

Randomized Controlled Trial of Levamisole Hydrochloride as Adjunctive Therapy in Severe Falciparum Malaria With High Parasitemia

PubMed Central

Maude, Richard J.; Silamut, Kamolrat; Plewes, Katherine; Charunwatthana, Prakaykaew; Ho, May; Abul Faiz, M.; Rahman, Ridwanur; Hossain, Md Amir; Hassan, Mahtab U.; Bin Yunus, Emran; Hoque, Gofranul; Islam, Faridul; Ghose, Aniruddha; Hanson, Josh; Schlatter, Joel; Lacey, Rachel; Eastaugh, Alison; Tarning, Joel; Lee, Sue J.; White, Nicholas J.; Chotivanich, Kesinee; Day, Nicholas P. J.; Dondorp, Arjen M.

2014-01-01

Background. Cytoadherence and sequestration of erythrocytes containing mature stages of Plasmodium falciparum are central to the pathogenesis of severe malaria. The oral anthelminthic drug levamisole inhibits cytoadherence in vitro and reduces sequestration of late-stage parasites in uncomplicated falciparum malaria treated with quinine. Methods. Fifty-six adult patients with severe malaria and high parasitemia admitted to a referral hospital in Bangladesh were randomized to receive a single dose of levamisole hydrochloride (150 mg) or no adjuvant to antimalarial treatment with intravenous artesunate. Results. Circulating late-stage parasites measured as the median area under the parasite clearance curves were 2150 (interquartile range [IQR], 0–28 025) parasites/µL × hour in patients treated with levamisole and 5489 (IQR, 192–25 848) parasites/µL × hour in controls (P = .25). The “sequestration ratios” at 6 and 12 hours for all parasite stages and changes in microvascular blood flow did not differ between treatment groups (all P > .40). The median time to normalization of plasma lactate (<2 mmol/L) was 24 (IQR, 12–30) hours with levamisole vs 28 (IQR, 12–36) hours without levamisole (P = .15). Conclusions. There was no benefit of a single-dose of levamisole hydrochloride as adjuvant to intravenous artesunate in the treatment of adults with severe falciparum malaria. Rapid parasite killing by intravenous artesunate might obscure the effects of levamisole. PMID:23943850

Targeting malaria parasite proteins to the erythrocyte.

PubMed

Templeton, Thomas J; Deitsch, Kirk W

2005-09-01

The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.

Functional characterization and comparison of Plasmodium falciparum proteins as targets of transmission-blocking antibodies.

PubMed

Nikolaeva, Daria; Illingworth, Joseph J; Miura, Kazutoyo; Alanine, Daniel Gw; Brian, Iona J; Li, Yuanyuan; Fyfe, Alex J; Da, Dari F; Cohuet, Anna; Long, Carole A; Draper, Simon J; Biswas, Sumi

2017-10-31

Plasmodium falciparum malaria continues to evade control efforts, utilizing highly specialized sexual-stages to transmit infection between the human host and mosquito vector. In a vaccination model, antibodies directed to sexual-stage antigens, when ingested in the mosquito blood meal, can inhibit parasite growth in the midgut and consequently arrest transmission. Despite multiple datasets for the Plasmodium sexual-stage transcriptome and proteome, there have been no rational screens to identify candidate antigens for transmission-blocking vaccine (TBV) development. This study characterizes 12 proteins from across the P. falciparum sexual-stages as possible TBV targets. Recombinant proteins are heterologously expressed as full-length ectodomains in a mammalian HEK293 cell system. The proteins recapitulate native parasite epitopes as assessed by indirect fluorescence assay and a proportion exhibits immunoreactivity when tested against sera from individuals living in malaria-endemic Burkina Faso and Mali. Purified IgG generated to the mosquito-stage parasite antigen enolase demonstrates moderate inhibition of parasite development in the mosquito midgut by the ex vivo standard membrane feeding assay. The findings support the use of rational screens and comparative functional assessments in identifying proteins of the P. falciparum transmission pathway and establishing a robust pre-clinical TBV pipeline. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

Agglutination Assays of the Plasmodium falciparum-Infected Erythrocyte.

PubMed

Tan, Joshua; Bull, Peter C

2015-01-01

The agglutination assay is used to determine the ability of antibodies to recognize parasite variant antigens on the surface of Plasmodium falciparum-infected erythrocytes. In this technique, infected erythrocytes are selectively labelled with a DNA-binding fluorescent dye and mixed with antibodies of interest to allow antibody-surface antigen binding. Recognition of surface antigens by the antibodies can result in the formation of agglutinates containing multiple parasite-infected erythrocytes. These can be viewed and quantified using a fluorescence microscope.

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.

Multiple essential functions of Plasmodium falciparum actin-1 during malaria blood-stage development.

PubMed

Das, Sujaan; Lemgruber, Leandro; Tay, Chwen L; Baum, Jake; Meissner, Markus

2017-08-15

The phylum Apicomplexa includes intracellular parasites causing immense global disease burden, the deadliest of them being the human malaria parasite Plasmodium falciparum, which invades and replicates within erythrocytes. The cytoskeletal protein actin is well conserved within apicomplexans but divergent from mammalian actins, and was primarily reported to function during host cell invasion. However, novel invasion mechanisms have been described for several apicomplexans, and specific functions of the acto-myosin system are being reinvestigated. Of the two actin genes in P. falciparum, actin-1 (pfact1) is ubiquitously expressed in all life-cycle stages and is thought to be required for erythrocyte invasion, although its functions during parasite development are unknown, and definitive in vivo characterisation during invasion is lacking. Here we have used a conditional Cre-lox system to investigate the functions of PfACT1 during P. falciparum blood-stage development and host cell invasion. We demonstrate that PfACT1 is crucially required for segregation of the plastid-like organelle, the apicoplast, and for efficient daughter cell separation during the final stages of cytokinesis. Surprisingly, we observe that egress from the host cell is not an actin-dependent process. Finally, we show that parasites lacking PfACT1 are capable of microneme secretion, attachment and formation of a junction with the erythrocyte, but are incapable of host cell invasion. This study provides important mechanistic insights into the definitive essential functions of PfACT1 in P. falciparum, which are not only of biological interest, but owing to functional divergence from mammalian actins, could also form the basis for the development of novel therapeutics against apicomplexans.

Detailed methodology for high resolution scanning electron microscopy (SEM) of murine malaria parasitized-erythrocytes.

PubMed

Hayakawa, Eri H; Matsuoka, Hiroyuki

2016-10-01

Scanning electron microscopy (SEM) is a powerful tool used to investigate object surfaces and has been widely applied in both material science and biology. With respect to the study of malaria, SEM revealed that erythrocytes infected with Plasmodium falciparum, a human parasite, display 'knob-like' structures on their surface comprising parasitized proteins. However, detailed methodology for SEM studies of malaria parasites is lacking in the literature making such studies challenging. Here, we provide a step-by-step guide to preparing Plasmodium-infected erythrocytes from two mouse strains for SEM analysis with minimal structural deterioration. We tested three species of murine malaria parasites, P. berghei, P. yoelii, and P. chabaudi, as well as non-parasitized human erythrocytes and P. falciparum-infected erythrocytes for comparisons. Our data demonstrated that the surface structures of parasitized erythrocytes between the three species of murine parasites in the two different strains of mice were indistinguishable and no surface alterations were observed in P. falciparum-erythrocytes. Our SEM observations contribute towards an understanding of the molecular mechanisms of parasite maturation in the erythrocyte cytoplasm and, along with future studies using our detailed methodology, may help to gain insight into the clinical phenomena of human malaria. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites

PubMed Central

Chen, Patty B.; Ding, Shuai; Zanghì, Gigliola; Soulard, Valérie; DiMaggio, Peter A.; Fuchter, Matthew J.; Mecheri, Salah; Mazier, Dominique; Scherf, Artur; Malmquist, Nicholas A.

2016-01-01

Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes. PMID:26902486

Streamlined, PCR-based testing for pfhrp2- and pfhrp3-negative Plasmodium falciparum.

PubMed

Parr, Jonathan B; Anderson, Olivia; Juliano, Jonathan J; Meshnick, Steven R

2018-04-02

Rapid diagnostic tests (RDTs) that detect histidine-rich protein 2 (PfHRP2) are used throughout Africa for the diagnosis of Plasmodium falciparum malaria. However, recent reports indicate that parasites lacking the pfhrp2 and/or histidine-rich protein 3 (pfhrp3) genes, which produce antigens detected by these RDTs, are common in select regions of South America, Asia, and Africa. Proving the absence of a gene is challenging, and multiple PCR assays targeting these genes have been described. A detailed characterization and comparison of published assays is needed to facilitate robust and streamlined testing approaches. Among six pfhrp2 and pfhrp3 PCR assays tested, the lower limit of detection ranged from 0.01 pg/µL to 0.1 ng/µL of P. falciparum 3D7 strain DNA, or approximately 0.4-4000 parasite genomes/µL. By lowering the elongation temperature to 60 °C, a tenfold improvement in the limit of detection and/or darker bands for all exon 1 targets and for the first-round reaction of a single exon 2 target was achieved. Additionally, assays targeting exon 1 of either gene yielded spurious amplification of the paralogous gene. Using these data, an optimized testing algorithm for the detection of pfhrp2- and pfhrp3-negative P. falciparum is proposed. Surveillance of pfhrp2- and pfhrp3-negative P. falciparum requires careful laboratory workflows. PCR-based testing methods coupled with microscopy and/or antigen testing serve as useful tools to support policy development. Standardized approaches to the detection of pfhrp2- and pfhrp3-negative P. falciparum should inform efforts to define the impact of these parasites.

In silico screening for Plasmodium falciparum enoyl-ACP reductase inhibitors

NASA Astrophysics Data System (ADS)

Lindert, Steffen; Tallorin, Lorillee; Nguyen, Quynh G.; Burkart, Michael D.; McCammon, J. Andrew

2015-01-01

The need for novel therapeutics against Plasmodium falciparum is urgent due to recent emergence of multi-drug resistant malaria parasites. Since fatty acids are essential for both the liver and blood stages of the malarial parasite, targeting fatty acid biosynthesis is a promising strategy for combatting P. falciparum. We present a combined computational and experimental study to identify novel inhibitors of enoyl-acyl carrier protein reductase ( PfENR) in the fatty acid biosynthesis pathway. A small-molecule database from ChemBridge was docked into three distinct PfENR crystal structures that provide multiple receptor conformations. Two different docking algorithms were used to generate a consensus score in order to rank possible small molecule hits. Our studies led to the identification of five low-micromolar pyrimidine dione inhibitors of PfENR.

Plasmodium falciparum infection in febrile Congolese children: prevalence of clinical malaria 10 years after introduction of artemisinin-combination therapies.

PubMed

Etoka-Beka, Mandingha Kosso; Ntoumi, Francine; Kombo, Michael; Deibert, Julia; Poulain, Pierre; Vouvoungui, Christevy; Kobawila, Simon Charles; Koukouikila-Koussounda, Felix

2016-12-01

To investigate the proportion of malaria infection in febrile children consulting a paediatric hospital in Brazzaville, to determine the prevalence of submicroscopic malaria infection, to characterise Plasmodium falciparum infection and compare the prevalence of uncomplicated P. falciparum malaria according to haemoglobin profiles. Blood samples were collected from children aged <10 years with an axillary temperature ≥37.5 °C consulting the paediatric ward of Marien Ngouabi Hospital in Brazzaville. Parasite density was determined and all samples were screened for P. falciparum by nested polymerase chain reaction (PCR) using the P. falciparum msp-2 marker to detect submicroscopic infections and characterise P. falciparum infection. Sickle cell trait was screened by PCR. A total of 229 children with fever were recruited, of whom 10% were diagnosed with uncomplicated malaria and 21% with submicroscopic infection. The mean parasite density in children with uncomplicated malaria was 42 824 parasites/μl of blood. The multiplicity of infection (MOI) was 1.59 in children with uncomplicated malaria and 1.69 in children with submicroscopic infection. The mean haemoglobin level was 10.1 ± 1.7 for children with uncomplicated malaria and 12.0 ± 8.6 for children with submicroscopic infection. About 13% of the children harboured the sickle cell trait (HbAS); the rest had normal haemoglobin (HbAA). No difference in prevalence of uncomplicated malaria and submicroscopic infection, parasite density, haemoglobin level, MOI and P. falciparum genetic diversity was observed according to haemoglobin type. The low prevalence of uncomplicated malaria in febrile Congolese children indicates the necessity to investigate carefully other causes of fever. © 2016 John Wiley & Sons Ltd.

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

PubMed Central

Manske, Magnus; Miotto, Olivo; Campino, Susana; Auburn, Sarah; Almagro-Garcia, Jacob; Maslen, Gareth; O’Brien, Jack; Djimde, Abdoulaye; Doumbo, Ogobara; Zongo, Issaka; Ouedraogo, Jean-Bosco; Michon, Pascal; Mueller, Ivo; Siba, Peter; Nzila, Alexis; Borrmann, Steffen; Kiara, Steven M.; Marsh, Kevin; Jiang, Hongying; Su, Xin-Zhuan; Amaratunga, Chanaki; Fairhurst, Rick; Socheat, Duong; Nosten, Francois; Imwong, Mallika; White, Nicholas J.; Sanders, Mandy; Anastasi, Elisa; Alcock, Dan; Drury, Eleanor; Oyola, Samuel; Quail, Michael A.; Turner, Daniel J.; Rubio, Valentin Ruano; Jyothi, Dushyanth; Amenga-Etego, Lucas; Hubbart, Christina; Jeffreys, Anna; Rowlands, Kate; Sutherland, Colin; Roper, Cally; Mangano, Valentina; Modiano, David; Tan, John C.; Ferdig, Michael T.; Amambua-Ngwa, Alfred; Conway, David J.; Takala-Harrison, Shannon; Plowe, Christopher V.; Rayner, Julian C.; Rockett, Kirk A.; Clark, Taane G.; Newbold, Chris I.; Berriman, Matthew; MacInnis, Bronwyn; Kwiatkowski, Dominic P.

2013-01-01

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. 1,2 Here we describe methods for large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short term culture. Analysis of 86,158 exonic SNPs that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome. PMID:22722859

Resisting and tolerating P. falciparum in pregnancy under different malaria transmission intensities.

PubMed

Ndam, Nicaise Tuikue; Mbuba, Emmanuel; González, Raquel; Cisteró, Pau; Kariuki, Simon; Sevene, Esperança; Rupérez, María; Fonseca, Ana Maria; Vala, Anifa; Maculuve, Sonia; Jiménez, Alfons; Quintó, Llorenç; Ouma, Peter; Ramharter, Michael; Aponte, John J; Nhacolo, Arsenio; Massougbodji, Achille; Briand, Valerie; Kremsner, Peter G; Mombo-Ngoma, Ghyslain; Desai, Meghna; Macete, Eusebio; Cot, Michel; Menéndez, Clara; Mayor, Alfredo

2017-07-17

Resistance and tolerance to Plasmodium falciparum can determine the progression of malaria disease. However, quantitative evidence of tolerance is still limited. We investigated variations in the adverse impact of P. falciparum infections among African pregnant women under different intensities of malaria transmission. P. falciparum at delivery was assessed by microscopy, quantitative PCR (qPCR) and placental histology in 946 HIV-uninfected and 768 HIV-infected pregnant women from Benin, Gabon, Kenya and Mozambique. Resistance was defined by the proportion of submicroscopic infections and the levels of anti-parasite antibodies quantified by Luminex, and tolerance by the relationship of pregnancy outcomes with parasite densities at delivery. P. falciparum prevalence by qPCR in peripheral and/or placental blood of HIV-uninfected Mozambican, Gabonese and Beninese women at delivery was 6% (21/340), 11% (28/257) and 41% (143/349), respectively. The proportion of peripheral submicroscopic infections was higher in Benin (83%) than in Mozambique (60%) and Gabon (55%; P = 0.033). Past or chronic placental P. falciparum infection was associated with an increased risk of preterm birth in Mozambican newborns (OR = 7.05, 95% CI 1.79 to 27.82). Microscopic infections were associated with reductions in haemoglobin levels at delivery among Mozambican women (-1.17 g/dL, 95% CI -2.09 to -0.24) as well as with larger drops in haemoglobin levels from recruitment to delivery in Mozambican (-1.66 g/dL, 95% CI -2.68 to -0.64) and Gabonese (-0.91 g/dL, 95% CI -1.79 to -0.02) women. Doubling qPCR-peripheral parasite densities in Mozambican women were associated with decreases in haemoglobin levels at delivery (-0.16 g/dL, 95% CI -0.29 to -0.02) and increases in the drop of haemoglobin levels (-0.29 g/dL, 95% CI -0.44 to -0.14). Beninese women had higher anti-parasite IgGs than Mozambican women (P < 0.001). No difference was found in the proportion of submicroscopic

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

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.

Amplification of a Gene Related to Mammalian mdr Genes in Drug-Resistant Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Wilson, Craig M.; Serrano, Adelfa E.; Wasley, Annemarie; Bogenschutz, Michael P.; Shankar, Anuraj H.; Wirth, Dyann F.

1989-06-01

The malaria parasite Plasmodium falciparum contains at least two genes related to the mammalian multiple drug resistance genes, and at least one of the P. falciparum genes is expressed at a higher level and is present in higher copy number in a strain that is resistant to multiple drugs than in a strain that is sensitive to the drugs.

Artemisinin-resistant Plasmodium falciparum malaria

PubMed Central

Fairhurst, Rick M.; Dondorp, Arjen M.

2016-01-01

For more than five decades, Southeast Asia (SEA) has been fertile ground for the emergence of drug-resistant Plasmodium falciparum malaria. After generating parasites resistant to chloroquine, sulfadoxine, pyrimethamine, quinine, and mefloquine, this region has now spawned parasites resistant to artemisinins – the world's most potent antimalarial drugs. In areas where artemisinin resistance is prevalent, artemisinin combination therapies (ACTs) – the first-line treatments for malaria – are failing fast. This worrisome development threatens to make malaria practically untreatable in SEA, and threatens to compromise global endeavors to eliminate this disease. A recent series of clinical, in-vitro, genomics, and transcriptomics studies in SEA have defined in-vivo and in-vitro phenotypes of artemisinin resistance; identified its causal genetic determinant; explored its molecular mechanism; and assessed its clinical impact. Specifically, these studies have established that artemisinin resistance manifests as slow parasite clearance in patients and increased survival of early ring-stage parasites in vitro; is caused by single nucleotide polymorphisms in the parasite's ‘K13’ gene; is associated with an upregulated “unfolded protein response” pathway that may antagonize the pro-oxidant activity of artemisinins; and selects for partner drug resistance that rapidly leads to ACT failures. In SEA, clinical studies are urgently needed to monitor ACT efficacy where K13 mutations are prevalent; test whether new combinations of currently-available drugs cure ACT failures; and advance new antimalarial compounds through preclinical pipelines and into clinical trials. Intensifying these efforts should help to forestall the spread of artemisinin and partner drug resistance from SEA to Sub-Saharan Africa, where the world's malaria transmission, morbidity, and mortality rates are highest. PMID:27337450

Artemisinin-Resistant Plasmodium falciparum Malaria.

PubMed

Fairhurst, Rick M; Dondorp, Arjen M

2016-06-01

For more than five decades, Southeast Asia (SEA) has been fertile ground for the emergence of drug-resistant Plasmodium falciparum malaria. After generating parasites resistant to chloroquine, sulfadoxine, pyrimethamine, quinine, and mefloquine, this region has now spawned parasites resistant to artemisinins, the world's most potent antimalarial drugs. In areas where artemisinin resistance is prevalent, artemisinin combination therapies (ACTs)-the first-line treatments for malaria-are failing fast. This worrisome development threatens to make malaria practically untreatable in SEA, and threatens to compromise global endeavors to eliminate this disease. A recent series of clinical, in vitro, genomics, and transcriptomics studies in SEA have defined in vivo and in vitro phenotypes of artemisinin resistance, identified its causal genetic determinant, explored its molecular mechanism, and assessed its clinical impact. Specifically, these studies have established that artemisinin resistance manifests as slow parasite clearance in patients and increased survival of early-ring-stage parasites in vitro; is caused by single nucleotide polymorphisms in the parasite's K13 gene, is associated with an upregulated "unfolded protein response" pathway that may antagonize the pro-oxidant activity of artemisinins, and selects for partner drug resistance that rapidly leads to ACT failures. In SEA, clinical studies are urgently needed to monitor ACT efficacy where K13 mutations are prevalent, test whether new combinations of currently available drugs cure ACT failures, and advance new antimalarial compounds through preclinical pipelines and into clinical trials. Intensifying these efforts should help to forestall the spread of artemisinin and partner drug resistance from SEA to sub-Saharan Africa, where the world's malaria transmission, morbidity, and mortality rates are highest.

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.

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

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.

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum.

PubMed

Abdi, Abdirahman; Eschenlauer, Sylvain; Reininger, Luc; Doerig, Christian

2010-10-01

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal "sterile alpha-motif" domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.

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.

Modeling metabolism and stage-specific growth of Plasmodium falciparum HB3 during the intraerythrocytic developmental cycle.

PubMed

Fang, Xin; Reifman, Jaques; Wallqvist, Anders

2014-10-01

The human malaria parasite Plasmodium falciparum goes through a complex life cycle, including a roughly 48-hour-long intraerythrocytic developmental cycle (IDC) in human red blood cells. A better understanding of the metabolic processes required during the asexual blood-stage reproduction will enhance our basic knowledge of P. falciparum and help identify critical metabolic reactions and pathways associated with blood-stage malaria. We developed a metabolic network model that mechanistically links time-dependent gene expression, metabolism, and stage-specific growth, allowing us to predict the metabolic fluxes, the biomass production rates, and the timing of production of the different biomass components during the IDC. We predicted time- and stage-specific production of precursors and macromolecules for P. falciparum (strain HB3), allowing us to link specific metabolites to specific physiological functions. For example, we hypothesized that coenzyme A might be involved in late-IDC DNA replication and cell division. Moreover, the predicted ATP metabolism indicated that energy was mainly produced from glycolysis and utilized for non-metabolic processes. Finally, we used the model to classify the entire tricarboxylic acid cycle into segments, each with a distinct function, such as superoxide detoxification, glutamate/glutamine processing, and metabolism of fumarate as a byproduct of purine biosynthesis. By capturing the normal metabolic and growth progression in P. falciparum during the IDC, our model provides a starting point for further elucidation of strain-specific metabolic activity, host-parasite interactions, stress-induced metabolic responses, and metabolic responses to antimalarial drugs and drug candidates.

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

Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction

PubMed Central

Goldfless, Stephen J.; Wagner, Jeffrey C.; Niles, Jacquin C.

2014-01-01

The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous, and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important out come given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum. PMID:25370483

A new method for culturing Plasmodium falciparum shows replication at the highest erythrocyte densities

NASA Technical Reports Server (NTRS)

Li, Tao; Glushakova, Svetlana; Zimmerberg, Joshua

2003-01-01

Plasmodium falciparum replicates poorly in erythrocyte densities greater than a hematocrit of 20%. A new method to culture the major malaria parasite was developed by using a hollow fiber bioreactor that preserves healthy erythrocytes at hematocrit up to 100%. P. falciparum replicated equally well at all densities studied. This method proved advantageous for large-scale preparation of parasitized erythrocytes (and potentially immunogens thereof), because high yields ( approximately 10(10) in 4 days) could be prepared with less cost and labor. Concomitantly, secreted proteins were concentrated by molecular sieving during culture, perhaps contributing to the parasitemic limit of 8%-12% with the 3D7 strain. The finding that P. falciparum can replicate at packed erythrocyte densities suggests that this system may be useful for study of the pathogenesis of fatal cerebral malaria, of which one feature is densely packed blood cells in brain microvasculature.

Therapeutic efficacy and safety of dihydroartemisinin-piperaquine versus artesunate-mefloquine in uncomplicated Plasmodium falciparum malaria in India

PubMed Central

2012-01-01

Background Resistance in Plasmodium falciparum to commonly used anti-malarial drugs, especially chloroquine, is being increasingly documented in India. By 2007, the first-line treatment for uncomplicated malaria has been revised to recommend artemisinin-based combination therapy (ACT) for all confirmed P. falciparum cases. Objective The objective of this study was to compare the efficacy, safety and tolerability between dihydroartemisinin-piperaquine (DP) and artesunate plus mefloquine (A + M) drug combinations in the treatment of uncomplicated P. falciparum malaria in India. Methods Between 2006 and 2007, 150 patients with acute uncomplicated P. falciparum malaria were enrolled, randomized to DP (101) or A + M (49) and followed up for 63 days as part of an open-label, non-inferiority, randomized, phase III multicenter trial in Asia. Results The heterogeneity analysis showed no statistically significant difference between India and the other countries involved in the phase III study, for both the PCR-corrected and uncorrected cure rates. As shown at the whole study level, both forms of ACT were highly efficacious in India. In fact, in the per protocol population, the 63-day cure rates were 100% for A + M and 98.8% for DP. The DP combination exerted a significant post-treatment prophylactic effect, and compared with A + M a significant reduction in the incidence of new infections for DP was observed (respectively 17.1% versus 7.5% of patients experienced new infection within follow up). Parasite and fever clearance was rapid in both treatment arms (median time to parasite clearance of one day for both groups). Both DP and A + M were well tolerated, with the majority of adverse events of mild or moderate severity. The frequencies of individual adverse events were generally similar between treatments, although the incidence of post treatment adverse events was slightly higher in patients who received A + M with respect to those treated with

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

Plasmodium falciparum Founder Populations in Western Cambodia Have Reduced Artemisinin Sensitivity In Vitro

PubMed Central

Amaratunga, Chanaki; Witkowski, Benoit; Dek, Dalin; Try, Vorleak; Khim, Nimol; Miotto, Olivo

2014-01-01

Reduced Plasmodium falciparum sensitivity to short-course artemisinin (ART) monotherapy manifests as a long parasite clearance half-life. We recently defined three parasite founder populations with long half-lives in Pursat, western Cambodia, where reduced ART sensitivity is prevalent. Using the ring-stage survival assay, we show that these founder populations have reduced ART sensitivity in vitro at the early ring stage of parasite development and that a genetically admixed population contains subsets of parasites with normal or reduced ART sensitivity. PMID:24867977

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

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.

Mitochondrial RNA polymerase is an essential enzyme in erythrocytic stages of Plasmodium falciparum.

PubMed

Ke, Hangjun; Morrisey, Joanne M; Ganesan, Suresh M; Mather, Michael W; Vaidya, Akhil B

2012-09-01

We have shown that transgenic Plasmodium falciparum parasites expressing the yeast DHODH (dihydroorotate dehydrogenase) are independent of the mtETC (mitochondrial electron transport chain), suggesting that they might not need the mitochondrial genome (mtDNA), since it only encodes three protein subunits belonging to the mtETC and fragmentary ribosomal RNA molecules. Disrupting the mitochondrial RNA polymerase (mtRNAP), which is critical for mtDNA replication and transcription, might then cause the generation of a ρ(0) parasite line lacking mtDNA. We made multiple attempts to disrupt the mtRNAP gene by double crossover recombination methods in parasite lines expressing yDHODH either episomally or integrated in the genome, but were unable to produce the desired knockout. We verified that the mtRNAP gene was accessible to recombination by successfully integrating a triple HA tag at the 3' end via single cross-over recombination. These studies suggest that mtRNAP is essential even in mtETC-independent P. falciparum parasites. Copyright © 2012 Elsevier B.V. All rights reserved.

Parasitic plants have increased rates of molecular evolution across all three genomes

PubMed Central

2013-01-01

Background Theoretical models and experimental evidence suggest that rates of molecular evolution could be raised in parasitic organisms compared to non-parasitic taxa. Parasitic plants provide an ideal test for these predictions, as there are at least a dozen independent origins of the parasitic lifestyle in angiosperms. Studies of a number of parasitic plant lineages have suggested faster rates of molecular evolution, but the results of some studies have been mixed. Comparative analysis of all parasitic plant lineages, including sequences from all three genomes, is needed to examine the generality of the relationship between rates of molecular evolution and parasitism in plants. Results We analysed DNA sequence data from the mitochondrial, nuclear and chloroplast genomes for 12 independent evolutionary origins of parasitism in angiosperms. We demonstrated that parasitic lineages have a faster rate of molecular evolution than their non-parasitic relatives in sequences for all three genomes, for both synonymous and nonsynonymous substitutions. Conclusions Our results prove that raised rates of molecular evolution are a general feature of parasitic plants, not confined to a few taxa or specific genes. We discuss possible causes for this relationship, including increased positive selection associated with host-parasite arms races, relaxed selection, reduced population size or repeated bottlenecks, increased mutation rates, and indirect causal links with generation time and body size. We find no evidence that faster rates are due to smaller effective populations sizes or changes in selection pressure. Instead, our results suggest that parasitic plants have a higher mutation rate than their close non-parasitic relatives. This may be due to a direct connection, where some aspect of the parasitic lifestyle drives the evolution of raised mutation rates. Alternatively, this pattern may be driven by an indirect connection between rates and parasitism: for example, parasitic

Parasitic plants have increased rates of molecular evolution across all three genomes.

PubMed

Bromham, Lindell; Cowman, Peter F; Lanfear, Robert

2013-06-19

Theoretical models and experimental evidence suggest that rates of molecular evolution could be raised in parasitic organisms compared to non-parasitic taxa. Parasitic plants provide an ideal test for these predictions, as there are at least a dozen independent origins of the parasitic lifestyle in angiosperms. Studies of a number of parasitic plant lineages have suggested faster rates of molecular evolution, but the results of some studies have been mixed. Comparative analysis of all parasitic plant lineages, including sequences from all three genomes, is needed to examine the generality of the relationship between rates of molecular evolution and parasitism in plants. We analysed DNA sequence data from the mitochondrial, nuclear and chloroplast genomes for 12 independent evolutionary origins of parasitism in angiosperms. We demonstrated that parasitic lineages have a faster rate of molecular evolution than their non-parasitic relatives in sequences for all three genomes, for both synonymous and nonsynonymous substitutions. Our results prove that raised rates of molecular evolution are a general feature of parasitic plants, not confined to a few taxa or specific genes. We discuss possible causes for this relationship, including increased positive selection associated with host-parasite arms races, relaxed selection, reduced population size or repeated bottlenecks, increased mutation rates, and indirect causal links with generation time and body size. We find no evidence that faster rates are due to smaller effective populations sizes or changes in selection pressure. Instead, our results suggest that parasitic plants have a higher mutation rate than their close non-parasitic relatives. This may be due to a direct connection, where some aspect of the parasitic lifestyle drives the evolution of raised mutation rates. Alternatively, this pattern may be driven by an indirect connection between rates and parasitism: for example, parasitic plants tend to be smaller than

Immunogenicity and in vitro Protective Efficacy of a Recombinant Multistage Plasmodium falciparum Candidate Vaccine

NASA Astrophysics Data System (ADS)

Shi, Ya Ping; Hasnain, Seyed E.; Sacci, John B.; Holloway, Brian P.; Fujioka, Hisashi; Kumar, Nirbhay; Wohlhueter, Robert; Hoffman, Stephen L.; Collins, William E.; Lal, Altaf A.

1999-02-01

Compared with a single-stage antigen-based vaccine, a multistage and multivalent Plasmodium falciparum vaccine would be more efficacious by inducing "multiple layers" of immunity. We have constructed a synthetic gene that encodes for 12 B cell, 6 T cell proliferative, and 3 cytotoxic T lymphocyte epitopes derived from 9 stage-specific P. falciparum antigens corresponding to the sporozoite, liver, erythrocytic asexual, and sexual stages. The gene was expressed in the baculovirus system, and a 41-kDa antigen, termed CDC/NIIMALVAC-1, was purified. Immunization in rabbits with the purified protein in the presence of different adjuvants generated antibody responses that recognized vaccine antigen, linear peptides contained in the vaccine, and all stages of P. falciparum. In vitro assays of protection revealed that the vaccine-elicited antibodies strongly inhibited sporozoite invasion of hepatoma cells and growth of blood-stage parasites in the presence of monocytes. These observations demonstrate that a multicomponent, multistage malaria vaccine can induce immune responses that inhibit parasite development at multiple stages. The rationale and approach used in the development of a multicomponent P. falciparum vaccine will be useful in the development of a multispecies human malaria vaccine and vaccines against other infectious diseases.

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.

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.

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

Autophagy-Related Protein ATG18 Regulates Apicoplast Biogenesis in Apicomplexan Parasites

PubMed Central

Bansal, Priyanka; Tripathi, Anuj; Thakur, Vandana; Mohmmed, Asif

2017-01-01

ABSTRACT Mechanisms by which 3′-phosphorylated phosphoinositides (3′-PIPs) regulate the development of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are poorly understood. The catabolic process of autophagy, which is dependent on autophagy-related proteins (ATGs), is one of the major targets of 3′-PIPs in yeast and mammals. In the present study, we identified autophagy-related protein ATG18 as an effector of 3′-PIPs in these parasites. P. falciparum ATG18 (PfATG18) and T. gondii ATG18 (TgATG18) interact with 3′-PIPs but exhibited differences in their specificity of interaction with the ligand PIP. The conditional knockdown of T. gondii or P. falciparum ATG18 (Tg/PfATG18) impaired replication of parasites and resulted in their delayed death. Intriguingly, ATG18 depletion resulted in the loss of the apicomplexan parasite-specific nonphotosynthetic plastid-like organelle apicoplast, which harbors the machinery for biosynthesis of key metabolites, and the interaction of ATG18 to phosphatidylinositol 3-phosphate (PI3P) was critical for apicoplast inheritance. Furthermore, ATG18 regulates membrane association and apicoplast localization of ATG8. These findings provide insights into a novel noncanonical role of ATG18 in apicoplast inheritance. This function of ATG18 in organelle biogenesis is unprecedented in any organism and may be conserved across most apicomplexan parasites. PMID:29089429

Investigation of volatile organic biomarkers derived from Plasmodium falciparum in vitro

PubMed Central

2012-01-01

Background There remains a need for techniques that improve the sensitive detection of viable Plasmodium falciparum as part of diagnosis and therapeutic monitoring in clinical studies and usual-care management of malaria infections. A non-invasive breath test based on P. falciparum-associated specific volatile organic compounds (VOCs) could fill this gap and provide insights into parasite metabolism and pathogenicity. The aim of this study was to determine whether VOCs are present in the headspace above in vitro P. falciparum cultures. Methods A novel, custom-designed apparatus was developed to enable efficient headspace sampling of infected and non-infected cultures. Conditions were optimized to support cultures of high parasitaemia (>20%) to improve the potential detection of parasite-specific VOCs. A number of techniques for VOC analysis were investigated including solid phase micro-extraction using two different polarity fibres, and purge and trap/thermal desorption, each coupled to gas chromatography–mass spectrometry. Each experiment and analysis method was performed at least on two occasions. VOCs were identified by comparing their mass spectra against commercial mass spectral libraries. Results No unique malarial-specific VOCs could be detected relative to those in the control red blood cell cultures. This could reflect sequestration of VOCs into cell membranes and/or culture media but solvent extractions of supernatants and cell lysates using hexane, dichloromethane and ethyl acetate also showed no obvious difference compared to control non-parasitized cultures. Conclusions Future in vivo studies analysing the breath of patients with severe malaria who are harbouring a parasite biomass that is significantly greater than achievable in vitro may yet reveal specific clinically-useful volatile chemical biomarkers. PMID:22958460

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.

South-East Asian strains of Plasmodium falciparum display higher ratio of non-synonymous to synonymous polymorphisms compared to African strains

PubMed Central

Singh, Gajinder Pal; Sharma, Amit

2016-01-01

Resistance to frontline anti-malarial drugs, including artemisinin, has repeatedly arisen in South-East Asia, but the reasons for this are not understood. Here we test whether evolutionary constraints on Plasmodium falciparum strains from South-East Asia differ from African strains. We find a significantly higher ratio of non-synonymous to synonymous polymorphisms in P. falciparum from South-East Asia compared to Africa, suggesting differences in the selective constraints on P. falciparum genome in these geographical regions. Furthermore, South-East Asian strains showed a higher proportion of non-synonymous polymorphism at conserved positions, suggesting reduced negative selection. There was a lower rate of mixed infection by multiple genotypes in samples from South-East Asia compared to Africa. We propose that a lower mixed infection rate in South-East Asia reduces intra-host competition between the parasite clones, reducing the efficiency of natural selection. This might increase the probability of fixation of fitness-reducing mutations including drug resistant ones. PMID:27853513

Small-molecule histone methyltransferase inhibitors display rapid antimalarial activity against all blood stage forms in Plasmodium falciparum

PubMed Central

Malmquist, Nicholas A.; Moss, Thomas A.; Mecheri, Salah; Scherf, Artur; Fuchter, Matthew J.

2012-01-01

Epigenetic factors such as histone methylation control the developmental progression of malaria parasites during the complex life cycle in the human host. We investigated Plasmodium falciparum histone lysine methyltransferases as a potential target class for the development of novel antimalarials. We synthesized a compound library based upon a known specific inhibitor (BIX-01294) of the human G9a histone methyltransferase. Two compounds, BIX-01294 and its derivative TM2-115, inhibited P. falciparum 3D7 parasites in culture with IC50 values of ∼100 nM, values at least 22-fold more potent than their apparent IC50 toward two human cell lines and one mouse cell line. These compounds irreversibly arrested parasite growth at all stages of the intraerythrocytic life cycle. Decrease in parasite viability (>40%) was seen after a 3-h incubation with 1 µM BIX-01294 and resulted in complete parasite killing after a 12-h incubation. Additionally, mice with patent Plasmodium berghei ANKA strain infection treated with a single dose (40 mg/kg) of TM2-115 had 18-fold reduced parasitemia the following day. Importantly, treatment of P. falciparum parasites in culture with BIX-01294 or TM2-115 resulted in significant reductions in histone H3K4me3 levels in a concentration-dependent and exposure time-dependent manner. Together, these results suggest that BIX-01294 and TM2-115 inhibit malaria parasite histone methyltransferases, resulting in rapid and irreversible parasite death. Our data position histone lysine methyltransferases as a previously unrecognized target class, and BIX-01294 as a promising lead compound, in a presently unexploited avenue for antimalarial drug discovery targeting multiple life-cycle stages. PMID:23011794

Major Threat to Malaria Control Programs by Plasmodium falciparum Lacking Histidine-Rich Protein 2, Eritrea

PubMed Central

Berhane, Araia; Anderson, Karen; Mihreteab, Selam; Gresty, Karryn; Rogier, Eric; Mohamed, Salih; Hagos, Filmon; Embaye, Ghirmay; Chinorumba, Anderson; Zehaie, Assefash; Dowd, Simone; Waters, Norman C.; Gatton, Michelle L.; Udhayakumar, Venkatachalam; Cunningham, Jane

2018-01-01

False-negative results for Plasmodium falciparum histidine-rich protein (HRP) 2–based rapid diagnostic tests (RDTs) are increasing in Eritrea. We investigated HRP gene 2/3 (pfhrp2/pfhrp3) status in 50 infected patients at 2 hospitals. We showed that 80.8% (21/26) of patients at Ghindae Hospital and 41.7% (10/24) at Massawa Hospital were infected with pfhrp2-negative parasites and 92.3% (24/26) of patients at Ghindae Hospital and 70.8% (17/24) at Massawa Hospital were infected with pfhrp3-negative parasites. Parasite densities between pfhrp2-positive and pfhrp2-negative patients were comparable. All pfhrp2-negative samples had no detectable HRP2/3 antigen and showed negative results for HRP2-based RDTs. pfhrp2-negative parasites were genetically less diverse and formed 2 clusters with no close relationships to parasites from Peru. These parasites probably emerged independently by selection in Eritrea. High prevalence of pfhrp2-negative parasites caused a high rate of false-negative results for RDTs. Determining prevalence of pfhrp2-negative parasites is urgently needed in neighboring countries to assist case management policies. PMID:29460730

Major Threat to Malaria Control Programs by Plasmodium falciparum Lacking Histidine-Rich Protein 2, Eritrea.

PubMed

Berhane, Araia; Anderson, Karen; Mihreteab, Selam; Gresty, Karryn; Rogier, Eric; Mohamed, Salih; Hagos, Filmon; Embaye, Ghirmay; Chinorumba, Anderson; Zehaie, Assefash; Dowd, Simone; Waters, Norman C; Gatton, Michelle L; Udhayakumar, Venkatachalam; Cheng, Qin; Cunningham, Jane

2018-03-01

False-negative results for Plasmodium falciparum histidine-rich protein (HRP) 2-based rapid diagnostic tests (RDTs) are increasing in Eritrea. We investigated HRP gene 2/3 (pfhrp2/pfhrp3) status in 50 infected patients at 2 hospitals. We showed that 80.8% (21/26) of patients at Ghindae Hospital and 41.7% (10/24) at Massawa Hospital were infected with pfhrp2-negative parasites and 92.3% (24/26) of patients at Ghindae Hospital and 70.8% (17/24) at Massawa Hospital were infected with pfhrp3-negative parasites. Parasite densities between pfhrp2-positive and pfhrp2-negative patients were comparable. All pfhrp2-negative samples had no detectable HRP2/3 antigen and showed negative results for HRP2-based RDTs. pfhrp2-negative parasites were genetically less diverse and formed 2 clusters with no close relationships to parasites from Peru. These parasites probably emerged independently by selection in Eritrea. High prevalence of pfhrp2-negative parasites caused a high rate of false-negative results for RDTs. Determining prevalence of pfhrp2-negative parasites is urgently needed in neighboring countries to assist case management policies.

Correlation between Cyclin Dependent Kinases and Artemisinin-Induced Dormancy in Plasmodium falciparum In Vitro

PubMed Central

Gray, Karen-Ann; Gresty, Karryn J.; Chen, Nanhua; Zhang, Veronica; Gutteridge, Clare E.; Peatey, Christopher L.; Chavchich, Marina; Waters, Norman C.; Cheng, Qin

2016-01-01

Background Artemisinin-induced dormancy provides a plausible explanation for recrudescence following artemisinin monotherapy. This phenomenon shares similarities with cell cycle arrest where cyclin dependent kinases (CDKs) and cyclins play an important role. Methods Transcription profiles of Plasmodium falciparum CDKs and cyclins before and after dihydroartemisinin (DHA) treatment in three parasite lines, and the effect of CDK inhibitors on parasite recovery from DHA-induced dormancy were investigated. Results After DHA treatment, parasites enter a dormancy phase followed by a recovery phase. During the dormancy phase parasites up-regulate pfcrk1, pfcrk4, pfcyc2 and pfcyc4, and down-regulate pfmrk, pfpk5, pfpk6, pfcrk3, pfcyc1 and pfcyc3. When entering the recovery phase parasites immediately up-regulate all CDK and cyclin genes. Three CDK inhibitors, olomoucine, WR636638 and roscovitine, produced distinct effects on different phases of DHA-induced dormancy, blocking parasites recovery. Conclusions The up-regulation of PfCRK1 and PfCRK4, and down regulation of other CDKs and cyclins correlate with parasite survival in the dormant state. Changes in CDK expression are likely to negatively regulate parasite progression from G1 to S phase. These findings provide new insights into the mechanism of artemisinin-induced dormancy and cell cycle regulation of P. falciparum, opening new opportunities for preventing recrudescence following artemisinin treatment. PMID:27326764

Antibodies against multiple merozoite surface antigens of the human malaria parasite Plasmodium falciparum inhibit parasite maturation and red blood cell invasion.

PubMed

Woehlbier, Ute; Epp, Christian; Hackett, Fiona; Blackman, Michael J; Bujard, Hermann

2010-03-18

Plasmodium falciparum merozoites expose at their surface a large protein complex, which is composed of fragments of merozoite surface protein 1 (MSP-1; called MSP-183, MSP-130, MSP-138, and MSP-142) plus associated processing products of MSP-6 and MSP-7. During erythrocyte invasion this complex, as well as an integral membrane protein called apical membrane antigen-1 (AMA-1), is shed from the parasite surface following specific proteolysis. Components of the MSP-1/6/7 complex and AMA-1 are presently under development as malaria vaccines. The specificities and effects of antibodies directed against MSP-1, MSP-6, MSP-7 on the growth of blood stage parasites were studied using ELISA and the pLDH-assay. To understand the mode of action of these antibodies, their effects on processing of MSP-1 and AMA-1 on the surface of merozoites were investigated. Antibodies targeting epitopes located throughout the MSP-1/6/7 complex interfere with shedding of MSP-1, and as a consequence prevent erythrocyte invasion. Antibodies targeting the MSP-1/6/7 complex have no effect on the processing and shedding of AMA-1 and, similarly, antibodies blocking the shedding of AMA-1 do not affect cleavage of MSP-1, suggesting completely independent functions of these proteins during invasion. Furthermore, some epitopes, although eliciting highly inhibitory antibodies, are only poorly recognized by the immune system when presented in the structural context of the intact antigen. The findings reported provide further support for the development of vaccines based on MSP-1/6/7 and AMA-1, which would possibly include a combination of these antigens.

Profiling the anti-protozoal activity of anti-cancer HDAC inhibitors against Plasmodium and Trypanosoma parasites.

PubMed

Engel, Jessica A; Jones, Amy J; Avery, Vicky M; Sumanadasa, Subathdrage D M; Ng, Susanna S; Fairlie, David P; Skinner-Adams, Tina; Andrews, Katherine T

2015-12-01

Histone deacetylase (HDAC) enzymes work together with histone acetyltransferases (HATs) to reversibly acetylate both histone and non-histone proteins. As a result, these enzymes are involved in regulating chromatin structure and gene expression as well as other important cellular processes. HDACs are validated drug targets for some types of cancer, with four HDAC inhibitors clinically approved. However, they are also showing promise as novel drug targets for other indications, including malaria and other parasitic diseases. In this study the in vitro activity of four anti-cancer HDAC inhibitors was examined against parasites that cause malaria and trypanosomiasis. Three of these inhibitors, suberoylanilide hydroxamic acid (SAHA; vorinostat(®)), romidepsin (Istodax(®)) and belinostat (Beleodaq(®)), are clinically approved for the treatment of T-cell lymphoma, while the fourth, panobinostat, has recently been approved for combination therapy use in certain patients with multiple myeloma. All HDAC inhibitors were found to inhibit the growth of asexual-stage Plasmodium falciparum malaria parasites in the nanomolar range (IC50 10-200 nM), while only romidepsin was active at sub-μM concentrations against bloodstream form Trypanosoma brucei brucei parasites (IC50 35 nM). The compounds were found to have some selectivity for malaria parasites compared with mammalian cells, but were not selective for trypanosome parasites versus mammalian cells. All compounds caused hyperacetylation of histone and non-histone proteins in P. falciparum asexual stage parasites and inhibited deacetylase activity in P. falciparum nuclear extracts in addition to recombinant PfHDAC1 activity. P. falciparum histone hyperacetylation data indicate that HDAC inhibitors may differentially affect the acetylation profiles of histone H3 and H4.

Hemoglobin consumption by P. falciparum in individual erythrocytes imaged via quantitative phase spectroscopy

NASA Astrophysics Data System (ADS)

Rinehart, Matthew T.; Park, Han Sang; Walzer, Katelyn A.; Chi, Jen-Tsan Ashley; Wax, Adam

2016-04-01

Plasmodium falciparum infection causes structural and biochemical changes in red blood cells (RBCs). To quantify these changes, we apply a novel optical technique, quantitative phase spectroscopy (QPS) to characterize individual red blood cells (RBCs) during the intraerythrocytic life cycle of P. falciparum. QPS captures hyperspectral holograms of individual RBCs to measure spectroscopic changes across the visible wavelength range (475-700 nm), providing complex information, i.e. amplitude and phase, about the light field which has interacted with the cell. The complex field provides complimentary information on hemoglobin content and cell mass, which are both found to dramatically change upon infection by P. falciparum. Hb content progressively decreases with parasite life cycle, with an average 72.2% reduction observed for RBCs infected by schizont-stage P. falciparum compared to uninfected cells. Infection also resulted in a 33.1% reduction in RBC’s optical volume, a measure of the cells’ non-aqueous components. Notably, optical volume is only partially correlated with hemoglobin content, suggesting that changes in other dry mass components such as parasite mass may also be assessed using this technique. The unique ability of QPS to discriminate individual healthy and infected cells using spectroscopic changes indicates that the approach can be used to detect disease.

Long term persistence of clonal malaria parasite Plasmodium falciparum lineages in the Colombian Pacific region.

PubMed

Echeverry, Diego F; Nair, Shalini; Osorio, Lyda; Menon, Sanjay; Murillo, Claribel; Anderson, Tim J C

2013-01-07

Resistance to chloroquine and antifolate drugs has evolved independently in South America, suggesting that genotype - phenotype studies aimed at understanding the genetic basis of resistance to these and other drugs should be conducted in this continent. This research was conducted to better understand the population structure of Colombian Plasmodium falciparum in preparation for such studies. A set of 384 SNPs were genotyped in blood spot DNA samples from 447 P. falciparum infected subjects collected over a ten year period from four provinces of the Colombian Pacific coast to evaluate clonality, population structure and linkage disequilibrium (LD). Most infections (81%) contained a single predominant clone. These clustered into 136 multilocus genotypes (MLGs), with 32% of MLGs recovered from multiple (2 - 28) independent subjects. We observed extremely low genotypic richness (R = 0.42) and long persistence of MLGs through time (median = 537 days, range = 1 - 2,997 days). There was a high probability (>5%) of sampling parasites from the same MLG in different subjects within 28 days, suggesting caution is needed when using genotyping methods to assess treatment success in clinical drug trials. Panmixia was rejected as four well differentiated subpopulations (FST = 0.084 - 0.279) were identified. These occurred sympatrically but varied in frequency within the four provinces. Linkage disequilibrium (LD) decayed more rapidly (r2 = 0.17 for markers <10 kb apart) than observed previously in South American samples. We conclude that Colombian populations have several advantages for association studies, because multiple clone infections are uncommon and LD decays over the scale of one or a few genes. However, the extensive population structure and low genotype richness will need to be accounted for when designing and analyzing association studies.

Long term persistence of clonal malaria parasite Plasmodium falciparum lineages in the Colombian Pacific region

PubMed Central

2013-01-01

Background Resistance to chloroquine and antifolate drugs has evolved independently in South America, suggesting that genotype - phenotype studies aimed at understanding the genetic basis of resistance to these and other drugs should be conducted in this continent. This research was conducted to better understand the population structure of Colombian Plasmodium falciparum in preparation for such studies. Results A set of 384 SNPs were genotyped in blood spot DNA samples from 447 P. falciparum infected subjects collected over a ten year period from four provinces of the Colombian Pacific coast to evaluate clonality, population structure and linkage disequilibrium (LD). Most infections (81%) contained a single predominant clone. These clustered into 136 multilocus genotypes (MLGs), with 32% of MLGs recovered from multiple (2 – 28) independent subjects. We observed extremely low genotypic richness (R = 0.42) and long persistence of MLGs through time (median = 537 days, range = 1 – 2,997 days). There was a high probability (>5%) of sampling parasites from the same MLG in different subjects within 28 days, suggesting caution is needed when using genotyping methods to assess treatment success in clinical drug trials. Panmixia was rejected as four well differentiated subpopulations (FST = 0.084 - 0.279) were identified. These occurred sympatrically but varied in frequency within the four provinces. Linkage disequilibrium (LD) decayed more rapidly (r2 = 0.17 for markers <10 kb apart) than observed previously in South American samples. Conclusions We conclude that Colombian populations have several advantages for association studies, because multiple clone infections are uncommon and LD decays over the scale of one or a few genes. However, the extensive population structure and low genotype richness will need to be accounted for when designing and analyzing association studies. PMID:23294725

In vitro selection of Plasmodium falciparum Pfcrt and Pfmdr1 variants by artemisinin.

PubMed

Njokah, Muturi J; Kang'ethe, Joseph N; Kinyua, Johnson; Kariuki, Daniel; Kimani, Francis T

2016-07-22

Anti-malarial drugs are the major focus in the prevention and treatment of malaria. Artemisinin-based combination therapy (ACT) is the WHO recommended first-line treatment for Plasmodium falciparum malaria across the endemic world. Also ACT is increasingly relied upon in treating Plasmodium vivax malaria where chloroquine is failing. The emergence of artemisinin drug-resistant parasites is a serious threat faced by global malaria control programmes. Therefore, the success of treatment and intervention strategies is highly pegged on understanding the genetic basis of resistance. Here, resistance in P. falciparum was generated in vitro for artemisinin to produce levels above clinically relevant concentrations in vivo, and the molecular haplotypes investigated. Genomic DNA was extracted using the QIAamp mini DNA kit. DNA sequences of Pfk13, Pfcrt and Pfmdr1 genes were amplified by PCR and the amplicons were successfully sequenced. Single nucleotide polymorphisms were traced by standard bidirectional sequencing and reading the transcripts against wild-type sequences in Codon code Aligner Version 5.1 and NCBI blast. Exposure of parasite strains D6 and W2 to artemisinin resulted in a decrease in parasite susceptibility to artemisinin (W2 and D6) and lumefantrine (D6 only). The parasites exhibited elevated IC50s to multiple artemisinins, with >twofold resistance to artemisinin; however, the resistance index obtained with standard methods was noticeably less than expected for parasite lines recovered from 50 µg/ml 48 h drug pressure. The change in parasite susceptibility was associated with Pfmdr-185K mutation, a mutation never reported before. The Pfcrt-CVMNK genotype (Pfcrt codons 72-76) was retained and notably, the study did not detect any polymorphisms reported to reduce P. falciparum susceptibility in vivo in the coding sequences of the Pfk13 gene. This data demonstrate that P. falciparum has the capacity to develop resistance to artemisinin derivatives in vitro

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.

Comparison between microscopic examination, ELISA and quantitative buffy coat analysis in the diagnosis of falciparum malaria in an endemic population.

PubMed

Tanpradist, S; Tharavanij, S; Yamokgul, P; Bualombai, P; Wongchotigul, V; Singhasivanon, P; Patarapotikul, J; Thammapalerd, N; Prasittisuk, C; Tantanasrikul, S

1995-03-01

Monoclonal antibody-based ELISA and QBC (quantitative buffy coat analysis) were tested in two endemic areas with low and high incidence of malaria in Kanchanaburi Province, West Thailand with annual parasite incidence in 1992 of 119 and 5 per 1,000 population, respectively. The numbers of individuals positive by thick blood film examination (TBF) for P. falciparum with or without P. vivax, and P. vivax only were 82 and 69, respectively. The detection limit of ELISA was 10 parasites/10(6) red blood cells (RBC) (0.001% parasitemia). Of 1,095 individuals involved in the study at the beginning of the study, ELISA showed sensitivity, specificity, positive predictive value and negative predictive value of 78.1%, 94.9%, 72% and 98.1%, respectively. Nine of 18 (50%) TBF-positive but ELISA-positive individuals had parasitemia of less than 10 parasites/10(6) RBC. High and low incidence areas did not affect the validity of our result. Regression analysis showed good correlation between log parasitemia and ELISA percent OD increase (Y = 0 + 64.9*logX, r = 0.65), and agreement between TBF and ELISA results was 95.9%. In a fortnightly follow-up, in 82 TBF-positive individuals, both ELISA and TBF positive rates correlatively declined with agreement of 96.3%. With samples taken on the first day of the study, the TBF and QBC results were also correlated with agreement of 95.8% for P. falciparum, 95.6% for P. vivax. During 8 week follow-up involving altogether 191 samples, agreement between TBF and QBC results were 87.4% for P. falciparum. QBC detected more cases with P. falciparum infections but detected smaller number of cases with P. vivax infections.

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.

Plasmodium falciparum sulfadoxine resistance is geographically and genetically clustered within the DR Congo

PubMed Central

Taylor, Steve M.; Antonia, Alejandro L.; Parobek, Christian M.; Juliano, Jonathan J.; Janko, Mark; Emch, Michael; Alam, Md Tauqeer; Udhayakumar, Venkatachalam; Tshefu, Antoinette K.; Meshnick, Steven R.

2013-01-01

Understanding the spatial clustering of Plasmodium falciparum populations can assist efforts to contain drug-resistant parasites and maintain the efficacy of future drugs. We sequenced single nucleotide polymorphisms (SNPs) in the dihydropteroate synthase gene (dhps) associated with sulfadoxine resistance and 5 microsatellite loci flanking dhps in order to investigate the genetic backgrounds, genetic relatedness, and geographic clustering of falciparum parasites in the Democratic Republic of the Congo (DRC). Resistant haplotypes were clustered into subpopulations: one in the northeast DRC, and the other in the balance of the DRC. Network and clonal lineage analyses of the flanking microsatellites indicate that geographically-distinct mutant dhps haplotypes derive from separate lineages. The DRC is therefore a watershed for haplotypes associated with sulfadoxine resistance. Given the importance of central Africa as a corridor for the spread of antimalarial resistance, the identification of the mechanisms of this transit can inform future policies to contain drug-resistant parasite strains. PMID:23372922

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

Assessment of the Plasmodium falciparum Preerythrocytic Antigen UIS3 as a Potential Candidate for a Malaria Vaccine.

PubMed

Longley, Rhea J; Halbroth, Benedict R; Salman, Ahmed M; Ewer, Katie J; Hodgson, Susanne H; Janse, Chris J; Khan, Shahid M; Hill, Adrian V S; Spencer, Alexandra J

2017-03-01

Efforts are under way to improve the efficacy of subunit malaria vaccines through assessments of new adjuvants, vaccination platforms, and antigens. In this study, we further assessed the Plasmodium falciparum antigen upregulated in infective sporozoites 3 (PfUIS3) as a vaccine candidate. PfUIS3 was expressed in the viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) and used to immunize mice in a prime-boost regimen. We previously demonstrated that this regimen could provide partial protection against challenge with chimeric P. berghei parasites expressing PfUIS3. We now show that ChAd63-MVA PfUIS3 can also provide partial cross-species protection against challenge with wild-type P. berghei parasites. We also show that PfUIS3-specific cellular memory responses could be recalled in human volunteers exposed to P. falciparum parasites in a controlled human malaria infection study. When ChAd63-MVA PfUIS3 was coadministered with the vaccine candidate P. falciparum thrombospondin-related adhesion protein (PfTRAP) expressed in the ChAd63-MVA system, there was no significant change in immunogenicity to either vaccine. However, when mice were challenged with double chimeric P. berghei - P. falciparum parasites expressing both PfUIS3 and PfTRAP, vaccine efficacy was improved to 100% sterile protection. This synergistic effect was evident only when the two vaccines were mixed and administered at the same site. We have therefore demonstrated that vaccination with PfUIS3 can induce a consistent delay in patent parasitemia across mouse strains and against chimeric parasites expressing PfUIS3 as well as wild-type P. berghei ; when this vaccine is combined with another partially protective regimen (ChAd63-MVA PfTRAP), complete protection is induced. Copyright © 2017 Longley et al.

Plasmodium falciparum Calcium-Dependent Protein Kinase 2 Is Critical for Male Gametocyte Exflagellation but Not Essential for Asexual Proliferation.

PubMed

Bansal, Abhisheka; Molina-Cruz, Alvaro; Brzostowski, Joseph; Mu, Jianbing; Miller, Louis H

2017-10-17

Drug development efforts have focused mostly on the asexual blood stages of the malaria parasite Plasmodium falciparum Except for primaquine, which has its own limitations, there are no available drugs that target the transmission of the parasite to mosquitoes. Therefore, there is a need to validate new parasite proteins that can be targeted for blocking transmission. P. falciparum calcium-dependent protein kinases ( Pf CDPKs) play critical roles at various stages of the parasite life cycle and, importantly, are absent in the human host. These features mark them as attractive drug targets. In this study, using CRISPR/Cas9 we successfully knocked out Pf CDPK2 from blood-stage parasites, which was previously thought to be an indispensable protein. The growth rate of the Pf CDPK2 knockout (KO) parasites was similar to that of wild-type parasites, confirming that Pf CDPK2 function is not essential for the asexual proliferation of the parasite in vitro The mature male and female gametocytes of Pf CDPK2 KO parasites become round after induction. However, they fail to infect female Anopheles stephensi mosquitoes due to a defect(s) in male gametocyte exflagellation and possibly in female gametes. IMPORTANCE Despite reductions in the number of deaths it causes, malaria continues to be a leading infectious disease of the developing world. For effective control and elimination of malaria, multiple stages of the parasite need to be targeted. One such stage includes the transmission of the parasite to mosquitoes. Here, we demonstrate the successful knockout of Pf CDPK2, which was previously thought to be indispensable for parasite growth in red blood cells. The Pf CDPK2 KO parasites are incapable of establishing an infection in mosquitoes. Therefore, our study suggests that targeting Pf CDPK2 may be a good strategy to control malaria transmission in countries with high transmission. Moreover, molecular understanding of the signaling pathway of Pf CDPK2 may provide additional

An investigation of the protective effect of alpha+-thalassaemia against severe Plasmodium falciparum amongst children in Kumasi, Ghana.

PubMed

Opoku-Okrah, C; Gordge, M; Kweku Nakua, E; Abgenyega, T; Parry, M; Robertson, C; Smith, C L

2014-02-01

Several factors influence the severity of Plasmodium falciparum; here, we investigate the impact of alpha+-thalassaemia genotype on P. falciparum parasitemia and prevalence of severe anaemia amongst microcytic children from Kumasi, Ghana. Seven hundred and thirty-two children (≤10 years) with P. falciparum were categorised into normocytic and microcytic (mean cell volume ≤76 fL). Microcytic individuals were genotyped for the -α(3.7) deletional thalassaemia mutation and parasite densities determined. Amongst microcytic patients both parasite densities and prevalence of severe malaria parasitemia (≥100 000/μL) were significantly lower (P < 0.001) in the presence of an alpha+-thalassaemia genotype compared with non-alpha+-thalassaemia genotype. There was no evidence that alpha+-thalassaemia protected against severe anaemia. The protection conferred by alpha-thalassaemia genotype against severe P. falciparum parasitemia did not change with increasing age. The severity of P. falciparum parasitemia was significantly lower in both the homozygous and heterozygous alpha+-thalassaemia groups compared with microcytic individuals with non-alpha+-thalassaemia genotype. The protective effect, from severe malaria, of the alpha+-thalassaemia allele does not alter with age. © 2013 John Wiley & Sons Ltd.

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.

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.

Polymorphisms in Plasmodium falciparum Chloroquine Resistance Transporter and Multidrug Resistance 1 Genes: Parasite Risk Factors that Affect Treatment Outcomes for P. falciparum Malaria after Artemether-Lumefantrine and Artesunate-Amodiaquine

PubMed Central

Venkatesan, Meera; Gadalla, Nahla B.; Stepniewska, Kasia; Dahal, Prabin; Nsanzabana, Christian; Moriera, Clarissa; Price, Ric N.; Mårtensson, Andreas; Rosenthal, Philip J.; Dorsey, Grant; Sutherland, Colin J.; Guérin, Philippe; Davis, Timothy M. E.; Ménard, Didier; Adam, Ishag; Ademowo, George; Arze, Cesar; Baliraine, Frederick N.; Berens-Riha, Nicole; Björkman, Anders; Borrmann, Steffen; Checchi, Francesco; Desai, Meghna; Dhorda, Mehul; Djimdé, Abdoulaye A.; El-Sayed, Badria B.; Eshetu, Teferi; Eyase, Frederick; Falade, Catherine; Faucher, Jean-François; Fröberg, Gabrielle; Grivoyannis, Anastasia; Hamour, Sally; Houzé, Sandrine; Johnson, Jacob; Kamugisha, Erasmus; Kariuki, Simon; Kiechel, Jean-René; Kironde, Fred; Kofoed, Poul-Erik; LeBras, Jacques; Malmberg, Maja; Mwai, Leah; Ngasala, Billy; Nosten, Francois; Nsobya, Samuel L.; Nzila, Alexis; Oguike, Mary; Otienoburu, Sabina Dahlström; Ogutu, Bernhards; Ouédraogo, Jean-Bosco; Piola, Patrice; Rombo, Lars; Schramm, Birgit; Somé, A. Fabrice; Thwing, Julie; Ursing, Johan; Wong, Rina P. M.; Zeynudin, Ahmed; Zongo, Issaka; Plowe, Christopher V.; Sibley, Carol Hopkins

2014-01-01

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 – 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36–17.97, P < 0.001) were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine. PMID:25048375

Wherever I may roam: protein and membrane trafficking in P. falciparum-infected red blood cells.

PubMed

Deponte, Marcel; Hoppe, Heinrich C; Lee, Marcus C S; Maier, Alexander G; Richard, Dave; Rug, Melanie; Spielmann, Tobias; Przyborski, Jude M

2012-12-01

Quite aside from its immense importance as a human pathogen, studies in recent years have brought to light the fact that the malaria parasite Plasmodium falciparum is an interesting eukaryotic model system to study protein trafficking. Studying parasite cell biology often reveals an overrepresentation of atypical cell biological features, possibly driven by the parasites' need to survive in an unusual biological niche. Malaria parasites possess uncommon cellular compartments to which protein traffic must be directed, including secretory organelles such as rhoptries and micronemes, a lysosome-like compartment referred to as the digestive vacuole and a complex (four membrane-bound) plastid, the apicoplast. In addition, the parasite must provide proteins to extracellular compartments and structures including the parasitophorous vacuole, the parasitophorous vacuolar membrane, the Maurer's clefts and both cytosol and plasma membrane of the host cell, the mature human red blood cell. Although some of these unusual destinations are possessed by other cell types, only Plasmodium parasites contain them all within one cell. Here we review what is known about protein and membrane transport in the P. falciparum-infected cell, highlighting novel features of these processes. A growing body of evidence suggests that this parasite is a real "box of tricks" with regards to protein traffic. Possibly, these tricks may be turned against the parasite by exploiting them as novel therapeutic targets. Copyright © 2012 Elsevier B.V. All rights reserved.

[Efficiency and specificity of the KAT-test for rapid diagnosis of falciparum malaria].

PubMed

Cong, Le Dinh; Sergiev, V P; Rabinovich, S A; Nhah, Doan Hanh; Huong, Nguyen Van; Morozov, E N; Kukina, I V; Thinh, Ta Thi; Maksakovskaia, E V; Dao, Le Minh; Chalyĭ, V F; To, Dang Thi; Fandeev, V A; Hoa, Ngo Viet; Due, Nguyen Thi

2002-01-01

A new rapid KAT Quick Malaria test for the diagnosis of falciparum malaria, which is based on the detection of a monoclonal antibody-antigen complex of malaria parasites, has been worked out by the KAT Medical CC in South Africa. The efficiency and specificity of the KAT test were compared with those of the microscopic method and with the ICT test for rapid diagnosis of P. falciparum and P. vivax. The polymerase chain reaction was used as a control test. Testing for malaria was performed on 98 blood samples from feverish patients in Vietnam and Tadjikistan and among the persons who had returned to Moscow from endemic regions. The efficiency of the KAT test for falciparum-malaria was found to be 100% versus 90.5% with ICT. The absence of cross-reactions with P. vivax and the presence of pseudopositive results of the KAT test for fever cases of non-malaria origin indicate its high specificity. There was no correlation between the rate of test line colouring and the level of parasitemia. The KAT test yielded positive results only when gametocytes were found in blood specimens.

Effective treatment with a tetrandrine/chloroquine combination for chloroquine-resistant falciparum malaria in Aotus monkeys

PubMed Central

2013-01-01

Background In vitro evidence indicates that tetrandrine (TT) can potentiate the action of chloroquine 40-fold against choloquine-resistant Plasmodium falciparum. The key question emanating from that study is “would tetrandine and chloroquine be highly effective in a live Aotus monkey model with chloroquine-resistant parasites”. This study was designed to closely mimic the pharmacological/anti-malarial activity in man. Methods The Vietnam Smith/RE strain of P. falciparum, which is chloroquine-resistant was used in this study. Previous experimental procedures were followed. Panamanian owl monkeys (Aotus) were inoculated with 5×106 erythrocytes parasitized with the CQ-resistant strain of P. falciparum. Oral drug treatment was with CQ (20 mg/kg) and/or tetrandrine at 15 mg/Kg, 30 mg/Kg or 60 mg/Kg or 25 mg/Kg depending on experimental conditions. Results and Discussion Parasitaemia was cleared rapidly with CQ and TT while CQ treatment alone was ineffective. Recrudescence of malaria occurred after seven days post-infection. However, four animals were treated orally with TT and CQ parasites were cleared. It is likely that monkeys were cured via a combination of both drug and host immune responses. A single Aotus monkey infected with P. falciparum and untreated with drugs, died. No side effects were observed with these drug treatments. Conclusions This combination of chloroquine and tetrandrine forms the basis of a new attack on chloroquine-resistant malaria - one based upon inhibition of the basis of chloroquine resistance, the multiple drug resistance pump. Previous studies demonstrated that the parasite MDR pump was found on parasite membranes using 3H azidopine photoaffinity labelling. Since MDR-based choloroquine resistance is induced by chloroquine, the basis of the action of tetrandrine is the following: 1) tetrandrine inhibits the MDR pump by stimulating MDR ATPase which limits the energy of the pump by depletion of parasite ATP, 2) tetrandrine blocks the

Treatment of acute uncomplicated falciparum malaria with artemether-lumefantrine in nonimmune populations: a safety, efficacy, and pharmacokinetic study.

PubMed

Hatz, Christoph; Soto, Jaime; Nothdurft, Hans Dieter; Zoller, Thomas; Weitzel, Thomas; Loutan, Louis; Bricaire, Francois; Gay, Frederick; Burchard, Gerd-Dieter; Andriano, Kim; Lefèvre, Gilbert; De Palacios, Patricia Ibarra; Genton, Blaise

2008-02-01

The efficacy and safety of artemether-lumefantrine for the treatment of malaria in nonimmune populations are not well defined. In this study, 165 nonimmune patients from Europe and non-malarious areas of Colombia with acute, uncomplicated falciparum malaria or mixed infection including P. falciparum were treated with the six-dose regimen of artemether-lumefantrine. The parasitologic cure rate at 28 days was 96.0% for the per protocol population (119/124 patients). Median times to parasite clearance and fever clearance were 41.5 and 36.8 hours, respectively. No patient had gametocytes after Day 7. Treatment was well tolerated; most adverse events were mild to moderate and seemed to be related to malaria. There were few serious adverse events, none of which were considered to be drug-related. No significant effects on ECG or laboratory parameters were observed. In conclusion, the six-dose regimen of artemether-lumefantrine was effective and well tolerated in the treatment of acute uncomplicated falciparum malaria in nonimmune patients.

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.

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

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

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

Resistance screening and trend analysis of imported falciparum malaria in NSW, Australia (2010 to 2016).

PubMed

Prosser, Christiane; Meyer, Wieland; Ellis, John; Lee, Rogan

2018-01-01

The World Health Organization currently recommends artemisinin (along with a partner drug) as the global frontline treatment for Plasmodium falciparum malaria. Artemisinin resistant P. falciparum are now found throughout the greater Mekong subregion of South East Asia. Several polymorphisms in the parasite's kelch gene have been demonstrated to confer artemisinin resistance. While genotypes within the greater Mekong subregion are thoroughly examined in the literature, P. falciparum populations within several areas that do not (yet) have endemic resistance are underrepresented. This investigation characterised the Pfkelch13 propeller domains from 153 blood samples of 140 imported cases of P. falciparum malaria in New South Wales from 2010 to 2016. A low level of propeller domain diversity was observed, including the C580Y coding mutation most strongly associated with artemisinin resistance in South East Asia. The resistance genotype was found in a sample originating in Papua New Guinea, where this mutation, or artemisinin treatment failure, have not been previously reported. Sequencing a panel of geographically informative polymorphisms within the organellar genomes identified the C580Y parasite as having Oceanic origins. Patient data analysis revealed that New South Wales, Australia, P. falciparum malaria cases often originated from regions with limited drug resistance screening. The C580Y finding from outside of the greater Mekong subregion supports the consensus to upscale molecular surveillance of artemisinin resistance outside of South East Asia. The genetic screening results identify a risk of importing resistant falciparum malaria to Australia, supporting an ongoing surveillance protocol to pre-empt treatment failure and contribute to global data gathering.

Transmission dynamics of co-endemic Plasmodium vivax and P. falciparum in Ethiopia and prevalence of antimalarial resistant genotypes

PubMed Central

Yewhalaw, Delenasaw; Nguyen, Jennifer; Kebede, Estifanos; Zemene, Endalew; Getachew, Sisay; Tushune, Kora; Zhong, Daibin; Zhou, Guofa; Petros, Beyene; Yan, Guiyun

2017-01-01

Ethiopia is one of the few African countries where Plasmodium vivax is co-endemic with P. falciparum. Malaria transmission is seasonal and transmission intensity varies mainly by landscape and climate. Although the recent emergence of drug resistant parasites presents a major issue to malaria control in Ethiopia, little is known about the transmission pathways of parasite species and prevalence of resistant markers. This study used microsatellites to determine population diversity and gene flow patterns of P. falciparum (N = 226) and P. vivax (N = 205), as well as prevalence of drug resistant markers to infer the impact of gene flow and existing malaria treatment regimes. Plasmodium falciparum indicated a higher rate of polyclonal infections than P. vivax. Both species revealed moderate genetic diversity and similar population structure. Populations in the northern highlands were closely related to the eastern Rift Valley, but slightly distinct from the southern basin area. Gene flow via human migrations between the northern and eastern populations were frequent and mostly bidirectional. Landscape genetic analyses indicated that environmental heterogeneity and geographical distance did not constrain parasite gene flow. This may partly explain similar patterns of resistant marker prevalence. In P. falciparum, a high prevalence of mutant alleles was detected in codons related to chloroquine (pfcrt and pfmdr1) and sulfadoxine-pyrimethamine (pfdhps and pfdhfr) resistance. Over 60% of the samples showed pfmdr1 duplications. Nevertheless, no mutation was detected in pfK13 that relates to artemisinin resistance. In P. vivax, while sequences of pvcrt-o were highly conserved and less than 5% of the samples showed pvmdr duplications, over 50% of the samples had pvmdr1 976F mutation. It remains to be tested if this mutation relates to chloroquine resistance. Monitoring the extent of malaria spread and markers of drug resistance is imperative to inform policy for evidence

Transmission dynamics of co-endemic Plasmodium vivax and P. falciparum in Ethiopia and prevalence of antimalarial resistant genotypes.

PubMed

Lo, Eugenia; Hemming-Schroeder, Elizabeth; Yewhalaw, Delenasaw; Nguyen, Jennifer; Kebede, Estifanos; Zemene, Endalew; Getachew, Sisay; Tushune, Kora; Zhong, Daibin; Zhou, Guofa; Petros, Beyene; Yan, Guiyun

2017-07-01

Ethiopia is one of the few African countries where Plasmodium vivax is co-endemic with P. falciparum. Malaria transmission is seasonal and transmission intensity varies mainly by landscape and climate. Although the recent emergence of drug resistant parasites presents a major issue to malaria control in Ethiopia, little is known about the transmission pathways of parasite species and prevalence of resistant markers. This study used microsatellites to determine population diversity and gene flow patterns of P. falciparum (N = 226) and P. vivax (N = 205), as well as prevalence of drug resistant markers to infer the impact of gene flow and existing malaria treatment regimes. Plasmodium falciparum indicated a higher rate of polyclonal infections than P. vivax. Both species revealed moderate genetic diversity and similar population structure. Populations in the northern highlands were closely related to the eastern Rift Valley, but slightly distinct from the southern basin area. Gene flow via human migrations between the northern and eastern populations were frequent and mostly bidirectional. Landscape genetic analyses indicated that environmental heterogeneity and geographical distance did not constrain parasite gene flow. This may partly explain similar patterns of resistant marker prevalence. In P. falciparum, a high prevalence of mutant alleles was detected in codons related to chloroquine (pfcrt and pfmdr1) and sulfadoxine-pyrimethamine (pfdhps and pfdhfr) resistance. Over 60% of the samples showed pfmdr1 duplications. Nevertheless, no mutation was detected in pfK13 that relates to artemisinin resistance. In P. vivax, while sequences of pvcrt-o were highly conserved and less than 5% of the samples showed pvmdr duplications, over 50% of the samples had pvmdr1 976F mutation. It remains to be tested if this mutation relates to chloroquine resistance. Monitoring the extent of malaria spread and markers of drug resistance is imperative to inform policy for evidence

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.

A PfRH5-Based Vaccine Is Efficacious against Heterologous Strain Blood-Stage Plasmodium falciparum Infection in Aotus Monkeys

PubMed Central

Douglas, Alexander D.; Baldeviano, G. Christian; Lucas, Carmen M.; Lugo-Roman, Luis A.; Crosnier, Cécile; Bartholdson, S. Josefin; Diouf, Ababacar; Miura, Kazutoyo; Lambert, Lynn E.; Ventocilla, Julio A.; Leiva, Karina P.; Milne, Kathryn H.; Illingworth, Joseph J.; Spencer, Alexandra J.; Hjerrild, Kathryn A.; Alanine, Daniel G.W.; Turner, Alison V.; Moorhead, Jeromy T.; Edgel, Kimberly A.; Wu, Yimin; Long, Carole A.; Wright, Gavin J.; Lescano, Andrés G.; Draper, Simon J.

2015-01-01

Summary Antigenic diversity has posed a critical barrier to vaccine development against the pathogenic blood-stage infection of the human malaria parasite Plasmodium falciparum. To date, only strain-specific protection has been reported by trials of such vaccines in nonhuman primates. We recently showed that P. falciparum reticulocyte binding protein homolog 5 (PfRH5), a merozoite adhesin required for erythrocyte invasion, is highly susceptible to vaccine-inducible strain-transcending parasite-neutralizing antibody. In vivo efficacy of PfRH5-based vaccines has not previously been evaluated. Here, we demonstrate that PfRH5-based vaccines can protect Aotus monkeys against a virulent vaccine-heterologous P. falciparum challenge and show that such protection can be achieved by a human-compatible vaccine formulation. Protection was associated with anti-PfRH5 antibody concentration and in vitro parasite-neutralizing activity, supporting the use of this in vitro assay to predict the in vivo efficacy of future vaccine candidates. These data suggest that PfRH5-based vaccines have potential to achieve strain-transcending efficacy in humans. PMID:25590760

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria

PubMed Central

Biagini, Giancarlo A.; Fisher, Nicholas; Shone, Alison E.; Mubaraki, Murad A.; Srivastava, Abhishek; Hill, Alisdair; Antoine, Thomas; Warman, Ashley J.; Davies, Jill; Pidathala, Chandrakala; Amewu, Richard K.; Leung, Suet C.; Sharma, Raman; Gibbons, Peter; Hong, David W.; Pacorel, Bénédicte; Lawrenson, Alexandre S.; Charoensutthivarakul, Sitthivut; Taylor, Lee; Berger, Olivier; Mbekeani, Alison; Stocks, Paul A.; Nixon, Gemma L.; Chadwick, James; Hemingway, Janet; Delves, Michael J.; Sinden, Robert E.; Zeeman, Anne-Marie; Kocken, Clemens H. M.; Berry, Neil G.; O’Neill, Paul M.; Ward, Stephen A.

2012-01-01

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc1. Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria. PMID:22566611

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

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

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

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro.

PubMed

Dubey, M L; Hegde, Ramakrishna; Ganguly, N K; Mahajan, R C

2003-04-01

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.

Treatment uptake by individuals infected with Plasmodium falciparum in rural Gambia, West Africa.

PubMed Central

von Seidlein, Lorenz; Clarke, Sian; Alexander, Neâl; Manneh, Fandingding; Doherty, Tom; Pinder, Margaret; Walraven, Gijs; Greenwood, Brian

2002-01-01

OBJECTIVE: To find out what proportion of Plasmodium falciparum infections are treated in rural Gambia. METHODS: Subjects from four villages in the Gambia were followed over nine months through visits to village health workers. Monthly cross-sectional malaria surveys measured the prevalence of P. falciparum infection. Linked databases were searched for treatment requests. Treated cases were individuals with parasitaemia who requested treatment during narrow or extended periods (14 or 28 days, respectively) before or after a positive blood film was obtained. FINDINGS: Parasite prevalence peaked in November 1998, when 399/653 (61%) individuals had parasitaemia. Parasite prevalence was highest throughout the study in children aged 5-10 years. Although access to treatment was better than in most of sub-Saharan Africa, only 20% of infected individuals sought medical treatment up to 14 days before or after a positive blood film. Within two months of a positive blood film, 199/726 (27%) individuals with parasitaemia requested treatment. Despite easy access to health care, less than half (42%) of those with parasite densities consistent with malaria attacks (5000/ l) requested treatment. High parasite density and infection during October-November were associated with more frequent treatment requests. Self-treatment was infrequent in study villages: in 3/120 (2.5%) households antimalarial drugs had been used in the preceding malaria season. CONCLUSION: Many P. falciparum infections may be untreated because of their subclinical nature. Intermittent presumptive treatment may reduce morbidity and mortality. It is likely that not all untreated infections were asymptomatic. Qualitative research should explore barriers to treatment uptake, to allow educational interventions to be planned. PMID:12471399

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

Use of buffy coat thick films in detecting malaria parasites in patients with negative conventional thick films.

PubMed

Duangdee, Chatnapa; Tangpukdee, Noppadon; Krudsood, Srivicha; Wilairatana, Polrat

2012-04-01

To determine the frequency of malaria parasite detection from the buffy coat blood films by using capillary tube in falciparum malaria patients with negative conventional thick films. Thirty six uncomplicated falciparum malaria patients confirmed by conventional thick and thin films were included in the study. The patients were treated with artemisinin combination therapy at Hospital for Tropical Diseases, Bangkok, Thailand for 28 day. Fingerpricks for conventional blood films were conducted every 6 hours until negative parasitemia, then daily fingerpricks for parasite checks were conducted until the patients were discharged from hospital. Blood samples were also concurrently collected in 3 heparinized capillary tubes at the same time of fingerpricks for conventional blood films when the prior parasitemia was negative on thin films and parasitemia was lower than 50 parasites/200 white blood cells by thick film. The first negative conventional thick films were compared with buffy coat thick films for parasite identification. Out of 36 patients with thick films showing negative for asexual forms of parasites, buffy coat films could detect remaining 10 patients (27.8%) with asexual forms of Plasmodium falciparum. The study shows that buffy coat thick films are useful and can detect malarial parasites in 27.8% of patients whose conventional thick films show negative parasitemia.

Ex Vivo Susceptibility of Plasmodium falciparum to Antimalarial Drugs in Western, Northern, and Eastern Cambodia, 2011-2012: Association with Molecular Markers

PubMed Central

Lim, Pharath; Dek, Dalin; Try, Vorleak; Eastman, Richard T.; Chy, Sophy; Sreng, Sokunthea; Suon, Seila; Mao, Sivanna; Sopha, Chantha; Sam, Baramey; Ashley, Elizabeth A.; Miotto, Olivo; Dondorp, Arjen M.; White, Nicholas J.; Su, Xin-zhuan; Char, Meng Chuor; Anderson, Jennifer M.; Amaratunga, Chanaki; Menard, Didier

2013-01-01

In 2008, dihydroartemisinin (DHA)-piperaquine (PPQ) became the first-line treatment for uncomplicated Plasmodium falciparum malaria in western Cambodia. Recent reports of increased treatment failure rates after DHA-PPQ therapy in this region suggest that parasite resistance to DHA, PPQ, or both is now adversely affecting treatment. While artemisinin (ART) resistance is established in western Cambodia, there is no evidence of PPQ resistance. To monitor for resistance to PPQ and other antimalarials, we measured drug susceptibilities for parasites collected in 2011 and 2012 from Pursat, Preah Vihear, and Ratanakiri, in western, northern, and eastern Cambodia, respectively. Using a SYBR green I fluorescence assay, we calculated the ex vivo 50% inhibitory concentrations (IC50s) of 310 parasites to six antimalarials: chloroquine (CQ), mefloquine (MQ), quinine (QN), PPQ, artesunate (ATS), and DHA. Geometric mean IC50s (GMIC50s) for all drugs (except PPQ) were significantly higher in Pursat and Preah Vihear than in Ratanakiri (P ≤ 0.001). An increased copy number of P. falciparum mdr1 (pfmdr1), an MQ resistance marker, was more prevalent in Pursat and Preah Vihear than in Ratanakiri and was associated with higher GMIC50s for MQ, QN, ATS, and DHA. An increased copy number of a chromosome 5 region (X5r), a candidate PPQ resistance marker, was detected in Pursat but was not associated with reduced susceptibility to PPQ. The ex vivo IC50 and pfmdr1 copy number are important tools in the surveillance of multidrug-resistant (MDR) parasites in Cambodia. While MDR P. falciparum is prevalent in western and northern Cambodia, there is no evidence for PPQ resistance, suggesting that DHA-PPQ treatment failures result mainly from ART resistance. PMID:23939897

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

Human Antibodies to a Mr 155,000 Plasmodium falciparum Antigen Efficiently Inhibit Merozoite Invasion

NASA Astrophysics Data System (ADS)

Wahlin, Birgitta; Wahlgren, Mats; Perlmann, Hedvig; Berzins, Klavs; Bjorkman, Anders; Patarroyo, Manuel E.; Perlmann, Peter

1984-12-01

IgG from a donor clinically immune to Plasmodium falciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, <1 μ g/ml) than the original IgG preparation. The major antibody in this eluate was directed against a parasite-derived antigen of Mr 155,000 (Pf 155) deposited by the parasite in the erythrocyte membrane in the course of invasion. A detailed study of IgG fractions from 11 donors with acute P. falciparum malaria or clinical immunity revealed the existence of an excellent correlation between their capacities to stain the surface of infected erythrocytes, their titers in reinvasion inhibition, and the presence of antibodies to Pf 155 as detected by immunoblotting. No such correlations were seen when the IgG fractions were analyzed for immunofluorescence of intracellular parasites or for the presence of antibodies to other parasite antigens as detected by immunoprecipitation of [35S]methionine-labeled and NaDodSO4/PAGE-separated parasite extracts. The results suggest that Pf 155 has an important role in the process of erythrocyte infection and that host antibodies to this antigen may efficiently interfere with this process.

Acute Kidney Injury in Children with Plasmodium falciparum Malaria: Determinants for Mortality.

PubMed

Prasad, Rajniti; Mishra, Om P

2016-01-01

♦ Acute kidney injury (AKI) in P. falciparum malaria infection is an important morbidity in children. The purpose of the present study was done to observe the renal involvement, associated morbidities and outcome. ♦ Out of 156 patients with severe P. falciparum malaria, diagnosed on the basis of compatible clinical presentations and positive malarial parasites in the peripheral blood smear and/or histidine rich protein 2 antigen, 31 had AKI at presentation and were analyzed. ♦ Of 31 (19.9%) patients with AKI, 4 were classified at risk, 11 injury, and 16 failure stage, as per pRIFLE criteria (pediatric version of RIFLE [R = risk, I = injury, F = failure, L = loss E = end-stage kidney disease]). Mean age of children with AKI was 7.7 ± 3.2 years. A significantly higher proportion of patients with AKI had hypoglycemia (41.9%), pulmonary edema (32.2%), and disseminated intravascular coagulation (DIC) (29.0%) compared to those without AKI (18.4%, 4.8%, and 3.2%, respectively). Twelve patients (38.7%) required peritoneal dialysis (PD), 8 (25.8%) died, and all were in failure stage. The non-survivors had significantly higher blood urea (p = 0.005) and serum creatinine levels (p = 0.042), lower glomerular filtration rate (p < 0.001), longer duration of illness (p = 0.003), and oliguria/anuria (p = 0.001) than survivors at admission. On logistic regression analysis, the disseminated intravascular coagulation (DIC), jaundice and parasite density (≥ 3+) were found to be significant factors contributing to mortality in children with AKI. ♦ Acute kidney injury in falciparum malaria is one of the severe systemic complications. Duration of illness and presence of comorbidities adversely affected the outcome. Copyright © 2016 International Society for Peritoneal Dialysis.

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

Coinfection with Plasmodium falciparum and Schistosoma haematobium: Additional Evidence of the Protective Effect of Schistosomiasis on Malaria in Senegalese Children

PubMed Central

Lemaitre, Magali; Watier, Laurence; Briand, Valérie; Garcia, André; Le Hesran, Jean Yves; Cot, Michel

2014-01-01

Parasitic infections are associated with high morbidity and mortality in developing countries. Several studies focused on the influence of helminth infections on malaria but the nature of the biological interaction is under debate. Our objective was to undertake a study to explore the influence of the measure of excreted egg load caused by Schistosoma haematobium on Plasmodium falciparum parasite densities. Ten measures of malaria parasite density and two measures of schistosomiasis egg urinary excretion over a 2-year follow-up period on 178 Senegalese children were considered. A linear mixed-effect model was developed to take data dependence into account. This work showed that children with a light S. haematobium infection (1–9 eggs/mL of urine) presented lower P. falciparum parasite densities than children not infected by S. haematobium (P < 0.04). Possible changes caused by parasite coinfections should be considered in the anti-helminth treatment of children and in malaria vaccination development. PMID:24323515

Coinfection with Plasmodium falciparum and Schistosoma haematobium: additional evidence of the protective effect of Schistosomiasis on malaria in Senegalese children.

PubMed

Lemaitre, Magali; Watier, Laurence; Briand, Valérie; Garcia, André; Le Hesran, Jean Yves; Cot, Michel

2014-02-01

Parasitic infections are associated with high morbidity and mortality in developing countries. Several studies focused on the influence of helminth infections on malaria but the nature of the biological interaction is under debate. Our objective was to undertake a study to explore the influence of the measure of excreted egg load caused by Schistosoma haematobium on Plasmodium falciparum parasite densities. Ten measures of malaria parasite density and two measures of schistosomiasis egg urinary excretion over a 2-year follow-up period on 178 Senegalese children were considered. A linear mixed-effect model was developed to take data dependence into account. This work showed that children with a light S. haematobium infection (1-9 eggs/mL of urine) presented lower P. falciparum parasite densities than children not infected by S. haematobium (P < 0.04). Possible changes caused by parasite coinfections should be considered in the anti-helminth treatment of children and in malaria vaccination development.

Falciparum Malaria Outbreak in Sabah Linked to an Immigrant Rubber Tapper.

PubMed

Jeffree, Saffree Mohammad; Ahmed, Kamruddin; Safian, Nazarudin; Hassan, Rohaizat; Mihat, Omar; Lukman, Khamisah Awang; Shamsudin, Shamsul Bahari; Kamaludin, Fadzilah

2018-01-01

Sabah is a Malaysian state situated in the northern part of Borneo, and it is endemic for malaria. The incidence of malaria is the lowest (0.05/1,000 population) in Penampang districts of Sabah. In June 26, 2012, two Plasmodium falciparum malaria cases were notified to public health department from a village in Penampang. Immediate investigation was initiated to identify the risk factors and to institute control measures. We performed active case finding by asking household members of all houses in the village regarding malaria symptoms and by examining blood smears. Environmental investigation was performed by collecting samples to detect mosquito breeding sites and to identify malaria transmitting vector mosquitoes. A case-control study with a ratio of 1:4 (11 cases and 44 controls) was conducted using self-administered questionnaire. The microscopic examination of blood smear for malarial parasite and entomology sampling was carried out. The malarial attack rate was 2.3%, 6/11 smears have gametocyte, and the case fatality rate was 9.1%. One case was a migrant rubber tapper from Indonesia which happened to be the first case with gametocyte positive. Overall, the incidence of malaria was higher (6/11) among rubber tappers. The odds of cases for those living nearby stagnant water were 7.3 [95% confidence interval: 1.2-43.5] times higher. In conclusion, an outbreak of P. falciparum malaria was introduced into a malaria-free village by a migrant rubber tapper, by whom the imported parasite was introduced to the community via vector Anopheles balabacensis . Living near stagnant water bodies was the risk factor in this outbreak.

Blood-Stage Parasitaemia and Age Determine Plasmodium falciparum and P. vivax Gametocytaemia in Papua New Guinea.

PubMed

Koepfli, Cristian; Robinson, Leanne J; Rarau, Patricia; Salib, Mary; Sambale, Naomi; Wampfler, Rahel; Betuela, Inoni; Nuitragool, Wang; Barry, Alyssa E; Siba, Peter; Felger, Ingrid; Mueller, Ivo

2015-01-01

A better understanding of human-to-mosquito transmission is crucial to control malaria. In order to assess factors associated with gametocyte carriage, 2083 samples were collected in a cross-sectional survey in Papua New Guinea. Plasmodium species were detected by light microscopy and qPCR and gametocytes by detection of pfs25 and pvs25 mRNA transcripts by reverse-transcriptase PCR (qRT-PCR). The parasite prevalence by PCR was 18.5% for Plasmodium falciparum and 13.0% for P. vivax. 52.5% of all infections were submicroscopic. Gametocytes were detected in 60% of P. falciparum-positive and 51% of P. vivax-positive samples. Each 10-fold increase in parasite density led to a 1.8-fold and 3.3-fold increase in the odds of carrying P. falciparum and P. vivax gametocytes. Thus the proportion of gametocyte positive and gametocyte densities was highest in young children carrying high asexual parasite densities and in symptomatic individuals. Dilution series of gametocytes allowed absolute quantification of gametocyte densities by qRT-PCR and showed that pvs25 expression is 10-20 fold lower than pfs25 expression. Between 2006 and 2010 parasite prevalence in the study site has decreased by half. 90% of the remaining infections were asymptomatic and likely constitute an important reservoir of transmission. However, mean gametocyte densities were low (approx. 1-2 gametocyte/μL) and it remains to be determined to what extent low-density gametocyte positive individuals are infective to mosquitos.

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.

The Central Role of cAMP in Regulating Plasmodium falciparum Merozoite Invasion of Human Erythrocytes

PubMed Central

More, Kunal R.; Siddiqui, Faiza Amber; Pachikara, Niseema; Ramdani, Ghania; Langsley, Gordon; Chitnis, Chetan E.

2014-01-01

All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K+ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca2+ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca2+ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria. PMID:25522250

Evolution of genetic polymorphisms of Plasmodium falciparum merozoite surface protein (PfMSP) in Thailand.

PubMed

Kuesap, Jiraporn; Chaijaroenkul, Wanna; Ketprathum, Kanchanok; Tattiyapong, Puntanat; Na-Bangchang, Kesara

2014-02-01

Plasmodium falciparum malaria is a major public health problem in Thailand due to the emergence of multidrug resistance. The understanding of genetic diversity of malaria parasites is essential for developing effective drugs and vaccines. The genetic diversity of the merozoite surface protein-1 (PfMSP-1) and merozoite surface protein-2 (PfMSP-2) genes was investigated in a total of 145 P. falciparum isolates collected from Mae Sot District, Tak Province, Thailand during 3 different periods (1997-1999, 2005-2007, and 2009-2010). Analysis of genetic polymorphisms was performed to track the evolution of genetic change of P. falciparum using PCR. Both individual genes and their combination patterns showed marked genetic diversity during the 3 study periods. The results strongly support that P. falciparum isolates in Thailand are markedly diverse and patterns changed with time. These 2 polymorphic genes could be used as molecular markers to detect multiple clone infections and differentiate recrudescence from reinfection in P. falciparum isolates in Thailand.

A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion.

PubMed

Baum, Jake; Tonkin, Christopher J; Paul, Aditya S; Rug, Melanie; Smith, Brian J; Gould, Sven B; Richard, Dave; Pollard, Thomas D; Cowman, Alan F

2008-03-13

Malaria parasites invade host cells using actin-based motility, a process requiring parasite actin filament nucleation and polymerization. Malaria and other apicomplexan parasites lack Arp2/3 complex, an actin nucleator widely conserved across eukaryotes, but do express formins, another type of actin nucleator. Here, we demonstrate that one of two malaria parasite formins, Plasmodium falciparum formin 1 (PfFormin 1), and its ortholog in the related parasite Toxoplasma gondii, follows the moving tight junction between the invading parasite and the host cell, which is the predicted site of the actomyosin motor that powers motility. Furthermore, in vitro, the PfFormin1 actin-binding formin homology 2 domain is a potent nucleator, stimulating actin polymerization and, like other formins, localizing to the barbed end during filament elongation. These findings support a conserved molecular mechanism underlying apicomplexan parasite motility and, given the essential role that actin plays in cell invasion, highlight formins as important determinants of malaria parasite pathogenicity.

Structure of Plasmodium falciparum ADP-ribosylation factor 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, William J.; Smith, Craig D.; Senkovich, Olga

Vesicular trafficking may play a crucial role in the pathogenesis and survival of the malaria parasite. ADP-ribosylation factors (ARFs) are among the major components of vesicular trafficking pathways in eukaryotes. The crystal structure of ARF1 GTPase from Plasmodium falciparum has been determined in the GDP-bound conformation at 2.5 {angstrom} resolution and is compared with the structures of mammalian ARF1s.

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

Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine

PubMed Central

Sá, Juliana Martha; Twu, Olivia; Hayton, Karen; Reyes, Sahily; Fay, Michael P.; Ringwald, Pascal; Wellems, Thomas E.

2009-01-01

Chloroquine (CQ) resistance (CQR) in Plasmodium falciparum originated from at least six foci in South America, Asia, and Oceania. Malaria parasites from these locations exhibit contrasting resistance phenotypes that are distinguished by point mutations and microsatellite polymorphisms in and near the CQR transporter gene, pfcrt, and the multidrug resistance transporter gene, pfmdr1. Amodiaquine (AQ), a 4-aminoquinoline related to CQ, is recommended and often used successfully against CQ-resistant P. falciparum in Africa, but it is largely ineffective across large regions of South America. The relationship of different pfcrt and pfmdr1 combinations to these drug-resistant phenotypes has been unclear. In two P. falciparum genetic crosses, particular pfcrt and pfmdr1 alleles from South America interact to yield greater levels of resistance to monodesethylamodiaquine (MDAQ; the active metabolite of AQ) than to CQ, whereas a pfcrt allele from Southeast Asia and Africa is linked to greater CQ than MDAQ resistance with all partner pfmdr1 alleles. These results, together with (i) available haplotype data from other parasites; (ii) evidence for an emerging focus of AQ resistance in Tanzania; and (iii) the persistence of 4-aminoquinoline-resistant parasites in South America, where CQ and AQ use is largely discontinued, suggest that different histories of drug use on the two continents have driven the selection of distinct suites of pfcrt and pfmdr1 mutations. Increasing use of AQ in Africa poses the threat of a selective sweep of highly AQ-resistant, CQ-resistant parasites with pfcrt and pfmdr1 mutations that are as advantaged and persistent as in South America. PMID:19884511

Renal related disorders in concomitant Schistosoma haematobium-Plasmodium falciparum infection among children in a rural community of Nigeria.

PubMed

Morenikeji, Olajumoke A; Eleng, Ituna E; Atanda, Omotayo S; Oyeyemi, Oyetunde T

2016-01-01

Schistosomiasis and malaria are two common parasitic diseases that are co-endemic in resource-poor communities of sub-Saharan Africa. This study aims to assess the effects of single and concomitant Plasmodium falciparum and Schistosoma haematobium infections on two indicators of renal injury in school children in a rural community of Nigeria. A cross-sectional epidemiological survey was carried out on a total of 173 schoolchildren between ages 6 and 18 years (mean age 11.4±2.6 years). Urine and blood samples were collected by standard methods for concurrent microscopic diagnosis of S. haematobium and P. falciparum infections. Urinary blood (hematuria) and protein were determined using a urinalysis dipstick. The prevalence of single infections was 75.1% and 78.2% for S. haematobium and P. falciparum, respectively. A total of 57.1% individuals were infected with the two parasites. The prevalence of hematuria was significantly higher in the co-infection status (63.8%) than in single S. haematobium (52.2%) and P. falciparum (43.7%) infection statuses (p=0.04), while no significant variation was recorded in proteinuria in the three infection statuses (p=0.53). The proportion of children with renal injury associated with the co-infection of these parasites is very high, particularly in young children, who seem to have a higher prevalence of hematuria. Copyright © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Crystal Structure Analyses of the Fosmidomycin-Target Enzyme from Plasmodium Falciparum

NASA Astrophysics Data System (ADS)

Umeda, Tomonobu; Kusakabe, Yoshio; Tanaka, Nobutada

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. Fosmidomycin has proved to be efficient in the treatment of P. falciparum malaria through the inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the non-mevalonate pathway of isoprenoid biosynthesis, which is absent in humans. Crystal structure analyses of P. falciparum DXR (PfDXR) revealed that (i) an intrinsic flexibility of the PfDXR molecule accounts for the induced-fit movement to accommodate the bound inhibitor in the active site, and (ii) a cis arrangement of the oxygen atoms of the hydroxamate group of the bound inhibitor is essential for tight binding of the inhibitor to the active site metal. We believe that our study will serve as a useful guide to develop more potent PfDXR inhibitors.

Test characteristics of the SD FK80 Plasmodium falciparum/Plasmodium vivax malaria rapid diagnostic test in a non-endemic setting

PubMed Central

2009-01-01

Background The SD FK80 P.f/P.v Malaria Antigen Rapid Test (Standard Diagnostics, Korea) (FK80) is a three-band malaria rapid diagnostic test detecting Plasmodium falciparum histidine-rich protein-2 (HRP-2) and Plasmodium vivax-specific lactate dehydrogenase (Pv-pLDH). The present study assessed its performance in a non-endemic setting. Methods Stored blood samples (n = 416) from international travellers suspected of malaria were used, with microscopy corrected by PCR as the reference method. Samples infected by Plasmodium falciparum (n = 178), Plasmodium vivax (n = 99), Plasmodium ovale (n = 75) and Plasmodium malariae (n = 24) were included, as well as 40 malaria negative samples. Results Overall sensitivities for the diagnosis of P. falciparum and P. vivax were 91.6% (95% confidence interval (CI): 86.2% - 95.0%) and 75.8% (65.9% - 83.6%). For P. falciparum, sensitivity at parasite densities ≥ 100/μl was 94.6% (88.8% - 97.6%); for P. vivax, sensitivity at parasite densities ≥ 500/μl was 86.8% (75.4% - 93.4%). Four P. falciparum samples showed a Pv-pLDH line, three of them had parasite densities exceeding 50.000/μl. Two P. vivax samples, one P. ovale and one P. malariae sample showed a HRP-2 line. For the HRP-2 and Pv-pLDH lines, respectively 81.4% (136/167) and 55.8% (43/77) of the true positive results were read as medium or strong line intensities. The FK80 showed good reproducibility and reliability for test results and line intensities (kappa values for both exceeding 0.80). Conclusion The FK80 test performed satisfactorily in diagnosing P. falciparum and P. vivax infections in a non-endemic setting. PMID:19930609

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds.

PubMed

Schuck, Desirée Cigaran; Ribeiro, Ramira Yuri; Nery, Arthur A; Ulrich, Henning; Garcia, Célia R S

2011-11-01

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds. Copyright © 2011 International Society for Advancement of Cytometry.

Evaluation of community-based systems for the surveillance of day three-positive Plasmodium falciparum cases in Western Cambodia.

PubMed

Cox, Jonathan; Dy Soley, Lek; Bunkea, Tol; Sovannaroth, Siv; Soy Ty, Kheang; Ngak, Song; Bjorge, Steven; Ringwald, Pascal; Mellor, Steven; Sintasath, David; Meek, Sylvia

2014-07-22

Delayed clearance of Plasmodium falciparum parasites is used as an operational indicator of potential artemisinin resistance. Effective community-based systems to detect P. falciparum cases remaining positive 72 hours after initiating treatment would be valuable for guiding case follow-up in areas of known resistance risk and for detecting areas of emerging resistance. Systems incorporating existing networks of village malaria workers (VMWs) to monitor day three-positive P. falciparum cases were piloted in three provinces in western Cambodia. Quantitative and qualitative data were used to evaluate the wider feasibility and sustainability of community-based surveillance of day three-positive P. falciparum cases. Of 294 day-3 blood slides obtained across all sites (from 297 day-0 positives), 63 were positive for P. falciparum, an overall day-3 positivity rate of 21%. There were significant variations in the systems implemented by different partners. Full engagement of VMWs and health centre staff is critical. VMWs are responsible for a range of individual tasks including preparing blood slides on day-0, completing forms, administering directly observed therapy (DOT) on days 0-2, obtaining follow-up slides on day-3 and transporting slides and paperwork to their supervising health centre. When suitably motivated, unsalaried VMWs are willing and able to produce good quality blood smears and achieve very high rates of DOT and day-3 follow-up. Community-based surveillance of day-3 P. falciparum cases is feasible, but highly intensive, and as such needs strong and continuous support, particularly supervision and training. The purpose and role of community-based day-3 surveillance should be assessed in the light of resource requirements; scaling-up would need to be systematic and targeted, based on clearly defined epidemiological criteria. To be truly comprehensive, the system would need to be extended beyond VMWs to other public and private health providers.

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

Plant Hormone Salicylic Acid Produced by a Malaria Parasite Controls Host Immunity and Cerebral Malaria Outcome.

PubMed

Matsubara, Ryuma; Aonuma, Hiroka; Kojima, Mikiko; Tahara, Michiru; Andrabi, Syed Bilal Ahmad; Sakakibara, Hitoshi; Nagamune, Kisaburo

2015-01-01

The apicomplexan parasite Toxoplasma gondii produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite Plasmodium spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in P. berghei ANKA and T. gondii cell lysates. However, addition of salicylic acid to P. falciparum and T. gondii culture had no effect. We transfected P. falciparum 3D7 with the nahG gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting Pseudomonas sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E2, which potentially modulates host immunity as an adaptive evolution of Plasmodium spp. To investigate the function of salicylic acid and prostaglandin E2 on host immunity, we established P. berghei ANKA mutants expressing nahG. C57BL/6 mice infected with nahG transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The nahG-transfectant also significantly increased the mortality rate of mice. Prostaglandin E2 reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the nahG transfectant. Thus, salicylic acid of Plasmodium spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E2.

Plant Hormone Salicylic Acid Produced by a Malaria Parasite Controls Host Immunity and Cerebral Malaria Outcome

PubMed Central

Matsubara, Ryuma; Aonuma, Hiroka; Kojima, Mikiko; Tahara, Michiru; Andrabi, Syed Bilal Ahmad; Sakakibara, Hitoshi; Nagamune, Kisaburo

2015-01-01

The apicomplexan parasite Toxoplasma gondii produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite Plasmodium spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in P. berghei ANKA and T. gondii cell lysates. However, addition of salicylic acid to P. falciparum and T. gondii culture had no effect. We transfected P. falciparum 3D7 with the nahG gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting Pseudomonas sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E2, which potentially modulates host immunity as an adaptive evolution of Plasmodium spp. To investigate the function of salicylic acid and prostaglandin E2 on host immunity, we established P. berghei ANKA mutants expressing nahG. C57BL/6 mice infected with nahG transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The nahG-transfectant also significantly increased the mortality rate of mice. Prostaglandin E2 reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the nahG transfectant. Thus, salicylic acid of Plasmodium spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E2. PMID:26466097

Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

PubMed Central

2010-01-01

Background Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level. Results Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was

Prevalence of PCR detectable malaria infection among febrile patients with a negative Plasmodium falciparum specific rapid diagnostic test in Zanzibar.

PubMed

Baltzell, Kimberly A; Shakely, Deler; Hsiang, Michelle; Kemere, Jordan; Ali, Abdullah Suleiman; Björkman, Anders; Mårtensson, Andreas; Omar, Rahila; Elfving, Kristina; Msellem, Mwinyi; Aydin-Schmidt, Berit; Rosenthal, Philip J; Greenhouse, Bryan

2013-02-01

We screened for malaria in 594 blood samples from febrile patients who tested negative by a Plasmodium falciparum-specific histidine-rich protein-2-based rapid diagnostic test at 12 health facilities in Zanzibar districts North A and Micheweni, from May to August 2010. Screening was with microscopy, polymerase chain reaction (PCR) targeting the cytochrome b gene (cytbPCR) of the four major human malaria species, and quantitative PCR (qPCR). The prevalence of cytbPCR-detectable malaria infection was 2% (12 of 594), including 8 P. falciparum, 3 Plasmodium malariae, and 1 Plasmodium vivax infections. Microscopy identified 4 of 8 P. falciparum infections. Parasite density as estimated by microscopy or qPCR was > 4,000 parasites/μL in 5 of 8 cytbPCR-detectable P. falciparum infections. The infections that were missed by the rapid diagnostic test represent a particular challenge in malaria elimination settings and highlight the need for more sensitive point-of-care diagnostic tools to improve case detection of all human malaria species in febrile patients.

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

Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift.

PubMed

Geislinger, Thomas M; Chan, Sherwin; Moll, Kirsten; Wixforth, Achim; Wahlgren, Mats; Franke, Thomas

2014-09-20

Understanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated. A simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker. The possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of ~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated. The method allows for the enrichment of red blood cells early after the invasion by P. falciparum parasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic- or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.

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

Genetic Diversity and Population Structure of Plasmodium falciparum in Lake Victoria Islands, A Region of Intense Transmission

PubMed Central

Mulenge, Felix M.; Hunja, Carol W.; Magiri, Esther; Culleton, Richard; Kaneko, Akira; Aman, Rashid A.

2016-01-01

Understanding the genetic structure and transmission dynamics of Plasmodium falciparum parasites in malaria-endemic regions is crucial before the implementation of interventions. Located in a high-transmission region of western Kenya where P. falciparum is the predominant species, the Lake Victoria islands are ideal for feasibility of malaria elimination studies. We analyzed genetic variation in eight microsatellite loci to examine parasite population structure and gene flow patterns across five sites. High levels of genetic diversity were measured throughout the region (mean heterozygosity index = 0.84). The overall fixation index value between the sites was 0.044, indicating that approximately 5% of the overall allelic variation is due to differences between the populations. Based on these results, we concluded that parasite population structure in the studied islands is shaped by human migration patterns that maintain extensive parasite gene flow between the sites. Consequently, any malaria elimination and interventions strategies in the study area will have to be carried out broadly on all four islands and adjoining mainland region. PMID:27601522

The anthraquinone emodin inhibits the non-exported FIKK kinase from Plasmodium falciparum.

PubMed

Lin, Benjamin C; Harris, Darcy R; Kirkman, Lucy M D; Perez, Astrid M; Qian, Yiwen; Schermerhorn, Janse T; Hong, Min Y; Winston, Dennis S; Xu, Lingyin; Lieber, Alexander M; Hamilton, Matthew; Brandt, Gabriel S

2017-12-01

The FIKK family of kinases is unique to parasites of the Apicomplexan order, which includes all malaria parasites. Plasmodium falciparum, the most virulent form of human malaria, has a family of 19 FIKK kinases, most of which are exported into the host red blood cell during malaria infection. Here, we confirm that FIKK 8 is a non-exported member of the FIKK kinase family. Through expression and purification of the recombinant kinase domain, we establish that emodin is a relatively high-affinity (IC 50 =2μM) inhibitor of PfFk8. Closely related anthraquinones do not inhibit PfFk8, suggesting that the particular substitution pattern of emodin is critical to the inhibitory pharmacophore. This first report of a P. falciparum FIKK kinase inhibitor lays the groundwork for developing specific inhibitors of the various members of the FIKK kinase family in order to probe their physiological function. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Persistence and immunogenicity of chemically attenuated blood stage Plasmodium falciparum in Aotus monkeys.

PubMed

De, Sai Lata; Stanisic, Danielle I; van Breda, Karin; Bellete, Bernadette; Harris, Ivor; McCallum, Fiona; Edstein, Michael D; Good, Michael F

2016-08-01

Malaria is a disease caused by a protozoan of the Plasmodium genus and results in 0.5-0.7million deaths per year. Increasing drug resistance of the parasite and insecticide resistance of mosquitoes necessitate alternative control measures. Numerous vaccine candidates have been identified but none have been able to induce robust, long-lived protection when evaluated in malaria endemic regions. Rodent studies have demonstrated that chemically attenuated blood stage parasites can persist at sub-patent levels and induce homologous and heterologous protection against malaria. Parasite-specific cellular responses were detected, with protection dependent on CD4+ T cells. To investigate this vaccine approach for Plasmodium falciparum, we characterised the persistence and immunogenicity of chemically attenuated P. falciparum FVO strain parasites (CAPs) in non-splenectomised Aotus nancymaae monkeys following administration of a single dose. Control monkeys received either normal red blood cells or wild-type parasites followed by drug treatment. Chemical attenuation was performed using tafuramycin A, which irreversibly binds to DNA. CAPs were detected in the peripheral blood for up to 2days following inoculation as determined by thick blood smears, and for up to 8days as determined by quantitative PCR. Parasite-specific IgG was not detected in monkeys that received CAPs; however, in vitro parasite-specific T cell proliferation was observed. Following challenge, the CAP monkeys developed an infection; however, one CAP monkey and the infection and drug-cure monkeys showed partial or complete resistance. These experiments lay the groundwork for further assessment of CAPs as a potential vaccine against malaria. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

PubMed

Davis, Mindy I; Patrick, Stephen L; Blanding, Walker M; Dwivedi, Varun; Suryadi, Jimmy; Golden, Jennifer E; Coussens, Nathan P; Lee, Olivia W; Shen, Min; Boxer, Matthew B; Hall, Matthew D; Sharlow, Elizabeth R; Drew, Mark E; Morris, James C

2016-10-01

Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 μM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Fitness of artemisinin-resistant Plasmodium falciparum in vitro

PubMed Central

Hott, Amanda; Tucker, Matthew S.; Casandra, Debora; Sparks, Kansas; Kyle, Dennis E.

2015-01-01

Objectives Drug resistance confers a fitness advantage to parasites exposed to frequent drug pressure, yet these mutations also may incur a fitness cost. We assessed fitness advantages and costs of artemisinin resistance in Plasmodium falciparum in vitro to understand how drug resistance will spread and evolve in a competitive environment. Methods Genotyping of SNPs, drug susceptibility assays and copy number determination were used to assess the impact of artemisinin resistance on parasite fitness. An artemisinin-resistant clone (C9) selected in vitro from an isogenic parental clone (D6) was used to conduct competitive growth studies to assess fitness of artemisinin resistance. The resistant and susceptible clones were mixed or grown alone in the presence and absence of drug pressure (dihydroartemisinin or pyrimethamine) to quantify the rate at which artemisinin resistance was gained or lost. Results We experimentally demonstrate for the first time that artemisinin resistance provides a fitness advantage that is selected for with infrequent exposure to drug, but is lost in the absence of exposure to artemisinin drugs. The best correlations with artemisinin resistance were decreased in vitro drug susceptibility to artemisinin derivatives, increased copy number of Pf3D7_1030100 and an SNP in Pf3D7_0307600. An SNP conferring an E208K mutation in the kelch gene (Pf3D7_1343700) was not associated with resistance. Furthermore, we observed second-cycle ring-stage dormancy induced by pyrimethamine, suggesting that dormancy is a fitness trait that provides an advantage for survival from antimalarial drug stress. Conclusions Artemisinin-resistant P. falciparum have a fitness advantage to survive and predominate in the population even in the face of infrequent exposure to artemisinin drugs. PMID:26203183

Fitness of artemisinin-resistant Plasmodium falciparum in vitro.

PubMed

Hott, Amanda; Tucker, Matthew S; Casandra, Debora; Sparks, Kansas; Kyle, Dennis E

2015-10-01

Drug resistance confers a fitness advantage to parasites exposed to frequent drug pressure, yet these mutations also may incur a fitness cost. We assessed fitness advantages and costs of artemisinin resistance in Plasmodium falciparum in vitro to understand how drug resistance will spread and evolve in a competitive environment. Genotyping of SNPs, drug susceptibility assays and copy number determination were used to assess the impact of artemisinin resistance on parasite fitness. An artemisinin-resistant clone (C9) selected in vitro from an isogenic parental clone (D6) was used to conduct competitive growth studies to assess fitness of artemisinin resistance. The resistant and susceptible clones were mixed or grown alone in the presence and absence of drug pressure (dihydroartemisinin or pyrimethamine) to quantify the rate at which artemisinin resistance was gained or lost. We experimentally demonstrate for the first time that artemisinin resistance provides a fitness advantage that is selected for with infrequent exposure to drug, but is lost in the absence of exposure to artemisinin drugs. The best correlations with artemisinin resistance were decreased in vitro drug susceptibility to artemisinin derivatives, increased copy number of Pf3D7_1030100 and an SNP in Pf3D7_0307600. An SNP conferring an E208K mutation in the kelch gene (Pf3D7_1343700) was not associated with resistance. Furthermore, we observed second-cycle ring-stage dormancy induced by pyrimethamine, suggesting that dormancy is a fitness trait that provides an advantage for survival from antimalarial drug stress. Artemisinin-resistant P. falciparum have a fitness advantage to survive and predominate in the population even in the face of infrequent exposure to artemisinin drugs. © The Author 2015. 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.

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

Retention of Plasmodium falciparum ring-infected erythrocytes in the slow, open microcirculation of the human spleen.

PubMed

Safeukui, Innocent; Correas, Jean-Michel; Brousse, Valentine; Hirt, Déborah; Deplaine, Guillaume; Mulé, Sébastien; Lesurtel, Mickael; Goasguen, Nicolas; Sauvanet, Alain; Couvelard, Anne; Kerneis, Sophie; Khun, Huot; Vigan-Womas, Inès; Ottone, Catherine; Molina, Thierry Jo; Tréluyer, Jean-Marc; Mercereau-Puijalon, Odile; Milon, Geneviève; David, Peter H; Buffet, Pierre A

2008-09-15

The current paradigm in Plasmodium falciparum malaria pathogenesis states that young, ring-infected erythrocytes (rings) circulate in peripheral blood and that mature stages are sequestered in the vasculature, avoiding clearance by the spleen. Through ex vivo perfusion of human spleens, we examined the interaction of this unique blood-filtering organ with P falciparum-infected erythrocytes. As predicted, mature stages were retained. However, more than 50% of rings were also retained and accumulated upstream from endothelial sinus wall slits of the open, slow red pulp microcirculation. Ten percent of rings were retained at each spleen passage, a rate matching the proportion of blood flowing through the slow circulatory compartment established in parallel using spleen contrast-enhanced ultrasonography in healthy volunteers. Rings displayed a mildly but significantly reduced elongation index, consistent with a retention process, due to their altered mechanical properties. This raises the new paradigm of a heterogeneous ring population, the less deformable subset being retained in the spleen, thereby reducing the parasite biomass that will sequester in vital organs, influencing the risk of severe complications, such as cerebral malaria or severe anemia. Cryptic ring retention uncovers a new role for the spleen in the control of parasite density, opening novel intervention opportunities.

Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker

PubMed Central

Tun, Kyaw M; Imwong, Mallika; Lwin, Khin M; Win, Aye A; Hlaing, Tin M; Hlaing, Thaung; Lin, Khin; Kyaw, Myat P; Plewes, Katherine; Faiz, M Abul; Dhorda, Mehul; Cheah, Phaik Yeong; Pukrittayakamee, Sasithon; Ashley, Elizabeth A; Anderson, Tim J C; Nair, Shalini; McDew-White, Marina; Flegg, Jennifer A; Grist, Eric P M; Guerin, Philippe; Maude, Richard J; Smithuis, Frank; Dondorp, Arjen M; Day, Nicholas P J; Nosten, François; White, Nicholas J; Woodrow, Charles J

2015-01-01

Summary Background Emergence of artemisinin resistance in southeast Asia poses a serious threat to the global control of Plasmodium falciparum malaria. Discovery of the K13 marker has transformed approaches to the monitoring of artemisinin resistance, allowing introduction of molecular surveillance in remote areas through analysis of DNA. We aimed to assess the spread of artemisinin-resistant P falciparum in Myanmar by determining the relative prevalence of P falciparum parasites carrying K13-propeller mutations. Methods We did this cross-sectional survey at malaria treatment centres at 55 sites in ten administrative regions in Myanmar, and in relevant border regions in Thailand and Bangladesh, between January, 2013, and September, 2014. K13 sequences from P falciparum infections were obtained mainly by passive case detection. We entered data into two geostatistical models to produce predictive maps of the estimated prevalence of mutations of the K13 propeller region across Myanmar. Findings Overall, 371 (39%) of 940 samples carried a K13-propeller mutation. We recorded 26 different mutations, including nine mutations not described previously in southeast Asia. In seven (70%) of the ten administrative regions of Myanmar, the combined K13-mutation prevalence was more than 20%. Geospatial mapping showed that the overall prevalence of K13 mutations exceeded 10% in much of the east and north of the country. In Homalin, Sagaing Region, 25 km from the Indian border, 21 (47%) of 45 parasite samples carried K13-propeller mutations. Interpretation Artemisinin resistance extends across much of Myanmar. We recorded P falciparum parasites carrying K13-propeller mutations at high prevalence next to the northwestern border with India. Appropriate therapeutic regimens should be tested urgently and implemented comprehensively if spread of artemisinin resistance to other regions is to be avoided. Funding Wellcome Trust–Mahidol University–Oxford Tropical Medicine Research

Genetic diversity of the merozoite surface protein-3 gene in Plasmodium falciparum populations in Thailand.

PubMed

Pattaradilokrat, Sittiporn; Sawaswong, Vorthon; Simpalipan, Phumin; Kaewthamasorn, Morakot; Siripoon, Napaporn; Harnyuttanakorn, Pongchai

2016-10-21

An effective malaria vaccine is an urgently needed tool to fight against human malaria, the most deadly parasitic disease of humans. One promising candidate is the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum. This antigenic protein, encoded by the merozoite surface protein (msp-3) gene, is polymorphic and classified according to size into the two allelic types of K1 and 3D7. A recent study revealed that both the K1 and 3D7 alleles co-circulated within P. falciparum populations in Thailand, but the extent of the sequence diversity and variation within each allelic type remains largely unknown. The msp-3 gene was sequenced from 59 P. falciparum samples collected from five endemic areas (Mae Hong Son, Kanchanaburi, Ranong, Trat and Ubon Ratchathani) in Thailand and analysed for nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity. The gene was also subject to population genetic analysis (F st ) and neutrality tests (Tajima's D, Fu and Li D* and Fu and Li' F* tests) to determine any signature of selection. The sequence analyses revealed eight unique DNA haplotypes and seven amino acid sequence variants, with a haplotype and nucleotide diversity of 0.828 and 0.049, respectively. Neutrality tests indicated that the polymorphism detected in the alanine heptad repeat region of MSP-3 was maintained by positive diversifying selection, suggesting its role as a potential target of protective immune responses and supporting its role as a vaccine candidate. Comparison of MSP-3 variants among parasite populations in Thailand, India and Nigeria also inferred a close genetic relationship between P. falciparum populations in Asia. This study revealed the extent of the msp-3 gene diversity in P. falciparum in Thailand, providing the fundamental basis for the better design of future blood stage malaria vaccines against P. falciparum.

A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

PubMed

Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

2016-11-01

Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

Plasmodium falciparum SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle

PubMed Central

Stallmach, Robert; Kavishwar, Manoli; Withers-Martinez, Chrislaine; Hackett, Fiona; Collins, Christine R; Howell, Steven A; Yeoh, Sharon; Knuepfer, Ellen; Atid, Avshalom J; Holder, Anthony A; Blackman, Michael J

2015-01-01

The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle. PMID:25599609

The use of mobile phone data for the estimation of the travel patterns and imported Plasmodium falciparum rates among Zanzibar residents.

PubMed

Tatem, Andrew J; Qiu, Youliang; Smith, David L; Sabot, Oliver; Ali, Abdullah S; Moonen, Bruno

2009-12-10

Malaria endemicity in Zanzibar has reached historically low levels, and the epidemiology of malaria transmission is in transition. To capitalize on these gains, Zanzibar has commissioned a feasibility assessment to help inform on whether to move to an elimination campaign. Declining local transmission has refocused attention on imported malaria. Recent studies have shown that anonimized mobile phone records provide a valuable data source for characterizing human movements without compromising the privacy of phone users. Such movement data in combination with spatial data on P. falciparum endemicity provide a way of characterizing the patterns of parasite carrier movements and the rates of malaria importation, which have been used as part of the malaria elimination feasibility assessment for the islands of Zanzibar. Records encompassing three months of complete mobile phone usage for the period October-December 2008 were obtained from the Zanzibar Telecom (Zantel) mobile phone network company, the principal provider on the islands of Zanzibar. The data included the dates of all phone usage by 770,369 individual anonymous users. Each individual call and message was spatially referenced to one of six areas: Zanzibar and five mainland Tanzania regions. Information on the numbers of Zanzibar residents travelling to the mainland, locations visited and lengths of stay were extracted. Spatial and temporal data on P. falciparum transmission intensity and seasonality enabled linkage of this information to endemicity exposure and, motivated by malaria transmission models, estimates of the expected patterns of parasite importation to be made. Over the three month period studied, 88% of users made calls that were routed only through masts on Zanzibar, suggesting that no long distance travel was undertaken by this group. Of those who made calls routed through mainland masts the vast majority of trips were estimated to be of less than five days in length, and to the Dar Es Salaam

Impairment of the Plasmodium falciparum erythrocytic cycle induced by angiotensin peptides.

PubMed