In malaria, rosetting is described as a phenomenon where an infected erythrocyte (IRBC) is attached to uninfected erythrocytes (URBC). In some studies, rosetting has been associated with malaria pathogenesis. Here, we have identified a new type of rosetting. Using a step-by-step approach, we identified IGFBP7, a protein secreted by monocytes in response to parasite stimulation, as a rosette-stimulator for Plasmodium falciparum- and P. vivax-IRBC. IGFBP7-mediated rosette-stimulation was rapid yet reversible. Unlike type I rosetting that involves direct interaction of rosetting ligands on IRBC and receptors on URBC, the IGFBP7-mediated, type II rosetting requires two additional serum factors, namely von Willebrand factor and thrombospondin-1. These two factors interact with IGFBP7 to mediate rosette formation by the IRBC. Importantly, the IGFBP7-induced type II rosetting hampers phagocytosis of IRBC by host phagocytes.
The habitats of Eurycoma longifolia Jack, a slender tree, are jungles in Malaysia and Indonesia. It belongs to the family Simaroubaceae and is a source of quassinoids with anabolic, antimalarial and cytostatic activity. In this study, conducted during 2008 in Mae Sot, Thailand, a standardized extract of E. longifolia containing three major quassinoids, eurycomanone (1), 13,21-dihydroeurycomanone (2) and 13alpha(21)-epoxyeurycomanone (3) was evaluated for antiplasmodial activity against Plasmodium falciparum and its activity has been compared with that of artemisinin, using 38 fresh parasite isolates and assessment of inhibition of schizont maturation. The IC(50), IC(90) and IC(99) values for artemisinin were 4.30, 45.48 and 310.97 microg/l, and those for the root extract from E. longifolia 14.72, 139.65 and 874.15 microg/l respectively. The GMCOC for artemisinin was 337.81 mug/l, and for the plant extract it was 807.41 microg/l. The log-concentration probit regressions were parallel. The inhibitory activity of the E. longifolia extract was higher than that expected from the three quassinoids isolated from the plant, suggesting synergism between the quassinoids or the presence of other unidentified compounds.
A double blind, randomized, controlled Phase 2 clinical trial was conducted to assess the safety, immunogenicity, and biologic impact of the vaccine candidate Apical Membrane Antigen 1-Combination 1 (AMA1-C1), adjuvanted with Alhydrogel. Participants were healthy children 2-3 years old living in or near the village of Bancoumana, Mali. A total of 300 children received either the study vaccine or the comparator. No impact of vaccination was seen on the primary endpoint, the frequency of parasitemia measured as episodes >3000/microL/day at risk. There was a negative impact of vaccination on the hemoglobin level during clinical malaria, and mean incidence of hemoglobin <8.5 g/dL, in the direction of lower hemoglobin in the children who received AMA1-C1, although these differences were not significant after correction for multiple tests. These differences were not seen in the second year of transmission.
Malaria, a mosquito-transmitted parasitic disease, has been targeted for elimination in many parts of the world. For many years, Plasmodium vivax, Plasmodium falciparum, Plasmodium ovale and Plasmodium malariae have been known to cause malaria in humans. Now, Plasmodium knowlesi is considered to be an important cause of malaria, especially in Southeast Asia. The emergence of P. knowlesi with zoonotic implication is a challenge in the elimination efforts of malaria in Southeast Asia. P. knowlesi is known to cause severe complicated malaria in humans. P. knowlesi parasite is transmitted between humans and wild macaque through mosquito vectors. It appears that the malaria disease severity and host immune evasion depend on antigenic variation exhibited at the surface of the infected erythrocyte. P. knowlesi is sensitive to antimalarial drug artemisinin. Identification of vector species, their biting behavior, timely correct diagnosis, and treatment are important steps in disease management and control. There is a need to identify and implement effective intervention measures to cut the chain of transmissions from animals to humans. The zoonotic malaria definitely poses a significant challenge in elimination and subsequent eradication of all types of malaria from this globe.
Heterologous prime-boost immunisation strategies can evoke powerful antibody responses and may be of value in developing an improved malaria vaccine. Herein, we show that an immunisation protocol that primes Balb/c mice with a recombinant Bacille Calmette-Guérin (rBCG) vaccine consisting of a plasmid encoding a synthetic fragment of the ESAT-6 epitope of Mycobacterium tuberculosis, the fragment 2 region II of erythrocyte-binding antigen (F2RIIEBA) and the three repeat sequences of the circumsporozoite protein (NANP)3 of Plasmodium falciparum before subsequently boosting the mice with either two doses of the rBCG clone or with a DNA vaccine expressing the native form of F2RIIEBA generating higher serum anti-F2RIIEBA antibody levels than an immunisation protocol that calls for a homologous prime-boost with two doses of rBCG. These results demonstrate the potential of DNA vaccination in boosting the antibody response to a recombinant vaccine expressing multiple epitopes.
Exploiting natural resources for bioactive compounds is an attractive drug discovery strategy in search for new anti-malarial drugs with novel modes of action. Initial screening efforts in our laboratory revealed two preparations of soil-derived actinomycetes (H11809 and FH025) with potent anti-malarial activities. Both crude extracts showed glycogen synthase kinase 3β (GSK3β)-inhibitory activities in a yeast-based kinase assay. We have previously shown that the GSK3 inhibitor, lithium chloride (LiCl), was able to suppress parasitaemia development in a rodent model of malarial infection. The present study aims to evaluate whether anti-malarial activities of H11809 and FH025 involve the inhibition of GSK3β. The acetone crude extracts of H11809 and FH025 each exerted strong inhibition on the growth of Plasmodium falciparum 3D7 in vitro with 50% inhibitory concentration (IC50) values of 0.57 ± 0.09 and 1.28 ± 0.11 µg/mL, respectively. The tested extracts exhibited Selectivity Index (SI) values exceeding 10 for the 3D7 strain. Both H11809 and FH025 showed dosage-dependent chemo-suppressive activities in vivo and improved animal survivability compared to non-treated infected mice. Western analysis revealed increased phosphorylation of serine (Ser 9) GSK3β (by 6.79 to 6.83-fold) in liver samples from infected mice treated with H11809 or FH025 compared to samples from non-infected or non-treated infected mice. A compound already identified in H11809 (data not shown), dibutyl phthalate (DBP) showed active anti-plasmodial activity against 3D7 (IC50 4.87 ± 1.26 µg/mL which is equivalent to 17.50 µM) and good chemo-suppressive activity in vivo (60.80% chemo-suppression at 300 mg/kg body weight [bw] dosage). DBP administration also resulted in increased phosphorylation of Ser 9 GSK3β compared to controls. Findings from the present study demonstrate that the potent anti-malarial activities of H11809 and FH025 were mediated via inhibition of host GSK3β. In addition, our study suggests that DBP is in part the bioactive component contributing to the anti-malarial activity displayed by H11809 acting through the inhibition of GSK3β.
A seroepidemiological study was carried out on Orang Asli (Aborigines) children who lead a semi-nomadic life in the deep jungles of Ulu Kelantan, Malaysia. Out of a total of about 190 children below 14 years, 143 were studied. Blood was collected from finger pricks on standard "strip type" filter papers for indirect fluorescent antibody (IFA) tests with Plasmodium falciparum antigen. A positive reaction at 1:10 dilution in infants and young children was considered positive and the reasons are given. The P. falciparum antibody prevalence rate was 84.6% compared to 81.8% spleen and 43.4% parasite rates. Both P. Falciparum and P. vivax were present in children. The age-specific patterns of antibody, spleen and parasite rates were those of a hyperendemic community. There was a positive correlation between antibody and spleen rates up to the age of 9 years. In older children, the antibody rates increased while the spleen and the parasite rates dropped.
Studies show that the pH of the malaria parasite's digestive vacuole (DV) plays a key role in the physiological functions of this organelle and antimalarial drug accumulation, and yet is technically difficult to measure. In this study, a flow cytometry-based technique was developed to measure the DV pH using a ratiometric pH indicator, FITC-dextran loaded into the DV of saponin-permeabilized parasites. To calculate the DV pH, a standard pH calibration curve was generated by incubating the saponin-permeabilized cells in buffers with different pH in the presence of an ionophore, CCCP. The measured average pH of the DV was 5.27 ± 0.03 that is approximately the same in the parasites observed microscopically by Hayward et al. (2006) (5.50 ± 0.14) using the same probe. The removal of glucose from the medium, causing a rapid depletion of parasite ATP, resulted in an alkalization of the DV. The DV was reacidified upon restoration of glucose to the medium. This technique provides a rapid, simple and quantitative measurement of the DV pH on a large number of cells. It will also be useful in future attempts to evaluate the effect of antimalarial drugs (i.e. chloroquine and artemisinin-based drugs) in pH changes of the DV.
An antiplasmodial screening of Phyllanthus debilis and Phyllanthus urinaria was carried out. The medicinal plants were extracted and evaluated for in vitro antiplasmodial activity against D10 (chloroquine-sensitive, CQS) and Gombak A (chloroquine-resistant, CQR) strains of Plasmodium falciparum. The methanolic crudes from the soxhlet extraction were active against both strains however, P. urinaria (IC50 8.9 μg/ml with CQR strain) exhibited better anti-malarial activity compared to P. debilis (IC50 12.2 μg/ml with CQR strain). Furthermore, the methanolic crude of P. urinaria obtained by the cold extraction has good anti-malarial activity towards CQS (IC50 4.1 μg/ml). The concentration of macronutrients (calcium and magnesium) and trace metals (copper, manganese, iron and zinc) from three Phyllanthus species i.e. P. debilis Klein ex Wild., Phyllanthus niruri L., P. urinaria L. and Alpinia conchigera Griff. were determined using microwave digestion method and analyzed by Flame Atomic Absorption Spectroscopy. Standard Reference Material 1547 (peach leaves) was used to validate the method throughout this study. The recovery values were in the range of 80% to 120% which were in very good agreement with the certified values. The three Phyllanthus species and leaves of A. conchigera showed the highest concentration of calcium compared to other metals and macronutrients studied. The significant presence of all the important macronutrients and trace metals which are essential for human health and well-being substantiate their use medicinally in traditional practices.
The Plasmodium falciparum serine repeat antigen (SERA) is one of the promising blood-stage malarial vaccine candidates. In this study, recombinant Mycobacterium bovis bacille Calmette-Guerin (rBCG) expressing the 22 kDa protein (SE22) from the 47 kDa Nterminal domain of serine repeat antigen (SERA), generated in favour of mycobacterium codon usage, elicited specific immune response in BALB/c mice with a mixed Th1/Th2 profile. Immunized sera containing high levels of specific IgG1 and IgG2a against the epitope (as determined by ELISA) were reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA). Furthermore, the lymphocyte proliferative response to SE22 antigen from rBCG-immunized mice was higher than that of controls. The expression of intracellular cytokines (IL-2, IL-4 and IFNγ) in CD4+- and CD8+-cells was also enhanced following in-vitro stimulation with SE22. These findings indicate that a rBCG-based vaccine candidate expressing a blood-stage antigen of P. falciparum could enhance both humoral and cellular immune responses, thus paving the way for the rational use of rBCG as a vaccine candidate against malaria.
According to the report of the Intergovernmental Panel on Climate Change (IPCC), Malaysia will experience an increase of 3-5°C in the future. As the development of the malaria parasite, Plasmodium falciparum, is sensitive to temperature, we investigated, using computer models, the effect of increase of 3º and 5ºC on the possible changes in the epidemiology of malaria transmission of P. falciparum in Malaysia. Four environmentally different locations were selected: Kuala Lumpur (KL), Cameron Highlands (CH), Kota Kinabalu (KK) and Kinabalu Park (KP). The extrinsic incubation period (EIP) was estimated using hourly temperatures and the mean daily temperatures. The EIP values estimated using the mean daily temperature were lower than those computed from hourly temperatures in warmer areas (KL, KK), but higher in the cooler areas (CH, KP). The computer simulations also indicated that the EIP will be decreased if the temperature was raised by 3º or 5ºC, with the effect more pronounced for the greater temperature increase, and for the cooler places. The vector cohort that is still alive at a time to transmit malaria (s(EIP)) also increased when the temperature was raised, with the increase more pronounced in the cooler areas. This study indicates an increase in temperature will have more significant effect in shortening the EIP in a cooler place (eg CH, KP), resulting in a greater s(EIP), and consequently increasing the transmission intensity and malaria risk. A temperature increase arising from the global climate change will likely affect the epidemiology of malaria in Malaysia, especially in the cooler areas.
Proteins on the surface of Plasmodium falciparum merozoites are good targets for vaccine development against malaria because they are accessible to antibodies in the plasma. The 19 kDa C-terminus of merozoite surface protein-1 (MSP-1(19)) has been shown to induce both inhibitory as well as blocking antibodies, the latter blocking the protective effects of the former. Inhibitory antibodies bind to MSP-1(19) and inhibit merozoite invasion of red blood cells (RBC) but the binding of blocking antibodies can prevent binding of inhibitory antibodies thereby allowing the parasite to invade RBC. We constructed a synthetic version of the MSP-1(19) of the P. falciparum using mycobacterium codon usage by assembly PCR. The synthetic MSP-1(19) was mutated at various sites to promote the production of inhibitory but not blocking antibodies as previously reported. The native and mutated MSP-1(19) were cloned and expressed in Mycobacterium bovis bacille Calmette-Guerin (BCG) and the expressions of the recombinant proteins were detected by specific monoclonal antibodies (mAbs) namely, 12.10 and 1E1 against MSP-1(19) using Western blotting. The mutated MSP-1(19) protein reacted with the inhibitory mAb, 12.10, but not the blocking mAb, 1E1, paving the way for the construction of a potential recombinant BCG (rBCG) blood stage vaccine against malaria.
Macrophages are involved in innate immunity against malaria due to their ability to phagocytose infected erythrocytes and produce inflammatory cytokines, which are important for controlling parasite growth during malaria infection. In this study, the ability of a recombinant BCG (rBCG) vaccine expressing the 19-kDa C-terminus of merozoite surface protein-1 (MSP1-C) of Plasmodium falciparum, to stimulate the phagocytic activity and secretion of pro-inflammatory cytokines by the macrophage cell line J774A.1 was measured at varying times. The results demonstrate the ability of the rBCG construct to activate the inflammatory action of macrophages, which is important as a first-line of defence in clearing malaria infections.
An epidemiological cross-sectional study was undertaken to determine the endemicity of malaria among the Orang Asli population of Raub, Pahang. Malaria endemicity was measured in terms of the prevalence of parasitaemia and splenomegaly. A total of 520 Orang Asli were examined. The point prevalence of malaria was 24.2% (95% CI 20.7-25.1), with Plasmodium falciparum (67.5%) being the predominant species. Children < 12 years were at least 3.7 times more likely to be parasitaemic compared to those older. The prevalence of malaria among children 2-<10 years was 38.1% (95% CI 31.6-50.0). Spleen rate among children 2-<10 years old was 22.3% (95% CI 17.1-28.3). The average enlarged spleen size was 1.2. These findings classify the study area as being mesoendemic. Malaria control activities among the Orang Asli should focus on protecting vulnerable subgroups like young children. Measuring the level of malaria endemicity at regular intervals is fundamental in evaluating the effectiveness of malaria control programs.
Seven Malaysian medicinal plants were screened for their antiplasmodial activities in vitro. These plants were selected based on their traditional claims for treatment or to relieve fever. The plant extracts were obtained from Forest Research Institute Malaysia (FRIM). The antiplasmodial activities were carried out using the pLDH assay to Plasmodium falciparum D10 strain (sensitive strain) while the cytotoxic activities were carried out towards Madin- Darby bovine kidney (MDBK) cells using MTT assay. The concentration of extracts used for both screening assays were from the highest concentration 64 microg/ml, two fold dilution to the lowest concentration 0.03 microg/ml. Goniothalamus macrophyllus (stem extract) showed more than 60% growth inhibition while Goniothalamus scortechinii root and stem extract showed a 90% and more than 80% growth inhibition at the last concentration tested, 0.03 microg/ml. The G. scortechini (leaves extract) showed an IC50 (50% growth inhibition) at 8.53 microg/ml, Ardisia crispa (leaves extract) demonstrated an IC50 at 5.90 +/- 0.14 microg/ml while Croton argyratus (leaves extract) showed a percentage inhibition of more than 60% at the tested concentration. Blumea balsamifera root and stem showed an IC50 at 26.25 +/- 2.47 microg/ml and 7.75 +/- 0.35 microg/ ml respectively. Agathis borneensis (leaves extract) demonstrated a 50% growth inhibition at 11.00 +/- 1.41 microg/ml. The study gives preliminary scientific evidence of these plant extracts in line with their traditional claims.
In the present study we examined the effect of E. longifolia methanol extract (TA164) on the GSH levels of P. falciparum infected erythrocytes and uninfected erythrocytes. Our study on parasite growth shows the IC50 and IC75 values of TA164 to be 0.17 g/ml and 6 g/ml respectively while for BSO was 25.5 g/ml and 46.5 g/ml respectively. About 95% to 100% growth inhibition of P. falciparum infected erythrocyte was observed when treated with TA164 and BSO at 16 g/ml and 64 g/ml respectively. The study on GSH contents indicated that non-infected erythrocytes treated with 6 g/ml (IC75 values) of TA164 at 24 hours incubation showed less GSH content as compared to non-treated erythrocytes. A similar observation was seen on treated trophozoite infected erythrocyte (10% parasitemia) when treated with 6 g/ml at 3 hours incubation. Analysis of the GSH contents of parasite compartments treated with TA164 at the same concentration (6 g/ml) for 3 hours incubation indicated a reduction of GSH contents. At the same concentration, TA164 did not affect the GSH contents of enriched trophozoite infected erythrocytes (60-70% parasitemia). TA164 did affect the GSH content of non-infected erythrocyte at 24 hours (accept IC50 value) as well as the parasite compartments (trophozoite infected erythrocyte and parasite itself) but fails to affect the GSH content of enriched trophozoite infected erythrocyte.
Matched MeSH terms: Plasmodium falciparum/drug effects*; Plasmodium falciparum/growth & development
Malaria is one of the most dangerous infectious diseases due to its high infection and mortality rates, especially in the tropical belt. Plasmodium falciparum (P. falciparum), the most virulent malaria parasite in humans, was recently reported to develop resistance against the final efficient antimalarial drug, artemisinin. Little is known about the resistance mechanisms, which further complicates the problem as a proper counteraction is unable to be taken. Hence, the understanding of drug mode of action and its molecular target is valuable knowledge that needs to be considered to develop the next generation of antimalarial drugs. P. falciparum protein kinase (Pf PK) is an attractive target for antimalarial chemotherapy due to its vital roles in all P. falciparum life stages. Moreover, overall structural differences and the presence of unique Pf PKs that are absent in human kinome, suggesting specific inhibition of Pf PK without affecting human cells is achievable. To date, at least 86 eukaryotic protein kinases have been identified in P. falciparum kinome, by which less than 40 were validated as potential targets at the erythrocytes stage. In this review, recent progress of the furthest validated Pf PKs; Pf Nek-1, Pf CDPK1, Pf CDPK4, Pf PKG, and Pf CLK-3 will be briefly discussed.
Chloroquine resistance transporter of Plasmodium falciparum (PfCRT) is a food vacuolar transmembrane protein that mediates susceptibility of the parasite to chloroquine. A mutation at K76T of the Pfcrt gene is a key determinant for chloroquine resistance phenotype. In the absence of drug pressure, in vitro growth rate of chloroquine-resistance parasites was outcompeted by wild-type parasites unless intragenic compensatory mutations occurred. Chloroquine-resistant P. falciparum bearing the Cam734 haplotype known to circulate in endemic areas of Cambodia bordering Thailand contains 9 mutations in Pfcrt and exhibits both chloroquine resistance and comparable growth rate to the chloroquine-sensitive 3D7 strain. To analyze the evolution of the Cam734 haplotype, codon-based analysis was performed by using the mixed effects model of evolution (MEME), branch-site random effects likelihood (BR-REL) and other related methods. Results revealed that the Cam734 haplotype has evolved distinctively from other known mutant haplotypes including the most common Dd2 haplotype in Southeast Asia. Evidence of episodic positive selection was detected at codon 144, characterized by c.[430G>T; 431C>T] (p.A144F), known to be indispensable for both chloroquine resistance and restoration of growth rate of the parasites. To survey the prevalence of mutations at codons 76 and 144 in Pfcrt among Thai isolates, restriction fragment analysis of 548 P. falciparum isolates collected from six endemic provinces of Thailand during 1991 and 2016 was performed. The 144F Pfcrt mutant was detected in 7 (1.28%) isolates. All Thai isolates analyzed herein harbored a mutation at codon 76 whilst the wild-type parasite was not found. The low prevalence of isolates bearing the mutation 144F in PfCRT could imply little or lack of survival advantage of this mutant in endemic areas of Thailand where the wild-type parasites seem to be absent or extremely rare.
Studies profiling community and zonal malaria entomological risk indices are required to identify high risk areas where targeted control resources are most needed or likely to have the greatest impact on reducing risk of malaria infection. This study presents a first report on malaria vector risk indices in two vegetation zones within Adamawa state, Nigeria. Endophilic mosquitoes were collected for one year in selected communities in the Guinea and Sudan savanna zones within the State. Plasmodium falciparum Sporozoite and human blood meal ELISA assays were carried out on the female Anopheles mosquitoes collected. Sibling species composition of the An. gambiae complex were determined using PCR assays. Mean numbers of mosquitoes in the Guinea savanna communities were significantly (t = 7.73, DF = 11, p < 0.001) higher than the Sudan. Man-biting rates (F = 2.76, p = 0.13) of Anopheles mosquitoes were higher in the Guinea but not significantly different from Sudan savanna. Sporozoite rates of mosquitoes within the Guinea savanna were 2.7 times higher than the Sudan. The predominant Anopheles coluzzii species encountered in the state had higher overall human blood indices (0.63) and sporozoite rates (6.9%) compared to An. gambiae (0.39, 1.9%) and An. arabiensis (0.58, 2.3%) respectively. Overall annual human blood indices (0.59) of mosquitoes in Adamawa were lower compared to reports from other States. Prevalence and higher transmission risks indices of endophilic An. coluzzii mosquitoes reveal the need for LLIN and management of relatively permanent An. coluzzii breeding sites in the State. Widespread cattle rearing lifestyle and lower human blood indices of mosquitoes in the study area suggest the need to investigate cattle blood indices of the mosquitoes in the state. Higher entomological risk indices in the Guinea Savanna zone provide baseline information for prioritization of malaria vector control supplies within the State.
The reduced efficacy of the mainstay antimalarial drugs due to the widespread of drugresistant Plasmodium falciparum has necessitated efforts to discover new antimalarial drugs with new targets. Quercus infectoria (Olivier) has long been used to treat various ailments including fever. The acetone extract of the plant galls has recently been reported to have a promising antimalarial activity in vitro. This study was aimed to determine the effect of the Q. infectoria gall acetone crude extract on pH of the digestive vacuole of Plasmodium falciparum. A ratiometric fluorescent probe, fluorescein isothiocyanate-dextran (FITC-dextran) was used to facilitate a quantitative measurement of the digestive vacuole pH by flow cytometry. Mid trophozoite stage malaria parasites grown in resealed erythrocytes containing FITC-dextran were treated with different concentrations of the acetone extract based on the 50% inhibitory concentration (IC50). Saponin-permeabilized parasites were analyzed to obtain the ratio of green/yellow fluorescence intensity (Rgy) plotted as a function of pH in a pH calibration curve of FITC-dextran. Based on the pH calibration curve, the pH of the digestive vacuole of the acetone extract-treated parasites was significantly altered (pH values ranged from 6.35- 6.71) in a concentration-dependent manner compared to the untreated parasites (pH = 5.32) (p < 0.001). This study provides a valuable insight into the potential of the Q. infectoria galls as a promising antimalarial candidate with a novel mechanism of action.