Theoretical analysis of mixed Plasmodium malariae and Plasmodium falciparum infections with partial cross-immunity

A deterministic model for assessing the dynamics of mixed species malaria infections in a human population is presented to investigate the effects of dual infection with Plasmodium malariae and Plasmodium falciparum. Qualitative analysis of the model including positivity and boundedness is performed. In addition to the disease free equilibrium, we show that there exists a boundary equilibrium corresponding to each species. The isolation reproductive number of each species is computed as well as the reproductive number of the full model. Conditions for global stability of the disease free equilibrium as well as local stability of the boundary equilibria are derived. The model has an interior equilibrium which exists if at least one of the isolation reproductive numbers is greater than unity. Among the interesting dynamical behaviours of the model, the phenomenon of backward bifurcation where a stable boundary equilibrium coexists with a stable interior equilibrium, for a certain range of the associated invasion reproductive number less than unity is observed. Results from analysis of the model show that, when cross-immunity between the two species is weak, there is a high probability of coexistence of the two species and when cross-immunity is strong, competitive exclusion is high. Further, an increase in the reproductive number of species i increases the stability of its boundary equilibrium and its ability to invade an equilibrium of species j. Numerical simulations support our analytical conclusions and illustrate possible behaviour scenarios of the model.     

Plasmodium knowlesi from archival blood films: Further evidence that human infections are widely distributed and not newly emergent in Malaysian Borneo

Human infections with Plasmodium knowlesi have been misdiagnosed by microscopy as Plasmodium malariae due to their morphological similarities. Although microscopy-identified P. malariae cases have been reported in the state of Sarawak (Malaysian Borno) as early as 1952, recent epidemiological studies suggest the absence of indigenous P. malariae infections. The present study aimed to determine the past incidence and distribution of P. knowlesi infections in the state of Sarawak based on archival blood films from patients diagnosed by microscopy as having P. malariae infections. Nested PCR assays were used to identify Plasmodium species in DNA extracted from 47 thick blood films collected in 1996 from patients in seven different divisions throughout the state of Sarawak. Plasmodium knowlesi DNA was detected in 35 (97.2%) of 36 blood films that were positive for Plasmodium DNA, with patients originating from all seven divisions. Only one sample was positive for P. malariae DNA. This study provides further evidence of the widespread distribution of human infections with P. knowlesi in Sarawak and its past occurrence. Taken together with data from previous studies, our findings suggest that P. knowlesi malaria is not a newly emergent disease in humans.     

Confocal microscopic findings of cysteine protease calpain in Plasmodium falciparum

Pf-calpain, a cysteine protease of Plasmodium falciparum, is believed to be one of the central mediators for essential parasitic activity. However, the roles of calpain on parasitic activity have not been determined in P. falciparum. In the present study, the localization of Pf-calpain was investigated using polyclonal antibodies (anti-Pf-calpain antibody A and B) against peptides that distinguished it from human calpain-7 and rat calpain-10 protein. Recombinant Pf-calpain (rPf-calpain) was identified as a 46 kDa protein using an anti-Pf-calpain antibody A, which can recognize the Pf-calpain binding site. Confocal microscopy revealed calpain within cytoplasmic localized parasites in the erythrocytic cycle. The findings suggested that the expression of Pf-calpain would be proportional to all different parasites in the erythrocytic cycle. On the other hand, anti-human calpain-7 antibody detected Pf-calpain in schizonts, and the immunofluorescence was stronger than with anti-rat calpain-10 antibody. However, the antibodies reacted with calpains in human red blood cells. These results show that anti-Pf-calpain antibody A and B specifically recognize only Pf-calpain. Taken together, the results suggest that Pf-calpain is expressed in all erythrocytic stages. In particular, the expression of Pf-calpain is increased much more when the late ring matures into the early trophozoite. Moreover, anti-Pf-calpain antibody A and B against synthetic peptides of the catalytic domain of Pf-calpain are useful to specifically detect Pf-calpain in all erythrocytic stages, while human and rat calpain antibody are not useful.     

Microsensor study of photosynthesis and calcification in the scleractinian coral, Galaxea fascicularis: active internal carbon cycle

Sources of inorganic carbon (Ci) for photosynthesis and calcification and the mechanisms involved in their uptake in scleractinian corals were investigated in microcolonies of Galaxea fascicularis. Direct measurements of Ca²⁺, pH and O₂ on the surface and inside the polyp's coelenteron were made with microsensors. Gross photosynthesis (Pg) and net photosynthesis (Pn) were measured on the surface. Light respiration (LR) was calculated from Pg and Pn. The effect of light/dark and dark/light switches on Ca²⁺ and pH dynamics on the surface and inside the coelenteron were followed. To evaluate the different sources of Ci for photosynthesis and calcification, Ci-free seawater and 6-Ethoxyzolamide and Acetazolamide, inhibitors for carbonic anhydrase (CA) were used.In normal seawater, Pg was about seven times higher than Pn, the LR was ca. 80-90% of the Pg. Thus, most of the O₂ produced in Pg are immediately consumed in respiration, indicating the presence of a highly active internal C-cycle. As the internal C-cycle is highly active, a large part of the Ci for calcification will have passed through the metabolism of the symbiont. The high LR provides ATP for energy requiring processes in light.Ci for photosynthesis and calcification can come from seawater in the form of free Ci, respiration of photosynthates (internal C-cycle) or respiration of the ingested plankton. These sources form a common carbon pool (C-pool) that is used for the different processes.In Ci-free seawater, Pg decreased by about 12.5%, indicating that most of the photosynthetically fixed Ci can temporarily be supplied from internal sources. The initial decalcification, observed directly upon the switch to Ci-free seawater, showed that the Ca-pools in the coral are exchangeable. Part of the Pg in Ci-free seawater may depend on this decalcification for its Ci supply.Three localities of CA were defined. One on the surface facing seawater and one on endodermal cells facing the coelenteron, while the third is intracellular. The inhibition of CA decreased Pg by about 30%, while it increased the concentration of Ca²⁺ as a result of a decrease in its precipitation. The reduction of photosynthesis and calcification by CA inhibition demonstrated that both processes need the enzyme for the supply of Ci. The pH on the surface and inside the coelenteron decreased upon 6-Ethoxyzolamide addition indicating a role of CA in pH control.     

Pfs 16 pivotal role in Plasmodium falciparum gametocytogenesis: A potential antiplasmodial drug target

Mature gametocytes, the sexual stage of Plasmodium falciparum, ensure the continued transmission of malaria from the human host to the mosquito vector. Even if gametocytes are not implicated in the malaria physiopathology it is crucial to the spread of malaria. Gametocytes are to be a key target for drugs used against Plasmodium in public health. The expression levels of 4 sexual-stage specific genes, Pfs 16, Pfs 25, Pfg 27and S 18S rRNA, during gametocytogenesis of various P. falciparum strains were analyzed by a real time PCR assay. The strains showed different capacities to produce mature gametocytes and in parallel different patterns of sexual gene expression. There was a correlation only between Pfs 16 cDNA overexpression in the first 48 h of the culture and the production of mature gametocytes. Pfs 16 is an early marker of the development of mature gametocytes in cultures and is therefore a potential target for new antimalarial drugs.     

Methotrexate and aminopterin lack in vivo antimalarial activity against murine malaria species

The antifolate anticancer drug methotrexate (MTX) has potent activity against Plasmodium falciparum in vitro. Experience of its use in the treatment of rheumatoid arthritis indicates that it could be safe and efficacious for treating malaria. We sought to establish a murine malaria model to study the mechanism of action and resistance of MTX and its analogue aminopterin (AMP). We used Plasmodium berghei, Plasmodium yoelii yoelii, Plasmodium chabaudi and Plasmodium vinckei. None of these species were susceptible to either drug. We have also tested the efficacy of pyrimethamine in combination with folic acid in P. berghei, and data indicate that folic acid does not influence pyrimethamine efficacy, which suggests that P. berghei may not transport folate. Since MTX and AMP utilise folate receptor/transport to gain access to cells, their lack of efficacy against the four tested murine malaria species may be the result of inefficiency of drug transport.     

Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine-pyrimethamine in Mali

Sulfadoxine-pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.     

Kinetic properties and specificity of trimeric Plasmodium falciparum and human dUTPases

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. Hydrolysis of other nucleotides similar in structure to dUTP would be physiologically negative and therefore high substrate specificity is essential. Binding and hydrolysis of nucleotides different to dUTP by the dUTPases from Plasmodium falciparum (PfdUTPase) and human (hdUTPase) was evaluated by applying isothermal titration calorimetry (ITC). The ribo and deoxyribonucleoside triphosphates dGTP, dATP, dCTP, dTTP, UTP, FdUTP and IdUTP have been analysed. dUTP and FdUTP were the most specific substrates for both enzymes. The specificity constants (k_cat/K_m) for the remaining ones, except for the IdUTP, were very similar for both enzymes, although PfdUTPase showed a slightly higher specificity for dCTP and UTP and the human enzyme for dTTP and dCTP. PfdUTPase was very efficient in using FdUTP as substrate indicating that small size substituents in the 5′ position are well tolerated. In addition product inhibition was assessed by binding studies with the nucleoside monophosphate derivatives and thermodynamic parameters were established. When FdUTP hydrolysis was monitored, Plasmodium dUTPase was more sensitive to end-product inhibition by FdUMP than the human enzyme. Taken together these results highlight further significant differences between the human and Plasmodium enzymes that may be exploitable in selective inhibitor design.     

Plasmodium falciparum: In vitro interaction of quassin and neo-quassin with artesunate, a hemisuccinate derivative of artemisinin

Quassia amara L. (Family Simaroubaceae) is known to have several medicinal properties including the activity against malaria. An HPLC method was employed for purification of the biologically active quassinoids; quassin (Q) and neo-quassin (NQ), further characterized by MALDI-TOF analyses. Purified Q, NQ and the crude bark extract (S1) along with artesunate (AS) were studied for their in vitro anti-plasmodial activity. The in vivo toxicity studies at intraperitoneal doses with higher concentrations of the crude bark extract (S1) in Balb/C mice ruled out the apprehension of toxicity. Interaction studies between the test compounds among themselves (Q + NQ) and individually with artesunate (AS + Q, AS + NQ), were carried out in vitro at four ratios (1:5, 1:2, 2:1 and 5:1) on chloroquine sensitive (MRC-pf-20) and resistant (MRC-pf-303) strains of Plasmodium falciparum. The crude bark extracts of Q. amara exhibited higher P. falciparum inhibitory activity (IC₅₀ = 0.0025 μg/ml) as compared to that of the isolated compounds, quassin (IC₅₀ = 0.06 μg/ml, 0.15 μM), neo-quassin (IC₅₀ = 0.04 μg/ml, 0.1 μM) and also to the positive control, artesunate (IC₅₀ = 0.02 μg/ml, 0.05 μM). The in vitro drug interaction study revealed the compounds, quassin and neo-quassin to be additive to each other. At lower ratios, artesunate was found to be a potential combination partner with both the compounds. It was interesting to note that none of the combinations exhibited antagonistic interactions. This phenomenon offers the opportunity for further exploration of novel therapeutic concentrations and combinations.     

Sulfadoxine-pyrimethamine impairs Plasmodium falciparum gametocyte infectivity and Anopheles mosquito survival

Sulfadoxine-pyrimethamine (SP) is currently the drug of choice for intermittent preventive treatment of Plasmodiumfalciparum both in pregnancy and infancy. A prolonged parasite clearance time conferred by dhfr and dhps mutations is believed to be responsible for increased gametocyte prevalence in SP treated individuals. However, using a direct feeding assay in Mali, we showed that gametocytes present in peripheral venous blood post-SP treatment had reduced infectivity for Anophelesgambiae sensu stricto (ss) mosquitoes. We investigated the potential mechanisms involved in the dhfr and dhps quintuple mutant NF-135 and the single dhps 437 mutant NF-54. Concentrations of sulfadoxine (S) and pyrimethamine (P) equivalent to the serum levels of the respective drugs on day 3 (S = 61 μg/ml, P = 154.7 ng/ml) day 7 (S = 33.8 μg/ml, P = 66.6 ng/ml) and day 14 (S = 14.2 μg/ml, P = 15.7 ng/ml) post-SP treatment were used to study the effect on gametocytogenesis, gametocyte maturation and infectivity to Anophelesstephensi mosquitoes fed through an artificial membrane. The drugs readily induced gametocytogenesis in the mutant NF-135 strain but effectively killed the wild-type NF-54. However, both drugs impaired gametocyte maturation yielding odd-shaped non-exflagellating mature gametocytes. The concomitant ingestion of both S and P together with gametocytemic blood-meal significantly reduced the prevalence of oocyst positivity as well as oocyst density when compared to controls (P < 0.001). In addition, day 3 concentrations of SP decreased mosquito survival by up to 65% (P < 0.001). This study demonstrates that SP is deleterious in vitro for gametocyte infectivity as well as mosquito survival.     

Molecular characterization and localization of Plasmodium falciparum choline kinase

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K_m values for choline and ATP are found to be 145 ± 20 μM and 2.5 ± 0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the α and β content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.     

Plasmodium falciparum: Effect of Solanum nudum steroids on thiol contents and β-hematin formation in parasitized erythrocytes

We studied the effects on total thiols glutathione (GSH) and cysteine contents in Plasmodium falciparum in vitro when treated with four steroid derivatives and a sapogenin (Diosgenone) extracted from Solanum nudum. We also determined their capacity to inhibit β-hematin formation. We showed that SN-1 (16α-acetoxy-26-hydroxycholest-4-ene-3,22-dione) increased total glutathione and cysteine concentrations while SN-4 (26-O-β-d-glucopyranosyloxy-16α-acetoxycholest-4-ene-3,22-dione) decreased the concentration of both thiols. Acetylation in C16 was crucial for the effect of SN-1 while type furostanol and terminal glucosidation were necessary for the inhibitory properties of SN-4. The combination of steroids and buthionine sulfoximine, a specific inhibitor of a step-limiting enzyme in GSH synthesis, did not modify the glutathione contents. Finally, we found that SN-1 inhibited more than 80% of β-hematin formation at 5.0 mM, while the other steroids did not show any effect.     

In vivo inhibition of Helicoverpa armigera gut pro-proteinase activation by non-host plant protease inhibitors

We evaluated 22 different host and non-host plant protease inhibitors (PIs) for in vivo inhibition of Helicoverpa armigera gut pro- and proteinases, and their biological activity against the pod borer, H. armigera, the most important pest of agriculture and horticultural crops worldwide. In vitro activation of H. armigera gut pro-proteinases (HaGPPs) in larvae fed on non-host plant PIs showed significant in vivo inhibition of HaGPPs activation in solution as well as in gel assays. The larvae fed on diet incorporated with Datura alba ness PIs showed highest inhibition of HaGPPs, followed by Psophocarpus tetragonolobus. Non-host plant PIs from Pongamia pinnata, Mucuna pruriens, Capsicum annuum, and Nigela sativa showed maximum inhibitory potential towards HaGPs in vivo, and also exhibited moderate level of inhibition of pro-proteinases. However, some of non-host plant PIs, such as those from Penganum harmala and Solanum nigrum, and the principal host plant PIs, viz., Cicer arietinum and Cajanus cajan did not inhibit HaGPP activity. Pro-proteinase level increased with the growth of the larvae, and maximum HaGPP activity was observed in the fifth-instars. Larvae fed on diets with D. alba ness PIs showed greater inhibition of HaGPPs as compared to the larvae fed on diets with P. tetragonolobus. Low concentrations of partially purified HaGPs treated with gut extract of larvae fed on D. alba ness showed that out of 10 proteinase isoforms, HaGPs 5 and 9 were activators of pro-proteinases. Larval growth and development were significantly reduced in the larvae fed on the non-host plant PIs, of which D. alba ness resulted in highest stunted growth of H. armigera larvae. The in vivo studies indicated that non-host plant PIs were good candidates as inhibitors of the HaGPs as well as HaGPPs. The PIs from the non-host plants can be expressed in genetically engineered plants to confer resistance to H. armigera.     

In vitro and in vivo screening of essential oils for the control of wet bubble disease of Agaricus bisporus

Proliferation of fungal pathogens, such as Mycogone perniciosa, can severely affect the yields of cultivated mushrooms, including that of the button mushroom, Agaricus bisporus. A reduction in the number of fungicidal products approved for commercial application is currently providing new challenges to the mushroom industry. Forty essential oils, seven pure terpenoids and one phenylpropanoid were screened in vitro to determine the abilities of these substances to inhibit the growth of M. perniciosa. The fungal growth medium of both A. bisporus and M. perniciosa was supplemented with each test substance at a concentration of 50 μL/L. Ten essential oils were further investigated at lower concentrations ranging from 5 to 40 μL/L. The main components of these oils were determined by GC–FID and GC–MS. Lemon verbena (Lippia citriodora), lemongrass (Cymbopogon citratus) and thyme (Thymus vulgaris) oils were found to substantially inhibit the growth of the pathogen, while demonstrating lower toxicity towards A. bisporus than any of the other oils tested. A preliminary in vivo trial using M. perniciosa-inoculated casings revealed that the preventative use of lemon verbena or thyme oils was able to control the development of the disease. A commercial trial using these oils, as well as two of their main components (nerol and thymol), at a concentration of 40 μL/L, revealed that none of these treatments were detrimental to the growth of the A. bisporus and an overall yield similar to that following application of a commercial fungicide (Chronos 450 SC) was obtained. These results suggest that essential oils or mixtures of selected pure components of essential oils may in future find application in button mushroom production, either as a substitute for synthetic fungicides or as an additional protective measure.     

Plasmodium knowlesi as a Threat to Global Public Health

Malaria is a tropical disease caused by protozoans of the Plasmodium genus. Delayed diagnosis and misdiagnosis are strongly associated with higher mortality. In recent years, a greater importance is attributed to Plasmodium knowlesi, a species found mainly in Southeast Asia. Routine parasitological diagnostics are associated with certain limitations and difficulties in unambiguous determination of the parasite species based only on microscopic image. Recently, molecular techniques have been increasingly used for predictive diagnosis. The aim of the study is to draw attention to the risk of travelling to knowlesi malaria endemic areas and to raise awareness among personnel involved in the therapeutic process.     

Plasmodium chabaudi: Expression of active recombinant chabaupain-1 and localization studies in Anopheles sp.

Plasmodium cysteine proteases have been shown to be immunogenic and are being used as malaria potential serodiagnostic markers and vaccine targets. Genes encoding two Plasmodium chabaudi cysteine proteases chabaupain-1 (CP-1) and chabaupain-2 (CP-2) were identified and further expressed in Escherichia coli. Solubilisation of recombinant CP-1 and CP-2 was achieved by decreasing the temperature of induction.Anopheles gambiae tissues infected with Plasmodium were analyzed by Western blotting using the anti-CP-1 antibody showing that CP-1 is only present in the A. gambiae midguts being absent from other infected mosquito biological material. Anti-CP-1 anti-serum recognized a 30 kDa band in P. chabaudi, Plasmodium berghei and Plasmodium yoelii lysates but does not recognize the recombinant CP-2 extracts suggesting high antibody specificity.