Reduced Susceptibility of Plasmodium falciparum to Artesunate in Southern Myanmar

Background

Plasmodium falciparum resistance to artemisinins, the first line treatment for malaria worldwide, has been reported in western Cambodia. Resistance is characterized by significantly delayed clearance of parasites following artemisinin treatment. Artemisinin resistance has not previously been reported in Myanmar, which has the highest falciparum malaria burden among Southeast Asian countries.

Methods

A non-randomized, single-arm, open-label clinical trial of artesunate monotherapy (4 mg/kg daily for seven days) was conducted in adults with acute blood-smear positive P. falciparum malaria in Kawthaung, southern Myanmar. Parasite density was measured every 12 hours until two consecutive negative smears were obtained. Participants were followed weekly at the study clinic for three additional weeks. Co-primary endpoints included parasite clearance time (the time required for complete clearance of initial parasitemia), parasite clearance half-life (the time required for parasitemia to decrease by 50% based on the linear portion of the parasite clearance slope), and detectable parasitemia 72 hours after commencement of artesunate treatment. Drug pharmacokinetics were measured to rule out delayed clearance due to suboptimal drug levels.

Results

The median (range) parasite clearance half-life and time were 4.8 (2.1–9.7) and 60 (24–96) hours, respectively. The frequency distributions of parasite clearance half-life and time were bimodal, with very slow parasite clearance characteristic of the slowest-clearing Cambodian parasites (half-life longer than 6.2 hours) in approximately 1/3 of infections. Fourteen of 52 participants (26.9%) had a measurable parasitemia 72 hours after initiating artesunate treatment. Parasite clearance was not associated with drug pharmacokinetics.

Conclusions

A subset of P. falciparum infections in southern Myanmar displayed markedly delayed clearance following artemisinin treatment, suggesting either emergence of artemisinin resistance in southern Myanmar or spread to this location from its site of origin in western Cambodia. Resistance containment efforts are underway in Myanmar.

Trial Registration

Australian New Zealand Clinical Trials Registry ACTRN12610000896077     

Quantitative Analysis of Immune Response and Erythropoiesis during Rodent Malarial Infection

Malarial infection is associated with complex immune and erythropoietic responses in the host. A quantitative understanding of these processes is essential to help inform malaria therapy and for the design of effective vaccines. In this study, we use a statistical model-fitting approach to investigate the immune and erythropoietic responses in Plasmodium chabaudi infections of mice. Three mouse phenotypes (wildtype, T-cell-deficient nude mice, and nude mice reconstituted with T-cells taken from wildtype mice) were infected with one of two parasite clones (AS or AJ). Under a Bayesian framework, we use an adaptive population-based Markov chain Monte Carlo method and fit a set of dynamical models to observed data on parasite and red blood cell (RBC) densities. Model fits are compared using Bayes'' factors and parameter estimates obtained. We consider three independent immune mechanisms: clearance of parasitised RBCs (pRBC), clearance of unparasitised RBCs (uRBC), and clearance of parasites that burst from RBCs (merozoites). Our results suggest that the immune response of wildtype mice is associated with less destruction of uRBCs, compared to the immune response of nude mice. There is a greater degree of synchronisation between pRBC and uRBC clearance than between either mechanism and merozoite clearance. In all three mouse phenotypes, control of the peak of parasite density is associated with pRBC clearance. In wildtype mice and AS-infected nude mice, control of the peak is also associated with uRBC clearance. Our results suggest that uRBC clearance, rather than RBC infection, is the major determinant of RBC dynamics from approximately day 12 post-innoculation. During the first 2–3 weeks of blood-stage infection, immune-mediated clearance of pRBCs and uRBCs appears to have a much stronger effect than immune-mediated merozoite clearance. Upregulation of erythropoiesis is dependent on mouse phenotype and is greater in wildtype and reconstitited mice. Our study highlights the informative power of statistically rigorous model-fitting techniques in elucidating biological systems.     

Clonal Variants of Plasmodium falciparum Exhibit a Narrow Range of Rolling Velocities to Host Receptor CD36 under Dynamic Flow Conditions

Cytoadhesion of Plasmodium falciparum parasitized red blood cells (pRBCs) has been implicated in the virulence of malaria infection. Cytoadhesive interactions are mediated by the protein family of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). The PfEMP1 family is under strong antibody and binding selection, resulting in extensive sequence and size variation of the extracellular domains. Here, we investigated cytoadhesion of pRBCs to CD36, a common receptor of P. falciparum field isolates, under dynamic flow conditions. Isogeneic parasites, predominantly expressing single PfEMP1 variants, were evaluated for binding to recombinant CD36 under dynamic flow conditions using microfluidic devices. We tested if PfEMP1 size (number of extracellular domains) or sequence variation affected the pRBC-CD36 interaction. Our analysis showed that clonal parasite variants varied ∼5-fold in CD36 rolling velocity despite extensive PfEMP1 sequence polymorphism. In addition, adherent pRBCs exhibited a characteristic hysteresis in rolling velocity at microvascular flow rates, which was accompanied by changes in pRBC shape and may represent important adaptations that favor stable binding.     

Rapid Response to Selection,Competitive Release and Increased Transmission Potential of Artesunate-Selected Plasmodium chabaudi Malaria Parasites

The evolution of drug resistance, a key challenge for our ability to treat and control infections, depends on two processes: de-novo resistance mutations, and the selection for and spread of resistant mutants within a population. Understanding the factors influencing the rates of these two processes is essential for maximizing the useful lifespan of drugs and, therefore, effective disease control. For malaria parasites, artemisinin-based drugs are the frontline weapons in the fight against disease, but reports from the field of slower parasite clearance rates during drug treatment are generating concern that the useful lifespan of these drugs may be limited. Whether slower clearance rates represent true resistance, and how this provides a selective advantage for parasites is uncertain. Here, we show that Plasmodium chabaudi malaria parasites selected for resistance to artesunate (an artemisinin derivative) through a step-wise increase in drug dose evolved slower clearance rates extremely rapidly. In single infections, these slower clearance rates, similar to those seen in the field, provided fitness advantages to the parasite through increased overall density, recrudescence after treatment and increased transmission potential. In mixed infections, removal of susceptible parasites by drug treatment led to substantial increases in the densities and transmission potential of resistant parasites (competitive release). Our results demonstrate the double-edged sword for resistance management: in our initial selection experiments, no parasites survived aggressive chemotherapy, but after selection, the fitness advantage for resistant parasites was greatest at high drug doses. Aggressive treatment of mixed infections resulted in resistant parasites dominating the pool of gametocytes, without providing additional health benefits to hosts. Slower clearance rates can evolve rapidly and can provide a strong fitness advantage during drug treatment in both single and mixed strain infections.     

Improved In Vitro Culture of Plasmodium falciparum Permits Establishment of Clinical Isolates with Preserved Multiplication,Invasion and Rosetting Phenotypes

To be able to robustly propagate P. falciparum at optimal conditions in vitro is of fundamental importance for genotypic and phenotypic studies of both established and fresh clinical isolates. Cryo-preserved P. falciparum isolates from Ugandan children with severe or uncomplicated malaria were investigated for parasite phenotypes under different in vitro growth conditions or studied directly from the peripheral blood. The parasite cultures showed a minimal loss of parasite-mass and preserved percentage of multiple infected pRBCs to that in peripheral blood, maintained adhesive phenotypes and good outgrowth and multiplication rates when grown in suspension and supplemented with gas. In contrast, abnormal and greatly fluctuating levels of multiple infections were observed during static growth conditions and outgrowth and multiplication rates were inferior. Serum, as compared to Albumax, was found necessary for optimal presentation of PfEMP1 at the pRBC surface and/or for binding of serum proteins (immunoglobulins). Optimal in vitro growth conditions of P. falciparum therefore include orbital shaking (50 rev/min), human serum (10%) and a fixed gas composition (5% O₂, 5% CO₂, 90% N₂). We subsequently established 100% of 76 frozen patient isolates and found rosetting with schizont pRBCs in every isolate (>26% schizont rosetting rate). Rosetting during schizogony was often followed by invasion of the bound RBC as seen by regular and time-lapse microscopy as well as transmission electron microscopy. The peripheral parasitemia, the level of rosetting and the rate of multiplication correlated positively to one another for individual isolates. Rosetting was also more frequent with trophozoite and schizont pRBCs of children with severe versus uncomplicated malaria (p<0.002; p<0.004). The associations suggest that rosetting enhances the ability of the parasite to multiply within the human host.     

Evasion of Immunity to Plasmodium falciparum: Rosettes of Blood Group A Impair Recognition of PfEMP1

The ABO blood group antigens are expressed on erythrocytes but also on endothelial cells, platelets and serum proteins. Notably, the ABO blood group of a malaria patient determines the development of the disease given that blood group O reduces the probability to succumb in severe malaria, compared to individuals of groups A, B or AB. P. falciparum rosetting and sequestration are mediated by PfEMP1, RIFIN and STEVOR, expressed at the surface of the parasitized red blood cell (pRBC). Antibodies to these antigens consequently modify the course of a malaria infection by preventing sequestration and promoting phagocytosis of pRBC. Here we have studied rosetting P. falciparum and present evidence of an immune evasion mechanism not previously recognized. We find the accessibility of antibodies to PfEMP1 at the surface of the pRBC to be reduced when P. falciparum forms rosettes in blood group A RBC, as compared to group O RBC. The pRBC surrounds itself with tightly bound normal RBC that makes PfEMP1 inaccessible to antibodies and clearance by the immune system. Accordingly, pRBC of in vitro cloned P. falciparum devoid of ABO blood group dependent rosetting were equally well detected by anti-PfEMP1 antibodies, independent of the blood group utilized for their propagation. The pathogenic mechanisms underlying the severe forms of malaria may in patients of blood group A depend on the ability of the parasite to mask PfEMP1 from antibody recognition, in so doing evading immune clearance.     

Pharmacokinetics and pharmacodynamics of intravenous artesunate during severe malaria treatment in Ugandan adults

ABSTRACT: BACKGROUND: Severe malaria is a medical emergency with high mortality. Prompt achievement of therapeutic concentrations of highly effective anti-malarial drugs reduces the risk of death. The aim of this study was to assess the pharmacokinetics and pharmacodynamics of intravenous artesunate in Ugandan adults with severe malaria. METHODS: Fourteen adults with severe falciparum malaria requiring parenteral therapy were treated with 2.4 mg/kg intravenous artesunate. Blood samples were collected after the initial dose and plasma concentrations of artesunate and dihydroartemisinin measured by solid-phase extraction and liquid chromatography-tandem mass spectrometry. The study was approved by the Makerere University Faculty of Medicine Research and Ethics Committee (Ref2010-015) and Uganda National Council of Science and Technology (HS605) and registered with ClinicalTrials.gov (NCT01122134). RESULTS: All study participants achieved prompt resolution of symptoms and complete parasite clearance with median (range) parasite clearance time of 17 (8-24) hours. Median (range) maximal artesunate concentration (Cmax) was 3260 (1020-164000) ng/mL, terminal elimination half-life (T1/2) was 0.25 (0.1-1.8) hours and total artesunate exposure (AUC) was 727 (290-111256) ngh/mL. Median (range) dihydroartemisinin Cmax was 3140 (1670-9530) ng/mL, with Tmax of 0.14 (0.6 - 6.07) hours and T1/2 of 1.31 (0.8-2.8) hours. Dihydroartemisinin AUC was 3492 (2183-6338) ngh/mL. None of the participants reported adverse events. CONCLUSIONS: Plasma concentrations of artesunate and dihydroartemisinin were achieved rapidly with rapid and complete symptom resolution and parasite clearance with no adverse events.     

Interaction of mouse dendritic cells and malaria-infected erythrocytes: uptake, maturation, and antigen presentation

Consistent with their seminal role in detecting infection, both mouse bone marrow-derived and splenic CD11c+ dendritic cells (DCs) exhibited higher levels of uptake of Plasmodium chabaudi-parasitized RBCs (pRBCs) than of noninfected RBCs (nRBCs) as determined by our newly developed flow cytometric technique using the dye CFSE to label RBCs before coculture with DCs. To confirm that expression of CFSE by CD11c+ cells following coculture with CFSE-labeled pRBCs represents internalization of pRBC by DCs, we showed colocalization of CFSE-labeled pRBCs and PE-labeled CD11c+ DCs by confocal fluorescence microscopy. Treatment of DCs with cytochalasin D significantly inhibited the uptake of pRBCs, demonstrating that uptake is an actin-dependent phagocytic process. The uptake of pRBCs by splenic CD11c+ DCs was significantly enhanced after infection in vivo and was associated with the induction of DC maturation, IL-12 production, and stimulation of CD4+ T cell proliferation and IFN-gamma production. These results suggest that DCs selectively phagocytose pRBCs and present pRBC-derived Ags to CD4+ T cells, thereby promoting development of protective Th1-dependent immune responses to blood-stage malaria infection.     

Post-treatment haemolysis in severe imported malaria after intravenous artesunate: case report of three patients with hyperparasitaemia

ABSTRACT: Parenteral artesunate has been shown to be a superior treatment option compared to parenteral quinine in adults and children with severe malaria. Little evidence, however, is available on long-term safety. Recently, cases of late-onset haemolysis after parenteral treatment with artesunate have been reported in European travellers with imported Plasmodium falciparum malaria. Therefore, an extended follow-up of adult patients treated for severe imported malaria was started in August 2011 at the University Medical Center Hamburg-Eppendorf. Until January 2012, three patients with hyperparasitaemia (range: 14-21%) were included for analysis. In all three patients, delayed haemolysis was detected in the second week after the first dose of intravenous artesunate. Reticulocyte production index remained inadequately low in the 7 - 14 days following the first dose of artesunate despite rapid parasite clearance. Post-treatment haemolysis after parenteral artesunate may be of clinical relevance in particular in imported severe malaria characterized by high parasite levels. Extended followup of at least 30 days including controls of haematological parameters after artesunate treatment seems to be indicated. Further investigations are needed to assess frequency and pathophysiological background of this complication.     

Is malarial anaemia homologous to neocytolysis after altitude acclimatisation?

Malaria patients frequently develop severe anaemia that can persist after Plasmodium infection has been cleared from the circulation. This puzzling phenomenon involves massive death of young uninfected erythrocytes at a time when parasitic infection is very low. We have observed striking similarities in erythrocyte homoeostasis during altitude acclimatisation and Plasmodium infection, both of which initially induce an increase in circulating erythropoietin (Epo). Decreasing levels of Epo after return to low altitudes induce the death of young erythrocytes, a phenomenon called neocytolysis. In a similar way, we propose that Epo, which peaks during acute malaria and decreases after parasite clearance, could be contributing to anaemia causing neocytolysis during recovery from Plasmodium infection.     

The decline of malaria in Finland – the impact of the vector and social variables

Background

Resistance to anti-malarial drugs hampers control efforts and increases the risk of morbidity and mortality from malaria. The efficacy of standard therapies for uncomplicated Plasmodium falciparum and Plasmodium vivax malaria was assessed in Chumkiri, Kampot Province, Cambodia.

Methods

One hundred fifty-one subjects with uncomplicated falciparum malaria received directly observed therapy with 12 mg/kg artesunate (over three days) and 25 mg/kg mefloquine, up to a maximum dose of 600 mg artesunate/1,000 mg mefloquine. One hundred nine subjects with uncomplicated vivax malaria received a total of 25 mg/kg chloroquine, up to a maximum dose of 1,500 mg, over three days. Subjects were followed for 42 days or until recurrent parasitaemia was observed. For P. falciparum infected subjects, PCR genotyping of msp1, msp2, and glurp was used to distinguish treatment failures from new infections. Treatment failure rates at days 28 and 42 were analyzed using both per protocol and Kaplan-Meier survival analysis. Real Time PCR was used to measure the copy number of the pfmdr1 gene and standard 48-hour isotopic hypoxanthine incorporation assays were used to measure IC₅₀ for anti-malarial drugs.

Results

Among P. falciparum infected subjects, 47.0% were still parasitemic on day 2 and 11.3% on day 3. The PCR corrected treatment failure rates determined by survival analysis at 28 and 42 days were 13.1% and 18.8%, respectively. Treatment failure was associated with increased pfmdr1 copy number, higher initial parasitaemia, higher mefloquine IC₅₀, and longer time to parasite clearance. One P. falciparum isolate, from a treatment failure, had markedly elevated IC₅₀ for both mefloquine (130 nM) and artesunate (6.7 nM). Among P. vivax infected subjects, 42.1% suffered recurrent P. vivax parasitaemia. None acquired new P. falciparum infection.

Conclusion

The results suggest that artesunate-mefloquine combination therapy is beginning to fail in southern Cambodia and that resistance is not confined to the provinces at the Thai-Cambodian border. It is unclear whether the treatment failures are due solely to mefloquine resistance or to artesunate resistance as well. The findings of delayed clearance times and elevated artesunate IC₅₀ suggest that artesunate resistance may be emerging on a background of mefloquine resistance.     

Sensitivity of Plasmodium falciparum to Antimalarial Drugs in Hainan Island,China

Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5±10.6 hr) and the mean parasite clearance time (27.3±12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P>0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (P<0.0001). The resistance intensities decreased as follows: chloroquine>piperaquine>pyronarididine>artesunate. The inhibitory dose 50 (IC₅₀) was 3.77×10^-6 mol/L, 2.09×10^-6 mol/L, 0.09×10^-6 mol/L, and 0.05×10^-6 mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60×10^-6 mol/L, 9.26×10^-6 mol/L, 0.55×10^-6 mol/L, and 0.07×10^-6 mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity.     

Sustainable malaria control: transdisciplinary approaches for translational applications

Background

Intravenous (IV) artesunate is the treatment of choice for severe malaria. In Europe, however, no GMP-manufactured product is available and treatment data in European travellers are scarce. Fortunately, artesunate became available in the Netherlands and Belgium through a named patient programme. This is the largest case series of artesunate treated patients with severe malaria in Europe.

Methods

Hospitalized patients treated with IV artesunate between November 2007 and December 2010 in the Netherlands and Belgium were retrospectively evaluated. Patient characteristics, treatment and clinical outcome were recorded on a standardized form and mortality, parasite clearance times and the occurrence of adverse events were evaluated.

Results

Of the 68 treated patients, including 55 with severe malaria, two patients died (2/55 = 3.6%). The mean time to 50% parasite clearance (PCT50), 90% and 99% were 4.4 hours (3.9 - 5.2), 14.8 hours (13.0 - 17.2), and 29.5 hours (25.9 - 34.4) respectively. Artesunate was well tolerated. However, an unusual form of haemolytic anaemia was observed in seven patients. The relationship with artesunate remains uncertain.

Conclusions

Data from the named patient programme demonstrate that IV artesunate is effective and well-tolerated in European travellers lacking immunity. However, increased attention needs to be paid to the possible development of haemolytic anaemia 2-3 weeks after start of treatment. Treatment of IV artesunate should be limited to the period that IV treatment is required and should be followed by a full oral course of an appropriate anti-malarial drug.     

Therapeutic efficacy of artesunate in the treatment of uncomplicated Plasmodium falciparum malaria and anti-malarial, drug-resistance marker polymorphisms in populations near the China- Myanmar border

ABSTRACT: BACKGROUND: The aim of this study was to evaluate the clinical outcome after seven-day artesunate monotherapy for uncomplicated Plasmodium falciparum malaria in Yingjiang County along the China-Myanmar border and investigate genetic polymorphisms in the P. falciparum chloroquine-resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps) and ATPase (pfatp6) genes. METHODS: Patients [greater than or equal to] one year of age with fever (axillary temperature [greater than or equal to]37.5 degreesC) or history of fever and P. falciparum mono-infection were included. Patients received anti-malarial treatment with artesunate (total dose of 16 mg/kg over seven days) by directly observed therapy. After a 28- day follow-up, treatment efficacy and effectiveness were assessed based on clinical and parasitological outcomes. Treatment failure was defined as recrudescence of the original parasite and distinguished with new infection confirmed by PCR. Analysis of gene mutation and amplification were performed by nested polymerase chain reaction. RESULTS: Sixty-five patients were enrolled; 10 withdrew from the study, and six were lost to follow-up. All but two patients demonstrated adequate clinical and parasitological response; 12 had detectable parasitaemia on day 3. These two patients were confirmed to be new infection by PCR. The efficacy of artesunate was 95.9 %. The pfcrt mutation in codon 76 was found in all isolates (100 %), and mutations in codons 71 and 72 were found in 4.8 % of parasite isolates. No mutation of pfmdr1 (codons 86 or 1246) was found. Among all samples, 5.1 % were wild type for pfdhfr, whereas the other samples had mutations in four codons (51, 59, 108 and 164), and mutations in pfdhps (codons 436, 437, 540 and 581) were found in all isolates. No samples had mutations in pfatp6 codons 623 or 769, but two new mutations (N683K and R756K) were found in 4.6 % and 9.2 % of parasite isolates, respectively. CONCLUSION: Plasmodium falciparum infection was associated with slow parasite clearance and suspected artemisinin resistance at the China-Myanmar border area. The prevalence of pfcrt 76 T and markers for SP resistance are still high. It should be strengthened further on parasite clearance time or clearance half life to confirm the resistance status, and molecular epidemiology should provide complementary information to assess the appropriateness of current policies based on artemisinin derivatives.     

Artesunate,a potential drug for treatment of Babesia infection

The effects of artesunate, a water-soluble artemisinin derivative, against Babesia species, including Babesia bovis, Babesia gibsoni and Babesia microti were studied. Cultures of B. bovis and B. gibsoni were treated with 0.26, 2.6, 26 and 260 μM artesunate, showing inhibition of parasite growth at concentrations equal to and greater than 2.6 μM artesunate by days 3 post-treatment for B. gibsoni and B. bovis in a dose-dependent manner. Consistent with in vitro experiments, artesunate was effective in the treatment of mice infected with B. microti at doses equal to and greater than 10 mg/kg of body weight on days 8–10 post-infection. Taken together, these results suggest that artesunate could be a potential drug against Babesia infection.     

Suppressive effects of the anti-oxidant N-acetylcysteine on the anti-malarial activity of artesunate

The anti-oxidant drug N-acetylcysteine (NAC) has been proposed as adjunctive treatment in severe falciparum malaria. However, this might inhibit the anti-malarial drug action of the artemisinins, which are thought to exert their parasitocidal action through oxidative damage. We studied the interaction between NAC and artesunate as well as quinine in an in vitro drug sensitivity assay. Combination with NAC reduced the parasitocidal effect of artesunate only within the first 6 h of incubation, whereas no interaction was observed with quinine. Pre-incubation of P. falciparum with NAC resulted in a similar inhibitory effect on the anti-malarial activity of artesunate, whereas no inhibition was observed when NAC was added 2 h after parasite exposure to artesunate. Assessment of parasite maturation inhibition by the standard Giemsa's staining was in accordance with the use of a vital staining. The results herein caution the use of adjunctive treatment for malaria infection. Combination of antagonistic drugs may lead to adverse effects.     

Resistance to Malaria by Enhanced Phagocytosis of Erythrocytes in LMP7-deficient Mice

General cellular functions of proteasomes occur through protein degradation, whereas the specific function of immunoproteasomes is the optimization of antigen processing associated with MHC class I. We and others previously reported that deficiency in subunits of immunoproteasomes impaired the activation of antigen-specific CD8⁺ T cells, resulting in higher susceptibility to tumor and infections. We demonstrated that CD8⁺ T cells contributed to protection against malaria parasites. In this study, we evaluated the role of immunoproteasomes in the course of infection with rodent malaria parasites. Unexpectedly, Plasmodium yoelii infection of mice deficient in LMP7, a catalytic subunit of immunoproteasomes, showed lower parasite growth in the early phase of infection and lower lethality compared with control mice. The protective characteristics of LMP7-deficient mice were not associated with enhanced immune responses, as the mutant mice showed comparable or diminished activation of innate and acquired immunity. The remarkable difference was observed in erythrocytes instead of immune responses. Parasitized red blood cells (pRBCs) purified from LMP7-deficient mice were more susceptible to phagocytosis by macrophages compared with those from wild-type mice. The susceptibility of pRBC to phagocytosis appeared to correlate with deformity of the membrane structures that were only observed after infection. Our results suggest that RBCs of LMP7-deficient mice were more likely to deform in response to infection with malaria parasites, presumably resulting in higher susceptibility to phagocytosis and in the partial resistance to malaria.     

Decreased reticuloendothelial system clearance and increased blood half-life and immune cell labeling for nano- and micron-sized superparamagnetic iron-oxide particles upon pre-treatment with Intralipid

Background

Superparamagnetic iron-oxide nanoparticles are useful as contrast agents for anatomical, functional and cellular MRI, drug delivery agents, and diagnostic biosensors. Nanoparticles are generally cleared by the reticuloendothelial system (RES), in particular taken up by Kupffer cells in the liver, limiting particle bioavailability and in-vivo applications. Strategies that decrease the RES clearance and prolong the circulation residence time of particles can improve the in-vivo targeting efficiency.

Methods

Intralipid 20.0%, an FDA approved nutritional supplement, was intravenously administered in rats at the clinical dose (2 g/kg) 1 h before intravenous injection of ultra-small superparamagnetic iron-oxide (USPIO) or micron-sized paramagnetic iron-oxide (MPIO) particles. Blood half-life, monocyte labeling efficiency, and particle biodistribution were assessed by magnetic resonance relaxometry, flow cytometry, inductively-coupled plasma MS, and histology.

Results

Pre-treatment with Intralipid resulted in a 3.1-fold increase in USPIO blood half-life and a 2-fold increase in USPIO-labeled monocytes. A 2.5-fold increase in MPIO blood half-life and a 5-fold increase in MPIO-labeled monocytes were observed following Intralipid pre-treatment, with a 3.2-fold increase in mean iron content up to 2.60 pg Fe/monocyte. With Intralipid, there was a 49.2% and 45.1% reduction in liver uptake vs. untreated controls at 48 h for USPIO and MPIO, respectively.

Conclusions

Intralipid pre-treatment significantly decreases initial RES uptake and increases in-vivo circulation and blood monocyte labeling efficiency for nano- and micron-sized superparamagnetic iron-oxide particles.

General significance

Our findings can have broad applications for imaging and drug delivery applications, increasing the bioavailability of nano- and micron-sized particles for target sites other than the liver.     

Plasmodium berghei: parasite clearance after treatment with dihydroartemisinin in an asplenic murine malaria model

Clinical reports indicate that malaria-infected asplenic patients have a reduced capacity for parasite clearance despite intensive antimalarial therapy. The aim of this study was to evaluate the efficacy of dihydroartemisinin in an asplenic murine malaria model. Mice were inoculated with Plasmodium berghei parasitised erythrocytes and received a single dose of dihydroartemisinin 56 h later, at 2-5% parasitaemia. Haematology, liver biochemistry and histopathology of key organs were performed to evaluate organ response to malaria infection. The nadir parasitaemia occurred 20 h after dihydroartemisinin administration, falling 2.8- to 6.0-fold and 2.7- to 6.9-fold in asplenic and intact mice, respectively, (10-100 mg/kg). Histopathology indicated increased stimulation of liver function/activity during malaria infection of asplenic mice (as compared to intact mice). Overall efficacy of single-dose dihydroartemisinin treatment in asplenic mice was similar to intact mice although the rate of recrudescence in asplenic mice was significantly greater than intact mice at 30 and 100 mg/kg. The asplenic murine malaria model could be used in pre-clinical studies of splenic function and clearance of malaria parasites, pathophysiological studies or antimalarial drug efficacy in asplenia.     

In Vitro and In Vivo High-Throughput Assays for the Testing of Anti-Trypanosoma cruzi Compounds

Background

The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.

Methods

In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC₅₀ lower than that of BZ.

Findings

This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.

Conclusion

These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.