Plasmodium falciparum malarial parasites from Brazil lack artemisinin resistance-associated mutations in the kelch13 gene

Abstract INTRODUCTION Mutations in the propeller domain of the Plasmodium falciparum kelch13 (k13) gene are associated with artemisinin resistance. METHODS: We developed a PCR protocol to sequence the pfk13 gene and determined its sequence in a batch of 50 samples collected from 2003 to 2016 in Brazil. RESULTS: We identified 1 K189T substitution located outside the propeller domain of the PfK13 protein in 36% of samples. CONCLUSIONS: Although the sample size is relatively small, these results suggest that P. falciparum artemisinin-resistant mutants do not exist in Brazil, thereby supporting the continuation of current treatment programs based on artemisinin-based combination therapy.

Highly active ozonides selected against drug resistant malaria

Ever increasing multi-drug resistance by Plasmodium falciparum is creating new challenges in malaria chemotherapy. In the absence of licensed vaccines, treatment and prevention of malaria is heavily dependent on drugs. Potency, range of activity, safety, low cost and ease of administration are crucial issues in the design and formulation of antimalarials. We have tested three synthetic ozonides NAC89, LC50 and LCD67 in vitro and in vivo against multidrug resistant Plasmodium. In vitro, LC50 was at least 10 times more efficient inhibiting P. falciparum multidrug resistant Dd2 strain than chloroquine and mefloquine and as efficient as artemisinin (ART), artesunate and dihydroartemisinin. All three ozonides showed high efficacy in clearing parasitaemia in mice, caused by multi-drug resistant Plasmodium chabaudi strains, by subcutaneous administration, demonstrating high efficacy in vivo against ART and artesunate resistant parasites.

Histological studies on the retina and cerebellum of Wistar rats treated with ArteetherTM

Introduction: Arteether TM, a derivative of artemisinin, is among the recent drugs that have given renewed hope for combating malarial menace. The present study investigated the effects of arteetherTM on the histology of the retina and cerebellum of Wistar rats. Materials and Methods: Twenty adult albino Wistar rats weighing 150-200 g, were randomly divided into four groups (A, B, C and D) of five animals each and used for this study. Group A rats were given intramuscular (i.m.) arteetherTM (3 mg/kg b.w.) daily for 3 days. Group B rats were given i.m. arteetherTM (6 mg/kg b.w.) daily for 3 days. Group C rats were also given i. m. of arteetherTM (3 mg/kg b. w.) daily for 3 days, and the same dose was repeated at two-weekly intervals for 4 further weeks; while Group D rats which received normal saline (0.9 % w/v, 3 ml/kg b.w.), served as controls. At the end of the experiment, the rats were sacrificed by cervical dislocation. The retina and cerebellum were excised and processed routinely for histopathology changes, using haematoxylin and eosin stain (H & E), as well as Nissl stain. Results: Results obtained showed normal cellular components of the retina and cerebellum in all groups, and no cyto-pathological changes were observed. Conclusion: Thus, this study showed that under light microscopic examination, therapeutic doses of arteetherTM caused no significant cyto-pathologic changes in the retina and cerebellum of Wistar rats.(AU)

In vitro susceptibility of Plasmodium falciparum Welch field isolates to infusions prepared from Artemisia annua L. cultivated in the Brazilian Amazon

Artemisinin is the active antimalarial compound obtained from the leaves of Artemisia annua L. Artemisinin, and its semi-synthetic derivatives, are the main drugs used to treat multi-drug-resistant Plasmodium falciparum (one of the human malaria parasite species). The in vitro susceptibility of P. falciparum K1 and 3d7 strains and field isolates from the state of Amazonas, Brazil, to A. annua infusions (5 g dry leaves in 1 L of boiling water) and the drug standards chloroquine, quinine and artemisinin were evaluated. The A. annua used was cultivated in three Amazon ecosystems (várzea, terra preta de índio and terra firme) and in the city of Paulínia, state of São Paulo, Brazil. Artemisinin levels in the A. annua leaves used were 0.90-1.13% (m/m). The concentration of artemisinin in the infusions was 40-46 mg/L. Field P. falciparum isolates were resistant to chloroquine and sensitive to quinine and artemisinin. The average 50% inhibition concentration values for A. annua infusions against field isolates were 0.11-0.14 μL/mL (these infusions exhibited artemisinin concentrations of 4.7-5.6 ng/mL) and were active in vitro against P. falciparum due to their artemisinin concentration. No synergistic effect was observed for artemisinin in the infusions.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii / Ação in vitro de drogas antiparasitárias, especialmente artesunato, contra Toxoplasma gondii

INTRODUCTION: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. METHODS: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. RESULTS: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. CONCLUSIONS: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

INTRODUÇÃO: Toxoplasmose é geralmente uma infecção benigna, exceto nos eventos de doença ocular, congênito e do sistema nervoso central, e particularmente quando o paciente é imunocomprometido. O tratamento consiste de drogas que frequentemente causam efeitos adversos, então novas drogas, mais efetivas são necessárias. Neste estudo, a possível atividade de artesunato, uma droga usada com sucesso no tratamento da malária, sobre o crescimento de Toxoplasma gondii em cultura celular é avaliado e comparado à ação de drogas que já estão sendo utilizadas contra este parasita. MÉTODOS: Células LLC-MK2 foram cultivadas em meio RPMI, mantidas em garrafas plásticas descartáveis e incubados a 36ºC com 5% CO2. Taquizoítos da cepa RH foram usados. As seguintes drogas foram testadas: artesunato, cotrimoxazol, pentamidina, pirimetamina, quinino e trimetoprima. Os efeitos dessas drogas sobre taquizoítos foram analisados por análise regressiva não linear com o software Prism 3.0. RESULTADOS: Artesunato mostrou uma concentração inibitória media (IC50) de 0,075µM e uma toxicidade sobre células LLC MK2 de 2,003µM. Pirimetamina foi efetiva a uma IC50 de 0,482µM e uma toxicidade de 11,178µM. Trimetoprima sozinha foi efetiva contra o parasita in vitro. Cotrimoxazol também foi efetivo contra o parasita, mas a concentrações mais altas que aquelas observadas para artesunato e pirimetamina. Pentamidina e quinino não tiveram efeitos inibitórios sobre os taquizoítos. CONCLUSÕES: Provou-se que artesunato in vitro pode ser uma alternativa útil para o tratamento da toxoplasmose, implicando um subsequente efeito in vivo e sugerindo o mecanismo desta droga contra o parasita.

Lamivudine-artesunate co-administration affects glucose metabolism in healthy and diseased wistar rats

HIV-Malaria co morbidity frequently requires the co administration of Lamivudine and Artesunate, in Malaria endemic areas where HIV is also a problem. This situation is a frequent occurrence in developing countries of the tropics, like Nigeria where the burden of Malaria and HIV is heavy. The co administration of these drugs may result in interactions with possible physiologic and/or therapeutic consequences. This study investigated the effect of Lamivudine-Artesunate co administration on body weight, relative organ weight and glucose tolerance in healthy and diseased [Plasmodium berghei infected and cyclophosphamide immunosuppressed] wistar rats. Animals received a cumulative 21 day treatment with Lamivudine [20 mg/kg] and/or 7 day Artesunate [10 mg/kg], with healthy or disease controls. Results showed that organ weights and body weights were not affected. Oral glucose was however affected in the combination and Artesunate groups in both disease and healthy rats. The study shows that glucose tolerance is altered with Lamivudine-Artesunate co administration, and may be beneficial, as hypoglycaemia is often a complication of Malaria therapy

Drug Resistance and in Vitro Susceptibility of Plasmodium falciparum in Thailand during 1988-2003

The aim of the present study was to investigate antimalarial drug pressure resulting from the clinical use of different antimalarials in Thailand. The phenotypic diversity of the susceptibility profiles of antimalarials, i.e., chloroquine (CQ), quinine (QN), mefloquine (MQ), and artesunate (ARS) in Plasmodium falciparum isolates collected during the period from 1988 to 2003 were studied. P. falciparum isolates from infected patients were collected from the Thai-Cambodian border area at different time periods (1988-1989, 1991-1992, and 2003), during which 3 different patterns of drug use had been implemented: MQ + sulphadoxine (S) + pyrimethamine (P), MQ alone and MQ + ARS, respectively. The in vitro drug susceptibilities were investigated using a method based on the incorporation of [3H] hypoxanthine. A total of 50 isolates were tested for susceptibilities to CQ, QN, MQ, and ARS. Of these isolates, 19, 16, and 15 were adapted during the periods 1988-1989, 1991-1993, and 2003, respectively. P. falciparum isolates collected during the 3 periods were resistant to CQ. Sensitivities to MQ declined from 1988 to 2003. In contrast, the parasite was sensitive to QN, and similar sensitivity profile patterns were observed during the 3 time periods. There was a significantly positive but weak correlation between the IC50 values of CQ and QN, as well as between the IC50 values of QN and MQ. Drug pressure has impact on sensitivity of P. falciparum to MQ. A combination therapy of MQ and ARS is being applied to reduce the parasite resistance, and also increasing the efficacy of the drug.

Host vascular endothelial growth factor is trophic for Plasmodium falciparum-infected red blood cells.

Plasmodium falciparum, the protozoan parasite responsible for severe malaria infection, undergoes a complex life cycle. Infected red blood cells (iRBC) sequester in host cerebral microvessels, which underlies the pathology of cerebral malaria. Using immunohistochemistry on post mortem brain samples, we demonstrated positive staining for vascular endothelial growth factor (VEGF) on iRBC. Confocal microscopy of cultured iRBC revealed accumulation of VEGF within the parasitophorous vacuole, expression of host VEGF-receptor 1 and activated VEGF-receptor 2 on the surface of iRBC, but no accumulation of VEGF receptors within the iRBC. Addition of VEGF to parasite cultures had a trophic effect on parasite growth and also partially rescued growth of drug treated parasites. Both these effects were abrogated when parasites were grown in serum-free medium, suggesting a requirement for soluble VEGF receptor. We conclude that P. falciparum iRBC can bind host VEGF-R on the erythrocyte membrane and accumulate host VEGF within the parasitophorous vacuole, which may have a trophic effect on parasite growth.

Gametocyte Clearance in Uncomplicated and Severe Plasmodium falciparum Malaria after Artesunate-Mefloquine Treatment in Thailand

Artemisinin-based combination therapy (ACT) is currently promoted as a strategy for treating both uncomplicated and severe falciparum malaria, targeting asexual blood-stage Plasmodium falciparum parasites. However, the effect of ACT on sexual-stage parasites remains controversial. To determine the clearance of sexual-stage P. falciparum parasites from 342 uncomplicated, and 217 severe, adult malaria cases, we reviewed and followed peripheral blood sexualstage parasites for 4 wk after starting ACT. All patients presented with both asexual and sexual stage parasites on admission, and were treated with artesunate-mefloquine as the standard regimen. The results showed that all patients were asymptomatic and negative for asexual forms before discharge from hospital. The percentages of uncomplicated malaria patients positive for gametocytes on days 3, 7, 14, 21, and 28 were 41.5, 13.1, 3.8, 2.0, and 2.0%, while the percentages of gametocyte positive severe malaria patients on days 3, 7, 14, 21, and 28 were 33.6, 8.2, 2.7, 0.9, and 0.9%, respectively. Although all patients were negative for asexual parasites by day 7 after completion of the artesunate-mefloquine course, gametocytemia persisted in some patients. Thus, a gametocytocidal drug, e.g., primaquine, may be useful in combination with an artesunate-mefloquine regimen to clear gametocytes, so blocking transmission more effectively than artesunate alone, in malaria transmission areas.

Effect of artesunate on Toxoplasma gondii: in vitro and in vivo studies

The effect of Artesunate [As] on Toxoplasma gondii [T. gondii] in vitro and in vivo was studied. In vitro, tachyzoites of RH strain were exposed to As in a concentration of 2 microg/ml for 72 hours. The assessment of As effect was carried out by studying the viability, infectivity and ultrastructure changes of treated tachyzoites by scanning electron microscope [SEM]. In the in vivo study, Swiss albino mice were infected intraperitoneally with tachyzoites of T. gondii RH strain, then orally treated with As in a dose of 200 mg/kg for five successive days. The effect of As was evaluated by the mortality rate and survival time of the treated mice. Parasite burden, viability, infectivity and ultrastructure changes of tachyzoites harvested from the peritoneal cavities of infected treated mice as compared with infected non-treated control mice were also studied. In vitro study demonstrated a significant reduction in viability and infectivity of tachyzoites exposed to As compared with untreated controls. In vivo study, showed that treatment of infected mice with As induced a significant decrease in mortality rate and increase in survival time. There was also a significant reduction in parasite burden in infected treated mice with significant reduction in viability and infectivity of tachyzoites harvested from them as compared with infected non-treated control. SEM showed distortion in tachyzoites' shape, peeling, erosions and discontinuity in areas of surface membrane of treated tachyzoites of both in vitro and in vivo studies. So, As proved an effective and promising drug in treating acute toxoplasmosis

Assessment of in vitro antimalarial interactions between dihydroartemisinin and fosmidomycin.

Malaria remains one of the leading causes of morbidity and mortality in the tropics with an annual estimate of 500 million clinical cases and 2 million deaths. The treatment and control of malaria is becoming increasingly difficult due to Plasmodium falciparum resistance to commonly used antimalarials. Combination therapy is currently the strategy for combating multi-drug resistant falciparum malaria, through exploiting pharmacodynamic synergistic effects and delaying the emergence of drug resistance. The combination of artemisinin derivatives with fosmidomycin, which have different modes of action, appears to be one of the most promising combinations. The objective of the present study was to investigate the antimalarial interactions between dihydroartemisinin and fosmidomycin in vitro, against chloroquine-resistant (K1) and chloroquine-sensitive (G112) P. falciparum strains. Concentration-response analysis was performed based on an in vitro schizont maturation inhibition test. The fixed concentration ratios of dihydroartemisinin: fosmidomycin used were 0:5,000, 2:4,500, 6:3,500, 10:2,500, 14:1,500, 18:500 and 20:0 nM. The highest final concentrations of dihydroartemisinin and fosmidomycin were 20 and 5,000 nM, respectively. Results showed IC50 (drug concentration which produced 50% schizont maturation inhibition) medians (range) for dihydroartemisinin against K1 and G112 strains to be 1.6 (1.2-2.0) and 2.5 (2.4-2.6) nM, respectively. The IC50 medians (range) for fosmidomycin against K1 and G112 strains were 1,347 (1,068-1,625) and 786 (737-834) nM, respectively. An isobologram revealed an increasing trend for the fraction IC50 (FIC), which indicates marked antagonism of this drug combination against both chloroquine resistant and chloroquine sensitive strains.

Antimalarial drugs disrupt ion homeostasis in malarial parasites

Plasmodium chabaudi malaria parasite organelles are major elements for ion homeostasis and cellular signaling and also target for antimalarial drugs. By using confocal imaging of intraerythrocytic parasites we demonstrated that the dye acridine orange (AO) is accumulated into P. chabaudi subcellular compartments. The AO could be released from the parasite organelles by collapsing the pH gradient with the K+/H+ ionophore nigericin (20 µM), or by inhibiting the H+-pump with bafilomycin (4 µM). Similarly, in isolated parasites loaded with calcium indicator Fluo 3-AM, bafilomycin caused calcium mobilization of the acidic calcium pool that could also be release with nigericin. Interestingly after complete release of the acidic compartments, addition of thapsigargin at 10 µM was still effective in releasing parasite intracellular calcium stores in parasites at trophozoite stage. The addition of antimalarial drugs chloroquine and artemisinin resulted in AO release from acidic compartments and also affected maintenance of calcium in ER store by using different drug concentrations.

Artesunate reduces proliferation, interferes DNA replication and cell cycle and enhances apoptosis in vascular smooth muscle cells / 华中科技大学学报（医学）（英德文版）

This study examined the effect of artesunate (Art) on the proliferation, DNA replication, cell cycles and apoptosis of vascular smooth muscle cells (VSMCs). Primary cultures of VSMCs were established from aortas of mice and artesunate of different concentrations was added into the medium. The number of VSMCs was counted and the curve of cell growth was recorded. The activity of VSMCs was assessed by using MTT method and inhibitory rate was calculated. DNA replication was evaluated by [3H]-TdR method and apoptosis by DNA laddering and HE staining. Flowmetry was used for simultaneous analysis of cell apoptosis and cell cycles. Compared with the control group, VSMCs proliferation in Art interfering groups were inhibited and [3H]-TdR incorprating rate were decreased as well as cell apoptosis was induced. The progress of cell cycle was blocked in G0/G1 by Art in a dose-dependent manner. It is concluded that Art inhibits VSMCs proliferation by disturbing DNA replication, inducing cell apoptosis and blocking cell cycle in G0/G1 phase.

Effect of dihydroartemisinin on the antioxidant capacity of P. falciparum-infected erythrocytes.

Many lines of evidence reveal that artemisinin, an antimalarial containing endoperoxide, generates free radicals to kill malaria parasites. The present study re-evaluated the antioxidants of P. falciparum-infected erythrocytes in the absence and presence of 0.25, 0.5 and 1.0 ng/ml of dihydroartemisinin (DHA), the active metabolite of artemisinin. The ratio of reduced to oxidized glutathione (GSH/GSSG) and activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were determined. The data indicated that malaria infection induced oxidative stress in erythrocytes that resulted in a significant lower GSH in parasitized cells compared to the non-parasitized. DHA showed no effect on the antioxidant levels of non-parasitized erythrocytes treated under similar conditions as P. falciparum-infected erythrocytes. However, significantly lower GSH as well as catalase and GPx activities in parasitized cells were seen at drug concentrations of 0.5 and 1.0 ng/ml (p < 0.05). GSH is the most sensitive indicator of oxidative stress in malaria-infected erythrocytes both in the absence and in the presence of DHA. Parasite GPx might play a more important role than catalase in the elimination of peroxide. Parasite viabilities in the presence of DHA were analyzed simultaneously and were affected to a greater extent than the antioxidant levels. The present observation showed that although DHA killed malaria parasites by generating free radicals from the endoperoxide bridge causing the reduction of antioxidants, but the depletion of parasite antioxidants is not a prerequisite for the parasite death.

In vitro effect of artesunate against Acanthamoeba spp.

The in vitro effects of artesunate, the antimalarial agent, and metronidazole against Acanthamoeba spp were studied. Acanthamoeba Group II and Acanthamoeba polyphaga-like were isolated from natural water courses in Buri Ram Province, northeastern Thailand. The trophozoites were axenically cultured in PPYG medium and treated with artesunate in a concentration of 5-700 microg/ml. Artesunate showed its ability to inhibit the growth of acanthamoeba trophozoites: 54% at 50 mg/ml (after six days of exposure) and 93.2% at 100 microg/ml (after two days). The 500-700 microg/ml concentration caused inhibition on the first day of more than 93.2%; excystation did not occur in drug-treated medium. The present study shows that artesunate is amebastatic rather than amebicidal in an axenic culture of trophozoites at the highest concentration of 100 microg/ml. Metronidazole, in concentrations of 5-1,000 microg/ml, had no effects on either trophozoites or cysts.

Use of Artemisinin (Qinghaosu) Derivatives in Treatment of Malaria

Derivatives of the Chinese herbal remedy ginghaosu (artemisinin) are useful in the treatment of multiple-drug resistant malaria. This review covers the discovery; development; clinical pharmacology and toxicology of these compounds; with emphasis on those derivatives currently in use in parts of Africa