In vitro and in vivo antitrypanosomal activity of the fresh leaves of Ranunculus Multifidus Forsk and its major compound anemonin against Trypanosoma congolense field isolate.

BACKGROUND: Animal trypanosomiasis is a major livestock problem due to its socioeconomic impacts in tropical countries. Currently used trypanocides are toxic, expensive, and the parasites have developed resistance to the existing drugs, which calls for an urgent need of new effective and safe chemotherapeutic agents from alternative sources such as medicinal plants. In Ethiopian traditional medicine fresh leaves of Ranunculus multifidus Forsk, are used for the treatment of animal trypanosomiasis. The present study aimed to evaluate the antitrypanosomal activity of the fresh leaves of R. multifidus and its major compound anemonin against Trypanosoma congolense field isolate. METHODS: Fresh leaves of R. multifidus were extracted by maceration with 80% methanol and hydro-distillation to obtain the corresponding extracts. Anemonin was isolated from the hydro-distilled extract by preparative TLC. For the in vitro assay, 0.1, 0.4, 2 and 4 mg/ml of the test substances were incubated with parasites and cessation or drop in motility of the parasites was monitored for a total duration of 1 h. In the in vivo assay, the test substances were administered intraperitoneally daily for 7 days to mice infected with Trypanosoma congolense. Diminazene aceturate and 1% dimethylsulfoxide (DMSO) were used as positive and negative controls, respectively. RESULTS: Both extracts showed antitrypanosomal activity although the hydro-distilled extract demonstrated superior activity compared to the hydroalcoholic extract. At a concentration of 4 mg/ml, the hydro-distilled extract drastically reduced motility of trypanosomes within 20 min. Similarly, anemonin at the same concentration completely immobilized trypanosomes within 5 min of incubation, while diminazene aceturate (28.00 mg/kg/day) immobilized the parasites within 10 min. In the in vivo antitrypanosomal assay, anemonin eliminates parasites at all the tested doses (8.75, 17.00 and 35.00 mg/kg/day) and prevented relapse, while in diminazene aceturate-treated mice the parasites reappeared on days 12 to 14. CONCLUSIONS: The current study demonstrated that the fresh leaves of R. multifidus possess genuine antitrypanosomal activity supporting the use of the plant for the treatment of animal trypanosomiasis in traditional medicine. Furthermore, anemonin appears to be responsible for the activity suggesting its potential as a scaffold for the development of safe and cost effective antitrypanosomal agent.

Ursolic acid-rich extract presents trypanocidal action in vitro but worsens mice under experimental acute Chagas disease.

Chagas disease is a neglected tropical disease with only two drugs available for treatment and the plant Cecropia pachystachya has several compounds with antimicrobial and anti-inflammatory activities. This study aimed to evaluate a supercritical extract from C. pachystachya leaves in vitro and in vivo against Trypanosoma cruzi. A supercritical CO2 extraction was used to obtain the extract (CPE). Cytotoxicity and immunostimulation ability were evaluated in macrophages, and the in vitro trypanocidal activity was evaluated against epimastigotes and trypomastigotes forms. In vivo tests were done by infecting BALB/c mice with blood trypomastigotes forms and treating animals orally with CPE for 10 days. The parasitemia, survival rate, weight, cytokines and nitric oxide dosage were evaluated. CPE demonstrated an effect on the epi and trypomastigotes forms of the parasite (IC50 17.90 ± 1.2 µg/mL; LC50 26.73 ± 1.2 µg/mL) and no changes in macrophages viability, resulting in a selectivity index similar to the reference drug. CPE-treated animals had a worsening compared to non-treated, demonstrated by higher parasitemia and lower survival rate. This result was attributed to the anti-inflammatory effect of CPE, demonstrated by the higher IL-10 and IL-4 values observed in the treated mice compared to the control ones. CPE demonstrated a trypanocidal effect in vitro and a worsening in the in vivo infection due to its anti-inflammatory activity.

Bioactivity-guided isolation of trypanocidal coumarins and dihydro-pyranochromones from selected Apiaceae plant species.

Bioactivity-guided isolation of natural products from plant matrices is widely used in drug discovery. Here, this strategy was applied to identify trypanocidal coumarins effective against the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease (American trypanosomiasis). Previously, phylogenetic relationships of trypanocidal activity revealed a coumarin-associated antichagasic hotspot in the Apiaceae. In continuation, a total of 35 ethyl acetate extracts of different Apiaceae species were profiled for selective cytotoxicity against T. cruzi epimastigotes over host CHO-K1 and RAW264.7 cells at 10 µg/mL. A flow cytometry-based T. cruzi trypomastigote cellular infection assay was employed to measure toxicity against the intracellular amastigote stage. Among the tested extracts, Seseli andronakii aerial parts, Portenschlagiella ramosissima and Angelica archangelica subsp. litoralis roots exhibited selective trypanocidal activity and were subjected to bioactivity-guided fractionation and isolation by countercurrent chromatography. The khellactone ester isosamidin isolated from the aerial parts of S. andronakii emerged as a selective trypanocidal molecule (selectivity index ∼9) and inhibited amastigote replication in CHO-K1 cells, though it was significantly less potent than benznidazole. The khellactone ester praeruptorin B and the linear dihydropyranochromones 3'-O-acetylhamaudol and ledebouriellol isolated from the roots of P. ramosissima were more potent and efficiently inhibited the intracellular amastigote replication at < 10 µM. The furanocoumarins imperatorin, isoimperatorin and phellopterin from A. archangelica inhibited T. cruzi replication in host cells only in combination, indicative of superadditive effects, while alloimperatorin was more active in fractions. Our study reports preliminary structure-activity relationships of trypanocidal coumarins and shows that pyranocoumarins and dihydropyranochromones are potential chemical scaffolds for antichagasic drug discovery.

Effectiveness of the repurposed drug isotretinoin in an experimental murine model of Chagas disease.

Benznidazole and nifurtimox are the drugs currently used for the treatment of Chagas disease, however its side effects may affect patient adherence. In the search for new alternative therapies, we previously identified isotretinoin (ISO), an FDA-approved drug widely used for the treatment of severe acne through a drug repurposing strategy. ISO shows a strong activity against Trypanosoma cruzi parasites in the nanomolar range, and its mechanism of action is through the inhibition of T. cruzi polyamine and amino acid transporters from the Amino Acid/Auxin Permeases (AAAP) family. In this work, a murine model of chronic Chagas disease (C57BL/6 J mice), intraperitoneally infected with T. cruzi Nicaragua isolate (DTU TcI), were treated with different oral administrations of ISO: daily doses of 5 mg/kg/day for 30 days and weekly doses of 10 mg/kg during 13 weeks. The efficacy of the treatments was evaluated by monitoring blood parasitemia by qPCR, anti-T. cruzi antibodies by ELISA, and cardiac abnormalities by electrocardiography. No parasites were detected in blood after any of the ISO treatments. The electrocardiographic study of the untreated chronic mice showed a significant decrease in heart rate, while in the treated mice this negative chronotropic effect was not observed. Atrioventricular nodal conduction time in untreated mice was significantly longer than in treated animals. Mice treated even with ISO 10 mg/kg dose every 7 days, showed a significant reduction in anti-T. cruzi IgG levels. In conclusion, the intermittent administration of ISO 10 mg/kg would improve myocardial compromise during the chronic stage.

Potential of sulfur-selenium isosteric replacement as a strategy for the development of new anti-chagasic drugs.

Current treatment for Chagas disease is based on only two drugs: benznidazole and nifurtimox. Compounds containing sulfur (S) in their structure have shown promising results in vitro and in vivo against Trypanosoma cruzi, the parasite causing Chagas disease. Notably, some reports show that the isosteric replacement of S by selenium (Se) could be an interesting strategy for the development of new compounds for the treatment of Chagas disease. To date, the activity against T. cruzi of three Se- containing groups has been compared with their S counterparts: selenosemicarbazones, selenoquinones, and selenocyanates. More studies are needed to confirm the positive results of Se compounds. Therefore, we have investigated S compounds described in the literature tested against T. cruzi. We focused on those tested in vivo that allowed isosteric replacement to propose their Se counterparts as promising compounds for the future development of new drugs against Chagas disease.

Probiotic administration enhanced antitrypanosomal effects of diminazene aceturate in dogs experimentally infected with Trypanosoma brucei brucei.

OBJECTIVES: This study demonstrated the enhancing actions of probiotic on the antitrypanosomal effects of diminazene aceturate in dogs experimentally infected with Trypanosoma brucei brucei. METHODS: Twenty (20) apparently healthy adult local dogs of both sexes were randomly divided into five groups each containing four dogs. Group I were uninfected and untreated while groups III, IV and V were infected. Groups II, III, IV and V were administered multispecies probiotic (MSP) and/or diminazene aceturate (DA). Parasitaemia was determined, clinical signs recorded and blood collected for haematology. RESULTS: Results revealed T. b. brucei prepatent periods of 4.75 ± 0.25, (4-5) days and significant decrease of parasitaemia, clinical signs and mortality in groups IV and V compared to group III. Mortalities of 100% (group III), 25% (group IV) and 0% (group V) were recorded. Mean packed cells volume, haemoglobin concentration and red blood cells count showed no significant difference in groups I, II, and V, but were significantly decreased in groups III and IV post-treatment. CONCLUSIONS: The administration of MSP to infected dogs enhanced the antitrypanosomal effects of diminazene aceturate.

Follow-up of dairy cattle naturally infected by Trypanosoma vivax after treatment with isometamidium chloride.

Trypanosoma vivax infections cause nonspecific clinical signs in cattle associated with aparasitemic intervals, making disease diagnosis a challenge. In Brazil, diminazene aceturate and isometamidium chloride (ISM) are available to treat bovine trypanosomosis. The objective of this study was to follow-up, by molecular and serological techniques, dairy cattle naturally infected by T. vivax after ISM treatment. Thirty cattle naturally infected with T. vivax received two applications of ISM, at a dosage of 1.0 mg/kg intramuscularly, on days 0 and 150. For T. vivax diagnosis, EDTA-blood and serum samples were evaluated on 0, 7, 15, 30, 60, 90, 120, 150, 180, 210, and 240 days after treatment PCR, Loop-mediated isothermal amplification (LAMP) and ELISA. Animals with persistent detection of T. vivax DNA by both PCR and LAMP were found and continuous detection of anti-T. vivax IgG antibodies by ELISA, suggesting the presence of T. vivax resistance to ISM. The combination of LAMP and ELISA tests can prevent misdiagnosis of the parasite clearance in treated cattle, contributing to better disease control. This is the first experiment that demonstrates the persistence infection of T. vivax under ISM treatment in a natural infected herd and evidence of ISM chemotherapy-resistant T. vivax in Brazil.

Eugenia uniflora and Syzygium samarangense extracts exhibit anti-trypanosomal activity: Evidence from in-silico molecular modelling, in vitro, and in vivo studies.

The parasite Trypanosoma brucei is the main cause of the sleeping sickness threatening millions of populations in many African countries. The parasitic infection is currently managed by some synthetic medications, most of them suffer limited activity spectrum and/or serious adverse effects. Some studies have pointed out the promising therapeutic potential of the plant extracts rich in polyphenols to curb down parasitic infections caused by T. brucei and other trypanosomes. In this work, the main components dominating Eugenia uniflora and Syzygium samarangense plant extracts were virtually screened, through docking, as inhibitors of seven T. brucei enzymes validated as potential drug targets. The in vitro and in vivo anti-T. brucei activities of the extracts in two treatment doses were evaluated. Moreover, the extract effects on the packed cell volume level, liver, and kidney functions were assessed. Five compounds showed strong docking and minimal binding energy to five target enzymes simultaneously and three other compounds were able to bind strongly to at least four of the target enzymes. These compounds represent lead hits to develop novel trypanocidal agents of natural origin. Both extracts showed moderate in vitro anti-trypanosomal activity. Infected animal groups treated over 5 days with the studied extracts showed an appreciable in vivo anti-trypanosomal activity and ameliorated in a dose dependent manner the anaemia, liver, and kidney damages induced by the infection. In conclusion, Eugenia uniflora and Syzygium samarangense could serve as appealing sources to treat trypanosomes infections.

In-vitro Anti-trypanosomal and Cytotoxicity Evaluation of 3-methyl-3,4-dihydroquinazolin-2(1H)-one Derivatives.

Sleeping sickness, caused by trypanosomes, is a debilitating, neglected tropical disease wherein current treatments suffer from several drawbacks such as toxicity, low activity, and poor pharmacokinetic properties, and hence the need for alternative treatment is apparent. To this effect, we screened in vitro a library of 2-quinazolinone derivatives for antitrypanosomal activity against T.b. brucei and cytotoxicity against HeLa cells. Seven compounds having no overt cytotoxicity against HeLa cells exhibited antitrypanosomal activity in the range of 0.093-45 µM were identified. The activity data suggests that the antitrypanosomal activity of this compound class is amenable to substituents at N1 and C6 positions. Compound 14: having a molecular weight of 238Da, ClogP value of 1 and a total polar surface area of 49 was identified as the most active, exhibiting an IC50 value of 0.093 µM Graphical Abstract.

Follow-up of dairy cattle naturally infected by Trypanosoma vivax after treatment with isometamidium chloride / Acompanhamento de bovinos leiteiros naturalmente infectados com Trypanosoma vivax após tratamento com cloreto de isometamidium

Abstract Trypanosoma vivax infections cause nonspecific clinical signs in cattle associated with aparasitemic intervals, making disease diagnosis a challenge. In Brazil, diminazene aceturate and isometamidium chloride (ISM) are available to treat bovine trypanosomosis. The objective of this study was to follow-up, by molecular and serological techniques, dairy cattle naturally infected by T. vivax after ISM treatment. Thirty cattle naturally infected with T. vivax received two applications of ISM, at a dosage of 1.0 mg/kg intramuscularly, on days 0 and 150. For T. vivax diagnosis, EDTA-blood and serum samples were evaluated on 0, 7, 15, 30, 60, 90, 120, 150, 180, 210, and 240 days after treatment PCR, Loop-mediated isothermal amplification (LAMP) and ELISA. Animals with persistent detection of T. vivax DNA by both PCR and LAMP were found and continuous detection of anti-T. vivax IgG antibodies by ELISA, suggesting the presence of T. vivax resistance to ISM. The combination of LAMP and ELISA tests can prevent misdiagnosis of the parasite clearance in treated cattle, contributing to better disease control. This is the first experiment that demonstrates the persistence infection of T. vivax under ISM treatment in a natural infected herd and evidence of ISM chemotherapy-resistant T. vivax in Brazil.

Resumo Em bovinos, infecções por Trypanosoma vivax geram sinais clínicos inespecíficos que, associados a intervalos aparasitêmicos, faz com que o diagnóstico da enfermidade seja desafiador. No Brasil, somente aceturato de diaminazeno e cloridrato de isometamidum (ISM) estão disponíveis para o tratamento da tripanossomose bovina. Este trabalho teve como objetivo acompanhar bovinos leiteiros naturalmente infectados por T. vivax, após o tratamento com ISM por meio de técnicas moleculares e sorológica. Foram utilizados 30 bovinos naturalmente infectados com T. vivax, sendo estes tratados com duas aplicações de ISM, na dosagem de 1,0 mg/kg por via intramuscular profunda, nos dias 0 e 150. Foram avaliadas, para diagnóstico de T. vivax, amostras de sangue acrescido de EDTA e soro, colhidas nos 0, 7, 15, 30, 60, 90, 120, 150, 180, 210 e 240 dias após os tratamentos pela reação em cadeia da polimerase (PCR), amplificação circular isotérmica do DNA (LAMP) e ensaio de imunoabsorção enzimático (ELISA). Verificou-se a presença de animais com persistência na detecção de DNA de T. vivax pela PCR e LAMP, bem como detecção contínua de anticorpos IgG anti-T. vivax pelo método de ELISA, sugerindo a presença de resistência de T. vivax ao ISM. A combinação dos testes LAMP e ELISA pode evitar falsos diagnósticos da eliminação do parasita nos bovinos tratados, contribuindo para um melhor controle da doença. Este é o primeiro experimento que demonstra infecção persistente do T. vivax em rebanho naturalmente infectado, tratado com ISM, e evidencia possível resistência ao quimioterápico no Brasil.

Re-evaluating pretomanid analogues for Chagas disease: Hit-to-lead studies reveal both in vitro and in vivo trypanocidal efficacy.

Phenotypic screening of a 900 compound library of antitubercular nitroimidazole derivatives related to pretomanid against the protozoan parasite Trypanosoma cruzi (the causative agent for Chagas disease) identified several structurally diverse hits with an unknown mode of action. Following initial profiling, a first proof-of-concept in vivo study was undertaken, in which once daily oral dosing of a 7-substituted 2-nitroimidazooxazine analogue suppressed blood parasitemia to low or undetectable levels, although sterile cure was not achieved. Limited hit expansion studies alongside counter-screening of new compounds targeted at visceral leishmaniasis laid the foundation for a more in-depth assessment of the best leads, focusing on both drug-like attributes (solubility, metabolic stability and safety) and maximal killing of the parasite in a shorter timeframe. Comparative appraisal of one preferred lead (58) in a chronic infection mouse model, monitored by highly sensitive bioluminescence imaging, provided the first definitive evidence of (partial) curative efficacy with this promising nitroimidazooxazine class.

Trypanocidal Essential Oils: A Review.

Trypanosomiases are diseases caused by parasitic protozoan trypanosomes of the genus Trypanosoma. In humans, this includes Chagas disease and African trypanosomiasis. There are few therapeutic options, and there is low efficacy to clinical treatment. Therefore, the search for new drugs for the trypanosomiasis is urgent. This review describes studies of the trypanocidal properties of essential oils, an important group of natural products widely found in several tropical countries. Seventy-seven plants were selected from literature for the trypanocidal activity of their essential oils. The main chemical constituents and mechanisms of action are also discussed. In vitro and in vivo experimental data show the therapeutic potential of these natural products for the treatment of infections caused by species of Trypanosoma.

Effects of Highly Diluted Drugs on Experimental Infection with Trypanosoma cruzi In Vivo: Systematic Review.

Objective: To investigate, through a systematic review, the effects of the use of highly diluted drugs in the treatment of experimental infection with Trypanosoma cruzi. Design: The authors searched for scientific publications in the databases PubMed, Web of Science, SCOPUS, LILACS, and the Google Scholar search system, from 2000 to 2018, following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement. According to the criteria established, a total of 22 studies were included. Settings/Location: The study took place at the State University of Maringá, Maringá, PR, Brazil. Subjects: Male mice (Mus musculus) or rats (Rattus norvegicus). Interventions: Highly diluted drugs. Outcome measures: The parameters evaluated in the studies were parasitological, clinical, immunological, histopathological and hematological. Results: The studies demonstrated that the effects of highly diluted drugs are related to their dynamizations, treatment regimen, and host susceptibility to T. cruzi infection, and depend on the initial information transmitted to the treated organism, making this information the "model" of how the treated organism will react. Regardless of the mechanism of action, these drugs provide a decrease in inflammation, which is one of the central phenomena of the pathogenesis of T. cruzi infection. Conclusions: This systematic review brings out the importance of the T. cruzi infection model as a reliable and valid model for studying different effects produced by highly diluted drugs. Considering the findings and in a broader perspective, this study contributes to considering these drugs as a possible way of dealing with "treatment" in general, presents the need to reexamine the biochemical model and develop a model for the effect of high dilutions in general, as well as for the treatment of parasitic infections.

Toosendanin relatives, trypanocidal principles from Meliae Cortex.

Africa Trypanosomiasis remains a serious health problem, but the approved drugs for this disease are so few that novel trypanocidal compounds are demanded. In search for trypanocidal principles from medicinal plants, we found MeOH extracts of Meliae Cortex with potent activity through the screening from about 300 kinds of methanolic extract. By bioassay-guided fractionation from this extract through the liquid-liquid partition and subsequent chromatographic technique using silica gel and ODS, finally we disclosed toosendanin (1) and its relatives as active principles. These active congeners showed not only potent trypanocidal activity but also little cytotoxicity to display the excellent selective index. Taking the isolated amount as well as trypanocidal activity into consideration, 1 was disclosed to be the responsible active principle in Meliae Cortex. Additionally, the derivatives of 1 were chemically prepared from 1 and bioactivity of them were also evaluated. Through the comparison with their trypanocidal activity among the isolated relatives and the synthesized derivatives of 1, the epoxide moiety was revealed to be essential for their potent trypanocidal activity. Furthermore, 3-O-acetyl group and 7-hydroxyl group were presumed to be important functional groups and introduction of methylpropionyl group into hemiacetal hydroxy moiety was clarified to enhance their typanocidal activity.

In Vitro Drug Efficacy Testing Against Trypanosoma brucei.

The recent endorsement of fexinidazole by the European Medicines Agency for the treatment of human African trypanosomiasis has demonstrated the high predictive value of cell-based assays for parasite chemotherapy. Here we describe three in vitro drug susceptibility tests with Trypanosoma brucei that have served as the basis for the identification of fexinidazole as a promising lead: (1) a standard assay with end-point measurement to determine drug efficacy; (2) a wash-out assay to test for reversibility and speed of drug action; (3) isothermal microcalorimetry for real-time measurement of onset of drug action and time to kill. Together, these assays allow to estimate pharmacodynamic parameters in vitro and to devise appropriate treatment regimens for subsequent in vivo experiments.

Lychnopholide in Poly(d,l-Lactide)-Block-Polyethylene Glycol Nanocapsules Cures Infection with a Drug-Resistant Trypanosoma cruzi Strain at Acute and Chronic Phases.

Chagas disease remains neglected, and current chemotherapeutics present severe limitations. Lychnopholide (LYC) at low doses loaded in polymeric poly(d,l-lactide)-block-polyethylene glycol (PLA-PEG) nanocapsules (LYC-PLA-PEG-NC) exhibits anti-Trypanosoma cruzi efficacy in mice infected with a partially drug-resistant strain. This study reports the efficacy of LYC-PLA-PEG-NC at higher doses in mice infected with a T. cruzi strain resistant to benznidazole (BZ) and nifurtimox (NF) treated at both the acute phase (AP) and the chronic phase (CP) of infection by the oral route. Mice infected with the T. cruzi VL-10 strain were treated by the oral route with free LYC (12 mg/kg of body weight/day), LYC-PLA-PEG-NC (8 or 12 mg/kg/day), or BZ at 100 mg/kg/day or were not treated (controls). Treatment efficacy was assessed by hemoculture (HC), PCR, enzyme-linked immunosorbent assay (ELISA), heart tissue quantitative PCR (qPCR), and histopathology. According to classical cure criteria, treatment with LYC-PLA-PEG-NC at 12 mg/kg/day cured 75% (AP) and 88% (CP) of the animals, while at a dose of 8 mg/kg/day, 43% (AP) and 43% (CP) were cured, showing dose-dependent efficacy. The negative qPCR results for heart tissue and the absence of inflammation/fibrosis agreed with the negative results obtained by HC and PCR. Thus, the mice treated with the highest dose could be considered 100% cured, in spite of a low ELISA reactivity in some animals. No cure was observed in animals treated with free LYC or BZ or the controls. These results are exceptional in terms of experimental Chagas disease chemotherapy and provide evidence of the outstanding contribution of nanotechnology in mice infected with a T. cruzi strain totally resistant to BZ and NF at both phases of infection. Therefore, LYC-PLA-PEG-NC has great potential as a new treatment for Chagas disease and deserves further investigations in clinical trials.

Activity of the sesquiterpene lactone goyazensolide against Trypanosoma cruzi in vitro and in vivo.

BACKGROUND: The current drugs for Chagas disease treatment present several limitations. METHODS: The sesquiterpene lactone goyazensolide (GZL) was evaluated regarding to cytotoxicity and trypanocidal activity against amastigotes, selectivity index (SI) in vitro, acute toxicity and anti-Trypanosoma cruzi activity in vivo. RESULTS: The in vitro cytotoxicity in H9c2 cells was observed at doses >250 ng mL-1 of GZL and the SI were of 52.82 and 4.85 (24 h) and of 915.00 and 41.00 (48 h) for GZL and BZ, respectively. Nephrotoxicity and hepatotoxicity were not verified. Treatment with GZL of mice infected with CL strain led to a significant decrease of parasitaemia and total survival at doses of 1 and 3 mg kg-1 day-1 by oral and IV, respectively. This last group cured 12.5% of the animals (negativation of HC, PCR, qPCR and ELISA). Animals infected with Y strain showed significant decrease of parasitaemia and higher negativation in all parasitological tests in comparison to BZ and control groups, but were ELISA reactive, as well as the BZ group, but mice treated with 5.0 mg kg-1 day-1 by oral were negative in parasitological tests and survived. CONCLUSION: GZL was more active against T. cruzi than benznidazole in vitro and presented important therapeutic activity in vivo in both T. cruzi strains.

Role of nucleic acid amplification assays in monitoring treatment response in chagas disease: Usefulness in clinical trials.

Chagas disease has become a global health problem due to migration of infected people out of Latin America to non-endemic countries. For more than 40 years, only the nitroimidazole compounds Benznidazole and Nifurtimox, have been used for specific treatment of Trypanosoma cruzi infection with disappointing results, specially due to the long duration of treatment and adverse events in the chronic phase. In the last years, ergosterol inhibitors have been also proposed for specific treatment. Different randomized clinical trials were performed for evaluating their treatment efficacy and safety. One of the greatest concerns in clinical trials is to provide an early surrogate biomarker of response to trypanocidal chemotherapy. Serological response is slow and the classical parasitological tests have poor sensitivity and are time-consuming. Nowadays, PCR is the most helpful tool for assessing treatment response in a short period of time. Different protocols of PCR have been developed, being quantitative real time PCR based on amplification of repetitive satellite or minicircle DNA sequences plus an internal amplification standard, the mostly employed strategies in clinical trials. Standardized protocols and the use of an external quality assessment ensure adequate technical procedures and reliable data. Clinical trials have shown a significant reduction in parasite loads, reaching undetectable DNA levels in bloodstream after specific treatment, however events of treatment failure have also been reported. Treatment failure could be due to inadequate penetrance of the drugs into the affected tissues, to the presence of primary or secondary drug resistance of the infecting strains as well as to the existence of dormant parasite variants reluctant to drug action. The early diagnosis of drug resistance would improve clinical management of Chagas disease patients, allowing dictating alternative therapies with a combination of existing drugs or new anti-T. cruzi agents. The aim of this review was to describe the usefulness of detecting T.cruzi DNA by means of real time PCR assays, as surrogate biomarker in clinical trials for evaluating new drugs for CD or new regimens of available drugs and the possibility to detect treatment failure.

Evaluation of antitrypanosomal activity of Pterocarpus santalinoides L'H'erit ex DC hydroethanol leaf extract in rats experimentally infected with Trypanosoma brucei.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Pterocarpus santalinoides are used alone or with other plants by traditional healers in some Southern Nigerian and Ivorian rural villages to treat blood parasitic infections including trypanosomiasis and malaria. However, their efficacy and safety remains doubtful. AIM: To evaluate the antitrypanosomal activity of Pterocarpus santalinoides hydroethanol leaf extract (PSELE) in vitro and in vivo. MATERIALS AND METHODS: The phytochemical and acute toxicity studies of PSELE were performed using standard methods. Eleven different concentrations of the extract were screened for in vitro antitrypanosomal activity. For the in vivo study, twenty-five female albino rats were randomly assigned into five groups of five rats each. Group A was the uninfected and untreated group while groups B - E were infected intraperitoneally with 106 trypanosomes. Group C rats were treated once on day 11 post infection (PI) with 7 mg/kg diminazene aceturate while groups D and E rats were also treated on same day with 200 mg/kg and 400 mg/kg PSELE respectively for seven days. The level of parasitaemia (LOP), survivability, packed cell volume (PCV), total leucocyte count (TLC), total erythrocyte count (TEC), body weight and rectal temperature were used to assess the antitrypanosomal effect of PSELE. RESULTS: Phytochemical analysis revealed the presence of flavonoids, tannins, carbohydrates and reducing sugar. No sign of toxicity or mortality was observed following acute toxicity study at a single dose of 2000 mg/kg. The LC50 of PSELE was 0.0625 mg/ml. Parasitaemia was first detected on day 8 and all infected rats became parasitaemic on day 10 (PI). PSELE significantly reduced (p < 0.05) LOP, improved PCV, TEC and prolonged survival time of the rats compared with the infected untreated group B. CONCLUSION: It was therefore concluded that PSELE is safe, possesses some antitrypanosomal activity and may serve as a lead for the development of an effective alternative antitrypanosomal drug.