Involvement of tryparedoxin peroxidase (TryP) and trypanothione reductase (TryR) in antimony unresponsive of Leishmania tropica clinical isolates of Iran.

Clinical resistance to pentavalent antimonial compounds has long been recognized as a major problem in the treatment of human leishmaniasis. Trypanothione metabolism, the main form of thiol, has shown to play a central role in antimony resistance of laboratory-generated resistant Leishmania spp. and field-isolated resistant L. donovani; but the mechanism of antimony resistance in the clinical isolates of L. tropica causing anthroponotic cutaneous leishmaniasis (ACL) is less studied. Patients were selected among confirmed positive ACL cases who referred to Pasteur Institute of Iran, Tehran, from endemic regions of north-east and south of Iran. L. tropica clinical isolates were collected from patients who were either treatment-responsive (MAS=S1 to S5) or unresponsive (MAR=R1 to R4) to Glucantime® (meglumine antimoniate=MA). Isolates were tested for sensitivity to trivalent antimony (SbIII) in promastigotes and to pentavalent antimony (SbV) in intracellular amastigotes stages. Intracellular thiol levels were assayed and trypanothione-dependent components, including trypanothione reductase (TR) and tryparedoxin peroxidase I (TryP) were analysed at protein level and enzymatic activity in isolates. The MAR isolates had an approximate two fold increase in the levels of intracellular thiols (P< 0.05) accompanied by an average 5-10 fold increase in in vitro resistance to antimony. TryP was amplified at the protein level in all MAR strains as compared to the MAS strains (range: 2.8-5.6 fold). All MAR isolates metabolized H2O2 at higher rates than MAS isolates (8.55±0.75 nmol/min/mg vs. 3.14±0.36 nmol/min/mg) (P< 0.05). In addition, levels of TryR protein were also markedly elevated in 3 out of 4 MAR isolates (range: 2.2-4.1 fold). This was accompanied by overexpressed TryR activity (mean level of 46.83±2.43 for extracts of MAR vs. 20.98±3.02 for MAS strains) (P< 0.05). Elevated levels of TryP, active enzyme in peroxide detoxification, were observed in MAR parasites resulting in an increased metabolism of H2O2. TryR activity was overexpressed on average in extracts of MAR strains, but not in all isolates. Enhanced anti-oxidant defenses through thiol metabolism may play a significant role in clinical resistance of ACL patients to Glucantime.

Eligibility for Local Therapies in Adolescents and Adults with Cutaneous Leishmaniasis from Southwestern Colombia: A Cross-Sectional Study.

Local therapies have been proposed as safe and effective alternatives to systemic drugs in cutaneous leishmaniasis (CL), especially among less severe cases. However, they are not widely available and used in endemic places, including Colombia, which has a high burden of disease. Further complicating the uptake of local therapies is that different treatment guidelines have been established by the World Health Organization (WHO) and Pan American Health Organization (PAHO). Using data from a large referral center in Colombia, we determined the proportion of patients who would be eligible for and potentially benefit from local therapies according to both international guidelines. The sample included 1,891 confirmed cases of CL aged ≥ 12 years, mostly infected with Leishmania Viannia panamensis (91%, n = 601/660), between 2004 and 2014. Overall, 57% of the sample had one lesion, whereas another 31% had two to three lesions. For 74% of patients, all lesions were in an area other than head or neck. The maximum lesion size was ≤ 3 cm for 58% and < 5 cm for 88% of the sample. Based on our data, up to 56% of patients could have been eligible for local therapies according to the WHO criteria. By contrast, only 23% were eligible according to the more restrictive PAHO criteria. Regardless, these data suggest that a substantial proportion of CL patients in Colombia may benefit from local therapies given their relatively mild presentation of disease and low risk of complications. Individualized risk-benefit assessment and guideline adjustments may increase local therapy eligibility and benefit a large number of patients.

Efficacy of pentavalent antimoniate intralesional infiltration therapy for cutaneous leishmaniasis: A systematic review.

BACKGROUND: The mainstays of cutaneous leishmaniasis (CL) treatment, in several world regions, are pentavalent antimony (Sbv) compounds administered parenterally, despite their recognized toxicity, which requires frequent laboratory monitoring and complicates their use in areas with scarce infrastructure. As result of these drawbacks, the WHO Expert Committee on leishmaniasis has expanded the recommendations for the use of local therapies, including Sbv intralesional infiltration (IL-Sbv), as CL therapy alternatives even in the New World. However, the efficacy of these approaches has never been compiled. The aim of this study was to critically and systematically assess the efficacy of IL-Sbv for CL treatment. METHODOLOGY: The PRISMA guidelines for systematic reviews and the Cochrane manual were followed. The sources used were the MEDLINE and LILACS databases and the International Clinical Trials Registry Platform of the World Health Organization. The outcome of interest was a clinical cure, defined as complete re-epithelialization of all lesions. The IL-Sbv pooled cure rate was estimated for several subgroups and direct comparisons were performed when possible. RESULTS: Thirty nine articles (40 studies) involving 5679 patients treated with IL-Sbv infiltration were included. In direct comparison, only three studies involving 229 patients compared IL-Sbv infiltration versus placebo and no difference was observed (OR: 1,9; 95%IC 0,93 to 3,82) based on cure rate 69.6% (95%CI 17.6-96.1%) and 83,2% (95%CI 66-92.7%) for placebo and IL-Sbv, respectively. In an alternative and non-comparative analysis, gathering all study arms using the intervention, the pooled IL-Sbv efficacy rate was 75% (95%CI 68-81%). In the Old World, the observed overall IL-Sbv efficacy rate was 75% (95%CI 66-82%), and the cure rates were significantly higher with sodium stibogluconate (SSG) than with meglumine antimoniate (MA): 83% (95%CI 75-90%) versus 68% (95%CI 54-79%), p = 0.03. Studies directly comparing IL-Sbv with topical 15% paromomycin ointment, IL hypertonic saline, radiofrequency-induced heat therapy, topical trichloroacetic acid and cryotherapy showed no significant difference in efficacy between the interventions. The analyses suggested a higher efficacy of IL-Sbv combined with cryotherapy (81.8%, 95%IC 62.4-92.4%) when compared with IL-Sbv alone (53.3%, 95%IC 46.1-66%), OR: 3.14 (95%CI 1.1-8.9), p = 0.03. In the New World, the global IL-Sbv efficacy was 77%(95%CI 66-85%). In contrast with the Old World, a significant difference favoring MA in relation to SSG was observed: 61% (95%CI 49-73%) versus 82% (95%CI 70-89%).By comparing IL infiltration schedules, it was determined that patients submitted to IL-Sbv treatments longer than 14 days had higher cure rates. CONCLUSIONS: Despite the high heterogeneity and low methodological quality of studies, an indirect comparison shows that the antimony infiltration efficacy rate is similar to that reported for antimony systemic use. The evidence gathered thus far is insufficient to identify the ideal IL therapeutic regime or estimate the rates of adverse events and mucosal late complications.

Modulation of inflammation response to murine cutaneous Leishmaniasis by homeopathic medicines: Antimonium crudum 30cH.

BACKGROUND: Leishmaniasis is a zoonotic disease caused by protozoan parasites of the mononuclear phagocytic system. The modulation activity of these cells can interfere in the host/parasite relationship and influences the prognosis. METHODS: We evaluated the effects of the homeopathic preparation Antimonium crudum 30cH on experimental infection induced by Leishmania (L.) amazonensis. Male Balb/c mice were inoculated with 2 × 10(6)Leishmania (L.) amazonensis promastigotes into the footpad and, after 48 h (acute phase) or 60 days (chronic phase), cell population of lymphocytes and phagocytes present in the peritoneal washing fluid and spleen were analyzed by flow cytometry and histopathology, with histometry of the subcutaneous primary lesion, local lymph node and spleen. Immunohistochemistry was performed to quantify CD3 (T lymphocyte), CD45RA (B lymphocyte) and CD11b (phagocytes) positive cells. RESULTS: In treated mice, during the acute phase, there was significant increase of the macroscopic lesion, associated to inflammatory edema, as well increase in the number of free amastigotes and B lymphocytes inside the lesion. Increase of B lymphocytes (predominantly B-2 cells) was also seen in the local lymph node, spleen and peritoneum. In the chronic phase, the inflammatory process in the infection focus was reduced, with reduced phagocyte migration and peritoneal increase of B-1a cells (precursors of B-2 immunoglobulin producers cells) and T CD8+ cells. CONCLUSION: The treatment of mice with Antimonium crudum 30cH induced a predominantly B cell pattern of immune response in Leishmania (L.) amazonensis experimental infection, alongside the increase of free amastigote forms number in the infection site. The clinical significance of this study is discussed, further studies are suggested.

Efficacy of anti-leishmania therapy in visceral leishmaniasis among HIV infected patients: a systematic review with indirect comparison.

OBJECTIVE: We conducted a systematic literature review with indirect comparison of studies evaluating therapeutic efficacy and toxicity associated to visceral leishmaniasis (VL) therapy among HIV infected individuals. MAIN OUTCOME MEASUREMENTS: The outcomes of interest were clinical and parasitological cure, mortality, and adverse events. METHODS: PRISMA guidelines for systematic reviews and Cochrane manual were followed. Sources were MEDLINE, LILACS, EMBASE, Web of Knowledge databases and manual search of references from evaluated studies. We included all studies reporting outcomes after VL treatment, regardless of their design. Study quality was evaluated systematically by using the Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomized studies in meta-analyses. Comprehensive Meta-Analysis software v.2.2.048 was used to perform one-group meta-analysis of study arms with the same drug to estimate global rates of success and adverse events with each drug. These estimates were used, when possible, to indirectly compare treatment options, adjusted for CD4 count. Direct comparison was pooled when available. RESULTS: Seventeen studies reporting five treatment regimens and outcome of 920 VL episodes occurring in HIV infected individuals were included. The main outstanding difference in outcome among the treatment regimens was observed in mortality rate: it was around 3 times higher with high-dose antimony use (18.4%, CI 95% 13.3-25%), indirectly compared to lipid formulations of amphotericin B treatment (6.1%, CI 95% 3.9-9.4%). It was observed, also by indirect comparison, higher rates of clinical improvement in study arms using amphotericin B than in study arms using pentavalent antimonial therapy (Sb(v)). The parasitological cure, an outcome that presented some degree of risk of selection and verification bias, had rates that varied widely within the same treatment arm, with high heterogeneity, hampering any formal comparison among drugs. One direct comparison of amphotericin and antimoniate was possible combining results of two studies and confirming the superiority of amphotericin. CONCLUSIONS: Available evidence suggests that amphotericin is superior to antimony treatment. Death rate using antimoniate high dose is unacceptably high. Randomized controlled trials are necessary to compare different formulations and doses of amphotericin, alternative therapies and drug combinations.

The use of azythromycin and N-methyl glucamine for the treatment of cutaneous Leishmaniasis caused by Leishmania (Leishmania) amazonensis in C57BL6 mice.

BACKGROUND: The first choice treatment for cutaneous Leishmaniasis is N-methyl glucamine: it has high toxicity, requires parenteral administration and cure is not always reached. Azythromycin showed in vitro action and controversial results in humans with the disease. OBJECTIVE: To verify if the association of N-methyl-glucamine - azythromycin is more effective than N-methyl-glucamine alone for the treatment of experimental Leishmaniasis. METHODS: Twenty-five C57BL/6 mice were inoculated with L. (L.) amazonensis strain and divided into two groups. One group was treated with 400 mgSbV/kg/day of N-methyl glucamine and 200mg/kg/day of azythromycin for 20 days and the other group received the same dose of N-methyl glucamine alone during the same period of time. Clinical and parasitological evaluations were submitted to statistical analyses. RESULTS: There was no statistical difference in clinical analysis, in amastigotes investigation and in cultures. There were significant differences in cultures using limiting dilution, which showed lower efficacy of the association N-methyl glucamine -azythromycin. CONCLUSION: N-methyl glucamine-azythromycin association was not more effective than N-methyl glucamine alone.

O uso da associação azitromicina e N-metil glucamina no tratamento da leishmaniose cutânea causada por Leishmania (Leishmania) amazonensis em camundongos C57BL6 / The use of azythromycin and N-methyl glucamine for the treatment of cutaneous Leishmaniasis caused by Leishmania (Leishmania) amazonensis in C57BL6 mice

FUNDAMENTOS: O tratamento de primeira escolha da leishmaniose tegumentar americana é a N-metil-glucamina que tem alta toxicidade, exige administração parenteral e nem sempre cura. A azitromicina mostrou ação in vitro e resultado contraditório na doença humana. OBJETIVO: Verificar se a associação N-metil-glucamina+azitromicina é mais eficaz do que N-metil-glucamina no tratamento da leishmaniose experimental. MÉTODOS: 25 camundongos inoculados com a cepa C57BL/6 de L. (L.) amazonensis foram divididos em dois grupos. Um foi tratado com 400mgSbV/kg/dia de N-metil-glucamina associado a 200mg/kg/dia de azitromicina durante 20 dias, e o outro com N-metil-glucamina, na mesma dose, durante o mesmo tempo. Foi feita avaliação clínica e parasitológica com análise estatística. RESULTADO: Na avaliação clínica, pesquisa de amastigotas e das culturas, não houve diferença estatística. Verificou-se, entretanto, diferença significante no resultado das culturas realizadas através de diluição limitante, que desfavoreceu a associação NMG+ azitromicina. CONCLUSÃO: A associação N-metil-glucamina e azitromicina não demonstrou mais eficácia do que o N-metil-glucamina em uso isolado.

BACKGROUND: The first choice treatment for cutaneous Leishmaniasis is N-methyl glucamine: it has high toxicity, requires parenteral administration and cure is not always reached. Azythromycin showed in vitro action and controversial results in humans with the disease. OBJECTIVE: To verify if the association of N-methyl-glucamine - azythromycin is more effective than N-methyl-glucamine alone for the treatment of experimental Leishmaniasis. METHODS: Twenty-five C57BL/6 mice were inoculated with L. (L.) amazonensis strain and divided into two groups. One group was treated with 400mgSbV/kg/day of N-methyl glucamine and 200mg/kg/day of azythromycin for 20 days and the other group received the same dose of N-methyl glucamine alone during the same period of time. Clinical and parasitological evaluations were submitted to statistical analyses. RESULTS: There was no statistical difference in clinical analysis, in amastigotes investigation and in cultures. There were significant differences in cultures using limiting dilution, which showed lower efficacy of the association N-methyl glucamine -azythromycin. CONCLUSION: N-methyl glucamine-azythromycin association was not more effective than N-methyl glucamine alone.

Efficacy of homeopathically potentized antimony on blood coagulation. A randomized placebo controlled crossover trial.

BACKGROUND: Homeopathically potentized antimony 6x is traditionally used in anthroposophic medicine for an alleged pro-coagulatory effect in bleeding disorders. However, the scientific evidence base is yet insufficient. Results of a previous in vitro study suggested a slight increase of maximal clot firmness (MCF) and a tendency towards a shorter clotting time (CT). The objective of this study was to investigate the pro-coagulatory effects of antimony in vivo, and possible unexpected or adverse events. PARTICIPANTS AND METHODS: A randomized placebo controlled double blind crossover study was carried out in 30 healthy volunteers (15 males, 15 females). Each participant received intravenously 10 ml of antimony 6x and placebo in a randomized order at an interval of 1 month. Thrombelastography (TEG) was carried out immediately before and 30 and 60 min after the injection. RESULTS: Statistically significant pro-coagulatory effects were observed 30 min after injection for CT in men (p = 0.0306), and for MCF in men and women combined (p = 0.0476). The effect of antimony was significantly larger on test day 1 than on test day 2, whereas the effect of placebo was similar on both test days. No unexpected adverse or adverse events causally related to antimony were observed. CONCLUSION: This study adds evidence to the hypothesis that homeopathically potentized antimony may be efficacious in vivo. The consistency of the results with previous in vitro results indicates an effect on MCF and CT. The in vivo application of antimony 6x is safe.

Produtos naturais com potencial leishmanicida / Natural products with leishmanicide potential

A leishmaniose é uma parasitose causada porprotozoários do gênero Leishmania. É uma doença endêmica que abrange mais de 80 países, incluindo alguns do continente europeu e, principalmente, países sub-desenvolvidos ou em desenvolvimento. Nesta revisão discorre-se sobre as opções terapêuticas tradicionais e atuais, cuja atividade leishmanicida pode conduzirao desenvolvimento racional de novos fármacos. Ressaltando-se o uso de produtos naturais na pesquisa e tratamento de Leishmaniose (ex. quinolonas, chalconase extratos brutos).

Does homeopathically potentized antimony stimulate coagulation? A summary of previous findings and results of an in vitro pilot study by means of thrombelastography.

BACKGROUND: Potentized antimony is traditionally used in anthroposophic medicine to enhance hemostasis in bleeding disorders, but evidence of its effectiveness is scarce. On the other hand, non-toxic and economic additional therapeutic options for hemostatic disorders are desirable. OBJECTIVES: We examined all available literature on the subject and performed a controlled pilot in vitro study to test the procoagulatory potency of antimony D 5. DESIGN: Freshly drawn citrated whole blood of 12 healthy volunteers and 12 patients with bleeding disorders was equally distributed into 344 portions, after which it was mixed with antimony D 5, or its potentized vehicle (lactose D 5) as control solution and tested with thrombelastography. The paired t-test and the Wilcoxon signed rank test were used for statistical analysis. In 5 of the 12 healthy donors, a second blood sample was drawn to assess individual variability and increase the total number of replicates. Thus three separate calculations were performed: for the 12 patients, the 12 healthy donors, and the 5 later samples from the same donors. The analysis was exploratory, and no Bonferroni correction was applied. RESULTS: In the antimony D5 samples of the 12 healthy subjects, but not the patients, there was a tendency toward a shorter clotting time (CT) (p = 0.074) and a trend for an increased clot firmness, expressed as maximal amplitude (MA) (p = 0.058). The increase of MA was significant (p = 0.011) when the later samples were included. No statistical difference was detected for the clot formation time and the clot lysis index. CONCLUSION: The exploratory results of this pilot study are inconclusive as to whether antimony D5 has a procoagulatory effect in vitro, although the results suggest an effect on MA and possibly CT. More research is warranted.

Treatment of rabies in mice and foxes with antiviral compounds.

Thirty four chemical compounds were injected into rabies infected mice by intramuscular (i.m.) route. Twenty four compounds such as well known therapeutic agents: amantadine, lipacids, phenol compounds, didemnin-B, procaine, nucleosides analogues (ribavirin, tiazofurin, pyrazofurin) had no effect. Two compounds had a slight effect not justifying to consider them as possible therapeutic agents: selenazofurin and an analogue of ribavirin (RTA). Eight heteropolyanions (HPA), which have a related chemical structure, were efficient providing 100% protection. Nineteen compounds were injected into rabies infected mice by the intracerebral (i.c.) route. Fourteen compounds such as ribavirin, RTA, selenazofurin, tiazofurin and 9 HPA compounds had no effect. Five other HPA compounds (HPA 23-39-46-51-56) were efficient preventing the development of clinical infection in some mice. Whatever was the treatment route, treated surviving mice developed rabies neutralizing antibodies. No proof of viral multiplication was found in their brains. As some HPA compounds did produce a therapeutic effect in mice, two of them HPA 23 and HPA 39 were administered to rabies-infected foxes. In foxes the compounds prolonged the mean survival time and increased the number of survivors. These data suggest that chemotherapy might be worthwhile when vaccination was impossible.

Leishmania donovani: oral efficacy and toxicity of formycin B in the infected hamster.

Formycin B, a structural analog of inosine, was evaluated as an orally administrable antileishmanial agent. Against Leishmania donovani in hamsters, it achieved an 85-92% reduction in numbers of parasites in livers of infected animals after oral administration at 13 mg/kg/day for 4 days. Its efficacy by oral administration was approximately four to eight times that by intramuscular administration and four times that of the positive control drug Glucantime by intramuscular administration. The levels of formycin B in serum after the final oral administration of 26 mg/kg/day were 1.4 micrograms/ml at 1 hr and 0.3 microgram/ml at 2 hr. The concentration in liver was greater (9.0 micrograms/ml at 1 hr) and declined more slowly. With this latter dosage or with 104 mg/kg/day there was no acute toxicity of formycin B to bone marrow or formed elements of the blood. The only statistically significant toxicity to the liver was a doubling of serum total bilirubin levels. Comparison of the in vivo efficacy of formycin B against L. donovani to the mild acute toxicity of the drug suggests that formycin B has potential as an oral agent against visceral leishmaniasis.

Action of p-(4-amidino-phenoxy)-benzaldehyde-p-amidino-phenylhydrazone dihydrochloride on Leishmania donovani infections in the golden hamster.

The chemotherapeutic effect of a new diamidine, HOE 668, the p-(4-amidino-phenoxy)-benzaldehyde-p-amidino-phenylhydrazone dihydrochloride, was compared with that of known anti-leishmanial drugs in golden hamsters infected with Leishmania donovani. The effect of HOE 668 against visceral leishmaniasis proved superior to that of pentamidine isethionate and the pentavalent antimonial drugs, sodium stibogluconate and N-methylglucamine antimoniate. However, HOE 668 can be used only experimentally because of its toxicity. Its very good anti-leishmanial action qualifies HOE 668 as a standard compound in screening tests.

A method for detecting therapeutic activity against Schistosoma mansoni in mice.

A simple and rapid assay, suitable for routine screening against Schistosoma mansoni in mice, can be achieved by using a reduction in the severity of hepatic lesions as the chief criterion of efficacy. Previous attempts to use this criterion were largely hampered by the use of inappropriate time schedules. Provided the timing of treatment and necropsy is restricted to a certain schedule, a mere glance at the opened abdomen of an infected mouse is sufficient to determine whether schistosome reproduction has been suppressed (by chemosterilization or by broader anthelmintic effects). The essence of the necessary schedule is treatment beginning at 4 weeks after infection and prolonged (continuously or intermittently) for 2 weeks, followed by necropsy at 8 weeks after infection. Using the methods described, two persons can easily examine mice for therapeutic response at the rate of 300 per hour. The assay has been shown to detect both schistosomaticidal and chemosterilizing compounds.