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Introduction: Resistance against anti-Leishmania drugs (DR) has been studied for years, giving important insights into long-term adaptations of these parasites to drugs, through genetic modifications. However, microorganisms can also survive lethal drug exposure by entering into temporary quiescence, a phenomenon called drug tolerance (DT), which is rather unexplored in Leishmania. Methods: We studied a panel of nine Leishmania braziliensis strains highly susceptible to potassium antimonyl tartrate (PAT), exposed promastigotes to lethal PAT pressure, and compared several cellular and molecular parameters distinguishing DT from DR. Results and discussion: We demonstrated in vitro that a variable proportion of cells remained viable, showing all the criteria of DT and not of DR: i) signatures of quiescence, under drug pressure: reduced proliferation and significant decrease of rDNA transcription; ii) reversibility of the phenotype: return to low IC50 after removal of drug pressure; and iii) absence of significant genetic differences between exposed and unexposed lineages of each strain and absence of reported markers of DR. We found different levels of quiescence and DT among the different L. braziliensis strains. We provide here a new in-vitro model of drug-induced quiescence and DT in Leishmania. Research should be extended in vivo, but the current model could be further exploited to support R&D, for instance, to guide the screening of compounds to overcome the quiescence resilience of the parasite, thereby improving the therapy of leishmaniasis.

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BACKGROUND: Control of cutaneous leishmaniasis (CL) relies on chemotherapy, yet gaps in our understanding of the determinants of therapeutic outcome impede optimization of antileishmanial drug regimens. Pharmacodynamic (PD) parameters of antimicrobials are based on the relationship between drug concentrations/exposure and microbial kill. However, viable Leishmania persist in a high proportion of individuals despite clinical resolution, indicating that determinants other than parasite clearance are involved in drug efficacy. METHODS: In this study, the profiles of expression of neutrophils, monocytes, Th1 and Th17 gene signatures were characterized in peripheral blood mononuclear cells (PBMCs) during treatment with meglumine antimoniate (MA) and clinical cure of human CL caused by Leishmania (Viannia). We explored relationships of immune gene expression with plasma and intracellular antimony (Sb) concentrations. RESULTS: Our findings show a rapid and orchestrated modulation of gene expression networks upon exposure to MA. We report nonlinear pharmacokinetic/pharmacodynamic (PK/PD) relationships of Sb and gene expression dynamics in PBMCs , concurring with a time lag in the detection of intracellular drug concentrations and with PK evidence of intracellular Sb accumulation. CONCLUSIONS: Our results quantitatively portray the immune dynamics of therapeutic healing, and provide the knowledge base for optimization of antimonial drug treatments, guiding the selection and/or design of targeted drug delivery systems and strategies for targeted immunomodulation.

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The immune mechanisms that contribute to the efficacy of treatment of cutaneous leishmaniasis (CL) are not fully understood. The aim of this study was to define immune correlates of the outcome of treatment of CL caused by Leishmania (Viannia) species during standard of care treatment with pentavalent antimonials. We conducted a comparative expression profiling of immune response genes in peripheral blood mononuclear cells (PBMCs) and lesion biopsy specimens obtained from CL patients before and at the end of treatment (EoT) with meglumine antimoniate. The ex vivo response of PBMCs to L (V) panamensis partially reflected that of lesion microenvironments. Significant downregulation of gene expression profiles consistent with local innate immune responses (monocyte and neutrophil activation and chemoattractant molecules) was observed at EoT in biopsy specimens of patients who cured (n = 8), compared to those from patients with treatment failure (n = 8). Among differentially expressed genes, pretreatment expression of CCL2 was significantly predictive of the therapeutic response (receiver operating characteristic [ROC] curve, area under the curve [AUC] = 0.82, P = 0.02). Polymorphisms in regulatory regions of the CCL2 promoter were analyzed in a pilot cohort of DNA samples from CL patients (cures, n = 20, and treatment failure, n = 20), showing putative association of polymorphisms rs13900(C/T) and rs2857656(G/C) with treatment outcome. Our data indicate that dampening gene expression profiles of monocyte and neutrophil activation characterize clinical cure after treatment of CL, supporting participation of parasite-sustained inflammation or deregulated innate immune responses in treatment failure.

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OBJECTIVES: There is a clear need for new strategies of leishmaniasis treatment. This work was conducted to evaluate the efficacy of the co-administration of tamoxifen and meglumine antimoniate (SbV ) in a phase II pilot clinical trial in localised cutaneous leishmaniasis patients. METHODS: A randomised controlled pilot clinical trial was conducted to evaluate the efficacy and safety of oral (40 mg/day for 20 days) or topical tamoxifen (0.1% tamoxifen citrate for 20 days) combined with meglumine antimoniate (20 mg SbV /kg/day for 20 days) vs. a standard SbV protocol (20 mg/kg/day for 20 days) for the treatment of cutaneous leishmaniasis. Primary outcome was complete epithelisation of the lesion 6 months after the end of treatment. Secondary outcomes were lesion healing 2 months after the end of treatment and frequency and severity of adverse events. RESULTS: A total of 38 subjects were included in the trial, 15 were treated with standard SbV and 23 with the combination of tamoxifen and SbV . Of the patients treated with the co-administration scheme, 12 received tamoxifen orally and 11 were treated with topical tamoxifen. Tamoxifen administered by the oral or topical routes was well tolerated. Cure rates 6 months after the end of treatment per intention to treat were 40% in the group treated with the standard SbV scheme, and 36.4% and 58%, respectively, for groups treated with SbV plus topical or oral tamoxifen. CONCLUSIONS: In the doses and schemes used in this study, co-administration of oral tamoxifen and SbV resulted in higher cure rates in comparison with the standard scheme of treatment, although not to statistically significant levels.

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Pentavalent antimonial (Sb5+) drugs such as meglumine antimoniate (MA) are the mainstay treatment of leishmaniases in developing countries. The effects of these compounds on drug-metabolizing enzymes have not been characterized and their potential pharmacokinetic interactions with other drugs are therefore unknown. The present study investigated whether treatment with MA (300 mg Sb5+/kg body weight/day, subcutaneously) for 24 days affected the activities of cytochrome P450 (CYP)1A (ethoxyresorufin- O-deethylase), CYP2A5 (coumarin 7-hydroxylase), CYP2E1 ( p-nitrophenol-hydroxylase), CYP2B9/10 (benzyloxy-resorufin- O-debenzylase), or CYP3A11 (erythromycin- N-demethylase) in the livers of Swiss Webster (SW) and DBA-2 male and female mice. The results showed that CYP2A5-, CYP2E1-, and CYP3A11-catalyzed reactions were unaffected by MA treatment. A decrease in CYP2B9/10 activity was noted in DBA-2 females (but not males) and was not observed in SW males or females. However, repeated MA administration reduced mouse liver CYP1A activity. CYP1A2 messenger RNA (mRNA) levels were not affected by MA and in vitro exposure of mouse liver microsomes to Sb3+ and Sb5+ did not reduce CYP1A activity. These findings suggested that in vivo treatment with Sb5+ drugs depressed CYP1A activity, without downregulating CYP1A2 mRNA expression. Since in vitro treatment of liver microsomes failed to inhibit CYP1A activity, this effect may require intact cells.

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Meglumine antimoniate (MA) and sodium stibogluconate are pentavalent antimony (SbV) drugs used since the mid-1940s. Notwithstanding the fact that they are first-choice drugs for the treatment of leishmaniases, there are gaps in our knowledge of their toxicological profile, mode of action and kinetics. Little is known about the distribution of antimony in tissues after SbV administration. In this study, we evaluated the Sb content of tissues from male rats 24 h and three weeks after a 21-day course of treatment with MA (300 mg SbV/kg body wt/d, subcutaneous). Sb concentrations in the blood and organs were determined by inductively coupled plasma-mass spectrometry. In rats, as with in humans, the Sb blood levels after MA dosing can be described by a two-compartment model with a fast (t1/2 = 0.6 h) and a slow (t1/2 >> 24 h) elimination phase. The spleen was the organ that accumulated the highest amount of Sb, while bone and thyroid ranked second in descending order of tissues according to Sb levels (spleen >> bone, thyroid, kidneys > liver, epididymis, lungs, adrenals > prostate > thymus, pancreas, heart, small intestines > skeletal muscle, testes, stomach > brain). The pathophysiological consequences of Sb accumulation in the thyroid and Sb speciation in the liver, thyroid, spleen and bone warrant further studies.

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The favorable outcome of the treatment of a disease is influenced by the adherence to therapy. Our objective was to assess factors associated with adherence to treatment of patients included in a clinical trial of equivalence between the standard and alternative treatment schemes with meglumine antimoniate (MA) in the treatment of cutaneous leishmaniasis (CL), in the state of Rio de Janeiro. Between 2008 and 2011, 57 patients with CL were interviewed using a questionnaire to collect socioeconomic data. The following methods were used for adherence monitoring: counting of vial surplus, monitoring card, Morisky test and modified Morisky test (without the question regarding the schedule); we observed 82.1% (vial return), 86.0% (monitoring card), 66.7% (Morisky test) and 86.0% (modified Morisky test) adherence. There was a strong correlation between the method of vial counting and the monitoring card and modified Morisky test. A significant association was observed between greater adherence to treatment and low dose of MA, as well as with a lower number of people sleeping in the same room. We recommend the use of the modified Morisky test to assess adherence to treatment of CL with MA, because it is a simple method and with a good performance, when compared to other methods.

O desfecho favorável ao tratamento de uma enfermidade é influenciado pela adesão à terapia. Objetivamos avaliar fatores associados à adesão ao tratamento dos pacientes incluídos em ensaio clínico de equivalência entre o esquema de tratamento padrão e alternativos com antimoniato de meglumina (AM) no tratamento da leishmaniose cutânea (LC) no estado do Rio de Janeiro. Entre 2008 e 2011, 57 pacientes com LC foram entrevistados através de questionário para coleta de dados socioeconômicos. Para monitorização da adesão foram utilizados os seguintes métodos: contagem de ampolas excedentes, cartão de acompanhamento, teste de Morisky e teste de Morisky modificado (sem a pergunta referente ao horário). Observou-se adesão de 82,1% (devolução de ampolas), 86,0% (cartão de acompanhamento), 66,7% (teste de Morisky) e 86,0% (teste de Morisky modificado). Houve forte concordância entre o método contagem de ampolas e cartão de acompanhamento, bem como teste de Morisky modificado. Verificou-se associação significativa entre maior adesão ao tratamento e baixa dose de AM, bem como com menor número de pessoas dormindo no mesmo quarto. Recomendamos a utilização do teste de Morisky modificado na avaliação da adesão ao tratamento da LC com AM por ser método simples e com bom desempenho quando comparado aos outros testes.

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La leishmaniosis es una enfermedad infecciosa parasitaria, endémica, de distribución mundial, que en Venezuela se presenta como leishmaniosis tegumentaria americana (cutánea localizada, cutáneomucosa y cutáneo difusa) además de la forma visceral (kala azar). La variedad cutánea localizada representa el 90 por ciento de los casos atendidos en la consulta de Endemias Rurales del Instituto de Medicina Tropical y en los Servicios de Pediatría Médica y Enfermedades Infecciosas del Adulto del Hospital Universitario de Caracas, y la mayoría son tratados ambulatoriamente con antimoniato de meglumina. En algunas situaciones clínicas es necesaria la hospitalización. Se describen ocho casos hospitalizados: 1) niña de dos años con leishmaniosis cutánea que había recibido inmunoterapia para la parasitosis; 2) paciente de 59 años con leishmaniosis de la mucosa nasal; 3) paciente de 64 años con úlcera de los chicleros en el pabellón auricular; 4) paciente de 36 años con falla terapéutica a la meglumina, quien había recibido inmunoterapia para leishmaniosis; 5) paciente de 59 años alcohólico, con ulceración extensa de cinco años de evolución; 6) mujer de 83 años con leishmaniosis cutánea en miembros inferiores; 7) paciente con leishmaniosis de la mucosa nasal y SIDA; 8) paciente de 37 años con kala azar. Se analizan las razones para la hospitalización y las dosis de antimoniato de meglumina empleadas para la solución terapéutica

Leishmaniasis is an endemic parasitic infectious disease of worldwide distribution. In Venezuela the main characteristics correspond to american tegumentary leishmaniasis (cutaneous localized, cutaneous-mucous, cutaneous diffuse) and visceral leishmaniasis (kala azar). The localized cutaneous variety represents 90 percent of the cases attended at the ambulatory medical clinic for rural endemic diseases at the Tropical Medicine Institute, Pediatric and Adult services for Infectious Diseases at the Universitary Hospital of Caracas, and these patients are treated with pentavalent antimonials. In some clinical conditions hospitalization is required. We describe eight cases of patients treated under hospitalization. Case 1) a two year old girl with cutaneous leishmaniasis who had received immunotherapy for this parasitic disease; case 2) fifty nine year old female with nasal leishmaniasis; case 3) sixty four year old male with a rubber tapper’s ulcer in the ear; case 4) thirty six year old male with treatment failure to pentavalent antimonials having received immunotherapy for leishmaniasis; case 5) fifty nine year old male alcoholic patient, with extended ulcer of five years evolution; case 6) eighty three year old female with cutaneous leishmanisis in the legs; case 7) thirty three year old male with leishmaniasis of the nasal mucosa and AIDS; case 8) thirty seven year old male with kala azar. We discuss the reasons for hospitalization and the dosage of pentavalent antimonials administered

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La leishmaniosis tegumentaria americana es un problema de salud en el medio rural venezolano. La Cátedra de Medicina Tropical (UCV) y el Hospital Universitario de Caracas reciben pacientes con diagnóstico presuntivo, se realiza el diagnóstico de certeza y son tratados ambulatoria u hospitalariamente con antimoniato de meglumina en series terapéuticas de 10 días, a dosis de 3 000 mg/día a 4 500 mg/día. Se evalúan los resultados de 221 pacientes, la mayoría adultos masculinos, con úlcera única en miembros inferiores, procedentes del medio rural del Estado Miranda. Predominó la variedad cutánea localizada, seguida de la cutáneo mucosa con 4 pacientes y cutáneo difusa con 3. Hubo cicatrización de las úlceras y desaparición de los nódulos cutáneos, excepto en 1 paciente (0,3 por ciento) que requirió anfotericina B por falla terapéutica. En el estudio participan estudiantes del curso regular de la cátedra, integrantes del programa: docencia en medicina tropical basada en publicaciones periódicas

American tegumentary leishmaniasis (ATL) is a health problem in the rural areas of Venezuela. Patients are referred to the Tropical Medicine Department (UCV) and to the University Hospital of Caracas with presumptive diagnosis of the disease, are studied to confirm the diagnosis and treated with antimoniate of meglumine in 10 days course at dose of 3 000 mg./day to 4 500 mg./day according to complications as outpatients or hospitalized. We report the results of 221 patients treated, mostly male adults from the rural area of the Miranda state with a sole ulcer in the limbs. Among the clinical presentation the cutaneous-localized form was predominating, followed by the localized muco-cutaneous with 4 and cutaneous-diffuse variety with 3. They responded with healing of the ulcers and disappearance of skin nodules, except one (0.3 percent) who required anfotericine B due to lack of response to meglumine therapy. In this investigation there was the participation of students from the Tropical Medicine course: learning based on periodic publications with students

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Os autores relatam um caso de leishmaniose cutâneo-mucosa em uma paciente de 89 anos, diabética e hipertensa, tratada inicialmente com alopurinol por 10 meses não havendo cicatrização das lesões. Posteriormente, recebeu antimoniato de N-metil glucamina (glucantime) por 4 dias, na dose total de 2.380mg do Sbv, mas desenvolveu cardiotoxicidade e hipocalemia, sendo suspenso o tratamento, entretanto, evoluiu com regressão clínica total das lesões, apesar de ter recebido pequena dose desta medicação.

The authors report a case of a 89 years-old woman with mucocutaneous leishmaniasis and previous diabetes mellitus and high blood pressure, who had been treated with allopurinol for 10 months without healing of lesions. Afterwards, she has been treated with meglumine antimonate, [quot ]glucantime[quot ] for 4 days, with a total dose 2,380 mg of Sbv, but developed cardiac side effects and hypokalemia, hence the treatment was withdrawn. However, this patient developed total clinical regression of lesions, in spite of she has been received low dose of this drug.

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