Amphotericin B resistance in Leishmania amazonensis: In vitro and in vivo characterization of a Brazilian clinical isolate.

In Brazil, Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The state of Maranhão in the Northeast of Brazil is prevalent for these clinical forms of the disease and also has high rates of HIV infection. Here, we characterized the drug susceptibility of a L. amazonensis clinical isolate from a 46-year-old man with diffuse cutaneous leishmaniasis coinfected with HIV from this endemic area. This patient underwent several therapeutic regimens with meglumine antimoniate, liposomal amphotericin B, and pentamidine, without success. In vitro susceptibility assays against promastigotes and intracellular amastigotes demonstrated that this isolate had low susceptibility to amphotericin B, when compared with the reference strain of this species that is considered susceptible to antileishmanial drugs. Additionally, we investigated whether the low in vitro susceptibility would affect the in vivo response to amphotericin B treatment. The drug was effective in reducing the lesion size and parasite burden in mice infected with the reference strain, whereas those infected with the clinical isolate and a resistant line (generated experimentally by stepwise selection) were refractory to amphotericin B treatment. To evaluate whether the isolate was intrinsically resistant to amphotericin B in animals, infected mice were treated with other drugs that had not been used in the treatment of the patient (miltefosine, paromomycin, and a combination of both). Our findings demonstrated that all drug schemes were able to reduce lesion size and parasite burden in animals infected with the clinical isolate, confirming the amphotericin B-resistance phenotype. These findings indicate that the treatment failure observed in the patient may be associated with amphotericin B resistance, and demonstrate the potential emergence of amphotericin B-resistant L. amazonensis isolates in an area of Brazil endemic for cutaneous leishmaniasis.

Successful treatment of diffuse cutaneous leishmaniasis caused by Leishmania amazonensis

Abstract Diffuse cutaneous leishmaniasis is a rare universal disease associated with an inadequate host cell immune response, caused by different species: infantum, aethiopica, major, mexicana, and others, which presents the challenge of a poor therapeutic response. In Brazil, it is caused by L. amazonensis. A case confirmed by histopathology with an abundance of vacuolated macrophages full of amastigotes and lymphocyte scarcity, identified by RFLP-ITS1PCR and in vitro decrease and exhaustion of the host cell immune response to L. amazonensis antigen, was treated early (3 months after the onset) with Glucantime (2 months) and allopurinol (29 months) with clinical cure, after a follow-up for 30 months after treatment.

Successful treatment of diffuse cutaneous leishmaniasis caused by Leishmania amazonensis.

Diffuse cutaneous leishmaniasis is a rare universal disease associated with an inadequate host cell immune response, caused by different species: infantum, aethiopica, major, mexicana, and others, which presents the challenge of a poor therapeutic response. In Brazil, it is caused by L. amazonensis. A case confirmed by histopathology with an abundance of vacuolated macrophages full of amastigotes and lymphocyte scarcity, identified by RFLP-ITS1PCR and in vitro decrease and exhaustion of the host cell immune response to L. amazonensis antigen, was treated early (3 months after the onset) with Glucantime (2 months) and allopurinol (29 months) with clinical cure, after a follow-up for 30 months after treatment.

Case Report: Coinfection by Leishmania amazonensis and HIV in a Brazilian Diffuse Cutaneous Leishmaniasis Patient.

Diffuse cutaneous leishmaniasis (DCL) is a rare type of leishmaniasis characterized by diffuse skin lesions. In Brazil, Leishmania (L.) amazonensis is the main etiological agent of this clinical form. The state of Maranhão has the highest prevalence of this disease in the country, as well as a high rate of HIV infection. Here, we report the first case of DCL/HIV of Brazil. A 46-year-old man from the Amazonian area of Maranhão state presented atypical lesion in the left upper limb and dissemination of diffuse erythematous nodules over his entire body. Histopathological examination confirmed the presence of intracellular amastigotes of Leishmania, and a polymerase chain reaction and molecular identification by restriction fragment profile identified L. (L.) amazonensis as the causative agent of the disease. The patient was also diagnosed with HIV virus after the leishmaniasis diagnosis. The initial treatments for leishmaniasis were liposomal amphotericin B (AmB-L) (4 mg/kg) for 10 days and prophylactic use of Glucantime® (10 mg/Sb+5/kg) for 2 months. After unsuccessful initial treatments, he was treated with a combination of AmB-L (4 mg/kg) alternated with pentamidine (4 mg/kg) for 10 days but failed in the first therapeutic cycle. Subsequently, he had a good response to treatment with pentamidine (4 mg/kg).

Miltefosina para o tratamento da leishmaniose tegumentar / Miltefosine for the treatment of cutaneous leishmaniasis

CONTEXTO: A leishmaniose tegumentar (LT) é uma doença infecciosa e não contagiosa, que acomete pele e mucosas, e é causada por protozoários do gênero Leshmania. A doença apresenta baixa letalidade, mas pode causar graves deformidades que impactam na autoestima dos pacientes. É considerada uma doença negligenciada por acometer principalmente populações de baixa renda e permanece como relevante problema de saúde pública, com consideráveis taxas de incidência e prevalência no País. Os medicamentos atualmente preconizados pelo Ministério da Saúde para o tratamento da LT são todos de uso sistêmico e apresentam um potencial hepato, cardio e nefrotóxico, sendo o antimoniato de meglumina, o tratamento de primeira escolha. A maior parte dos casos de LT ocorre em áreas de difícil acesso, o que dificulta tanto a aplicação parenteral da droga, como o monitoramento de seus efeitos colaterais. A miltefosina é um medicamento de uso oral e eficaz no tratamento da LT, mas ainda não é disponibilizada no SUS. A disponibilização de um medicamento de uso oral e efetivo contra a leishmaniose aumentaria a adesão ao tratamento nas áreas mais pobres e remotas do Brasil. TECNOLOGIA: Miltefosina. PERGUNTA: O uso da miltefosina é eficaz e seguro no tratamento da leishmaniose tegumentar quando comparado ao uso do medicamento recomendado como primeira linha de tratamento pelo Ministério da Saúde, o antimoniato de meglumina? EVIDÊNCIAS CIENTÍFICAS: O Parecer Técnico Científico (PTC) da Fiocruz "Miltefosina para tratamento de leishmaniose tegumentar americana: Evidências de eficácia e segurança" foi utilizado como base das evidências científicas. Na busca realizada nesse PTC, foram selecionados 6 ensaios clínicos randomizados comparando a miltefosina via oral com o antimoniato de meglumina via parenteral. Os estudos mostraram taxas similares de cura em 6 meses com os 2 medicamentos e eventos adversos de menor gravidade com a miltefosina. AVALIAÇÃO DE IMPACTO ORÇAMENTÁRIO: Estima-se que impacto orçamentário anual com a incorporação da miltefoseina varie de R$ 561.863,89 a R$ 2.809.319,45, no primeiro ano, e de R$ 8.427.958,35 a R$ 19.665.236,16, ao longo dos próximos cinco anos. O cálculo do impacto orçamentário não levou em conta os custos que seriam economizados com a incorporação da miltefosina: custos diretos, relacionados à administração parenteral dos medicamentos, e os indiretos, relacionados ao deslocamento diário do paciente, sozinho ou acompanhado de cuidadores ou outros membros da família, de sua residência até uma unidade de saúde para receber a administração parenteral dos medicamentos. Como a maioria desses pacientes residem em áreas rurais com difícil acesso aos serviços de saúde, esse deslocamento resulta em perda de trabalho. RECOMENDAÇÃO PRELIMINAR: A CONITEC, em 10/11/2016 recomendou a incorporação no SUS de miltefosina para o tratamento de pacientes com leishmaniose tegumentar. CONSULTA PÚBLICA: O tema foi colocado em consulta pública nº 40, realizada entre os dias 30/11/16 a 19/12/2016. Foram recebidas 06 contribuições de cunho técnico-científico e 3 contribuições de experiência ou opinião. Foram feitas alterações de texto e nenhuma argumentação foi considerada suficiente para alterar a recomendação preliminar. RECOMENDAÇÃO FINAL: Os membros da CONITEC em 09/05/2018 deliberaram, por unanimidade, por recomendar a incorporação no SUS de miltefosina para o tratamento de leishmaniose tegumentar em primeira linha de tratamento. DECISÃO: Incorporar a miltefosina para o tratamento da leishmaniose tegumentar em primeira linha de tratamento, no âmbito do Sistema Único de Saúde - SUS. Dada pela Portaria nº 60 de 31 de outubro de 2018, publicada no Diário Oficial da União nº 210, de 31 de outubro de 2018, seção 1, página 40.

PD-L1 May Mediate T-Cell Exhaustion in a Case of Early Diffuse Leishmaniasis Caused by Leishmania (L.) amazonensis.

Introduction: Diffuse cutaneous leishmaniasis (DCL) is a rare disease form associated with Leishmania (L.) amazonensis in South America. It represents the "anergic" pole of American Tegumentary Leishmaniasis, and the explanation for its resistance to treatment remains elusive. We aimed to study some possible immunological mechanisms involved in the poor DCL treatment response by evaluating some cell surface molecules obtained from a patient with DCL by flow cytometry. Case presentation: A 65-year-old DCL patient who initially failed to respond to the standard treatment for the disease showed vacuolated macrophages filled with amastigotes in lesion biopsy, and L. (L.) amazonensis was identified through ITS1PCR amplification. The Leishmania skin test and indirect immunofluorescence analysis revealed negative results. Peripheral blood from the patient was collected after a few months of treatment, when the patient presented with no lesion. Peripheral blood mononuclear cells were analyzed ex vivo and in vitro after 48 h of stimulation with soluble L. (L.) amazonensis antigen (SLA). Cell death, surface molecules, and intracellular molecules, such as IFN-γ and granzyme B, were analyzed in the cells using flow cytometry. Analysis of the surface markers showed an increased expression of the inhibitory molecule programmed death ligand 1 (PD-L1) in the monocytes restimulated with SLA (approximately 65%), whereas the negative controls were 35% positive for PD-L1. Conversely, compared with the negative controls, we observed a decrease in CD4+IFN-γ+ T cells (8.32 versus 1.7%) and CD8+IFN-γ+ T cells (14% versus 1%). We also observed a relevant decrease in the granzyme B levels in the CD8+ T cells, from 31% in the negative controls to 5% after SLA restimulation. Conclusion: The dysfunctional activation of PD-L1 inhibitory pathway after Leishmania antigen stimulation and reduced levels of IFN-gamma and granzyme B-producing cells could be closely related to unresponssiveness to standard drug treatment of DCL patient.

Case Report: Diffuse Cutaneous Leishmaniasis by Leishmania infantum in a Patient Undergoing Immunosuppressive Therapy: Risk Status in an Endemic Mediterranean Area.

This case report highlights the risk of severe cutaneous leishmaniasis (CL) by Leishmania infantum in patients undergoing immunosuppressant therapy who either live in an endemic area or are visiting in the transmission season. The case patient, resident in Majorca (Balearic Islands), presented 12 disseminated erythematous skin lesions, 1-6 cm in diameter, located on the scalp, cheek, umbilical region, and lower extremities 8 years after undergoing anti-tumor necrosis factor (TNF) therapy. Parasite presence in peripheral blood and high levels of specific antibodies were also observed, indicating a possible risk of CL shifting toward a visceral infection. However, once CL was diagnosed, anti-TNF therapy was discontinued and liposomal amphotericin B was administered, resulting in a complete healing of lesions, no Leishmania DNA detection in blood, and an important serological decrease in antibodies. The lack of data on the supposed epidemiological association between leishmaniasis and immunosuppressive therapy highlights the importance of implementing surveillance systems in endemic areas. No obvious relationship was found based on the data provided by the Balearic Islands Epidemiological System, in contrast with data reported in nearby endemic areas. This indicates that if the suspected association is to be clarified, greater efforts are needed to report information about concomitant diseases and therapies in leishmaniasis patients.

Disseminated Autochthonous Dermal Leishmaniasis Caused by Leishmania siamensis (PCM2 Trang) in a Patient from Central Thailand Infected with Human Immunodeficiency Virus.

AbstractSeveral case reports of autochthonous leishmaniasis in Thailand have been published since 1996. Most of the previous cases presented with visceral leishmaniasis (VL) and were mostly reported in southern part of Thailand. Recently, it has been evident that Leishmania martiniquensis is the main cause of Leishmania infection in Thailand. However, Leishmania siamensis (PCM2 Trang isolate) was found to be of a separate lineage with restricted distribution in southern Thailand and also a cause of disseminated dermal and visceral leishmaniasis in one published case. Here we report the first patient from central Thailand with human immunodeficiency virus infection presenting with disseminated dermal leishmaniasis. Polymerase chain reaction and DNA sequencing analysis (large subunit of RNA polymerase II and 18S ribosomal RNA internal transcribed spacer 1) from the tissue biopsy sample revealed the pathogen sequences to be highly homologous to PCM2 Trang strain previously reported from southern Thailand.

Coinfection of Leishmania guyanensis and Human Immunodeficiency Virus-Acquired Immune Deficiency Syndrome: Report of a Case of Disseminated Cutaneous Leishmaniasis in Ecuador.

AbstractReported herein is the first case of Leishmania-human immunodeficiency virus (HIV) coinfection in Ecuador. In Ecuador, HIV infections overlap endemic areas of leishmaniasis. Immunosuppression is a well-established risk factor for developing severe disease. This is a severe case of a 32-year-old man presenting with disseminated pleomorphic ulcers, papules, and cutaneous plaque-like lesions over his whole body. Numerous amastigotes were observed in both skin scrapings and biopsies. The sequence of the cytochrome b gene confirmed the presence of Leishmania guyanensis. The patient was treated but failed to respond to meglumine antimoniate and amphotericin B. Six months later, the patient died due to bacterial septic shock.

The antifungal compound butenafine eliminates promastigote and amastigote forms of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis.

The production of ergosterol lipid, important for the Leishmania membrane homeostasis, involves different enzymes. This pathway can be blocked to azoles and allylamines drugs, such as Butenafine. The aim of the present work was to evaluate the anti-leishmanicidal activity of this drug in 2 major species of Leishmania responsible for causing the American tegumentar leishmaniasis (L. (L.) amazonensis and L. (V.) braziliensis). Butenafine eliminated promastigote forms of L. amazonensis and L. braziliensis with efficacy similar to miltefosine, a standard anti-leishmania drug. In addition, butenafine induced alterations in promastigote forms of L. amazonensis that resemble programmed cell death. Butenafine as well as miltefosine presented mild toxicity in peritoneal macrophages, however, butenafine was more effective to eliminate intracellular amastigotes of both L. amazonensis and L. braziliensis, and this effect was not associated with elevated levels of nitric oxide or hydrogen peroxide. Taken together, data presented herein suggests that butenafine can be considered as a prototype drug able to eliminate L. amazonensis and L. braziliensis, etiological agents of anergic diffuse and mucocutaneous leishmaniasis, respectively.

New World and Old World Leishmania Infections: A Practical Review.

Leishmaniasis is a parasitic infection endemic to more than 90 countries worldwide. As travel to endemic areas increases, dermatologists need to keep this entity in the differential for any chronic skin lesion in persons who may have had a possible exposure for any duration. It can be difficult to diagnose because manifestations are varied and sometimes subclinical. This article discusses the current state of epidemiology, pathogenesis, clinical presentation, diagnosis, and treatment options. A special focus is placed on cutaneous manifestations and their treatment.

In vitro and in vivo miltefosine susceptibility of a Leishmania amazonensis isolate from a patient with diffuse cutaneous leishmaniasis.

Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo. This study contributed to establish that the miltefosine transporter is essential for drug activity in L. amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.

In vitro and in vivo antileishmania activity of sesquiterpene lactone-rich dichloromethane fraction obtained from Tanacetum parthenium (L.) Schultz-Bip.

The discovery of new treatments for neglected diseases, including leishmaniasis, is a substantial challenge for scientific research. Plant extracts have shown potential in the selective treatment of tropical diseases. The present study evaluated the in vitro and in vivo antileishmania effects of a sesquiterpene lactone-rich dichloromethane fraction (DF) obtained from the aerial parts of Tanacetum parthenium (L.) Schultz-Bip. In vitro studies of the DF indicated an IC50 of 2.40±0.76 µg mL(-1) against the promastigote form and 1.76±0.25 µg mL(-1) against the axenic amastigote form of Leishmania amazonensis. In vivo intramuscular treatment with DF decreased the growth and size of footpad lesions in mice. The DF also significantly decreased the parasite population compared with animals that were treated with the reference drug. Plasma malondialdehyde levels were increased slightly by the DF, attributable to its parthenolide-rich composition that causes cellular apoptosis, compared with the control group, demonstrating treatment efficacy without toxicity or genotoxicity. Because the isolation and purification of plant compounds are costly and time-consuming and generate low yields, extract fractions, such as the DF studied herein, represent a promising alternative for the treatment of leishmaniasis.

Case report: Transient success using prolonged treatment with miltefosine for a patient with diffuse cutaneous leishmaniasis infected with Leishmania mexicana mexicana.

Leishmania sp. is an intracellular parasite that causes a variable degree of clinical manifestations, especially in the skin. We present the case of a 38-year-old male with a chronic history of mucocutaneous disease present since childhood that generated deformity, loss of cartilage in the ears and nose, and scarring that limited his range of motion. The parasite was identified as L. mexicana mexicana. The patient was treated with a 3-month course of oral miltefosine with overwhelming results.

A rare case of diffuse cutaneous leishmaniasis in an immunocompetent patient from sub-Himalayan India.

Diffuse cutaneous leishmaniasis (DCL) is a rare variant of cutaneous leishmaniasis (CL) characterised by multiple (≥10), widespread, slowly progressive, cosmetically disfiguring, non-ulcerating nodules without visceral involvement. The disease is resistant to chemotherapy and characterized by relapses. We present a rare case of a patient with DCL who had extensive lesions (282) and who was HIV negative from a new focus of leishmaniasis in sub-Himalayan India. A favourable response with a regression of the lesions was observed after a month of treatment with intravenous and intralesional sodium stibogluconate with oral pentoxiphyline. Familiarity with such exceptional cases in immunocompetent individuals may facilitate diagnosis and a promising treatment outcome.

Evaluación del transporte de glucosa, fármacos y del potencial de membrana como métodos pronósticos de quimioterapia en Leishmania spp / Evaluation of glucose transport, drugs and membrane potential as prognostic methods of chemotherapy in Leishmania spp

El fracaso terapéutico en leishmaniasis a menudo esta asociado a resistencia a los medicamentos por parte de los parásitos. Hasta ahora no se ha evaluado sistemáticamente si este fenotipo compromete u optimiza el metabolismo o la efectividad en leishmania, es decir, su competencia y adaptabilidad. Durante su ciclo de vida los parásitos deben ajustarse a condiciones de vida extremas, lo cual no es gratuito. Al presentarse conflictos que comprometen propiedades de leishmania esenciales para su supervivencia, surge un costo de adaptación. Sin embargo, tales compensaciones son el precio a pagar que garantizan la co-evolucion del binomio hospedero-parásito y el mantenimiento de la diversidad genética de leishmania. Comprender ese costo es imprescindible a fin de diseñar medidas de prognosis y control exitosas. Adicionalmente, el método vigente y confiable para evaluar resistencia en leishmania es el método in vitro macrofago-amastigote, el cual es oneroso y laborioso. Como parte de un proyecto sobre quimo-resistencia en Leismania, planteamos evaluar posibles parámetros bioquímicos que pudieran servir como marcadores celulares a ser usados para identificar parásitos con fenotipo quimioresistentes en aislados de pacientes y compararlos con cepas de referencia. Los resultados sugieren que algunos aislados:a) tienen incrementada la expresión transportadores ABC, b) utilizan glucosa de forma diferencial, c) tienen un potencial de membrana menos polarizado y d) expresan diferente sensibilidad a inhibidores clásicos de la función mitocondrial. En conjunto, los datos indican que los aislados estudiados expresan los mismos cambios fisiológicos ya descritos en parásitos de referencia quimioresistentes. Es decir, que los cambios explorados podrían constituir un patrón general asociado a este fenómeno leishmania, lo cual los valida como marcadores celulares de resistencia. En conclusion, se propone un nuevo enfoque al problema del tratamiento de la enfermedad ya que, además de las estrategias clásicas, se añadirían herramientas de pronostico del éxito de la quimioterapia.