Detection of Leishmania RNA Virus in Clinical Samples from Cutaneous Leishmaniasis Patients Varies according to the Type of Sample.

Leishmania RNA virus (LRV) is a double-stranded RNA virus belonging to the Totiviridae family detected as cytoplasmic inclusions in some strains of the human parasite Leishmania spp. Experimental evidence supports the hypothesis that human coinfection with Leishmania spp.-LRV triggers an exacerbated immune response in the host that can be responsible for the observed complicated outcomes in cutaneous leishmaniasis (CL), such as mucosal leishmaniasis (ML) and treatment failure of CL. However, the reported frequencies of LRV associated with complicated outcomes in patient's series are highly variable, diminishing the relevance on the virus presence in the pathogenesis of the disease. To assess whether or not the inconsistent information about the frequency of LRV associated with CL complicated outcomes could be related to the virus detection approach, the present study evaluated the LRV presence in clinical samples using a diagnostic algorithm according to the type of the sample. In 36 samples with diagnosis of complicated forms of CL (15 of ML and 21 of CL antimony treatment failure) and six samples with non-Leishmania spp. infection, the LRV presence was assessed by RT-PCR, RT-qPCR, and nested RT-PCR. Viral load was estimated in parasite clinical isolates. By combining the methods, LRV1 presence was confirmed in 45% (9/20) of isolates and 37.5% (6/16) of the incisional biopsies. Remarkably, in some cases (4/8), LRV1 was undetectable in the isolates but present in their respective biopsies, and less frequently, the opposite was observed (1/8), suggesting the possibility of loss of parasites harboring LRV1 during the in vitro growth.

Modelo macrófago humano-amastigote para la identificación de aislados quimio-resistentes de leishmania, prueba de concepto / A human macrophage-amastigote model for chemo-resistant isolates identification in Leishmania, a proof of concept

Este trabajo utilizó un modelo macrófago humano-amastigote como herramienta para recrear in vitro la infección causada por aislados de pacientes con fracaso terapéutico y valorar su utilidad en la identificación de aislados de Leishmania con fenotipo quimio-resistente. Objetivos: (1) Evaluar un modelo in vitro de macrófago humano-amastigote y (2) Determinar su utilidad en la identificación de aislados de Leishmania con fenotipo quimio-resistente. Métodos: Se evaluó un protocolo de purificación basado en la capacidad de los monocitos de adherirse al plástico. Monocitos purificados de sangre humana fueron infectados con promastigotes metacíclicos de especies de referencia y aislados de Leishmania de tres pacientes con falla terapéutica a antimoniales. Se determinó el porcentaje de infección inicial y el efecto leishmanicida de glucantime, anfotericina­B y pentamidina; se correlacionó la capacidad leishmanicida con los niveles de producción de óxido nítrico en cada condición estudiada. Resultados: Los resultados sugieren que el modelo macrófago humano-amastigote empleado recrea in vitro la infección causada por especies de referencia, o con aislados de pacientes con fracaso terapéutico. Adicionalmente sugieren que en monocitos infectados (1) con el aislado VE98MR no puede definirse una IC50 para glucantime ni para pentamidina y (2) con el aislado VE96ZC no puede definirse una IC50 para glucantime mas si para pentamidina. De igual forma, se evidencia una disminución efectiva del porcentaje de infección susceptible a anfotericina-B, para todos los aislados y cepas de referencia. El efecto leishmanicida no se correlaciona con aumentos significativos de la producción de óxido nítrico. Conclusiones: El modelo macrófago humano-amastigote empleado constituye una prueba de concepto que permitió identificar como aislados potencialmente quimio-resistentes a L. (L.) amazonensis (VE98MR) y L. (L.) mexicana (VE96ZC), mas no al aislado L. (L.) amazonensis (VE2000MM)(AU)

This work used a human-amastigote macrophage model as a tool to recreate in vitro infection caused by isolates from patient's with therapeutic failure and assess its usefulness in the identification of chemo-resistant Leishmania isolates. Objectives: (1) Evaluate in vitro a human-amastigote macrophage model and (2) determine its usefulness in the identification of Leishmania isolates with chemo-resistant phenotype. Methods: A purification protocol based on the ability of monocytes to adhere to plastic was evaluated. Monocytes purified from human blood were infected with metacyclic promastigotes of reference species and Leishmania isolates from three patients with antimonial therapeutic failure. The percentage of initial infection and the leishmanicidal effect of glucantime, amphotericin-B and pentamidine were determined; the leishmanicidal capacity was correlated with the levels of nitric oxide production in each condition studied. Results: Results suggest that the human-amastigote macrophage model recreates in vitro the infection caused by reference species, or isolates from patients with therapeutic failure. In addition, they suggest that (1) an effective IC50 for glucantime and pentamidine could not be defined in monocytes infected with the isolate VE98MR and (2) an effective IC50 for pentamidine but nor for glucantime could be defined in monocytes infected with the isolate VE96ZC. On the contrary, an effective decrease in the percentage of infection susceptible to amphotericin-B was observed for all isolates and reference strains. The leishmanicidal effect did not correlate with significant increases in nitric oxide production. Conclusion: The human-amastigote macrophage model used constitutes a proof of concept to identify as potentially chemo-resistant isolates L. (L.) amazonensis (VE98MR) and L. (L.) mexicana (VE96ZC), but not L (L.) amazonensis (VE2000MM)(AU)

Severe Cutaneous Leishmaniasis in a Human Immunodeficiency Virus Patient Coinfected with Leishmania braziliensis and Its Endosymbiotic Virus.

Leishmania parasites cause a broad range of disease, with cutaneous afflictions being, by far, the most prevalent. Variations in disease severity and symptomatic spectrum are mostly associated to parasite species. One risk factor for the severity and emergence of leishmaniasis is immunosuppression, usually arising by coinfection of the patient with human immunodeficiency virus (HIV). Interestingly, several species of Leishmania have been shown to bear an endogenous cytoplasmic dsRNA virus (LRV) of the Totiviridae family, and recently we correlated the presence of LRV1 within Leishmania parasites to an exacerbation murine leishmaniasis and with an elevated frequency of drug treatment failures in humans. This raises the possibility of further exacerbation of leishmaniasis in the presence of both viruses, and here we report a case of cutaneous leishmaniasis caused by Leishmania braziliensis bearing LRV1 with aggressive pathogenesis in an HIV patient. LRV1 was isolated and partially sequenced from skin and nasal lesions. Genetic identity of both sequences reinforced the assumption that nasal parasites originate from primary skin lesions. Surprisingly, combined antiretroviral therapy did not impact the devolution of Leishmania infection. The Leishmania infection was successfully treated through administration of liposomal amphotericin B.

A Potential Role for Mononuclear Phagocytes in Cutaneous Ulcer Development in Human Immunodeficiency Virus-Leishmania braziliensis Coinfection.

Skin ulcer development in cutaneous leishmaniasis due to Leishmania braziliensis infection is associated with a mononuclear cell infiltrate and high levels of tumor necrosis factor (TNF). Herein, we show that despite the absence of Leishmania-driven TNF, a cutaneous leishmaniasis patient with acquired immunodeficiency syndrome developed a skin ulcer. The presence of mononuclear phagocytes and high levels of TNF, chemokine (C-C motif) ligand 2 (CCL2), and metalloproteinase-9 in tissue are identified as potential contributors to immunopathology observed in L. braziliensis-infected patients.

Leishmanicidas potenciais: síntese de fármacos dirigidos derivados de inibidores de diidrofolato redutase e pteridina redutase, de ação seletiva em macrófagos / Potentials Leishmanicidas: synthesis targeted drugs of inhibitors DHFR and PTR, on macrophage in selectivity action

A leishmaniose é doença amplamente distribuída no mundo, afetando mais de 80 países. A quimioterapia desta doença caracteriza-se pela falta de fármacos de ação específica e eficiente, sendo o tratamento de escolha baseado em compostos antimoniais, cuja ação tóxica sobre o organismo do hospedeiro é intensa. Com base na ação das enzimas DHFR e PTR1, envolvidas na biossíntese dos folatos, e na existência de receptores de manose na superficíe dos macrófagos, células parasitadas pela Leishmania sp., foram sintetizados fármacos dirigidos de pirimetamina, fármaco inibidor da DHFR e da PTR1. Para liberação seletiva nos macrófagos, utilizaram-se, como transportadores, derivados de dextrano e manana...

Short report: detection of Leishmaniavirus in human biopsy samples of leishmaniasis from Peru.

Leishmaniavirus is a double-stranded RNA virus that persistently infects some strains of the protozoan parasite Leishmania. There is considerable interest in the possibility that the presence of this virus alters parasite phenotype and may affect disease pathogenesis. If so, the virus marker could provide a valuable prognostic indicator for human leishmaniasis, particularly in those cases caused by New World parasite strains. The virus has been detected in cultured L. braziliensis, L. b. guyanensis, and L. major. To date there has been no information as to the extent of infection in samples prior to culturing in the laboratory. This study demonstrates, through the reverse transcription-polymerase chain reaction, that Leishmaniavirus exists in human biopsy samples of leishmaniasis prior to manipulation in culture.

Infecções naturais de mamíferos silvestres em área endêmica de leishmaniose tegumentar do Estado de São Paulo, Brasil / Natural infection of silvatic mammals of cutaneous leishmaniasis in São Paulo State, Brazil

Descreve-se a presença de foco de leismanioses tegumentar no vale do rio Moji-Guaçú, em região comum aos municípios de Luiz Antonio, São Carlos, Rincão e Santa Rita do Passa Quatro, no Estado de São Paulo, Brasil Os casos humanos apresentavam formas clínicas caracterizadas por lesões úlcero-vegetantes, de evolução lenta e pobre em parasitos. As investigações sobre infecção natural em animais silvestres levaram ao isolamento de roedores, de três cepas em cultura, dias procedentes de Akodon arviculoides e uma de Oryzomys nigripes. As provas de inoculação em hamsters foram, até o momento, positivas para duas delas, mas com evolução lenta, com manifestações clínicas muito discretas e pobres em parasitos. Pelos dados disponíveis até o momento, parecem tratar-se de cepas filiáveis à raça “lenta”, à qual se atribui papel na etiologia da forma cutâneo-mucosa da leishmaniose.