María Clara Echeverry

Leishmaniasis Visceral Canina: pruebas diagnósticas no identifican Estados Reales de la infección

Objetivos Establecer la capacidad de las pruebas serologicas de Inmunofluorescencia indirecta (IFAT) e Inmunoensayo ligado a enzimas (ELISA), para detectar el estado real de la infeccion en leishmaniasis visceral canina (LVC). Metodos Un total de 211 perros criollos localizados en el sur del Departamento del Tolima, zona endemica para la leishmaniasis visceral, fueron evaluados por medio de examen clinico y serologico, usando como antigeno la cepa colombiana de Leishmania infantum (infantum) MHOM/COL/CL044B. Asi mismo, con la finalidad de establecer reacciones cruzadas o coinfecciones, se evaluo la reactividad de los sueros ante antigenos especificos de Trypanosoma cruzi, por medio de la prueba Western Blot (WB). La frecuencia de LVC fue del 44,1 % (93/211) y 50,2 % (103/211) mediante IFAT y ELISA respectivamente. Resultados La seroreactividad ante antigenos especificos de T. cruzi fue del 1,42 % (3/211). La concordancia de las tecnicas serologicas fue baja (K=12,1 %) y a pesar de la presencia de signos clinicos en los animales evaluados, las razones de prevalencia halladas, demostraron que no hay asociacion entre la ocurrencia de las manifestaciones clinicas y la seropositividad a las pruebas diagnosticas. Conclusiones La baja asociacion de las pruebas serologicas utilizadas en el diagnostico de Leishmania infantum, sugiere que es necesario desarrollar estudios que permitan establecer un algoritmo de pruebas diagnosticas en el pais para confirmar el estado real de la infeccion de los animales y de esta forma orientar eficientemente los recursos de Salud Publica destinados al control de la enfermedad .

Agonist mediated internalization of M 2 mAChR is β-arrestin-dependent

Background Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is β-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted internalization of M2 mAChRs is a β-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous β-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M2 mAChR in mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockout mice that lack expression of either one or both isoforms of β-arrestin (β-arrestin 1 and 2). Results In wild type MEF cells transiently expressing M2 mAChRs, 40% of surface M2 mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M2 mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF β-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either β-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M2 mAChRs led to a stable co-localization with GFP-tagged β-arrestin within endocytic structures in multiple cell lines; the compartment to which β-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M2 mAChRs was moderately rescued in MEF β-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (β-arrestin 2 ΔLIELD), AP-2 (β-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of β-arrestin 1 (319–418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells. Conclusion In summary, this study demonstrates that agonist-promoted internalization of M2 mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles.

Clinical and Parasitological Features of Patients with American Cutaneous Leishmaniasis that Did Not Respond to Treatment with Meglumine Antimoniate

Background American cutaneous leishmaniasis (ACL) is a complicated disease producing about 67.000 new cases per year. The severity of the disease depends on the parasite species; however in the vast majority of cases species confirmation is not feasible. WHO suggestion for ACL produced by Leishmania braziliensis, as first line treatment, are pentavalent antimonial derivatives (Glucantime or Sodium Stibogluconate) under systemic administration. According to different authors, pentavalent antimonial derivatives as treatment for ACL show a healing rate of about 75% and reasons for treatment failure are not well known. Methods In order to characterise the clinical and parasitological features of patients with ACL that did not respond to Glucantime, a cross-sectional observational study was carried out in a cohort of 43 patients recruited in three of the Colombian Army National reference centers for complicated ACL. Clinical and paraclinical examination, and epidemiological and geographic information were recorded for each patient. Parasitological, histopathological and PCR infection confirmation were performed. Glucantime IC50 and in vitro infectivity for the isolated parasites were estimated. Results Predominant infecting Leishmania species corresponds to L. braziliensis (95.4%) and 35% of the parasites isolated showed a significant decrease in in vitro Glucanatime susceptibility associated with previous administration of the medicament. Lesion size and in vitro infectivity of the parasite are negatively correlated with decline in Glucantime susceptibility (Spearman: r = (-)0,548 and r = (-)0,726; respectively). Conclusion A negative correlation between lesion size and parasite resistance is documented. L. braziliensis was found as the main parasite species associated to lesion of patients that underwent treatment failure or relapse. The indication of a second round of treatment in therapeutic failure of ACL, produced by L. braziliensis, with pentavalent antimonial derivatives is discussable.

Improving Leishmania Species Identification in Different Types of Samples from Cutaneous Lesions

ABSTRACT The discrimination of Leishmania species from patient samples has epidemiological and clinical relevance. In this study, different gene target PCR-restriction fragment length polymorphism (RFLP) protocols were evaluated for their robustness as Leishmania species discriminators in 61 patients with cutaneous leishmaniasis. We modified the hsp70-PCR-RFLP protocol and found it to be the most reliable protocol for species identification.

Toward an objective diagnostic for detection of Leishmania RNA virus (LRV) in clinical samples from cutaneous leishmaniasis patients.

Leishmania RNA virus (LRV) is a double strand 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 co-infection 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 cutaneous leishmaniasis (TFCL). However, the reported frequencies of LRV associated to complicated outcomes in patients’ series are highly variable, diminishing the relevance on the virus presence in the pathogenesis of the disease. For determining if the apparent inconsistent information about the frequency of LRV associated to CL complicated outcomes could be connected with the virus detection approach, this study tested previously described methods for LRV detection in clinical samples of patients according with the type of sample. In 36 samples with diagnosis of complicated forms of CL (15 ML, 21 TFCL) and 6 samples with non-Leishmania spp infection, LRV presence was assessed by RT-PCR, RT-qPCR and nested-PCR. By combining methods, LRV1 presence was confirmed in 45% (9/20) of isolates and 75% (12/16) of biopsies. The predominant LRV1-infected Leishmania species in this study was Leishmania (Viannia) braziliensis and, for first time, Leishmania (Viannia) panamensis was found infected in clinical samples. In a number of cases, LRV1 was undetectable in the isolates but present in their respective biopsies, suggesting the possibility of underreporting of LRV1 presence in studies focused in parasites isolates exclusively.

Curvas de fusión de regiones genómicas específicas: una herramienta prometedora para el diagnóstico y tipificación de las especies causantes de la leishmaniasis cutánea en Colombia

Introducción. La leishmaniasis cutánea es una enfermedad causada por parásitos del género Leishmania que tiene gran incidencia en Colombia. El diagnóstico y la identificación de la especie infecciosa son factores críticos en el momento de escoger e iniciar el tratamiento. Actualmente, los métodos de diagnóstico y tipificación requieren procedimientos complejos, por lo que es necesario validar nuevos marcadores moleculares y métodos que simplifiquen el proceso.Objetivo. Desarrollar una herramienta basada en la reacción en cadena de la polimerasa (PCR) con curvas de fusión (High Resolution Melting; PCR-HRM) para el diagnóstico y tipificación de las tres especies de Leishmania de importancia epidemiológica en casos de leishmaniasis cutánea en Colombia.Materiales y métodos. Los genomas de Leishmania panamensis, L. braziliensis y L. guyanensis se compararon mediante métodos bioinformáticos. Las regiones específicas de especie identificadas se validaron mediante PCR. Para los marcadores seleccionados se diseñó una PCR-HRM y se estimaron algunos parámetros de validez y seguridad usando aislamientos de pacientes colombianos caracterizados previamente mediante PCR y análisis de polimorfismos en la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism - RFLP; PCR-RFLP) del gen hsp70.Resultados. El análisis genómico comparativo mostró 24 regiones específicas de especie. Sin embargo, la validación mediante PCR solo identificó un marcador específico para cada especie de Leishmania. Los otros marcadores mostraron amplificación cruzada. El límite de detección para los tres marcadores seleccionados fue de un parásito, mientras que la sensibilidad, la especificidad, el valor predictivo positivo y el negativo fueron de 91,4, 100, 100 y 75 %, respectivamente.Conclusiones. Las tres regiones seleccionadas pueden emplearse como marcadores moleculares en el diagnóstico y tipificación de las especies causantes de la leishmaniasis cutánea en Colombia.