Evaluación de la susceptibilidad a la anfotericina B de parásitos de Leishmania del subgénero Viannia con resistencia adquirida in vitro a miltefosina
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Castaño Rodríguez, Marcela
Director
Echeverry Gaitán, María Clara
Ovalle Bracho, Clemencia Elena
Tipo de contenido
Trabajo de grado - Maestría
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EspañolFecha de publicación
2020-03-19
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Dentro de los fármacos usados como segunda línea de tratamiento para la leishmaniasis
están la anfotericina B y la miltefosina. Los dos medicamentos interfieren con la
composición de lípidos en la membrana parasitaria y existe evidencia tanto in vitro como
en aislamientos clínicos de resistencia cruzada entre estos medicamentos en especies
pertenecientes al subgénero Leishmania Leishmania spp. En Suramérica las especies
predominantes pertenecen al subgénero Leishmania Viannia spp. El presente estudio
evaluó la ocurrencia de disminución de susceptibilidad a anfotericina B in vitro en parásitos
de Leishmania Viannia spp con previa exposición a miltefosina. Los parásitos evaluados
correspondían a clones inducidos resistentes y a aislamientos de pacientes tratados con
miltefosina. El establecimiento de dosis efectivas 50 (EC50) se hizo por el método de
rezarsurina, la cuantificación de mRNA de genes involucrados en la biosíntesis y
transporte de lípidos se realizó por RT-qPCR. En los parásitos con inducción experimental
de resistencia a miltefosina se observó concomitantemente el aumento en la tolerancia a
anfotericina B; acompañado por una disminución en la expresión de enzimas involucradas
en la biosíntesis de esterol, esterol metil transferasa y latosterol oxidasa; de una proteína
de unión endosoma-lisosoma vamp y del transportador de miltefosina. El limitado número
de aislamientos clínicos no presentó una correlación entre los valores EC50 calculados
para los dos fármacos. Lo anterior permite concluir que es posible generar resistencia
cruzada entre miltefosina y anfotericina b en parásitos del subgénero Leishmania Viannia
y se requiere estudios con mayor número de aislamiento
Among the second-line drugs used to treat Leishmaniasis are amphotericin B and miltefosine, both drugs interfere with the lipid composition of the parasite membrane, there is cross-resistance evidence in vitro and in clinical isolates in species of the Leishmania subgenus, Leishmania leishmania spp. In South America the predominant species belong to the subgenus Leishmania viannia spp. The aim of the study was to evaluate the susceptibility to amphotericin B in vitro in parasites of Leishmania viannia spp with previous exposition to miltefosine. The evaluated parasites were clones induce resistant to miltefosine and clinical isolates that have been previously expose to miltefosine in Leishmaniasis treatment. Resarzurin method was used to stablish the EC50, mRNA quantification of genes involve in sterol biosynthesis was done through a RT-qPCR. It was observed in induce resistant clones to miltefosine a higher tolerance to amphotericin B, accompany by a loss of expression of enzymes involved in the sterol biosynthesis, sterol methyl transferase and lathosterol oxidase, of a protein involve in membrane fusion of endosome-lysosome, vamp and miltefosina transporter. The limit number of clinical isolates did not follow the change in susceptibility observed for the induce resistant clones in both drugs. As a conclusion, is possible to induce cross-resistance between amphotericin B and miltefosina in Leishmania viannia parasites and a bigger sample size of clinical isolates is needed to assess cross-resistance.
Among the second-line drugs used to treat Leishmaniasis are amphotericin B and miltefosine, both drugs interfere with the lipid composition of the parasite membrane, there is cross-resistance evidence in vitro and in clinical isolates in species of the Leishmania subgenus, Leishmania leishmania spp. In South America the predominant species belong to the subgenus Leishmania viannia spp. The aim of the study was to evaluate the susceptibility to amphotericin B in vitro in parasites of Leishmania viannia spp with previous exposition to miltefosine. The evaluated parasites were clones induce resistant to miltefosine and clinical isolates that have been previously expose to miltefosine in Leishmaniasis treatment. Resarzurin method was used to stablish the EC50, mRNA quantification of genes involve in sterol biosynthesis was done through a RT-qPCR. It was observed in induce resistant clones to miltefosine a higher tolerance to amphotericin B, accompany by a loss of expression of enzymes involved in the sterol biosynthesis, sterol methyl transferase and lathosterol oxidase, of a protein involve in membrane fusion of endosome-lysosome, vamp and miltefosina transporter. The limit number of clinical isolates did not follow the change in susceptibility observed for the induce resistant clones in both drugs. As a conclusion, is possible to induce cross-resistance between amphotericin B and miltefosina in Leishmania viannia parasites and a bigger sample size of clinical isolates is needed to assess cross-resistance.