In vitro effectivity of three approved drugs and their synergistic interaction against Leishmania infantum / Efectividad in vitro de tres fármacos aprobados y su interacción sinérgica contra Leishmania infantum

Introduction: Leishmaniasis remains one of the neglected tropical diseases. Repurposing existing drugs has proven to be successful for treating neglected tropical diseases while combination therapy is a strategic alternative for the treatment of infectious diseases. Auranofin, lopinavir/ritonavir, and sorafenib are FDA approved drugs used in the treatment of diverse diseases by acting on different essential biological enzymes. Objective: To evaluate the effects of monotherapy and combined therapies with the three drugs against Leishmania infantum. Materials and methods: We compared the leishmanicidal effects of the three drugs on promastigotes in vitro as regards the parasite count, the drug concentration providing a half-maximal response, and the ultrastructural changes of the parasite. We determined the fractional inhibitory concentration index of combined drugs in two ways, as well as the activity of the three drugs together to establish their synergetic effect. Results: The monotherapy with the three drugs was effective with auranofin showing the best leishmanicidal effect (EC50=1.5 µM), whereas sorafinib reduced parasite growth at EC50=2.5 µM. The scanning electron microscopy of promastigotes from all treated media showed distortion in the shape with loss of flagella and bleb formation. Acidocalcinosis was evident by transmission electron microscopy with all treatments suggesting apoptosis. Treatment with lopinavir/ritonavir showed signs of autophagy. The two-way combination of the drugs led to additive interactions while the combination of the three drugs showed synergistic action. Conclusion: Each drug when used as monotherapy against Leishmania spp. was effective, but the combination therapy was more effective than the individual drugs due to the additive or synergistic effects.

Introducción. La leishmaniasis sigue siendo una de las enfermedades tropicales desatendidas. La reutilización de medicamentos existentes ha demostrado ser exitosa para tratar enfermedades tropicales desatendidas y la terapia combinada es una alternativa estratégica para el tratamiento de enfermedades infecciosas. Auranofin, lopinavir/ritonavir y sorafenib son medicamentos aprobados por la Food and Drug Administration (FDA) de Estados Unidos utilizados en el tratamiento de diversas enfermedades, pues actúan sobre diferentes enzimas biológicas esenciales. Objetivo. Evaluar los efectos terapéuticos de la monoterapia y de los tres fármacos combinados contra Leishmania infantum. Materiales y métodos. Los efectos leishmanicidas de los tres fármacos sobre los promastigotes se compararon in vitro en cuanto al recuento de parásitos, la concentración del fármaco que proporcionara una respuesta semimáxima y los cambios ultraestructurales del parásito. Se calculó el índice de concentración inhibitoria de fracciones de fármacos combinados de dos maneras y la actividad de los tres fármacos juntos para determinar el efecto sinérgico. Resultados. La monoterapia con los tres medicamentos fue efectiva, pero la auranofina tuvo el mejor efecto antileishmanicida con un CE50 de 1,5 µM, en tanto que el sorafinib redujo el crecimiento del parásito con un CE50 de 2,5 µM. La microscopía electronica de barrido de promastigotes de todos los medios tratados mostró una distorsión en la forma, con pérdida de flagelos y formación de ampollas. La acidocalcinosis fue evidente por microscopía electrónica de transmisión con todos los tratamientos, lo que sugiere apoptosis. El tratamiento con lopinavir/ritonavir mostró signos de autofagia. La combinación de dos medicamentos condujo a interacciones aditivas, mientras que la combinación de las tres drogas produjo una acción sinérgica. Conclusión. Los tres medicamentos usados como monoterapia contra Leishmania spp. fueron efectivos, pero el tratamiento combinado lo fue en mayor medida debido a los efectos aditivos o sinérgicos.

Developmental stages and viability of Toxocara canis eggs outside the host / Desarrollo y viabilidad de huevos de Toxocara canis

ABSTRACT Introduction: Toxocariasis is a soil-transmitted zoonotic disease caused mainly by ingestion of larvated eggs of Toxocara canis. Objectives: To study the morphology of the intraovular developmental stages of Toxocara canis in culture, characterize non-viable eggs and the sequences of larval molting and compare the viability of eggs at the early stages of division and at reaching full maturation. Material and methods: Observation of developing embryos and characterization of non-viable eggs were done using light microscope. The proportions of viable eggs during embryonation were compared to the proportions of viable mature eggs. Results: Cell division commenced after 24 hours of cultivation. Early stages were found to be present over a period of 3-5 days. The developmental stages identified were eggs with: One cell, two cells, three cells, four cells, early morula, late morula, blastula, gastrula, tadpole, pre-larva, first, second and third stage larva. Two larval molts occurred. Non-viable eggs had degenerated cytoplasm, thin or collapsed shell and the larvae did not move after exposure to light. No significant differences were found between the proportions of viable eggs from day five to day 21 as compared to viability of fully mature eggs (30 days). Conclusion: Developing embryos in the environment may be considered as a potential threat to the public health. The precise identification of developmental stages and the clear differentiation of viable and non-viable eggs can help in determining an accurate baseline rate of development that could be used in studies of ovicidal compounds.

RESUMEN Introducción. La toxocariasis es una enfermedad zoonótica transmitida por contacto con el suelo contaminado y causada principalmente por la ingestión de huevos larvados de Toxocara canis. Objetivos. Estudiar la morfología de los estadios intraovulares en desarrollo de T. canis en cultivo, caracterizar los huevos no viables y las secuencias de las mudas larvarias, y comparar la viabilidad de los huevos en las etapas tempranas de división y al alcanzar la maduración completa. Materiales y métodos. Se observó el desarrollo de los embriones y se caracterizaron los huevos no viables, mediante microscopía de luz. Se comparó la proporción de huevos viables con embrión con la de huevos maduros viables. Resultados. La división celular comenzó 24 horas después de iniciado el cultivo. Los estadios tempranos estuvieron presentes por un periodo de tres a cinco días. Los estadios de desarrollo identificados fueron: huevos con una célula, con dos células, con tres células y con cuatro células;mórula temprana, mórula tardía, blástula, gástrula, renacuajo, prelarva, primer, segundo y tercer estado larvario. Se presentaron dos mudas larvarias. Los huevos no viables tenían el citoplasma degradado, cubierta exterior delgada o colapsada, y su larva no se movía al exponerla a la luz. No se encontraron diferencias significativas entre la proporción de huevos viables del día 5 al día 21, al compararla con la viabilidad de los huevos completamente maduros (30 días). Conclusión. Los embriones en desarrollo en el medio ambiente pueden considerarse como un riesgo potencial para la salud pública. La identificación precisa de los estadios de desarrollo y la clara diferenciación de huevos viables y no viables, pueden ayudar a determinar con exactitud una tasa basal de desarrollo, la cual sería útil en el estudio de compuestos ovicidas.

Impact of the age of Biomphalaria alexandrina snails on Schistosoma mansoni transmission: modulation of the genetic outcome and the internal defence system of the snail

Of the approximately 34 identified Biomphalariaspecies,Biomphalaria alexandrinarepresents the intermediate host of Schistosoma mansoniin Egypt. Using parasitological and SOD1 enzyme assay, this study aimed to elucidate the impact of the age of B. alexandrinasnails on their genetic variability and internal defence against S. mansoniinfection. Susceptible and resistant snails were reared individually for self-reproduction; four subgroups of their progeny were used in experiment. The young susceptible subgroup showed the highest infection rate, the shortest pre-patent period, the highest total cercarial production, the highest mortality rate and the lowest SOD1 activity. Among the young and adult susceptible subgroups, 8% and 26% were found to be resistant, indicating the inheritance of resistance alleles from parents. The adult resistant subgroup, however, contained only resistant snails and showed the highest enzyme activity. The complex interaction between snail age, genetic background and internal defence resulted in great variability in compatibility patterns, with the highest significant difference between young susceptible and adult resistant snails. The results demonstrate that resistance alleles function to a greater degree in adults, with higher SOD1 activity and provide potential implications for Biomphalariacontrol. The identification of the most susceptible snail age enables determination of the best timing for applying molluscicides. Moreover, adult resistant snails could be beneficial in biological snail control.