Anti-leishmanial activity of 29 daily sessions of intralesional-pentavalent antimony administration on Leishmania (Viannia)-infected BALB/c mice / Actividad antileishmania de 29 sesiones diarias de antimonio pentavalente intralesional en ratones BALB/c infectados con Leishmania (Viannia)

Abstract Introduction: Intralesional-pentavalent antimonials (IL-SbV) are recommended for simple cutaneous leishmaniasis (CL). Few treatment sessions (1-5) and drug volumes (1-5 ml each), relative to lesion size (LS), are recommended. There is not a validated IL-SbV protocol using doses calculated as mg/kg body weight and administered over a large number of IL-sessions, with small injection volumes. Objective: The study aim was to determine the efficacy of different concentrations of IL-SbV administered in 29 daily sessions of 100 μL each, on CL infected mice. Methods: Leishmania (Viannia) panamensis and L. (V.) braziliensis-infected mice (N = 6) were treated with 150, 50, and 16.6 mgSbV/kg/day x 29 days. Percentage of lesion area reduction, aesthetic and final (no lesions, no parasites) efficacy and effective dose (ED)50 were determined. In vitro-SbV activity against parasites was evaluated for both species. Results: The ED50 values were 72.2 and 66.3 (at the end of treatment), 54.3 and 37.7 (15-days pt.), and 145.3 and 148.6 (60-days pt.) for each species, respectively. Differences were observed between Leishmania species at 15-days pt., but not later. At 60-day pt., IL-SbV-150 mg showed final cure rates of 66.6 % for L. (V.) panamensis and 33.3 % for L. (V.) braziliensis-infected mice. After 15 days pt., lesion reactivation was observed in some "aesthetically cured" mice. Glucantime was not active in in vitro assays. Conclusions: The IL-SbV use with a dose calculated as mg/kg body weight and administered over a large number of IL-sessions, with small injection volumes each day could be effective against L. (V.) panamensis and L. (V.) braziliensis-CL infection. An appropriate SbV-dose (higher than 150 mg/kg/day x less than 29 days) must be evaluated.

Resumen Introducción: Los antimoniales pentavalentes aplicados intralesionalmente (IL-SbV) se recomiendan para el tratamiento de la leishmaniasis cutánea (LC) simple. Se recomiendan pocas sesiones (1-5) y volúmenes (1-5 ml cada uno) en relación con el tamaño de la lesión (LS). No existe un protocolo de IL-SbV validado que utilice dosis calculadas según el peso corporal (en mg/kg) y administradas durante varias sesiones en pocos volúmenes de inyección. Objetivo: El objetivo del estudio fue determinar la eficacia de diferentes concentraciones de IL-SbV administradas en 29 sesiones diarias de 100 μL cada una, en ratones con LC. Métodos: Ratones infectados con L. (V.) panamensis y L. (V.) braziliensis (N = 6) fueron tratados intralesionalmente con 150, 50 y 16,6 mg SbV/kg/día x 29 días. Se determinó el porcentaje de reducción del área de la lesión, la eficacia estética y final (sin lesiones, sin parásitos) y la dosis efectiva (DE)50. Adicionalmente de evaluó la actividad in vitro del SbV. Resultados: Los valores de DE50 fueron 72.2 y 66.3 (al final del tratamiento), 54.3 y 37.7 (15 días pt) y 145.3 y 148.6 (60 días pt) para cada especie. Se encontraron diferencias entre las especies sólo a los 15 días pt. La eficacia del tratamiento IL-SbV-150 mg, 60 días pt., fue de 66.6 y 33.3 % en ratones infectados con L. (V.) panamensis L. (V.) braziliensis respectivamente. Después de 15 días pt., se observó reactivación de la lesión en algunos ratones "estéticamente curados". Glucantime no fue activo in vitro. Conclusiones: El uso intralesional de SbV con una dosis calculada en mg/kg de peso corporal y administrada durante varias sesiones, con pequeños volúmenes de inyección cada día, podría ser eficaz en LC por L. (V.) panamensis y L. (V.) braziliensis. Dosis adecuadas de SbV (superiores a 150 mg/kg/día x 20) deben evaluarse.

Evaluation of cytochrome b sequence to identify Leishmania species and variants: the case of Panama

BACKGROUND The genetic heterogeneity of Leishmania parasites is a major factor responsible for the wide variety of Leishmania-associated manifestations. Consequently, understanding the genetic make-up of Leishmania species using suitable molecular markers is an important component of realising local and regional scale disease risk. The cytochrome b (cytb) is frequently used to type New World Leishmania species. However, its potential to discriminate Leishmania species and variants requires further evaluation. OBJECTIVES To explore the capacity of cytb gene to identify New World Leishmania species and variants and to develop an approach able to type local Leishmania species and variants. METHODS We retrieved 360 partial and complete Leishmania cytb gene sequences publicly available in GenBank database to study all single nucleotide polymorphisms (SNPs) across the cytb gene that differentiate New World Leishmania species. This information was used to develop an approach based upon the polymorphisms found in a DNA segment of 948bp. We also compared the typing results found with this technique with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiling obtained using HSP70 gene as target. One hundred Panamanian isolates were used to both typed Leishmania species and assess local genetic variability. FINDINGS We found complete agreement between our cytb approach and the PCR-RFLP profiling method based on HSP70 for Leishmania species identification. Ninety-two isolates were identified as L. panamensis, although other Viannia species were found circulating at a lower frequency. Three L. panamensis haplotypes were identified in Panamanian provinces. We also provide an initial report of L. guyanensis haplotypes circulating in Panama. MAIN CONCLUSIONS Cytb gene sequence encompasses key main SNPs that aid to identify Leishmania species. The cytb approach developed with this information was able to identify and assess genetic variability of local Leishmania species found in this study.

In vitro and in vivo antileishmanial activity of Artemisia annua L. leaf powder and its potential usefulness in the treatment of uncomplicated cutaneous leishmaniasis in humans

ABSTRACT INTRODUCTION: Cutaneous leishmaniasis (CL) is a tropical disease that affects millions of individuals worldwide. The current drugs for CL may be effective but have serious side effects; hence, alternatives are urgently needed. Although plant-derived materials are used for the treatment of various diseases in 80% of the global population, the validation of these products is essential. Gelatin capsules containing dried Artemisia annua leaf powder were recently developed as a new herbal formulation (totum) for the oral treatment of malaria and other parasitic diseases. Here, we aimed to determine the usefulness of A. annua gel capsules in CL. METHODS: The antileishmanial activity and cytotoxicity of A. annua L. capsules was determined via in vitro and in vivo studies. Moreover, a preliminary evaluation of its therapeutic potential as antileishmanial treatment in humans was conducted in 2 patients with uncomplicated CL. RESULTS: Artemisia annua capsules showed moderate in vitro activity in amastigotes of Leishmania (Viannia) panamensis; no cytotoxicity in U-937 macrophages or genotoxicity in human lymphocytes was observed. Five of 6 (83.3%) hamsters treated with A. annua capsules (500mg/kg/day) for 30 days were cured, and the 2 examined patients were cured 45 days after initiation of treatment with 30g of A. annua capsules, without any adverse reactions. Both patients remained disease-free 26 and 24 months after treatment completion. CONCLUSION: Capsules of A. annua L. represent an effective treatment for uncomplicated CL, although further randomized controlled trials are needed to validate its efficacy and safety.

Compuestos sintéticos del tipo de estirilquinolinas con actividades leishmanicida y citotóxica / Styrylquinolines-type synthetic compounds with leishmanicidal and cytotoxic activities

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Leishmania (Viannia) panamensis expresses a nuclease with molecular and biochemical features similar to the Endonuclease G of higher eukaryotes / Leishmania (Viannia) panamensis expresa una nucleasa molecular y bioquímicamente similar a la Endonucleasa G de eucariotas superiores

Objective: To characterize the molecular and biochemical features of the Endonuclease G of Leishmania (Viannia) panamensis.Methods: The gene of the putative L. (V.) panamensis Endonuclease G was amplified, cloned, and sequenced. The recombinant protein was produced in a heterologous expression system and biochemical assays were run to determine its ion, temperature, and pH preferences.Results: The L. (V.) panamensis rENDOG has biochemical features similar to those found in other trypanosomatids and higher eukaryotes. In addition, phylogenetic analysis revealed a possible evolutionary relationship with metazoan ENDOG.Conclusions: L. (V.) panamensis has a gene that codifies an ENDOG homologous to those of higher organisms. This enzyme can be produced in Escherichia coli and is able to degrade covalently closed circular double-stranded DNA. It has a magnesium preference, can be inhibited by potassium, and is able to function within a wide temperature and pH range.

Objetivo: Caracterizar molecular y bioquímicamente la Endonucleasa G (EndoG) de Leishmania (Viannia) panamensis.Métodos: El gen de la putativa Endonucleasa G de L. (V.) panamensis fue amplificado, clonado y secuenciado. La proteína recombinante se produjo en un sistema de expresión heterólogo y la proteína activa se sometió a pruebas bioquímicas para determinar la preferencia de iones, temperatura y pH.Resultados: La rEndoG de L. (V.) panamensis muestra características bioquímicas similares a aquellas descritas en otros trypanosomatidos y en eucariotas superiores. Además, los análisis filogenéticos muestran una posible relación evolutiva con la Endonucleasa G de metazoos.Conclusiones: Leishmania (V.) panamensis posee un gen que codifica para una endonucleasa homóloga a la EndoG de otros organismos superiores, que se puede producir de forma recombinante en Escherichia coli y que es capaz de degradar ADN circular cerrado de doble cadena. Tiene una preferencia por los iones magnesio y manganeso para usarlos como cofactor y es inhibida por el potasio. Además, funciona en un amplio rango de pH y temperatura.

Evaluación de la actividad citotóxica y leishmanicida de extractos y fracciones de Piper cumanense y Piper holtonii / Evaluation of the cytotoxic and leishmanicidal activity of extracts and fractions of Piper cumanense and Piper holtonii

Especies del género Piper son reportadas como promisorias para el tratamiento de enfermedades tropicales. Este estudio evalúa la actividad citotóxica y leishmanicida de extractos y fracciones de diferente polaridad obtenidas de las especies vegetales Piper cumanense (P. cumanense) y Piper holtonii (P. holtonii); se emplearon macrófagos murinos J774 y promastigotes de Leishmania panamensis MHOM/CO/87/UA140. La fracción hexánica (PcH) presentó un efecto leishmanicida con una selectividad de 2 en los modelos in vitro empleados. Esta selectividad permite sugerir una potencial actividad antileishmanial, que amerita seguir siendo explorada.

Piper genus’ species are reported as promissory as tropical diseases treatment. This research showed the cytotoxic and leishmanicidal activity of extracts and fractions of different polarity derived from Piper cumanense (P. cumanense) and Piper holtonii (P. holtoni) on murine macrophages J774 and L. panamensis promastigotes (MHOM/ CO/87/UA140). Hexanic fraction (PcH) exhibited leishmanicidal effect with 2-fold index selectivity in this in vitro model used. These results suggest a potential antileishmanial activity which should be more studied.

Morfología y citoquímica de cultivos celulares de Aedes aegypti (Diptera: Culicidae) y susceptibilidad a Leishmania panamensis (Kinetoplastida: Trypanosomatidae)

Morphology and cytochemistry of Aedes aegypti’s cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae). The first cellular line of Aedes aegypti was developed by Grace in 1966; afterwards, other cellular lines of this species have been generated. These have been used for the study of pathogenic organisms like viruses, bacteria and parasites, which demonstrates their importance in biomedical applications. This research describes, for the first time, some cytochemical characteristics of A. aegypti cell cultures, that were infected with (MHOM/CO/87CL412) strain of Leishmania panamensis. A morphological study of the cell culture was also carried out. Maintenance of the cell culture, parasites and infection in vitro were carried out in the Laboratory of Entomology, Cell Biology and Genetics of the Universidad de La Salle. The cell cultures infected with the parasite were maintained in a mixture of mediums Grace/L15, supplemented with 10 % fetal bovine serum (FBS) at pH 6.8 and a temperature of 26 ºC, during 3, 6 and 9 post-infection days. After this, these cell cultures were processed through High Resolution Light Microscopy (HRLM) and Transmission Electron Microscopy (TEM) based on standard protocols defined by the Group of Microscopy and Image Analyses of the Instituto Nacional de Salud. Semi-fine slices of 1 µm colored with toluidine blue were used for the morphological analysis of the culture, and ultra fine cuts of 60 to 90 nm stained with uranyl acetate and lead citrate where used for the ultrastructural study. In addition, PAS and peroxidase staining was carried out in cells fixed with methanol. The morphometric study was analyzed with software ImageJ (NIH). In the semi-fine slices, small cells were observed showing fibroblastic appearance 10.84±2.54 µm in length and 5.31±1.26 µm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and vacuolated cytoplasm, 23.04±4,00 µm in length and 13.96±3.70 µm wide. These last ones predominated over the ones with fibroblastic appearance. Regarding the PAS coloration, 7.08 % of the cells presented abundant PAS positive cytoplasmatic granules which indicated polysaccharides presence. The peroxidase test gave a negative result. The greatest percentage of infection (18.90 %) of one total of 101 cells, turned up by day 6. Some cells analyzed by TEM presented a vacuolated aspect cytoplasm; some contained parasites, other fibrillar material and others were empty. The results indicate that A. aegypti cell culture can support the internalization and transformation of the parasite, which demonstrates the capacity that these cell cultures have to be infected with L. panamensis and to maintain the infection for approximately one week. Rev. Biol. Trop. 56 (2): 447-458. Epub 2008 June 30.

La primera línea celular de Aedes aegypti fue establecida por Grace en 1966 y desde entonces se han utilizado para el estudio de virus, bacterias y parásitos. En el presente trabajo se describen, por primera vez, algunas características citoquímicas de los cultivos celulares de A. aegypti, infectados con la cepa (MHOM/CO/87CL412) de Leishmania panamensis. También se realizó un estudio morfológico de las células del cultivo. Se observaron 30 células pequeñas con apariencia fibrolastoide de 10.84±2.54 µm de largo y 5.31±1.26 µm de ancho; otras 30 presentaron apariencia epitelioide con 23.04±4.00 µm de largo y 13.96±3.70 µm de ancho; éstas últimas predominaron sobre las de apariencia fibroblastoide. De 113 células, un 7.08%, presentaron abundantes gránulos citoplasmáticos positivos con la coloración de PAS, indicando presencia de polisacáridos. La prueba de peroxidasa dio un resultado negativo. El mayor porcentaje de infección (18.90%), de un total de 101 células, se presentó el día 6. Ultraestructuralmente, las células presentaron un citoplasma con aspecto vacuolado; algunas contenían parásitos, otras material fibrilar y otras estaban vacías. Los resultados indican que los cultivos celulares de A. aegypti pueden ser infectados por L. panamensis y mantener dicho proceso por aproximadamente una semana.

Sand flies fauna involved in the transmission of cutaneous leishmaniasis in Afro-Colombian and Amerindian communities of Choco, Pacific Coast of Colombia / Fauna flebotomínea involucrada en la transmisión de leishmaniasis cutánea en las comunidades negra e indígena de la Costa Pacífica del Choco, Colombia

A prospective study was conducted on the diversity of phlebotomine in the pacific coast of Colombia and its relationship with the eco-epidemiology of leishmaniasis. A total of 5,365 phlebotomines were collected on light traps, in human baits also standing still on walls, trees and mammalian caves. In total, 42 phlebotomine species were identified belonging to the genera Lutzomyia, Brumptomyia and Warileya. Among these, the highly anthropophilic vector species Lutzomyia gomezi (Nitzulescu), Lu. hartmanni (Fairchild & Hertig) and Lu. trapidoi (Fairchild & Hertig). Leishmania panamensis was the main parasite species affecting the population, with a prevalence of 57 percent in Indians and 26 percent in black people. In the Indian community no difference was found in the skin test (Montenegro) between sexes, being positive in 57 percent of indian males and 58.5 percent in indian females. In contrast, 39 percent of black males and 10 percent black females were found to be positive in the skin test. These results confirm that the transmission of L. panamensis in the studied area is restricted to the rural areas and linked to people activities and the location of their homes

Se llevo a cabo un estudio prospectivo sobre diversidad de fauna flebotomínea en la Costa Pacifica colombiana y su relación con la epidemiología de la leishmaniosis, donde se capturó un total de 5.365 ejemplares en trampas de luz, madrigueras de mamíferos, en reposo y en cebo humano. Se colectó un total de 42 especies de flebotomíneos pertenecientes a los géneros Lutzomyia, Brumptomyia y Warileya e igualmente se encontraron especies altamente antropofilicas e incriminadas en la capacidad vectorial como Lutzomyia gomezi (Nitzulescu), Lu. hartmanni (Fairchild & Hertig) y Lu. trapidoi (Fairchild & Hertig). Además se encontró que Leishmania panamensis es la especie de parásito que está afectando a la población en general siendo la prevalencia de infección de la enfermedad un 57 por ciento, para los indígenas, mientras en la población negra sólo el 26 por ciento ha tenido contacto con el parásito. En los indígenas no hay diferencias en la positividad para la prueba de Montenegro entre los sexos: 57 por ciento de los hombres y 58.5 por ciento de las mujeres son positivas. Mientras que en la comunidad negra 39 por ciento de los hombres son positivos para esta prueba, frente a 10 por ciento de las mujeres, reflejando esto las diferencias en el comportamiento de las dos etnias frente al bosque. Estos resultados confirman que la transmisión de L. panamensis en el área de estudio está restringida a las áreas rurales y ligada a la actividad de las personas y a la ubicación con respecto al bosque de las viviendas