Dilema ético y epidemiológico sobre el tratamiento de perros para la leishmaniasis visceral en América Latina

In the Americas there are between 4,500 and 6,800 annual cases of severe visceral leishmaniasis, and mortality is estimated to range between 7 and 10%. However, underreporting and subclinical infections mask the real epidemiological importance of visceral leishmaniasis. Control efforts, which have typically focused on insecticide spraying of sand fly vectors and dog culling, have yielded disparate results. Nevertheless, thousands of dogs are sacrificed each year in countries endemic for visceral leishmaniasis. Additionally, current guidelines of leishmaniasis control programs have banned dog treatment with drugs of human use while therapy with other drugs resulted in high rates of relapses. Society requires that control programs take a more humanitarian approach aimed at limiting dog culling. There is an urgent need to promote responsible dog-ownership and support research on: a) novel veterinary therapies, b) low-cost molecular diagnosis of canine visceral leishmaniasis, and c) determination of dog infectivity threshold for proper reservoir management.En las Américas hay entre 4.500 y 6.800 casos anuales de leishmaniasis visceral grave y se estima que la mortalidad varía entre 7 y 10 %. Sin embargo, el subregistro y las infecciones subclínicas enmascaran la importancia epidemiológica real de la leishmaniasis visceral. Los esfuerzos de control que típicamente se han enfocado en la aspersión de insecticidas contra los flebotomíneos vectores y el sacrificio de perros, han arrojado resultados dispares. No obstante, miles de perros se sacrifican cada año en países endémicos para leishmaniasis visceral. Además, los lineamientos actuales de los programas de control de la leishmaniasis han prohibido el tratamiento de perros con medicamentos de uso humano, mientras que otras drogas resultan en altas tasas de recaída. La sociedad requiere que los programas de control tengan un manejo más humanitario enfocado a limitar el sacrificio canino. Hay una necesidad urgente de promover la tenencia responsable de los perros y apoyar la investigación en: a) terapias veterinarias novedosas, b) diagnósticos moleculares de bajo costo y c) determinación de los umbrales de capacidad infecciosa canina para el manejo adecuado del reservorio.

Spatial and Temporal Patterns of Phlebotominae Sand Flies (Diptera: Psychodidae) in a Cutaneous Leishmaniaisis Focus in Northern Argentina

Phlebotomine sand ßies (Diptera: Psychodidae) were captured in an area of Argentina endemic for American cutaneous leishmaniasis (ACL). A total of 44,944 ßies were collected during a 130-wk interepidemic period from 1990 through 1993. These sand ßies included Lutzomyia neivai (Pinto) (97.8%), Lutzomyia migonei (Franc¸a) (1.2%), Lutzomyia cortelezzii (Bre`thes) (0.8%), Lutzomyia shannoni (Dyar) (0.1%), and Lutzomyia punctigeniculata (Floch and Abonnenc) (0.1%). Lutzomyia neivai was more abundant in secondary forests and peridomestic environments associated with human cases than in primary forest or xeric thorn scrub areas. Time series analyses of species densities suggested a bimodal or trimodal annual pattern related to rainfall peaks, a 5-wk reproductive cycle, and peridomestic local populations that were located adjacent to secondary forests. In general, sand ßy abundance was correlated with the rainfall of the previous year. Lutzomyia neivai spatial distributions were consistent with ACL incidence patterns during the study and in the recent outbreaks in Argentina. However, Lu. migonei also may be involved in peridomestic transmission. Our results suggest that there is a need for improved, long-term surveillance of sand ßies and ACL cases, as well as development of effective intervention strategies.Fil: Salomón, Oscar Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Wilson, M. l.. University of Michigan; Estados UnidosFil: Munstermann, L. E.. University of Yale; Estados UnidosFil: Travi, B. l.. Centro Internacional de Investigaciones Medicas; Colombi

Cuantificación de citocinas caninas mediante reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real

Introduction. Canines are the principal domestic reservoirs of visceral leishmaniasis in both the Old and New World. The development of highly sensitive and quantitative methods, such as real time reverse transcriptase polymerase chain reaction for measurement of canine cytokines has not been exploited in studies of visceral leishmaniasis.Objective. To standardize the relative quantification of canine IFN-g, IL-4, IL-10, IL-12p40 and IL-12p35 using real time reverse transcriptase polymerase chain reaction.Materials and methods. RNA was isolated from PBMCs from 1 year–old foxhounds and cultured with or without Con A, LPS orStaphylococcus aureus extract. This RNA was used in one-step real time reverse transcriptase polymerase chain reaction to optimize the concentrations of the cytokine primers and probes, generate standard curves for each cytokine, confirm equivalent amplification efficiency of cytokine and normalizer (18S rRNA) RNA, and to quantify the expression of the cytokine RNA. The comparative Ct method was used to determine the relative levels of gene expression in the samples, expressed as the fold-increase relative to the control samples.Results. The regression coefficient for the standard curves and the amplification efficiencies of the cytokine and normalizer RNA indicated that the quantification was reliable over a broad concentration range of input RNA. Relative to control cells, activation of PBMCs led to increased expression of IFN-g (132-fold), IL-4 (8.8-fold), IL-10 (7.2-fold), and IL-12p40 (275-fold). Basal expression of IL-12p35 was also detected.Conclusion. This approach provides several advantages over conventional assays for cytokine measurement and can be exploited in the study of the immunopathogenesis and immunity in canine leishmaniasis.Introducción. Los caninos son el principal reservorio domestico de la leishmaniasis visceral en el Nuevo y Viejo mundo. La reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real para la medición de citocinas caninas no ha sido implementada para el estudio de la leishmaniasis visceral.Objetivo. Estandarizar la cuantificación relativa de IFN-g, IL-4, IL-10, IL-12p40 y IL-12p35 caninas utilizando reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real.Materiales y métodos. Células mononucleares de sangre periférica de perros Fox-Hound fueron estimuladas con ConA, LPS y extracto de Staphylococcus aureus. El ARN fue utilizado en la reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real de un solo paso para optimizar las concentraciones de iniciadores y sondas especificas de cada citocina, generar curvas estándar, confirmar la eficiencia de amplificación de las citocinas y del normalizador (18S ARNr) y cuantificar la expresión de ARN. El método comparativo Ct fue utilizado para determinar los niveles relativos de expresión de ARN en las muestras, expresado como el incremento en el número de veces comparado con los controles.Resultados. El coeficiente de regresión para las curvas estándar y las eficiencias de amplificación de las citocinas y el normalizador, indicaron que la cuantificación fue confiable en un amplio rango de concentraciones de ARN. La activación de células mononucleares de sangre periférica resultó en un incremento en la expresión de IFN-g (132), IL-4 (8.8), IL-10 (7,2), y IL-12p40 (275), relativo a células control. La expresión basal de IL-12p35 fue también detectada.Conclusión. Esta metodología, comparada con los métodos convencionales disponibles para la medición de citocinas, ofrece varias ventajas y podría ser utilizada en estudios sobre inmunopatogenia e inmunidad en leishmaniasis visceral canina

Éxito de la alimentación de Lutzomyia evansi (Diptera: Psychodidae) expuestos experimentalmente a reservorios mamíferos pequeños en un foco endémico de Leishmania chagasi en el norte de Colombia

Lutzomyia evansi is the vector of Leishmania chagasi in northern Colombia. Differences in feeding success were revealed, when this phlebotomine sand fly was fed on five species of small mammal hosts from an endemic focus of visceral leishmaniasis. In each trial, 50 female sand flies were provided access to similar-sized depilated areas of the hind foot of each of 44 individual mammals and allowed to feed for 30 minutes. The number of engorged sand flies was counted at the end of each trial and compared among host species by analysis of variance and Tukey's multiple comparisons test. Sand flies fed least successfully on Sciurus granatensis, a common squirrel in the endemic area. It has not been found infected with L. chagasi. Intermediate numbers of sand flies engorged on Heteromys anomalus and Zygodontomys brevicauda, but these two mammals have not been found infected with L. chagasi and are not expected to be important in transmission. Sand flies fed most successfully on Didelphis marsupialis and Proechimys canicollis. These are the two most abundant mammals in the endemic area and frequently are infected. Results provided further evidence that these two species are the wild mammals with the greatest impact on transmission of L. chagasi in northern Colombia.Un método sencillo de laboratorio reveló diferencias en el éxito de alimentación de Lutzomyia evansi, el vector de Leishmania chagasi en el norte de Colombia, cuando se alimentó sobre cinco especies de pequeños mamíferos de un foco endémico de leishmaniasis visceral, en los que éstos podrían actuar como reservorios. En cada ensayo, a 50 flebótomos hembra se les permitió alimentarse durante 30 minutos sobre un área similar de piel depilada de la pata posterior en 44 mamíferos. El número de flebótomos alimentados se comparó entre especies a través de un análisis de varianza y de la prueba de Tukey de comparaciones múltiples. Los flebótomos escasamente se alimentaron sobre Sciurus granatensis, una ardilla común en el área endémica que no se ha encontrado infectada con Leishmania chagasi. En otros dos mamíferos que hasta el presente han sido negativos para L. chagasi (Heteromys anomalus y Zygodontomys brevicauda), los flebótomos se alimentaron en bajos números. En cambio, los flebótomos se alimentaron en altas proporciones sobre Didelphis marsupialis y Proechimys canicollis, los dos mamíferos más abundantes en el área endémica y que se hallan infectados con L. chagasi. Los resultados aportaron evidencia adicional que estas dos especies de mamíferos silvestres serían una fuente de sangre común para los flebótomos y que, por lo tanto, pueden tener gran impacto sobre la transmisión de L. chagasi en el norte de Colombia

Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis

<p>Abstract</p> <p>Background</p> <p>The Syrian hamster, <it>Mesocricetus auratus</it>, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules.</p> <p>Results</p> <p>Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-γ, TNF-α, IL-10, IL-12p40, TGF-β, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2^-ΔΔCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and <it>Leishmania panamensis </it>infected hamsters.</p> <p>Conclusions</p> <p>The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.</p

¿Oncocercosis en Colombia? Una revaluación del foco de López de Micay

Human onchocerciasis was first described as existing in Colombia in 1965 in a small focus in Lopez de Micay on the Pacific Coast. Subsequent follow-up of the focus 12 years later showed that infection-prevalence had fallen from 15 to 7.5%. Since no patients were reported thereafter, the focus was considered to have been extinguished until 1989, when a child with occular keratitis was referred to the University Hospital in Cali. Onchocerciasis was confirmed by skin,snip examination. In July 1989 a multidisciplinaty team conducted a new survey in the area. Skin biopsies were obtained from 170 individuals. Infection-prevalence detected by skin-snip examination was 4.1% (71170). Ten percent of the surveyed males and 0.9% of the females had detectable microfilariae (mf) in skin. Microfilariae density in skin varied between 0.5 mf per milligram of skin to 47 mflmg and was directly related to the patients' age. Neither palpable subcutaneous nodules nor dermal alterations attributable to the parasite were detected in any of the patients. Occular pathology was found in two patients, consisting of bilateral keratitis and retinal degeneration, respectively. Simuliid activity at the time of the survey was very low and collection was not attempted. We hypothesise that active transmission may be taking place further upstream, where vector activity is greater. An increase in non-immune human settlement in the area is expected, due to the future construction of hydroelectrical plants and related connecting highways. Questions are therefore raised concerning the impact of immigration of naive population to this hypoendemic focus.En 1965 se describió el primer foco de oncocercosis humana en Colombia en López de Micay, en la costa pacífica. Doce años después, una visita de seguimiento mostró que la prevalencia de infección había disminuido del 15 al 7,5%. Ya que no se volvieron a reportar pacientes, se consideró extinguido el foco hasta 1989, cuando un niño con queratitis ocular fue remitido al Hospital Universitario de Cali. Se confirmó el diagnóstico de oncocercosis con una biopsia de piel. En julio de 1989 se hizo una nueva visita al área y se tomaron muestras a 170 personas. La prevalencia de infección detectada por biopsia de piel fue de 4,1% (71170). Se detectaron microfilarias (mf) en piel en 10% de los hombres muestreados y 0,9% de las mujeres. La microfilarodermia varió entre 0,5 mf por miligramo de piel y 47mflmg y estaba directamente relacionada con la edad. No se encontraron nódulos subcutáneos palpables ni alteraciones dérmicas atribuibles al parásito en ningún paciente. Dos pacientes presentaron cambios oculares: queratitis bilateral y degeneración de la retina, respectivamente. Durante la visita, la actividad de los simúlidos fue muy baja y no se intentaron capturas. Es probable que la transmisión ocurra en sitios río arriba, donde la actividad de los vectores es mayor. Con la construcción de vías de acceso y una planta hidroeléctrica se espera un aumento de la población no inmune en el área. Cabe preguntar, cúal será el impacto de esta población susceptible sobre este foco hipoendémico

Heterogeneities in leishmania infantum infection : using skin parasite burdens to identify highly infectious dogs

Background: The relationships between heterogeneities in host infection and infectiousness (transmission to arthropod vectors) can provide important insights for disease management. Here, we quantify heterogeneities in Leishmania infantum parasite numbers in reservoir and non-reservoir host populations, and relate this to their infectiousness during natural infection. Tissue parasite number was evaluated as a potential surrogate marker of host transmission potential. Methods: Parasite numbers were measured by qPCR in bone marrow and ear skin biopsies of 82 dogs and 34 crab-eating foxes collected during a longitudinal study in Amazon Brazil, for which previous data was available on infectiousness (by xenodiagnosis) and severity of infection. Results: Parasite numbers were highly aggregated both between samples and between individuals. In dogs, total parasite abundance and relative numbers in ear skin compared to bone marrow increased with the duration and severity of infection. Infectiousness to the sandfly vector was associated with high parasite numbers; parasite number in skin was the best predictor of being infectious. Crab-eating foxes, which typically present asymptomatic infection and are non-infectious, had parasite numbers comparable to those of non-infectious dogs. Conclusions: Skin parasite number provides an indirect marker of infectiousness, and could allow targeted control particularly of highly infectious dogs

Of cattle, sand flies and men : a systematic review of risk factor analyses for South Asian visceral leishmaniasis and implications for elimination

Background: Studies performed over the past decade have identified fairly consistent epidemiological patterns of risk factors for visceral leishmaniasis (VL) in the Indian subcontinent. Methods and Principal Findings: To inform the current regional VL elimination effort and identify key gaps in knowledge, we performed a systematic review of the literature, with a special emphasis on data regarding the role of cattle because primary risk factor studies have yielded apparently contradictory results. Because humans form the sole infection reservoir, clustering of kala-azar cases is a prominent epidemiological feature, both at the household level and on a larger scale. Subclinical infection also tends to show clustering around kala-azar cases. Within villages, areas become saturated over a period of several years; kala-azar incidence then decreases while neighboring areas see increases. More recently, post kalaazar dermal leishmaniasis (PKDL) cases have followed kala-azar peaks. Mud walls, palpable dampness in houses, and peridomestic vegetation may increase infection risk through enhanced density and prolonged survival of the sand fly vector. Bed net use, sleeping on a cot and indoor residual spraying are generally associated with decreased risk. Poor micronutrient status increases the risk of progression to kala-azar. The presence of cattle is associated with increased risk in some studies and decreased risk in others, reflecting the complexity of the effect of bovines on sand fly abundance, aggregation, feeding behavior and leishmanial infection rates. Poverty is an overarching theme, interacting with individual risk factors on multiple levels. Conclusions: Carefully designed demonstration projects, taking into account the complex web of interconnected risk factors, are needed to provide direct proof of principle for elimination and to identify the most effective maintenance activities to prevent a rapid resurgence when interventions are scaled back. More effective, short-course treatment regimens for PKDL are urgently needed to enable the elimination initiative to succeed

Inflammatory stimuli alter bone marrow composition and compromise bone health in the malnourished host

Inflammation has a role in the pathogenesis of childhood malnutrition. We investigated the effect of malnutrition and inflammatory challenge on bone marrow composition and bone health. We studied an established murine model of moderate acute malnutrition at baseline and after acute inflammatory challenge with bacterial lipopolysaccharide (LPS), a surrogate of Gram-negative bacterial sepsis, or Leishmania donovani, the cause of visceral leishmaniasis. Both of these infections cause significant morbidity and mortality in malnourished children. Of the 2 stimuli, LPS caused more pronounced bone marrow changes that were amplified in malnourished mice. LPS challenge led to increased inflammatory cytokine expression (Il1b, Il6, and Tnf), inflammasome activation, and inflammatory monocyte accumulation in the bone marrow of malnourished mice. Depletion of inflammatory monocytes in Csfr1-LysMcre-DT malnourished mice significantly reduced the inflammasome activation and IL1-ß production after LPS challenge. The inflammatory challenge also led to increased expansion of mesenchymal stem cells (MSCs), bone marrow adiposity, and expression of genes (Pparg, Adipoq, and Srbp1) associated with adipogenesis in malnourished mice. This suggests that inflammatory challenge promotes differentiation of BM MSCs toward the adipocyte lineage rather than toward bone-forming osteoblasts in the malnourished host. Concurrent with this reduced osteoblastic potential there was an increase in bone-resorbing osteoclasts, enhanced osteoclast activity, upregulation of inflammatory genes, and IL-1B involved in osteoclast differentiation and activation. The resulting weakened bone formation and increased bone resorption would contribute to the bone fragility associated with malnutrition. Lastly, we evaluated the effect of replacing lipid rich in omega-6 fatty acids (corn oil) with lipid-rich in omega-3 fatty acids (fish oil) in the nutrient-deficient diet. LPS-challenged malnourished mice that received dietary fish oil showed decreased expression of inflammatory cytokines and Rankl and reduced osteoclast differentiation and activation in the bone marrow. This work demonstrates that the negative effect of inflammatory challenge on bone marrow is amplified in the malnourished host. Increasing dietary intake of omega-3 fatty acids may be a means to reduce inflammation and improve bone health in malnourished children