ESTUDIO PRELIMINAR DE CITOCINAS EN SUERO DE PERROS CON LEISHMANIASIS VISCERAL EN UNA ZONA ENDÉMICA VENEZOLANA

Diversos estudios en modelos de ratones han demostrado que la inmunidad protectora frente a Leishmania es mediada por linfocitos T, células presentadoras de antígeno y citocinas. Sin embargo, pocos estudios se han realizado para evaluar citocinas en perros infectados en forma natural con Leishmania infantum/chagasi. El perro doméstico es el principal reservorio del parásito, en tal sentido, el objetivo de este trabajo fue determinar las citocinas en suero de 33 perros con Leishmaniasis Visceral Canina (LVC), provenientes del estado Nueva Esparta, Venezuela (foco endémico). Los perros fueron clasificados en sintomáticos o asintomáticos, de acuerdo al análisis de los signos clínicos de la enfermedad, coincidentes con los títulos de anticuerpos contra las kinesinas recombinantes de Leishmania rK39 y rK26. Otros dos grupos incluyeron: 10 perros de la misma zona endémica como grupo control endémico (CE) y 10 perros de la zona no endémica como control sano (CS). Las concentraciones (pg/mL) de las citocinas solubles IFN-g, TNF-a, IL-10, IL-6, IL-4 e IL-2 se determinaron por citometría de flujo (Kit CBA Hu Th1/Th2, BDTM). Los resultados mostraron concentraciones estadísticamente mayores (p<0,05) de IFN-g (69,93±7,46), IL-4 (7,51±2,68), TNF-a3,86±1,46) e IL-2 (39,85±3,84) en el grupo de perros asintomáticos, con respecto a los perros sintomáticos (60,8±10,6; 5,28±0,80; 2,76±0,72 y 36,04±3,61, respectivamente) y los perros sanos (51±14; 4,65±0,2; 3,21±0,89 y 32,65±5,86, respectivamente). Los perros asintomáticos también presentaron mayor concentración de IL-6 (4,9±0,55) que los CS (4,02±0,64) (p<0,01). Estos resultados demuestran que los perros en estado asintomático exhiben mayor proporción de citocinas de activación celular y proinflamatorias. Los resultados señalan a la medición de citocinas séricas como reflejo del estado inmunológico de los caninos en futuros estudios orientados a vacunación o terapia.   (Palabras clave: Perro; citocinas; suero sanguíneo;  Leishmaniasis visceral; Células Th1; Células Th2)   Abstract   Different experimental murine models have shown that protective immunity against Lesihmania depends upon T cells, cytokines, and antigen presenting cells. However, the role of cytokines in naturally-infected hosts like domestic dogs is controversial. Few studies have evaluated cytokines in dogs naturally-infected with Leishmania infantum/chagasi. Since the domestic dog is the main reservoir of the parasite, a study was conducted to determine cytokines in serum of 33 dogs with Canine Visceral Lesihmaniasis from endemic areas of the State of Nueva Esparta, Venezuela. Dogs were classified as symptomatic (SD) and asymptomatic (AD), according to the expression of three or more clinical signs and levels of  antibodies for rK39 and rK26. Ten non-infected, rK39 negative controls were included from an endemic area (EA) and ten dogs from a non-endemic area were used as healthy controls (HC). The following cytokines (pg/mL) were measured in serum by flow cytometry (CBA Hu Th1/Th 2, BDTM kit): IFN-g, TNF-a, IL-10, IL-6, IL-4 and IL-2. Results show a higher  concentration (P<0.05) of  IFN-g (69.93±7.46), IL-4 (7.51±2.68), TNF-a (3.86±1.46), and IL-2 (39.85±3.84) in AD when compared with SD (60.8±10.6; 5.28±0.80; 2.76± 0.72; and 36.04±3.61, respectively); and HC (51±14; 4.65±0.2; 3.21±0.89, and 32.65±5.86, respectively). The AD also showed higher levels (P<0.01) of IL-6 (4.9±0.55) compared with HC (4.02±0.64). Results show that AD exhibit a higher proportion of cellular activation and proinflammatory cytokines. Results indicate that measuring of serum cytokines could reflect the immunological status in dogs in future clinical trials oriented to either vaccination or therapy.   (Key words: Dog; cytokines; blood serum; Leishmaniasis visceral; Th1 cells; /Th2 cells

The direct agglutination test as an alternative method for the diagnosis of canine and human visceral leishmaniasis

Publicado en la Revista Biomédica 27, 2007. Pp [email protected], [email protected]@gmail.co

Epidemiological aspects of human and canine visceral leishmaniasis in Venezuela

OBJECTIVE: To report recent data on the distribution of human and canine visceral leishmaniasis (VL) in Venezuela, and to highlight problems associated with effective control measures. METHODS: We report the number of cases, incidence rate, age and sex distribution, and mortality rates for human VL (HVL) for the period of 1995 through 2000, based on National Registry of Leishmaniasis data. We carried out serological studies on a total of 3 025 domestic dogs from the 12 states in Venezuela reporting cases of human VL in this 1995-2000 period and also from the state of Yaracuy, where cases were reported earlier during the decade of the 1990s. RESULTS: From 1995 through 2000, 242 cases of HVL were reported from 12 states, in various sections of Venezuela. There was a relatively stable national incidence rate of 0.2 cases per 100 000 persons per year. Of the 242 cases, 26.0% were from Margarita Island, one of the three islands that make up the state of Nueva Esparta (Margarita Island was the only one of the Nueva Esparta islands that had HVL cases). Over the 1995-2000 period, the annual incidence rates for Nueva Esparta ranged from 1.7 to 3.8 cases per 100000 population. Males in Venezuela were more frequently affected (59.5%) than were females (40.5%). In terms of age, 67.7% of the VL patients were < 4 years of age, and 80.6% were younger than 15 years. The mortality rate among the persons with VL was 7.85% during the 1995-2000 period. Serological screening with rK39 antigen of 1217 dogs from Margarita Island found a 28.5% positivity rate (testing of dogs was not done on the two other islands of Nueva Esparta). In contrast, the rate was 2.8% in the 1 808 samples from dogs from 12 states on the mainland. CONCLUSIONS: Human and canine VL are unevenly distributed in Venezuela. The distribution may reflect such factors as differences among the states in human population density, vector density, and the presence or absence of other trypanosomatidae. Particularly high infection rates in very young children as well as in domestic dogs occur in semiurban communities of Nueva Esparta, where other human-infecting trypanosomatidae have not been reported. Control measures related to limiting canine infection might contribute to disease control where VL infections are frequent. Reducing VL mortality requires increased awareness among medical professionals of the possibility of VL in the differential diagnosis of hepato-splenic syndromes, particularly in children

Evaluación de una prueba de aglutinación directa como método alternativo para el diagnóstico de leishmaniasis visceral canina y humana en Venezuela

Introduction. Visceral leishmaniasis is the most severe clinical form of leishmaniasis and is often fatal without proper treatment. Therefore, early and accurate diagnosis is important, butoften difficult in endemic areas.Objective. The aim was to evaluate a direct agglutination test as a potential visceral leishmaniasis diagnostic method in endemic areas of Venezuela.Materials and methods. The performance of the direct agglutination test, based on freezedried Leishmania donovani antigen was evaluated under laboratory conditions using serum samples of humans and dogs from several Venezuelan visceral leishmaniasis endemic areas: Nueva Esparta (Margarita Island), Lara, Anzoátegui and Trujillo Status. The study included confirmed visceral leishmaniasis patients (n=30), visceral leishmaniasis suspected subjects (n=4), healthy controls (n=19) and patients with other confirmed diseases (n=20). In addition, 24 serum samples from dogs with confirmed visceral leishmaniasis and 18 healthy control dogs were tested.Results. All serum samples of visceral leishmaniasis patients, either active or recovered, were positive. They showed anti-L. donovani titers above 1:1600. Three out of four suspected visceral leishmaniasis cases were also positive, while serum samples from endemic controls and patients with other diseases had titers lower than 1:800. A sensitivity of 100% was obtained for all threshold levels under consideration and 100% specificity at the threshold titer of 1:800 (95% confidence interval: 91-100%). A 93% sensitivity (95% confidence interval: 76-99%) was observed in dog samples, with 100% specificity (95% confidence interval: 79-100%) at the threshold titer of 1:200.Conclusion. The direct agglutination test seems suitable for use in epidemiological studies and for serological diagnosis of human visceral leishmaniasis and canine visceral leishmaniasis.Introducción. La leishmaniasis visceral es la forma clínica más grave de la leishmaniasis. &Eacute;sta puede ser fatal si no se administra el tratamiento adecuado. Por ello, el diagnostico temprano es importante, pero a menudo difícil, en las áreas endémicas.Objetivo. Evaluar el potencial de la prueba de aglutinación directa como un método para el diagnóstico de leishmaniasis visceral en zonas endémicas de Venezuela. Materiales y métodos. La efectividad de la prueba de aglutinación directa con el antígeno congelación-descongelación de Leishmania donovani fue evaluada bajo condiciones de laboratorio usando muestras de sueros de humanos y perros provenientes de diferentes regiones endémicas de leishmaniasis visceral de Venezuela: Nueva Esparta (Isla de Margarita), Estados Lara, Anzoátegui y Trujillo. Se incluyeron pacientes con diagnóstico confirmado de leishmaniasis visceral (n=30), sospecha de leishmaniasis visceral (n=4), voluntarios sanos (n=19) y pacientes con otras enfermedades (n=20). Además, se evaluaron 24 muestras de suero de perros con leishmaniasis visceral y 18 controles.Resultados. Todas las muestras de los pacientes con leishmaniasis visceral activa o curada fueron positivas. Mostraron títulos anti-L. donovani por encima de 1:1.600. Tres de cuatro casos con sospecha de leishmaniasis visceral también resultaron positivos a la prueba, mientras que los sueros controles y los de los pacientes con otras patologías dieron títulos por debajo de 1:800. Se obtuvo una sensibilidad de 100% a todos los puntos de corte considerados y una especificidad de 100% al punto de corte de 1:800 (intervalo de confianza de 95%, IC95%: 90,97%-100%). Las muestras de perros mostraron una sensibilidad de 92,59% (IC95%: 75,69%-99,09%) y 100% de especificidad (IC95%: 79,42%-100%) al punto de corte de 1:200. Conclusión. En general, nuestros resultados indican que el uso de la prueba de aglutinación directa es apropiado para la realización de estudios epidemiológicos y para el diagnóstico serológico de leishmaniasis visceral humana y canina en las áreas rurales de Venezuela

Herramientas no invasivas en Venezuela: comparación entre las pruebas inmunoserológicas DAT, rK26 y rK39 en el diagnóstico de leishmaniasis visceral

Human visceral leishmaniasis is a serious public health problem in endemic countries because of its high potential lethality, particularly in children. Rapid diagnosis is essential to early treatment and control of visceral leishmaniasis. The aim was to compare three serodiagnostic tools for human visceral leishmaniasis. Three methods were compared: the rK39 dipstick (Kalazar detection test, Inbios International Inc.), ELISA rK26 and direct agglutination test (DAT) (KIT Biomedical Research). Fifty serum samples from patients positive for rK39 ELISA were compared from four endemic provinces in Venezuela: Nueva Esparta (Margarita island), Lara, Anzoátegui and Trujillo. Additional serum samples from 17 healthy volunteers and 25 patients with other diseases were included. The rK39 ELISA served as the baseline standard method. Sensitivity, specificity, positive predictive value, negative predictive value and likelihood ratio were calculated for each test. All methods had a positive correlation with rK39 ELISA (p <0.0001). They showed high sensitivity and specificity. The direct agglutination test and the rK39 dipstick showed high sensitivity values, 89.7% (95% CI: 81.34.0-98.2%) and 94.2% (95% CI: 87.7-100%), respectively, and high specificity, 81.0% (95% CI: 80.0-99.5%) and 100%. The rK26 ELISA showed good specificity, 99% (95% CI: 95.2-100%), but a very low sensitivity, 37% (95% CI: 23.4-50.2%). Overall results indicated that DAT and rK39 dipstick have the highest specificity and sensitivity. Both are simple, cost-effective and field applicable tests. Therefore, they are recommended for early and accurate diagnosis of visceral leishmaniasi

Epidemiological and Immunological Aspects of Human Visceral Leishmaniasis on Margarita Island, Venezuela

Sixty-five patients were diagnosed with visceral leishmaniasis (VL) on Margarita Island in the decade from 1990 to1999; 86.2% were £ 3 years old. All were leishmanin-negative at diagnosis. Evaluation of 23 cured patients in 1999 revealed that 22/23 had converted to leishmanin-positive; five had persisting antibodies to rK39 antigen, with no clinical evidence of disease. Leishmanin tests were positive in 20.2% of 1,643 healthy individuals from 417 households in endemic areas. Of the positive reactors, 39.8% were identified in 35 (8.4%) of the households, 15 of which had an antecedent case of VL, a serologically positive dog or both. Weak serological activity to rK39 antigen was detected in 3 of 488 human sera from the endemic areas. The presence of micro-foci of intense peri-urban transmission and the apparent absence of other Trypanosomatidae causing human disease offer a unique opportunity for the study of reservoirs, alternative vectors and evaluation of control measures on the Island

Seroprevalencia de la Leishmaniasis Visceral Canina Mediante Elisa con rK39 en Focos Endémicos de Venezuela

En el presente trabajo se presenta la seroprevalencia de la leishmaniasis visceral canina (LVC), zoonosis causada por Leishmania infantum/chagasi. Se realizaron pruebas serol&oacute;gicasy examen cl&iacute;nico a 15.822 perros de 13 entidades federales end&eacute;micas para la leishmaniasis visceral en Venezuela, en el periodo 2004-2012. Los sueros fueron analizados mediante ELISA con el ant&iacute;geno recombinante rK39. Los resultados muestran que 14,8% de la poblaci&oacute;n de caninos son positivos para LV. Los estados Lara (19%) y Gu&aacute;rico (18%) mostraron una mayor prevalencia de la enfermedad. Sin embargo, para los a&ntilde;os 2010-2012, la prevalencia de la LVC para las entidades federales como Anzo&aacute;tegui, Aragua, Carabobo, Cojedes, Nueva Esparta y Sucre se mantuvieron entre un 3% y un 31%. Los caninos seropositivos (67,1% machos y 32,9% hembras) ten&iacute;an edades promedio de 4,8&plusmn;2,9 a&ntilde;os. El 81% de los caninos seropositivos encontrados en estas zonas, no present&oacute; signos cl&iacute;nicos caracter&iacute;sticos de LVC, mientras que la cl&iacute;nica presentada por el resto fueron ulceraciones cut&aacute;neas (8,5%), alopecia (9,4%) y onicogrifosis (19,2%). Este reporte muestra la distribuci&oacute;n geogr&aacute;fica (tanto en zonas rurales como urbanas) y caracter&iacute;sticas cl&iacute;nicas m&aacute;s resaltantes de perros parasitados en las diferentes regiones end&eacute;micas del pa&iacute;s, con el fin de tomar medidas estrat&eacute;gicas que fortalezcan los programas de control y prevenci&oacute;n de esta zoonosis problema de salud p&uacute;blica.AbstractThis study discloses the seroprevalence of canine visceral leishmaniasis (CVL), a zoonotic disease caused by Leishmania infantum/chagasi. In this study, serological tests and clinical examinations were performed in 15,822 dogs from 13 federal entities endemic for visceral leishmaniasis in Venezuela, during the period 2004-2012. Serum samples were analysed by ELISA against the recombinant antigen rK39. Results demonstrateda prevalence of 14.8% of positive dogs for VL. Lara (19%) and Gu&aacute;rico (18%) states showed the highest seroprevalence of the disease. However, for the years 2010-2012, the prevalence of CVL for federal entities as Anzo&aacute;tegui, Aragua, Carabobo, Cojedes, Nueva Esparta, and Sucre remained between 3% and 31%. The seropositive canines (67.1% males and 32.9% females) average 4.8&plusmn;2.9 years of age and 81% of the dogs found in these endemic areas did not show clinical signs characteristic of LVC, while clinical symptoms presented by the rest were skin ulceration (8.5%), alopecia (9.4%) and onychogryphosis (19.2%). This report demonstrates the geographical distribution (both rural and urban) and most striking clinical features of parasitized dogs in different endemic regions of the country, in order to take strategic actions to strengthen the control and prevention programs of this public health problem