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La leishmaniasis es una infección parasitaria que afecta a humanos, animales domésticos y silvestres. Es causada por los miembros del género Leishmania que realizan parte de su ciclo biológico en un hospedador vertebrado, en forma aflagelar o amastigote. Y completan su desarrollo en el tubo digestivo del hospedador invertebrado, en su forma flagelar o promastigote, estos son flebótomos del género Phlebotomus en el Viejo Mundo y Lutzomyia en el Nuevo Mundo (Miró, 2007b; Baneth, 2006, 2007). En varios países de América el principal vector de leishmaniasis visceral es Lutzomyia longipalpis, su distribución abarca desde el sur de México hasta el norte de Argentina y es capturada dentro y fuera de las viviendas humanas (Milleron et al., 2004; Lainson y Rangel, 2005; González et al., 2006; Ramírez et al., 2006; Dantas, 2008; Diniz et al., 2008). La leishmaniasis está distribuida en el sur de Europa, África, Asia, América del sur, centro y recientemente en Estados Unidos y Canadá (Baneth, 2007). En el mundo hay 14 millones de personas infectadas y cada año se registran 2 millones de casos nuevos. De ellos 500,000 viscerales, que provoca más de 50,000 defunciones; y 1´500,000 casos cutáneos. La población en riesgo se eleva a 350 millones de personas y sólo en 33 de los 88 países endémicos la leishmaniasis es una enfermedad de notificación obligatoria (OMS, 2007). Los cambios ecológicos, demográficos y medioambientales relacionados con nuevos proyectos de desarrollo, urbanización y grandes movimientos de población están conduciendo a un aumento a escala mundial de consecuencias sanitarias adversas donde el desarrollo del flebótomo vector se ve favorecido, de tal manera que la aparición de casos parece estar relacionada con la continua deforestación y la expansión urbana, que se ha intensificado en los últimos años. Posicionándose como un problema de salud pública cada vez más acusado en muchas regiones de Latinoamérica, especialmente en Brasil, Colombia y Venezuela, donde anteriormente no se encontraba (Desjeux, 2002; Rondón, 2006; ENY-740S, 2007; Sousa y Pearson, 2009). El rol del perro (Canis familiaris), como reservorio en la transmisión doméstica y peri doméstica de la leishmaniasis humana ha sido reconocida desde que Charles Nicoles, el ganador al premio Nobel, descubrió la enfermedad en perros en Tunisia en 1908. El número de perros infectados en Sud América es estimado en millones con una alta tasa de infección en algunas áreas de Brasil y Venezuela (Killick-Kendrick, 1999; Baneth, 2006, 2007; Sousa y Pearson, 2009). La leishmaniasis canina es una enfermedad zoonótica que existe en cerca de 50 de los 88 países donde la leishmaniasis humana está presente y resulta frecuentemente mortal en humanos y perros no tratados (Baneth, 2006; Coura et al., 2006). En la población canina afectada los signos clínicos son muy variables, podemos observar desde animales aparentemente sanos, hasta otros que manifiestan varios signos clínicos. Esto se debe a la complejidad de los mecanismos patogénicos dependientes del parásito y a la marcada individualidad de la respuesta inmunitaria del hospedador (Miró, 2007b). Los términos que utilizaremos como abreviación para leishmaniasis canina será Lcan, en esta especie la piel se ve afectada en el transcurso de la diseminación de la enfermedad a los órganos internos, manifestándose ambas formas de la enfermedad a la vez (Killick-Kendrick, 1999; Baneth, 2006; Miró, 2007b; OIE, 2008). Para designar la enfermedad según sus manifestaciones clínicas en humanos, usaremos las siguientes abreviaciones: leishmaniasis cutánea (LC), leishmaniasis mucocutánea (LMC) y leishmaniasis visceral (LV) (Killick-Kendrick, 1999; Baneth, 2006). La presente monografía pretende ser un documento actualizado donde se revisa la enfermedad en todos sus aspectos, para aumentar los conocimientos existentes sobre esta zoonosis parasitaria de creciente interés en los últimos veinte años.

Leishmaniasis, a protozoan infection caused by Leishmania parasites is a neglected disease affecting millions across the world. It is exhibited by diverse clinical presentations that broadly classified as visceral (VL) and cutaneous (CL) leishmaniasis. Both CL and VL are endemic to Ethiopia, which the later is generally considered as fatal, if left untreated. Leishmania donovani is the sole agent of Ethiopian human VL. In Africa, the worst VL affected regions are found in Sudan and Ethiopia. VL is considered as an endemic and at the same time emerging disease in north, northwest, south and southwest with sporadic cases in Eastern Ethiopia. The epidemiology is more or less associated with seasonal migration to endemic areas and HIV/AIDS. The transmission of CL in Ethiopia is known to involve zoonotic cycle while VL transmission isn't clearly understood despite traditional generalization of anthroponosis in East African platform. The aim of this dissertation is to determine VL transmission cycle and study variability L. donovani and P. orientalis in Ethiopia. Studies on human and non-human hosts were conducted to determine the transmission dynamics. To assess the role of symptomatic and asymptomatic L. donovani infected persons in the epidemiology of VL, a community based cohort was conducted. As the...

Leishmania are protozoan parasites responsible for the range of infections collectively known as leishmaniasis. Currently, drugs represent the only course of treatment. The nitroaromatic prodrugs are a class of agents that are used clinically used to treat trypanosomatid diseases; and in the parasites Trypanosoma brucei and Trypanosoma cruzi these compounds undergo reduction activation by enzymes homologous with bacterial type I nitroreductases (NTRs). From the Leishmania major genome database, we have identified a protein (LmNTR) that could catalyse this reaction. Based on co-factor, oxygen-insensitive activity and substrate range, we demonstrate that the LmNTR displays many characteristics of its bacterial counterparts. Gene deletion studies revealed that LmNTR is essential to the insect and mammalian stages of L. major. Null mutant parasites could not be generated while loss of a single LmNTR allele in the insect form conferred resistance to a range of nitroaromatic compounds without affecting their growth rate or ability to differentiate into infectious forms. Heterozygous lines could not establish an infection in vitro using a tissue culture model, or in vivo, in mice. As NTRs are absent from most eukaryotes, with trypanosomatids being a major exception, we exploit this difference to evaluate a library of nitroaromatic compounds against L. major parasites. Biochemical screens against the purified enzyme revealed that several compounds were effective substrates for LmNTR, generating higher activities than nifurtimox, the clinically used nitro-based agent that targets trypanosomes. Using phenotypic screens, we demonstrated that growth inhibition mirrored enzyme activity, with the most potent compounds generating IC50’s <100 nM whilst having little effect on mammalian cells. L. major NTR was shown to play a key role in parasite killing as heterozygous lines displayed resistance to the compounds. In conclusion, we have shown that LmNTR is essential to the parasite and by exploiting its prodrug activating properties, identified several novel agents that provide new lead structures to treat leishmanial infections.

De acordo com dado da OrganizaÃÃo Mundial de SaÃde (OMS) existem cerca de 12 milhÃes de pessoas no mundo com leishmaniose. No Brasil o agente etiolÃgico da Leishmaniose visceral (LV) à a Leishmania chagasi, e a principal forma de transmissÃo do parasita ao homem e outros hospedeiros mamÃferos à por meio da picada do mosquito fÃmea de insetos dÃpteros da famÃlia Psychodidae, cujo vetor principal à o Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva). O calazar ou leishmaniose visceral à uma doenÃa tropical que se caracteriza pela presenÃa de febre, anemia, hepatoesplenomegalia, manifestaÃÃes hemorrÃgicas, alÃm de linfoadenomegalia, perda de peso, taquicardia, tosse seca, diarrÃia, febre, pancitopenia e hipergamaglobulinemia. O envolvimento hematolÃgico à comum nos pacientes com LV, sendo a anemia a anormalidade mais freqÃente decorrente de natureza multifatorial e com prevalÃncia variÃvel conforme a populaÃÃo analisada. Consiste em avaliar o padrÃo das alteraÃÃes hematolÃgicas perifÃricas dos pacientes com Leishmaniose Visceral. As amostras de sangue foram colhidas apÃs o diagnÃstico laboratorial de pesquisa de formas amastigotas de Leishmania sp em aspirado medular nos pacientes (n=30) do Hospital SÃo Josà de DoenÃas Infecciosas, em Fortaleza, CearÃ, no perÃodo de Janeiro de 2007 a Julho de 2008 e de um grupo controle (n=30) constituÃdo de doadores de sangue do HEMOCE. As anÃlises laboratoriais foram realizadas no LaboratÃrio de AnÃlises ClÃnicas do Hospital Geral de Fortaleza e no LaboratÃrio Louis Pasteur Medicina DiagnÃstica. As contagens dos elementos sanguÃÃneos foram realizadas em contador hematolÃgico Pentra 120 ABX, seguido de avaliaÃÃo citomorfolÃgica. As dosagens bioquÃmicas foram realizadas em Analisador bioquÃmico HITACHI Modular P800 e Modulo E/RocheÂ. A comparaÃÃo entre os grupos, em relaÃÃo à mÃdia das variÃveis quantitativas foi realizada atravÃs do Teste t de Student, para os dados com hipÃtese de distribuiÃÃo normal e Mann-Whitney, no caso de nÃo normalidade dos dados. Foram considerados estatisticamente significantes valores de p < 0,05. Das anÃlises (n=30) pacientes, observou-se que 63%(19) dos pacientes sÃo do interior do Estado do Cearà e 37%(11) pacientes sÃo da capital, Fortaleza. Que 80%(24) dos pacientes sÃo do sexo masculino e 20(8%) do sexo feminino. A maioria dos pacientes, 57% apresentavam-se na faixa etÃria entre 31 a 45 anos de idade. Com relaÃÃo ao tempo da doenÃa atà a data do diagnÃstico laboratorial, 47%(14) dos pacientes jà apresentavam sinais clÃnicos da doenÃa com 30 a 90 dias. Obtivemos para o sexo masculino contagem das HemÃcias, uma mÃdia de 3,69 0,6644 (x106/mm3), Hemoglobina de 9,383Â1,3002 (g/dL), HematÃcrito de 28,6708Â4,0687(%),para o sexo feminino contagem das HemÃcias, uma mÃdia de 3,525 0,6233(x106/mm3), Hemoglobina de 8,5666Â1,6561(g/dL), HematÃcrito de 26,9666Â3,9026(%). Com relaÃÃo aos Ãndices hematimÃticros observamos para ambos os sexo, um Volume Corpuscular MÃdio(VCM) de 78,1Â7,0 (fL), Hemoglobina Corpuscular MÃdia(HCM) de 25,42Â2,51 (pg), ConcentraÃÃo de Hemoglobina Corpuscular MÃdia(CHCM) de 32 2,15 (%), DistribuiÃÃo de amplitude das hemÃcias(RDW) de 15,337Â2,435 (%), Contagem de reticulÃcitos de 20.636,63Â20.300,39 (/mm3). Das anÃlises citomorfolÃgica, Das anÃlises morfolÃgicas evidenciou-se um perfil laboratorial caracterÃstico de anemia do tipo microcÃtica e hipocrÃmica seguido de normocÃtica e normocrÃmica e em 65% das amostras observamos a presenÃa de ârouleauxâ. Para as determinaÃÃes do Ferro sÃrico obtivemos uma mÃdia de 32,53Â17,31 (Âg/dL), Transferrina de 146,83Â42,19 (mg/dL), Ferritina de 1339,47Â599,05 (ng/mL), Vitamina B12 de 573,5Â253,94 (pg/mL). Ãcido FÃlico de 10Â3,47 (ng/mL). Para as contagens dos leucÃcitos a mÃdia observada foi de 2802,33 Â2322,16 (nÂ/mm3), neutrÃfilos de 1.426,33Â1.348,88(nÂ/mm3), linfÃcitos de 982,60Â576,47x(nÂ/mm3), monÃcitos de 350,13Â283,47(nÂ/mm3), plaquetas de 120,233,33Â90.640,89 (nÂ/mm3). Para as determinaÃÃes do VHS ao diagnÃstico, os pacientes apresentaram uma mÃdia de 81,77 mm na primeira hora de observaÃÃo. As determinaÃÃes do TAP nos mostrou que ao diagnÃstico os pacientes apresentaram uma mÃdia de 67,87% de atividade de protrombina. Das anÃlises observou-se que na LV existe um comprometimento na hematopoese traduzindo no sangue perifÃrico por uma pancitopenia a favor principalmente da linhagem eritrÃide, sendo a anemia nestes pacientes de natureza provavelmente crÃnica / According to World Health Organization (WHO) data there are some 12 million people worldwide with leishmaniasis. In Brazil the etiological agent of visceral leishmaniasis (VL) is Leishmania chagasi, and is the main means of transmission from the parasite to human beings and other hosts mammals is through the biting of the female mosquito of the order diptera insects belonging to the Psychodidae family, whose main vector is the Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva). The visceral leishmaniasis or kala azar is a tropical disease that is characterized by the presence of fever, anemia, hepatosplenomegaly, hemorrhagic manifestations, and linfoadenomegaly, weight loss, tachycardia, dry cough, diarrhea, fever, pancytopenia and hypergammaglobulinemia. The hematological involvement is common in patients with VL, with anemia the most frequent abnormality due to its multifactor nature and prevalence varies depending on the population being studied. It is meant to evaluate the pattern of peripheral alterations of patients with hematological Visceral Leishmaniasis. Blood samples were collected after the diagnosis of researches seeking ways of amastigotes Leishmania sp in bone marrow aspirate in patients (n = 30), Hospital of Infectious Diseases SÃo JosÃ, in Fortaleza, in the state of CearÃ, in the period from January 2007 to July 2008 and a control group (n = 30) consists of blood donors from HEMOCE. Laboratory tests were performed at the Clinical Analysis Laboratory of the General Hospital of Fortaleza and the Louis Pasteur Medical Diagnosis Laboratory. The counting of blood elements were measured with an ABX blood Pentre 60 gauge, followed by cytomorphology assessment. The biochemical measurements were made in a HITACHI biochemical analyzer Module P800 and Roche  Modular I. The comparison between the groups, for the average of quantitative variables was performed by Student t-test, for the normal distribution and Mann â Whitney hypothesis data, in case of non-normality of the data. We have statistically considered meaningful values of 0.05. The analysis (n = 30) patients, the study showed that 63% (19) of the patients are from within the State of Cearà and 37% (11)of the patients are from the capital of the state of CearÃ, Fortaleza. That 80% (24) of patients are male and 20 (8%) were females. Most of the patients, 57% were aged between 31 to 45 years old. In what concerns the period of time of the disease until the date of laboratory diagnosis, 47% (14) of the patients already had signs of clinical disease with 30 to 90 days. We have to count the male of red blood cells, an average of 3.69  0.6644 (x106/mm3), hemoglobin of 9383  1.3002 (g/dL), Hematocrit of 28.6708  4.0687 (%) for the female count of red blood cells, an average of 3525  0.6233 (x106/mm3), hemoglobin from 8.5666  1.6561 (g/dL), Hematocrit of 26.9666  3.9026 (%) . With regard to RBC indices observed for both sex, cell volume Medium (MCV) of 78.1  7.0 (fL), mean corpuscular hemoglobin (MCH) of 25.42  2.51 (pg), mean corpuscular hemoglobin concentration (MCHC) of 32  2.15 (%), distribution of amplitude of red blood cells (RDW) of 15,337  2435 (%), reticulocyte count of 20.636.63  20.300.39 (/ mm3). Of the morphological analysis showed up a laboratory profile characteristic of the type of anemia and microcytic normocytic and hypochromic followed by normochromic and 65% of the samples found the presence of "rouleax". For the determinations of serum iron had a average of 32.53  17.31 (Âg/dL), Transferrin of 146.83  42.19 (mg/dL), Ferritin of 1.339.47  599.05 (ng /ml), Vitamin B12 from 573.5  253.94 (pg/mL), Folic Acid, 10  3.47 (ng/mL). For the average counts of leukocytes observed was 2802.33  2322.16 (nÂ/mm3), of neutrophils 1426.33  1348.88 (nÂ/mm3), lymphocytes from 982.60  576.47 (nÂ/ mm3); monocytes of 350.13  283.47 (nÂ/mm3), platelet count 120.233.33  90640.89 (n Â/mm3). For the determinations of the VHS diagnosis, the patients had an average of 81.77 mm in the first hours of observation. The determination of TAP showed us that the diagnosis of the patients showed an average of 67.87% of activity prothrombin. The analysis it was observed that there is an impairment in VL hematopoese in translating in peripheral blood by a pancytopenia mainly in favor of the erythroid lineage, and the anemia in patients with chronic nature probably

Determina si existe asociación entre la forma clínica y la especie de Leishmania identificada mediante la técnica de PCR-HRM, en pacientes atendidos en el Laboratorio de Leishmaniosis del Instituto Nacional de Salud. / Tesis

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Investigation of the molecular alterations of macrophage function during intracellular infection by Leishmania donovani revealed both adverse and positive influences of this protozoan on host cell function. Chapter I delineates a negative effect which this parasite has on signal transduction pathways in its host cell. In macrophages put in contact, or infected with L. donovani, c-fos gene expression mediated through protein kinase A was unaffected under conditions where there was an impairment of protein kinase C-mediated c-fos gene expression. Selective impairment of protein kinase C-, or, calmodulin-dependent protein kinase-mediated signal transduction in the macrophage was found to influence the establishment of infection. Chapters two and three describe a positive enhancement of macrophage function by L. donovani. Intramacrophage infection with L. donovani was shown to enhance host cell viability in the absence of growth factor. This was attributable to the elaboration of a soluble factor(s) by infected macrophages into the cell culture supernatant, which enhanced macrophage viability in a manner independent of cell replication. Further characterization of the mechanism of this enhancement revealed that L. donovani infection, and lipophosphoglycan, inhibited macrophage death by apoptosis. Cell supernatants derived from L. donovani infected cells were also capable of inhibiting macrophage apoptosis. To identify the active factor in infected cell supernatants, the cytokine gene expression profile of L. donovani infected macrophages was delineated and possible candidate cytokines were further investigated. Levels of TNF-$ alpha$ capable of causing an abrogation of apoptosis were found to be produced by infected macrophages. However, antibody neutralization of TNF in infected cell cultures could not reverse the inhibition of apoptosis by L. donovani, implicating the involvement of multiple factors in the abrogation of apoptosis by L. donovani.

The cellular transformation of the Leishmania protozoan parasite from the promastigote to the amastigote stages takes place in the phagolysosomal compartment of vertebrate host macrophage cells. This cytodifferentiation is a prerequisite for parasite survival and for the establishment of the infection in the mammalian host. The differential screening of a L. donovani amastigote cDNA library using stage-specific cDNA probes allowed the identification of the A2 genes, which are only expressed by the parasite as an amastigote. A2 specific transcripts are developmentally expressed in Leishmania in response to a combination of pH and temperature shifts, conditions associated with the transfer from the insect vector to the phagolysosomal compartment in macrophages. The coding genes are clustered on a 850 kb chromosome and arranged in tandem arrays with copies of another gene, the A2rel gene. The developmental expression is mediated post-transcriptionally and involves elements located in the 3$ prime$ untranslated region; accurate processing by trans splicing is essential for the A2 mRNAs developmental accumulation in cells transferred into phagolysosomal conditions. The A2 protein product is composed mostly of highly repeated domains, carries a functional signal peptide at its amino-terminal end, and shares sequence homology with other developmentally expressed proteins in unrelated parasites of humans. Using A2 locus sequences, vectors and transfection systems were designed and developed to differentially express reporter or selectable markets specifically in the amastigote stage.