American visceral leishmaniasis dissociated from Lutzomyia longipalpis (Diptera, Psychodidae) in the State of Espírito Santo, Brazil / Leishmaniose visceral americana dissociada da presença de Lutzomyia longipalpis (Diptera, Psychodidae) no Estado do Espírito Santo, Brasil

The occurrence of American visceral leishmaniasis in the State of Espírito Santo, Brazil has always been associated with the presence of the Lutzomyia longipalpis vector. The geographic distribution of this vector in this state is related to the presence of specific geoclimatic characteristics, such as a dry climate, low elevations (< 450m), steep slopes and rocky outcrops. The occurrence of human autochthonous cases of American visceral leishmaniasis in municipalities without these geoclimatic characteristics justifies the present study and our main goal was to test the association between the occurrence of American visceral leishmaniasis and the presence of the Lu. longipalpis. Sand flies were captured monthly from July 2006 to August 2007 using Shannon and CDC traps in two municipalities with records of autochthonous American visceral leishmaniasis and one with no record. We captured 13,112 sand flies, but no Lu. longipalpis was found. The absence of Lu. longipalpis and the possible role of another American visceral leishmaniasis vector in these localities were discussed.

A ocorrência de leishmaniose visceral americana no Estado do Espírito Santo, Brasil, sempre foi associada à presença do vetor Lutzomyia longipalpis. A distribuição geográfica desse vetor no estado está relacionada à ocorrência de características geoclimáticas específicas, tais como clima seco, baixas altitudes, relevo acidentado e afloramentos rochosos A ocorrência de casos humanos autóctones de leishmaniose visceral americana em municípios sem essas características geoclimáticas justificou o presente estudo, com objetivo de testar a associação entre a ocorrência de leishmaniose visceral americana e a presença de Lu. longipalpis. Flebotomíneos foram capturados mensalmente de julho de 2006 a agosto de 2007 com uso de armadilhas de Shannon e CDC em dois municípios com registros de leishmaniose visceral americana autóctone e um sem registros. Foram capturados 13.112 flebotomíneos, mas nenhum Lu. longipalpis foi encontrado. A ausência de Lu. longipalpis e a possível participação de outro vetor de leishmaniose visceral americana nessas localidades foram discutidas.

The utility of rhesus monkey (Macaca mulatta) and other non-human primate models for preclinical testing of Leishmania candidate vaccines

Leishmaniasis causes significant morbidity and mortality, constituting an important global health problem for which there are few effective drugs. Given the urgent need to identify a safe and effective Leishmania vaccine to help prevent the two million new cases of human leishmaniasis worldwide each year, all reasonable efforts to achieve this goal should be made. This includes the use of animal models that are as close to leishmanial infection in humans as is practical and feasible. Old world monkey species (macaques, baboons, mandrills etc.) have the closest evolutionary relatedness to humans among the approachable animal models. The Asian rhesus macaques (Macaca mulatta) are quite susceptible to leishmanial infection, develop a human-like disease, exhibit antibodies to Leishmania and parasite-specific T-cell mediated immune responses both in vivo and in vitro, and can be protected effectively by vaccination. Results from macaque vaccine studies could also prove useful in guiding the design of human vaccine trials. This review summarizes our current knowledge on this topic and proposes potential approaches that may result in the more effective use of the macaque model to maximize its potential to help the development of an effective vaccine for human leishmaniasis.

Mucosal leishmaniasis ("espundia") responsive to low dose of N-methyl glucamine (Glucantime) in Rio de Janeiro, Brazil

Response to treatment with antimonial drugs varies considerably depending on the parasite strain involved, immune status of the patient and clinical form of the disease. Therapeutic regimens with this first line drug have been frequently modified both, in dose and duration of therapy. A regimen of 20 mg/kg/day of pentavalent antimony (Sb5+) during four weeks without an upper limit on the daily dose is currently recommended for mucosal disease ("espundia"). Side-effects with this dose are more marked in elderly patients, more commonly affected by this form of leishmaniasis. According to our experience, leishmaniasis in Rio de Janeiro responds well to antimony and, in cutaneous disease, high cure rates are obtained with 5 mg/kg/day of Sb5+ during 30 to 45-days. In this study a high rate of cure (91.4 percent) employing this dose was achieved in 36 patients with mild disease in this same geographic region. Side-effects were reduced and no antimony refractoriness was noted with subsequent use of larger dose in patients that failed to respond to initial schedule

Atypical Mucocutaneous Leishmaniasis Caused by Leishmania braziliensis in an Acquired Immunodeficiency Syndrome Patient: T-cell Responses and Remission of Lesions Associated with Antigen Immunotherapy

An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B). Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens). Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR). Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4). After the first course of combined therapy it became positive (SI=4.17). The antigen responding cells were predominantly T-cells (47.5 percent) most of them with CD8+ phenotype (33 percent). Very low CD4+ cells (2.2 percent) percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g) production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells

Characterization of Endotrypanum (Kinetoplastida: Trypanosomatidae), a unique parasite infecting the neotropical tree sloths (Edentata)

This article reviews current concepts of the biology of Endotrypanum spp. Data summarized here on parasite classification and taxonomic divergence found among these haemoflagellates come from our studies of molecular chacterization of Endotrypanum stocks (representing an heterogenous population of reference strains and isolates from the Brazilian Amazon region) and from scientific literature. Using numerical zymotaxonomy we have demonstrated genetic diversity among these parasites. The molecular trees obtained revealed that there are , at least, three groups (distinct species?) of Endotrypanum, which are distributed in Central and South America. In concordance with this classification ofthe parasites there are further newer molecular data obtained using distinct markers. Moreover, comparative studies (based on the molecular genetics of the organisms) have shown the phylogenetic relationships between some Endotrypanum and related kinetoplastid lineages.

Genetic diversity in natural populations of New World Leishmania

Our results have shown the wide diversity of parasites within New World Leishmania. Biochemical and molecular characterization of species within the genus has revealed that much of the population heterogeneity has a genetic basis. The source of genetic diversity among Leishmania appears to arise from predominantly asexual, clonal reproduction, although occasional bouts of sexual reproduction can be ruled out. Genetic variation is extensive with some clones widely distributed and others seemingly unique and localized to a particular endemic focus. Epidemiological studies of leishmaniasis has been directed to the ecology and dynamics of Leishmania species/variants, particularly in localized areas. Future research using molecular techniques should aim to identify and following Leishmania types in nature and correlate genetic typing with important clinical characteristics such as virulence, pathogenicity, drug resistance and antigenic variation. The epidemiological significance of such variation not only has important implications for the control of the leishmaniases, but would also help to elucidate the evolutionary biology of the causative agents.

Genetic data showing evolutionary links between Leishmania and Endotrypanum

Striking similarities at the morphological, molecular and biological levels exist between many trypanosomatids isolated from sylvatic insects and/or vertebrate reservoir hosts that make the identification of medically important parasites demanding. Some molecular data have pointed to the relationship between some Leishmania species and Endotrypanum, which has an important epidemiological significance and can be helpful to understand the evolution of those parasites. In this study, we have demonstrated a close genetic relationship between Endotrypanum and two new leishmanial species, L. (V.) colombiensis and L. (V.) equatoriensis. we have used (a) numerical zymotaxonomy and (b) the variability of the internal transcribed spacers of the rRNA genes to examine relationship in this group. The evolutionary trees obtained revealed high similarity between L. (V.) colombiensis, L. (V.) equatoriensis and Endotrypanum, forming a tight cluster of parasites. Based on further results of (c) minicircle kDNA heterogeneity analysis and (d) measurement of the sialidase activity these parasites were also grouped together.

Response to heterologous leishmanins in cutaneous leishmaniasis in Nigeria - discovery of a new focus

A pilot study was undertaken to preliminary illustrate the leishmanin skin test (LST) positivity to distinct antigen preparations (derived from promastigote of either Leishmania major or L. amazonensis, or pooled L. mexicana, L. amazonensis and L. guyanensis) in cutaneous leishmaniasis (CL) patients and healthy subjects living in two endemic foci in Nigeria. The study was designed to provide insights into whether cross-species leishmanin, such as that prepared from New World Leishmania could be useful to detect cases of Old World leishmanial infection and to compare the results with LST using L. major-derived leishmanin. The overall LST positivity in individuals from Keana tested with the cross-major-derived leishmanin was 28.7 per cent (27/94), while the positivity rate in the subjects from Kanana tested with the same leishmanin was 54.5 per cent (6/11). Lower positivity values were obtained when L. major (12.5 per cent; 11/88) or L. amazonensis (15.8 per cent; 9/57) was tested as antigen in grossly comparable populations. Moreover, the pooled leishmanin identified most of the subjects (13/14; 92.9 per cent) with active or healed CL, and the maximum reaction sizes were found among positive subjects in this group. No healthy controls (10 total) showed specific DTH response. The LST was useful for assessing the prevalence of subclinical infection and for measuring CL transmission over time. We report for the first time the occurrence of CL in Kanana village of Langtang South local government area of Plateau State.

Characterization of Endotrypanum parasites using specific monoclonal antibodies

A large number of Endotrypanum stocks (representing an heterogeneous population of strains) have been screened against a panel of monoclonal antibodies (MAbs) derived for selected species of Endotrypanum or Leishmania, to see wether this approach could be used to group/differentiate further among these parasites. Using different immunological assay systems, MAbs considered specific for the genus Endotrypanum (E-24, CXXX-3G5-F12) or strain M6159 of E. schaudinni (E-2, CXIV-3C7-F5) reacted variably according to the test used but in the ELISA or immunoflurescence assay both reacted with all the strains tested. Analysis using these MAbs showed antigenic diversity occuring among the Endotrypanum strains, but no qualitative or quantitative reactivity pattern could be consistently related toparasite origin (i.e., host species involved) or geographic area of isolation. Western blot analysis of the parasites showed that these MAbs recognized multiple components. Differences existed either in the epitope density or molecular forms associated with the antigenic determinants and therefore allowed the assignment of the strains to specific antigenic groups. Using immunoflurescence or ELISA assay, clone E-24 produced reaction with L. equatorensis (which is a parasite of sloth and rodent), but not with other trypanosomatids examined. Interestingly, the latter parasite and the Endotrypanum strains cross-reacted with a number of MAbs that were produced against members of the L. major-L. tropica complex.

Monoclonal antibodies for the identification of new world Leishmania species

Monoclonal antibodies specific for selected species complexes of Leishmania have been employed for the characterization of several representative strains of Leishmania isolated from different hosts and localities in the Americas. In the past 15 years, data have been accumulated concerning (i) the specificities of a number of these monoclonal antibodies and (ii) the antigenic variation (level of the expressed antigenic determinants) ocurring among New World Leishmania species or strain variants as recognized by the monoclonal antibodies. This report is an attempt to summarize in brief the data accumulated to date on these points and to indicate the directions for future applications of these specific monoclonal antibodies for identification of leishmanial isolates.

Molecular karyotype analysis and mapping of housekeeping genes to chromosomes of selected species complexes of Leishmania

The molecular karyotypes for 20 reference strais of species complexes of Leishmania were determined by contour-clamped homogeneous eletric field (CHEF) electrosphoresis. Determination of number/position of chromosome-sized bands and chromosomal DNA locations of house-keeping genes were the two criteria used for differentiating and classifying the Leishmania species. We have established two gel running conditions of optimal separation of chromosomes, wich resolved DNA molecules as large as 2,500 kilobase pairs (kb). Chromosomes were polymorphic in number (22-30) and size (200-2,500 kb) of bands among members of five complexes of Leishmania. Although each stock had a distinct karyotype, in general the differences found between strains and/or species within each complex were not clear enough for parasite identification. However, each group showed a specific number of size-concordant DNA molecules, wich allowed distinction among the Leishmania complex parasites. Clear differences between the Old and New world groups of parasites or among some New World Leishmania species were also apparent in relation to the chromosome locations of beta-tubulin genes. Based on these results as well as data from other published studies the potencial of using DNA karyotype for identifying and classifying leishmanial field isolates is discussed

Disseminated American muco-cutaneous leishmaniasis caused by Leishmania brasiliensis brasiliensis in a patient with AIDS: a case report

The authors report a case of culture-proven disseminated American muco-cutaneous leishmaniasis caused by Leishmania brasiliensis brasiliensis in an HIV positive patient. Lesions began in the oropharynx and nasal mucosa eventually spreading to much of the skin surface. The response to a short course of glucantime therapy was good

Description of Leishmania equatorensis sp. n. (Kinetoplastida: Trypanosomatidae), a new parasite infecting arboreal mammals in Ecuador

Characterization is given of a new parasite, Leishmania equatoriensis sp.n. wich was isolated from the viscera of a sloth (Choloepus hoffmanni) and a squirrel (Sciurus granatensis), captured in humid tropical forest onthe Pacific Coast of Ecuador. Data based on biological and molecular criteria, as well as numerical zymotaxonomical analysis, indicate that this parasite is a new species of the L. brasiliensis complex. L. equatoriensis is cleary distinguishable form all other known species within this complex, using the following molecular criteria: reactivity patterns with specific monoclonal antibodies, isoenzyme electrophoresis, and restriction-endonuclease fragment patterns of kinetoplast DNA (k-DNA)

Antigenic differences among Leishmania amazonensis isolates and their relationship with distinct clinical forms of the disease

Immunoblot analysis was used to investigate antigenic differences among clinical isolates of Leishmania amazonensis and their role in the etiology of the diseases. Western blots of promastigote homogenates were analyzed with either monoclonal antibodies (MAbs) specific for the L. mexicana complex (M-4, M-6, M-9 and M-11) or polyclonal sera from L. amazonensis infected patients with the various forms of clinical disease. In the case of the MAbs, no significant variation was observed among the strains of L. amazonensis, isolated from cases of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), visceral leishmaniasis (VL) or post kala-azar dermal leishmaniasis (PKDL), in either the relative morbility (Mr) or the quantitative amount (intensity) of the antigenic determinats. In the case of the sera of the infected patients, the patterns of antigenic reactivity of these strains revealed that, despite showing the presence of shared antigens, differences were observed between some of the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum. Differences were also demonstrated between the antigenic determinants of a single isolate of L. amazonensis that were recognized by the different patient's sera. No apparent association was consistently found, however, between the Mr components identified in these isolates and clinical form of the disease or the geographical area of isolation. In addition, the spectrum of antigens recognized by the sera from patients with the same clinical form were not identical; although in some instances, similar Mr antigens were shared. These results indicate that isolates of L. amazonensis are not antigenically identical (homogeneous) and that the immune responses (antibodies) observed among infected patients are heterogeneous

Immunopathology of american cutaneous leishmaniasis. Modulation of MHC class II gene products by Keratinocytes before and after glucantime therapy

Epidermal changes from 32 cutaneous and 3 mucosal American leishmaniasis (ACL) active lesions were studied for HLA-DR, -DP expression, Lanerhans cells and lymphocyte infiltration. In addition to a DR and DQ positivity at the surface of the cells of the inflammatory infiltrate, a strong reaction for DR antigens was detected on keratinocytes. Hyperplasia of Langerhans cells was present in al cutaneous lesions and epidermis was infiltrated by T lymphocytes. When healed lesions of 14 of these subjects were re-biopsied 1 to 12 months after the end of pentavalent antimonial therapy, MHC class antigens could no longer be seen on keratinocytes. Our data represrn evidence for hhe reversibility of the abnormal HLA-DR expression by keratinocytes in ACL after Glucantime therapy or spontaneous scar formation, demonstrating that this expresion is restricted to the period of active lesions. The present findings can be regarded as an indirect evidence that keratinocytes may be involved in the immunopathology of ACL

An outbreak of american cutaneous leishmaniasis (Leishmania braziliensis braziliensis) in a periurban area of Rio de Janeiro city, Brazil: clinical and epidemiological studies

De julho de 1984 a setembro de 1986, 105 casos de leishmaniose cutânea foram estudados numa localidade situada na imediata vizinhança da área urbanizada da regiäo metropolitana da cidade do Rio de Janeiro. A ocupaçäo do sítio deu-se aproximadamente há 20 anos, mas os primeiros casos foram registrados somente seis meses antes do início de nosso estudo. Os casos eram quase que exclusivamente cutâneos, da forma clínica ulcerada com um e seis meses de evoluçäo. O teste de Montenegro foi positivo em todos os casos e anticorpos antileishmania foram detectados por imunofluorescência indireta em 74,3% dos pacientes. A demonstraçäo do parasito foi obtida em 69,5%. Animais domésticos infectados foram facilmente encontrados: 32% dos cäes examinados e 30,8% dos equinos mostravam presença de leishmânia em lesöes ulceradas. Parasitos isolados, tanto de casos humanos como de cäes e equinos, foram imulogicamente caracterizados e identificados com L. b. braziliensis. Da populaçäo de flebotomíneos encontrados 73% eram de Lutzomyia intermedia capturados principalmente com iscas humanas e de animais domésticos. Nossas observaçöes sugerem que esta é uma área de estabelecimento recente da infecçäo por L. b. braziliensis e que a transmissäo ocorre provavelmente tanto no peridomicílio como no interior das habitaçöes