INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index). Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

REPRODUCIBILITY OF ALKALINE ANTIGENS OF Leishmania major-LIKE AND Leishmania (V.) braziliensis EVALUATED BY IgG-ELISA. COMPARISON OF ANTIGENS ADDED OF A PROTEIN INHIBITOR (PMSF) OR NOT

This paper deals with the analysis of 10 batches of L.major-like and L.(V.) braziliensis antigens added or not of a proteases inhibitor evaluated by means of an IgG-ELISA on three consecutive days using positive standard sera from patients with diagnosis of American Leishmaniasis previously tested for the presence of IgG antibodies by means of ELISA. The statistical analysis showed that for L. (V.) braziliensis the PMSF-containing antigen did not show any difference among batches or days of testing; the L.(V.) braziliensis antigen without PMSF showed statistical significance for differences among batches and a two-way ANOVA showed significant differences between antigens. L.major-like antigen prepared with or without PMSF showed differences among batches; all 3 days of testing displayed differences for the PMSF antigen but only for days 1 and 2 for the antigen without inhibitor. A two-way ANOVA showed differences among batches of the antigens but not for antigens with and without the protein inhibitor. According to the statistical analysis the L.major-like antigen added or not of PMSF has shown that it is the choice antigen for mucocutaneous leishmaniasis serology.

Prevalence of Trypanosoma cruzi and Leishmania chagasi infection and risk factors in a Colombian indigenous population

This study was carried out in order to obtain base-line data concerning the epidemiology of American Visceral Leishmaniasis and Chagas’ Disease in an indigenous population with whom the government is starting a dwelling improvement programme. Information was collected from 242 dwellings (1,440 people), by means of house to house interviews about socio-economic and environmental factors associated with Leishmania chagasi and Trypanosoma cruzi transmission risk. A leishmanin skin test was applied to 385 people and 454 blood samples were collected on filter paper in order to detect L. chagasi antibodies by ELISA and IFAT and T. cruzi antibodies by ELISA. T. cruzi seroprevalence was 8.7% by ELISA, L. chagasi was 4.6% and 5.1% by IFAT and ELISA, respectively. ELISA sensitivity and specificity for L. chagasi antibodies were 57% and 97.5% respectively, as compared to the IFAT. Leishmanin skin test positivity was 19%. L. chagasi infection prevalence, being defined as a positive result in the three-immunodiagnostic tests, was 17.1%. Additionally, 2.7% of the population studied was positive to both L. chagasi and T. cruzi, showing a possible cross-reaction. L. chagasi and T. cruzi seropositivity increased with age, while no association with gender was observed. Age (p<0.007), number of inhabitants (p<0.05), floor material (p<0.03) and recognition of vector (p<0.01) were associated with T. cruzi infection, whilst age ( p<0.007) and dwelling improvement (p<0.02) were associated with L. chagasi infection. It is necessary to evaluate the long-term impact of the dwelling improvement programme on these parasitic infections in this community.

Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-g by lymphocyte from subjects vaccinated with Leishvacinâ . The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 mg of each protein at 15 days interval. One hundred mg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 105 infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57.14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7.60) and 10.77UI/ml of IFN-g production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis.

Investigation of trypanothione synthetase of Leishmania infantum as a potential target for new anti-parasitic drugs

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Diversidade genética da população parasitária de leishmania em Portugal

RESUMO O presente estudo teve como principal objectivo avaliar a diversidade genética de uma população parasitária de Leishmania em isolados portugueses de hospedeiros humanos, caninos, vulpinos e do vector, aplicando dois marcadores moleculares: kDNA e microssatélites. No Capítulo 1 fez-se uma revisão bibliográfica sobre as leishmanioses incluindo a epidemiologia da infecção nos países da bacia mediterrânica nomeadamente Portugal. Deu-se especial relevo à epidemiologia molecular que nos últimos anos tem vindo a ser desenvolvida. No Capítulo 2 efectuou-se um inquérito de leishmaniose canina que abrangeu 374 cães provenientes da Região Metropolitana de Lisboa. Foi encontrada uma prevalência total de 19,2%, com a prevalência de 18,4% nos cães com dono e 21,6% nos cães sem dono ou vadios. Os resultados obtidos evidenciaram a importância dos cães vadios na transmissão do parasita e disseminação da doença. A partir dos 72 cães infectados, foram isolados 49 estirpes de Leishmania, tendo estas sido tipadas como L. infantum zimodeme MON-1. Estas estirpes, em conjunto com outras amostras isoladas a partir de humanos, vector e outros canídeos, foram utilizadas para avaliar a diversidade genética. No Capítulo 3 foram desenvolvidas sequências iniciadoras cinetoplastideais, MC1 e MC2, tendo-se estas revelado específicas e sensíveis para a identificação do complexo L. donovani isolados em cultura ou directamente a partir de amostras clínicas. Aplicou-se a metodologia de kDNA-PCR-RFLP na análise de 161 amostras de DNA, das quais 134 eram provenientes de isolados portugueses de L. infantum. Foram identificados 16 genótipos na totalidade das amostras, tendo 13 sido identificados nas amostras portuguesas. Observou-se a predominância do genótipo A, observado exclusivamente na população parasitária portuguesa. Em termos geográficos esta metodologia mostrou estar de acordo com a tipagem isoenzimática, e outros marcadores moleculares, individualizando as amostras provenientes de África num único genótipo. No entanto não se observou individualização ao nível das regiões de Portugal estudadas, sugerindo a existência de fluxo genético entre as diferentes áreas geográficas. No Capítulo 4 aplicou-se a análise de 13 loci de microssatélites, polimórficos para L. infantum, em 154 amostras, das quais 128 eram provenientes de diferentes regiões geográficas de Portugal e de diferentes hospedeiros e vector. Obteve-se um maior grau de polimorfismo com estes marcadores do que com o kDNA, identificando-se 85 genótipos. Observou-se uma maior diversidade molecular nas amostras provenientes do Algarve e Alto Douro e, relativamente ao hospedeiro, estes alvos moleculares mostraram ser muito mais polimórficos no hospedeiro humano que o canino, indo ao encontro dos resultados de tipagem isoenzimática que se conhecem até à actualidade. Foi individualizado um agrupamento de amostras não MON-1 e dentro deste, um sub-agrupamento das amostras de África Oriental (Etiópia e Sudão), como anteriormente sugerido por outros autores. No Capítulo 5 discutiram-se os resultados obtidos permitindo verificar que a variabilidade dos parasitas Leishmania no nosso país é maior do que tem sido considerada até ao presente. Possibilitaram também o conhecimento de que há genótipos predominantes em Portugal e que a variabilidade genética no hospedeiro humano e no vector é superior à do reservatório doméstico e silvático.

Visceral leishmaniasis caused by Leishmania (Viannia) braziliensis in a patient infected with human immunodeficiency virus

The current article reports the case of a 19-month-old-girl, from the state of Minas Gerais, Brazil, with visceral leishmaniasis, by Leishmania (Viannia) braziliensis, and Human Immunodeficiency Virus (HIV) co-infection. The child's mother and father, aged 22 and 27 years old, respectively, were both HIV positive. The child was admitted to the General Pediatric Center, in Belo Horizonte, presenting high fever, fatigue, weight loss and enlargement of liver and spleen. Indirect immunofluorescent test revealed a titer of 1:320 for Leishmania. Such result was confirmed by the presence of amastigotes in bone marrow aspirate samples and culture of promastigote forms. Parasites were identified as being Leishmania (Viannia) braziliensis through PCR, using a L. braziliensis complex primer and a generic primer, followed by hibridization. Specific leishmaniasis therapy (GlucantimeÒ antimonial) was intravenously administered.

Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae)

DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).

Chronic interstitial pneumonitis in dogs naturally infected with Leishmania (Leishmania) chagasi: a histopathological and morphometric study

Eighteen mongrel dogs of unknown age and naturally infected with Leishmania (Leishmania) chagasi, were obtained from the City Hall of Belo Horizonte, Brazil. Four dogs were used as control. Lung samples were obtained and immediately fixed in formalin. The histopathological picture of all lung tissue sections was a chronic and diffuse interstitial pneumonitis. The thickened inter-alveolar septa were characterized by the cellular exudate (mostly macrophages, lymphocytes and plasmocytes) associated with collagen deposition. Morphometric analysis showed greater septal thickness in the infected animals than in controls. In fact, the morphometric study of collagen stained with ammoniac silver confirmed a larger deposition of collagen in the infected animals. The parasitologic method was carried out during the study of the lesions on the slides. However, we did not observe any correlation between the histopathologic and morphometric data and the clinical status of the animals. We conclude that the pulmonary lesions observed in all naturally infected dogs were correlated with the disease and that the morphometric method used was satisfactory for the analysis of septal thickness and of increased collagen deposition, confirming the presence of fibrosis.

Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

Leishmaniose Cutânea Causada por Leishmania Infantum Zimodeme Mon-1 em Portugal

Leishmania infantum zimodeme MON-1 foi isolado a partir de uma lesão cutânea da face de uma criança, residente em Lisboa e que nunca saíu do país. Após biópsia excisional não houve recorrência da lesão. Este é o primeiro caso em que este agente é identificado como responsável pela leishmaniose cutãnea em Portugal.

Single step polymerase chain reaction (PCR) for the diagnosis of the Leishmania (Viannia) subgenus

In Brazil, the main etiologic agent of Leishmaniasis that frequently presents with mucosal involvement belongs to the Viannia subgenus. The therapeutic conduct in this disease depends on the parasitological diagnosis, and classical methods are restricted in identifying the agent. In this paper we describe a polymerase chain reaction (PCR), which uses primers designed from mini-exons repetitive sequences. The PCR amplifies a 177bp fragment that can distinguish (Viannia) from (Leishmania) subgenus. This test could be a useful diagnostic tool.