Aspectos ecológicos da leishmaniose tegumentar americana: 8. avaliação da atividade enzoótica de Leishmania (Viannia) braziliensis, em ambiente florestal e peridomiciliar, região do Vale do Ribeira, Estado de São Paulo, Brasil

A evidência da transmissão extraflorestal da leishmaniose cutâneo-mucosa na região do Vale do Ribeira ensejou o presente estudo epidemiológico prospectivo, visando avaliar a atividade enzoótica de L. (V.) braziliensis. A pesquisa paratisológica da infecção natural em pequenos mamíferos e população canina foi complementada com o teste de imunofluorescência indireta (IFI) para cães e captura de flebotomíneos em ambiente florestal e peridomiciliar. A positividade para o teste sorológico e exame parasitológico somente foi observada para cães residentes e com taxas de 5,6 e 2,4%, respectivamente. Entre animais silvestres e sinantrópicos capturados, destacam-se os pertencentes a Oryzomys (Oligoryzomys) e Rattus rattus, ambos assinalados em proporções equivalentes (29,3%), em ambiente peridomiciliar. Foram capturados apenas 166 exemplares femininos de Lutzomyia intermedia, fato atribuído à borrifação das habitações humanas e anexos com DDT. No contexto epidemiológico mais amplo, discute-se a fragilidade do ciclo extraflorestal da L. (V.) braziliensis; o papel do cão e de pequenos mamíferos, como fonte de infecção domiciliar, além de analisar o potencial deles na dispersão do parasita na área estudada.

Isolation of Leishmania sp. from aqueous humor of a patient with cutaneous disseminated leishmaniasis and bilateral iridocyclitis (preliminary report)

The Authors report an uncommon case of leishmaniasis with disseminated cutaneous lesions, systemic manifestations and ocular involvement, the latter being characterized by bilateral nongranulomatous iridocyclitis. The severity of the oph-thalmologic lesions and its unresponsiveness to therapy (in spite of satisfactory regression of both systemic and cutaneous manifestations) lead to a needle aspiration of the anterior eye chamber, content. From this material Leishmania sp was isolated. To our knowledge this is the first time that Leishmania has been shown into the ocular globe.

Comportamiento experimental del Sporothrix schenckii y la Leishmania mexicana en el hamster

La descripción macroscópica del proceso de patogénesis en hamsters inoculados subcutáneamente en nariz con Sporothrix schenckii ó Leishmania mexicana spp. proporcionó bases para diferenciar estos dos microorganismos en un modelo animal utilizado comunmente para estudiarlos. Observaciones secuenciales durante 150 días permitieron afirmar que en las infecciones causadas por estos patógenos se presentaron edema y eritema como signos primarios, seguidos de alopecia, necrosis y ulceración. La producción de pus fué una característica distintiva para el S. schenckii. Estos signos clínicos se observaron más temprano en la esporotricosis que en la infección por L. mexicana, mostrando diferencias estadísticas significantes en días promedio de aparición. El presente trabajo muestra que las lesiones producidas tanto por el S. schenckii como la L. mexicana en este modelo experimental comparten signos clínicos, pero el tiempo de aparición de los mismos y su frecuencia relativa permiten diferenciarlas. Las condiciones de inoculación como: cepa de los microorganismos, dosis del inóculo, sitio y vía de inoculación, deben tenerse presentes en la evaluación de su comportamiento experimental.

Leishmaniose cutânea experimental. III- Aspectos histopatológicos do comportamento evolutivo da lesão cutânea produzida em Cebus apella (Primates: Cebidae) por Leishmania (Viannia) lainsoni, L. (V.) braziliensis e L. (Leishmania) amazonensis

Estudaram-se os aspectos histopatológicos relativos à evolução da infecção experimental produzida em Cebus apella (Primates: Cebidae) por Leishmania (V.) lainsoni, L. (V.) braziliensis e L. (L.) amazonensis. O exame microscópico de biópsias seqüênciais, obtidas dos animais a intervalos definidos de tempo (a primeira, às 48 ou 72 horas após a inoculação, e as seguintes, a cada 30 dias), mostrou que o desenvolvimento das lesões, independentemente da espécie de Leishmania inoculada, passa por uma seqüência de etapas a nível tecidual - 1) infiltrado inespecífico crônico; 2) nódulo macrofágico (com numerosos parasitas); 3) necrose das células parasitadas; 4) granuloma epitelióide; 5) absorção da área necrosada (às vezes formando granuloma de corpo estranho); 6) infiltrado inespecífico crônico residual); e 7) cicatrização - que representaria a formação e a resolução das lesões. Discutiram-se também os prováveis mecanismos imunopatológicos que determinam esta seqüência de eventos e sua possível semelhança com a evolução das lesões na leishmaniose tegumentar humana.

Leishmaniose cutânea na Amazônia: isolamento de Leishmania (Viannia) lainsoni do roedor Agouti paca (Rodentia: Dasyproctidae), no estado do Pará, Brasil

Fez-se o registro, pela primeira vez, do isolamento de Leishmania (V.) lainsoni de um mamífero silvestre, o roedor Agouti paca (Rodentia: Dasyproctidae), no Estado do Pará, Brasil. As amostras do parasita foram isoladas da pele, aparentemente íntegra, de 3 espécimes desse roedor, capturados no município de Tucuruí (ilha de Tocantins), em área que seria inundada pela formação do lago da hidrelétrica construída naquele município. Nenhum isolamento foi obtido de vísceras de qualquer dos animais. A identificação das amostras de L. (V.) lainsoni baseou-se na morfologia de amastigotas e promastigotas, no comportamento da infecção em "hamsters", na análise bioquímica de isoenzimas e, ainda, através de testes com anticorpos monoclonais. A natureza inaparente da infecção nos animais faz supor que o mamífero em questão possa representar um hospedeiro definitivo do parasita na região Amazônica.

Clonal variation within a mucosal isolate derived from a patient with Leishmania (Viannia) braziliensis infection

Three isolates over 5 years from a patient with persistent relapsing mucosal leishmaniasis due to Leishmania (Viannia) braziliensis and 7 clones from one of these isolates were studied by zymodemes and scrodemes analysis. Results showed evidences of clonal phenotypic variation. Eight isoenzymes markers demonstrated clear differences on Cellulose Acetate (CA) and thin starch gel electrophoresis. Also a panel of specific monoclonal antibodies showed such differences. Our observations provide additional evidence that Leishmania (Viannia) braziliensis is composed by subpopulations of parasites with peculiar biochemical and antigenic characteristics.

Comparison on the performance of Leishmania major-like and Leishmania braziliensis braziliensis as antigen for new world leishmaniasis IgG-immunofluorescence test

The performance of an antigen of L. major-like promastigotes for the serological diagnosis of mucocutaneous leishmaniasis in the IgG-immunofluorescent test was compared to that of an antigen of L. braziliensis braziliensis. Each antigen was used to test two hundred and twenty-four sera of etiologies such as mucocutaneous leishmaniasis, deep mycoses, toxoplasmosis, malaria, Chagas' disease, visceral leishmaniasis, anti-nuclear factor, schistosomaiasis, rheumatoid factor and normal controls. Agreement between responses to each antigen was high: 77.2% of leishmaniases sera agreed on a positive or a negative result to both antigens and 91.1 % of control sera. Cross reactivity was restricted to Chagas' disease sera, visceral leishmaniasis, anti-nuclear factor and paracoccidiodomycosis. The quantitative response of leishmaniasis and Chagas' disease sera to both antigens was evaluated by a linear regression; although the y-intercept and the slope were different for each antigen, neither was better than the other in the disclosure of anti-Leishmania antibodies. In the case of Chagas' disease sera the L. major-like antigen was better than L. b. braziliensis' to disclose cross-reacting antibodies.

Histopatologia da forma localizada de leishmaniose cutânea por Leishmania (Leishmania) amazonensis

São descritas as alterações microscópicas presentes na forma localizada (ulcerada) da Leishmaniose cutânea produzida por Leishmania (Leishmania) amazonensis. Nesse tipo de manifestação, menos conhecido do que a forma anérgica ou difusa devida ao mesmo agente, as lesões são clinicamente idênticas às de leishmaniose cutânea causada por espécies outras de Leishmania, pertencentes ao subgênero Viannia. Na infecção localizada por L. (L.) amazonensis, entretanto, há um aspecto peculiar, só recentemente conhecido, ou seja, cerca de 50% dos indivíduos atingidos não reagem ao teste de Montenegro. A principal característica histológica observada foi a acumulação na derme, quase sempre focal, de numerosos macrófagos contendo no citoplasma um grande vacúolo cheio de amastigotas. O quadro é semelhante ao da forma difusa, porém sem o aspecto histiocitomatóide, próprio da última. Afora esses grupos de macrófagos, vêem-se também, na forma localizada, muitas células mononucleares da inflamação, principalmente plasmócitos e macrófagos não parasitados. Os acúmulos de macrófagos com amastigotas, quando volumosos, podem sofrer necrose na parte central; os parasitos, contidos nas células, são destruídos com elas ou liberados, e sua eliminação através da úlcera deve contribuir para a cura do processo. Esse tipo de necrose nunca foi descrito em casos da forma difusa. Não houve grande diferença, no quadro histológico, entre pacientes Montenegro-negativos e positivos. Apenas em alguns casos, do grupo Montenegro-positivo, havia granulomas formados por histiócitos epitelióides sem parasitos. Quanto à persistência das células com parasitos nas lesões, observou-se que aos seis meses ou mais de evolução, em ambos os grupos, ainda estavam elas presentes. Tal achado não é comum na leishmaniose tegumentar por L. (V.) braziliensis.

Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American Cutaneous Leishmaniasis

Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL). The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO) was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100ºC for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.

Leishmania (Viannia) panamensis - induced cutaneous leishmaniasis in susceptible and resistant mouse strains

We studied the susceptibility to Leishmania (Viannia) panamensis in strains of mice. The C57BL/6 strain was resistant and showed self-controlled lesion at the injected foot pad. The BALB/c and DBA/2J strains were susceptible and showed a foot swelling that started day 20 post-infection and progressed to a tumour-like lesion in later period of observation. The CBA/HJ strain was found to be of intermediary resistance. In contrast to other known cutaneous leishmaniasis in mice, the lesion in L. (V.) panamensis-infected mice was restricted to the inoculation site in the skin. In addition, we studied the development of cellular response and antibodies against Leishmania antigen in BALB/c and C57BL/6 strains. The proliferative response of lymph node cells against L. (V.) panamensis antigen was biphasic in both strains. An initial response was seen on day 20, followed by a refractory period between 40 and 80 days and a second response around fourth month post-infection. The response in the latter period was higher in C57BL/6 strain than in BALB/c strain. BALB/c strain presented much higher anti-Leishmania antibody level than C57BL/6 strain. The model and the correlation of immunological variables and the course of the infection are discussed.

Histopathology and immunocytochemical study of type 3 and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi

The objective of this study was to compare the histopathological changes and expression of CR3 and CR4 in the liver and spleen of dogs naturally and experimentally infected with L. chagasi. The basic histopathological lesions observed mainly in naturally infected dogs were: epithelioid hepatic granulomas, hyperplasia and hypertrophy of Kupffer cells, Malpigui follicles and mononucleated cells of the red pulp of the spleen. Sections from the liver and spleen by immunocytochemistry technique showed the presence of CD11b,c\CD 18 antigens in the control and infected animals and no qualitative or quantitative differences in the liver. Nevertheless, CD18 was always increased in the spleen of naturally and experimentally infected dogs. These results indicate that there is a difference in the activaton of CD 18 in both experimental and natural cases of canine visceral leishmaniasis that should play an important role in the immunological response to Leishmania chagasi infection.

Cross-reactivity of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania (Viannia) braziliensis

We have detected antibodies, in the sera of Chagas disease, Kala-azar and Mucocutaneous leishmaniasis patients, that bind multiple antigens shared between the three causative agents. The Chagas disease sera showed 98 to 100% positive results by ELISA when the Leishmania braziliensis and Leishmania chagasi antigens were used, respectively. The Kala-azar sera showed 100% positive results with Trypanosoma cruzi or L. braziliensis antigens by immunofluorescence assays. The antibodies in the sera of Mucocutaneous leishmaniasis patients showed 100% positive results by ELISA assays with T. cruzi or L. chagasi antigens. Furthermore, the direct agglutination of L. chagasi promastigotes showed that 95% of Kala-azar and 35% of Mucocutaneous leishmaniasis sera agglutinated the parasite in dilutions above 1:512. In contrast, 15% of Chagas sera agglutinated the parasite in dilutions 1:16 and below. Western blot analysis showed that the Chagas sera that formed at least 24 bands with the T. cruzi also formed 13 bands with the L. chagasi and 17 bands with the L. braziliensis. The Kala-azar sera that recognized at least 29 bands with the homologous antigen also formed 14 bands with the T. cruzi and 10 bands with the L. braziliensis antigens. Finally, the Mucocutaneous leishmaniasis sera that formed at least 17 bands with the homologous antigen also formed 10 bands with the T. cruzi and four bands with the L. chagasi antigens. These results indicate the presence of common antigenic determinants in several protozoal proteins and, therefore, explain the serologic cross-reactions reported here.

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.