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.

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.

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.

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.

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.

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.

BODY WEIGHT AS A DETERMINANT OF CLINICAL EVOLUTION IN HAMSTERS (Mesocricetus auratus) INFECTED WITH Leishmania (Viannia) panamensis

SUMMARY The clinical outcome of infection with Leishmania species of the subgenus Viannia in hamster model (Mesocricetus auratus) has shown to be different depending on experimental protocol. Body weight has been a relevant determinant of the clinical outcome of the infection in hamsters with visceral leishmaniasis but its importance as a clinical parameter in hamsters with cutaneous leishmaniasis is not known. In this study, the clinical evolution of infection with L. (V) panamensis was evaluated in juvenile and adult male hamsters during 11 weeks by comparing clinical parameters such as attitude, temperature, respiratory rate, appearance of the stool, and body weight between infected and non-infected groups. Results showed that body weight decreased in adult hamsters after infection by L. (V) panamensis; this observation supports the use of body weight as an additional parameter to define the management or treatment of cutaneous leishmaniasis in infected adult hamsters used as an animal experimental model for leishmaniasis.

DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL

Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility.

GENOTYPE CHARACTERIZATION OF Leishmania (Viannia) braziliensis ISOLATED FROM HUMAN AND CANINE BIOPSIES WITH AMERICAN CUTANEOUS LEISHMANIASIS

Introduction:American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.) braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo.Methods:Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil (36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from biopsies. The primer set amplified a specific region of Leishmania internal transcribed spacers of the ribosomal DNA locus.Results:Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among 14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample.Conclusion:These results suggest that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of Brazil, showing great variability among the natural populations of endemic foci.

Leishmaniose causada por Leishmania viannia braziliensis (Lvb) um caso de evolução atípica

Os autores relatam o caso de uma criança portadora de Leishmaniose Tegumentar Americana causada por Leishmania braziliensis braziliensis que foi infectada durante a amamentação, desenvolvendo lesão infiltrativa e nodular nos lábios, com posterior disseminação para os seios da face, fossas nasais e pavilhão auricular e cuja evolução clinica pós-terapêutica caracterizou-se por períodos sucessivos de regressão e de reativação da lesão. Enfatizam a gravidade do caso, e as dificuldades terapêuticas com a utilização dos antimoniais pentavalentes, antimoniato-N-metil glucamina (Glucantime) e o stibogluconato de sódio(Pentostam).