USE OF THE POLYMERASE CHAIN REACTION FOR THE DIAGNOSIS OF ASYMPTOMATIC Leishmania INFECTION IN A VISCERAL LEISHMANIASIS-ENDEMIC AREA / Uso da reação em cadeia da polimerase para o diagnóstico de infecção assintomática por Leishmania em área endêmica de leishmaniose visceral

The diagnosis of asymptomatic infection with Leishmania (Leishmania) chagasi has become more important over recent years. Expansion of visceral leishmaniasis might be associated with other routes of transmission such as transfusion, congenital or even vector transmission, and subjects with asymptomatic infection are potential reservoirs. Moreover, the identification of infection may contribute to the management of patients with immunosuppressive conditions (HIV, transplants, use of immunomodulators) and to the assessment of the effectiveness of control measures. In this study, 149 subjects living in a visceral leishmaniasis endemic area were evaluated clinically and submitted to genus-specific polymerase chain reaction (PCR), serological testing, and the Montenegro skin test. Forty-nine (32.9%) of the subjects had a positive PCR result and none of them developed the disease within a follow-up period of three years. No association was observed between the results of PCR, serological and skin tests. A positive PCR result in subjects from the endemic area did not indicate a risk of progression to visceral leishmaniasis and was not associated with a positive result in the serological tests.

O diagnóstico de infecção assintomática por Leishmania (Leishmania) chagasi tem assumido crescente importância nos últimos anos. A expansão da leishmaniose visceral pode estar associada a outras vias de transmissão tais como transfusional, congênita, ou mesmo vetorial, sendo os indivíduos com infecção assintomática, potenciais reservatórios. Ademais, a identificação da infecção poderia auxiliar na condução dos pacientes com condições de imunossupressão (HIV, transplante, uso de imunomoduladores) e na avaliação da efetividade das medidas de controle. Neste estudo, foram avaliados clinicamente 149 indivíduos residentes em área endêmica de leishmaniose visceral e realizada a reação em cadeia da polimerase (PCR) gênero-específica, testes sorológicos e teste de Montenegro. Destes, 49 (32,9%) apresentaram PCR positiva, dos quais nenhum evoluiu com clínica de leishmaniose visceral nos três anos subsequentes. Não houve associação entre o resultado da PCR, dos exames sorológicos e do teste cutâneo. A positividade da PCR em indivíduos da área endêmica estudada não indicou risco de progressão para leishmaniose visceral e também não foi associada à maior positividade dos testes sorológicos.

Leishmaniose visceral: estudo de flebotomíneos e infecção canina em Montes Claros, Minas Gerais / Visceral leishmaniasis: a study on phlebotomine sand flies and canine infection in Montes Claros, State of Minas Gerais

A leishmaniose visceral no Brasil estava inicialmente associada a áreas rurais, mas devido às diversas alterações no ambiente como, desmatamentos, urbanização e intenso processo migratório, ocorreu a expansão das áreas endêmicas, levando à urbanização da doença, principalmente nas regiões Sudeste e Centro Oeste do país. No município de Montes Claros, situado ao norte de Minas Gerais, foi feito um estudo para verificação da situação da LV. No ano de 2002 foi realizado inquérito sorológico canino e no período de setembro de 2002 a agosto de 2003 foi feito levantamento entomológico, utilizando armadilhas luminosas de CDC. A prevalência da LV canina apresentou taxa média de infecção em torno de 5 por cento. A fauna de flebotomíneos estimada foi de 16 espécies, totalizando 1043 exemplares. Lutzomyia longipalpis foi a espécie predominante com 74 por cento, o que sugere a sua participação na transmissão de LV em Montes Claros.

Phlebotomine sand flies in Porteirinha, an area of American visceral leishmaniasis transmission in the State of Minas Gerais, Brazil

A study of the phlebotomine sand fly fauna was carried out in an endemic area of American visceral leishmaniasis (AVL) in the municipality of Porteirinha, in the Brazilian state of Minas Gerais. Captures were performed with CDC light traps in 7 districts, 5 days per month, during 2 consecutive years (January 2000 to December 2001). A total of 3240 sand flies were captured and identified. Sixteen species were found, among which 15 belonged to the genus Lutzomyia and one to the genus Brumptomyia. Lutzomyia longipalpis, a proven vector of AVL, was the predominant species (71.85 percent) throughout the time period. The interference of climatic factors (temperature, humidity, and rainfall) over the populational dynamics of the sand flies was determined. Statistical analysis of the data showed a significant correlation among the number of phlebotomine sand flies collected, rainfall, and humidity, whereas the effect of temperature was negligible, in that particular region. The amount of collected phlebotomine, the number of human cases, and the prevalence of canine AVL in the districts of Porteirinha are discussed.

Identification and purification of immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine (Leishvacin®)

Immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine against American tegumentary leishmaniasis (Leishvacin®), produced by Biobrás (Biochemistry of Brazil ), Montes Claros, State of Minas Gerais, Brazil, were identified and purified by polyacrylamide electrophoresis gel and electroelution. C57BL/10 mice were vaccinated with proteins with estimated molecular weights of 42, 46, 63, 66, 73, 87, 97, and 160kDa in three doses of 30æg of each protein at 15-day intervals combined with 250æg of Corynebacterium parvum followed by a challenge infection with 10(5) infective promastigotes from Leishmania (Leishmania) amazonensis. The ability of these proteins to induce immune response and protection was analyzed. No statistical difference was observed in the level of IFN-g induced by proteins in vaccinated groups in comparison with control groups. Six months after challenge infection, protection levels of 28.57; 42.86; 57.14; 42.86; 42.86, 57.14; 42.86 and 57.14 percent were demonstrated for each purified protein

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-gamma by lymphocyte from subjects vaccinated with Leishvacin. The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 mu/g of each protein at 15 days interval. One hundred mu/g 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 percent 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 percent of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43 percent 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-gamma production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14 percent of protection was found after the first challenge and 28.57 percent 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

Leishmaniose visceral canina estudo parasitológico e histopatológico em olhos de cäes: parte I / Canine visceral leishmaniosis parasitological and histopathological study in dog's eyes: part I

Vários autores têm relatado o envolvimento ocular externo em cäes naturalmente infectados com Leihmania infantum e Leishmania donovani. Entretanto, ainda näo foram realizados estudos dos olhos de cäes no nosso meio. Objetivando investigar a possibilidade de parasitismo ocular, quatro cäes controle e 12 cäes com leishmaniose visceral foram examinados, todos provinientesda regiäo metropolitana de Belo Horizonte, MG, Brasil.O diagnóstico foi realizado através de sorologia convencional (RIFI e ELISA) e exame parasilógico de aspirado de medula óssea. Para o isolamento do parasito foi realizada a inoculaçäo de aproximadamente 100 ml de humor aquoso e/ou corpo vítreo em meio de cultura de NNN/LIT, que foi mantido a ...

Leishmaniose visceral canina estudo imunológico em olhos de cäes: parte II / Canine visceral leishmaniasis immunological study in dog's eyes

O quadro clínico da leishmaniose visceral canina se assemelha muito ao quadro clínico da leishmaniose visceral humana. Embora estas alteraçöes sejam amplamente abordadas em praticamente todos os sistemas no cäo, o estudo das alteraçöes imunológicas näo têm sido profundamente estudados. Aproximadamente 20ml de humor aquoso e corpo vítreo foram utilizados para exames imunológicos através do ensaio imunoenzimático (ELISA) para detecçäo de anticorpos anti leishmania (IgM e IgG) e 10ml de humor aquoso foram utilizados para dosagem de proteína pela micro técnica de Lowry. Detectou se anticorpos anti leishmania (IgM e IgG) em níveis que variaram de 1:80 a 1:2560 no humor aquoso e/ou corpo vítreo dos animais infectados e menores que 1:5 nos animais controle...

Assessment of immunity induced in mice by glycoproteins derived from different strains and species of Leishamnia

A comparative study was undertaken on the immunogenic properties of 63kDa glycoproteins obtained from five different strains/species of Leishmania and assessed in C57BL/10 mice. The humoral immune response was assessed by ELISA against the five different antigens of the immunized animals. The cellular immune response was derived from Leishmania. The response was found to be species-specific in all of determined by means of the cytokine profiles secreted by the spleen cells of immunized animals. The presence of y-IFN and IL-2, and the absence of IL-4 in the supernatants of cells stimulated by L. amazonensis antigen established that the cellular response is of Th1 type. The five glycoproteins tested were equally effective in protecting C57BL/10 mice against challenge by L. amazonensis. About 50 por cento of the immunized animals were protected for six months.

Leishmaniose tegumentar canina: infecçäo experimental / Canine cutaneous leishmaniases: experimental infection

Os autores promoveram inoculaçöes de promastigotas de Leishmania braziliensis em cäes, verificando que o inóculo de 10 elevado a 5 formas foi capaz de infectar 100 por cento dos animais. As lesöes, que eram inicialmente nodulares, ulceravam após aproximadamente 70 dias da infecçäo. Parasitos foram demonstrados nas lesöes, 6 meses após a inoculaçäo.

Desenvolvimento da leishmania mexicana amazonensis em diferentes cepas de camundongos isogênicos / Development of Leishmania mexicana amazonensis in different strains of isogenic mice

Acompanhou-se o desenvolvimento da Leishmania mexicana amazonensis em sete cepas de camundongos isogênicos (BALB/c, C3H, DBA/2, CBA, ABY, ASW e C57BL/10). Todas foram susceptíveis ao parasito, com 100 por cento de animais infectados. Maiores lesöes foram observadas no CBA e BALB/c enquanto no ABY ocorreu menor desenvolvimento, näo sendo observado auto-cura. Sugere-se o uso das cepas CBA e BALB/c para trabalhos com leishmaniose difusa e as demais para estudos com vacinas.

Estudo comparativo entre meios de cultura para o isolamento de leishmania / A comparative study of culture media for the isolation of leishmania

Os autores propöem a associaçäo do meio de NNN com o meio de LIT para o isolamento e manutençäo de amostras de Leishmania. Os resultados apresentados mostraram que a associaçäo proposta NNN/LIT, foi capaz de revelar 100 por cento dos casos estudados, sendo o mais eficaz. O meio de LIT mostrou-se ineficaz para isolamentos enquanto que o meio de NNN revelou 88,2 por cento dos casos.