PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

THE PROCESS OF Leishmania INFECTION - DISEASE AND NEW PERSPECTIVES OF PALEOPARASITOLOGY

Species of the genus Leishmania (Kinetoplastida, Trypanosomatidae) are causative agents of leishmaniasis, a complex disease with variable clinical spectrum and epidemiological diversity, constituting, in some countries, a serious public health problem. The origin and evolution of leishmaniasis has been under discussion regarding some clinical and parasitological aspects. After the introduction of paleoparasitology, molecular methods and immunodiagnostic techniques have been applied allowing the recovery of parasite remains, as well as the diagnosis of past infections in humans and other hosts. The dating of archaeological samples has allowed the parasitological analysis in time and space. This manuscript presents the state of the art of leishmaniasis and prospects related to paleoparasitology studies and their contribution to the evolutionary and phylogenetic clarification of parasites belonging to the genus Leishmania, and the leishmaniasis caused by them.

Leishmania infantum AS A CAUSATIVE AGENT OF CUTANEOUS LEISHMANIASIS IN THE STATE OF MATO GROSSO DO SUL, BRAZIL

Cutaneous leishmaniasis is caused by different species of theLeishmania genus. Leishmania(Leishmania) infantum, causing cutaneous leishmaniasis, has been described in patients living in areas where visceral leishmaniasis is endemic. In this study, it was possible to characterize this species in seven slides from cutaneous tissue imprints from patients with cutaneous leishmaniasis in the State of Mato Grosso do Sul, Brazil.

Tentativa de transmissão da Leishmania donovani pela picada do Lutzomyia longipalpis entre cães

Os Autores apresentam dados sôbre tentativas de transmissão experimental da Leishmania donovani pela picada de Lutzomyia longipalpis entre cães. Dois cães jovens sadios foram picados respectivamente por dois e sete flebótomos ricamente infectados e não adquiriram leishmaniose.

Estado actual de nuestros conocimientos sobre la enfermedad de chagas en la Republica Mexicana

Se presentan los datos obtenidos de la revisión minuciosa en relación a enfermedad de Chagas de la literatura médica de la República Mexicana y extranjera desde el afio de 1939 en que por primeira vez se reporta a Trypanosoma cruzi en México, hasta el ano de 1976. Mediante mapas, tablas y cuadros se senalan las localidades en donde se han encontrado casos humanos, reservorios no humanos y transmisores. Se hacen comentários acerca de los resultados obtenidos y se seffala la importancia de incrementar los estúdios sobre enfermedades de Chagas en nuestro país.

Human mucocutaneous leishmaniasis in Três Braços, Bahia - Brazil: an area of Leishmania braziliensis braziliensis transmission. I. Laboratory diagnosis

Leishmanial parasites were detected in 71.2% of patients with cutaneous disease and 48% of patients with mucosal disease, using principally scanning of imprints mears and histological sections and hamster inoculation. Parasites were more frequent in early cutaneous lesions (p < 0.005) o fless than two month duration. Also they were more common in multiple than single mucosal lesions (p < 0.02) in spite of considerable prior glucan time therapy in the former group. 93% of cutaneous lesions had a positive leishmanin skin test and most of the negatives occurred in patients with lesions of less than one month duration. 97% of patients with single mucosal lesion and 79% with multiple mucosal lesions had a positive skin test. 86% of cutaneous disease and 90% of mucosal disease was associated with a positive indirect immunofluorescent antibody test at a ≥ 1/20 dilution. In both groups multiple lesions were associated with higher titres and titres were significantly higher in patients with mucosal disease compared with cutaneous disease (p < 0.01).

Human mucocutaneous leishmaniasis in Três Braços, Bahia - Brazil: an area of Leishmania braziliensis braziliensis transmission. II. Cutaneous disease. Presentation and evolution

The clinical records of 182 patients with cutaneous leishmaniasis probably due to Leishmania braziliensis braziliensis are analysed. 68% had a single lesion which was usually an ulceron the lower anterior tibial third. Many had short histories of one to two months and all age groups were represented 13% had closed lesions of a verrucose or plaque like nature. Evolution of these skin lesions after treatment was related to the regularity of antimony therapy. Although healing usually occurred in three months, the time to scarring after commencing treatment was variable and related to the size ofthe lesion (p < 0.01). Usually if sufficient antimony treatment was given the lesion closed. Seven of the ten patients with initially negative leishmanin skin tests converted to positive after treatment. A significant decline of indirect fluorescent antibody titres occurred in patients followed, during and after therapy.

Leishmaniose mucocutânea humana em Três Braços, Bahia - Brasil: uma área de transmissão de Leishmania braziliensis braziliensis. III. Comprometimento mucoso e evolução inicial

Numa análise de 57 pacientes o acometimento da mucosa foi mais comumente observado em homens (77%) na terceira década de vida, embora fosse grande a variação das idades e ocorrendo mesmo o acometimento de duas crianças. Com a exceção de nove pacientes (16%) todos os outros tinham sinais de leishmaniose cutânea sendo que em somente oito (14%) de lesão era ativa. O acometimento do nariz foi observado em 100% de 19 pacientes que apresentavam lesões múltiplas e em 92% de 38 pacientes apresentando uma única lesão. A faringe, palato, laringe e lábio superior foram afetados nesta experiência. 42% dos pacientes com lesões múltiplas apresentavam acometimento da laringe sendo que em dois pacientes a única lesão existente apresentava-se neste ponto. Não foi observada qualquer diferença relacionada com a idade no que se referia à existencia de lesões únicas ou múltiplas. A duração do acometimento da mucosa variou de menos de 4 até 264 meses. Somente 7% desenvolveram o acometimento da mucosa após mais de dez anos o desenvolvimento da lesão cutânea. Os pacientes usualmente responderam ao tra-tamento adequado por antimonial embora em algumas exceções fosse usada amphotericina B. Morreram três pacientes que se recusaram a colaborar no tratamento. Dois anos após o tratamento observou-se positividade de anticorpos fluorescentes em somente 18% dos pacientes entre aqueles acompanhados.

Ensaio terapêutico com glucantime em sagüis (Callithrix jacchus) infectados com uma cepa de Leishmania donovani aparentemente resistente ao tratamento

Os autores relatam o resultado de um ensaio terapêutico em sagüis com uma cepa de Leishmania donovani isolada de um caso humano fatal de calazar, clinicamente resistente aos antimoniais (Glucantime e Pentostam), Anfotericina B ePentamidina. Os testes cutâneos realizados no paciente para avaliação da imunidade celular foram negativos à exceção do DNCB a 2%. Quatro sangüis adultos (Callithrix jacchus) foram inoculados por via intraperitoneal com uma suspensão deformas amastigotas de L. donovani. Duzentos e dez dias após, todos os animais mostravam formas amastigotas do protozoário, evidenciadas através de punção hepática por aspiração. Iniciou-se então um esquema terapêutico com glucantime (28 mgSb v/kg/dia) em três séries de 10 dias com cinco dias de intervalo entre elas em três dosprimatas,ficando o quarto animal como controle. Nos três animais tratados houve curaparasitológica da doença, o mesmo não ocorrehdo com o controle. O fato de a amostra de L. donovani ter sido, no paciente, resistente aos vários tratamentos e ter sido sensível em modelo experimental à terapêutica com glucantime, sugere a possibilidade de que fatores imunológicos do paciente possam ter contribuído para a evolução fatal da doença.

El método de cultivo "in vitro" como instrumento práctico en el diagnóstico y el aislamiento primário de Leishmania braziliensis braziliensis I. Observaciones de laboratório

Comparamos diferentes medios artificiales y procedimientos de cultivo in vitro de Leishmania braziliensis braziliensis (Lbb) aislada de lesiones de hamsteres experimentalmente infectados. Observamos que la aspiración de material de la lesión, usando jeringa de 5 mlyaguja 22g(25 x 8mm), mostró resultados satisfactorios siendo un procedimiento conveniente de colecta. El medio de Agar Sangre Difco (DAB) es un medio eficiente para el aislamiento de Lbb. El medio de Evans demostro similares resultados al DAB, mientras que el medio líquido de Schneider y el químicamente definido AR-103 fueron nitidamente inferiores. En la propagación in vitro de Lbb, el medio de Schneider permitió un buen crecimiento del flagelado, alcanzando densidades de 0,21 - 2,57 x 10(8) promastigotes por ml ya, en el 5.° dia de cultivo. La adición de 5-fluorocytosine a los tubos de cultivo (150/jg/ml)para evitar la contaminación por hongos, no inhibió el crecimiento de Lbb.