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.

Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-g, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasis

LARVICIDAL ACTIVITY OF Bacillus sphaericus 2362 AGAINST Anopheles nuneztovari, Anopheles darlingi AND Anopheles braziliensis (DIPTERA, CULICIDAE)

In this present study, preliminary data was obtained regarding the mortality rate of the Amazonian anophelines, Anopheles nuneztovari, Anopheles darlingi and Anopheles braziliensis when subjected to treatment with Bacillus sphaericus strain 2362, the WHO standard strain. Initially, experiments were conducted to test the mortality rate of the three species of anopheline larvae. The third larval instar of An. nuneztovari and the second and third larval instars of An. darlingi proved to be the least susceptible. In other experiments, the same three mosquito species were tested with the standard strain 2362, An. nuneztovari was the least susceptible to this insect pathogen, while An. braziliensis was the most susceptible. This latter species showed a difference in the level of LC50 concentration, when compared to the former, of 2.4, 2.5 and 1.8 in readings taken 24, 48 and 72 hours after exposure to the bacillus.

FIRST REPORT OF CUTANEOUS LEISHMANIASIS CAUSED BY Leishmania (Leishmania) infantum chagasi IN AN URBAN AREA OF RIO DE JANEIRO, BRAZIL

SUMMARY American tegumentary leishmaniasis (ATL) is an infectious disease caused by protozoa of the genus Leishmania, and transmitted by sandflies. In the state of Rio de Janeiro, almost all of the cases of American tegumentary leishmaniasis (ATL) are caused by Leishmania (Viannia) braziliensis, while cases of visceral leishmaniasis (VL) are caused by Leishmania (Leishmania) infantum chagasi. The resurgence of autochthonous VL cases in Rio de Janeiro is related to the geographic expansion of the vector Lutzomyia longipalpis and its ability to adapt to urban areas. We report the first case of leishmaniasis with exclusively cutaneous manifestations caused by L. (L.) infantum chagasi in an urban area of Rio de Janeiro. An eighty-one-year-old woman presented three pleomorphic skin lesions that were not associated with systemic symptoms or visceromegalies. Multilocus enzyme electrophoresis identified L. (L.) infantum chagasi, but direct smear and PCR of bone narrow were negative for Leishmania sp. (suggesting exclusively cutaneous involvement). We discuss the different dermatological presentations of viscerotropic leishmaniasis of the New and Old World, and the clinical and epidemiological importance of the case. Etiologic diagnosis of ATL based upon exclusive clinical criteria may lead to incorrect conclusions. We should be aware of the constant changes in epidemiological patterns related to leishmaniases.

Sobre a sensibilidade da cultura de leucócitos circulantes na detecção de Leishmania no sangue periférico de pacientes com leishmaniose tegumentar

Foi investigada a presença de Leishmania, através da cultura de leucócitos circulantes, no sangue periférico de 60 pacientes portadores de leishmaniose tegumentar americana, nas suas diferentes formas clínicas, assim como nas principais fases evolutivas da doença. Biópsias de lesões cutâneas e/ou de mucosa desses pacientes foram obtidas com a finalidade de isolar e caracterizar os parasitas, através da técnica de anticorpos monoclonais. Dos 60 pacientes examinados, foram isoladas 40 amostras de Leishmania das lesões biopsiadas, sendo 5 de Leishmania (V.) brasiliensis, 3 de L. (V.) guyanensis, 1 de L. (V.) lainsoni, 13 de L. (L.) amazonensis e 18 não puderam ser caracterizados a nível específico, porém, reagiram com anticorpos monoclonais do grupo braziliensis. Quanto àpesquisa através das culturas de leucócitos circulantes, esta revelou resultados completamente negativos. Com base nesses achados, os autores concluíram ser pouco consistente atribuir valor à cultura de leucócitos para o diagnóstico da leishmaniose tegumentar.

Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160), whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.

Evaluation of Leishmania antigens preparation and storage for use in enzyme immunoassays

The influence of time and temperature on the storage of an alkaline antigen of L.major-like and L.(V.) braziliensis promastigotes added or not of a proteases inhibitor (PMSF) was evaluted by means of an IgG-ELISA. Antibodies in assays using L. major-like antigen stored at -20oC for 6 monsths had a statistically lower geometric mean titer (GMT) and different 95% confidence interval limits (CL) than antigens stored otherwise, as assessed by the "t" statistic. The PMSF L. major-like antigen after storage for 6 months at a temperature of 4oC had the same GMT and 95% CL displayed at time zero as well as when storage for 4 and 6 months at -20oC. Significant diferences were not found when L.(V.) braziliensis antigens were stored at times and temperatures mentioned; the PMSF antigen stored for 2 months at -70oC resulted in a lower serum GMT and 95% CL than any other, as assessed by the "t" statistic. Antigen performance did not show any statistical difference associated to the addition of PMSF within the same species; the largest difference between antigens was that between PMSF-L. (V.) braziliensis and L. major-like without PMSF.

Identificação de espécies de Leishmania isoladas de casos humanos em Mato Grosso do Sul por meio da reação em cadeia da polimerase

As leishmanioses são zoonoses endêmicas em Mato Grosso do Sul e têm por agentes etiológicos nessa região Leishmania (Leishmania) chagasi, Leishmania (Leishmania) amazonensis e Leishmania (Viannia) braziliensis. Como método para identificação de espécies de Leishmania, a reação em cadeia da polimerase é uma ferramenta com elevada especificidade e sensibilidade. Analisaram-se 39 isolados de Leishmania criopreservados, obtidos por meio de aspirado medular e/ou biópsia de lesão, conforme a suspeita clínica. Os isolados foram submetidos à extração de DNA e à reação em cadeia da polimerase com os iniciadores: RV1/RV2 para Leishmania (Leishmania) chagasi, a1/a2 para a identificação de Leishmania (Leishmania) amazonensis e b1/b2 para Leishmania (Viannia) braziliensis. Leishmania (Leishmania) chagasi foi a única espécie identificada em 37 casos de leishmaniose visceral. Leishmania (Leishmania) amazonensis foi identificada em dois isolados de pacientes com diagnóstico de leishmaniose tegumentar. Os resultados obtidos confirmam a possibilidade do uso dos três pares de iniciadores como uma ferramenta na caracterização de isolados de Leishmania.

Studies on the relationship between lectin binding carbohydrates and different strains of Leishmania from the New World

The culture forms of L. mexicana pifanoi (LRC L-90), L. mexicana mexicana (LRC L-94, M-379); L. braziliensis braziliensis (LRC L-77, L-1, M-2903, H-LSS) and L. mexicana amazonensis (H-JMMO, M-JOF, H-21, H-PLL,M-1696) were tested with the following lectins: Canavalia ensiformis, Ricinus communis-120, Axinella polypoides, Phaseolus vulgaris, Evonymus europaeus, lotus tetragonolobus, Dolichos biflorus, Aaptos papillata II, Laburnum alpinum, Ulex europaeus, Arachis hypogaea and Soja hispida. All examined strains of Leishmania were agglutinated by C. ensiformis, R. communis-120 and A. popypoides. No agglutination reactions were observed with P. vulgaris, D.biflorus, A. papillata II, E. europaeus and L. tetragonolobus. Only L. m. pifanoi and the L. m. amazonensis strains H-JMMO and MJOF showed agglutination reactions with S. hispida, U. europaeus, L. alpinum and A. hypogaea, while L. m. mexicana (LRC L-94; M-379) strains, L. b. braziliensis H. LSS, LRC L-77; L-1; M-2903 and the L. m. amazonensis strains, H-PLL, H-21, M-1696 showed no agglutination reactions with these four lectins.

Morfometria amastigotica y desarrollo de cuatro aislados de Leishmania mexicana pifanoi de Venezuela em Lutzomyia townsendi

Utilizando parámetros biométricos de la fase amastigota y el tipo de desarrollo de los parásitos en el flebótomo Lutzomyia townsendi, se han estudiado cuatro aislados de Leishmania obtenidos de pacientes con leishmaniasis cutánea difusa, comparándolos con otros aislados de L. mexicana mexicana y L. braziliensis. Los resultados muestran una estrecha semejanza entre los cuatro aislados de pcientes anérgicos y la L. mexicana mexicana y sugieren que el nombre de L. mexicana pifanoi no parece sostenible; los cuatro aislados, por el contrario, podrían identificarse como L. mexicana amazonensis.

Bases experimentales para la identificacion de Leishmania spp. de America por morfometria de amastigotos

Estudios morfométricos sobre los amastigotos de dieciocho poblaciones de cuatro aislados pertenecientes a dos especies venezolanas de Leishmania (L. braziliensis y L. garnhami) indican que la posición del einetoplasto nose modifica en modo estadísticamente significativo cuando los parásitos son sometidos a pasajes por hamsteres y ratones,cultivos en medio NNN o por infección en un vector (Lu. townsendi). La posición del cinetoplasto, medido como la distancia entre el extremo posterior del amastigoto al cinetoplasto, dividido entre la distancia del organoide al extremo anterior de la célula, permite diferenciar a L. braziliensis de L. mexicana y L. garnhami. Los otros parámetros morfométricos no son tan confiables.

Ensayos metodologicos para la investigacion de reservorios de Leishmania spp en los Andes venezolanos

Se describen dos técnicas, presuntiva y confirmativa, para la investigación de mamíferos que pudieran ser reservorios de Leishmania que parasitan al hombre. Se investigan los cambios en los títulos de inmovilización y aglutinación de promastigotos de cultivo por los sueros de animales normales y expuestos una o varias veces a la inoculación intradérmica de pequeñas dosis de promastigotos vivos. Se registra una caída de los títulos de aglutinación en los sueros de hamsteres, de Holochilus venezuelae y de Didelphis marsupialis después de la inoculación con L. mexicana mexicana de Panamá y de L. gamhami de la región de los Andes venezolanos. Se discute la natureza de estos fenómenos. Se han hecho xenodiagnósticos con Lutzomyia townsendi en Holochilus venezuelae y Sigmodon hispidus infectados experimentalmente com L. mexicana mexicana, L. mexicana amazonensis, L. braziliensis y L. garnhami. Las pruebas fueron leidas mediante el examen microscópico de las gotitas de heces excretadas entre las 108 y 132 horas después de la ingesta infectante, tras colorearlas con Giemsa. Se obtuvieron resultados positivos en 23% de los experimentos usando mamíferos con lesiones localizadas, dejando a los flebótomos ingurgitarse libremente sobre animales anestesiados que poseian una hasta varias lesiones localizadas.

El "Status" taxonomico de leishmania garnhami, indicado por su patron de desarrollo en el Vector

Bajo condiciones experimentales, se estabelece el de desarrollo primario de Leishmania garnhami em su vector Lutzomyia townsendi, siguiendo disecciones diarias entre el Iro y 12mo dia post-imnfección. Se demuestra que L. garnhami puede lograr el estabelecimiento inicial de la infección en cualquiera de las regiones del intestino y en los túbulos de Malpighi del insecto vector. Un comportamiento similar fue observado en ejemplares de Lu. longipalpis. Se discute la susceptibilidad de Lu. townsendi a las infecciones por L. mexicana, L. braziliensis y L. garnhami. Se concluye que L. garnhami, a juzgar por su comportamiento en el vector, debe considerarse distinta a las especies del complejo L.mexicana y que su patrón de desarrollo difiere del de las especies de la Sección Suprapylaria, ya que invaden el triângulo pilórico, y del de la Peripylaria por su invasión a los túbulos de Malpighi.

Susceptibility of laboratory-reared female Lutzomyia longipalpis (Lutz & Neiva, 1912) to infection by different species and strains of Leishmania Ross, 1903

A study was undertaken to compare the susceptibility of laboratory-reared female Lutzomyia longipalpis to infection by different species or strains of New World Leishmania. The sand flies proved to be highly susceptible to infection by a strain of Le. guyanensis, with flagellates developing in all (18/18) of the specimens examined. A lower infection rate of 37 per cents (10/27) was recorded in flies exposed to infection by a strain of Le. amazonensis. Flagellates developed in 13 per cents (6/46) of the sand flies that glood fed on dogs in the earlly stage of experimental infection with an old laboratory strain of Le. chagasi. In contrast, promastigotes did not develop in sand flies that blood fed on dogs with naturally acquired Le. chagasi. The naturally infected dogas were in an advanced stage of disease. Flagellates developed in 9// (3/32) of the sand flies that blood fed on lesions of hamsters infected with a strain of Le. braziliensis and in 9 per cents (3/34) of those that fed on hamsters with lesions due to a parasite fo the mexicana complex (strain MHOM/BR/73/BH121). Sand flies did not develop flagellate infections after blood feeding on hamsters bearing lesions induced by strain MHOM/BR/71/BR49. Factors influencing the susceptibility of Lu. longipalpis to infection by New World species of Leishmania are discussed.

Ecology of phlebotomine sand flies (Diptera: Psychodidae)in a focus of Leishmania (Viannia) brasiliensis in northeastern Colombia

The phlebotomine sand fly fauna of two coffee plantations in a Leishmania-endemic area of Norte de Santander, Colombia was studied. Regular insect collections using a variety of methods were made for three and a half years. Information was obtained on diurnal resting sites, host range and seasonal abundance for 17 species, of wich five (Lutzomyia spinicrassa, Lu. serrana,Lu. shannoni, Lu. ovallesi and Lu. gomezi) were far more numerous than the others, anthropophilic and present throughout the year. The behaviour of these and the remaining 12 species is discussed in relation to their potential role in transmission of Leishmania (Viannia) brasiliensis in the area.