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There are several gaps in our knowledge on the origin and spread of Leishmania (Viannia) braziliensis, an etiological agent of cutaneous and mucocutaneous or American tegumentary leishmaniasis, to different biomes, hosts, and vectors, with important epidemiological implications, including the possible existence of an anthroponotic component. Historical, biological, and epidemiological evidence suggests that Leishmania (V.) braziliensis and its variants were preexistent in Amazonia with great genetic variability, where they dispersed with less variability to other regions (clonal expansion). During pre-Columbian times the parasite may have been transported by migrating humans and probably also their dogs, from western Amazonia to the high inter-Andean valleys and from there to other regions of South America. The same thing could have happened later, in the same way, when it spread to non-Amazonian regions of Brazil and other countries of South and Central America, between the late 19th and early 20th centuries, during the so-called Rubber Boom and construction of the Madeira-Mamoré Railway in the Brazilian Amazon, by migrant workers who later returned to their places of origin, transporting the agent. The parasite’s dispersal in genetic correlated clusters, involving unexpectedly distinct ecosystems in Brazil (Amazonian, Cerrado, Caatinga and Atlantic Forest biomes), has continued until the present through human displacement. The infection of certain species of domestic, synanthropic and even wild animals, could be secondary to anthropogenic introduction of L. (V.) braziliensis in new environments. We admit the same phenomena happening in the probable transference of Leishmania infantum (visceral leishmaniasis), and of Yersinia pestis (plague) from the Old world to the New world, generating domestic and wild enzotic cycles from these agents. These assumptions associated with human infections, chronicity and parasite persistence with possibility of recovery of Leishmania in peripheral blood, skin and scars of cured or asymptomatic patients, (that may provide an alternative blood meal), along with the sand flies’ adaptation to the peri-domicile and the high susceptibility of domestic dogs, horses, mules and cats to the parasite, can reinforce the evidence of anthropogenic spread of L. (V.) braziliensis.

Background We identified the species of Leishmania isolated from traveling and migrant patients attended in a reference center from 2000 to 2015, we performed the georeferencing of these species in Rio de Janeiro (RJ) state and we had knowledge about the human flows between the likely location of infection (LLI) and place of residence (PR) in RJ state, Brazil. Methodology/Principal findings This is a retrospective cross-sectional study including 171 patients diagnosed with ATL. Google Maps, OpenStreetMap, and Bing Maps were tools used to georeference LLI and PR. For etiological identification, we used isoenzyme electrophoresis, polymerase chain reaction-restriction fragment length polymorphism (molecular target hsp70C with restriction enzymes HaeIII and BstUI), and sequencing of the internal transcribed spacer of ribosomal DNA. ARCGIS software was used to create maps of the geographic distribution of Leishmania species in the state and municipality of RJ, together with flows between the LLI and PR. Isolates from 104 patients were identified as: L. (Viannia) braziliensis (80.8%), L. (V.) naiffi (7.7%), L. (V.) guyanensis (6.7%), L. (Leishmania) amazonensis (1%), and genetic variants of L. (V.) braziliensis (3.8%). The flow maps showed that the LLI included 4 countries, 19 Brazilian states, and 18 municipalities of RJ state. The Brazilian states with the highest density of cases were Amazonas (n = 32), Bahia (n = 18), and Ceará (n = 15). Conclusions/Significance This work is the first contribution to the knowledge of the routes of Leishmania species introduced in RJ state by migrants and travelers patients. L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi, L. (L.) amazonensis, and genetic variants of L. (V.) braziliensis were identified in RJ state. To determine whether the autochthonous transmission of these imported species is possible it is necessary the adaptation of these species to environmental conditions as well as the presence of reservoirs and phlebotomine vectors in this region., Author summary Leishmaniasis is one of the most important neglected parasitic diseases in humans worldwide. American Tegumentary Leishmaniasis (ATL) is a disease caused by protozoa of the genus Leishmania, which are injected into the skin by small insects known by sandflies. Many different Leishmania species may cause ATL, which can affect the skin and/or oral and nasal mucosa. In this study, we analyzed flows between the likely location of infection (LLI) and the place of residence (PR) in Rio de Janeiro (RJ) state and identified Leishmania species isolated from travelers and migrants attended between 2000 and 2015 in a reference hospital for the care of patients with ATL. This work is the first contribution to knowledge about the routes of Leishmania species introduced to RJ state by migrant and traveling patients. In addition to Leishmania (Viannia) braziliensis, which was the most common species, other species have also been introduced to RJ state, including L. (V.) guyanensis, L. (V.) naiffi, L. (Leishmania) amazonensis, and genetic variants of L. (V.) braziliensis. Knowledge of the migratory flow and geographic location of these imported Leishmania species may contribute to the surveillance and control of ATL in RJ state, Brazil.

espanolHay un consenso en torno al reconocimiento del cine como medio de adquisicion de una alfabetizacion mediatica. La problematica del cine como expresion artistica y de la promocion de la interdisciplinaridad entre las areas de la comunicacion, del cine y la educacion y de la educacion artistica en particular que se han establecido en el ambito de la ensenanza de las ciencias de la comunicacion, como Lobo (1999), Pacheco (2016), Migliorin (2015) y en el caso de la formacion de gestores en las areas de la educacion superior, en la Escuela Superior de Educacion, del Instituto Politecnico de Viana do Castelo (ESE-IPVC), a muchas otras areas, como es el caso de la formacion de gestores en las areas de las artes y de la cultura, de los especialistas de los cursos de luz, sonido e imagen y en la formacion de profesores (Moura et al, 2017). En este contexto, el objetivo de este articulo es analizar un proyecto de investigacion que se repite anualmente, cuando enfrentan a los estudiantes con el cine documental y sus conceptos, la alfabetizacion cinematografica, el analisis de imagenes fijas y en movimiento, resultando en la creacion de peliculas documentales a la pelicula desde el texto de Almada Negreiros "Manifiesto Anti Dantas". EnglishThere is a consensus around the recognition of cinema as a means of acquiring media literacy. The problematization of cinema as an artistic expression and the promotion of interdisciplinarity between the areas of communication, cinema and education and artistic education in particular that have been established in the field of Communication Science teaching, such as Lobo (1999), Pacheco (2016), Migliorin (2015) and others argue, have been applied to many other areas, such as the training of managers in the areas of higher education, in the School of Education of the Polytechnic Institute of Viana do Castelo (ESE-IPVC). the arts and culture, specialists in light, sound and image courses and teacher training (Moura et al, 2017). In this context, the objective of this article is to analyze a research project that is repeated annually, when students are confronted with the documentary cinema and its concepts, the cinematographic literacy, the analysis of fixed and moving images, resulting in the creation of documentary films at from the text of Almada Negreiros "Manifesto Anti Dantas". portuguesExiste um consenso em torno do reconhecimento do cinema como meio de aquisicao de uma literacia mediatica. A problematizacao do cinema como expressao artistica e da promocao da interdisciplinaridade entre as areas da comunicacao, do cinema e educacao e da educacao artistica em particular que tem sido estabelecidas no âmbito do ensino das Ciencias da Comunicacao, tal como Lobo (1999), Pacheco (2016), Migliorin (2015) e outros argumentam, tem vindo a ser aplicadas na Escola Superior de Educacao, do Instituto Politecnico de Viana do Castelo (ESE-IPVC), a muitas outras areas, como e o caso da formacao de gestores nas areas das artes e da cultura, dos especialistas dos cursos de luz, som e imagem e na formacao de professores (Moura et al, 2017). Neste contexto, o objetivo deste artigo e analisar um projeto de investigacao que se repete anualmente, quando confrontam os estudantes com o cinema documental e seus conceitos, a literacia cinematografica, a analise de imagens fixas e em movimento, resultando na criacao de filmes documentais a partir do texto de Almada Negreiros “Manifesto Anti Dantas”.

There is a broad consensus on the recognition of cinema as a means of acquiring media literacy. The problematization of cinema as an artistic expression and the promotion of interdisciplinarity between the areas of communication, cinema and education and artistic education in particular that have been established in the field of communication science teaching. In this context, the purpose of this article is to describe action-research projects that are repeated annually, when students are confronted with cinema, audiovisual language, analysis of still and moving images, the follow-up of documentary creation with students from different courses, and their involvement in the dynamization of an audiovisual space from the text of a Portuguese writer, Almada Negreiros "Manifesto Anti Dantas" or interviews in rural communities, using video cameras, within the International Project Rural 3.0 Service-Learning for Rural Development, which is an international transversal project, funded in July 2018 by E+ Knowledge Alliances, coordinated by the School of Education of the Polytechnic Institute of Viana do Castelo, involving sixteen partners from eight European countries. Erasmus + Programme of the European Union, under the development of the project entitled “3.0- Service Learning for the Rural Development” | Promotor: Instituto Politécnico de Viana do Castelo info:eu-repo/semantics/publishedVersion

INTRODUCTION: Favorable responses in American tegumentary leishmaniasis (ATL) patients to treatment with 5 mg Sbv/kg/day meglumine antimoniate (MA) has been reported in Rio de Janeiro, but little is known regarding the therapeutic response to low doses in patients from other locations. METHODS: A retrospective review of medical records was conducted to compare the therapeutic response to 5 mg Sbv/kg/day MA treatment among 36 patients who acquired ATL in Brazilian states other than Rio de Janeiro (OS group) and 72 patients from Rio de Janeiro (RJ group). RESULTS: One course of 5 mg Sbv/kg/day MA cured 72.8% of 81 cutaneous (CL) and 66.6% of 27 mucosal (ML) leishmaniasis-infected patients: 70% in the CL/RJ group, 81% in the CL/OS group, 50% in the ML/RJ group, and 80% in the ML/OS group. After up to two additional treatment courses at the same dose, 88.9% and 85.2% of the CL and ML patients were cured, respectively. Adverse events were observed in 40% of patients in the CL/RJ group, 57% of the CL/OS group, 58% of the ML/RJ group, and 80% of the ML/OS group. No significant differences were observed in the cure rates or adverse effects between the RJ and OS groups. No patients required permanent discontinuation of treatment due to adverse events. CONCLUSIONS: Patients with ATL acquired in both RJ and OS may respond to low-dose MA. While high-dose MA should remain the standard treatment for ATL, low-dose MA might be preferred when toxicity is a primary concern.

Trypanosoma caninum is a parasite isolated from domestic dogs, of which several biological aspects remain unknown, including evolutive forms found in vertebrate hosts. The objective of this study was to evaluate co-cultures of T. caninum with different cell lines as feeder layers to monitor the differentiation process and investigate infective potential. The study was performed using DH-82, MDCK, and Lulo cell lines. T. caninum from axenic culture was added to the cultured adherent cells. At intervals over 30 days, aliquots of the supernatant were collected for quantification and assessment of differentiation. Infectivity assays were performed on the aforementioned cell lines seeded on glass coverslips and evaluated after 6, 24, and 72 h. In the supernatant of the feeder layer, T. caninum presented similar growth profiles, with epimastigote and trypomastigote forms in binary and multiple divisions. During co-culture with DH-82 and MDCK cells, a higher level of differentiation to trypomastigotes was observed. This study shows that the differentiation process of this parasite can vary according to culture conditions and that DH-82 and MDCK lineages could be applied to the study of trypomastigote forms. All forms of T. caninum described until now (aflagellar epimastigotes, typical epimastigotes, or trypomastigotes) were unable to infect the cell line Finally, this study provides additional data about morphobiological aspects. Although the biological cycle of T. caninum has not been established, the present data suggest the importance of feeder layers in promoting the growth and differentiation of this new parasite.

There is a broad consensus on the recognition of cinema as a means of acquiring media literacy. The problematization of cinema as an artistic expression and the promotion of interdisciplinarity between the areas of communication, cinema and education and artistic education in particular that have been established in the field of communication science teaching. In this context, the purpose of this article is to describe an action-research that is repeated annually, when students are confronted with cinema, audio-visual language, analysis of still and moving images, the follow-up of documentary creation with students from a cinema course and the dynamization of an audio-visual space from the text of a Portuguese writer, Almada Negreiros "Manifesto Anti Dantas". The findings provide useful information on the contribution critical analysis strategies bring to art in general and cinema in particular and that the study of visual culture in terms of economy, business, technology, experiences provide a more critical understanding of society through projects that guarantee a free and flexible teaching-learning program and stimulate much discussion and polemic.

The present study was developed in the urban area of Paracatu, an endemic city for the American visceral leishmaniasis in Brazil. A six-month canine survey was performed with 6295 domiciled dogs in 28 districts in that area and showed that 4.2% of those (267 dogs) were positive for VL by ELISA and IFAT serum assays. Prevalence ratios for canine VL varied between 1.2% and 16.1%, depending on the district under investigation. Fifteen dogs - 80% of which were clinically asymptomatic for VL - were submitted to a more detailed study that comprised direct parasitological examination and Leishmania kDNA amplification of tissue samples as well as two PCR-RFLP methods using myelocultures. Leishmania amastigotes or Leishmania DNA were detected in all dogs but one. The infecting species of Leishmania was identified in about 50% (7/15) of the sample dogs: Leishmania (Leishmania) chagasi in two of them and, unexpectedly, Leishmania (Leishmania) amazonensis in the remaining five. Three months after the end of confiscation and elimination of the VL-seropositive dogs in the 28 districts of Paracatu, a systematic entomological survey was performed in five of them. Six hundred and sixty five (665) phlebotomine sand flies were captured in total, from which 89.5% were identified as Lutzomyia longipalpis. The population density of that species increased during the rainy season. Other thirteen (13) species of phlebotomine sand flies were captured at varying percentages from 0.2 to 5.0%. It is worth noting that L. longipalpis females were predominantely intradomicile when compared to males, suggesting that the VL transmission cycle in Paracatu may be occurring inside home.

Analyses of MLEE, RAPD and LSSP-PCR were used to compare the panel of american tegumentary leishmaniasis (ATL) isolates obtained from lesions of patients with rare clinical manifestations of the disease and typical lesions. All of the 34 samples analyzed by MLEE demonstrated similar electromorphic profiles with Leishmania (Viannia) braziliensis reference strain. Through the RAPD analysis, nine genetic profiles (genotypes) were identified. LSSP-PCR corroborates the initial screening and phenetic analysis has grouped the isolates into two major clusters comprising the nine different genotypes. Prevalent genotype defined as LbmtDNAgen1 was detected in the largest number of isolates. There was no association between genotypes and clinical symptoms. However, two different genotypes could be identified in the initial (LbmtDNAGen9) and reactivated lesion (LbmtDNAGen3) of the same patient. Our results support the idea of a less pronounced genotypic diversity among L. (V.) braziliensis circulating in the State of Rio de Janeiro and demonstrate the useful application of these molecular markers in genetics variability studies.

Different molecular markers have been employed for typing Trypanosoma cruzi strains from endemic areas of Chagas disease. The low-stringency single specific primer-polymerase chain reaction (LSSP-PCR) has been a sensitive and informative technique that uses the variable region of kinetoplast DNA minicircles as a genetic marker, allowing detection of DNA sequence variation. In the present study, we analyzed the intra-lineage genetic variability of the T. cruzi strains obtained from triatomine feces collected on filter paper FTA card by LSSP-PCR. The hybridization of the PCR products with a probe for the subgenus Schizotrypanum and a clone-specific probe from Dm28c confirmed the subgenus as T. (S.) cruzi and respective lineages as T. cruzi I. Phenetic analysis showed the presence of three clusters that diverged by different coefficients of similarity. Thirteen T. cruzi I genotypes were observed circulating among Triatoma pseudomaculata and Rhodnius nasutus from peridomiciliary and natural environments in five peri-urban and urban localities of Jaguaruana, Ceará, Brazil. These data indicate the importance of the circulation of T. cruzi I genotypes among T. pseudomaculata and R. nasutus in different environments and the possible risk of Chagas disease domestic transmission.

Randomly amplified polymorphic DNA (RAPD) and nuclear ribosomal DNA sequence analyses were used to assess the genetic population structure of the South American triatomine species Triatomo rubrovario throughout its geographical distribution. To investigate the genetic variability at both intraspecific and intrapopulational levels the RAPD profiles and the nucleotide sequences of the rDNA intergenic spacers, ITS-1 and ITS-2, were analysed and compared. The phenetic analysis based on RAPD profiles show three distinct clusters diverging by similarity coefficients ranging from 0.62 to 0.96. The ITS-1 and ITS-2 sequence variability detected may be considered very high, suggesting reproductive isolation between populations. A total of seven composite haplotypes (CH) were found, among which three are specific for Brazil, other three for Uruguay, and the last one common for the three countries studied. The population studied in Argentina does not represent an independent CH. Sequence analyses proved that the five populations studied are easily differentiable and that there is heterogeneity within each one. True mutations and indels are the responsible of sequence differences between haplotypes and populations, suggesting that divergence processes may presently go on within this species. The large intraspecific variability detected may underlie the known plasticity of T. rubrovaria, making it a potential intradomiciliary invader and consequently an appropriate vector for Chagas disease transmission. Therefore, this triatomine species must be continuously monitored throughout.

We report here the first case of co-infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil. Isoenzyme characterisation identified the parasites isolated in culture from the cutaneous lesion as L. (V.) braziliensis and the isolates from blood and lymph node as L. (L.) chagasi. PCR analysis using specific primers followed by molecular hybridisation for direct Leishmania species identification in tissue fragments confirmed the presence of L. (V.) braziliensis DNA in the cutaneous lesion and of L. (L.) chagasi DNA in spleen and popliteal lymph node fragments. This report emphasises the importance of identification of Leishmania species infecting seropositive dogs in endemic areas, and the consequent re-assessment of control and epidemiological surveillance measures for the control of leishmaniasis, as is the case in Brazil.

Eighteen Trypanosoma cruzi strains isolated from naturally infected triatomines were studied genetically. The majority of the strains were from Triatoma brasiliensis, the principal vector of Chagas disease in the northeast of Brazil. Multilocus enzyme electrophoresis (MLEE) and randomly amplified polymorphic DNA (RAPD) analyses were used to investigate the genotypic diversity and the spread of the T. cruzi genotypes in different environments. MLEE clearly distinguished two distinct isoenzyme profiles, and RAPD analysis revealed 10 different genotypes circulating in rural areas. The strains could be typed as isoenzyme variants of the T. cruzi principal zymodeme Z1 (T. cruzi I). An effective program of epidemiological vigilance is required to prevent the spread of T. cruzi I strains into human dwellings.

Triatoma rubrovaria has become the most frequently captured triatomine species since the control of T. infestans in the state of Rio Grande do Sul (RS), Brazil. The aim of this study was to evaluate aspects of the vectorial competence of T. rubrovaria using nymphs raised in laboratory under environmental conditions of temperature and humidity and fed on mice. The average developmental period of T. rubrovaria was 180.1 days. The percentage of defecation shortly after feeding was still higher than previous studies in which samples of T. rubrovaria subjected to a slight starvation period before the blood meal were used. The obtained results support former indication that T. rubrovaria presents bionomic characteristics propitious to be a good vector of Trypanosoma cruzi to man. Therefore its domiciliary invasion process must be continuously monitored.

Three cases of Trypanosoma cruzi-HIV co-infected haemophiliacs are described. Parasitological (xenodiagnosis, haemoculture, PCR) and immunological (CD4+ and CD8+ T cell counts, in vitro lymphoproliferative responses) studies were performed. Hybridization of isolated parasites with a specific probe confirmed the T. cruzi aetiology. We observed that despite the high parasitaemia, no clinical or parasitological evidence of T. cruzi reactivation was detected. CD4+ T cells decreased with time in two patients and the lymphocyte proliferative response to T. cruzi was very low in all patients. These data suggest that T. cruzi infection may have a long silent course in immunosuppressed HIV patients. Therefore, this parasitic infection should be investigated in any AIDS patient coming from areas endemic for Chagas' disease.

Data from the Chagas Disease Control Program indicate a growing domiciliary and peridomiciliary invasion of Triatoma rubrovaria in the State of Rio Grande do Sul, where it has become the most frequent triatomine species captured there since the control of T. infestans. Bionomic characteristics that could influence the vectorial capacity of T. rubrovaria as vector of Trypanosoma cruzi were evaluated: patterns of (i) feeding, (ii) defecation, and (iii) resistance to starvation, using insects fed on mice. Fifty three percent of the females showed a defecation pattern conducive to chagasic transmission, defecating either on or near the bite site. The averages of the resistance to starvation varied from 48.1 to 179 days, for the first and fifth nymphal stages, respectively. Our study shows that with respect to the patterns of feeding, defecation and resistance to fasting, T. rubrovaria presented similar rates to the ones observed for other effective vector species, such as T. infestans. Thus, based on our studies we conclude that T. rubrovaria has biological characteristics that can positively influence its capacity to become infected and transmit T. cruzi, and also to keep residual populations after chemical control interventions.

Triatoma rubrovaria has become the most frequently captured triatomine species after the control of T. infestans in the State of Rio Grande do Sul (RS), Brazil. Isoenzymatic and chromatic studies indicate the existence of, at least, two distinct phenotypic patterns of T. rubrovaria in RS. The geographic variation noted through molecular tools may also result in distinct profiles of vectorial potentiality. In order to enhance our understanding of the bionomic knowledge of T. rubrovaria separate batches of the species were collected from different municipalities of RS distant from 72 to 332 km: Santana do Livramento (natural ecotope), Santana do Livramento (artificial ecotope), Santiago (natural ecotope), Cangu u (peridomicile) and Encruzilhada do Sul (natural ecotope). A total of 285 specimens were collected, 85 specimens kept sufficient fecal material in their guts for the precipitin analysis. The results indicated the food eclecticism for this species and the anti-rodent serum showed the highest positivity in most localities. From the total of analyzed samples, only 1.3% of unique positivity for human blood was registered, all of them for Santiago population. This reactivity to human blood may be associated to pastures activities in the field.

We detected an outbreak of American cutaneous leishmaniasis in the Jequitinhonha River Valley, Minas Gerais, Brazil. Clinical and epidemiological aspects were studied for a period of two years. Data include results of physical examinations, Montenegro skin test and serology. In total 72 of the 299 individuals evaluated presented active lesions. Only one case out of these 72 patients showed the mucosal form of the disease. The precarious sanitary conditions, low educational level and low income found in the population studied demonstrated that, as with the other parasitic diseases, cutaneous leishmaniasis occurs with greater frequency in needy populations. A canine serological survey detected 20.3% (30/148) of dogs reactive to the Leishmania antigen. Lutzomyia intermedia was the predominant phlebotomine species and the majority of the specimens (84.9%) were captured in the peridomicile. Four samples from human and three from canine cases were isolated and characterised by PCR and isoenzymes as being Leishmania (Viannia) braziliensis. The peridomiciliary nature of the disease is discussed.

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.

Tegumentary leishmaniasis (TL) is a zoonotic disease that affects humans and domestic dogs. In Brazil, TL is considered endemic, and Leishmania (Viannia) braziliensis is the prevalent species causing this disease. There is debate about the role of dogs (Canis familiaris) as domestic reservoirs in the transmission cycle of TL. To date, classical parasitological techniques, including parasite isolation in culture media, have been able to detect parasites only from cutaneous lesions. In this study, we detected L. (V.) braziliensis DNA in intact skin fragments collected from 3 naturally infected dogs from the state of Rio de Janeiro, Brazil, with the use of PCR techniques associated with molecular hybridization. The detection of parasitic DNA in this anatomical site is an important finding vis-a-vis the importance of the domestic dogs in endemic areas of TL.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:45:29Z No. of bitstreams: 1 S0074-02762001000700018.pdf: 105482 bytes, checksum: cd29c6a368a5a65027789323f124d4ea (MD5) Made available in DSpace on 2013-08-22T18:45:29Z (GMT). No. of bitstreams: 1 S0074-02762001000700018.pdf: 105482 bytes, checksum: cd29c6a368a5a65027789323f124d4ea (MD5) Previous issue date: 2001-10-01 Made available in DSpace on 2013-09-30T19:34:34Z (GMT). No. of bitstreams: 2 S0074-02762001000700018.pdf: 105482 bytes, checksum: cd29c6a368a5a65027789323f124d4ea (MD5) S0074-02762001000700018.pdf.txt: 46307 bytes, checksum: a1ac4def2e6c4164b2ddcd4894a65fc7 (MD5) Previous issue date: 2001-10-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:09:24Z No. of bitstreams: 2 S0074-02762001000700018.pdf: 105482 bytes, checksum: cd29c6a368a5a65027789323f124d4ea (MD5) S0074-02762001000700018.pdf.txt: 46307 bytes, checksum: a1ac4def2e6c4164b2ddcd4894a65fc7 (MD5) Made available in DSpace on 2014-05-20T15:09:24Z (GMT). No. of bitstreams: 2 S0074-02762001000700018.pdf: 105482 bytes, checksum: cd29c6a368a5a65027789323f124d4ea (MD5) S0074-02762001000700018.pdf.txt: 46307 bytes, checksum: a1ac4def2e6c4164b2ddcd4894a65fc7 (MD5) Previous issue date: 2001-10-01 Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected. Fiocruz Centro de Pesquisa Hospital Evandro Chagas Universidade Estadual de São Paulo Departamento de Microbiologia e Imunologia Fiocruz Instituto Oswaldo Cruz Departamento de Bioquímica e Biologia Molecular Fiocruz Instituto Oswaldo Cruz Departamento de Protozoologia Universidade do Estado do Rio de Janeiro Departamento de Patologia Hospital Geral de Bonsucesso Serviço de Endoscopia Peroral

In the last few years the number of human cases of American visceral leishmaniasis in the Metropolitan Region of Belo Horizonte (MRBH), Minas Gerais, Brazil has increased, indicating an elevation in the transmission rate of the disease. The total number of notified human cases in the MRBH since 1994, when the first case was identified, up to 1999 was 345 of which 223 (65%) were from the city itself, indicating an urbanization of the disease in this region of Minas Gerais. The age distribution of visceral leishmaniasis cases in the MRBH shows a higher prevalence in children from 0-4 years old, responsible for 28.9% of the notifications. Clinical and immunological findings from dogs infected with Leishmania chagasi are described. The majority of these animals showed no sign of the disease. Sera from all infected dogs showed detectable Leishmania-induced high titles of antibodies based on the results of an indirect fluorescent antibody test. Samples of isolated Leishmania from human and dogs were characterized as L. (L.) chagasi by biochemical and molecular techniques.

An HIV positive patient presenting a clinical picture of visceral leishmaniasis co-infection was submitted to a bone marrow aspiration after admission to hospital. Amastigotes forms were seen in the bone marrow aspirate and the parasite grew in culture as promastigotes. Molecular analyses showed that the flagellates isolated did not belong to the genera Leishmania, Trypanosoma or Sauroleishmania. It was not possible to establish infection in laboratory animals. In vitro culture of mouse peritoneal macrophages revealed the invasion of the host cells by the flagellates and their killing 48 hr after infection. Opportunistic infection with an insect trypanosomatid was suspected. Further hybridization analyses against a pannel of different monoxenous and heteroxenous trypanosomatids showed kDNA cross-homology with Leptomonas pulexsimulantis a trypanosomatid found in the dog's flea

Two former patients treated for the cutaneous form of American tegumentary leishmaniasis were reviewed eight and 11 years, respectively, following clinical cure. We were able to isolate Leishmania parasites in a culture of material from the two scar biopsies, and in one of them the parasite was characterized as Leishmania (Viannia) braziliensis. In both cases, the histopathology revealed discreet hyperceratosis and a slight infiltrate of mononuclear cells surrounding and on the walls of the surface and deep dermal vessels. No amastigotes were seen on immunohistochemical or histopathologic examination. The Montenegro skin test result and the in vitro lymphoproliferative response to Leishmania antigen were positive, but no specific IgG and IgM antibodies were detected. Otorhinolaryngologic examination showed no macroscopic alteration in the mucosae. These findings are important for the evaluation and criteria of post-treatment cure.

Triatoma brasiliensis is considered as one of the most important Chagas disease vectors in the northeastern Brazil. This species presents chromatic variations which led to descriptions of subspecies, synonymized by Lent and Wygodzinsky (1979). In order to broaden bionomic knowledge of these distinct colour patterns of T. brasiliensis, captures were performed at different sites, where the chromatic patterns were described: Caicó, Rio Grande do Norte (T. brasiliensis brasiliensis Neiva, 1911), it will be called the "brasiliensis population"; Espinosa, Minas Gerais (T. brasiliensis melanica Neiva & Lent 1941), the "melanica population" and Petrolina, Pernambuco (T. brasiliensis macromelasoma, Galvão 1956), the "macromelasoma population". A fourth chromatic pattern was collected in Juazeiro, Bahia the darker one in overall cuticle coloration, the "Juazeiro population". At the sites of Caicó, Petrolina and Juazeiro, specimens were captured in peridomiciliar ecotopes and in wilderness. In Espinosa the specimens were collected only in wilderness, even though several exhaustive captures have been performed in peridomicile at different sites of this municipality. A total of 298 specimens were captured. The average registered infection rate was 15% for "brasiliensis population" and of 6.6% for "melanica population". Specimens of "macromelasoma" and of "Juazeiro populations" did not present natural infection. Concerning trophic resources, evaluated by the precipitin test, feeding eclecticism for the different colour patterns studied was observed, with dominance of goat blood in household surroundings as well as in wilderness

In the past few years, new aspects of the immunopathology of Chagas' disease have been described in immunosuppressed patients, such as fatal central nervous system lesions related to the reactivation of the parasite. This article is the first description of the genotypic characterization, at the strain level, of Trypanosoma cruzi isolated from a patient with Chagas' disease/AIDS co-infection. The presence of four hypodense lesions was observed in the cranial compute tomographic scan. the diagnosis of AIDS was assessed by the detection of anti-HIV antibodies using enzyme-linked immunosorbent assay (ELISA) and Western blot techniques. The CD4+ lymphocyte counts were maintained under 200 cells/mm3 during one year demonstrating the severity of the state of immunosuppression. Chagas' disease was confirmed by serological and parasitological methods. Trypomastigote forms were visualized in a thick blood smear. The parasite isolated is genotypically similar to the CL strain. The paper reinforces that cerebral Chagas' disease can be considered as another potential opportunistic infection in AIDS resulting from the reactivation of a dormant T. cruzi infection acquired years earlier.

A survey for canine tegumentary leishmaniasis (CTL) has been carried out between 1986 and 1993 in seven endemic localities for American cutaneous leishmaniasis in the State of Rio de Janeiro. 270 dogs have been examined for their clinical aspects, the development of delayed hypersensitivity (DHS) with Immunoleish antigen and with immunofluorescent antibody research of IgG (IF). 28.2% of them had ulcer lesions and 3.3% had scars. The lesions consisted of single (39.5%) and mucocutaneous lesions (31.6%), multiple cutaneous (25.0%) and mucocutaneous lesions associated with cutaneous ulcers (4.0%). Twelve (15.8%) isolates from biopsies were analyzed by zimodeme and schizodeme and identified as L. (V.) braziliensis. The overall prevalence of canine infection that was evaluated with the skin test was of 40.5% and with IF it was of 25.5%. Both tests showed a high positive rate with relation to the animals with mucosal lesions, as in the case of human mucocutaneous leishmaniasis. The comparison of the two tests showed the skin test to have a better performance although there was no statistical difference (p>0.05) between them. The proportional sensitivity and specificity was of 84.0% and 74.0%, respectively. The Immunoleish skin test and IF are useful tools to be employed in CTL field epidemiological surveys.Um inquérito epidemiológico em população canina foi realizado em 7 localidades endêmicas de Leishmaniose Tegumentar Americana (LTA) entre os anos de 1986 a 1993, no Estado do Rio de Janeiro. Duzentos e setenta cães foram examinados, segundo os parâmetros: clínicos, desenvolvimento de hipersensibilidade tardia e dosagem de anticorpos por imunofluorescência indireta (IFI). 28,2% dos animais possuíam lesões e 3,3% eram portadores de cicatrizes compatíveis com infecção prévia de Leishmania sp. De um total de 98 lesões ulceradas detectadas, 39,5% eram cutâneas únicas, 25,0% lesões cutâneas múltiplas, 31,6% lesões de mucosa e 4,0% lesões de mucosa associadas a lesões cutâneas. Doze amostras (15,8%) colhidas de fragmentos de lesões cutâneas foram analisadas por zimodema e esquizodema e identificadas como Leishmania (V.) braziliensis . A prevalência da infecção canina avaliada pelo teste cutâneo e pela IFI foram respectivamente de 40,5% e 25,5%. A comparação entre as reações mostrou que o teste cutâneo foi superior na detecção da infecção e da doença canina, embora, sem diferença estatística significante (p> 0,05). A sensibilidade e a especificidade proporcional foram respectivamente de 84,0% e 74,0%. A utilização de ambas as reações, cutânea e sorológica mostraram-se úteis como instrumento de diagnóstico epidemiológico em áreas de LTC.

Different molecular-genetic methods were used to identify a cohort of Leishmania strains from natural foci of zoonotic cutaneous leishmaniasis located in Central Asia, on the former USSR territory. The results obtained using isoenzymes, PCR, restriction fragment length polymorphisms of kDNA and molecular hybridization techniques are discussed in terms of their applicability, discrimination power and feasibility for answering questions related to molecular epidemiological research and for detecting mixed Leishmania infections.

Introduction During a diagnostic evaluation of canine visceral leishmaniasis (VL), two of seventeen dogs were found to be co-infected by Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi. Methods Specific polymerase chain reaction (PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR) assays were performed. Results PCR assays for Leishmania subgenus identification followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confirmed the presence of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in those fragments. Conclusions This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.

Little is known regarding the internal dissemination of initial cutaneous lesions and tissue tropism ofLeishmania (Viannia) braziliensispopulations in naturally infected dogs. The aim of this study was to investigate genetic polymorphisms ofL. (V.) braziliensispopulations in different anatomic sites of naturally infected dogs by using polymerase chain reaction (PCR) and low-stringency single specific primer-PCR (LSSP-PCR) techniques. The amplified products were analyzed by LSSP-PCR to investigate the genetic variability of the parasite populations present in different anatomical sites. Twenty-three out of the 52 samples gave PCR-positive results. The existence ofL. (V.) braziliensisstrains that remained restricted to cutaneous lesions and others showing characteristics of dissemination to internal organs and healthy skin was observed. LSSP-PCR and numerical analyses revealed that parasite populations that do not disseminate were genetically similar and belonged to a separate phenetic cluster. In contrast, populations that showed spreading to internal organs displayed a more polymorphic genetic profile. Despite the heterogeneity,L. (V.) braziliensispopulations with identical genetic profiles were observed in popliteal and cervical lymph nodes of the same animal. Our results indicate that infection in dogs can be manifested by dissemination and tissue tropism of genetically distinct populations ofL. (V.) braziliensis.

Com o objetiiro de identificar a espécie de Leishmania envolvida nas afecções cutâneas de indivíduos residentes nos municípios de Cosmópolis e Indaiatuba, região de Campinas, Estado de São Paulo, correlacionamos dados clínicos, histopatológicos e testes de bibridização in situ. Os expressivos índices de incidência de leishmaniose tegumentar americana obsewados nesses municípios nos levaram a iniciar esses estudos. Nesse trabalho, apresentamos os dados relativos a seis indivíduos. As características das lesões, ulceradas, de dificil cicatrização e presentes em locais expostos do corpo sugeriram quadros de leishmaniose tegumentar americana. Os testes de intradermoreação de Montenegro, positivos em cinco dos seis pacientes analisados também reforçaram a suspeita de leishmaniose tegumentar americana. Os padrões histopatológicos, como reação exsudativa celular e granulomatosa e a dificuldade de isolamento dos parasitas obtidos de biópsias de lesões são compatíveis com aqueles descritos para Leishmania (Viannia) braziliensis. Em testes de bibridização in situ do DNA do cinetoplasto de amastigotas das lesões dos seis pacientes, obseivamos que quatro deles apresentavam sinais de bibridização com a sonda de L. (Viannia) braziliensis, confirmando as suspeitas de que a Leishmania responsável pelas afecções cutâneas nos pacientes analisados era do subgenêro Viannia e do complexo braziliensis.A study was earned out to identify Leishmania species involved in skin lesions of patients from Cosmõpolis and Indaiatuba, State of São Paulo, Brazil. The epidemiological data of cutaneous leishmaniasis in two cities suggested a epidemic situation in 1994. The lesions were clinically characteristic of cutaneous leishmaniasis and five out six patients responded positively to Montenegro's intradermal test. The histopathology of skin lesions were characterized by two patterns: exsudative- cellular reaction and exsudative granulomatous reaction. The clinical and histopathological parameters suggested Leishmania (Viannia) braziliensis as the possible etiologic agent. In agreement, it was difficult to isolate and mantain the parasite in the laboratory. Characterization by in situ hybridization with kDNA amastigotes from lesions fragments confirmed that Leishmania (Viannia) braziliensis was the parasite responsible for the studied cutaneous lesions.

Molecular karyotype and kDNA restriction analyses were utilized to examine the genetic heterogeneity and plasticity of the Leishmania (Viannia) guyanensis strain WHI/BR/78/M5313, composed of metastatic and non-metastatic populations. Cloning revealed that the strain was constituted by multiple closely related populations that were distinguishable by restriction fragment polymorphisms in kDNA. Size polymorphisms in molecular karyotype were not detected. Passage of clones in hamsters and recovery of parasites from cutaneous metastatic lesions yielded evidence of further genetic heterogeneity among some of the progeny populations. Overall, six kDNA minicircle restriction patterns or schizodemes were observed among clones, subclones and progeny. Although the possibility that population heterogeneity was not resolved by cloning cannot be ruled out, subcloning and kDNA restriction analysis to determine whether the putative clones consisted of homogeneous populations showed the schizodeme of subclones of 3 out of 4 clones to be identical to the clone of origin, while a subclone of the fourth had a co-efficient of similarity of 0.95. Metastasis did not segregate with a particular schizodeme: all six restriction profiles were represented among populations isolated from metastatic lesions and some clones with the same restriction profile did not produce metastatic lesions. The strain from which the clones, subclones and progeny were derived had a kDNA restriction pattern identical to the most prevalent schizodeme (38%) among these subpopulations. This finding together with the reappearance of the repertoire of schizodemes found among clones in the populations recovered from metastatic lesions in hamsters inoculated with a single clone, suggest that sequence polymorphisms in kDNA can emerge during infection.

The aim of this study was to investigate genetic polymorphism inLeishmania braziliensispopulation previously typed through isoenzyme electrophoresis, isolated from the same patient in two different moments: (A) before the beginning of treatment and (B) after treatment failure to meglumine antimoniate or reactivation after successful initial treatment. Fifteen pairs of isolates were assessed using the polymorphic molecular marker LSSP-PCR and following the phenetic analysis. The genetic profiles of the 30 samples were grouped in four clusters. Only two patients presented total identity in the A and B isolates. Most isolates presented similarity coefficients varying from 0.63 to 0.91. In this group of patients genetic polymorphisms could be observed indicating low similarity between the pairs of isolates. The results demonstrate the existence of genetic polymorphism between the samples isolated before treatment and after reactivation or treatment failure, suggesting a possible differentiation of the structure of the original parasite population which could be involved in the mechanisms of resistance to treatment or reactivation of lesions in the ATL. This phenomenon is important, although other factors also could be involved in this context and are discussed in this paper.

SUMMARYTwo domestic guinea-pigs (Cavia porcellus), bought in Pinheiros, São Paulo State, Brazil, were taken by their owners a farm in the rural district of Capão Bonito, close to the Atlantic Forest, São Paulo, where they both developed tumour-like and ulcerating lesions on the ears. The causative agent was identified as Leishmania (L.) enriettii, based on biological characters and isoenzyme profiles. Sources of the parasite in wild mammals, and the possible sandfly vector species discussed.

Cutaneous disseminated lesions caused by Leishmania sp. were found in a pregnant mare (Equus cabalus) from a rural city in the State of Rio de Janeiro, Brazil. Before delivering, treatment was undertaken by immunotherapy followed by chemotherapy. Histopatology and serology were performed during treatment, as well as the biochemical characterization of the parasite (L. braziliensis) that was isolated from one of the lesions.

A flagellte trypanosomatid was isolated in culture from the digestive tract of the mosquito Culex saltanensis Dyar, 1928. It grows exuberantly in liver infusion in the form of epimastigotes and choanomastigotes typical of the genus Crithidia. The trypanosomatid was compared to C. deanei, C. fasciculata, C. luciliae, C. oncopelti and C. guilhermei. The techniques used for comparison were electron transmission microscopy, isoenzymes and kDNA restriction profiles. No endosymbionts were found at electron microscopy. Results for the biochemical methods employd indicate that the trypanosomatid isolated from C. saltanensis is a new species of Crithidia. The name C. ricardoi sp. n. is proposed for thes new species.

Chagas disease remains a public health concern in Brazil and other Latin American countries, mainly due to the potential domiciliation of native triatomine species. We analyzed the genetic variability of Triatoma pseudomaculata in sylvatic and peridomestic ecotopes throughout three localities in the northeastern state of Bahia, Brazil. We studied polymorphisms generated by random amplified polymorphic DNA (RAPD) and isoenzyme electrophoresis analyses. Based on RAPD analysis, each specimen was assigned to one of three genetic clusters. Although all sylvatic specimens from one locality were grouped into the same cluster, sylvatic and peridomestic specimens from the other two localities were broadly distributed between the remaining two clusters, suggesting that geographic population structuring was not occurring. Furthermore, isoenzyme analysis suggested that distinct populations were in Hardy-Weinberg equilibrium. Low statistical values for Wright's Fst index also supported the absence of population structuring and suggested the occurrence of panmixia. We conclude that genetic flow occurs between sylvatic and peridomestic T. pseudomaculata populations, probably as a consequence of passive and active dispersion of the insects, associated with deforestation and anthropic transformations.

Triatoma carcavalloi is considered a rare Chagas disease vector often collected inside domiciles in Rio Grande do Sul State. In this Brazilian state, T. carcavalloi has been collected in the same ecotope (rock piles) with two other species (T. rubrovaria and T. circummaculata), with which it also shares morphological characteristics. Previous morphological studies placed T. carcavalloi in the same species complex ("infestans complex") and subcomplex ("rubrovaria subcomplex") as T. rubrovaria, whereas T. circummaculata was placed in the "circummaculata complex." The phylogeny of a group composed of 16 species of triatomines was reevaluated with the inclusion of T. carcavalloi by Bayesian analysis using mtDNA sequences of subunits 12S and 16S of the ribosomal RNA, and the cytochrome oxidase I (COI) genes. The phenotypic relationship among T. carcavalloi and related triatomines was also inferred from morphometrics. Phylogenetic results indicate that T. carcavalloi is a sister species of T. rubrovaria, and both were recovered as closely related to T. circummaculata. Morphometric studies confirmed the closeness among T. carcavalloi, T. rubrovaria, and T. circummaculata, prompting the placement of the latter species in the "infestans complex" and "rubrovaria subcomplex."

The genetic polymorphism of Leishmania (Viannia) braziliensis detected in cases of mucosal leishmaniasis (ML) from HIV-infected and non HIV-infected patients was evaluated. Nine samples from three HIV-infected patients and five samples from five non HIV-infected patients were analysed by polymerase chain reaction (PCR), low-stringency single-specific primer PCR (LSSP-PCR) and phenetic analysis. The presence of L. (V.) braziliensis DNA was detected in all samples by specific PCR assay. The intraspecific polymorphism of the variable region of L. (V.) braziliensis kDNA minicircles was investigated by LSSP-PCR. Phenetic analysis grouped the genetic profiles into two distinct clusters, which discriminated between samples obtained from HIV-infected and non HIV-infected patients. In two HIV-infected patients, identical genetic profiles were detected in lesions biopsied at different times after the treatment of the initial lesion. Interestingly, genetically divergent profiles were detected in the cutaneous and mucosal lesions of the same HIV-infected patient collected at the same time. This is the first work comparing genetic polymorphism of L. (V.) braziliensis in cases of mucosal leishmaniasis from HIV-infected and non HIV-infected patients.

In this study diarrheagenic and uropathogenic Escherichia coli (UPEC) strains were comparatively characterized according to serotype, hemolytic activity, protein polymorphism among housekeeping enzymes, phylogenetic group and urovirulence genes. Intra-serogroup/serotype variations were observed. Hemolytic activity was detected in 100%, 93%, 67% and 39% of UPEC, EAEC, EPEC and ETEC strains, respectively. The alpha-hemolytic phenotype was observed in all pathogenic groups while beta-hemolytic phenotype was less frequent. PCR phylotyping revealed higher prevalence of diarrheagenic E. coli in groups A and D while uropathogenic strains were mainly found in subgroup B2. Amplification assays revealed that 74%, 45% and 22% of UPEC, EAEC and EPEC strains, respectively, carried at least one of the urovirulence sequences. The molecular typing system revealed a pathotype-specific clonal group distribution and showed a closer relationship between the EAEC and UPEC. Additionally, the occurrence of urovirulence traits, especially those related to iron acquisition, was more frequent among EAEC and UPEC than among the other E. coli pathotypes. This observation is of special value considering that the EAEC pathotype constitutes an emerging group of enteropathogens, particularly, in developing countries, and information on their pathogenic and phylogenetic characteristics is still scarce.

Polymerase chain reaction (PCR) and low-stringency single-specific primer PCR (LSSP-PCR) analyses were used to detect Leishmania (Viannia) braziliensis DNA and investigate kDNA signatures of parasite populations present in oral and nasal mucosa lesions from mucosal leishmaniasis patients. A total of 25 samples from 22 patients were processed by specific PCR/hybridization assays. Parasite DNA was detected in all samples analyzed. The intraspecific polymorphism of the variable region of L. (V.) braziliensis kDNA minicircles was also investigated by LSSP-PCR. Similar kDNA signatures were observed in parasites recovered from nasal and oral mucosa lesions of the same patient. In contrast, genetically divergent profiles were detected in lesions from patients biopsied at different times within a period of 1 year. This is the first work to report genetic typing of L. (V.) braziliensis directly from human oral and nasal mucosal lesions.

Aims: The genetic diversity of Beauveria bassiana was investigated by comparing isolates of this species to each other (49 from different geographical regions of Brazil and 4 from USA) and to other Beauveria spp. Methods and Results: The isolates were examined by multilocus enzyme electrophoresis (MLEE), amplified fragment length polymorphism (AFLP), and rDNA sequencing. MLEE and AFLP revealed considerable genetic variability among B. bassiana isolates. Several isolates from South and Southeast Brazil had high similarity coefficients, providing evidence of at least one population with clonal structure. There were clear genomic differences between most Brazilian and USA B. bassiana isolates. A Mantel test using data generated by AFLP provided evidence that greater geographical distances were associated with higher genetic distances. AFLP and rDNA sequencing demonstrated notable genotypic variation between B. bassiana and other Beauveria spp. Conclusion: Geographical distance between populations apparently is an important factor influencing genotypic variability among B. bassiana populations in Brazil. Significance and Impact of the Study: This study characterized many B. bassiana isolates. The results indicate that certain Brazilian isolates are considerably different from others and possibly should be regarded as separate species from B. bassiana sensu latu. The information on genetic variation among the Brazilian isolates, therefore, will be important to comprehending the population structure of B. bassiana in Brazil.

Trypanosoma cruzi, the etiological agent ofChagas disease, exists in nature as a complex ofheterogeneous populations. There is a wide spec-trum of human disease manifestations (Z Brener1987 Mem Inst Oswaldo Cruz 82: 205-212) andthe crucial problem in the study of Chagas diseaseis a lack of understanding of the factors involvedin its pathology. The imposition of natural and ar-tificial pressures can result in the selection of asubset of the population. The marked heterogene-ity of T. cruzi leads to a search for a relationshipbetween functional parameters of parasites isolatedfrom patients or specific geographical areas andthe manifestation of the disease itself. Anothercrucial, and someway, unsolved question is theinfluence of this heterogeneity on the stability ofthe parasite population (JA Dvorak 1984 J CellBiochem 24: 357-371, RW Finley & JA Dvorak1987 J Protozool 34: 409-415).In essence, the fundamental problems associ-ated with the attempts to understanding Chagasdisease were reported by Carlos Chagas in 1909(Mem Int Oswaldo Cruz 1: 159-218) and most ofthe phenomena currently under study were identi-fied by himself. As the disease can be manifestedin different clinical forms from symptomless to anacute fulminate infection or to severe or even in-apparent chronic infection, FL Lambrecht (1965Rev Inst Med Trop Sao Paulo 7: 346-352) advancedthe premise that T. cruzi may be composed of agenetically heterogeneous population of parasitesand that such heterogeneity may be one of the fac-tors that modulate the disease process. In fact, dif-ferences in isolates or strains from wide geographi-cal and host ranges have already been well docu-mented (Z Brener 1965 Ann Trop Med Parasitol59: 19-26, SG Andrade et al. 1970 Gaz Med Ba-hia 1: 32-42, Z Brener 1980 Advances Parasitol18: 247-292, WE Gutteridge 1981 Trans R SocTrop Med Hyg 75: 484-492).With the argument of population heterogene-ity in mind, some years ago, researchers from Bra-zil and outside begun to develop new biologicaland biochemical approaches to investigate this in-triguing parasite. Restriction analyses of kDNA(CM Morel et al 1980 Proc Natl Acad Sci USA 77:6810-6814) have shown a considerable degree ofheterogeneity in natural T. cruzi populations andimportant findings such as the occurrence of mixedinfection in human, reinfection and selection ofsubpopulations have been reported (CM Morel1984 TDR Series 5 : 333-375, MP Deane et al. 1984J Protozool 31: 276-280, CM Morel et al. 1986Parasitol Today 2: 97-101). Isoenzymatic analy-ses also confirmed the heterogeneity although ini-tial studies revealed discrete variability showingthree or four principal zymodemes (MA Miles etal. 1977 Trans R Soc Trop Med Hyg 71: 217-225,1980 Trans R Soc Trop Med Hyg 74: 221-237, AJRomanha 1979 Comp Biochem Physiol 62: 139-142). In recent times, new concepts, models andmethods have been introduced to elucidate the com-plexity of the population dynamics of T. cruzi. In1986, Tibayrenc and co-workers ( Proc Natl AcadSci USA 83: 115-119) have proposed that naturalpopulations of T. cruzi have a complex multiclonalstructure. Although T. cruzi is diploid (Tibayrencloc. cit , WC Gibson & MA Miles 1986 EMBO J 5:1299-1305) its reproduction is primarily clonal andsexual recombination is rare or even absent. Anextensive zymodeme diversity among distinctclones was discovered indicating that they couldhardly be clustered in a few groups (M Tibayrenc& FJ Ayala 1988 Evolution 42 : 277-292). The termclonet (M Tibayrenc 1990 Proc Natl Acad Sci USA87: 2414-2418) was introduced to explain the in-tragroup or infraspecific variation. On that ac-count, how many relevant phylogenetic subdivi-sion are there? (M Tibayrenc 1995 Adv Parasitol36: 47-115, RP Souto et al. 1996 Mol BiochemParasitol 83: 141-152). It seems that there is an

The Triatoma brasiliensis genetic structure was analyzed using the Cyt B gene in different geographic locations and ecotopes after a short and long period after insecticide treatment. Four different localities (16-40 km apart) in the state of Paraíba, Brazil, were sampled. Analysis of molecular variance (AMOVA) showed that grouping populations according to the geographic location or ecotope resulted in a higher variance among populations within groups (Phi(SC) ranging from 0.15 to 0.17) than among groups (Phi(CT) ranging from 0.04 to 0.07). The percentage of variation was reduced among populations within groups and increased among groups (Phi(SC) = 0.08, Phi(CT) = 0.16) by grouping 1) the domiciliary populations from each village and 2) all wild populations. These data indicated that T. brasiliensis is genetically structured both ecologically and at a smaller geographic scale for domiciliary populations. Re-infestations after insecticide treatment were composed of distinct populations, pointing to variable population sources for domiciliary infestations.