First report of diffuse cutaneous leishmaniasis and Leishmania amazonensis infection in Rio de Janeiro State, Brazil.

Diffuse cutaneous leishmaniasis (DCL) is characterised by multiple and progressive cutaneous lesions, resistance to chemotherapy and Leishmania-specific T-cell anergy. We report the first autochthonous DCL case and the first human infection with Leishmania amazonensis in Rio de Janeiro State, Brazil, where only L. braziliensis is considered to be the causative agent of cutaneous leishmaniasis. Leishmania amazonensis was identified by multilocus enzyme electrophoresis and PCR-RFLP. Our case was diagnosed as DCL according to clinical, parasitological, histopathological and immunological criteria. These observations indicate that L. amazonensis is increasing its geographical distribution in Brazil, accounting for unusual clinical presentations in new transmission areas.

A zymographic study of metalloprotease activities in extracts and extracellular secretions of Leishmania (Viannia) braziliensis strains.

Proteolytic activities of 5 strains of Leishmania (Viannia) braziliensis isolated from Brazilian and Colombian patients, presenting distinct clinical manifestations, were characterized and compared using whole-promastigote extracts and extracellular secretions. Zymographic assays concerning whole-cell extracts and supernatants resulted in the detection of high molecular weight bands, ranging from 50 to 125 kDa. Proteolytic activities from both whole-cell extracts and supernatants were optimal in a pH range 5.5 to 9.0 for all analysed strains. Such protease activities were inhibited when 10 mM 1,10-phenanthroline was assayed, strongly suggesting that the enzymes responsible for hydrolysis of the substrate belong to the metalloproteases class. Distinct profiles of metalloproteases were observed among the studied L. (V.) braziliensis strains. Differences among the microorganisms might be related to the geographical origin of the strains and/or to the clinical presentation.

Isolation and isoenzyme characterization of Leishmania (Viannia) braziliensis from a case of human cutaneous leishmaniasis in northeast centre of the state of São Paulo

The diagnosis of human cutaneous leishmaniasis in small towns is sometimes made without the species identification of the Leishmania, even in areas without previous epidemiological surveys. Here we report the isolation of a Leishmania strain from a patient of Rincão, state of São Paulo, that was identified by isoenzyme characterization as L. (Viannia) braziliensis. Sand fly collections were made in the area where the patient live in order to investigate the likely vector species.

Micro-geographical variation among male populations of the sandfly, Lutzomyia (Nyssomyia) intermedia, from an endemic area of American cutaneous leishmaniasis in the state of Rio de Janeiro, Brazil.

The genetic relationships among male Lutzomyia (Nyssomyia) intermedia (Lutz & Neiva) (Diptera: Psychodidae) from three populations from the same endemic area of American cutaneous leishmaniasis (ACL) in the state of Rio de Janeiro, Brazil, were compared. The sandflies were collected in three ecologically different habitats: domestic, extra-domestic and sylvatic over a total range of 800 m. Three molecular markers were employed to assess population variation. Based on MLEE markers, it could not be concluded that the three populations do not belong to the same gene pool (F(st) = 0.005). No within-population departure from Hardy-Weinberg equilibrium was detected (P < 0.05) and they presented the same level of gene variation. The number of migrants (Nm) indicated that at least 50 individuals per generation migrated between the three habitats. RAPD-PCR markers revealed that, except for the primer five, all were polymorphic. Phenetic analysis of the genotypes showed the presence of two principal clusters corresponding to: (1) domestic plus extra-domestic and (2) sylvatic. Unique genotypes were observed in each population. The sylvatic population was the most polymorphic, showing the largest number of genotypes and low level of similarity between them. Three mtDNA gene markers were studied by SSCP analysis. The most frequent haplotype for each marker ranged in frequency from 60 to 87% and individuals with unique haplotypes varied from 1 to 5%. Interestingly, the SSCP analysis showed a low level of polymorphism within populations. The disagreement between the different molecular markers observed and the hypothesis that L. intermedia could be participating in the transmission cycle of Leishmania (Viannia) braziliensis in environments ranging from the interior of human dwellings to the forest, are discussed.

Isolation and isoenzyme characterization of Leishmania (Viannia) braziliensis from a case of human cutaneous leishmaniasis in northeast centre of the state of São Paulo.

The diagnosis of human cutaneous leishmaniasis in small towns is sometimes made without the species identification of the Leishmania, even in areas without previous epidemiological surveys. Here we report the isolation of a Leishmania strain from a patient of Rincão, state of São Paulo, that was identified by isoenzyme characterization as L. (Viannia) braziliensis. Sand fly collections were made in the area where the patient live in order to investigate the likely vector species.

Leishmania (Viannia) braziliensis-induced chronic granulomatous cutaneous lesions affecting the nasal mucosa in the rhesus monkey (Macaca mulatta) model.

The present studies on infections with Leishmania (Viannia) braziliensis in rhesus macaques were made to characterize the evolution of different parasite strains and the immune responses they elicited in this experimental host. A standardized inoculum of promastigotes was injected intradermally either above the eyelid or on the forearm of each monkey. Sixteen infected monkeys developed longstanding infections which lasted until the end of the observation period (33 months). The time required for lesion development was very variable, not only for the isolates showing molecular differences but also for individual animals in groups infected with the same parasite strain. The inocula produced lesions of variable severity, ranging from localized cutaneous leishmaniasis (CL) with a tendency to spontaneous healing to non-healing disease. One infected animal developed persistent metastatic skin and mucosal lesions. Anti-Leishmania antibodies and parasite-specific T-cell responses were induced by the experimental infections. As the granulomatous inflammatory response found at the lesions in L. (V.) braziliensis-infected M. mulatta was similar to that in patients with CL, this primate model could be useful for studying the pathophysiology and immunoregulatory events associated with disease evolution, as well as for the evaluation of new drugs or candidate vaccines.

Epidemiological and clinical features of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in the State of Espírito Santo, Brazil

Between 1985 and 2000, epidemiological surveys of the American tegumentary leishmaniasis (ATL) were carried out in several rural and urban communities in Espírito Santo, Brazil. A total of 100 stocks of Leishmania (comprising isolates from both human and canine hosts with ATL) were identified by two methods of molecular characterization, using specific monoclonal antibodies and multilocus enzyme electrophoresis. Parasite isolates from 19 municipalities were found to belong to the same zymodeme and serodeme type as of the Leishmania (Viannia) braziliensis reference strain. In contrast, our genotyping studies have shown intra-specific variation among these parasites (comparisons of the variability of the internal transcribed spacers between the small and large subunits of the rRNA genes of the 22 stocks studiedrevealed at least 11 genotypes). Two main clusters of L. (V.) braziliensis genotypes were observed, representing parasites collected from different endemic regions in the state, where transmission reflects distinct eco-epidemiological features. Infection with this pathogen was associated with the characteristic disease forms, but neither the clinical outcome nor the response to treatment could be related to the genetic polymorphism of the isolates, as defined by using the proposed methodology.

Epidemiological and clinical features of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in the State of Espírito Santo, Brazil.

Between 1985 and 2000, epidemiological surveys of the American tegumentary leishmaniasis (ATL) were carried out in several rural and urban communities in Espírito Santo, Brazil. A total of 100 stocks of Leishmania (comprising isolates from both human and canine hosts with ATL) were identified by two methods of molecular characterization, using specific monoclonal antibodies and multilocus enzyme electrophoresis. Parasite isolates from 19 municipalities were found to belong to the same zymodeme and serodeme type as of the Leishmania (Viannia) braziliensis reference strain. In contrast, our genotyping studies have shown intra-specific variation among these parasites (comparisons of the variability of the internal transcribed spacers between the small and large subunits of the rRNA genes of the 22 stocks studied revealed at least 11 genotypes). Two main clusters of L. (V.) braziliensis genotypes were observed, representing parasites collected from different endemic regions in the state, where transmission reflects distinct eco-epidemiological features. Infection with this pathogen was associated with the characteristic disease forms, but neither the clinical outcome nor the response to treatment could be related to the genetic polymorphism of the isolates, as defined by using the proposed methodology.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease.

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-gamma) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79 percent in attenuated parasite-vaccinated monkeys, versus 75 percent in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-g) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application

The rarity of infection with Leishmania (Viannia) braziliensis among patients from the Manaus region of Amazonas state, Brazil, who have cutaneous leishmaniasis.

The frequency of Leishmania ( Viannia) braziliensis infection was assessed in 79 of the 138 patients with cutaneous leishmaniasis who attended a reference outpatient unit in Manaus, Amazonas state, between the August and December of 1997. The disease was characterized by one or more cutaneous ulcers, the skin lesions being frequently associated with satellite lymph-node enlargement. All parasite isolates were identified using monoclonal antibodies and enzyme electrophoresis. Only two (2.8%) of the 71 patients from whom parasites were successfully isolated were found to be infected with L. ( V.) braziliensis, the other 69 isolates being identified, from their isoenzyme profiles, as L. ( V.) guyanensis. In the Manaus region, therefore, almost all human cutaneous leishmaniasis is the result of infection with L. (V.) guyanensis, and L. ( V.) braziliensis is a relatively rare cause of the disease.

Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron.

Trypanosoma cruzi is currently classified into 2 major phylogenetic lineages, T. cruzi I and II, that correlate with the formerly described zymodeme 1 and 2, respectively. Another isoenzymic group (zymodeme 3-Z3) was also described. In this study, we analysed the genetic diversity among Z3 isolates of the Brazilian Amazon by restriction fragment length polymorphism of the intergenic transcribed spacers (ITSs) of the ribosomal RNA cistron and the size of the divergent domain D7 of the 24Salpha rRNA gene. DNAs from 12 T. cruzi Z3 isolates obtained from humans (2), Panstrongylus geniculatus (1), and Rhodnius brethesi (9) were submitted to PCR amplification of the ITSs plus the 5.8S rDNA. The PCR products were digested with 4 distinct endonucleases and the profiles analysed by a numerical methodology. The phenetic dendrogram revealed a clear dichotomy in the Z3 group, defining 2 groups that were named Z3-A and Z3-B. Dimorphism was also found in the band sizes of the amplified D7 divergent domain of the 24Salpha rDNA, which showed a perfect correlation with the ITSs clustering. The organization of the ribosomal cistron was investigated by Southern blotting and shown to be conserved in the genome of the 2 Z3 groups. This study shows that the rDNA cistron allows the definition of 2 distinct subclusters in Z3 isolates.

The genetic diversity of Brazilian Trypanosoma cruzi isolates and the phylogenetic positioning of zymodeme 3, based on the internal transcribed spacer of the ribosomal gene.

There is a high level of genetic diversity among the clinical isolates of the taxon Trypanosoma cruzi. The results of recent studies on well defined gene loci have revealed that T. cruzi can be divided into two major phylogenetic lineages, designated T. cruzi I and T. cruzi II. Further intra-lineage polymorphisms were explored, in clinical isolates from five representative regions of Brazil, using the internal transcribed spacers that flank the 5.8S ribosomal RNA gene. These regions were amplified in a PCR and the products were digested with six restriction enzymes before being subjected to RFLP analysis in polyacrylamide gels. The results were used to construct a complex evolutionary tree, in which the isolates were clustered into two major phylogenetic lineages with a low level of similarity. To investigate the evolutionary relationships between the isolates, the PCR products were cloned and sequenced. The results of the subsequent phylogenetic analysis confirmed the presence of the two major groups of T. cruzi and showed that zymodeme 3, a third iso-enzymatic sub-group, is closer to T. cruzi I than to T. cruzi II.

A first case of cutaneous leishmaniasis due to Leishmania (Viannia) lainsoni in Bolivia.

We present the first known case of cutaneous leishmaniasis due to Leishmania (Viannia) lainsoni detected in Bolivia. The parasite was isolated from a young girl living in the subtropical region of Carrasco (900-1000 m above sea level, Caranavi Province, Department of La Paz, Bolivia). The parasite identification was confirmed by multilocus enzyme electrophoresis.

Sensitivity of the polymerase chain reaction for the diagnosis of cutaneous leishmaniasis due to Leishmania (Viannia) guyanensis.

The sensitivity of the polymerase chain reaction (PCR) in 35 consecutive outpatients with cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis was evaluated using, as gold standard, the in vitro isolation of the parasite through culture of aspirates of the cutaneous ulcers. All isolates were identified using electrophoretic enzyme analysis. Patients were mainly young males with recent onset disease without prior specific treatment. PCR was performed using DNA extracted from fresh frozen biopsies of cutaneous ulcers. The reaction used a pair of oligonucleotides that amplify the conserved region of the minicircle molecule. PCR showed 100% sensitivity (95% CI from 90.0 to 100.0). These results were similar to the visualization of amastigotes in imprint preparations of cutaneous biopsy tissue and the inoculation of biopsy material in golden hamsters. Despite the high sensitivity of the PCR, in this particular clinical setting of cutaneous leishmaniasis caused by L. (V.) guyanensis in the Brazilian Amazon, it appears that the method of choice for diagnosis should be the direct visualization of amastigotes using imprint preparations and the PCR reserved for those patients with negative imprint results.

Speculations on the origin and evolution of the genus Leishmania.

Recently two hypotheses have been proposed for the evolution of Leishmania involving respectively a Neotropical or Paleartic origin for the species. Here an alternative proposal on the phylogeny of Leishmania based on the major divisions within the genus is presented. In this hypothesis a Neotropic origin is retained for L. (Viannia) and Paraleishmania, a recently described section within the genus Leishmania, while an African origin is proposed for L. (Leishmania) and possibly Sauroleishmania. The current distribution of Leishmania in the Neotropics is explained as the product of multiple introductions of Leishmania parasites into the New World. Problems with organismal identity in Sauroleishmania and the use of molecular sequence data in inferring phylogenies are also discussed.

Trypanosoma cruzi in the sylvatic environment: distinct transmission cycles involving two sympatric marsupials.

Thirty-five specimens of Philander frenata and 36 Didelphis marsupialis were captured in the same Atlantic forest area of Brazil between 1992 and 1994. Haemocultures showed that 50% of P. frenata and 60% of D. marsupialis were infected with Trypansoma cruzi. Biological, biochemical and molecular characterization of the isolates suggested 2 distinct transmission cycles of T. cruzi occurred between these 2 sympatric didelphids. The T. cruzi isolates could be distinguished according to their association with each marsupial species. Biochemical characterization (multilocus enzyme electrophoresis) revealed 15 zymodemes; more variability was observed among the P. frenata isolates than among the isolates from D. marsupialis. The course of natural and experimental infection in D. marsupialis and P. frenata was different and suggested that D. marsupialis was more resistant to infection than P. frenata. In the studied area, P. frenata seems to be a more important reservoir of T. cruzi than D. marsupialis, since 40% of the characterized isolates from P. frenata belonged to the T. cruzi II group, which is associated with human infections.