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E Cupolillo

Publications and source records attributed to E Cupolillo.

At least 19 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2005

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.

Animals↗
PubMed2005

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.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

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.

Adolescent↗
PubMed2002

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.

Animals↗
PubMed2002

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.

Adolescent↗
PubMed2002

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.

Animals↗
PubMed2002

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.

Animals↗
PubMed2001

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.

Adolescent↗
PubMed1998

American cutaneous leishmaniasis due to Leishmania (Viannia) guyanensis as an initial clinical presentation of human immunodeficiency virus infection.

The authors present the first report of Leishmania (Viannia) guyanensis (L.(V.) guyanenesis) associated with human immunodeficiency virus (HIV) in a Brazilian heterosexual man. It is also the first case of HIV infection associated with American cutaneous leishmaniasis in Brazilian Western Amazonia. The patient had cutaneous and mucous lesions with a negative Montenegro skin test. Histopathology showed large numbers of amastigotes, even in a lesion which had clinically healed. L.(V.) guyanenesis was typed by an immunoenzymatic technique. Various therapies were attempted, but the patient relapsed after each episode of treatment.

AIDS-Related Opportunistic Infections↗
PubMed1997

Genetic diversity among Leishmania (Viannia) parasites.

The mode of reproduction in, and population structure of Leishmania (Viannia) parasites were deduced from the nature and extent of genetic variation in the parasites. Forty-seven zymodemes belonging to the Viannia subgenus were analysed by enzyme electrophoresis and the profiles compared with those of two zymodemes belonging to the Leishmania subgenus. The results revealed extensive genetic variation in Viannia. The widespread occurrence of certain zymodemes, the level of heterozygosity and the great similarity observed among the samples are indicative of a clonal population structure. However, the large number of recurrent mutations needed to explain the variation observed and the presence of hybrid parasites indicate that occasional bouts of genetic exchange may occur.

Animals↗
PubMed1995

Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution of Leishmania.

In the New World, Leishmania of the Viannia subgenus cause both cutaneous and mucocutaneous disease. These parasites show considerable intra-species genetic diversity and variation, which complicates taxonomic classification and epidemiology. We have used the variability of the transcribed noncoding regions between the small and large subunit rRNA genes to examine relationships in this group. In a method termed intergenic region typing (IRT), PCR amplification products were obtained for the rapidly evolving 1-1.2-kb internal transcribed spacers (ITS) between the SSU and LSU rRNAs, from 50 parasites isolated from different hosts and geographic areas. Amplified DNAs were digested with 10 different enzymes, and fragment patterns compared after acrylamide gel electrophoresis. High levels of intra- and inter-specific variation were observed, and quantitative similarity comparisons were used to associate different lineages. A complex evolutionary tree was obtained. Some species formed tight clusters (L. equatorensis, L. panamensis, L. guyanensis, L. shawi), while L. braziliensis was highly polymorphic and L. naiffi showed intraspecific distances comparable to the largest obtained within all Viannia. L. colombiensis, L. equatorensis and L. lainsoni clearly represent distinct lineages. Good agreement was obtained with molecular trees based upon isoenzyme or mini-exon repeat sequence comparisons. Overall, IRT appears to be a superb method for epidemiological and taxonomic studies of Leishmania, being sensitive, rapid and quantitative while simultaneously revealing considerable molecular diversity. IRT could also be applied to other nonconserved intergenic regions, including those separating protein-coding genes.

Animals↗
PubMed1995

Discrimination of Leishmania isolates using a limited set of enzymatic loci.

The characterization of more than 200 Leishmania isolates, using 18 enzymatic loci, indicated that only a limited number of enzymes produces diagnostic alleles which are potentially useful in the identification of species or zymodemes. 6-Phospho-gluconate dehydrogenase was the most polymorphic enzyme, containing enzyme, containing the majority of diagnostic alleles, and could be used to separate strains of the subgenus Leishmania from those of Viannia. It appears that just a few enzymatic loci, chosen a priori, are all that are needed in many taxonomic and epidemiological studies of Leishmania.

Alleles↗