RESUMO
American Tegumentary Leishmaniasis is caused by parasites of the genus Leishmania, and causes significant health problems throughout the Americas. In Panama, Leishmania parasites are endemic, causing thousands of new cases every year, mostly of the cutaneous form. In the last years, the burden of the disease has increased, coincident with increasing disturbances in its natural sylvatic environments. The study of genetic variation in parasites is important for a better understanding of the biology, population genetics, and ultimately the evolution and epidemiology of these organisms. Very few attempts have been made to characterize genetic polymorphisms of parasites isolated from Panamanian patients of cutaneous leishmaniasis. Here we present data on the genetic variability of local isolates of Leishmania, as well as specimens from several other species, by means of Amplified Fragment Length Polymorphisms (AFLP), a technique seldom used to study genetic makeup of parasites. We demonstrate that this technique allows detection of very high levels of genetic variability in local isolates of Leishmania panamensis in a highly reproducible manner. The analysis of AFLP fingerprints generated by unique selective primer combinations in L. panamensis suggests a predominant clonal mode of reproduction. Using fluorescently labeled primers, many taxon-specific fragments were identified which may show potential as species diagnostic fragments. The AFLP permitted a high resolution genetic analysis of the Leishmania genus, clearly separating certain groups among L. panamensis specimens and highly related species such as L. panamensis and L. guyanensis. The phylogenetic networks reconstructed from our AFLP data are congruent with established taxonomy for the genus Leishmania, even when using single selective primer combinations. Results of this study demonstrate that AFLP polymorphisms can be informative for genetic characterization in Leishmania parasites, at both intra and inter-specific levels.
Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Leishmania/genética , Leishmaniose Cutânea/epidemiologia , Evolução Molecular , Humanos , Leishmania/classificação , Tipagem de Sequências Multilocus , Doenças Negligenciadas/epidemiologia , Panamá/epidemiologia , FilogeniaRESUMO
Previous kDNA polymorphism-based reports have revealed the existence of two Trypanosoma rangeli genotypes (KP1+ and KP1-): SL and SSU rRNA gene polymorphism-based studies have revealed that five genotypes (A-E) are distributed throughout different Latin-American countries. Some evidence has shown that the genotypes' biogeographical distribution is associated with sympatric Rhodnius species. 12 T. rangeli isolates from humans and reservoirs from El Salvador, Guatemala, Honduras, Costa Rica and Panama were characterised by kDNA and mini-exon gene intergene spacer analysis and compared to 12 previously characterised isolates from humans and vectors from Colombia, Guatemala, Honduras and Venezuela. Central American isolates corresponded to genotypes called KP1(+) or lineage A and KP1(-) or lineage C. Such dimorphism was corroborated by randomly amplified polymorphic DNA (RAPD) in 22 selected isolates; a dendrogram was thus produced having two defined branches. One branch grouped KP1(-) or lineage C strains isolated from Rhodnius colombiensis (Colombia), humans (Panama), Procyon lotor and Choloepus hoffmanni (Costa Rica). The other group was formed by KP1(+) or lineage A strains isolated from Rhodnius prolixus (Colombia, Venezuela) and humans (El Salvador, Guatemala, Honduras). These results present evidence that both groups infect different mammals (humans, domestic and silvatic animals) having no association with any particular vertebrate species; however, T. rangeli KP1(+) or (A) strains have been isolated in Central America in areas where R. prolixus circulate (Honduras, El Salvador and Guatemala) and KP1(-) or (C) strains have been isolated in areas where Rhodnius pallescens is the main vector (Panama and Costa Rica) indicating a parasite-vector association. The same lineages circulate in Andean countries (Colombia, Venezuela, Ecuador and Peru), KP1+ being associated with members of the prolixus group (R. prolixus and Rhodnius robustus) and KP1- with members of the pallescens group (R. pallescens, R. colombiensis and Rhodnius ecuadoriensis).
Assuntos
Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , Rhodnius/parasitologia , Trypanosoma/genética , Animais , América Central/epidemiologia , Doença de Chagas/transmissão , DNA de Cinetoplasto/análise , DNA de Cinetoplasto/genética , Evolução Molecular , Éxons , Variação Genética , Genoma de Protozoário , Interações Hospedeiro-Parasita , Humanos , Insetos Vetores/parasitologia , Filogenia , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
BACKGROUND: Among Chagas disease triatomine vectors, the largest genus, Triatoma, includes species of high public health interest. Triatoma dimidiata, the main vector throughout Central America and up to Ecuador, presents extensive phenotypic, genotypic, and behavioral diversity in sylvatic, peridomestic and domestic habitats, and non-domiciliated populations acting as reinfestation sources. DNA sequence analyses, phylogenetic reconstruction methods, and genetic variation approaches are combined to investigate the haplotype profiling, genetic polymorphism, phylogeography, and evolutionary trends of T. dimidiata and its closest relatives within Triatoma. This is the largest interpopulational analysis performed on a triatomine species so far. METHODOLOGY AND FINDINGS: Triatomines from Mexico, Guatemala, Honduras, Nicaragua, Panama, Cuba, Colombia, Ecuador, and Brazil were used. Triatoma dimidiata populations follow different evolutionary divergences in which geographical isolation appears to have had an important influence. A southern Mexican-northern Guatemalan ancestral form gave rise to two main clades. One clade remained confined to the Yucatan peninsula and northern parts of Chiapas State, Guatemala, and Honduras, with extant descendants deserving specific status. Within the second clade, extant subspecies diversity was shaped by adaptive radiation derived from Guatemalan ancestral populations. Central American populations correspond to subspecies T. d. dimidiata. A southern spread into Panama and Colombia gave the T. d. capitata forms, and a northwestern spread rising from Guatemala into Mexico gave the T. d. maculipennis forms. Triatoma hegneri appears as a subspecific insular form. CONCLUSIONS: The comparison with very numerous Triatoma species allows us to reach highly supported conclusions not only about T. dimidiata, but also on different, important Triatoma species groupings and their evolution. The very large intraspecific genetic variability found in T. dimidiata sensu lato has never been detected in a triatomine species before. The distinction between the five different taxa furnishes a new frame for future analyses of the different vector transmission capacities and epidemiological characteristics of Chagas disease. Results indicate that T. dimidiata will offer problems for control, although dwelling insecticide spraying might be successful against introduced populations in Ecuador.
Assuntos
Doença de Chagas/transmissão , Variação Genética/genética , Insetos Vetores/genética , Filogenia , Triatoma/classificação , Triatoma/genética , Animais , América Central , DNA Ribossômico/genética , Haplótipos , Insetos Vetores/classificação , Dados de Sequência MolecularRESUMO
Mutations in the 3' untranslated region of calmodulin gene have recently been reported to be specific to different Trypanosoma cruzi lineages. In the present report, this molecular marker was used to genotype 24 T. cruzi isolates from humans and vectors from different endemic areas in Panama. The finding of solely T. cruzi I genotype might explain the low morbidity of Chagas' disease in the region when compared to other countries in Latin America.
Assuntos
Regiões 3' não Traduzidas/genética , Calmodulina/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Animais , Sequência de Bases , Sequência Conservada , Genótipo , Panamá , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
Five commercially available enzyme-linked immunosorbent assays (ELISAs), one in-house ELISA, and two hemagglutination assays were evaluated to determine their diagnostic accuracy for Chagas' disease in two studies. In study 1, ELISA kits showed 100% sensitivity, but specificities ranged from 82.84% to 100% when leishmaniasis cases were included and from 95.57% to 100% when leishmaniasis cases were excluded. Kits using recombinant antigens or synthetic peptides are more specific than those using crude extracts from Trypanosoma cruzi epimastigote forms. Kits evaluated in Panama, in study 2, showed 75% to 100% sensitivity and 97.12% to 100% specificity. These data were obtained by using a Western blot assay with T. cruzi trypomastigote excreted-secreted antigens as a reference test to confirm T. cruzi infection.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doença de Chagas/diagnóstico , Leishmania/imunologia , Kit de Reagentes para Diagnóstico , Trypanosoma cruzi/imunologia , Trypanosoma/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Trypanosoma/classificaçãoRESUMO
The present work provides information on Trypanosoma cruzi genotype circulating in endemic areas of Chagas disease in Panama. A total of 26 crude stocks of T. cruzi, isolated from the blood of persons with different clinical profiles of Chagas disease were collected and crio-conserved until used. Most of the stocks had been characterized by means of isoenzyme electrophoresis on cellulose acetate membranes. The clinical profiles of infected persons included 9 (34.6%) asymptomatic and 17 acute (65.4%) including 5 (19.2%) fatal cases, 2 under 5 years old and 3 adults. A multiplex-PCR assay based on the amplification of the non-transcribed spacer of the mini-exon gene was performed. All stocks of T. cruzi included in the study were found to correspond to Tc I group. This result supports the predominance of T. cruzi-I in the transmission cycles affecting the human population in the Republic of Panama.
Assuntos
Doença de Chagas/parasitologia , Doenças Endêmicas , Trypanosoma cruzi/genética , Doença Aguda , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Eletroforese em Acetato de Celulose , Éxons/genética , Genes de Protozoários/genética , Humanos , Lactente , Isoenzimas/análise , Pessoa de Meia-Idade , Panamá , Reação em Cadeia da Polimerase , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
The present work provides information on Trypanosoma cruzi genotype circulating in endemic areas of Chagas disease in Panama. A total of 26 crude stocks of T. cruzi, isolated from the blood of persons with different clinical profiles of Chagas disease were collected and crio-conserved until used. Most of the stocks had been characterized by means of isoenzyme electrophoresis on cellulose acetate membranes. The clinical profiles of infected persons included 9 (34.6 percent) asymptomatic and 17 acute (65.4 percent) including 5 (19.2 percent) fatal cases, 2 under 5 years old and 3 adults. A multiplex-PCR assay based on the amplification of the non-transcribed spacer of the mini-exon gene was performed. All stocks of T. cruzi included in the study were found to correspond to Tc I group. This result supports the predominance of T. cruzi-I in the transmission cycles affecting the human population in the Republic of Panama.
Assuntos
Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Humanos , Lactente , Pessoa de Meia-Idade , Doença de Chagas/parasitologia , Doenças Endêmicas , Trypanosoma cruzi/genética , Doença Aguda , Eletroforese em Acetato de Celulose , Éxons/genética , Genes de Protozoários/genética , Isoenzimas/análise , Panamá , Reação em Cadeia da Polimerase , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
A total of 33 crude and cloned Trypanosoma rangeli stocks found as natural infections in human from Panama and other endemic areas of Central and South America were evaluated as producers of sialidase (SA) activity through the MU-NANA fluorescence test. Negative results were observed in 6 of the isolates: Panama (4), Honduras (1), and Brazil (1). In addition, an immunoblotting analysis confirm the presence of the SA antigen in these stocks without enzymatic activity. These findings must be considered in the interpretation of the biological significance of T. rangeli SA and in the proper characterization and identification of this parasite.
Assuntos
Neuraminidase/biossíntese , Trypanosoma/enzimologia , Animais , Fluorescência , Humanos , Immunoblotting , América Latina , Neuraminidase/imunologia , Trypanosoma/imunologiaRESUMO
A total of 33 crude and cloned Trypanosoma rangeli stocks found as natural infections in human from Panama and other endemic areas of Central and South America were evaluated as producers of sialidase (SA) activity through the MU-NANA fluorescence test. Negative results were observed in 6 of the isolates: Panama (4), Honduras (1), and Brazil (1). In addition, an immunoblotting analysis confirm the presence of the SA antigen in these stocks without enzymatic activity. These findings must be considered in the interpretation of the biological significance of T. rangeli SA and in the proper characterization and identification of this parasite.
Assuntos
Animais , Humanos , Neuraminidase/biossíntese , Trypanosoma/enzimologia , Fluorescência , Immunoblotting , América Latina , Neuraminidase/imunologia , Trypanosoma/imunologiaRESUMO
The Trypanosoma rangeli-secreted sialidase was purified by bovine submaxillary gland mucin-sepharose affinity chromatography. In immunoblotting analysis, antibodies raised against this molecule recognized polypeptides of 73 kDa in T. rangeli medium supernatant (TrSialr) and of 70 kDa in the cell lysates of T. rangeli (TrSials) and T. cruzi (TcSialL) epimastigotes. TrSialr, TrSials, and TcSialL were subjected to proteolytic cleavage with papain; the resultant peptide pattern displayed differences in the immunoblotting profiles. TrSials was purified by immunoprecipitation, and this protein band was recognized by sera from T. cruzi-infected chronic mice and Chagas' disease patients. In contrast, TrSialr was not recognized by these sera. The antibodies from the infected mice also recognized a band of 70 kDa present in the medium. These preliminary observations imply that the released and somatic sialidases are partially different molecules, with probably different biological roles. The related proteins recognized in T. rangeli and T. cruzi epimastigotes share many antigenic characteristics but have some structural differences, probably related to their function in the parasitic cell. On the basis of the strong antigenicity of TrSials, this molecule is proposed as the antigen for the detection of antibodies arising during T. cruzi infection.