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Vol. 15. Núm. 4.
Páginas 227-234 (diciembre 2011)
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Vol. 15. Núm. 4.
Páginas 227-234 (diciembre 2011)
Open Access
Evaluación de la variabilidad genética de aislamientos colombianos de Trypanosoma cruzi mediante marcadores microsatélites
Genetic variability evaluation of Trypanosoma cruzi Colombian isolates based on microsatellite DNA markers
Visitas
2430
María Clara Duque1, Juan David Ramírez1, Lina María Rendón1, Felipe Guhl1,
Autor para correspondencia
fguhl@uniandes.edu.co

Correspondencia: Apartado aéreo 4978, Carrera 1 N° 18-A-10, Bogotá, D.C., Colombia Tel.:éfono: (571) 332-4540; fax: (571) 332-4540.
1 Centro de Investigaciones en Microbiología y Parasitología Tropical, Departamento de Ciencias Biológicas, Facultad de Ciencias, Universidad de los Andes, Bogotá, D.C., Colombia
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Información del artículo
Resumen
Introducción

La enfermedad de Chagas, cuyo agente causal es Trypanosoma cruzi, constituye una antropozoonosis ampliamente distribuida en América Latina. Los estudios moleculares y los perfiles genéticos han demostrado que el parásito presenta una gran variabilidad y han permitido la clasificación de T. cruzi en seis unidades discretas de tipificación (I-VI), de las cuales, TcII a TcVI han sido ampliamente caracterizadas por distintos marcadores moleculares.

Objetivo

Evaluar la variabilidad genética de TcI, mediante el uso de marcadores de microsatélites.

Materiales y métodos

Se evaluaron siete microsatélites en quince aislamientos colombianos obtenidos mediante PCR a partir de reservorios, vectores y humanos, y se analizaron en un gel para desnaturalizar de poliacrilamida de un secuenciador láser fluorescente automático (ALF). Los datos se analizaron en un software para análisis de genética de poblaciones (Arlequin® 3.1 y Microsat®).

Resultados

Los resultados demostraron variabilidad dentro de TcI al obtener 24 alelos, de los cuales, 12 se reportan por primera vez. Aunque se encontraron genotipos asociados a la infección humana y al ciclo selvático de transmisión, ningún locus permitió comprobar la presencia de los genotipos previamente reportados. Los parámetros de heterocigocidad observada y esperada, permitieron determinar la presencia de dos poblaciones (aislamientos domésticos y selváticos); asimismo, el desequilibrio de ligamento facilitó la creación de dos mapas físicos para los loci analizados.

Conclusiones

Se corrobora la gran variabilidad genética presente en TcI, lo que sugiere un patrón de variación intraespecífica en Colombia.

Palabras clave:
microsatélites
genotipos
Trypanosoma cruzi
unidades discretas de tipificación
Abstract
Introduction

Chagas disease which is caused by the protozoan Trypanosoma cruzi, is a major public health problem in Latin American countries with a different distribution of the parasite across the continent in which nearly 15 million people are infected and 28 million are at risk. Genetic profiling of T. cruzi has shown great diversity and variability of the parasite allowing its classification into six discrete typing units (DTUs I-VI) in which TcII to TcVI are well characterized by different molecular markers. However, the presence of four subgroups according to the transmission cycle has only been reported in DTU T. cruzi I.

Objective

To evaluate the genetic variability evidenced within TCI by the use of microsatellite markers.

Materials and methods

Seven microsatellite loci were tested in fifteen Colombian isolates from vectors, reservoirs, and humans by means of PCR and automatic laser fluorescent sequencer (ALF). Data were analyzed using a population genetic data analysis software (Arlequin® 3.1 and Microsat®).

Results

Variability among the isolates was demonstrated with 24 alleles, from which twelve had never been reported before. However, none of the microsatellite loci were able to support the idea of genotypes within TcI strains. The parameters of expected and observed heterozygocity allowed us to determine presence of two populations (domestic and sylvatic isolates); likewise, the linkage disequilibrium helped with the construction of two physical maps for the loci analyzed.

Conclusions

We corroborated the high genetic variability displayed by TcI populations suggesting a pattern of intraspecific variation in Colombia.

Keywords:
Discrete typing units
microsatellites
genotypes
Trypanosoma cruzi
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Referencias
[1.]
A. Rassi Jr., A. Rassi, J.A. Marin-Neto.
Chagas disease.
Lancet, 375 (2010), pp. 1388-1402
[2.]
G.A. Schmunis, Z.E. Yadon.
Chagas disease: A Latin American health problem becoming a world health problem.
Acta Tropica, 115 (2010), pp. 14-21
[3.]
OMS.
Reporte sobre la enfermedad de Chagas.
Buenos Aires, (2007),
[4.]
F. Guhl.
Chagas disease in Andean countries.
Mem Inst Oswaldo Cruz, 102 (2007), pp. 29-38
[5.]
S. Brisse, C. Barnabé, M. Tibayrenc.
Identification of six Trypanosoma cruzi phylogenetic lineages by random amplified polymorphic DNA and multilocus enzyme electrophoresis.
Int J Parasitol, 30 (2000), pp. 35-44
[6.]
A. Falla, C. Herrera, A. Fajardo, M. Montilla, G.A. Vallejo, F. Guhl.
Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans.
Acta Tropica, 110 (2009), pp. 15-21
[7.]
M.L. Gomes, A. Macedo, S. Pena, E. Chiari.
Genetic relationships between Trypanosoma cruzi strains isolated from chronic chagasic patients in southern Brazil as revealed by RAPD and SSR-PCR analysis.
Acta Tropica, 69 (1998), pp. 99-109
[8.]
C. Herrera, M.D. Bargues, A. Fajardo, M. Montilla, O. Triana, et al.
Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia. Infection.
Genetics and Evolution, 7 (2007), pp. 535-539
[9.]
M. Rozas, S. De Doncker, X. Coronado, C. Barnabé, M. Tibayrenc, et al.
Evolutionary history of Trypanosoma cruzi according to antigen genes.
Parasitology, 135 (2008), pp. 1157-1164
[10.]
R.P. Souto, O. Fernandes, A. Macedo, D. Campbell, B. Zingales.
DNA markers define two major phylogenetic lineages of Trypanosoma cruzi.
Mol Biochem Parasitol, 83 (1996), pp. 141-152
[11.]
B. Zingales, R.P. Souto, R.H. Mangla, C.V. Lisboa, D.A. Campbell, et al.
Molecular epidemiology of American trypanosomiasis in Brazil based on dimorphisms of rRNA and mini-exon gene sequences.
Int J Parasitol, 28 (1998), pp. 105-112
[12.]
B. Zingales, S.G. Andrade, M. Briones, D.A. Campbell, E. Chiari, et al.
A new consensus for Trypanosoma cruzi intraspecific nomenclature: Second revision meeting recommends TcI to TcVI.
Mem Inst Oswaldo Cruz, 104 (2009), pp. 1051-1054
[13.]
J.M. Burgos, M. Diez, C. Vigilano, M. Bisio, M. Risso, et al.
Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation.
Clin Infect Dis, 51 (2010), pp. 485-495
[14.]
C.I. Cura, A.M. Mejía-Jaramillo, T. Duffy, J.M. Burgos, M. Rodriguero, et al.
Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes.
Int J Parasitol, 40 (2010), pp. 1599-1607
[15.]
F. Guhl, J.D. Ramírez.
Trypanosoma cruzi I diversity: Towards the need of genetic subdivision?.
[16.]
A.M. Mejía-Jaramillo, V.H. Peña, O. Triana-Chávez.
Trypanosoma cruzi: Biological characterization of lineages I and II supports the predominance of lineage I in Colombia.
Exper Parasitol, 121 (2009), pp. 83-91
[17.]
J.D. Ramírez, M.C. Duque, F. Guhl.
Phylogenetic reconstruction based on cytochrome b (Cytb) gene sequences reveals distinct genotypes within Colombian Trypanosoma cruzi I populations.
Acta Tropica, 119 (2011), pp. 61-65
[18.]
G. Zafra, J.C. Mantilla, J. Jácome, A.M. Macedo, C.I. González.
Direct analysis of genetic variability in Trypanosoma cruzi populations from tissues of Colombian chagasic patients.
Human Pathol, 42 (2011), pp. 1159-1168
[19.]
G. Zafra, J.C. Mantilla, C. Jácome, A. Macedo, C.I. González.
Evidence of Trypanosoma cruzi II infection in Colombian chagasic patients.
Parasitol Res, 103 (2008), pp. 731-734
[20.]
J.M. Freitas, E. Lages-Silva, E. Crema, S. Pena, A. Macedo.
Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues.
Int J Parasitol, 35 (2005), pp. 411-417
[21.]
N.R. Sturm, N. Vargas, S. Westenberger, D.A. Campbell, B. Zingales.
Evidence for multiple hybrid groups in Trypanosoma cruzi.
Int J Parasitol, 33 (2003), pp. 269-279
[22.]
M. Yeo, N. Acosta, M. Llewellyn, H. Sánchez, S. Adamson, et al.
Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids.
Int J Parasitol, 35 (2005), pp. 225-233
[23.]
M.S. Llewellyn, M.A. Miles, H. Carrasco, M. Lewis, M. Yeo, et al.
Genome-scale multilocus microsatellite typing of Trypanosoma cruzi discrete typing unit i reveals phylogeographic structure and specific genotypes linked to human infection.
[24.]
A. Salazar, A.G. Schijman, O. Triana-Chávez.
High variability of Colombian Trypanosoma cruzi lineage I stocks as revealed by lowstringency single primer-PCR minicircle signatures.
Acta Tropica, 100 (2006), pp. 110-118
[25.]
A.M. Macedo, C.R. Machado, C.P. Oliveira, S.P. Pena.
Trypanosoma cruzi: Genetic structure of populations and relevance of genetic variability to the pathogenesis of Chagas disease.
Mem Inst Oswaldo Cruz, 99 (2004), pp. 1-12
[26.]
R.P. Oliveira, N. Broude, A. Macedo, C.R. Cantor, C. Smith, et al.
Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites.
Proc Nat Acad Sci USA, 95 (1998), pp. 3776-3780
[27.]
H.M.S. Valadares, J. Ramos Pimenta, J.M. de Freitas, T. Duffy, D. Bartholomeu, et al.
Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites.
Int J Parasitol, 38 (2008), pp. 839-850
[28.]
C. Britto, M.A. Cardoso, D. Wincker, C.M. Morel.
A simple protocol for the physical cleavage of Trypanosoma cruzi kinetoplast DNA present in blood samples and its use in polymerase chain reaction (PCR)-based diagnosis of chronic Chagas disease.
Mem Inst Oswaldo Cruz, 88 (1993), pp. 171-172
[29.]
J.D. Ramirez, F. Guhl, F. Rosas, C. Morillo, F. Marin-Neto, S. Restrepo.
Evaluation of adult chronic Chagas’ heart disease diagnosis by molecular and serological methods.
J Clin Microbiol, 47 (2009), pp. 3945-3951
[30.]
L.G.L. Excoffier, S. Schneider.
Arlequin ver. 3.0: An integrated software package for population genetics data analysis.
Evolutionary Bioinformatics Online, 1 (2005), pp. 47-50
[31.]
E. Minch, A. Ruiz-Linares, D. Goldstein, M. Feldman, L.L. Cavalli-Sforza.
Microsat, version 1. 4d.
A computer program for calculating various statistics on microsatellite allele data, Stanford University Medical Center, (1995),
[32.]
K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0, et al.
Molecular biology and evolution, 24 (2007), pp. 1596-1599
[33.]
D.A. D’Avila, A. Macedo, H.M. Valadares, E. Dias, A.M. de Castro, et al.
Probing population dynamics of Trypanosoma cruzi during progression of the chronic phase in chagasic patients.
J Clin Microbiol, 47 (2009), pp. 1718-1725
[34.]
M.S. Llewellyn, J. Rivett-Carnac, S. Fitzpatrick, M. Lewis, M. Yeo, et al.
Extraordinary Trypanosoma cruzi diversity within single mammalian reservoir hosts implies a mechanism of diversifying selection.
Int J Parasitol, 41 (2011), pp. 609-614
[35.]
A. Marcili, L. Lima, V. Valente, S. Valente, J. Batista, et al.
Trypanosoma cruzi in Brazilian Amazonia: Lineages TCI and TCIIa in wild primates, Rhodnius spp. and in humans with Chagas disease associated with oral transmission.
Int J Parasitol, 39 (2009), pp. 615-623
[36.]
M. Yeo, M. Lewis, M. Carrasco, N. Acosta, M.S. Llewellyn, et al.
Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium.
Intl J Parasitol, 37 (2007), pp. 111-120
[37]
Barnabé, C., De Meeus, T., Francois, N., Bosseno, M.F., Monje, E.M., et al. Trypanosoma cruzi discrete typing units (DTUs): Microsatellite loci and population genetics of DTUs TcV and TcI in Bolivia and Peru. Infection, Genetics and Evolution. In Press, Corrected Proof.
[38.]
J.M. Burgos, J. Althech, N. Petrucelli, M. Bisio, M. Levin, et al.
Molecular diagnosis and treatment monitoring of congenital transmission of Trypanosoma cruzi to twins of a triplet delivery.
Diagn Microbiol Infect Dis, 65 (2009), pp. 58-61
[39.]
J.D. Ramírez, F. Guhl, L.M. Rendón, F. Rosas, C. Morillo, et al.
Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic chagasic patients.
PLoS Negl Trop Dis, 4 (2010), pp. e899
[40.]
N.M. El-Sayed, P. Myler, D. Bartholomeu, D. Nilsson, G. Aggarwal, et al.
The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease.
Science, 309 (2005), pp. 409-415
Copyright © 2011. Asociación Colombiana de Infectología (ACIN)
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