Validation of a poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations

Trypanosoma cruzi is the causative agent of American trypanosomiasis, a complex zoonotic disease that affects more than 10 million people in the Americas. Strains of this parasite possess a significant amount of genetic variability and hence can be divided into at least six discrete typing units (DT...

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Autores Principales: Ramírez, Juan David, Herrera, Claudia, Bogotá, Yizeth, Duque, María Clara, Suárez-Rivillas, Alejandro, Guhl, Felipe
Formato: Artículo (Article)
Lenguaje:Inglés (English)
Publicado: Elsevier 2013
Materias:
Acceso en línea:https://repository.urosario.edu.co/handle/10336/27183
https://doi.org/10.1016/j.mimet.2012.11.002
id ir-10336-27183
recordtype dspace
spelling ir-10336-271832022-05-02T12:37:16Z Validation of a poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations Validación de un ensayo de dilución limitante (LDA) distribuido por Poisson para una resolución rápida y precisa de infecciones multiclonales en poblaciones naturales de Trypanosoma cruzi Ramírez, Juan David Herrera, Claudia Bogotá, Yizeth Duque, María Clara Suárez-Rivillas, Alejandro Guhl, Felipe Multiclonality Chagas disease Trypanosoma cruzi Aneuploidy Infrapopulation Superinfection Trypanosoma cruzi is the causative agent of American trypanosomiasis, a complex zoonotic disease that affects more than 10 million people in the Americas. Strains of this parasite possess a significant amount of genetic variability and hence can be divided into at least six discrete typing units (DTUs). The life cycle of this protist suggests that multiclonal infections may emerge due to the likelihood of contact of triatomine insects with more than 100 mammal species. To date, there have been a few studies on but no consensus regarding standardised methodologies to identify multiclonal infections caused by this parasite. Hence, the aim of this study was to develop and validate a limiting dilution assay (LDA) to identify multiclonal infections in T. cruzi populations by comparing the feasibility and reliability of this method with the widely applied solid phase blood agar (SPBA) methodology. We cloned reference strains belonging to three independent genotypes (TcI, TcII, and TcIV) and mixed infections (TcI + TcII) using LDA and SPBA; the comparison was conducted by calculating the feasibility and reliability of the methods employed. Additionally, we implemented LDA in strains recently isolated from Homo sapiens, Rhodnius prolixus, Triatoma venosa, Panstrongylus geniculatus, Tamandua tetradactyla, Rattus rattus, Didelphis marsupialis and Dasypus novemcinctus, with the aim of resolving multiclonal infections using molecular characterization employing SL-IR (spliced leader intergenic region of mini-exon gene), the 24S? rDNA gene and microsatellite loci. The results reported herein demonstrate that LDA is an optimal methodology to distinguish T. cruzi subpopulations based on microsatellite markers by showing the absence of multiple peaks within a single locus. Conversely, SPBA showed patterns of multiple peaks within a single locus suggesting multiclonal events. The biological consequences of these results and the debate between multiclonality and aneuploidy are discussed. 2013-02-15 2020-08-19T14:41:16Z info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion ISSN: 0167-7012 EISSN: 1872-8359 https://repository.urosario.edu.co/handle/10336/27183 https://doi.org/10.1016/j.mimet.2012.11.002 eng info:eu-repo/semantics/restrictedAccess application/pdf Elsevier instname:Universidad del Rosario
institution EdocUR - Universidad del Rosario
collection DSpace
language Inglés (English)
topic Multiclonality
Chagas disease
Trypanosoma cruzi
Aneuploidy
Infrapopulation
Superinfection
spellingShingle Multiclonality
Chagas disease
Trypanosoma cruzi
Aneuploidy
Infrapopulation
Superinfection
Ramírez, Juan David
Herrera, Claudia
Bogotá, Yizeth
Duque, María Clara
Suárez-Rivillas, Alejandro
Guhl, Felipe
Validation of a poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations
description Trypanosoma cruzi is the causative agent of American trypanosomiasis, a complex zoonotic disease that affects more than 10 million people in the Americas. Strains of this parasite possess a significant amount of genetic variability and hence can be divided into at least six discrete typing units (DTUs). The life cycle of this protist suggests that multiclonal infections may emerge due to the likelihood of contact of triatomine insects with more than 100 mammal species. To date, there have been a few studies on but no consensus regarding standardised methodologies to identify multiclonal infections caused by this parasite. Hence, the aim of this study was to develop and validate a limiting dilution assay (LDA) to identify multiclonal infections in T. cruzi populations by comparing the feasibility and reliability of this method with the widely applied solid phase blood agar (SPBA) methodology. We cloned reference strains belonging to three independent genotypes (TcI, TcII, and TcIV) and mixed infections (TcI + TcII) using LDA and SPBA; the comparison was conducted by calculating the feasibility and reliability of the methods employed. Additionally, we implemented LDA in strains recently isolated from Homo sapiens, Rhodnius prolixus, Triatoma venosa, Panstrongylus geniculatus, Tamandua tetradactyla, Rattus rattus, Didelphis marsupialis and Dasypus novemcinctus, with the aim of resolving multiclonal infections using molecular characterization employing SL-IR (spliced leader intergenic region of mini-exon gene), the 24S? rDNA gene and microsatellite loci. The results reported herein demonstrate that LDA is an optimal methodology to distinguish T. cruzi subpopulations based on microsatellite markers by showing the absence of multiple peaks within a single locus. Conversely, SPBA showed patterns of multiple peaks within a single locus suggesting multiclonal events. The biological consequences of these results and the debate between multiclonality and aneuploidy are discussed.
format Artículo (Article)
author Ramírez, Juan David
Herrera, Claudia
Bogotá, Yizeth
Duque, María Clara
Suárez-Rivillas, Alejandro
Guhl, Felipe
author_facet Ramírez, Juan David
Herrera, Claudia
Bogotá, Yizeth
Duque, María Clara
Suárez-Rivillas, Alejandro
Guhl, Felipe
author_sort Ramírez, Juan David
title Validation of a poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations
title_short Validation of a poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations
title_full Validation of a poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations
title_fullStr Validation of a poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations
title_full_unstemmed Validation of a poisson-distributed limiting dilution assay (LDA) for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations
title_sort validation of a poisson-distributed limiting dilution assay (lda) for a rapid and accurate resolution of multiclonal infections in natural trypanosoma cruzi populations
publisher Elsevier
publishDate 2013
url https://repository.urosario.edu.co/handle/10336/27183
https://doi.org/10.1016/j.mimet.2012.11.002
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