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...
Published in: | Journal of Microbiological Methods |
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ftunivrosario:oai:repository.urosario.edu.co:10336/27183 2023-05-15T18:05:43+02:00 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 2013-02-15 application/pdf https://repository.urosario.edu.co/handle/10336/27183 https://doi.org/10.1016/j.mimet.2012.11.002 eng eng Elsevier ISSN: 0167-7012 EISSN: 1872-8359 https://repository.urosario.edu.co/handle/10336/27183 https://doi.org/10.1016/j.mimet.2012.11.002 info:eu-repo/semantics/restrictedAccess instname:Universidad del Rosario Multiclonality Chagas disease Trypanosoma cruzi Aneuploidy Infrapopulation Superinfection info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 2013 ftunivrosario https://doi.org/10.1016/j.mimet.2012.11.002 2022-08-01T07:02:32Z 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. Article in Journal/Newspaper Rattus rattus Universidad del Rosario, Bogotá: E-docUR Journal of Microbiological Methods 92 2 220 225 |
institution |
Open Polar |
collection |
Universidad del Rosario, Bogotá: E-docUR |
op_collection_id |
ftunivrosario |
language |
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 |
topic_facet |
Multiclonality Chagas disease Trypanosoma cruzi Aneuploidy Infrapopulation Superinfection |
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 |
Article in Journal/Newspaper |
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 |
genre |
Rattus rattus |
genre_facet |
Rattus rattus |
op_source |
instname:Universidad del Rosario |
op_relation |
ISSN: 0167-7012 EISSN: 1872-8359 https://repository.urosario.edu.co/handle/10336/27183 https://doi.org/10.1016/j.mimet.2012.11.002 |
op_rights |
info:eu-repo/semantics/restrictedAccess |
op_doi |
https://doi.org/10.1016/j.mimet.2012.11.002 |
container_title |
Journal of Microbiological Methods |
container_volume |
92 |
container_issue |
2 |
container_start_page |
220 |
op_container_end_page |
225 |
_version_ |
1766177213733928960 |