title: Optimized multilocus sequence typing (MLST) scheme for Trypanosoma cruzi.
abstract: Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.
authors.affiliation: Unidad de Epidemiología Molecular (UEM), Instituto de Patología Experimental, CONICET- Universidad Nacional de Salta, Salta, Argentina.
authors.forename: Patricio
authors.initials: P
authors.lastname: Diosque
authors.affiliation: Unidad de Epidemiología Molecular (UEM), Instituto de Patología Experimental, CONICET- Universidad Nacional de Salta, Salta, Argentina.
authors.forename: Nicolás
authors.initials: N
authors.lastname: Tomasini
authors.affiliation: Unidad de Epidemiología Molecular (UEM), Instituto de Patología Experimental, CONICET- Universidad Nacional de Salta, Salta, Argentina.
authors.forename: Juan José
authors.initials: JJ
authors.lastname: Lauthier
authors.affiliation: Faculty of Infectious and Tropical Diseases, Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Louisa Alexandra
authors.initials: LA
authors.lastname: Messenger
authors.affiliation: Unidad de Epidemiología Molecular (UEM), Instituto de Patología Experimental, CONICET- Universidad Nacional de Salta, Salta, Argentina.
authors.forename: María Mercedes
authors.initials: MM
authors.lastname: Monje Rumi
authors.affiliation: Unidad de Epidemiología Molecular (UEM), Instituto de Patología Experimental, CONICET- Universidad Nacional de Salta, Salta, Argentina.
authors.forename: Paula Gabriela
authors.initials: PG
authors.lastname: Ragone
authors.affiliation: Unidad de Epidemiología Molecular (UEM), Instituto de Patología Experimental, CONICET- Universidad Nacional de Salta, Salta, Argentina.
authors.forename: Anahí Maitén
authors.initials: AM
authors.lastname: Alberti-D'Amato
authors.affiliation: Unidad de Epidemiología Molecular (UEM), Instituto de Patología Experimental, CONICET- Universidad Nacional de Salta, Salta, Argentina.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Pérez Brandán
authors.affiliation: Maladies Infectieuses et Vecteurs Ecologie, Génétique, Evolution et Contrôle, MIVEGEC (IRD 224-CNRS 5290-UM1-UM2), IRD Center, Montpellier, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Barnabé
authors.affiliation: Maladies Infectieuses et Vecteurs Ecologie, Génétique, Evolution et Contrôle, MIVEGEC (IRD 224-CNRS 5290-UM1-UM2), IRD Center, Montpellier, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Tibayrenc
authors.affiliation: Faculty of Infectious and Tropical Diseases, Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Michael David
authors.initials: MD
authors.lastname: Lewis
authors.affiliation: Faculty of Infectious and Tropical Diseases, Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Martin Stephen
authors.initials: MS
authors.lastname: Llewellyn
authors.affiliation: Faculty of Infectious and Tropical Diseases, Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Michael Alexander
authors.initials: MA
authors.lastname: Miles
authors.affiliation: Faculty of Infectious and Tropical Diseases, Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Matthew
authors.initials: M
authors.lastname: Yeo
mesh_terms: D005838:Genotype; D058885:Multilocus Sequence Typing / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D014349:Trypanosoma cruzi / Q000235:genetics*