title: Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.
abstract: BACKGROUND
  Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).
  
  
  RESULTS
  The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.
  
  
  CONCLUSIONS
  Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.
authors.affiliation: Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"-INGEBI-CONICET, Buenos Aires, Argentina.
authors.forename: Carolina I
authors.initials: CI
authors.lastname: Cura
authors.affiliation: Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"-INGEBI-CONICET, Buenos Aires, Argentina.
authors.forename: Tomas
authors.initials: T
authors.lastname: Duffy
authors.affiliation: Instituto de Medicina Regional, Universidad Nacional del Nordeste, Resistencia, Chaco, Argentina.
authors.forename: Raúl H
authors.initials: RH
authors.lastname: Lucero
authors.affiliation: Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"-INGEBI-CONICET, Buenos Aires, Argentina.
authors.forename: Margarita
authors.initials: M
authors.lastname: Bisio
authors.affiliation: Laboratoire Hospitalier et Universitaire-CH Andrée Rosemon, Cayenne, French Guiana, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Péneau
authors.affiliation: Laboratorio Biología Celular, Centro de Investigaciones Regionales "Dr. Hideyo Noguchi", Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico.
authors.forename: Matilde
authors.initials: M
authors.lastname: Jimenez-Coello
authors.affiliation: Servicio de Medicina Interna, Hospital Politécnico LA FE, Valencia, Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: Calabuig
authors.affiliation: Servicio de Microbiología, Hospital Universitario y Politécnico LA FE, Valencia, Spain.
authors.forename: María J
authors.initials: MJ
authors.lastname: Gimenez
authors.affiliation: Laboratorio de Investigación en Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Edward
authors.initials: E
authors.lastname: Valencia Ayala
authors.affiliation: Department of Biology, University of Minnesota Duluth, Duluth, Minnesota, United States of America.
authors.forename: Sonia A
authors.initials: SA
authors.lastname: Kjos
authors.affiliation: Laboratorio de Parasitología, Instituto Nacional de Laboratorios en Salud, Ministerio de Salud y Deportes de Bolivia, La Paz, Bolivia.
authors.forename: José
authors.initials: J
authors.lastname: Santalla
authors.affiliation: Southwest National Primate Research Center and Department of Genetics, Texas Biomedical Research Institute, San Antonio, Texas, United States of America.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Mahaney
authors.affiliation: Instituto de Biología de la Altura, Universidad Nacional de Jujuy, Jujuy, Argentina.
authors.forename: Nelly M
authors.initials: NM
authors.lastname: Cayo
authors.affiliation: Epidemiología e Infectología Clínica, Hospital Universitario Fundación Favaloro, Buenos Aires, Argentina.
authors.forename: Claudia
authors.initials: C
authors.lastname: Nagel
authors.affiliation: Sección Infectología, Servicio de Clínica Médica, Hospital Italiano, Buenos Aires, Argentina.
authors.forename: Laura
authors.initials: L
authors.lastname: Barcán
authors.affiliation: Laboratorio de Investigación en Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Edith S
authors.initials: ES
authors.lastname: Málaga Machaca
authors.affiliation: Laboratorio Biología Celular, Centro de Investigaciones Regionales "Dr. Hideyo Noguchi", Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico.
authors.forename: Karla Y
authors.initials: KY
authors.lastname: Acosta Viana
authors.affiliation: Institut de Recherche pour le Développement and University Paris Descartes, UMR 216, Mother and Child Facing Tropical Diseases, Paris, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Brutus
authors.affiliation: Instituto de Biología de la Altura, Universidad Nacional de Jujuy, Jujuy, Argentina.
authors.forename: Susana B
authors.initials: SB
authors.lastname: Ocampo
authors.affiliation: Laboratoire Hospitalier et Universitaire-CH Andrée Rosemon, Cayenne, French Guiana, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Aznar
authors.affiliation: Parasitologia Médica e Biologia de Vetores, Área de Patologia, Faculdade de Medicina, Universidade de Brasilia, Brasilia DF, Brazil.
authors.forename: Cesar A
authors.initials: CA
authors.lastname: Cuba Cuba
authors.affiliation: Laboratorio de Eco-Epidemiología, Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Ricardo E
authors.initials: RE
authors.lastname: Gürtler
authors.affiliation: Centro Regional de Investigación en Salud Pública, Instituto Nacional de Salud Pública, Tapachula, Chiapas, Mexico.
authors.forename: Janine M
authors.initials: JM
authors.lastname: Ramsey
authors.affiliation: Drugs and Neglected Diseases Initiative, Genève, Switzerland.
authors.forename: Isabela
authors.initials: I
authors.lastname: Ribeiro
authors.affiliation: Southwest National Primate Research Center and Department of Genetics, Texas Biomedical Research Institute, San Antonio, Texas, United States of America.
authors.forename: John L
authors.initials: JL
authors.lastname: VandeBerg
authors.affiliation: Pan American Health Organization (PAHO), World Health Organization (WHO), Washington, D.C., United States of America.
authors.forename: Zaida E
authors.initials: ZE
authors.lastname: Yadon
authors.affiliation: Institute of Biotechnology, Molecular Parasitology Group, University of Granada, Granada, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Osuna
authors.affiliation: Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"-INGEBI-CONICET, Buenos Aires, Argentina.
authors.forename: Alejandro G
authors.initials: AG
authors.lastname: Schijman
mesh_terms: D000293:Adolescent; D000328:Adult; D001681:Biological Assay / Q000379:methods; D014355:Chagas Disease / Q000175:diagnosis* / Q000235:genetics / Q000469:parasitology; D002648:Child; D002675:Child, Preschool; D060085:Coinfection; D005260:Female; D014644:Genetic Variation / Q000235:genetics; D005838:Genotype; D006801:Humans; D008297:Male; D058889:Molecular Typing / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000145:classification* / Q000235:genetics*