Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/16262
Title: International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients
Authors: Schijman, Alejandro G.
Bisio, Margarita
Orellana, Liliana
Sued, Mariela
Duffy, Tomás
Mejia Jaramillo, Ana M.
Cura, Carolina
Auter, Frederic
Veron, Vincent
Qvarnstrom, Yvonne
Deborggraeve, Stijn
Hijar, Gisely
Zulantay, Inés
Lucero, Raúl Horacio
Velazquez, Elsa
Tellez, Tatiana
Sanchez Leon, Zunilda
Galvão, Lucia
Nolder, Debbie
Monje Rumi, María
Levi, José E.
Ramirez, Juan D.
Zorrilla, Pilar
Flores, María
Jercic, Maria I.
Crisante, Gladys
Añez, Néstor
Castro, Ana M. de
Gonzalez, Clara I.
Acosta Viana, Karla
Yachelini, Pedro
Torrico, Faustino
Robello, Carlos
Diosque, Patricio
Triana Chavez, Omar
Aznar, Christine
Russomando, Graciela
Büscher, Philippe
Assal, Azzedine
Guhl, Felipe
Sosa Estani, Sergio
Silva, Alexandre da
Britto, Constança
Luquetti, Alejandro
Ladzins, Janis
Affilliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh). Buenos Aires, Argentina.
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh). Buenos Aires, Argentina.
Universidad de Buenos Aires (UBA). Instituto de Cálculo. Buenos Aires, Argentina.
Universidad de Buenos Aires (UBA). Instituto de Cálculo. Buenos Aires, Argentina.
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh). Buenos Aires, Argentina.
Universidad de Antioquia. Grupo Chagas. Medellin, Colombia.
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh). Buenos Aires, Argentina.
French Blood Services. La Plaine Saint Denis, Paris, France.
Universidad de Parasitología. Laboratorio Hospitalario. Cayene, French Guiana.
Centers for Disease Control. Department of Parasitic Diseases. Atlanta, Georgia, USA.
Institute of Tropical Medicine. Antwerp, Belgium.
Instituto Nacional de Salud. Lima, Peru.
Facultad de Medicina. Santiago de Chile, Chile.
Universidad Nacional del Nordeste. Chaco, Argentina.
Instituto Nacional de Chagas. Fatala Chabénn, Buenos Aires, Argentina.
Universidad Mayor de San Simon. Facultad de Medicina. Centro Universitario de Medicina Tropical. Cochabamba, Bolivia.
Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud. Asunción del Paraguay, Paraguay.
Faculdade de Farmácia. Natal, RN, Brasil.
Hospital for Tropical Diseases. Department of Clinical Parasitology. London School of Tropical Medicine and Hygiene. London, United Kingdom.
Universidad Nacional de Salta. Laboratorio de Patología Experimental. Salta, Argentina.
Hospital Sirio Libanês. Banco de Sangue. São Paulo, SP, Brasil.
Universidad de los Andes. Centro de Investigaciones en Microbiología y Parasitología Tropical. Bogotá, Colombia.
Instituto Pasteur. Montevideo, Uruguay.
Instituto de Salud Carlos III. Centro Nacional de Microbiologia. Centro de Mahahonda. Madrid, España.
Instituto Nacional De Salud. Sección Parasitología. Santiago de Chile, Chile.
Universidad de los Andes. Centro de Investigaciones Parasitológicas ‘‘J.F. Torrealba’’. Mérida, Venezuela.
Universidad de los Andes. Centro de Investigaciones Parasitológicas ‘‘J.F. Torrealba’’. Mérida, Venezuela.
Universidade Federal de Goiás. Instituto de Patologia Tropical e Saúde Pública (IPTSP). Goiânia, GO, Brasil.
Universidad Industrial de Santander. Facultad de Salud. Grupo de Inmunologı´a y Epidemiologı´a Molecular (GIEM). Bucaramanaga, Colombia.
Universidad Autónoma de Yucatán. Centro de Investigaciones Regionales (CIR) ‘‘Dr Hideyo Noguchi’. Laboratorio de Biología Celular. Departamento de Biomedicina de Enfermedades Infecciosas y Parasitarias. Yucatán, México.
Universidad Católica de Santiago del Estero. Instituto de Biomedicina. Santiago del Estero, Argentina.
Universidad Mayor de San Simon. Facultad de Medicina. Centro Universitario de Medicina Tropical. Cochabamba, Bolivia.
Instituto Pasteur. Montevideo, Uruguay.
Universidad Nacional de Salta. Laboratorio de Patología Experimental. Salta, Argentina.
Universidad de Antioquia. Grupo Chagas. Medellin, Colombia.
Universidad de Parasitología. Laboratorio Hospitalario. Cayene, French Guiana.
Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud. Asunción del Paraguay, Paraguay.
Institute of Tropical Medicine. Antwerp, Belgium.
French Blood Services. La Plaine Saint Denis, Paris, France.
Universidad de los Andes. Centro de Investigaciones en Microbiología y Parasitología Tropical. Bogotá, Colombia.
Centro Nacional de Diagnóstico e Investigación de Endemoepidemias (CeNDIE) ANLIS Dr. Carlos G. Malbrán. Buenos Aires, Argentina.
Centers for Disease Control. Department of Parasitic Diseases. Atlanta, Georgia, USA.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.
Laboratório de Pesquisa de Doença de Chagas. Goiânia, GO, Brasil.
World Health Organization (WHO). Special Programme for Research and Training in Tropical Diseases (TDR). Geneve, Switzerland.
Abstract: Background: A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings: An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/ mL whereas specific kDNA tests detected 5.1023 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/ml of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/ml for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. Conclusion/Significance: This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.
Keywords: Trypanosoma cruzi
Chagas Disease
PCR Methods
human blood samples
keywords: Trypanosoma cruzi
Doença de Chagas
Reação em Cadeia da Polimerase
Métodos
Amostras de sangue humano
Issue Date: 2011
Publisher: Public Library of Science
Citation: SCHIJMAN, Alejandro G. et al. International Study to Evaluate PCR Methods for Detection of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients. PLoS Negl Trop Dis, v. 5, n.1, e931, 13p,Jan. 2011.
DOI: 10.1371/journal.pntd.0000931
ISSN: 1935-2727
Copyright: open access
Appears in Collections:IOC - Artigos de Periódicos

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