Artículo

Schijman, A.G.; Bisio, M.; Orellana, L.; Sued, M.; Duffy, T.; Mejia Jaramillo, A.M.; Cura, C.; Auter, F.; Veron, V.; Qvarnstrom, Y.; Deborggraeve, S.; Hijar, G.; Zulantay, I.; Lucero, R.H.; Velazquez, E.; Tellez, T.; Leon, Z.S.; Galvão, L. (...) Ladzins, J. "International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients" (2011) PLoS Neglected Tropical Diseases. 5(1)
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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 5.10-3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/μl 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/μl 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.

Registro:

Documento: Artículo
Título:International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients
Autor:Schijman, A.G.; Bisio, M.; Orellana, L.; Sued, M.; Duffy, T.; Mejia Jaramillo, A.M.; Cura, C.; Auter, F.; Veron, V.; Qvarnstrom, Y.; Deborggraeve, S.; Hijar, G.; Zulantay, I.; Lucero, R.H.; Velazquez, E.; Tellez, T.; Leon, Z.S.; Galvão, L.; Nolder, D.; Rumi, M.M.; Levi, J.E.; Ramirez, J.D.; Zorrilla, P.; Flores, M.; Jercic, M.I.; Crisante, G.; Añez, N.; de Castro, A.M.; Gonzalez, C.I.; Viana, K.A.; Yachelini, P.; Torrico, F.; Robello, C.; Diosque, P.; Chavez, O.T.; Aznar, C.; Russomando, G.; Büscher, P.; Assal, A.; Guhl, F.; Estani, S.S.; DaSilva, A.; Britto, C.; Luquetti, A.; Ladzins, J.
Filiación:Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh), Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Buenos Aires, Argentina
Instituto de Cálculo, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
Grupo Chagas, Universidad de Antioquia, Medellín, Colombia
French Blood Services, La Plaine Saint Denis, Paris, France
Laboratorio Hospitalario, Universidad de Parasitología, Cayene, French Guiana
Department of Parasitic Diseases, Centers for Disease Control, Atlanta, GA, United States
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 Chabén, Buenos Aires, Argentina
Centro Universitario de Medicina Tropical, Facultad de Medicina, Universidad Mayor de San Simon, Cochabamba, Bolivia
Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asunción, Asunción del Paraguay, Paraguay
Faculdade de Farmácia, Petrópolis, Natal, Rio Grande do Norte, Brazil
London School of Tropical Medicine, Hygiene Department of Clinical Parasitology, Hospital for Tropical Diseases, London, United Kingdom
Laboratorio de Patología Experimental, Universidad Nacional de Salta, Salta, Argentina
Blood Bank, Hospital Sirio Libanês, São Paulo, Brazil
Centro de Investigaciones en Microbiología y Parasitología Tropical, Universidad de los Andes, Bogotá, Colombia
Instituto Pasteur, Montevideo, Uruguay
Centro de Mahahonda, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Madrid, Spain
Sección Parasitología, Instituto Nacional De Salud, Santiago de Chile, Chile
Centro de Investigaciones Parasitológicas, J.F. Torrealba, Universidad de los Andes, Mérida, Venezuela
Instituto de Patologia Tropical e Saúde Pública (IPTSP), Universidade Federal de Goiás, Goiânia, Brazil
Grupo de Inmunología y Epidemiología Molecular (GIEM), Facultad de Salud, Universidad Industrial de Santander, Bucaramanga, Colombia
Departamento de Biomedicina de Enfermedades Infecciosas y Parasitarias Lab. de Biologia Celular, Centro de Investigaciones Regionales (CIR), Dr Hideyo Noguchi, Universidad Autónoma de Yucatán, Yucatán, Mexico
Instituto de Biomedicina, Universidad Católica de Santiago del Estero, Santiago del Estero, Argentina
Centro Nacional de Diagnostico e Investigacion de Endemoepidemias (CeNDIE) ANLIS, Buenos Aires, Argentina
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, Brazil
Laboratório de Pesquisa de Doença de Chagas, Goiãnia, Brazil
Special Programme for Research and Training in Tropical Diseases (TDR), World Health Organization (WHO), Geneve, Switzerland
Palabras clave:protozoal DNA; protozoal DNA; analytic method; article; blood analysis; Chagas disease; DNA determination; DNA extraction; DNA purification; gene amplification; gene dosage; human; methodology; nonhuman; parasite identification; polymerase chain reaction; real time polymerase chain reaction; sensitivity and specificity; serodiagnosis; single nucleotide polymorphism; Trypanosoma cruzi; validation process; blood; Chagas disease; clinical trial; comparative study; evaluation; genetics; international cooperation; isolation and purification; multicenter study; parasitology; Chagas Disease; DNA, Protozoan; Humans; International Cooperation; Parasitology; Polymerase Chain Reaction; Sensitivity and Specificity; Trypanosoma cruzi
Año:2011
Volumen:5
Número:1
DOI: http://dx.doi.org/10.1371/journal.pntd.0000931
Título revista:PLoS Neglected Tropical Diseases
Título revista abreviado:PLoS. Negl. Trop. Dis.
CAS:DNA, Protozoan
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_NIS27444_v5_n1_p_Schijman

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Citas:

---------- APA ----------
Schijman, A.G., Bisio, M., Orellana, L., Sued, M., Duffy, T., Mejia Jaramillo, A.M., Cura, C.,..., Ladzins, J. (2011) . International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Neglected Tropical Diseases, 5(1).
http://dx.doi.org/10.1371/journal.pntd.0000931
---------- CHICAGO ----------
Schijman, A.G., Bisio, M., Orellana, L., Sued, M., Duffy, T., Mejia Jaramillo, A.M., et al. "International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients" . PLoS Neglected Tropical Diseases 5, no. 1 (2011).
http://dx.doi.org/10.1371/journal.pntd.0000931
---------- MLA ----------
Schijman, A.G., Bisio, M., Orellana, L., Sued, M., Duffy, T., Mejia Jaramillo, A.M., et al. "International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients" . PLoS Neglected Tropical Diseases, vol. 5, no. 1, 2011.
http://dx.doi.org/10.1371/journal.pntd.0000931
---------- VANCOUVER ----------
Schijman, A.G., Bisio, M., Orellana, L., Sued, M., Duffy, T., Mejia Jaramillo, A.M., 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. 2011;5(1).
http://dx.doi.org/10.1371/journal.pntd.0000931