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International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.

Alejandro G Schijman 1, * Margarita Bisio 1 Liliana Orellana 2 Mariela Sued 2 Tomás Duffy 1 Ana M Mejia Jaramillo 3 Carolina Cura 1 Frederic Auter 4 Vincent Veron 5 yvonne Qvarnstrom 6 Stijn Deborggraeve 7 Gisely Hijar 8 Inés Zulantay 9 Raúl Horacio Lucero 10 Elsa Velazquez 11 Tatiana Tellez 12 Zunilda Sanchez Leon 13 Lucia Galvão 14 Debbie Nolder 15 María Monje Rumi 16 José E Levi 17 Juan D Ramirez 18 Pilar Zorrilla 19 María Flores 20 Maria I Jercic 21 Gladys Crisante 22 Néstor Añez 22 Ana M de Castro 23 Clara I Gonzalez 24 Karla Acosta Viana 25 Pedro yachelini 26 Faustino Torrico 12 Carlos Robello 19 Patricio Diosque 16 Omar Triana Chavez 3 Christine Aznar 5 Graciela Russomando 13 Philippe Büscher 7 Azzedine Assal 4 Felipe Guhl 18 Sergio Sosa Estani 27 Alexandre Dasilva 6 Constança Britto 28 Alejandro Luquetti 29 Janis Ladzins 30 
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.
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Submitted on : Wednesday, April 11, 2012 - 4:57:27 PM
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Alejandro G Schijman, Margarita Bisio, Liliana Orellana, Mariela Sued, Tomás Duffy, 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, Public Library of Science, 2011, 5 (1), pp.e931. ⟨10.1371/journal.pntd.0000931⟩. ⟨pasteur-00685052⟩



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