TY - JOUR
T1 - International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients
AU - Schijman, Alejandro G.
AU - Bisio, Margarita
AU - Orellana, Liliana
AU - Sued, Mariela
AU - Duffy, Tomás
AU - Mejia Jaramillo, Ana M.
AU - Cura, Carolina
AU - Auter, Frederic
AU - Veron, Vincent
AU - Qvarnstrom, Yvonne
AU - Deborggraeve, Stijn
AU - Hijar, Gisely
AU - Zulantay, Inés
AU - Lucero, Raúl Horacio
AU - Velazquez, Elsa
AU - Tellez, Tatiana
AU - Leon, Zunilda Sanchez
AU - Galvão, Lucia
AU - Nolder, Debbie
AU - Rumi, María Monje
AU - Levi, José E.
AU - Ramirez, Juan D.
AU - Zorrilla, Pilar
AU - Flores, María
AU - Jercic, Maria I.
AU - Crisante, Gladys
AU - Añez, Néstor
AU - de Castro, Ana M.
AU - Gonzalez, Clara I.
AU - Viana, Karla Acosta
AU - Yachelini, Pedro
AU - Torrico, Faustino
AU - Robello, Carlos
AU - Diosque, Patricio
AU - Chavez, Omar Triana
AU - Aznar, Christine
AU - Russomando, Graciela
AU - Büscher, Philippe
AU - Assal, Azzedine
AU - Guhl, Felipe
AU - Estani, Sergio Sosa
AU - DaSilva, Alexandre
AU - Britto, Constança
AU - Luquetti, Alejandro
AU - Ladzins, Janis
N1 - Copyright:
Copyright 2011 Elsevier B.V., All rights reserved.
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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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U2 - 10.1371/journal.pntd.0000931
DO - 10.1371/journal.pntd.0000931
M3 - Research Article
C2 - 21264349
AN - SCOPUS:79951548355
SN - 1935-2727
VL - 5
JO - PLoS Neglected Tropical Diseases
JF - PLoS Neglected Tropical Diseases
IS - 1
M1 - e931
ER -