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

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dc.creatorSchijman, Alejandro Gabriel
dc.creatorBisio, Margarita
dc.creatorOrellana, Liliana
dc.creatorSued, Mariela
dc.creatorDuffy, Tomás
dc.creatorJaramillo, Ana Maria Mejia
dc.creatorCura, Carolina
dc.creatorAuter, Frederic
dc.creatorVeron, Vincent
dc.creatorQvarnstrom, Yvonne
dc.creatorDeborggraeve, Stijn
dc.creatorHijar, Gisely
dc.creatorZulantay, Inés
dc.creatorLucero, Raúl Horacio
dc.creatorVelazquez, Elsa
dc.creatorTellez, Tatiana
dc.creatorLeon, Zunilda Sanchez
dc.creatorGalvão, Lucia
dc.creatorNolder, Debbie
dc.creatorRumi, María Monje
dc.creatorLevi, José Eduardo
dc.creatorRamirez, Juan David
dc.creatorZorrilla, Pilar
dc.creatorFlores, María
dc.creatorJercic, Maria Isabel
dc.creatorCrisante, Gladys
dc.creatorAñez, Néstor
dc.creatorCastro, Ana Maria de
dc.creatorGonzalez, Clara Isabel
dc.creatorViana, Karla Acosta
dc.creatorYachelini, Pedro
dc.creatorTorrico, Faustino
dc.creatorRobello, Carlos
dc.creatorDiosque, Patricio
dc.creatorChavez, Omar Triana
dc.creatorAznar, Christine
dc.creatorRussomando, Graciela
dc.creatorBüscher, Philippe
dc.creatorAssal, Azzedine
dc.creatorGuhl, Felipe
dc.creatorEstani, Sergio Sosa
dc.creatorDaSilva, Alexandre
dc.creatorBritto, Constança
dc.creatorOstermayer, Alejandro Luquetti
dc.creatorLadzins, Janis
dc.date.accessioned2018-04-02T13:18:49Z
dc.date.available2018-04-02T13:18:49Z
dc.date.issued2011-01
dc.description.abstractBackground: 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.
dc.identifier.citationSCHIJMAN, Alejandro G.; BISIO, Margarita; ORELLANA, Liliana; SUED, Mariela; DUFFY, Tomás; JARAMILLO, Ana M. Mejia; CURA, Carolina; AUSTER, Frederic; VERON, Vincent; QVARNSTROM, Yvonne; DEBORGGRAEVE, Stijn; HIJAR, Gisely; ZULANTAY, Inés; LUCERO, Raúl Horacio; VELAZQUEZ, Elsa; TELLEZ, Tatiana; LEON, Zunilda Sanchez; GALVÃO, Lucia; NOLDER, Debbie; RUMI, María Monje; LEVI, José E.; RAMIREZ, Juan D.; ZORRILA, Pilar; FLORES, María; JERCIC, Maria I.; CRISANTE, Gladys; AÑEZ, Néstor; CASTRO, Ana M. de; GONZALEZ, Clara I.; VIANA, Karla Acosta; YACHELINI, Pedro; TORRICO, Faustino; ROBELLO, Carlos; DIOSQUE, Patricio; CHAVEZ, Omar Triana; AZNAR, Christine; RUSSOMANDO, Graciela; BÜSCHER, Philippe; ASSAL, Azzedine; GUHL, Felipe; ESTANI, Sergio Sosa; DASILVA, Alexandre; BRITTO, Constança; LUQUETTI, Alejandro; LADZINS, Janis. International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. Plos Neglected Tropical Diseases, San Francisco, v. 5, n. 1, p. e931, Jan. 2011.pt_BR
dc.identifier.doi10.1371/journal.pntd.0000931
dc.identifier.issn1935-2727
dc.identifier.issne- 1935-2735
dc.identifier.urihttp://repositorio.bc.ufg.br/handle/ri/14263
dc.language.isoengpt_BR
dc.publisher.countryEstados unidospt_BR
dc.publisher.departmentInstituto de Patologia Tropical e Saúde Pública - IPTSP (RG)pt_BR
dc.rightsAcesso Abertopt_BR
dc.titleInternational study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patientspt_BR
dc.typeArtigopt_BR

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