International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients
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 performan...
| Autores: | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
|---|---|
| Tipo de recurso: | artículo |
| Fecha de publicación: | 2011 |
| País: | España |
| Institución: | Instituto de Salud Carlos III (ISCIII) |
| Repositorio: | Repisalud |
| Idioma: | inglés |
| OAI Identifier: | oai:repisalud.isciii.es:20.500.12105/6943 |
| Acceso en línea: | http://hdl.handle.net/20.500.12105/6943 |
| Access Level: | acceso abierto |
| Palabra clave: | Chagas Disease DNA, Protozoan Humans International Cooperation Parasitology Polymerase Chain Reaction |
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oai:repisalud.isciii.es:20.500.12105/6943 |
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España |
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| dc.title.none.fl_str_mv |
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients |
| title |
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients |
| spellingShingle |
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients Schijman, Alejandro G Chagas Disease DNA, Protozoan Humans International Cooperation Parasitology Polymerase Chain Reaction |
| title_short |
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients |
| title_full |
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients |
| title_fullStr |
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients |
| title_full_unstemmed |
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients |
| title_sort |
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients |
| dc.creator.none.fl_str_mv |
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-Chavez, Maria Jercic, Maria I Crisante, Gladys Añez, Néstor De Castro, Ana M 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 DaSilva, Alexandre Britto, Constança Luquetti, Alejandro Ladzins, Janis |
| author |
Schijman, Alejandro G |
| author_facet |
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-Chavez, Maria Jercic, Maria I Crisante, Gladys Añez, Néstor De Castro, Ana M 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 DaSilva, Alexandre Britto, Constança Luquetti, Alejandro Ladzins, Janis |
| author_role |
author |
| author2 |
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-Chavez, Maria Jercic, Maria I Crisante, Gladys Añez, Néstor De Castro, Ana M 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 DaSilva, Alexandre Britto, Constança Luquetti, Alejandro Ladzins, Janis |
| author2_role |
author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author author |
| dc.contributor.none.fl_str_mv |
Organización Panamericana de la Salud United Nations University Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina) World Health Organization (WHO/OMS) |
| dc.subject.none.fl_str_mv |
Chagas Disease DNA, Protozoan Humans International Cooperation Parasitology Polymerase Chain Reaction |
| topic |
Chagas Disease DNA, Protozoan Humans International Cooperation Parasitology Polymerase Chain Reaction |
| description |
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. |
| publishDate |
2011 |
| dc.date.none.fl_str_mv |
2011 2011-01-11 2011 2011-01-11 2018 2018-12-26 |
| dc.type.none.fl_str_mv |
research article http://purl.org/coar/resource_type/c_2df8fbb1 VoR http://purl.org/coar/version/c_970fb48d4fbd8a85 |
| dc.type.openaire.fl_str_mv |
info:eu-repo/semantics/article |
| format |
article |
| dc.identifier.none.fl_str_mv |
http://hdl.handle.net/20.500.12105/6943 |
| url |
http://hdl.handle.net/20.500.12105/6943 |
| dc.language.none.fl_str_mv |
Inglés eng |
| language_invalid_str_mv |
Inglés |
| language |
eng |
| dc.rights.none.fl_str_mv |
open access http://purl.org/coar/access_right/c_abf2 Atribución 4.0 Internacional http://creativecommons.org/licenses/by/4.0/ |
| dc.rights.openaire.fl_str_mv |
info:eu-repo/semantics/openAccess |
| rights_invalid_str_mv |
open access http://purl.org/coar/access_right/c_abf2 Atribución 4.0 Internacional http://creativecommons.org/licenses/by/4.0/ |
| eu_rights_str_mv |
openAccess |
| dc.format.none.fl_str_mv |
application/pdf |
| dc.publisher.none.fl_str_mv |
Public Library of Science (PLOS) |
| publisher.none.fl_str_mv |
Public Library of Science (PLOS) |
| dc.source.none.fl_str_mv |
reponame:Repisalud instname:Instituto de Salud Carlos III (ISCIII) |
| instname_str |
Instituto de Salud Carlos III (ISCIII) |
| reponame_str |
Repisalud |
| collection |
Repisalud |
| repository.name.fl_str_mv |
|
| repository.mail.fl_str_mv |
|
| _version_ |
1869423616024641536 |
| spelling |
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patientsSchijman, Alejandro GBisio, MargaritaOrellana, LilianaSued, MarielaDuffy, TomásMejia Jaramillo, Ana MCura, CarolinaAuter, FredericVeron, VincentQvarnstrom, YvonneDeborggraeve, StijnHijar, GiselyZulantay, InésLucero, Raúl HoracioVelazquez, ElsaTellez, TatianaSanchez Leon, ZunildaGalvão, LuciaNolder, DebbieMonje Rumi, MaríaLevi, José ERamirez, Juan DZorrilla, PilarFlores-Chavez, MariaJercic, Maria ICrisante, GladysAñez, NéstorDe Castro, Ana MGonzalez, Clara IAcosta Viana, KarlaYachelini, PedroTorrico, FaustinoRobello, CarlosDiosque, PatricioTriana Chavez, OmarAznar, ChristineRussomando, GracielaBüscher, PhilippeAssal, AzzedineGuhl, FelipeSosa Estani, SergioDaSilva, AlexandreBritto, ConstançaLuquetti, AlejandroLadzins, JanisChagas DiseaseDNA, ProtozoanHumansInternational CooperationParasitologyPolymerase Chain ReactionBACKGROUND: 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.Public Library of Science (PLOS)Organización Panamericana de la SaludUnited Nations UniversityConsejo Nacional de Investigaciones Científicas y Técnicas (Argentina)World Health Organization (WHO/OMS)20182018-12-2620112011-01-1120112011-01-11research articlehttp://purl.org/coar/resource_type/c_2df8fbb1VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/20.500.12105/6943reponame:Repisaludinstname:Instituto de Salud Carlos III (ISCIII)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Atribución 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:repisalud.isciii.es:20.500.12105/69432026-06-12T12:43:37Z |
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15,811543 |