Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients

Autores
Duffy, Tomás; Bisio, Margarita María Catalina; Altcheh, Jaime Marcelo; Burgos, Juan Miguel; Diez, Mirta; Levin, Mariano Jorge; Favaloro, Roberto Rene; Freilij, Hector León; Schijman, Alejandro Gabriel
Año de publicación
2009
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Background: This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence. Methodology/Principal Findings: The Q-PCR has a detection limit of 0.1 and 0.01 parasites/mL, with a dynamic range of 106 and 107 for Silvio X10 cl1 (T. cruzi I) and Cl Brener stocks (T. cruzi IIe), respectively, an efficiency of 99%, and a coefficient of determination (R2) of 0.998. In order to express accurately the parasitic loads: (1) we adapted a commercial kit based on silica-membrane technology to enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimize PCR inhibition; (2) results were normalized incorporating a linearized plasmid as an internal standard of the whole procedure; and (3) a correction factor according to the representativity of satellite sequences in each parasite lineage group was determined using a modified real-time PCR protocol (Lg-PCR). The Q-PCR strategy was applied (1) to estimate basal parasite loads in 43 pediatric Chagas disease patients, (2) to follow-up 38 of them receiving treatment with benznidazole, and (3) to monitor three chronic Chagas heart disease patients who underwent heart-transplantation and displayed events of clinical reactivation due to immunosupression. Conclusion/Significance: All together, the high analytical sensitivity of the Q-PCR strategy, the low levels of intra- and interassay variations, as well as the accuracy provided by the Lg-PCR based correction factor support this methodology as a key laboratory tool for monitoring clinical reactivation and etiological treatment outcome in Chagas disease patients.
Fil: Duffy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Bisio, Margarita María Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Altcheh, Jaime Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina
Fil: Burgos, Juan Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Diez, Mirta. Fundación Favaloro; Argentina
Fil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Favaloro, Roberto Rene. Fundación Favaloro; Argentina
Fil: Freilij, Hector León. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina
Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Materia
REAL TIME PCR
PARASITIC LOAD
TRYPANOSOMA CRUZI
CHAGAS DISEASE
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/79634
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/79634
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patientsDuffy, TomásBisio, Margarita María CatalinaAltcheh, Jaime MarceloBurgos, Juan MiguelDiez, MirtaLevin, Mariano JorgeFavaloro, Roberto ReneFreilij, Hector LeónSchijman, Alejandro GabrielREAL TIME PCRPARASITIC LOADTRYPANOSOMA CRUZICHAGAS DISEASEhttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3Background: This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence. Methodology/Principal Findings: The Q-PCR has a detection limit of 0.1 and 0.01 parasites/mL, with a dynamic range of 106 and 107 for Silvio X10 cl1 (T. cruzi I) and Cl Brener stocks (T. cruzi IIe), respectively, an efficiency of 99%, and a coefficient of determination (R2) of 0.998. In order to express accurately the parasitic loads: (1) we adapted a commercial kit based on silica-membrane technology to enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimize PCR inhibition; (2) results were normalized incorporating a linearized plasmid as an internal standard of the whole procedure; and (3) a correction factor according to the representativity of satellite sequences in each parasite lineage group was determined using a modified real-time PCR protocol (Lg-PCR). The Q-PCR strategy was applied (1) to estimate basal parasite loads in 43 pediatric Chagas disease patients, (2) to follow-up 38 of them receiving treatment with benznidazole, and (3) to monitor three chronic Chagas heart disease patients who underwent heart-transplantation and displayed events of clinical reactivation due to immunosupression. Conclusion/Significance: All together, the high analytical sensitivity of the Q-PCR strategy, the low levels of intra- and interassay variations, as well as the accuracy provided by the Lg-PCR based correction factor support this methodology as a key laboratory tool for monitoring clinical reactivation and etiological treatment outcome in Chagas disease patients.Fil: Duffy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bisio, Margarita María Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Altcheh, Jaime Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Burgos, Juan Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Diez, Mirta. Fundación Favaloro; ArgentinaFil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Favaloro, Roberto Rene. Fundación Favaloro; ArgentinaFil: Freilij, Hector León. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaPublic Library of Science2009-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/79634Duffy, Tomás; Bisio, Margarita María Catalina; Altcheh, Jaime Marcelo; Burgos, Juan Miguel; Diez, Mirta; et al.; Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients; Public Library of Science; PLoS Neglected Tropical Diseases; 3; 4; 4-2009; 419-4291935-2735CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2667272/info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pntd.0000419info:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0000419info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:16:02Zoai:ri.conicet.gov.ar:11336/79634instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:16:02.391CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients
title Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients
spellingShingle Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients
Duffy, Tomás
REAL TIME PCR
PARASITIC LOAD
TRYPANOSOMA CRUZI
CHAGAS DISEASE
title_short Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients
title_full Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients
title_fullStr Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients
title_full_unstemmed Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients
title_sort Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients
dc.creator.none.fl_str_mv Duffy, Tomás
Bisio, Margarita María Catalina
Altcheh, Jaime Marcelo
Burgos, Juan Miguel
Diez, Mirta
Levin, Mariano Jorge
Favaloro, Roberto Rene
Freilij, Hector León
Schijman, Alejandro Gabriel
author Duffy, Tomás
author_facet Duffy, Tomás
Bisio, Margarita María Catalina
Altcheh, Jaime Marcelo
Burgos, Juan Miguel
Diez, Mirta
Levin, Mariano Jorge
Favaloro, Roberto Rene
Freilij, Hector León
Schijman, Alejandro Gabriel
author_role author
author2 Bisio, Margarita María Catalina
Altcheh, Jaime Marcelo
Burgos, Juan Miguel
Diez, Mirta
Levin, Mariano Jorge
Favaloro, Roberto Rene
Freilij, Hector León
Schijman, Alejandro Gabriel
author2_role author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv REAL TIME PCR
PARASITIC LOAD
TRYPANOSOMA CRUZI
CHAGAS DISEASE
topic REAL TIME PCR
PARASITIC LOAD
TRYPANOSOMA CRUZI
CHAGAS DISEASE
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv Background: This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence. Methodology/Principal Findings: The Q-PCR has a detection limit of 0.1 and 0.01 parasites/mL, with a dynamic range of 106 and 107 for Silvio X10 cl1 (T. cruzi I) and Cl Brener stocks (T. cruzi IIe), respectively, an efficiency of 99%, and a coefficient of determination (R2) of 0.998. In order to express accurately the parasitic loads: (1) we adapted a commercial kit based on silica-membrane technology to enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimize PCR inhibition; (2) results were normalized incorporating a linearized plasmid as an internal standard of the whole procedure; and (3) a correction factor according to the representativity of satellite sequences in each parasite lineage group was determined using a modified real-time PCR protocol (Lg-PCR). The Q-PCR strategy was applied (1) to estimate basal parasite loads in 43 pediatric Chagas disease patients, (2) to follow-up 38 of them receiving treatment with benznidazole, and (3) to monitor three chronic Chagas heart disease patients who underwent heart-transplantation and displayed events of clinical reactivation due to immunosupression. Conclusion/Significance: All together, the high analytical sensitivity of the Q-PCR strategy, the low levels of intra- and interassay variations, as well as the accuracy provided by the Lg-PCR based correction factor support this methodology as a key laboratory tool for monitoring clinical reactivation and etiological treatment outcome in Chagas disease patients.
Fil: Duffy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Bisio, Margarita María Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Altcheh, Jaime Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina
Fil: Burgos, Juan Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Diez, Mirta. Fundación Favaloro; Argentina
Fil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Favaloro, Roberto Rene. Fundación Favaloro; Argentina
Fil: Freilij, Hector León. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina
Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
description Background: This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence. Methodology/Principal Findings: The Q-PCR has a detection limit of 0.1 and 0.01 parasites/mL, with a dynamic range of 106 and 107 for Silvio X10 cl1 (T. cruzi I) and Cl Brener stocks (T. cruzi IIe), respectively, an efficiency of 99%, and a coefficient of determination (R2) of 0.998. In order to express accurately the parasitic loads: (1) we adapted a commercial kit based on silica-membrane technology to enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimize PCR inhibition; (2) results were normalized incorporating a linearized plasmid as an internal standard of the whole procedure; and (3) a correction factor according to the representativity of satellite sequences in each parasite lineage group was determined using a modified real-time PCR protocol (Lg-PCR). The Q-PCR strategy was applied (1) to estimate basal parasite loads in 43 pediatric Chagas disease patients, (2) to follow-up 38 of them receiving treatment with benznidazole, and (3) to monitor three chronic Chagas heart disease patients who underwent heart-transplantation and displayed events of clinical reactivation due to immunosupression. Conclusion/Significance: All together, the high analytical sensitivity of the Q-PCR strategy, the low levels of intra- and interassay variations, as well as the accuracy provided by the Lg-PCR based correction factor support this methodology as a key laboratory tool for monitoring clinical reactivation and etiological treatment outcome in Chagas disease patients.
publishDate 2009
dc.date.none.fl_str_mv 2009-04
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/79634
Duffy, Tomás; Bisio, Margarita María Catalina; Altcheh, Jaime Marcelo; Burgos, Juan Miguel; Diez, Mirta; et al.; Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients; Public Library of Science; PLoS Neglected Tropical Diseases; 3; 4; 4-2009; 419-429
1935-2735
CONICET Digital
CONICET
url http://hdl.handle.net/11336/79634
identifier_str_mv Duffy, Tomás; Bisio, Margarita María Catalina; Altcheh, Jaime Marcelo; Burgos, Juan Miguel; Diez, Mirta; et al.; Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients; Public Library of Science; PLoS Neglected Tropical Diseases; 3; 4; 4-2009; 419-429
1935-2735
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2667272/
info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pntd.0000419
info:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0000419
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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