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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/79634
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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 Técnicas2025-09-29T09:47:11Zoai: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:34982025-09-29 09:47:11.432CONICET 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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1844613470162518016 |
score |
13.070432 |