Analytical validation of quantitative real-time PCR methods for quantification of trypanosoma cruzi DNA in blood samples from chagas disease patients

Título traducido de la contribución: Validación analítica de métodos cuantitativos de PCR en tiempo real para la cuantificación del ADN de trypanosoma cruzi en muestras de sangre de pacientes con enfermedad de Chagas

Juan Carlos Ramírez, Carolina Inés Cura, Otacilio Da Cruz Moreira, Eliane Lages-Silva, Natalia Juiz, Elsa Velázquez, Juan David Ramírez, Anahí Alberti, Paula Pavia, María Delmans Flores-Chávez, Arturo Muñoz-Calderón, Deyanira Pérez-Morales, José Santalla, Paulo Marcos Da Matta Guedes, Julie Peneau, Paula Marcet, Carlos Padilla, David Cruz-Robles, Edward Valencia, Gladys Elena Crisante & 24 otros Gonzalo Greif, Inés Zulantay, Jaime Alfredo Costales, Miriam Alvarez-Martínez, Norma Edith Martínez, Rodrigo Villarroel, Sandro Villarroel, Zunilda Sánchez, Margarita Bisio, Rudy Parrado, Lúcia Maria Da Cunha Galvão, Antonia Cláudia Jácome Da Câmara, Bertha Espinoza, Belkisyole Alarcón De Noya, Concepción Puerta, Adelina Riarte, Patricio Diosque, Sergio Sosa-Estani, Felipe Guhl, Isabela Ribeiro, Christine Aznar, Constança Britto, Zaida Estela Yadón, Alejandro G. Schijman

Resultado de la investigación: Contribución a RevistaArtículo

54 Citas (Scopus)

Resumen

Un estudio internacional fue realizado por 26 laboratorios de PCR experimentados de 14 países para evaluar el rendimiento de las estrategias de PCR cuantitativa en tiempo real a doble cara (qPCR) sobre la base de sondas TaqMan para la detección y cuantificación de cargas parasitarias en muestras de sangre periférica de pacientes con enfermedad de Chagas. Se estudiaron dos métodos: ADN satelital (ADN satelital) qPCR y ADN kinetoplástido (ADN kDNA) qPCR. Ambos métodos incluían un control interno de amplificación. El rango reportable, la sensibilidad analítica, los límites de detección y cuantificación, y la precisión fueron estimados de acuerdo con las directrices internacionales. Además, se estimó la inclusividad y exclusividad con el ADN de las poblaciones que representaban las diferentes unidades de tipificación discreta de Trypanosoma cruzi y Trypanosoma rangeli y Leishmania spp. ambos métodos se impugnaron contra 156 muestras de sangre proporcionadas por los laboratorios participantes, incluidas muestras de pacientes agudos y crónicos con hallazgos clínicos variados, infectados por vía oral o transmisión vectorial. kDNA qPCR mostró una mejor sensibilidad analítica que SatDNA qPCR, con límites de detección de equivalentes de parásitos de 0,23 y 0,70/ml, respectivamente. Los análisis de muestras clínicas revelaron una alta concordancia en términos de sensibilidad y cargas parasitarias determinadas por las qPCRs de ADN satelital y kDNA. Este esfuerzo es un paso importante hacia la validación internacional de los métodos de qPCR para la cuantificación del ADN de T. cruzi en muestras de sangre humana, con el objetivo de proporcionar un biomarcador sustituto preciso para el seguimiento del diagnóstico y tratamiento de pacientes con la enfermedad de Chagas.
Idioma originalEnglish (US)
Páginas (desde-hasta)605-615
Número de páginas11
PublicaciónJournal of Molecular Diagnostics
DOI
EstadoPublished - ene 1 2015
Publicado de forma externa

Huella dactilar

Chagas Disease
Trypanosoma cruzi
Satellite DNA
Real-Time Polymerase Chain Reaction
Parasite Load
DNA
Limit of Detection
Trypanosoma rangeli
Molecular Pathology
Leishmania
Physiologic Monitoring
Parasites
Biomarkers
Guidelines
Polymerase Chain Reaction
Therapeutics

Citar esto

Ramírez, Juan Carlos ; Cura, Carolina Inés ; Da Cruz Moreira, Otacilio ; Lages-Silva, Eliane ; Juiz, Natalia ; Velázquez, Elsa ; Ramírez, Juan David ; Alberti, Anahí ; Pavia, Paula ; Flores-Chávez, María Delmans ; Muñoz-Calderón, Arturo ; Pérez-Morales, Deyanira ; Santalla, José ; Marcos Da Matta Guedes, Paulo ; Peneau, Julie ; Marcet, Paula ; Padilla, Carlos ; Cruz-Robles, David ; Valencia, Edward ; Crisante, Gladys Elena ; Greif, Gonzalo ; Zulantay, Inés ; Costales, Jaime Alfredo ; Alvarez-Martínez, Miriam ; Martínez, Norma Edith ; Villarroel, Rodrigo ; Villarroel, Sandro ; Sánchez, Zunilda ; Bisio, Margarita ; Parrado, Rudy ; Maria Da Cunha Galvão, Lúcia ; Da Câmara, Antonia Cláudia Jácome ; Espinoza, Bertha ; De Noya, Belkisyole Alarcón ; Puerta, Concepción ; Riarte, Adelina ; Diosque, Patricio ; Sosa-Estani, Sergio ; Guhl, Felipe ; Ribeiro, Isabela ; Aznar, Christine ; Britto, Constança ; Yadón, Zaida Estela ; Schijman, Alejandro G. / Analytical validation of quantitative real-time PCR methods for quantification of trypanosoma cruzi DNA in blood samples from chagas disease patients. En: Journal of Molecular Diagnostics. 2015 ; pp. 605-615.
@article{e591d6610e474c9b8e1e643e57123183,
title = "Analytical validation of quantitative real-time PCR methods for quantification of trypanosoma cruzi DNA in blood samples from chagas disease patients",
abstract = "{\circledC} 2015 American Society for Investigative Pathology and the Association for Molecular Pathology.An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.",
author = "Ram{\'i}rez, {Juan Carlos} and Cura, {Carolina In{\'e}s} and {Da Cruz Moreira}, Otacilio and Eliane Lages-Silva and Natalia Juiz and Elsa Vel{\'a}zquez and Ram{\'i}rez, {Juan David} and Anah{\'i} Alberti and Paula Pavia and Flores-Ch{\'a}vez, {Mar{\'i}a Delmans} and Arturo Mu{\~n}oz-Calder{\'o}n and Deyanira P{\'e}rez-Morales and Jos{\'e} Santalla and {Marcos Da Matta Guedes}, Paulo and Julie Peneau and Paula Marcet and Carlos Padilla and David Cruz-Robles and Edward Valencia and Crisante, {Gladys Elena} and Gonzalo Greif and In{\'e}s Zulantay and Costales, {Jaime Alfredo} and Miriam Alvarez-Mart{\'i}nez and Mart{\'i}nez, {Norma Edith} and Rodrigo Villarroel and Sandro Villarroel and Zunilda S{\'a}nchez and Margarita Bisio and Rudy Parrado and {Maria Da Cunha Galv{\~a}o}, L{\'u}cia and {Da C{\^a}mara}, {Antonia Cl{\'a}udia J{\'a}come} and Bertha Espinoza and {De Noya}, {Belkisyole Alarc{\'o}n} and Concepci{\'o}n Puerta and Adelina Riarte and Patricio Diosque and Sergio Sosa-Estani and Felipe Guhl and Isabela Ribeiro and Christine Aznar and Constan{\cc}a Britto and Yad{\'o}n, {Zaida Estela} and Schijman, {Alejandro G.}",
year = "2015",
month = "1",
day = "1",
doi = "10.1016/j.jmoldx.2015.04.010",
language = "English (US)",
pages = "605--615",
journal = "Journal of Molecular Diagnostics",
issn = "1525-1578",
publisher = "Association of Molecular Pathology",

}

Ramírez, JC, Cura, CI, Da Cruz Moreira, O, Lages-Silva, E, Juiz, N, Velázquez, E, Ramírez, JD, Alberti, A, Pavia, P, Flores-Chávez, MD, Muñoz-Calderón, A, Pérez-Morales, D, Santalla, J, Marcos Da Matta Guedes, P, Peneau, J, Marcet, P, Padilla, C, Cruz-Robles, D, Valencia, E, Crisante, GE, Greif, G, Zulantay, I, Costales, JA, Alvarez-Martínez, M, Martínez, NE, Villarroel, R, Villarroel, S, Sánchez, Z, Bisio, M, Parrado, R, Maria Da Cunha Galvão, L, Da Câmara, ACJ, Espinoza, B, De Noya, BA, Puerta, C, Riarte, A, Diosque, P, Sosa-Estani, S, Guhl, F, Ribeiro, I, Aznar, C, Britto, C, Yadón, ZE & Schijman, AG 2015, 'Analytical validation of quantitative real-time PCR methods for quantification of trypanosoma cruzi DNA in blood samples from chagas disease patients', Journal of Molecular Diagnostics, pp. 605-615. https://doi.org/10.1016/j.jmoldx.2015.04.010

Analytical validation of quantitative real-time PCR methods for quantification of trypanosoma cruzi DNA in blood samples from chagas disease patients. / Ramírez, Juan Carlos; Cura, Carolina Inés; Da Cruz Moreira, Otacilio; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Marcos Da Matta Guedes, Paulo; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Maria Da Cunha Galvão, Lúcia; Da Câmara, Antonia Cláudia Jácome; Espinoza, Bertha; De Noya, Belkisyole Alarcón; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G.

En: Journal of Molecular Diagnostics, 01.01.2015, p. 605-615.

Resultado de la investigación: Contribución a RevistaArtículo

TY - JOUR

T1 - Analytical validation of quantitative real-time PCR methods for quantification of trypanosoma cruzi DNA in blood samples from chagas disease patients

AU - Ramírez, Juan Carlos

AU - Cura, Carolina Inés

AU - Da Cruz Moreira, Otacilio

AU - Lages-Silva, Eliane

AU - Juiz, Natalia

AU - Velázquez, Elsa

AU - Ramírez, Juan David

AU - Alberti, Anahí

AU - Pavia, Paula

AU - Flores-Chávez, María Delmans

AU - Muñoz-Calderón, Arturo

AU - Pérez-Morales, Deyanira

AU - Santalla, José

AU - Marcos Da Matta Guedes, Paulo

AU - Peneau, Julie

AU - Marcet, Paula

AU - Padilla, Carlos

AU - Cruz-Robles, David

AU - Valencia, Edward

AU - Crisante, Gladys Elena

AU - Greif, Gonzalo

AU - Zulantay, Inés

AU - Costales, Jaime Alfredo

AU - Alvarez-Martínez, Miriam

AU - Martínez, Norma Edith

AU - Villarroel, Rodrigo

AU - Villarroel, Sandro

AU - Sánchez, Zunilda

AU - Bisio, Margarita

AU - Parrado, Rudy

AU - Maria Da Cunha Galvão, Lúcia

AU - Da Câmara, Antonia Cláudia Jácome

AU - Espinoza, Bertha

AU - De Noya, Belkisyole Alarcón

AU - Puerta, Concepción

AU - Riarte, Adelina

AU - Diosque, Patricio

AU - Sosa-Estani, Sergio

AU - Guhl, Felipe

AU - Ribeiro, Isabela

AU - Aznar, Christine

AU - Britto, Constança

AU - Yadón, Zaida Estela

AU - Schijman, Alejandro G.

PY - 2015/1/1

Y1 - 2015/1/1

N2 - © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology.An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

AB - © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology.An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

U2 - 10.1016/j.jmoldx.2015.04.010

DO - 10.1016/j.jmoldx.2015.04.010

M3 - Article

SP - 605

EP - 615

JO - Journal of Molecular Diagnostics

JF - Journal of Molecular Diagnostics

SN - 1525-1578

ER -