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

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; 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

doi: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

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

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^*

^*Corresponding author for this work

Faculty of Exact and Natural Sciences

Research output: Contribution to journal › Article › peer-review

176 Scopus citations

Abstract

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.

Original language	English
Pages (from-to)	605-615
Number of pages	11
Journal	Journal of Molecular Diagnostics
Volume	17
Issue number	5
DOIs	https://doi.org/10.1016/j.jmoldx.2015.04.010
State	Published - 2015

Bibliographical note

Publisher Copyright:
© 2015 American Society for Investigative Pathology and the Association for Molecular Pathology.

Funding

Supported by CDR/Small Grant Programme PAHO/WHO (A.G.S.) and partially by CONICET grant PIP 112-200801-02915 and the National Agency of Science and Technology grant PICT 33955 .

Funders	Funder number
Small
Consejo Nacional de Investigaciones Científicas y Técnicas	PIP 112-200801-02915
Agencia Nacional de Promoción Científica y Tecnológica	PICT 33955
Centre for Dengue Research, University of Sri Jayewardenepura

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10.1016/j.jmoldx.2015.04.010

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Ramírez, J. C., Cura, C. I., Da Cruz Moreira, O., Lages-Silva, E., Juiz, N., Velázquez, E., Ramírez, J. D., Alberti, A., Pavia, P., Flores-Chávez, M. D., Muñoz-Calderón, A., Pérez-Morales, D., Santalla, J., Marcos Da Matta Guedes, P., Peneau, J., Marcet, P., Padilla, C., Cruz-Robles, D., Valencia, E., ... Schijman, A. G. (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, 17(5), 605-615. https://doi.org/10.1016/j.jmoldx.2015.04.010

@article{64ade9ea2ed34672980f5c6c0818c31d,

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

abstract = "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{\c c}a Britto and Yad{\'o}n, \{Zaida Estela\} and Schijman, \{Alejandro G.\}",

note = "Publisher Copyright: {\textcopyright} 2015 American Society for Investigative Pathology and the Association for Molecular Pathology.",

year = "2015",

doi = "10.1016/j.jmoldx.2015.04.010",

language = "English",

volume = "17",

pages = "605--615",

journal = "Journal of Molecular Diagnostics",

issn = "1525-1578",

publisher = "Elsevier B.V.",

number = "5",

}

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, vol. 17, no. 5, 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 et al.
In: Journal of Molecular Diagnostics, Vol. 17, No. 5, 2015, p. 605-615.

Research output: Contribution to journal › Article › peer-review

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

Y1 - 2015

N2 - 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 - 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.

UR - https://www.scopus.com/pages/publications/84952662398

U2 - 10.1016/j.jmoldx.2015.04.010

DO - 10.1016/j.jmoldx.2015.04.010

M3 - Article

C2 - 26320872

AN - SCOPUS:84952662398

SN - 1525-1578

VL - 17

SP - 605

EP - 615

JO - Journal of Molecular Diagnostics

JF - Journal of Molecular Diagnostics

IS - 5

ER -

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

Abstract

Bibliographical note

Funding

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