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.
N1 - Funding Information:
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 .
Publisher Copyright:
© 2015 American Society for Investigative Pathology and the Association for Molecular Pathology.
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.
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U2 - 10.1016/j.jmoldx.2015.04.010
DO - 10.1016/j.jmoldx.2015.04.010
M3 - Research 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 -
