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Title: Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients
Authors: Ramírez, Juan Carlos 
Cura, Carolina Inés 
da Cruz Moreira, Otacilio 
Lages-Silva, Eliane 
Juiz, Natalia A. 
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 L. 
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 
Sanchez Leon, Zunilda 
Bisio, Margarita 
Parrado, Rudy 
Maria da Cunha Galvão, Lúcia 
Jácome da Câmara, Antonia Cláudia 
Espinoza, Bertha 
Alarcón de Noya, Belkisyole 
Puerta, Concepción 
Riarte, Adelina 
Diosque, Patricio 
Sosa-Estani, Sergio 
Guhl, Felipe 
Ribeiro, Isabela 
Aznar, Christine 
Britto, Constança 
Yadón, Zaida Estela 
Schijman, Alejandro G. 
Issue Date: Sep-2015
Journal: The Journal of molecular diagnostics : JMD 
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.
DOI: 10.1016/j.jmoldx.2015.04.010
Appears in Collections:Publicaciones INP

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