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 CrisanteGonzalo 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 una revistaArtículorevisión exhaustiva

91 Citas (Scopus)

Resumen

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.

Idioma originalInglés estadounidense
Páginas (desde-hasta)605-615
Número de páginas11
PublicaciónJournal of Molecular Diagnostics
Volumen17
N.º5
DOI
EstadoPublicada - 2015
Publicado de forma externa

All Science Journal Classification (ASJC) codes

  • Patología y medicina forense
  • Medicina molecular

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