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.
authors: Laboratory of Molecular Biology of Chagas Disease (LaBMECh), Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina.
authors: Juan Carlos
authors: JC
authors: Ramírez
authors: Laboratory of Molecular Biology of Chagas Disease (LaBMECh), Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina.
authors: Carolina Inés
authors: CI
authors: Cura
authors: Laboratory of Molecular Biology of Endemic Diseases, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Brazil.
authors: Otacilio
authors: O
authors: da Cruz Moreira
authors: Laboratory of Discipline of Parasitology, Universidade Federal do Triângulo Mineiro, Uberaba, Brazil.
authors: Eliane
authors: E
authors: Lages-Silva
authors: Laboratory of Molecular Biology of Chagas Disease (LaBMECh), Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina.
authors: Natalia
authors: N
authors: Juiz
authors: National Institute of Parasitology "Dr. Mario Fatala Chabén", Buenos Aires, Argentina.
authors: Elsa
authors: E
authors: Velázquez
authors: Center for Research in Tropical Microbiology and Parasitology, Universidad de los Andes, Bogota, Colombia.
authors: Juan David
authors: JD
authors: Ramírez
authors: Institute of Experimental Pathology, CONICET-Universidad Nacional de Salta, Salta, Argentina.
authors: Anahí
authors: A
authors: Alberti
authors: Laboratory of Molecular Parasitology, Pontificia Universidad Javeriana, Bogota, Colombia.
authors: Paula
authors: P
authors: Pavia
authors: National Center for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.
authors: María Delmans
authors: MD
authors: Flores-Chávez
authors: Institute of Tropical Medicine, Universidad Central de Venezuela, Caracas, Venezuela.
authors: Arturo
authors: A
authors: Muñoz-Calderón
authors: Biomedical Research Institute, Universidad Nacional Autónoma de México, Mexico DF, Mexico.
authors: Deyanira
authors: D
authors: Pérez-Morales
authors: Laboratory of Parasitology and Molecular Biology, Instituto Nacional de Laboratorios en Salud, La Paz, Bolivia.
authors: José
authors: J
authors: Santalla
authors: Department of Microbiology and Parasitology, Universidade Federal do Rio Grande do Norte, Natal, Brazil.
authors: Paulo
authors: P
authors: Marcos da Matta Guedes
authors: Hospital and University Laboratory-CH AndrÃ©e Rosemon, Cayenne, French Guiana.
authors: Julie
authors: J
authors: Peneau
authors: Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia.
authors: Paula
authors: P
authors: Marcet
authors: National Center for Public Health, Instituto Nacional de Salud, Lima, Peru.
authors: Carlos
authors: C
authors: Padilla
authors: Laboratory of Genomics, Instituto Nacional de Cardiología "Ignacio Chávez", Mexico DF, Mexico.
authors: David
authors: D
authors: Cruz-Robles
authors: Laboratory for Research in Infectious Diseases, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors: Edward
authors: E
authors: Valencia
authors: Department of Biology, Universidad de los Andes, Merida, Venezuela.
authors: Gladys Elena
authors: GE
authors: Crisante
authors: Molecular Biology Unit, Instituto Pasteur de Montevideo, Montevideo, Uruguay.
authors: Gonzalo
authors: G
authors: Greif
authors: Basic Clinical Parasitology Laboratory, Universidad de Chile, Santiago, Chile.
authors: Inés
authors: I
authors: Zulantay
authors: Research Center for Infectious Diseases, Pontificia Universidad Católica de Ecuador, Quito, Ecuador.
authors: Jaime Alfredo
authors: JA
authors: Costales
authors: Microbiology Department, Hospital Clinic and Barcelona Centre for International Health Research (CRESIB), Barcelona, Spain.
authors: Miriam
authors: M
authors: Alvarez-Martínez
authors: State Laboratory of Public Health, Acapulco, Mexico.
authors: Norma Edith
authors: NE
authors: Martínez
authors: Biomedical Department, Instituto de Salud PÃºblica, Santiago, Chile.
authors: Rodrigo
authors: R
authors: Villarroel
authors: Laboratory of Molecular Biology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
authors: Sandro
authors: S
authors: Villarroel
authors: Research Institute for Health Sciences, Universidad Nacional de Asunción, Asuncion, Paraguay.
authors: Zunilda
authors: Z
authors: Sánchez
authors: Laboratory of Molecular Biology of Chagas Disease (LaBMECh), Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina.
authors: Margarita
authors: M
authors: Bisio
authors: Laboratory of Molecular Biology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
authors: Rudy
authors: R
authors: Parrado
authors: Department of Microbiology and Parasitology, Universidade Federal do Rio Grande do Norte, Natal, Brazil.
authors: Lúcia
authors: L
authors: Maria da Cunha Galvão
authors: Department of Microbiology and Parasitology, Universidade Federal do Rio Grande do Norte, Natal, Brazil.
authors: Antonia Cláudia
authors: AC
authors: Jácome da Câmara
authors: Biomedical Research Institute, Universidad Nacional Autónoma de México, Mexico DF, Mexico.
authors: Bertha
authors: B
authors: Espinoza
authors: Institute of Tropical Medicine, Universidad Central de Venezuela, Caracas, Venezuela.
authors: Belkisyole
authors: B
authors: Alarcón de Noya
authors: Laboratory of Molecular Parasitology, Pontificia Universidad Javeriana, Bogota, Colombia.
authors: Concepción
authors: C
authors: Puerta
authors: National Institute of Parasitology "Dr. Mario Fatala Chabén", Buenos Aires, Argentina.
authors: Adelina
authors: A
authors: Riarte
authors: Institute of Experimental Pathology, CONICET-Universidad Nacional de Salta, Salta, Argentina.
authors: Patricio
authors: P
authors: Diosque
authors: National Institute of Parasitology "Dr. Mario Fatala Chabén", Buenos Aires, Argentina.
authors: Sergio
authors: S
authors: Sosa-Estani
authors: Center for Research in Tropical Microbiology and Parasitology, Universidad de los Andes, Bogota, Colombia.
authors: Felipe
authors: F
authors: Guhl
authors: Drugs for Neglected Diseases Initiative (DNDi), Geneva, Switzerland.
authors: Isabela
authors: I
authors: Ribeiro
authors: Hospital and University Laboratory-CH AndrÃ©e Rosemon, Cayenne, French Guiana.
authors: Christine
authors: C
authors: Aznar
authors: Laboratory of Molecular Biology of Endemic Diseases, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Brazil.
authors: Constança
authors: C
authors: Britto
authors: Communicable Diseases and Health Analysis Department, Pan American Health Organization/World Health Organization (PAHO/WHO), Rio de Janeiro, Brazil.
authors: Zaida Estela
authors: ZE
authors: Yadón
authors: Laboratory of Molecular Biology of Chagas Disease (LaBMECh), Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina. Electronic address: schijman@dna.uba.ar.
authors: Alejandro G
authors: AG
authors: Schijman
mesh_terms: D014355:Chagas Disease / Q000097:blood* / Q000175:diagnosis / Q000235:genetics / Q000469:parasitology; D016054:DNA, Protozoan / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D007391:International Cooperation; D059021:Laboratory Proficiency Testing; D058889:Molecular Typing; D018512:Parasitemia / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D016054:DNA, Protozoan