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J Mol Diagn. 2015 Sep;17(5):605-15. 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.

Author information

1
Laboratory of Molecular Biology of Chagas Disease (LaBMECh), Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina.
2
Laboratory of Molecular Biology of Endemic Diseases, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Brazil.
3
Laboratory of Discipline of Parasitology, Universidade Federal do Triângulo Mineiro, Uberaba, Brazil.
4
National Institute of Parasitology "Dr. Mario Fatala Chabén", Buenos Aires, Argentina.
5
Center for Research in Tropical Microbiology and Parasitology, Universidad de los Andes, Bogota, Colombia.
6
Institute of Experimental Pathology, CONICET-Universidad Nacional de Salta, Salta, Argentina.
7
Laboratory of Molecular Parasitology, Pontificia Universidad Javeriana, Bogota, Colombia.
8
National Center for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.
9
Institute of Tropical Medicine, Universidad Central de Venezuela, Caracas, Venezuela.
10
Biomedical Research Institute, Universidad Nacional Autónoma de México, Mexico DF, Mexico.
11
Laboratory of Parasitology and Molecular Biology, Instituto Nacional de Laboratorios en Salud, La Paz, Bolivia.
12
Department of Microbiology and Parasitology, Universidade Federal do Rio Grande do Norte, Natal, Brazil.
13
Hospital and University Laboratory-CH Andrée Rosemon, Cayenne, French Guiana.
14
Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia.
15
National Center for Public Health, Instituto Nacional de Salud, Lima, Peru.
16
Laboratory of Genomics, Instituto Nacional de Cardiología "Ignacio Chávez", Mexico DF, Mexico.
17
Laboratory for Research in Infectious Diseases, Universidad Peruana Cayetano Heredia, Lima, Peru.
18
Department of Biology, Universidad de los Andes, Merida, Venezuela.
19
Molecular Biology Unit, Instituto Pasteur de Montevideo, Montevideo, Uruguay.
20
Basic Clinical Parasitology Laboratory, Universidad de Chile, Santiago, Chile.
21
Research Center for Infectious Diseases, Pontificia Universidad Católica de Ecuador, Quito, Ecuador.
22
Microbiology Department, Hospital Clinic and Barcelona Centre for International Health Research (CRESIB), Barcelona, Spain.
23
State Laboratory of Public Health, Acapulco, Mexico.
24
Biomedical Department, Instituto de Salud Pública, Santiago, Chile.
25
Laboratory of Molecular Biology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
26
Research Institute for Health Sciences, Universidad Nacional de Asunción, Asuncion, Paraguay.
27
Drugs for Neglected Diseases Initiative (DNDi), Geneva, Switzerland.
28
Communicable Diseases and Health Analysis Department, Pan American Health Organization/World Health Organization (PAHO/WHO), Rio de Janeiro, Brazil.
29
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.

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.

PMID:
26320872
PMCID:
PMC4698797
DOI:
10.1016/j.jmoldx.2015.04.010
[Indexed for MEDLINE]
Free PMC Article

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