Toward the Establishment of a Single Standard Curve for Quantification of Trypanosoma cruzi Natural Populations Using a Synthetic Satellite Unit DNA Sequence.

Autor:	Muñoz-Calderón A; Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Ingeniería Genética y Biología Molecular 'Dr Héctor Torres' (INGEBI), Buenos Aires, Argentina., Silva-Gomes NL; Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil., Apodaca S; Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Ingeniería Genética y Biología Molecular 'Dr Héctor Torres' (INGEBI), Buenos Aires, Argentina., Alarcón de Noya B; Sección de Inmunologia, Instituto de Medicina Tropical 'Dr Félix Pifano,' Universidad Central de Venezuela, Caracas, Venezuela., Díaz-Bello Z; Sección de Inmunologia, Instituto de Medicina Tropical 'Dr Félix Pifano,' Universidad Central de Venezuela, Caracas, Venezuela., Souza LRQ; Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil., Costa ADT; Laboratório de Ciências e Tecnologias Aplicadas à Saúde (LaCTAS), Instituto Carlos Chagas (ICC), Fundação Oswaldo Cruz, Curitiba, Brazil., Britto C; Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil., Moreira OC; Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. Electronic address: otacilio@ioc.fiocruz.br., Schijman AG; Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Ingeniería Genética y Biología Molecular 'Dr Héctor Torres' (INGEBI), Buenos Aires, Argentina. Electronic address: schijman@dna.uba.ar.
Jazyk:	angličtina
Zdroj:	The Journal of molecular diagnostics : JMD [J Mol Diagn] 2021 May; Vol. 23 (5), pp. 521-531. Date of Electronic Publication: 2021 Feb 04.
DOI:	10.1016/j.jmoldx.2021.01.007
Abstrakt:	Accurate diagnostic tools and surrogate markers of parasitologic response to treatment are needed for managing Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (TcI) and CL-Brener (TcVI) stocks similar 95% limit of detection values [0.903 (0.745 to 1.497) and 0.667 (CI, 0.113 to 3.927) copy numbers/μL, respectively] when synthetic DNA was the standard for quantification, allowing direct comparison of loads in samples infected with different discrete typing units. This standard curve was evaluated in 205 samples (38 acute oral and 19 chronic Chagas disease patients) from different geographical areas infected with various genotypes, including samples obtained during treatment follow-up; high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase 3 clinical trials, to follow up patients under treatment or at risk of reactivation, and in experimental models using different parasite strains. (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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