TY - JOUR
T1 - Towards the establishment of a consensus real-time qPCR to monitor Trypanosoma cruzi parasitemia in patients with chronic Chagas disease cardiomyopathy
T2 - A substudy from the BENEFIT trial
AU - Moreira, Otacilio C.
AU - Ramírez, Juan David
AU - Velázquez, Elsa
AU - Melo, Myllena F.A.Dias
AU - Lima-Ferreira, Carolina
AU - Guhl, Felipe
AU - Sosa-Estani, Sergio
AU - Marin-Neto, Jose Antonio
AU - Morillo, Carlos A.
AU - Britto, Constança
N1 - Funding Information:
We thank Maria Angelica Cardoso for the excellent technical assistance for the BENEFIT Workshop organization and real-time PCR experiments, as well as the Program for Technological Development in Tools for Health (PDTIS-Fiocruz) for the facilities and support on the Real Time PCR-based platform. We also thank Dr. A. Avezum, A.C. Mattos and the team from the Instituto Dante Pazzanese, São Paulo, for coordinating the collection of samples from patients for the BENEFIT Network in Brazil; the National Coordination for the collection of samples from 15 sites of BENEFIT Network for the recruitment of patients in Argentina; F. Quiroz and M. Florez from the Fundación Cardiovascular, San Gil, Santander; S. Navarrete from the Hospital de la Policia, Bogotá, as well as, F. Rosas and J. Betancourt from the Clínica Abood Shaio, Bogotá, for the collection of samples from patients for the BENEFIT project in Colombia. We are also grateful to B.A.S. Pereira for carefully revising the English grammar. Special acknowledgement to CIHR and TDR/WHO, as well as to PHRI, for funding the BENEFIT trial project [CIHR BENEFIT #: 102704; WHO TDR #: A30755].
PY - 2013/1
Y1 - 2013/1
N2 - Quantitative real-time PCR (qPCR) is an accurate method to quantify Trypanosoma cruzi DNA and can be used to follow-up parasitemia in Chagas disease (CD) patients undergoing chemotherapy. The Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) study is an international, multicenter, randomized, double-blinded and placebo-controlled clinical trial to evaluate the efficacy of benznidazole (BZ) treatment in patients with chronic Chagas cardiomyopathy (CCC). One important question to be addressed concerns the effectiveness of BZ in reducing overall parasite load in CCC patients, even in the absence of parasitological cure. This report describes the evaluation of multiple procedures for DNA extraction and qPCR-based protocols aiming to establish a standardized methodology for the absolute quantification of T. cruzi DNA in Guanidine-EDTA blood (GEB) samples. A panel of five primer sets directed to the T. cruzi nuclear satellite DNA repeats (Sat-DNA) and to the minicircle DNA conserved regions (kDNA) was compared in either SYBR Green or TaqMan systems. Standard curve parameters such as, amplification efficiency, coefficient of determination and intercept were evaluated, as well as different procedures to generate standard samples containing pre-established T. cruzi DNA concentration. Initially, each primer set was assayed in a SYBR Green qPCR to estimate parasite load in GEB samples from chronic Chagas disease patients. The results achieved from Bayesian transmutability analysis elected the primer sets Cruzi1/Cruzi2 (p=0.0031) and Diaz7/Diaz8 (p=0.0023) coupled to the QIAamp DNA Kit extraction protocol (silica gel column), as the most suitable for monitoring parasitemia in these patients. Comparison between the parasite burden of 150 GEB samples of BENEFIT patients from Argentina, Brazil and Colombia, prior to drug/placebo administration, was performed using Cruzi1/Cruzi2 primers in a SYBR Green approach. The median parasitemia found in patients from Argentina and Colombia (1.93 and 2.31 parasite equivalents/mL, respectively) was around 20 times higher than the one estimated for the Brazilian patients (0.1 parasite equivalents/mL). This difference could be in part due to the complexity of T. cruzi genetic diversity, which is a factor possibly implicated in different clinical presentations of the disease and/or influencing parasitemia levels in infected individuals from different regions of Latin America. The results of SYBR Green qPCR assays herein presented prove this methodology to be more cost efficient than the alternative use of internal fluorogenic probes. In addition, its sensitivity and reproducibility are shown to be adequate to detect low parasitemia burden in patients with chronic Chagas disease.
AB - Quantitative real-time PCR (qPCR) is an accurate method to quantify Trypanosoma cruzi DNA and can be used to follow-up parasitemia in Chagas disease (CD) patients undergoing chemotherapy. The Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) study is an international, multicenter, randomized, double-blinded and placebo-controlled clinical trial to evaluate the efficacy of benznidazole (BZ) treatment in patients with chronic Chagas cardiomyopathy (CCC). One important question to be addressed concerns the effectiveness of BZ in reducing overall parasite load in CCC patients, even in the absence of parasitological cure. This report describes the evaluation of multiple procedures for DNA extraction and qPCR-based protocols aiming to establish a standardized methodology for the absolute quantification of T. cruzi DNA in Guanidine-EDTA blood (GEB) samples. A panel of five primer sets directed to the T. cruzi nuclear satellite DNA repeats (Sat-DNA) and to the minicircle DNA conserved regions (kDNA) was compared in either SYBR Green or TaqMan systems. Standard curve parameters such as, amplification efficiency, coefficient of determination and intercept were evaluated, as well as different procedures to generate standard samples containing pre-established T. cruzi DNA concentration. Initially, each primer set was assayed in a SYBR Green qPCR to estimate parasite load in GEB samples from chronic Chagas disease patients. The results achieved from Bayesian transmutability analysis elected the primer sets Cruzi1/Cruzi2 (p=0.0031) and Diaz7/Diaz8 (p=0.0023) coupled to the QIAamp DNA Kit extraction protocol (silica gel column), as the most suitable for monitoring parasitemia in these patients. Comparison between the parasite burden of 150 GEB samples of BENEFIT patients from Argentina, Brazil and Colombia, prior to drug/placebo administration, was performed using Cruzi1/Cruzi2 primers in a SYBR Green approach. The median parasitemia found in patients from Argentina and Colombia (1.93 and 2.31 parasite equivalents/mL, respectively) was around 20 times higher than the one estimated for the Brazilian patients (0.1 parasite equivalents/mL). This difference could be in part due to the complexity of T. cruzi genetic diversity, which is a factor possibly implicated in different clinical presentations of the disease and/or influencing parasitemia levels in infected individuals from different regions of Latin America. The results of SYBR Green qPCR assays herein presented prove this methodology to be more cost efficient than the alternative use of internal fluorogenic probes. In addition, its sensitivity and reproducibility are shown to be adequate to detect low parasitemia burden in patients with chronic Chagas disease.
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U2 - 10.1016/j.actatropica.2012.08.020
DO - 10.1016/j.actatropica.2012.08.020
M3 - Research Article
C2 - 22982466
AN - SCOPUS:84868672506
SN - 0001-706X
VL - 125
SP - 23
EP - 31
JO - Acta Tropica
JF - Acta Tropica
IS - 1
ER -
