Article
Towards the establishment of a consensus real-time qPCR to monitor Trypanosoma cruzi parasitemia in patients with chronic Chagas disease cardiomyopathy: A substudy from the BENEFIT trial
Registro en:
MOREIRA, Octacilio C,; et al. Towards the establishment of a consensus real-time qPCR to monitor Trypanosoma cruzi parasitemia in patients with chronic Chagas disease cardiomyopathy: A substudy from the BENEFIT trial. Acta Tropica, v.125, n.1, p. 23– 31, 2013.
0001-706X
10.1016/j.actatropica.2012.08.020
Autor
Moreira, Octacílio C.
Ramirez, Juan David
Velásquez, Elsa
Melo, Myllena F. A. Dias
Ferreira, Carolina Lima
Guhl, Felipe
Sosa-Estani, Sergio
Marin Neto, José Antonio
Resumen
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.