Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro, Rio de Janeiro, Brazil.
Acta Trop. 2013 Jan;125(1):23-31. doi: 10.1016/j.actatropica.2012.08.020. Epub 2012 Sep 12.
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.
实时荧光定量 PCR（qPCR）是一种准确的方法，可以定量检测克氏锥虫 DNA，并可用于监测接受化疗的恰加斯病（CD）患者的寄生虫血症。Benznidazole Evaluation for Interrupting Trypanosomiasis（BENEFIT）研究是一项国际性、多中心、随机、双盲、安慰剂对照的临床试验，旨在评估贝那唑（BZ）治疗慢性恰加斯心肌病（CCC）患者的疗效。一个重要的问题是，BZ 治疗是否能有效降低 CCC 患者的总体寄生虫负荷，即使没有寄生虫学治愈。本报告描述了对多种 DNA 提取程序和基于 qPCR 的方案进行评估，旨在为胍-EDTA 血液（GEB）样本中克氏锥虫 DNA 的绝对定量建立标准化方法。比较了五组针对克氏锥虫核卫星 DNA 重复序列（Sat-DNA）和小环 DNA 保守区（kDNA）的引物组，分别在 SYBR Green 或 TaqMan 系统中进行。评估了扩增效率、决定系数和截距等标准曲线参数，以及生成包含预定义克氏锥虫 DNA 浓度的标准样品的不同程序。最初，在 SYBR Green qPCR 中检测每个引物组，以估计慢性 CD 患者 GEB 样本中的寄生虫负荷。贝叶斯转导可变性分析的结果选择了引物组 Cruzi1/Cruzi2（p=0.0031）和 Diaz7/Diaz8（p=0.0023）与 QIAamp DNA 试剂盒提取方案（硅胶柱）结合，最适合监测这些患者的寄生虫血症。使用 Cruzi1/Cruzi2 引物在 SYBR Green 方法中，对来自阿根廷、巴西和哥伦比亚的 BENEFIT 患者的 150 个 GEB 样本进行药物/安慰剂给药前的寄生虫负担比较。来自阿根廷和哥伦比亚的患者的中位寄生虫血症（分别为 1.93 和 2.31 个寄生虫当量/mL）约为巴西患者估计值（0.1 个寄生虫当量/mL）的 20 倍。这种差异可能部分归因于克氏锥虫遗传多样性的复杂性，这可能是导致疾病不同临床表现和/或影响拉丁美洲不同地区感染个体寄生虫血症水平的一个因素。本文中呈现的 SYBR Green qPCR 检测结果证明，该方法比使用内部荧光探针更具成本效益。此外，其灵敏度和重现性足以检测慢性恰加斯病患者的低寄生虫血症负担。
