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

Abstract

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.

Infection With Trypanosoma cruzi and Progression to Cardiomyopathy

“…As discussed, recent evidence obtained by real-time polymerase chain reaction at baseline from the BENEFIT trial indicates that patients with mild cardiomyopathy from Colombia and Argentina have a 20-fold higher parasite load compared with Brazilian patients. 14 The TcII strain is the most prevalent in both Argentina and Brazil and TcI is most prevalent in Colombia, suggesting that other factors besides strain may play a role in determining both parasite load and pathogenicity. It remains unclear whether higher T cruzi parasite load indeed accelerates both the progression and severity of the disease.…”

“…14 Recent substudies from the Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) trial 13,14 show a striking difference in parasite load among patients infected with T cruzi from Argentina, Brazil, and Colombia with early Chagas cardiomyopathy. The median parasite load in patients from Argentina and Colombia was ≈20 times higher than that observed in Brazilian patients.…”

Different Therapeutic Outcomes of Benznidazole and VNI Treatments in Different Genders in Mouse Experimental Models of Trypanosoma cruzi Infection

“…However, one animal from the male Bz-treated group, although displaying positive relapse (only one parasite counted into 50 fields by light microscopy), had no detectable parasite DNA in the corresponding qPCR (Fig. 2B), possibly because the DNA amount was under the limit of detection of the qPCR assay (13). It is worth mentioning that despite the higher strain Y inoculum (10 4 parasites/mouse versus 5 ϫ 10 3 parasites/mouse in the case of Colombiana) and shorter period of treatment (30 versus 60 days), animals treated with Bz (both male and female) exhibited lower parasite loads, as determined by qPCR, when strain Y was used, confirming the naturally resistant profile of the Colombiana strain (Fig.…”

Antitrypanosomal Activity of Sterol 14α-Demethylase (CYP51) Inhibitors VNI and VFV in the Swiss Mouse Models of Chagas Disease Induced by the Trypanosoma cruzi Y Strain

“…For qPCR, 500 l of blood was diluted 1:2 in a volume of guanidine solution (6 M guanidine-HCl, 0.2 M EDTA) and heated for 90 s in boiling water. Guanidine-EDTA blood (GEB) samples were processed with the QIAamp DNA minikit (Qiagen) as previously described (39). Multiplex real-time qPCR assays targeting the T. cruzi satellite nuclear DNA and the exogenous internal amplification control (plasmid pZErO-2 containing an insert from the Arabidopsis thaliana aquaporin gene, 40 amplification cycles) were performed as previously described (40).…”