Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients

Abstract

Background: This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence.

Methodology/principal findings: The Q-PCR has a detection limit of 0.1 and 0.01 parasites/mL, with a dynamic range of 10(6) and 10(7) for Silvio X10 cl1 (T. cruzi I) and Cl Brener stocks (T. cruzi IIe), respectively, an efficiency of 99%, and a coefficient of determination (R(2)) of 0.998. In order to express accurately the parasitic loads: (1) we adapted a commercial kit based on silica-membrane technology to enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimize PCR inhibition; (2) results were normalized incorporating a linearized plasmid as an internal standard of the whole procedure; and (3) a correction factor according to the representativity of satellite sequences in each parasite lineage group was determined using a modified real-time PCR protocol (Lg-PCR). The Q-PCR strategy was applied (1) to estimate basal parasite loads in 43 pediatric Chagas disease patients, (2) to follow-up 38 of them receiving treatment with benznidazole, and (3) to monitor three chronic Chagas heart disease patients who underwent heart-transplantation and displayed events of clinical reactivation due to immunosupression.

Conclusion/significance: All together, the high analytical sensitivity of the Q-PCR strategy, the low levels of intra- and inter-assay variations, as well as the accuracy provided by the Lg-PCR based correction factor support this methodology as a key laboratory tool for monitoring clinical reactivation and etiological treatment outcome in Chagas disease patients.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Dynamic range of the T. cruzi satellite DNA based Q-PCR.

Results are expressed as the number of parasites per milliliter of blood and represent the average of 5 independent experiments. Slope = −3.35. Efficiency = 99%. Dynamic range: 0.01–105 p/mL. R square: 0.998. C(t): cycle threshold.

Figure 2. Melting curve analysis of satellite amplicons from reference stocks.

Group I satellite amplicons show melting temperatures above 85°C, whereas Group II render amplification products with melting temperatures below 85°C.

Figure 3. Parasitic loads in peripheral blood samples from pediatric patients.

(A) Association between basal parasitic loads and patients' ages in 43 pediatric cases. Coefficient of correlation: −0.5832; P

Figure 4. Follow-up of Chronic Chagas heart disease patients after heart transplantation.

Parasitic loads in peripheral blood samples of Chronic Chagas heart disease patients with clinical reactivation due to immunosupression after heart transplantation. * Time of diagnosis of clinical reactivation and etiological treatment.