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

PLoS Negl Trop Dis. 2009;3(4):e419. doi: 10.1371/journal.pntd.0000419. Epub 2009 Apr 21.


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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Animals
  • Blood / parasitology*
  • Chagas Disease / parasitology*
  • Child
  • Child, Preschool
  • DNA, Protozoan / blood*
  • Female
  • Humans
  • Infant
  • Infant, Newborn
  • Male
  • Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Trypanosoma cruzi / isolation & purification*


  • DNA, Protozoan