Development of a Real-Time PCR Assay for Trypanosoma Cruzi Detection in Blood Samples

Acta Trop. 2007 Sep;103(3):195-200. doi: 10.1016/j.actatropica.2007.05.019. Epub 2007 Jun 23.

Abstract

The aim of this study was to develop a real-time PCR technique to detect Trypanosoma cruzi DNA in blood of chagasic patients. Analytical sensitivity of the real-time PCR was assessed by two-fold serial dilutions of T. cruzi epimastigotes in seronegative blood (7.8 down to 0.06 epimastigotes/mL). Clinical sensitivity was tested in 38 blood samples from adult chronic chagasic patients and 1 blood sample from a child with an acute congenital infection. Specificity was assessed with 100 seronegative subjects from endemic areas, 24 seronegative subjects from non-endemic area and 20 patients with Leishmania infantum-visceral leishmaniosis. Real-time PCR was designed to amplify a fragment of 166 bp in the satellite DNA of T. cruzi. As internal control of amplification human RNase P gene was coamplified, and uracil-N-glycosylase (UNG) was added to the reaction to avoid false positives due to PCR contamination. Samples were also analysed by a previously described nested PCR (N-PCR) that amplifies the same DNA region as the real-time PCR. Sensitivity of the real-time PCR was 0.8 parasites/mL (50% positive hit rate) and 2 parasites/mL (95% positive hit rate). None of the seronegative samples was positive by real-time PCR, resulting in 100% specificity. Sixteen out of 39 patients were positive by real-time PCR (41%). Concordance of results with the N-PCR was 90%. In conclusion, real-time PCR provides an optimal alternative to N-PCR, with similar sensitivity and higher throughput, and could help determine ongoing parasitaemia in chagasic patients.

Publication types

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

MeSH terms

  • Animals
  • Chagas Disease / diagnosis*
  • DNA, Protozoan / blood*
  • Humans
  • Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Trypanosoma cruzi / genetics
  • Trypanosoma cruzi / isolation & purification*

Substances

  • DNA, Protozoan