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


RESULTS
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.


CONCLUSIONS
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.
Duffy
Tomas
T
Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina.
Bisio
Margarita
M
Altcheh
Jaime
J
Burgos
Juan Miguel
JM
Diez
Mirta
M
Levin
Mariano Jorge
MJ
Favaloro
Roberto Rene
RR
Freilij
Hector
H
Schijman
Alejandro Gabriel
AG
D000293:Adolescent; D000818:Animals; D001769:Blood / Q000469:parasitology*; D014355:Chagas Disease / Q000469:parasitology*; D002648:Child; D002675:Child, Preschool; D016054:DNA, Protozoan / Q000097:blood*; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000302:isolation & purification*
D016054:DNA, Protozoan