Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples.
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.
Piron
Maria
M
Transfusion Safety Laboratory, Banc de Sang i Teixits, Barcelona, Spain. mpiron@vhebron.net
Fisa
Roser
R
Casamitjana
Natalia
N
López-Chejade
Paulo
P
Puig
Lluís
L
Vergés
Mireia
M
Gascón
Joaquim
J
Gómez i Prat
Jordi
J
Portús
Montserrat
M
Sauleda
Sílvia
S
D000818:Animals; D014355:Chagas Disease / Q000175:diagnosis*; D016054:DNA, Protozoan / Q000097:blood*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification*
D016054:DNA, Protozoan