Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.
BACKGROUND
The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy.


RESULTS
We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject.


CONCLUSIONS
The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular Dr. Héctor N. Torres (INGEBI-CONICET), Buenos Aires, Argentina.
Tomas
T
Duffy
Carolina I
CI
Cura
Juan C
JC
Ramirez
Teresa
T
Abate
Nelly M
NM
Cayo
Rudy
R
Parrado
Zoraida Diaz
ZD
Bello
Elsa
E
Velazquez
Arturo
A
Muñoz-Calderon
Natalia A
NA
Juiz
Joaquín
J
Basile
Lineth
L
Garcia
Adelina
A
Riarte
Julio R
JR
Nasser
Susana B
SB
Ocampo
Zaida E
ZE
Yadon
Faustino
F
Torrico
Belkisyole Alarcón
BA
de Noya
Isabela
I
Ribeiro
Alejandro G
AG
Schijman
D000328:Adult; D014355:Chagas Disease / Q000469:parasitology*; D002648:Child; D002675:Child, Preschool; D004276:DNA, Satellite / Q000235:genetics*; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D059208:Parasite Load / Q000379:methods* / Q000592:standards; D011247:Pregnancy; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000235:genetics*
D004276:DNA, Satellite