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ANALYTICAL VALIDATION OF QUANTITATIVE REAL-TIME PCR METHODS FOR QUANTIFICATION OF TRYPANOSOMA CRUZI DNA IN BLOOD SAMPLES FROM CHAGAS DISEASE PATIENTS
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Abstract
An international study was performed by 26 experienced PCR laboratories from 14 countries to
assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of
TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples
from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and
kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control.
Reportable range, analytical sensitivity, limits of detection and quantification, and precision were
estimated according to international guidelines. In addition, inclusivity and exclusivity were
estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing
units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156
blood samples provided by the participant laboratories, including samples from acute and chronic
patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA
qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and
0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high
concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA
qPCRs. This effort is a major step toward international validation of qPCR methods for the
quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate
biomarker for diagnosis and treatment monitoring for patients with Chagas disease.
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