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INTERNATIONAL STUDY TO EVALUATE PCR METHODS FOR DETECTION OF TRYPANOSOMA CRUZI DNA IN BLOOD SAMPLES FROM CHAGAS DISEASE PATIENTS
Doença de Chagas
Reação em Cadeia da Polimerase
Métodos
Amostras de sangue humano
Author
Affilliation
Abstract
Background: A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate
diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field.
The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an
external quality evaluation.
Methodology/Findings: An international collaborative study was launched by expert PCR laboratories from 16 countries.
Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete
typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA
blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR
tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the
remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better
specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better
specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/
mL whereas specific kDNA tests detected 5.1023 par/mL. Sixteen specific and coherent methods had a Good Performance in
both sets A and B (10 fg/ml of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities
and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets
A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets
of samples, detecting at least 10 fg/ml for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of
85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing
tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent
extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction
followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction
followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method
(LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These
four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the
performance obtained by the participating laboratories.
Conclusion/Significance: This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.
Keywords in Portuguese
Trypanosoma cruziDoença de Chagas
Reação em Cadeia da Polimerase
Métodos
Amostras de sangue humano
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