International Study to Evaluate PCR Methods for Detection of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

SCHIJMAN, A.G.; BISIO, M.; ORELLANA, L.; SUED, M-; DUFFY, T.

PLOS NEGLECTED TROPICAL DISEASES

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2011 vol. 5 p. 931 - 931

1935-2735

AbstractBackground: A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accuratediagnostics 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 anexternal 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 discretetyping units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTAblood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCRtests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and theremainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed betterspecificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented betterspecificity 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 inboth sets A and B (10 fg/ml of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificitiesand accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing SetsA and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three setsof samples, detecting at least 10 fg/ml for each DNA stock, 0.5 par/mL and a sensitivity between 83.3?94.4%, specificity of85?95%, accuracy of 86.8?89.5% and kappa index of 0.7?0.8 compared to consensus PCR reports of the 16 good performingtests and 63?69%, 100%, 71.4?76.2% and 0.4?0.5, respectively compared to serodiagnosis. Method LbD2 used solventextraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extractionfollowed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extractionfollowed 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). Thesefour methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming theperformance obtained by the participating laboratories.Conclusion/Significance: This study represents a first crucial step towards international validation of PCR procedures fordetection of T. cruzi in human blood samples.