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

Abstract

BackgroundA 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/FindingsAn 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 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl 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/µl 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/SignificanceThis study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

Combined parasitological and molecular-based diagnostic tools improve the detection of Trypanosoma cruzi in single peripheral blood samples from patients with Chagas disease

“…This positivity may still be related to several factors, such as the number of transient trypomastigotes in the peripheral circulation at the time of blood collection, appropriate storage conditions of the samples, DNA extraction procedures, amplification targets, the use of the same thermal cycler, and the ability to detect minimal quantities of parasite DNA 7,22,28 . PCR is more convenient than BC since it requires the collection of a lower blood volume, has a shorter processing time, and allows analysis of multiple samples simultaneously 55 .…”

“…Importantly, recent data have demonstrated the standardization and validation of PCR for the diagnosis of chronic ChD. Despite this, the absence of a reliable method to detect and quantify parasitemia is still an obstacle for improving our understanding of the impact of persistent parasitemia in the natural history of ChD and to characterize the parasite load in order to evaluate prognosis and therapy 28 . Recent findings have demonstrated a reliable protocol based on real-time PCR for validation and quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment in patients with ChD 56 .…”

“…PCR assays were performed according to previously optimized protocols 7 that were standardized and validated for the diagnosis of chronic ChD 28 . Blood samples (5mL) were collected from each patient and immediately transferred to sterile tubes containing guanidine/ethylenediaminetetraacetic acid buffer (pH 8.0, 5mL) 29 .…”

Enfermedades parasitarias congénitas

“…Detection of T. cruzi infection was carried out by PCR targeted to kDNA according to standardized protocols (Schijman, et al, 2011).…”

Experiencia terapéutica en un centro para la enfermedad de Chagas

“…13 , utilizando los partidores 121-122 que tienen como secuencia blanco el kinetoplasto del Trypanosoma cruzi. La serología convencional (ELISA IgG e Inmunofluorescencia indirecta IFI) se hace en el Laboratorio de Parasitología Clínica, Sección Parasitología del mismo Instituto usando antígenos solubles para ELISA y figurado para IFI, obtenido de cultivo in vitro de la cepa Tulahuén, origen chileno.…”