International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.
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.


RESULTS
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 5.10(-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.


CONCLUSIONS
This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.
Laboratorio de Biología Molecular de Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina. schijman@dna.uba.ar
Alejandro G
AG
Schijman
Margarita
M
Bisio
Liliana
L
Orellana
Mariela
M
Sued
Tomás
T
Duffy
Ana M
AM
Mejia Jaramillo
Carolina
C
Cura
Frederic
F
Auter
Vincent
V
Veron
Yvonne
Y
Qvarnstrom
Stijn
S
Deborggraeve
Gisely
G
Hijar
Inés
I
Zulantay
Raúl Horacio
RH
Lucero
Elsa
E
Velazquez
Tatiana
T
Tellez
Zunilda
Z
Sanchez Leon
Lucia
L
Galvão
Debbie
D
Nolder
María
M
Monje Rumi
José E
JE
Levi
Juan D
JD
Ramirez
Pilar
P
Zorrilla
María
M
Flores
Maria I
MI
Jercic
Gladys
G
Crisante
Néstor
N
Añez
Ana M
AM
De Castro
Clara I
CI
Gonzalez
Karla
K
Acosta Viana
Pedro
P
Yachelini
Faustino
F
Torrico
Carlos
C
Robello
Patricio
P
Diosque
Omar
O
Triana Chavez
Christine
C
Aznar
Graciela
G
Russomando
Philippe
P
Büscher
Azzedine
A
Assal
Felipe
F
Guhl
Sergio
S
Sosa Estani
Alexandre
A
DaSilva
Constança
C
Britto
Alejandro
A
Luquetti
Janis
J
Ladzins
D014355:Chagas Disease / Q000175:diagnosis* / Q000469:parasitology; D016054:DNA, Protozoan / Q000097:blood*; D006801:Humans; D007391:International Cooperation; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification*
D016054:DNA, Protozoan
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