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spelling paper:paper_NIS27444_v5_n1_p_Schijman2023-06-08T16:40:05Z International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients protozoal DNA protozoal DNA analytic method article blood analysis Chagas disease DNA determination DNA extraction DNA purification gene amplification gene dosage human methodology nonhuman parasite identification polymerase chain reaction real time polymerase chain reaction sensitivity and specificity serodiagnosis single nucleotide polymorphism Trypanosoma cruzi validation process blood Chagas disease clinical trial comparative study evaluation genetics international cooperation isolation and purification multicenter study parasitology Chagas Disease DNA, Protozoan Humans International Cooperation Parasitology Polymerase Chain Reaction Sensitivity and Specificity Trypanosoma cruzi 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 5.10-3 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/Significance:This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples. 2011 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_NIS27444_v5_n1_p_Schijman http://hdl.handle.net/20.500.12110/paper_NIS27444_v5_n1_p_Schijman
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-134
collection Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic protozoal DNA
protozoal DNA
analytic method
article
blood analysis
Chagas disease
DNA determination
DNA extraction
DNA purification
gene amplification
gene dosage
human
methodology
nonhuman
parasite identification
polymerase chain reaction
real time polymerase chain reaction
sensitivity and specificity
serodiagnosis
single nucleotide polymorphism
Trypanosoma cruzi
validation process
blood
Chagas disease
clinical trial
comparative study
evaluation
genetics
international cooperation
isolation and purification
multicenter study
parasitology
Chagas Disease
DNA, Protozoan
Humans
International Cooperation
Parasitology
Polymerase Chain Reaction
Sensitivity and Specificity
Trypanosoma cruzi
spellingShingle protozoal DNA
protozoal DNA
analytic method
article
blood analysis
Chagas disease
DNA determination
DNA extraction
DNA purification
gene amplification
gene dosage
human
methodology
nonhuman
parasite identification
polymerase chain reaction
real time polymerase chain reaction
sensitivity and specificity
serodiagnosis
single nucleotide polymorphism
Trypanosoma cruzi
validation process
blood
Chagas disease
clinical trial
comparative study
evaluation
genetics
international cooperation
isolation and purification
multicenter study
parasitology
Chagas Disease
DNA, Protozoan
Humans
International Cooperation
Parasitology
Polymerase Chain Reaction
Sensitivity and Specificity
Trypanosoma cruzi
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients
topic_facet protozoal DNA
protozoal DNA
analytic method
article
blood analysis
Chagas disease
DNA determination
DNA extraction
DNA purification
gene amplification
gene dosage
human
methodology
nonhuman
parasite identification
polymerase chain reaction
real time polymerase chain reaction
sensitivity and specificity
serodiagnosis
single nucleotide polymorphism
Trypanosoma cruzi
validation process
blood
Chagas disease
clinical trial
comparative study
evaluation
genetics
international cooperation
isolation and purification
multicenter study
parasitology
Chagas Disease
DNA, Protozoan
Humans
International Cooperation
Parasitology
Polymerase Chain Reaction
Sensitivity and Specificity
Trypanosoma cruzi
description 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 5.10-3 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/Significance:This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.
title International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients
title_short International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients
title_full International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients
title_fullStr International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients
title_full_unstemmed International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients
title_sort international study to evaluate pcr methods for detection of trypanosoma cruzi dna in blood samples from chagas disease patients
publishDate 2011
url https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_NIS27444_v5_n1_p_Schijman
http://hdl.handle.net/20.500.12110/paper_NIS27444_v5_n1_p_Schijman
_version_ 1769810416451452928