Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples

Background: The recent development of novel Polymerase Chain Reaction (PCR) technologies that confer theoretical advantages over quantitative PCR has considerable potential in the diagnosis of low load infections, such as Trypanosoma cruzi in the chronic phase of Chagas disease. We evaluated the uti...

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Autores:
Tipo de recurso:
Fecha de publicación:
2018
Institución:
Universidad del Rosario
Repositorio:
Repositorio EdocUR - U. Rosario
Idioma:
eng
OAI Identifier:
oai:repository.urosario.edu.co:10336/22821
Acceso en línea:
https://doi.org/10.1371/journal.pntd.0007063
https://repository.urosario.edu.co/handle/10336/22821
Palabra clave:
Adult
Blood sampling
Chagas disease
Child
Controlled study
Diagnostic test accuracy study
Dna extraction
Droplet digital polymerase chain reaction
Enzyme linked immunosorbent assay
Epimastigote
Fever
Gene dosage
Hemagglutination inhibition test
Hemagglutination test
Hepatomegaly
Human
Immunofluorescence test
Life cycle stage
Limit of detection
Nonhuman
Parasite load
Parasitological parameters
Predictive value
Real time polymerase chain reaction
Reproducibility
Sensitivity and specificity
Splenomegaly
Tandem repeat
Trypanosoma cruzi
Trypomastigote
Blood
Chagas disease
Classification
Diagnostic test
Evaluation study
Genetics
Isolation and purification
Parasitology
Procedures
Trypanosoma cruzi
Protozoal dna
Chagas disease
Humans
Real-time polymerase chain reaction
Sensitivity and specificity
Trypanosoma cruzi
Routine
Protozoan
Diagnostic tests
Dna
Rights
License
Abierto (Texto Completo)
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dc.title.spa.fl_str_mv Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples
title Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples
spellingShingle Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples
Adult
Blood sampling
Chagas disease
Child
Controlled study
Diagnostic test accuracy study
Dna extraction
Droplet digital polymerase chain reaction
Enzyme linked immunosorbent assay
Epimastigote
Fever
Gene dosage
Hemagglutination inhibition test
Hemagglutination test
Hepatomegaly
Human
Immunofluorescence test
Life cycle stage
Limit of detection
Nonhuman
Parasite load
Parasitological parameters
Predictive value
Real time polymerase chain reaction
Reproducibility
Sensitivity and specificity
Splenomegaly
Tandem repeat
Trypanosoma cruzi
Trypomastigote
Blood
Chagas disease
Classification
Diagnostic test
Evaluation study
Genetics
Isolation and purification
Parasitology
Procedures
Trypanosoma cruzi
Protozoal dna
Chagas disease
Humans
Real-time polymerase chain reaction
Sensitivity and specificity
Trypanosoma cruzi
Routine
Protozoan
Diagnostic tests
Dna
title_short Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples
title_full Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples
title_fullStr Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples
title_full_unstemmed Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples
title_sort Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples
dc.subject.keyword.spa.fl_str_mv Adult
Blood sampling
Chagas disease
Child
Controlled study
Diagnostic test accuracy study
Dna extraction
Droplet digital polymerase chain reaction
Enzyme linked immunosorbent assay
Epimastigote
Fever
Gene dosage
Hemagglutination inhibition test
Hemagglutination test
Hepatomegaly
Human
Immunofluorescence test
Life cycle stage
Limit of detection
Nonhuman
Parasite load
Parasitological parameters
Predictive value
Real time polymerase chain reaction
Reproducibility
Sensitivity and specificity
Splenomegaly
Tandem repeat
Trypanosoma cruzi
Trypomastigote
Blood
Chagas disease
Classification
Diagnostic test
Evaluation study
Genetics
Isolation and purification
Parasitology
Procedures
Trypanosoma cruzi
Protozoal dna
Chagas disease
Humans
Real-time polymerase chain reaction
Sensitivity and specificity
Trypanosoma cruzi
topic Adult
Blood sampling
Chagas disease
Child
Controlled study
Diagnostic test accuracy study
Dna extraction
Droplet digital polymerase chain reaction
Enzyme linked immunosorbent assay
Epimastigote
Fever
Gene dosage
Hemagglutination inhibition test
Hemagglutination test
Hepatomegaly
Human
Immunofluorescence test
Life cycle stage
Limit of detection
Nonhuman
Parasite load
Parasitological parameters
Predictive value
Real time polymerase chain reaction
Reproducibility
Sensitivity and specificity
Splenomegaly
Tandem repeat
Trypanosoma cruzi
Trypomastigote
Blood
Chagas disease
Classification
Diagnostic test
Evaluation study
Genetics
Isolation and purification
Parasitology
Procedures
Trypanosoma cruzi
Protozoal dna
Chagas disease
Humans
Real-time polymerase chain reaction
Sensitivity and specificity
Trypanosoma cruzi
Routine
Protozoan
Diagnostic tests
Dna
dc.subject.keyword.eng.fl_str_mv Routine
Protozoan
Diagnostic tests
Dna
description Background: The recent development of novel Polymerase Chain Reaction (PCR) technologies that confer theoretical advantages over quantitative PCR has considerable potential in the diagnosis of low load infections, such as Trypanosoma cruzi in the chronic phase of Chagas disease. We evaluated the utility of the digital droplet (dd)PCR platform in the detection of T. cruzi infection. Methodology/Principal findings: We imported a validated qPCR assay targeting the T. cruzi satellite tandem repeat (TcSTR) region to the ddPCR platform. Following optimization, we tested and repeated a standard curve of TcI epimastigotes to characterise the analytical performance of the assay on the ddPCR platform. We compared this to published qPCR performance data, and the performance of the qPCR assay in our own testing. We subsequently tested a panel of 192 previously characterized DNA specimens, extracted from the blood of individuals with and without T. cruzi infection. The assay performed well on the ddPCR platform, showing a limit of detection of 5 copies/?L or 1 parasite/mL. This was higher than the published limit of detection for qPCR, which was 0.46 parasites/mL. The ddPCR platform was not significantly more accurate than qPCR at any concentration tested. However, the clinical sensitivity and specificity of the assay were both 100% with perfect agreement between qPCR and ddPCR positive and negative result calling in clinical specimens. An average of 9,286 copies of TcSTR were detected per parasite. Conclusions/Significance: The use of the ddPCR platform to run this assay was comparable, but not superior in terms of performance, to the qPCR platform. © 2018 Ramírez et al. http://creativecommons.org/licenses/by/4.0/.
publishDate 2018
dc.date.created.spa.fl_str_mv 2018
dc.date.accessioned.none.fl_str_mv 2020-05-25T23:58:12Z
dc.date.available.none.fl_str_mv 2020-05-25T23:58:12Z
dc.type.eng.fl_str_mv article
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dc.type.spa.spa.fl_str_mv Artículo
dc.identifier.doi.none.fl_str_mv https://doi.org/10.1371/journal.pntd.0007063
dc.identifier.issn.none.fl_str_mv 19352727
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dc.identifier.uri.none.fl_str_mv https://repository.urosario.edu.co/handle/10336/22821
url https://doi.org/10.1371/journal.pntd.0007063
https://repository.urosario.edu.co/handle/10336/22821
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dc.language.iso.spa.fl_str_mv eng
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dc.relation.citationIssue.none.fl_str_mv No. 12
dc.relation.citationTitle.none.fl_str_mv PLoS Neglected Tropical Diseases
dc.relation.citationVolume.none.fl_str_mv Vol. 12
dc.relation.ispartof.spa.fl_str_mv PLoS Neglected Tropical Diseases, ISSN:19352727, 19352735, Vol.12, No.12 (2018)
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institution Universidad del Rosario
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dc.source.reponame.spa.fl_str_mv reponame:Repositorio Institucional EdocUR
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spelling 101171611860007f5f951-ff31-4b0f-94f0-7492d0a86f05520857956003a39d4a1-8a8d-431d-951a-63c54f374456453006a5-2ec3-4faf-8e32-a9d7075d519d1ba4f510-30ed-403d-b3df-d6d598932a1d81002983-a1d5-453d-983d-72b289a34d022020-05-25T23:58:12Z2020-05-25T23:58:12Z2018Background: The recent development of novel Polymerase Chain Reaction (PCR) technologies that confer theoretical advantages over quantitative PCR has considerable potential in the diagnosis of low load infections, such as Trypanosoma cruzi in the chronic phase of Chagas disease. We evaluated the utility of the digital droplet (dd)PCR platform in the detection of T. cruzi infection. Methodology/Principal findings: We imported a validated qPCR assay targeting the T. cruzi satellite tandem repeat (TcSTR) region to the ddPCR platform. Following optimization, we tested and repeated a standard curve of TcI epimastigotes to characterise the analytical performance of the assay on the ddPCR platform. We compared this to published qPCR performance data, and the performance of the qPCR assay in our own testing. We subsequently tested a panel of 192 previously characterized DNA specimens, extracted from the blood of individuals with and without T. cruzi infection. The assay performed well on the ddPCR platform, showing a limit of detection of 5 copies/?L or 1 parasite/mL. This was higher than the published limit of detection for qPCR, which was 0.46 parasites/mL. The ddPCR platform was not significantly more accurate than qPCR at any concentration tested. However, the clinical sensitivity and specificity of the assay were both 100% with perfect agreement between qPCR and ddPCR positive and negative result calling in clinical specimens. An average of 9,286 copies of TcSTR were detected per parasite. Conclusions/Significance: The use of the ddPCR platform to run this assay was comparable, but not superior in terms of performance, to the qPCR platform. © 2018 Ramírez et al. http://creativecommons.org/licenses/by/4.0/.application/pdfhttps://doi.org/10.1371/journal.pntd.00070631935272719352735https://repository.urosario.edu.co/handle/10336/22821engPublic Library of ScienceNo. 12PLoS Neglected Tropical DiseasesVol. 12PLoS Neglected Tropical Diseases, ISSN:19352727, 19352735, Vol.12, No.12 (2018)https://www.scopus.com/inward/record.uri?eid=2-s2.0-85059538668&doi=10.1371%2fjournal.pntd.0007063&partnerID=40&md5=c2f74fcf24de4d1fc116e61ac4e88245Abierto (Texto Completo)http://purl.org/coar/access_right/c_abf2instname:Universidad del Rosarioreponame:Repositorio Institucional EdocURAdultBlood samplingChagas diseaseChildControlled studyDiagnostic test accuracy studyDna extractionDroplet digital polymerase chain reactionEnzyme linked immunosorbent assayEpimastigoteFeverGene dosageHemagglutination inhibition testHemagglutination testHepatomegalyHumanImmunofluorescence testLife cycle stageLimit of detectionNonhumanParasite loadParasitological parametersPredictive valueReal time polymerase chain reactionReproducibilitySensitivity and specificitySplenomegalyTandem repeatTrypanosoma cruziTrypomastigoteBloodChagas diseaseClassificationDiagnostic testEvaluation studyGeneticsIsolation and purificationParasitologyProceduresTrypanosoma cruziProtozoal dnaChagas diseaseHumansReal-time polymerase chain reactionSensitivity and specificityTrypanosoma cruziRoutineProtozoanDiagnostic testsDnaEvaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samplesarticleArtículohttp://purl.org/coar/version/c_970fb48d4fbd8a85http://purl.org/coar/resource_type/c_6501Ramírez, Juan DavidHerrera, GiovannyGalindo Hernández, CarolinaCruz-Saavedra, LissaMuñoz, MarinaFlórez, CarolinaButcher, RobertORIGINALjournal-pntd-0007063.pdfapplication/pdf1659607https://repository.urosario.edu.co/bitstreams/af8ec8bd-4ca2-4a44-8f95-af6d1f425b1e/download113d72e01dea7e7015a3baded52376d7MD51TEXTjournal-pntd-0007063.pdf.txtjournal-pntd-0007063.pdf.txtExtracted texttext/plain55528https://repository.urosario.edu.co/bitstreams/1209956e-240f-46f0-b537-6ea134df5b99/downloadca6e220c3b2bf5d1616a0af518903b6aMD52THUMBNAILjournal-pntd-0007063.pdf.jpgjournal-pntd-0007063.pdf.jpgGenerated Thumbnailimage/jpeg4539https://repository.urosario.edu.co/bitstreams/25770059-4f67-4473-b586-cb3107bf3c2f/downloadd243ecc016df9c45f2f3a079fcd78763MD5310336/22821oai:repository.urosario.edu.co:10336/228212022-08-27 13:05:58.984https://repository.urosario.edu.coRepositorio institucional EdocURedocur@urosario.edu.co