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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| Lenguaje: | Inglés (English) |
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Public Library of Science
2018
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| Acceso en línea: | https://repository.urosario.edu.co/handle/10336/22821 https://doi.org/10.1371/journal.pntd.0007063 |
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ir-10336-228212022-08-27T18:05:58Z Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples Ramírez, Juan David Herrera, Giovanny Galindo-Hernández, Carolina Cruz-Saavedra, Lissa Muñoz, Marina Flórez, Carolina Butcher, Robert 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 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/. 2018 2020-05-25T23:58:12Z info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 19352727 19352735 https://repository.urosario.edu.co/handle/10336/22821 https://doi.org/10.1371/journal.pntd.0007063 eng info:eu-repo/semantics/openAccess application/pdf Public Library of Science instname:Universidad del Rosario |
| institution |
EdocUR - Universidad del Rosario |
| collection |
DSpace |
| language |
Inglés (English) |
| 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 |
| spellingShingle |
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 Ramírez, Juan David Herrera, Giovanny Galindo-Hernández, Carolina Cruz-Saavedra, Lissa Muñoz, Marina Flórez, Carolina Butcher, Robert Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples |
| 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/. |
| format |
Artículo (Article) |
| author |
Ramírez, Juan David Herrera, Giovanny Galindo-Hernández, Carolina Cruz-Saavedra, Lissa Muñoz, Marina Flórez, Carolina Butcher, Robert |
| author_facet |
Ramírez, Juan David Herrera, Giovanny Galindo-Hernández, Carolina Cruz-Saavedra, Lissa Muñoz, Marina Flórez, Carolina Butcher, Robert |
| author_sort |
Ramírez, Juan David |
| 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 |
| 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 |
| publisher |
Public Library of Science |
| publishDate |
2018 |
| url |
https://repository.urosario.edu.co/handle/10336/22821 https://doi.org/10.1371/journal.pntd.0007063 |
| _version_ |
1743431084577652736 |
| score |
12,131701 |