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

Juan David Ramírez, Giovanny Herrera, Carolina Hernández, Lissa Cruz-Saavedra, Marina Muñoz, Carolina Flórez, Robert Butcher

Resultado de la investigación: Contribución a RevistaArtículo

Resumen

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/μL 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.

Idioma originalEnglish (US)
Número de artículoe0007063
PublicaciónPLoS Neglected Tropical Diseases
Volumen12
N.º12
DOI
EstadoPublished - dic 1 2018

All Science Journal Classification (ASJC) codes

  • Public Health, Environmental and Occupational Health
  • Infectious Diseases

Citar esto

@article{5af10fbe69354230a96c3c6ef240abcc,
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",
abstract = "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/μL 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.",
author = "Ram{\'i}rez, {Juan David} and Giovanny Herrera and Carolina Hern{\'a}ndez and Lissa Cruz-Saavedra and Marina Mu{\~n}oz and Carolina Fl{\'o}rez and Robert Butcher",
year = "2018",
month = "12",
day = "1",
doi = "10.1371/journal.pntd.0007063",
language = "English (US)",
volume = "12",
journal = "PLoS Neglected Tropical Diseases",
issn = "1935-2727",
publisher = "Public Library of Science",
number = "12",

}

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; Hernández, Carolina; Cruz-Saavedra, Lissa; Muñoz, Marina; Flórez, Carolina; Butcher, Robert.

En: PLoS Neglected Tropical Diseases, Vol. 12, N.º 12, e0007063, 01.12.2018.

Resultado de la investigación: Contribución a RevistaArtículo

TY - JOUR

T1 - Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples

AU - Ramírez, Juan David

AU - Herrera, Giovanny

AU - Hernández, Carolina

AU - Cruz-Saavedra, Lissa

AU - Muñoz, Marina

AU - Flórez, Carolina

AU - Butcher, Robert

PY - 2018/12/1

Y1 - 2018/12/1

N2 - 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/μL 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.

AB - 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/μL 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.

UR - http://www.scopus.com/inward/record.url?scp=85059538668&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85059538668&partnerID=8YFLogxK

U2 - 10.1371/journal.pntd.0007063

DO - 10.1371/journal.pntd.0007063

M3 - Article

VL - 12

JO - PLoS Neglected Tropical Diseases

JF - PLoS Neglected Tropical Diseases

SN - 1935-2727

IS - 12

M1 - e0007063

ER -