Acceso Abierto

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

Título de la revista
Ramírez, Juan David
Herrera, Giovanny
Galindo Hernández, Carolina
Cruz-Saavedra, Lissa
Muñoz, Marina
Flórez, Carolina
Butcher, Robert



ISSN de la revista
Título del volumen
Public Library of Science

Buscar en:

Métricas alternativas

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/.
Palabras clave
Adult , Routine , Protozoan , 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 , Diagnostic tests , Dna , Humans , Real-time polymerase chain reaction , Sensitivity and specificity , Trypanosoma cruzi
Buscar en: