Abstract
BackgroundThe 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 findingsWe 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/SignificanceThe use of the ddPCR platform to run this assay was comparable, but not superior in terms of performance, to the qPCR platform.
Highlights
Chagas disease, caused by Trypanosoma cruzi, is a complex chronic pathology that affects around 8 million people worldwide and represents a serious public health problem [1]
Chagas disease is a complex pathology caused by the parasite Trypanosoma cruzi
Digital dropletPCR is a next-generation Polymerase Chain Reaction (PCR) technology that enables the absolute quantitation of nucleic acids
Summary
Chagas disease, caused by Trypanosoma cruzi, is a complex chronic pathology that affects around 8 million people worldwide and represents a serious public health problem [1]. In the acute phase of infection and disease, the parasitic load is sufficiently high that direct methods of parasitological observation such as a blood smear and hematocrit are recommended to diagnose the disease [6,7,8]. The chronic phase is characterized by a low and intermittent parasitic load that is not likely to be detected using direct parasitological methods. Serological assays such as indirect immunofluorescence assays, indirect hemagglutination test and enzyme linked immunosorbent assays (ELISA) are recommended to diagnose the disease [9,10,11,12]. We evaluated the utility of the digital droplet (dd)PCR platform in the detection of T. cruzi infection
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call