Droplet digital PCR as a sensitive tool to assess exposure pressure from Echinococcus multilocularis in intermediate hosts

Massolo, Alessandro; Gerber, Amanda; Umhang, Gérald; Nicholas, Colin; Liccioli, Stefano; Mori, Kensuke; Klein, Claudia

doi:10.1016/j.actatropica.2021.106078

Cited by 4 publications

(7 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After testing on four different human body fluids, the authors found the faecal samples to be a valuable source of parasite material for surveillance in areas where schistosomiasis incidence (or re-occurrence) is a concern. Similarly, probe-based Echinococcus multilocularis assays were used on > 100 liver samples from rodent intermediate hosts, most of which had no macroscopic lesions [ 20 ]. When comparing the ddPCR and qPCR results, it was found that the latter underestimated the exposure pressure.…”

Section: Resultsmentioning

confidence: 99%

Digital PCR: modern solution to parasite diagnostics and population trait genetics

Baltrušis

Höglund

2023

Parasites Vectors

View full text Add to dashboard Cite

The use of polymerase chain reaction (PCR)-based diagnostic approaches has steadily increased in the field of parasitology in recent decades. The most recent large-scale technological modification of the PCR formula, also known as third-generation PCR, came in the form of digital PCR (dPCR). Currently, the most common form of dPCR on the market is digital droplet PCR (ddPCR). Unlike quantitative real-time PCR (qPCR), the digital format allows for highly sensitive, absolute quantification of nucleic acid targets and does not require external standards to be included in the developed assays. Dividing each sample into thousands of compartments and using statistical models also eliminates the need for technical replicates. With unprecedented sensitivity and enforcement of binary endpoint reactions, ddPCR not only allows the use of tiny sample volumes (especially important when working with limited amounts of DNA) but also minimises the impact of variations in amplification efficiency and the presence of inhibitors. As ddPCR is characterised by excellent features such as high throughput, sensitivity and robust quantification, it is widely used as a diagnostic tool in clinical microbiology. Due to recent advances, both the theoretical background and the practical, current applications related to the quantification of nucleic acids of eukaryotic parasites need to be updated. In this review, we present the basics of this technology (particularly useful for new users) and consolidate recent advances in the field with a focus on applications to the study of helminths and protozoan parasites. Graphical Abstract

show abstract

Section: Resultsmentioning

confidence: 99%

Digital PCR: modern solution to parasite diagnostics and population trait genetics

Baltrušis

Höglund

2023

Parasites Vectors

View full text Add to dashboard Cite

show abstract

“…Massolo et al applied a ddPCR assay and assessed its suitability for detecting E. multilocularis DNA in liver samples from intermediate hosts independently of macroscopic lesions. The ddPCR assay provided the highest positivity rates for E. multilocularis DNA (15.09%) compared to qPCR (4.72%) and the presence of macroscopic lesions (1.88%) [12]. Applying such high-sensitivity molecular methods in prevalence studies can significantly increase the accuracy of the prevalence estimations.…”

Section: Cestodes Echinococcus Multilocularismentioning

confidence: 99%

“…High cost No reference samples required Inability to sequence the PCR amplic No standard curve required Better performance in the presence $$$$$$of inhibitors High sensitivity Among the different dPCR forms, the droplet dPCR (ddPCR) is the most comm used in veterinary parasitology [4,7,12]. The general principles and technical advan have been described in detail elsewhere and are beyond the scope of this review a [2,13,14].…”

Section: Absolute Quantificationmentioning

confidence: 99%

“…In the case of E. multilocularis, infection in intermediate hosts is regularly detected through the macroscopic examination of the target organs, like the liver, for lesions [53]. However, the macroscopic examination may fail to detect exposure to the parasite, thus leading to an underestimation of the true infection prevalence [12]. Consequently, macroscopic examination is typically followed by histopathology, immunohistochemistry, or molecular tests [53].…”

Section: Cestodes Echinococcus Multilocularismentioning

confidence: 99%

See 1 more Smart Citation

Current Applications of Digital PCR in Veterinary Parasitology: An Overview

Tsokana,

Symeonidou,

Sioutas

et al. 2023

Parasitologia

View full text Add to dashboard Cite

Digital PCR (dPCR) is an emerging technology that enables the absolute quantification of the targeted nucleic acids. The body of research on the potential applications of this novel tool is growing in human and veterinary medicine. Most of the research on dPCR applications in veterinary parasitology is concentrated on developing and validating new assays to detect and quantify parasites of great financial impact in the food-producing animal industry. Several studies describe the utility of dPCR for individualized medicine in companion animals. Most frequently, dPCR performance is assessed compared to quantitative PCR or Next Generation Sequencing platforms, while others also compare the accuracy of dPCR with traditional parasitological techniques considered gold standard methods. Other researchers describe dPCR assays for surveillance purposes, species identification, and quantification in mixed parasitic infections, the detection of mutations indicative of anthelmintic resistance, and the identification of new targets for drug development. This review provides an overview of the studies that employed dPCR in investigating animal parasites and parasitic diseases from a veterinary perspective and discusses how this novel technology could advance and facilitate diagnosis, surveillance, and the monitoring of response to treatment, or shed light on current gaps in our knowledge of the epidemiology of significant veterinary parasitic diseases.

show abstract

“…Thus, the obstacle of faint band intensity measurement found in cPCR can be eliminated, allowing ddPCR to produce precise and accurate results without external standards, even compared to quantitative real-time PCR, as used in previous parasitic studies [24,28,53,64]; it also led to better detection, when low amounts of DNA target were present [60]. The ddPCR technique has been used for the detection of medical important parasites, including Echinococcus multilocularis [39], Plasmodium spp. [37], Leishmania spp.…”

Section: Introductionmentioning

confidence: 99%

A newly developed droplet digital PCR for <i>Ehrlichia canis</i> detection: comparisons to conventional PCR and blood smear techniques

Wichianchot

Hongsrichan

Maneeruttanarungroj

et al. 2022

The Journal of Veterinary Medical Science

View full text Add to dashboard Cite

Canine monocytic ehrlichiosis caused by Ehrlichia canis infection is a life-threatening vector-borne disease in dogs worldwide. Routine blood smear has very low sensitivity and cannot accurately provide a quantitative result. Conventional PCR (cPCR) and real-time PCR (qPCR) are widely used as molecular methods for E. canis detection. qPCR is quantitative but relies on standard curves of known samples.To overcome this difficulty, this study developed a new E. canis quantitative detection method, using droplet digital polymerase chain reaction (ddPCR). ddPCR was evaluated against cPCR and blood smears. PCR amplicons and genomic DNA (gDNA) from 12 microscopic positive samples were used to identify the limits of detection (LODs) in ddPCR and cPCR. Our ddPCR was assessed in 92 field samples, it was compared with cPCR and blood smears. ddPCR showed LOD = 1.6 copies/reaction, or 78 times more sensitive than cPCR (LOD = 126 copies/reaction), using PCR amplicons as a template, whereas both ddPCR and cPCR had equal LODs at 0.02 ng gDNA/reaction. In addition, ddPCR had 100% sensitivity and 75% specificity for E. canis detection compared to cPCR and no cross-reaction with other blood pathogens was observed. ddPCR identified more positive samples than cPCR and blood smear. ddPCR improved the overall performance of E. canis detection, with a better LOD and comparable sensitivity and specificity to cPCR. The technique might be helpful for diagnosis of E. canis in light infection, evaluating the number of E. canis and follow-up after treatment.

show abstract

Droplet digital PCR as a sensitive tool to assess exposure pressure from Echinococcus multilocularis in intermediate hosts

Cited by 4 publications

References 31 publications

Digital PCR: modern solution to parasite diagnostics and population trait genetics

Digital PCR: modern solution to parasite diagnostics and population trait genetics

Current Applications of Digital PCR in Veterinary Parasitology: An Overview

A newly developed droplet digital PCR for <i>Ehrlichia canis</i> detection: comparisons to conventional PCR and blood smear techniques

Contact Info

Product

Resources

About