Evaluation of Droplet Digital PCR Assay for the Diagnosis of Candidemia in Blood Samples.

Biao Chen,Shaodong Bian,Wenqiang Li,Chen Liu,Yufeng Jiang,Xiaojie Zhang,Yingguang Xie,Yahui Feng,Ning Zhang,Dongmei Shi,Dongmei Li,Zhiya Yang,Renzhe Li

doi:10.3389/fmicb.2021.700008

Abstract

Numerous studies have shown that droplet digital PCR (ddPCR) is a promising tool for the diagnosis of pathogens, especially in samples with low concentrations of pathogenic DNA. An early diagnosis of candidemia is critical for the effective treatment of patients. In this study, we evaluated the sensitivity and specificity of ddPCR assay for Candida DNA detection both in vitro by mixing fungal cells with human blood and in vivo by analyzing blood samples from infected mice and patients with suspected candidemia. The results showed that ddPCR assay could detect a minimum of 4.5 DNA copies per reaction in blood samples. ddPCR showed higher sensitivity and specificity for Candida DNA detection than traditional culture and quantitative PCR (qPCR) methods and also exhibited significantly better positive and negative predictive values than the culture and qPCR methods that were commonly used in clinical practice. Hence, our study demonstrates that ddPCR assay is a promising method for the timely diagnosis of candidemia and could be useful for monitoring the treatment of candidemia.

Highlights

Candidemia is a leading cause of fungal infections among neonates and infants, and it affects immunocompromised adults (Golan et al, 2005; Mantadakis et al, 2018)
The specificity of droplet digital PCR (ddPCR) for C. albicans DNA detection was evaluated with the DNA extractions from C. tropicalis, C. parapsilosis, C. krusei, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Klebsiella pneumoniae using the same primer set for C. albicans assay
Designed ddPCR Assay Is Specific to C. albicans

Summary

INTRODUCTION

Candidemia is a leading cause of fungal infections among neonates and infants, and it affects immunocompromised adults (Golan et al, 2005; Mantadakis et al, 2018). The traditional procedure for diagnosing candidemia relies on blood cultures for the isolation of Candida spp. This method has low sensitivity and requires large volumes of blood (Clancy and Nguyen, 2013; Pappas et al, 2018). Quantitative PCR (qPCR)-based methods for DNA detection have become more popular than other non-culture methods They facilitate the rapid diagnosis of bloodstream fungal infections, allowing for the initiation of species-oriented therapy as soon as 6 h after the onset of sepsis (Mcmullan et al, 2008). To further optimize the ddPCR assay for the detection of Candida spp., the diagnostic performance of ddPCR assay was compared with conventional culture and qPCR assays using blood samples from mice with experimental candidemia and patients with suspected candidemia. To avoid the risk of false positive results due to laboratory contamination, all the experimental setups were performed in a biosafety cabinet

MATERIALS AND METHODS

Ethics Statement

RESULTS

DISCUSSION

ETHICS STATEMENT