Development of a Novel Method for the Clinical Visualization and Rapid Identification of Multidrug-Resistant Candida auris

Abstract

Outbreaks of multidrug-resistant Candida auris infections, associated with a mortality rate of 30% to 60%, are of serious global concern. Candida auris demonstrates high transmission rates in hospital settings; however, its rapid and accurate identification using currently available clinical identification techniques is challenging. In this study, we developed a rapid and effective method for detecting C. auris based on recombinase-aided amplification combined with lateral flow strips (RAA-LFS). We also screened the appropriate reaction conditions. Furthermore, we investigated the specificity and sensitivity of the detection system and its ability to distinguish other fungal strains. Candida auris was accurately identified and differentiated from related species at 37°C within 15 min. The minimum detection limit was 1 CFU (or 10 fg/reaction) and was not affected by high concentrations of related species or host DNA. The simple and cost-efficient detection method established in this study exhibited high specificity and sensitivity and successfully detected C. auris in simulated clinical samples. Compared with other traditional detection methods, this method greatly reduces the time and cost of testing and is thus suitable for hospitals or clinics in remote underfunded areas for screening C. auris infection and colonization. IMPORTANCE Candida auris is a highly lethal, multidrug-resistant, invasive fungus. However, conventional methods of C. auris identification are time-consuming and laborious and have low sensitivity and high error rates. In this study, a new molecular diagnostic method based on recombinase-aided amplification combined with lateral flow strips (RAA-LFS) was developed, and accurate results could be obtained by catalyzing the reaction at body temperature for 15 min. This method can be used for rapid clinical detection of C. auris, consequently saving valuable treatment time for patients.

Keywords: Candida auris; lateral flow strip; rapid detection; recombinase-aided isothermal amplification assay.

FIG 1

Screening of primer pairs according to their RAA performance. Agarose gel image analysis illustrates the amplicons of primer pairs targeting the ITS gene. Each primer pair is labeled above each lane. The DNA ladder band size is indicated. Primer dimers are indicated by white arrows. NTC, no-template control (numbered for each respective primer pair).

FIG 2

RAA-LFS test results using ITS-10′ primer-probe sets. Lateral flow strip (LFS) results of recombinase-mediated isothermal nucleic acid amplification (RAA) using different primer-probe sets. The Candida auris genomic DNA was used as the template, and reactions were performed at 39°C for 15 min. The name of each primer-probe set is indicated at the top of each strip. NTC, no-template control. The positions of test and control lines are marked on the right.

FIG 3

Fragments targeted by various primer-probe combinations. The ITS sequence fragments of three closely related strains, Candida pseudohaemulonii, C. duobushaemulonii, and C. haemulonii, were compared with the ITS sequence of C. auris. Primer and probe sequences are indicated under the aligned ITS sequences. Arrow lines indicate the extension direction of primers and probes. Tetrahydrofuran (THF) sites are indicated by the letter “H.”

FIG 4

Detection of standard reference strains of Candida auris. LFS results of RAA of different genomic DNA templates. Strain names are indicated on top of each strip. NTC, no-template control. The positions of control and test lines are indicated on the right of the image. All reactions were performed at 37°C for 15 min.

FIG 5

Differentiation among common pathogens. Results of RAA-LFS detection of Candida haemulonii, C. pseudohaemulonii, and C. duobushaemulonii strains (A) and other related species and common pathogenic bacteria (B). The strains are indicated at the top of each strip. NTC, no-template control. The positions of control and test lines are indicated on the right of the image. All reactions were performed at 37°C for 15 min.

FIG 6

Testing the RAA-LFS system using human blood samples. (A) Detection limits of the RAA-LFS system for Candida auris mixed into human blood samples. (B) The detection limit of C. auris DNA by the RAA-LFS system after mixing with human blood DNA at a 1:10 ratio. NTC, no-template control. The positions of the control and test lines are indicated on the right of the image. All reactions were performed at 37°C for 15 min.