Development and Validation of a Real-Time PCR Assay for Rapid Detection of Candida auris from Surveillance Samples

Abstract

Candida auris is an emerging multidrug-resistant yeast causing invasive health care-associated infection with high mortality worldwide. Rapid identification of C. auris is of primary importance for the implementation of public health measures to control the spread of infection. To achieve these goals, we developed and validated a TaqMan-based real-time PCR assay targeting the internal transcribed spacer 2 (ITS2) region of the ribosomal gene. The assay was highly specific, reproducible, and sensitive, with the detection limit of 1 C. auris CFU/PCR. The performance of the C. auris real-time PCR assay was evaluated by using 623 surveillance samples, including 365 patient swabs and 258 environmental sponges. Real-time PCR yielded positive results from 49 swab and 58 sponge samples, with 89% and 100% clinical sensitivity with regard to their respective culture-positive results. The real-time PCR also detected C. auris DNA from 1% and 12% of swab and sponge samples with culture-negative results, indicating the presence of dead or culture-impaired C. auris The real-time PCR yielded results within 4 h of sample processing, compared to 4 to 14 days for culture, reducing turnaround time significantly. The new real-time PCR assay allows for accurate and rapid screening of C. auris and can increase effective control and prevention of this emerging multidrug-resistant fungal pathogen in health care facilities.

Keywords: Candida auris; TaqMan chemistry; assay validation; real-time PCR assay; surveillance samples.

FIG 1

Real-time PCR assay sensitivity. Candida auris cell dilution series containing 10⁻¹ to 10⁵ CFU/PCR reaction were run in duplicate on three different days. The slopes and correlation coefficients (R²) are shown. The detection limit of the assay was 1 C. auris CFU/PCR reaction.

FIG 2

ROC curve analysis of C. auris real-time PCR assay for patient (swab) surveillance samples. A ROC curve was plotted by calculating the sensitivity and specificity of the real-time PCR C_T values compared to culture results for swabs.

FIG 3

ROC curve analysis of C. auris real-time PCR assay for environmental (sponge) surveillance samples. A ROC curve was plotted by calculating the sensitivity and specificity of the real-time PCR C_T values compared to culture results for sponges.

FIG 4

C. auris DNA detection in swab and sponge samples by real-time PCR assay. Crude DNA from surveillance samples was extracted by the bead-beating method, and 5 μl of extracted DNA was subjected to the real-time PCR assay. Results are shown as a scatter plot based on C_T counts for surveillance samples either positive or negative by culture. Error bars indicate SDs. The mean C_T values between culture-positive and culture-negative samples for swabs and sponges were statistically significant (P < 0.05).