A high-throughput and rapid method for accurate identification of emerging multidrug-resistant Candida auris

Abstract

Candida auris is an emerging multidrug-resistant yeast associated with invasive infection in healthcare settings. Recently, C auris cases in the United States have been detected in 11 states with the majority of cases in New York, New Jersey and Illinois. Rapid and accurate identification of C auris is critical for patient care and the implementation of public health measures to control the spread of infection. Our aim was to develop and validate a rapid DNA extraction method using the Roche MagNA Pure 96 instrument and a TaqMan real-time PCR assay for reliable, high-throughput identification of C auris. We evaluated 247 patient dermal swab samples previously analysed by culture/MALDI-TOF. The diagnostic sensitivity and specificity were 93.6% and 97.2%, respectively. The assay was highly reproducible with a detection limit of 1 C auris CFU/10 μL. A receiver operating characteristic curve analysis of the real-time PCR data showed an area of 0.982 under the curve, with a C_T cut-off value of ≤37.0. The turnaround time from DNA extraction to real-time PCR results was approximately 200 samples/day. In conclusion, we successfully validated a rapid and high-throughput method for accurate and reproducible identification of C auris with a significantly reduced turnaround time compared to culture/MALDI-TOF based methods.

Keywords: Candida auris; TaqMan real-time PCR; high-throughput rapid DNA extraction.

FIGURE 1

Instrument response: evaluation of instrument response for real-time PCR assay. Five dilutions (from 5 × 10⁶ to 5 × 10² CFU/mL) were prepared for evaluation of the instrument response and analytical sensitivity of real-time PCR assay. The instruments showed mean 3.30-fold (standard deviation: 0.415) increase in C_T value with every 10-fold dilution

FIGURE 2

Assay sensitivity: real-time PCR assay sensitivity. Five dilutions (from 5 × 10³ to 10 CFU/mL) were evaluated with twenty replicates of each dilutions. The assay was highly reproducible with a limit of detection (LOD) of 100 Candida auris CFU/mL or 1 C auris CFU/10 μL

FIGURE 3

Distribution of C_T value: C_T value distribution for Candida auris positive and negative samples. A centerline across the boxes indicates the median. Lower and upper boxes indicating the 25th percentile to the 75th percentile. Whiskers caps represent the maximum and minimum C_T values

FIGURE 4

Receiver operating characteristic (ROC) curve: a ROC curve analysis of the real-time PCR data showed an area of 0.982 under the curve, with a C_T value cut-off of ≤37.0