Development and evaluation of a TaqMan based qPCR assay for specific quantification of Candida auris

Hadis Jafarian1, Hossein Khodadadi1*, Parisa Badiee2
1Department of Medical Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; 2 Prof. Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
*Correspondence: Hossein Khodadadi: h_khodadadi@sums.ac.ir 

ABSTRACT
Objective: Candida auris is multidrug-resistant yeast causing invasive nosocomial infections. This emerging opportunistic pathogen has been rapidly spread across the world. Although standard microbiologic methods commonly misidentify C.auris as other yeast, Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method has made precise identification of the yeast possible. In the lack of access to the MALDI-TOF in routine clinical laboratories, rapid and accurate methods are demanded for detecting and identifying of C. auris.  Thus, we developed and validated a quantitative real-time PCR (qPCR) assay targeting the internal transcribed spacer 2 (ITS2) region of the ribosomal gene of C. auris. 
Method: 
The internal transcribed spacer2 (ITS2) region of the nuclear ribosomal DNA of C. auris and other related yeasts were analyzed for finding an amplifiable specific target in C.auris. A123 base pair target was selected and primers and probe designed according to TaqMan chemistry. Serial dilutions of counted targets containing from 105 to 100 CFU of the yeast were used to establish a standard curve for quantifying the yeast. The qPCR reaction was based on the simultaneous detection of a specific ITS target and also contained an internal control to compensate for variations in DNA extraction and the various compounds from human that inhibit PCR.
Results:
The qPCR assay was able to identify and quantify C.auris with the detection limit of one C. auris CFU per reaction. The specificity was confirmed by the lack of amplification of other candida species, other yeast and molds, bacteria and human DNA.  A qPCR using DNA extracted from a suspension contains one CFU of C. auris resulted in steady Ct-value (Ct) of 34. The assays resulted in a standard curve showed a highly significant linearity between the Ct-values and the dilution rates (R2 = 0.99; slope = −3.32).
 
Conclusion: The TaqMan qPCR assay could rapidly and accurately identify and quantify emerging opportunistic C. auris from a wider variety of specimen.  The Assay time considering sample processing and DNA extraction would take less than 4 h with greater sensitivity and specificity in comparison with microbiological based identification and conventional PCR.  This method shows great promise as a tool for rapid diagnosing exposures to C. auris in clinical laboratories.