Background: Candida auris is an emerging multidrug resistant yeast species causing fatal outbreaks throughout the world. In routine laboratories, this pathogen is normally neglected and misidentified as C. haemulonii, C. famata, C. sake, C. parapsilosis etc, due to the lack of specificity of the used phenotypic assays. Additionally, there are no FDA approved spectra for the public MALDI-TOF databases. Herein, we present a SYBR Green I-based qPCR assay to identify and distinguish C. auris, C. haemulonii, C. duobushaemulonii, and C. pseudohaemulonii by using melt-curve analysis.

Methods: In order to design species-specific primers, target regions were retrieved from the NCBI database. Validation and performance of the optimized assay was carried out using 150 C. auris strains from different geographical regions, 25 C. haemulonii strains, 6 C. duobushaemulonii, and 3 C. pseudohaemulonii strains. Afterwards, a broad range of yeast species, Aspergillus spp., and human DNA were utilized for specificity testing. Limit of detection (LOD), Efficiency, and R2 values were calculated.

Results: Our developed qPCR successfully distinguished and identified C. auris, C. haemulonii, C. duobushaemulonii, and C. pseudohaemulonii (Figure 1). No cross-reactivity with other yeast species, Aspergillus spp., and human DNA was observed, resulting in 100% specificity. The multiplex qPCR successfully distinguished one C. haemulonii strain, that had been misreported as C. pseudohaemulonii (CBS 12371). Analytical sensitivity revealed that the four species could be detected at the level of 10 CFU.

Conclusion: Due to the validation with an extensive number of C. auris strains and specificity testing by a broad range of yeast species and Aspergillus spp., our qPCR assay has the potential to be exploited in routine laboratories. Additionally, the high difference in melting temperatures among the four species, low limit of detection, and no cross-reactivity with human DNA and other fungi, suggest that it may be used for diagnosis of relevant species from clinical samples such as blood.

1 / Multiplex qPCR for rapid identification of Candida auris, C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii