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Published on: December 28, 2017
Identification of Candida auris and related species by multiplex PCR based on unique GPI protein-encoding genes
María Alvarado1, Joaquín Bartolomé Álvarez2, Shawn R Lockhart3
1Regional Center for Biomedical Research, Castilla-La Mancha Science & Technology Park, University of Castilla-La Mancha, Albacete, Spain.
Background:
The pathogen Candida auris is rapidly gaining clinical importance because of its resistance to antifungal treatments and its persistence in hospital environments. Early and accurate diagnosis of C. auris infections is crucial, and however, the fungus has often been misidentified by commercial systems.
Objectives:
To develop conventional and real-time PCR methods for accurate and rapid identification of C. auris and its discrimination from closely related species by exploiting the uniqueness of certain glycosylphosphatidylinositol (GPI)-modified protein-encoding genes.
Methods:
Species-specific primers for two unique putative GPI protein-encoding genes per species were designed for C. auris, C. haemulonii, C. pseudohaemulonii, C. duobushaemulonii, C. lusitaniae and C. albicans. Primers were blind tested for their specificity and efficiency in conventional and real-time multiplex PCR set-up.
Results:
All primers combinations showed excellent species specificity. In multiplex mode, correct identification was aided by different-sized amplicons for each species. Efficiency of the C. auris primers was validated using a panel of 155 C. auris isolates, including all known genetically diverse clades. In real-time multiplex PCR, different melting points of the amplicons allowed the distinction of C. auris from four related species. C. auris limit of detection was 5 CFU/reaction with a threshold value of 32. The method was also able to detect C. auris in spiked blood and serum.
Conclusions:
PCR identification based on unique GPI protein-encoding genes allows for accurate and rapid species identification of C. auris and related species without need for expensive equipment when applied in conventional PCR set-up.
Insights
This study developed a rapid and accurate PCR method for identifying Candida auris, a drug-resistant pathogen. The new technique effectively distinguishes C. auris from related species, aiding in timely diagnosis and treatment.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Candida auris is an emerging multidrug-resistant fungal pathogen causing significant clinical concern.
- Accurate and early diagnosis of C. auris is critical due to its high persistence in healthcare settings and resistance to antifungals.
- Conventional diagnostic systems frequently misidentify C. auris, hindering effective patient management.
Purpose of the Study:
- To develop novel conventional and real-time PCR assays for the precise and swift identification of Candida auris.
- To differentiate C. auris from closely related species by targeting unique glycosylphosphatidylinositol (GPI)-modified protein-encoding genes.
- To establish a reliable diagnostic tool for C. auris detection in clinical samples.
Main Methods:
- Design and synthesis of species-specific primers targeting unique GPI protein-encoding genes for C. auris and related species (C. haemulonii, C. pseudohaemulonii, C. duobushaemulonii, C. lusitaniae, C. albicans).
- Validation of primer specificity and efficiency using conventional and real-time multiplex PCR.
- Testing the C. auris primers against a diverse panel of 155 isolates and in spiked blood and serum samples.
Main Results:
- All primer sets demonstrated excellent species specificity in both conventional and real-time multiplex PCR formats.
- Multiplex PCR enabled correct identification through distinct amplicon sizes for each species.
- Real-time PCR distinguished C. auris from four related species based on melting point differences, with a limit of detection as low as 5 CFU/reaction.
- The method successfully detected C. auris in spiked blood and serum specimens.
Conclusions:
- PCR-based identification utilizing unique GPI protein-encoding genes provides accurate and rapid species identification for C. auris and related fungi.
- The developed conventional PCR method is cost-effective, requiring no specialized equipment.
- This diagnostic approach facilitates timely and reliable detection of C. auris infections, crucial for infection control and patient care.
