Identification of Candida auris and related species by multiplex PCR based on unique GPI protein-encoding genes

Abstract

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.

Keywords: Candida; Candida auris; C. haemulonii complex; PCR identification; candidosis; yeasts.

FIGURE 1

Specificity testing of PCR amplification using primers designed for unique GPI protein-encoding genes. (A) Colony PCR verification of the specificity of C. auris primers using a set of different 17 Candida species, S. cerevisiae and Y. lipolytica. (B-F) PCR cross-reactivity testing of GPI protein gene primers designed for C. haemulonii(B), C. pseudohaemulonii (C), C. duobushaemulonii (D), C. lusitaniae (E) and C. albicans (F). Each PCR reaction contains two species-specific primer pairs and generic Candida/Saccharomyces 5.8S rDNA primers for DNA template quality control

FIGURE 2

Efficiency testing of C. auris-specific primers. PCR was performed using both C. auris-specific primer pairs in a duplex colony PCR. (A and B) Examples of clinical C. auris isolates from a Spanish hospital: (A) bloodstream isolates, (B) isolates from other clinical samples. (C) Examples of C. auris isolates originating from patients in different countries. C. haemulonii was used as negative control. (D, left panel) Multiplex PCR amplification of C. auris and related species using a combination of six species-specific primer pairs that produce different-sized amplicons, and including 5.8S rDNA primers as control. (D, right panel) Identification of negative strains from (A) and (B) using multiplex PCR. (1) was correctly identified as C. albicans, (2) as C. pseudohaemulonii, (3) as C. duobushaemulonii and (4) as C. haemulonii

FIGURE 3

Melting profiles and sensitivity analysis of real-time PCR based on GPI protein-encoding gene amplicons. (A) Different melting temperatures of 0.2 kb amplicons of C. auris and related speciesC. haemulonii,C. pseudohaemulonii,C. duobushaemuloniiandC. lusitaniae. (B) Multiplex real-time colony PCR using a combination of five primer pairs. Shown are representative curves obtained with lysates for the five species tested. (C) Melting curves of a set of representative C. auris isolates from the Spanish outbreak (red curves: blood isolates, n = 17; blue curves: other clinical origin, n = 11) and from different countries (black curves, n = 11). All Tm values are within a window of 79.00°C ± 0.44°C. (D) Real-time PCR sensitivity analysis using dilution series of colony PCR lysates. (E) Real-time PCR sensitivity analysis of dilution series of C. auris lysates in blood, serum or H₂0