Multidrug-Resistant Candida auris Misidentified as Candida haemulonii: Characterization by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and DNA Sequencing and Its Antifungal Susceptibility Profile Variability by Vitek 2, CLSI Broth Microdilution, and Etest Method

Abstract

Candida auris is a multidrug-resistant yeast that causes a wide spectrum of infections, especially in intensive care settings. We investigated C. auris prevalence among 102 clinical isolates previously identified as Candida haemulonii or Candida famata by the Vitek 2 system. Internal transcribed spacer region (ITS) sequencing confirmed 88.2% of the isolates as C. auris, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) easily separated all related species, viz., C. auris (n = 90), C. haemulonii (n = 6), C. haemulonii var. vulnera (n = 1), and Candida duobushaemulonii (n = 5). The in vitro antifungal susceptibility was determined using CLSI broth microdilution (CLSI-BMD), the Vitek 2 antifungal susceptibility test, and the Etest method. C. auris isolates revealed uniformly elevated fluconazole MICs (MIC50, 64 μg/ml), and an alarming percentage of isolates (37%) exhibited elevated caspofungin MICs by CLSI-BMD. Notably, 34% of C. auris isolates had coexisting elevated MICs (≥2 μg/ml) for both fluconazole and voriconazole, and 10% of the isolates had elevated coexisting MICs (≥2 μg/ml) to two additional azoles, i.e., posaconazole and isavuconazole. In contrast to reduced amphotericin B MICs by CLSI-BMD (MIC50, 1 μg/ml) for C. auris, elevated MICs were noted by Vitek 2 (MIC50, 8 μg/ml), which were statistically significant. Candida auris remains an unnoticed pathogen in routine microbiology laboratories, as 90% of the isolates characterized by commercial identification systems are misidentified as C. haemulonii. MALDI-TOF MS proved to be a more robust diagnostic technique for rapid identification of C. auris. Considering that misleading elevated MICs of amphotericin B by the Vitek AST-YS07 card may lead to the selection of inappropriate therapy, a cautionary approach is recommended for laboratories relying on commercial systems for identification and antifungal susceptibility testing of rare yeasts.

FIG 1

Phylogenetic tree based on partial ITS sequences of Indian C. auris (n = 90), C. duobushaemulonii (n = 5), C. haemulonii (n = 6), and C. haemulonii var. vulnera (n = 1) isolates using neighbor-joining analysis with 2,000 bootstrap replications. Sequences of reference strains of Candida haemulonii (CBS 5150, Portugal; CBS 7801, United States), C. duobushaemulonii (CBS 7799, United States), and C. haemulonii var. vulnera (CNMCL-7462, Spain) along with Japanese (JCM 15448^T) and Korean (KCTC-17809 and KCTC-17810) C. auris isolates were retrieved from GenBank for the analysis. Bootstrap values are shown above the branches.

FIG 2

Score-oriented dendrogram of the main spectra (MALDI Biotyper 3.1; Bruker Daltonics) by using average linkages clustering the MALDI-TOF spectra of Indian Candida auris (n = 90) along with the C. haemulonii (n = 6), C. duobushaemulonii (n = 5), and C. haemulonii var. vulnera (n = 1) isolates. Candida auris (DSM 21092^T, KCTC-17809, and KCTC-17810) and C. pseudohaemulonii (JCM 12453^T and CBS 10004), C. haemulonii (CBS 5150 and CBS 5149^T), C. haemulonii var. vulnera (CL-7073), and C. duobushaemulonii (CBS 7800 and CBS 7799) were added to make the clustering in MALDI-TOF MS more robust. The isolates were classified into 4 phylogroups. The inset depicts the variation among Indian, Japanese, and Korean C. auris isolates leading to two clusters in phylogroup 4.