Reverse Transcription-Quantitative Real-Time PCR (RT-qPCR) Assay for the Rapid Enumeration of Live Candida auris Cells from the Health Care Environment.

Ongoing health care-associated outbreaks of the multidrug-resistant yeast Candida auris have prompted the development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcription (RT)-quantitative real-time PCR (RT-qPCR) assay to rapidly detect live C. auris in health care environments. This assay targeted the internal transcribed spacer 2 (ITS2) ribosomal gene by obtaining pure RNA followed by reverse transcription (ITS2 cDNA) and qPCR. ITS2 cDNA was not detectable in bleach-killed cells but was detectable in heat- and ethanol-killed C. auris cells. The assay was highly sensitive, with a detection limit of 10 CFU per RT-qPCR. Validation studies yielded positive cycle threshold (CT) values from sponge matrix samples spiked with 102 to 105 CFU of live C. auris, while dead (bleach-killed) C. auris (105/mL) or other live Candida species (105/mL) had no CT values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT-qPCR assay, confirming the concordance between culture and the PCR assay. The RT-qPCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples. The Candida auris RT-qPCR assay could be an invaluable tool in surveillance efforts to control the spread of live C. auris in health care environments.

A description of the first Candida auris-colonized individuals in New York State, 2016-2017.

Candida auris (C. auris) is a globally emerging multidrug-resistant yeast. New York State (NYS) first detected C. auris in July 2016 and is the state most affected. This brief report describes characteristics of the first 114 individuals colonized with C. auris identified through active surveillance/screening by NYS Department of Health. "Colonized/screened" individuals were old (median age, 74 year), had extensive health care exposures and underlying conditions (multiple health care facility admissions in the 90 days prior with more than 80% requiring mechanical ventilation), and had 30- and 90-day mortality rates of 17.5% and 37.7%, respectively (with approximately 60% expired in the 2-year follow-up period). This description is helpful to inform additional prevention measures and add to the collective understanding of C. auris in the United States.

Laboratory Analysis of an Outbreak of Candida auris in New York from 2016 to 2018: Impact and Lessons Learned.

Candida auris is a multidrug-resistant yeast which has emerged in health care facilities worldwide; however, little is known about identification methods, patient colonization, environmental survival, spread, and drug resistance. Colonization on both biotic (patients) and abiotic (health care objects) surfaces, along with travel, appear to be the major factors for the spread of this pathogen across the globe. In this investigation, we present laboratory findings from an ongoing C. auris outbreak in New York (NY) from August 2016 through 2018. A total of 540 clinical isolates, 11,035 patient surveillance specimens, and 3,672 environmental surveillance samples were analyzed. Laboratory methods included matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast isolate identification, real-time PCR for rapid surveillance sample screening, culture on selective/nonselective media for recovery of C. auris and other yeasts from surveillance samples, antifungal susceptibility testing to determine the C. auris resistance profile, and Sanger sequencing of the internal transcribed spacer (ITS) and D1/D2 regions of the ribosomal gene for C. auris genotyping. Results included (a) identification and confirmation of C. auris in 413 clinical isolates and 931 patient surveillance isolates as well as identification of 277 clinical cases and 350 colonized cases from 151 health care facilities, including 59 hospitals, 92 nursing homes, 1 long-term acute care hospital (LTACH), and 2 hospices, (b) successful utilization of an in-house developed C. auris real-time PCR assay for the rapid screening of patient and environmental surveillance samples, (c) demonstration of relatively heavier colonization of C. auris in nares than in the axilla/groin, and (d) predominance of the South Asia clade I with intrinsic resistance to fluconazole and elevated MIC to voriconazole (81%), amphotericin B (61%), flucytosine (5FC) (3%), and echinocandins (1%). These findings reflect greater regional prevalence and incidence of C. auris and the deployment of better detection tools in an unprecedented outbreak.

Assessment of environmental and occupational exposure while working with multidrug resistant (MDR) fungus Candida auris in an animal facility.

In less than a decade since its identification in 2009, the emerging fungal pathogen Candida auris has become a major public health threat due to its multidrug resistant (MDR) phenotype, high transmissibility, and high mortality. Unlike other Candida species, C. auris has acquired high levels of resistance to an already limited arsenal of antifungals. As an emerging pathogen, there are currently a limited number of documented murine models of C. auris infection. These animal models use inoculums as high as 107-108 cells per mouse, and the environmental and occupational exposure of working with these models has not been clearly defined. Using real-time quantitative polymerase chain reaction (PCR) and culture, we monitored the animal holding room as well as the procedure room for up to 6 months while working with an intravenous model of C. auris infection. This study determined that shedding of the organism is dose-dependent, as detectable levels of C. auris were detected in the cage bedding when mice were infected with 107 and 108 cells, but not with doses of 105 and 106 cells. Autoclaving bedding in closed micro-isolator cages was found to be an effective way to minimize exposure for animal caretakers. We found that tissue necropsies of infected mice were also an important source of potential source exposure to C. auris. To mitigate these potential exposures, we implemented a rigorous "buddy system" workflow and a disinfection protocol that uses 10% bleach followed by 70% ethanol and can be used in any animal facility when using small animal models of C. auris infection.