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Reverse Transcription-Quantitative real-time PCR (RT-qPCR) Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment.

Authors
  • Freitas, Bryanna Lexus1
  • Leach, Lynn1
  • Chaturvedi, Vishnu1
  • Chaturvedi, Sudha1, 2
  • 1 Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, NY 12208.
  • 2 Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY 12222.
Type
Published Article
Journal
Journal of Clinical Microbiology
Publisher
American Society for Microbiology
Publication Date
Dec 08, 2021
Identifiers
DOI: 10.1128/JCM.00779-21
PMID: 34878804
Source
Medline
Language
English
License
Unknown

Abstract

Ongoing healthcare-associated outbreaks of 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 detect live C. auris in healthcare environments rapidly. 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 detectable in heat- and ethanol-killed C. auris cells. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT-qPCR reaction. Validation studies yielded positive 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 cycle threshold (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. Candida auris RT-qPCR assay could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.

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