María Alvarado1, Joaquín Bartolomé Álvarez2, Shawn R Lockhart3, Eulogio Valentín4, Alba Cecilia Ruiz-Gaitán5, Elena Eraso6, Piet W J de Groot1. 1. Regional Center for Biomedical Research, Castilla-La Mancha Science & Technology Park, University of Castilla-La Mancha, Albacete, Spain. 2. Complejo Hospitalario Universitario de Albacete, Servicio de Salud de Castilla-La Mancha, Albacete, Spain. 3. Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA. 4. GMCA Research Unit, Departamento de Microbiología y Ecología, Universidad de Valencia, Burjassot, Spain. 5. Severe Infection Research Group, Health Research Institute La Fe, Valencia, Spain. 6. Departamento de Inmunología, Microbiología y Parasitología, Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), Bilbao, Spain.
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.
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.
Authors: Hala Najeeb; Sarush Ahmed Siddiqui; Zahra Anas; Syed Hasan Ali; Shajie Ur Rehman Usmani; Fareeha Jawed; Hafsa Nazir Jatoi Journal: Diseases Date: 2022-08-29