Use of a new PCR to identify Candida species isolated directly from a positive blood culture

Abstract number: P724

Roselló E., Loaiza N., Rodriguez V., Codina G., Andreu A., Planes A.M.

Objective: Evaluation of a multiplex real-time PCR to identify Candida species from positive blood cultures.

Materials and Methods: A PCR technique to identify Candida was evaluated using: a) the fungus-specific universal primer pair, ITS3 and ITS4, to amplify a rDNA region (a portion of the 5.8S region, the ITSII region and a portion of the 28S region); b) five species-specific TaqMan probes to identify the most frequent species isolated in clinical samples (C. albicans, C. parapsilosis, C. glabrata, C. tropicalis and C. krusei); and c) a generic Candida TaqMan probe. The PCR was used on the supernatant of positive blood cultures shown to contain yeasts by Gram staining. DNA extraction was made using the EZ1 automatic system (Blood DNA protocol, Quiagen). For the PCR reaction, 5 ml from the extraction were transferred to a tube containing C. albicans, C. parapsilosis and C. glabrata species-specific TaqMan probes; if negative, the procedure was repeated with C. tropicalis and C. krusei species-specific TaqMan probes. Finally, if there was no amplification, the reaction was repeated with the generic Candida TaqMan probe.

Results: 73 blood cultures from 63 patients (32 males/31 females; 51 adults/12 children) were used. Patients were admitted to ICU (43%), surgery (28.5%), haematology and oncology (8%) and internal medicine wards (20.6%). Positive blood cultures were subcultured on blood agar, Sabouraud agar and CHROMagar Candida. Subcultures resulted in: 61 pure cultures, 12 mixed (yeast and bacteria) and 1 mixed to two Candida species. The species identified from different cultures were: 29 C. albicans, 19 C. parapsilosis, 18 C. glabrata, 6 C. tropicalis, 1 C. lipolytica and 1 C. guilliermondii. The first PCR (C. albicans, C. parapsilosis and C. glabrata species-specific TaqMan probes) correctly identified all C. albicans and C. parapsilosis, and 16 out of 18 C. glabrata. The second PCR identified all 6 C. tropicalis. The universal Candida PCR detected the remaining two species (C. lipolytica and C. guilliermondii). This PCR detected and identified, directly from blood culture, 94.6% of yeast causing fungaemia.

Conclusion: The proven real-time PCR permits the detection and identification of species causing candidaemia in less than three hours, while the conventional systems take at least 48 hours.