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J Med Microbiol 56 (2007), 964-970; DOI: 10.1099/jmm.0.47149-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR

L. Metwally1,{dagger}, G. Hogg1, P. V. Coyle1, R. J. Hay2, S. Hedderwick3, B. McCloskey4, H. J. O'Neill1, G. M. Ong1, G. Thompson1,4, C. H. Webb1 and R. McMullan1

1 Department of Medical Microbiology, Royal Victoria Hospital, Belfast, Northern Ireland

2 Queen's University of Belfast, School of Medicine and Dentistry, Belfast, Northern Ireland

3 Department of Infectious Diseases, Royal Victoria Hospital, Belfast, Northern Ireland

4 Regional Intensive Care Unit, Royal Victoria Hospital, Belfast, Northern Ireland

Correspondence
L. Metwally
lobna.metwally{at}bll.n-i.nhs.uk

Received 3 January 2007
Accepted 5 March 2007

In view of both the delay in obtaining identification by conventional methods following blood-culture positivity in patients with candidaemia and the close relationship between species and fluconazole (FLC) susceptibility, early speciation of positive blood cultures has the potential to influence therapeutic decisions. The aim was to develop a rapid test to differentiate FLC-resistant from FLC-sensitive Candida species. Three TaqMan-based real-time PCR assays were developed to identify up to six Candida species directly from BacT/Alert blood-culture bottles that showed yeast cells on Gram staining at the time of initial positivity. Target sequences in the rRNA gene complex were amplified, using a consensus two-step PCR protocol, to identify Candida albicans, Candida parapsilosis, Candida tropicalis, Candida dubliniensis, Candida glabrata and Candida krusei; these are the most commonly encountered Candida species in blood cultures. The first four of these (the characteristically FLC-sensitive group) were identified in a single reaction tube using one fluorescent TaqMan probe targeting 18S rRNA sequences conserved in the four species. The FLC-resistant species C. krusei and C. glabrata were detected in two further reactions, each with species-specific probes. This method was validated with clinical specimens (blood cultures) positive for yeast (n=33 sets) and the results were 100 % concordant with those of phenotypic identification carried out concomitantly. The reported assay significantly reduces the time required to identify the presence of C. glabrata and C. krusei in comparison with a conventional phenotypic method, from ~72 to <3 h, and consequently allows optimization of the antifungal regimen at an earlier stage.

Abbreviations: FLC, fluconazole; ITS, internal transcribed spacer; PNA-FISH, peptide nucleic acid fluorescent in situ hybridization.

{dagger}Present address: Regional Virus Laboratory, Royal Hospitals, Grosvenor Road, Belfast BT12 6BA, UK.




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L. Metwally, D. J. Fairley, P. V. Coyle, R. J. Hay, S. Hedderwick, B. McCloskey, H. J. O'Neill, C. H. Webb, W. Elbaz, and R. McMullan
Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR
J. Med. Microbiol., March 1, 2008; 57(3): 296 - 303.
[Abstract] [Full Text] [PDF]


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