1887

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Volume 56, Issue 7

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

Abstract

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 species. Three TaqMan-based real-time PCR assays were developed to identify up to six 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 , , , , and ; these are the most commonly encountered 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 and were detected in two further reactions, each with species-specific probes. This method was validated with clinical specimens (blood cultures) positive for yeast (=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 and in comparison with a conventional phenotypic method, from ∼72 to <3 h, and consequently allows optimization of the antifungal regimen at an earlier stage.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): FLC, fluconazole , ITS, internal transcribed spacer and PNA-FISH, peptide nucleic acid fluorescent in situ hybridization
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2007-07-01
2024-05-01
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Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR
J. Med. Microbiol. 56, 964 (2007); https://doi.org/10.1099/jmm.0.47149-0
/content/journal/jmm/10.1099/jmm.0.47149-0
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