This investigation provides further evidence for the role of ET biofilm in VAP. The difficulty in eradicating an established microbial biofilm using antibiotics implies that increased attention must be directed towards modification of the ET to prevent or substantially reduce biofilm formation.

These data suggest that the described assays perform well in this population for enhancing the diagnosis of candidemia. The extent to which they may affect clinical outcomes, prescribing practice, and cost-effectiveness of care remains to be ascertained.

No abstract

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood. INTRODUCTIONBlood culture remains the accepted 'gold standard' method for diagnosis of candidiasis and can be highly sensitive. However, in some settings, culture can fail to detect Candida spp. in more than 50 % of patients with chronic disseminated candidiasis MoreiraOliveira et al., 2005). Molecular methods, particularly realtime PCR, are increasingly used alongside conventional microbiological techniques for the diagnosis of systemic candidiasis (Klingspor & Jalal, 2006; Kasai et al., 2006; Löffler et al., 2000;Schabereiter-Gurtner et al., 2007;Bretagne & Costa., 2005;White et al., 2006;Maaroufi et al., 2003; Moreira-Oliveira et al., 2005). However, two major limitations of PCR are the difficulty associated with breaking yeast cell walls to release Candida spp. DNA suitable for amplification (Maaroufi et al., 2004; Löffler et al., 1997) and limited sensitivity when the assays are adapted for testing clinical specimens, such as blood samples, in which the amounts of fungal DNA may be very low (White et al., 2003; Löffler et al., 2000;Ahmad et al., 2002;Challier et al., 2004). As a consequence, these tests are not yet recognized as part of the consensus diagnostic criteria, in contrast to some antigenaemia detection kits (Ascioglu et al., 2002).The yeast cell is notoriously difficult to lyse due to a highly complex cell wall structure that provides rigidity (Müller et al., 1998). Moreover, it has been suggested that the fungal load in blood from ...

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 (n533 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.